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Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods

Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods Abstract Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute’s Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing. genotoxicity, genetic toxicology, nanoparticles, test battery, mutagenicity, clastogenicity, testing strategy Nanomaterials (NMs) generally refer to nano-objects and particles with one or more dimensions in the nanometer size range [a diversity of definitions exists]. NMs present challenges in safety evaluation owing to small size, relatively large surface area, and unknown disposition in biological systems. A working group of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC) has been addressing the genotoxicity assessment of NMs. The group’s evaluation is anchored by a critical review of published primary data that evaluates potential genotoxic effects of NMs in the standard genotoxicity assays. The assays include the bacterial reverse mutation (Ames) assay; in vitro mammalian assays for mutations, chromosomal damage, micronucleus (MN) induction, or DNA strand breaks; and in vivo assays for genetic damage in various target tissues (MN, comet and transgenic mutation assays). This review follows and extends the UK’s Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM)’s review of genotoxicity assessment of NMs and experimental considerations in publications identified between 1992 and early 2012 (COM, 2012). It also builds on the Environmental Mutagenesis and Genomics Society (EMGS) 2010 workshop report (Pfuhler et al., 2013) in which issues related to assay utility and mechanistic endpoints were also addressed. Since our review began, other authors have come to similar assessments of the issues related to genotoxicity testing, using different analyses (Golbamaki et al., 2015; Magdolenova et al., 2014; Swedish Chemicals Agency, 2016). Our work provides a summary and an extensive critique of genotoxicity test data, methods validity, and potential conclusions about mechanisms. From this context we make recommendations for future testing of NMs (summarized in the Roadmap, Figure 1). Figure 1. Open in new tabDownload slide Nanomaterial genotoxicity testing roadmap. Figure 1. Open in new tabDownload slide Nanomaterial genotoxicity testing roadmap. MATERIALS AND METHODS For each genotoxicity assay, the published literature on NMs was analyzed by a team of genetic toxicologists experienced in that assay, who are the coauthors of this article. At a minimum, the teams conducted exhaustive literature searches to identify papers published after COM (2012) and through the end of 2014, using search terms designed to identify papers in diverse biological, physical, and nanotechnology journals. The combination of multiple assays without definition other than “genotoxicity” in many papers made searching difficult, particularly in the more recent literature. For the assays with fewer citations (except in vitro MN), earlier and more recent papers were often included in the analysis; each assay section below explains the range considered and any additional criteria that were part of the analysis. The following issues were considered in the literature evaluation: NM evaluated Type and size Characterization and sample preparation Test system uptake Test systems/assays used Standard methods or other Organization for Economic Co-operation and Development (OECD) Test Guidelines (TG) compliance Metabolic activation Positive controls for the assay Toxicity measurements Test validity (see Tables 1 and 2) Table 1. Summary of Literature Using in Vitro Genotoxicity Tests In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant a Including (as appropriate) S9, CB, No. cells treated, No. replicates/repeats, No. cells scored. b S. typhimurium strains TA1535, TA 97, TA98, TA100, TA102. c For example no significant increase in MN with positive control or data not provided. d CB = cytochalasin B Open in new tab Table 1. Summary of Literature Using in Vitro Genotoxicity Tests In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant a Including (as appropriate) S9, CB, No. cells treated, No. replicates/repeats, No. cells scored. b S. typhimurium strains TA1535, TA 97, TA98, TA100, TA102. c For example no significant increase in MN with positive control or data not provided. d CB = cytochalasin B Open in new tab Table 2. Summary of Literature Using In Vivo Genotoxicity Tests In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. Open in new tab Table 2. Summary of Literature Using In Vivo Genotoxicity Tests In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. Open in new tab Appropriateness of the system for assessment of NMs Need for methods alteration for NM assessment Results (positive or negative) Critique of result based on acceptance criteria and comparison with a concurrent positive and negative control If positive, dynamic range of effect Consistency of results with particular NMs in diverse published papers Insight into potential modes of action (MoAs) In the analysis, experiments were evaluated for adherence to established guidelines/guidance, including the OECD TG, an approach that is critical for interpreting results in a regulatory context. We recognized that sample characterization is of major importance for the assays, particularly analysis of the sample in vehicle and in the test system. However, other than noting whether samples were characterized, comments have not been made on the adequacy of sample preparation or characterization. RESULTS Literature evaluation and methods recommendations for genotoxicity assays are summarized in Tables 1 and 2 (for in vitro and in vivo genotoxicity tests, respectively) and presented by assay in the following sections. Bacterial (Ames) Genotoxicity Assays For the bacterial (Ames) genotoxicity assessment, 26 papers published from 2009 to 2016 (including COM, 2012) were considered (Table 3), and findings summarized in Table 1. Nanomaterials tested included metal oxides (Al-, Cu-, Ti-, Zn-, Ce-, In- [indium], IN tin, Dy- [dysprosium], W- [tungsten]), Fe- (magnetic), TiSi-, elemental metals (Mo [molybdenum], Ag, Cu, gold nanorods) as well as carbon-based (single-walled carbon nanotubes [SWCNTs] and multi-walled carbon nanotube[MWCNTs]), quantum dots, and complex or combination NMs (Au-PMA-ATTO, coated Zn oxides, asbestos, diesel exhaust, polymeric nanocapsules, phospholipids), and organic NMs. Most tests included assessment with and without an exogenous metabolic activation system (S9). Although S9 would usually not be expected to have an effect, the proteins in S9 could affect uptake of the nanoparticles. Some changes in results were reported in the presence of S9 (Gomaa et al., 2013; Hasegawa et al., 2012; Kumar et al., 2011; Liu et al., 2014; Lopes et al., 2012), but these were not consistent among NMs, within a lab, or within a discernible context, eg, metal oxides. Table 3. Bacterial (Ames) Genotoxicity Assay: Papers Evaluated Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Open in new tab Table 3. Bacterial (Ames) Genotoxicity Assay: Papers Evaluated Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Open in new tab Papers were not subjected to acceptance criteria, but were analyzed individually as described in the “Methods” section. Most studies used the standard set of Salmonella typhimurium and/or Escherichia coli tester strains or a subset consisting of TA98 and TA100. All papers incorporated a positive control, but positive and negative controls were often not adequate to determine the impact of methods variations. Most papers utilized a preincubation method, but this varied between 20 min and 20 h. Each paper was analyzed for validity individually, based on bacterial-specific methods required for mutation development, and on response outcomes typical for mutagens. Of major importance for these assays was the question of whether any studies with positive results seem valid. In total 5 out of 26 papers measured uptake of the particles into S. typhimurium standard tester strains (Butler et al., 2014, 2015; Clift et al., 2013; Kumar et al., 2011; Woodruff et al., 2012). The positive results in Kumar et al. (2011) with TiO2 were not replicated in Butler et al. (2014). Photographic evidence for uptake may not be definitive. Eight papers reported positive bacterial mutagenicity results (Clift et al., 2013; Gomaa et al., 2013; Jomini et al., 2012; Kumar et al., 2011; Liu et al., 2014; Lopes et al., 2012; Pan et al., 2010; Sadiq et al., 2015); however, this call was variously based on a 2-fold increase in revertant colonies over the control, a lesser increase that might be called marginal, or a greater increase under unacceptable conditions, ie, starvation (Jomini et al., 2012) or treatment using agar at 55° (Liu et al., 2014), likely to induce heat shock. The analysis provided here finds a lack of convincing validity for most if not all of the reported positives. For example, both Pan et al. (2010) and Kumar et al. (2011) report positive results that were borderline (approximately 2-fold over the control). More concerning is the pattern of response—a constant revertant colony number over a dose range encompassing 3 orders of magnitude, ie, an elevation without a dose response. Lopes et al. (2012) reported background colony counts with TA98 minus S9 as 10 times higher than the values plus S9 (200 vs approximately 20). Clearly there was a systematic error here with the minus S9 cultures. Gomaa et al. (2013) and Sadiq et al. (2015) respectively found a borderline positive only at the highest dose or doses, a limited but more credible result. Jomini et al. (2012) observed positive results with a fluctuation assay after preincubation up to 20 h in saline, a nonnutritive medium designed to minimize conditions impacting agglomeration and enhance association of particles with bacteria. There might be an important concept developed in this article regarding the ionic interactions of particles and cells. However, positive and negative controls did not appear to be subjected to the long preincubation time and thus the effect on the bacteria of a significant departure from standard methods, including starvation conditions, cannot be evaluated (discussed further below). Most concerning is a rationale for all 5 S. typhimurium tester strains testing positive with 1 agent (Dy2O3, in Hasegawa et al., 2012; iron oxide in Liu et al., 2014). Liu et al. (2014) found positive results with 10 nm polyethylene glycol (PEG)-coated iron oxide particles in all 5 strains of S. typhimurium (TA1535, TA97, TA98, TA100, and TA102) plus or minus S9, or with the 30 nm PEG-coated NM in the bacteria only with S9. This is problematic because the strains are designed to respond to different types of DNA interactions leading to different DNA sequence changes; there is little precedent for all of the tester strains responding similarly to one agent. These results indicate a systematic origin of positive responses that is unexplained. There are other issues in the papers reporting positive responses, eg, background mutant colony numbers that are not acceptable (eg, >200 for E. coli WP2) (Pan et al., 2010) and for TA98 as noted above (Lopes et al., 2012), and the lack of a rationale for positives in TA98 but not TA100 (Kumar et al., 2011), ie, results indicating sequence-specific alterations. Few of these studies appeared to confirm the results in a second experiment, a very important requirement for borderline or questionable positive results. When these results were reported, even though they are outside the norms of expectations, they were not discussed as such in the papers. It is possible that a few positives, eg, Cu metallic nanoparticles that showed a borderline dose response at high concentrations (Sadiq et al., 2015) perturb DNA replication, which could result in DNA sequence alterations. Our conclusion is that none of the studies reviewed were entirely credible with regard to induction of mutations in bacteria, and most were clearly not credible. As the scientific community has recognized that the standard bacterial Ames assay testing approach may not be adequate as a component of a test battery for NMs, method variations, including preincubation, expanded exposure times, and concentrated exposure of bacteria in small volumes, have been tried but did not change the negative outcome, eg, Butler et al. (2015). Some methods alterations, such as preincubation for a day in saline, ie, nongrowth medium at 37°, gave positive responses (Jomini et al., 2012), but the method alteration (without informative controls) compromised the validity of the test. A starvation condition induces stress responses and other responses designed to save the population from a nonviable condition. These conditions may lead to mutation generation that is not a result of classical DNA damage (Foster and Cairns, 1992; Wright, 2004). One paper used a nonstandard preincubation method in microtiter plates with generally negative results except for diesel exhaust particulates, which have tested positive in previous studies (Clift et al., 2013), likely due to the leaching of genotoxic polycyclic aromatic hydrocarbons. As noted previously by others (Doak et al., 2012; EFSA Scientific Committee, 2011; Landsiedel et al., 2009; Warheit and Donner, 2010), the gram-negative strains of bacteria used in the standard assays do not appear to have the capability for nanoparticle uptake, lacking mammalian mechanisms of endocytosis, pinocytosis, and phagocytosis. The lack of uptake is considered to be the reason for generally negative outcomes following nanoparticle exposure to bacteria. However, there is also the possibility that bacteria are not capable of the type of response that leads to positive effects with NMs in mammalian cells (eg, an indirect response to oxidative stress involving mitochondria). The negative result of AgNO3 in experiments with Ag nanoparticles indicated that the negative results were not necessarily due to the lack of uptake (Butler et al., 2015). Thus, the current standard bacterial mutagenicity test (Ames Assay) is not recognized as an informative component of a genotoxicity test battery for assessment of NMs. This conclusion is consistent with other reviews (COM, 2012; Vandebriel and De Jong, 2012) and discussions (Doak et al., 2012; Gonzalez et al., 2008; Landsiedel et al., 2009; Oesch and Landsiedel, 2012; Pfuhler et al., 2013), but we have provided an extensive analysis that goes well beyond previous work. In Vitro MN Assay The in vitro MN assay is a standard test system in the genotoxicity battery; however, it has been widely recognized over recent years that simply applying this test according to the OECD test guideline 487 is not wholly appropriate for nanomaterials. Thus, papers applying the in vitro MN assay to assess NMs from 1997 to 2014 (including the COM [2012] analysis) were carefully evaluated for their methodological approaches, as summarized in Table 1. Seventy-nine papers were identified (Table 4), in which 27 NMs had been evaluated consisting of metal oxides (eg, Al-, Cu-, Ce-, Fe-, Si-, Ti-, Y-, and Zn-oxides); metals (Au, Ag); carbon based (fullerenes, SWCNTs, MWCNTs), imogolite); and other combination materials (such as quantum dots, and WC). Table 4. In Vitro MN Assay: Papers Evaluated Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Open in new tab Table 4. In Vitro MN Assay: Papers Evaluated Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Open in new tab For the in vitro MN assay, there were few papers that had evaluated a similar material, making it difficult to draw general conclusions about the ability of NMs to reproducibly induce chromosomal damage in vitro. Both TiO2 and silver Nanoparticles (NPs) were most often reported to show significant increases in micronuclei, but these findings were not consistent and comparisons were difficult since dose ranges, cells used and exposure durations were too variable to reach a definitive conclusion. For most materials assessed, where reports indicated a significant increase in genotoxicity, this was usually only approximately 2- to 3-fold times over negative control values. An inherent problem with trying to reach conclusions regarding NM effects in the in vitro MN assay was the substantial variation in the methodologies applied. As seen in Table 1, numerous cell lines, variations in methods involving cytochalasin B (CB) and cytotoxicity measurements, treatment times, and endpoint analyses were used with the in vitro MN assay. Given the substantial variation in the methodology applied, the protocols used in each paper were critically evaluated and the following criteria were applied for excluding tests from this analysis: CB treatment: studies were excluded if simultaneous cotreatment with the test NM and CB was the only treatment regimen as this is known to hinder cellular uptake (Doak et al., 2009, 2012; Haynes et al., 1996). Reports with negative controls that had MN frequencies above 2%, or those where background MN frequency was not provided. Inappropriate positive controls where no increase in MN frequency was observed with the positive control, or where positive controls were included but data was not provided to allow evaluation of approach (not all studies included positive controls, but this was not used as a specific exclusion criterion as it would have substantially reduced the remaining papers making evaluation of test approaches very limited). Studies using excessively high doses (>500 μg/ml). [The issue of dose selection is considered in the Discussion; the acceptability of dose limits was not universally applied in the data review]. Studies without concurrent toxicity evaluation, an inappropriate toxicity test, or genotoxicity assessed at toxic doses ie, >50% cytotoxicity. Cell number scored was too low or an inappropriate scoring methodology applied. Missing experimental information that prevented full evaluation of methodology applied. From this evaluation, 36 studies were classified as acceptable (46%) (Table 5). However, this includes studies that conducted an evaluation of cytotoxicity using nonstandard approaches; if those studies that did not conduct approved cytotoxicity tests for the in vitro MN assay were removed from consideration, this number would be reduced to 19 acceptable studies (24%) (Table 6). Table 5. In Vitro MN Assay: Acceptable Studies Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Open in new tab Table 5. In Vitro MN Assay: Acceptable Studies Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Open in new tab Table 6. In Vitro MN Assay: Acceptable Studies With Approved Cytotoxicity Tests Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Open in new tab Table 6. In Vitro MN Assay: Acceptable Studies With Approved Cytotoxicity Tests Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Open in new tab When assessing the number of studies that evaluated uptake of the NM into the cell, 34 studies out of the total 79 had considered whether the NMs assessed had been internalized within the test cells (Table 7). However, only 9 of the 19 studies that had conducted an “acceptable” in vitro MN had evaluated uptake of the test NM (Table 8). Some examples of studies following an appropriate study design included Könczöl et al. (2012), Singh et al. (2012), and Migliore et al. (2010). Even with the exclusion criteria applied, there were very few studies that evaluated similar NMs, with the exception of silica and silver. Silica nanoparticles (Downs et al., 2012; Gonzalez et al., 2010; Guidi et al., 2013; Uboldi et al., 2012; Wang et al., 2007b,c) were largely negative (4 out of 6 studies) (Downs et al., 2012; Gonzalez et al., 2010; Guidi et al., 2013; Uboldi et al., 2012), while silver (Jiang et al., 2013; Kim et al., 2011; Li et al., 2012; Nymark et al., 2013) was positive for MN induction (3 out of 4 studies) (Jiang et al., 2013; Kim et al., 2011; Li et al., 2012). Thus, these studies were not unanimous in their findings and no 2 studies were conducted using the same cell line; 32 different cell lines were applied in the 79 papers reviewed. The tremendous variation in approach made it almost impossible to detect trends in the capacity for groups of NMs to induce genotoxicity. It was therefore apparent that standardization in the approaches applied to evaluate NMs is urgently needed. Table 7. In Vitro MN References: Studies That Considered NM Uptake Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Open in new tab Table 7. In Vitro MN References: Studies That Considered NM Uptake Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Open in new tab Table 8. In Vitro MN Assay: Acceptable Studies That Evaluated Uptake Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Open in new tab Table 8. In Vitro MN Assay: Acceptable Studies That Evaluated Uptake Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Open in new tab A relatively new development is the 3D human reconstructed skin MN assay (Aardema et al., 2010). This test is growing in importance as it allows evaluation of topically applied compounds on a reconstructed human 3D skin model. It is proving to be particularly useful for assessing the genotoxic potential of cosmetics products that can no longer be tested in vivo (Pfuhler et al., 2014; Wills et al., 2015). In Vitro Chromosomal Aberration Assay A total of 11 papers were considered (Table 9), recapped in Table 1, including 3 published from 2012 to 2014 and 8 references previously reviewed by COM (2012). Nanomaterials tested included TiO2, ZnO, SWCNT and MWCNT, fullerene, Ag, hydroxyapatite, and FePt capped with 2-aminoethanethiol. Chinese hamster lung (CHL) or Chinese hamster ovary (CHO) cell lines were most commonly used (8 out of 11 publications: Dufour et al., 2006; Honma et al., 2012; Kwon et al., 2014; Maenosono et al., 2009; Mrđanović et al., 2009; Shinohara et al., 2009; Theogaraj et al., 2007; Warheit et al., 2007). Human lymphocytes were also used (Turkez et al., 2014). In addition, there were single papers describing chromosomal aberrations in a mouse macrophage cell line (Di Giorgio et al., 2011) and a human mesenchymal stem cell line (Hackenberg et al., 2011). One study was excluded that did not have any cytotoxicity measure and where excessive dose levels, ie, >500 μg/ml, were used (Maenosono et al., 2009). Table 9. In Vitro Chromosomal Aberration Assay (CA): Papers Evaluated Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Open in new tab Table 9. In Vitro Chromosomal Aberration Assay (CA): Papers Evaluated Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Open in new tab Results in 3 chromosomal aberration papers were considered positive, involving tests of ZnO, carbon nanotubes, and Ag (Di Giorgio et al., 2011; Dufour et al., 2006; Hackenberg et al., 2011). ZnO had a maximum increase of total aberrations to 16% (Dufour et al., 2006). Carbon nanotubes generated 25% and 40% acentric fragments at 48 and 72 h, respectively, and were accompanied by detection of reactive oxygen species (ROS) by dichlorofluorescein in a mouse macrophage cell line (Di Giorgio et al., 2011). Ag NMs induced a relatively weak positive response (up to 10% aberrant cells), but this result was based only on 50 cells scored (Hackenberg et al., 2011). Positive controls were included in 9 out of 11 papers reviewed (Di Giorgio et al., 2011; Dufour et al., 2006; Hackenberg et al., 2011; Kwon et al., 2014; Maenosono et al., 2009; Mrđanović et al., 2009; Shinohara et al., 2009; Theogaraj et al., 2007; Turkez et al., 2014). The duration of treatment varied between 24 and 72 h. S9 was included in 5 out of 11 papers (Honma et al., 2012; Kwon et al., 2014; Maenosono et al., 2009; Shinohara et al., 2009; Warheit et al., 2007); no studies indicated that S9 affected the results. The way the chromosome aberrations results were expressed varied greatly throughout the papers, from a summary % of total chromosome aberrations to the detailed analysis of chromosome and chromatid breaks, acentric fragments and centromeric fusions. A paper that followed an OECD-compliant method for the standard chromosome aberration tests was that of Shinohara et al. (2009). The methods described a standard chromosome aberration test in a well-known cell line according to OECD TG 473. The results were well displayed to ensure that the types of aberrations scored were clear, indicating a knowledge of the assay. The NPs were characterized for primary and secondary size, but nuclear or cellular uptake was not measured; these data would have made this paper more informative. Two out of 11 papers did show nuclear or cellular uptake (Di Giorgio et al., 2011; Hackenberg et al., 2011), using electron microscopy and TEM respectively. Both of these papers showed positive results with carbon nanotubes and silver respectively. In Vitro Comet Assay Twenty-two papers on tests of metal oxides and carbon nanotubes in the in vitro comet assay published from 2012 to 2014 (postCOM, 2012) were considered (Table 10). Findings are summarized in Table 1. Naturally occurring and “soft” NMs, those that contain components such as polymers, gels, and biomaterials, were not considered. No papers were excluded from the analysis, but acceptability criteria were applied: the presence of a positive control, assessment of cytotoxicity, and evaluation of material uptake. There were 2 major methods used in these studies—the standard alkaline comet and the comet with the addition of formamidopyrimidine-DNA glycosylase (FPG) in order to cause strand breaks at sites of oxidative damage in the DNA, thereby providing evidence of the presence of oxidative lesions. Some studies compared both methods, thereby in principle providing information on the proportion of strand breaks caused by oxidative damage lesions. Table 10. In Vitro Comet Assay: Papers Evaluated Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab Table 10. In Vitro Comet Assay: Papers Evaluated Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab A diversity of cell lines was used in the studies, as follows: FPG method: A549 human lung alveolar carcinoma (2 papers) (Ursini et al., 2012, 2014); other cells used included BEAS 2B normal human bronchial epithelial cells, C3A human hepatoblastoma cell line, and HK-2 human renal proximal tubule epithelial cells (Kermanizadeh et al., 2012, 2013; Ursini et al., 2014). Standard method: BEAS 2B normal human bronchial epithelial (Chatterjee et al., 2014; Lindberg et al., 2013), HK-2 human renal proximal tubule epithelial cells (Kermanizadeh et al., 2013), MeT-5A human mesothelial (Lindberg et al., 2013), C2C12 -mouse myoblast (Cancino et al., 2013), A549 human lung alveolar carcinoma cells (De Marzi et al., 2013; Mu et al., 2012;, V79 hamster fibroblast cells (Chen et al., 2014), HepG2 (Alarifi et al., 2013; De Marzi et al., 2013; Kermanizadeh et al., 2012), Caco2 (De Marzi et al., 2013), Hacat (Mu et al., 2012), AGS (human gastric epithelial cancer cells) (Botelho et al., 2014), primary human lymphocytes (Battal et al., 2015; Moche et al., 2014), RAW 264.7 macrophages (Wilhelmi et al., 2013), HEK293 cells (Demir et al., 2014), NIH/3T3 cells (Demir et al., 2014; Ould-Moussa et al., 2014), IMR-90 human fibroblasts (Lim et al., 2012), M059K human glioblastoma cells and their PKC deficient counterpart (M059J cells) (Lim et al., 2012), CHO AA8 and their PKC deficient counterpart (CHO V33 cells) (Lim et al., 2012), mouse oocytes (Courbiere et al., 2013), and rat neurons (Zha et al., 2012). The type of cell used may have an impact on the outcome of the assay as different cells may have different internalization capacity, as well as differences in DNA repair and metabolic capability. Seventeen papers (Table 11) dealt with metal oxides, 2 with FPG (Kermanizadeh et al., 2012, 2013), and the remaining with the standard method. Seven papers tested carbon NMs, 3 with the FPG-modified method (Kermanizadeh et al., 2012; Ursini et al., 2012, 2014), 4 with the standard method (Cancino et al., 2013; Chatterjee et al., 2014; Kermanizadeh et al., 2013; Lindberg et al., 2013). Responses ranged from 2- to 7-fold increase in % tail DNA over the negative control level, with most in the 2- to 3-fold range. The 7-fold increase was seen for carbon nanotubes using the FPG-modified method (Ursini et al., 2012); it should be noted that the standard method was tested simultaneously and a negative response was observed. Table 11. In Vitro Comet Assay References: Papers With Metal Oxides Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab Table 11. In Vitro Comet Assay References: Papers With Metal Oxides Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab Few studies measured uptake of the NPs: 3 of 7 total studies for carbon nanotubes (Lindberg et al., 2013; Ursini et al., 2012, 2014); 2 of 3 with FPG (Ursini et al., 2012, 2014); 1 of 4 for the standard method (Lindberg et al., 2013); and 6 of 17 studies for metal oxides (Alarifi et al., 2013; Courbiere et al., 2013; Mu et al., 2012; Ould-Moussa et al., 2014; Wan et al., 2012; Wilhelmi et al., 2013), all using the standard method. There were considerable variations in methods in the studies reviewed. The doses ranged from 0.01 to 760 µg/ml (Cancino et al., 2013; Chatterjee et al., 2014; Kermanizadeh et al., 2012, 2013; Lindberg et al., 2013; Ursini et al., 2012, 2014) for carbon nanotubes and from 0.001 to 1000 µg/ml for metal oxides (Table 11). Treatment ranged from 2 to 72 h, with a 24-h treatment being most common. Most positive responses were seen at 24 h. Different sample sizes ranging from 50 to 300 cells were analyzed, and not all studies used a positive control. From the studies reviewed, it proves difficult to draw conclusions on the genotoxic profile of any specific NM using the in vitro comet assay. Most of the studies on metal oxides and carbon nanotubes tended to yield positive results using either the FPG method or the standard method (Kermanizadeh et al., 2012, 2013; Ursini et al., 2012, 2014). Although TiO2 and WC-Co generated mixed results (Botelho et al., 2014; Chen et al., 2014; Kermanizadeh et al., 2012; Moche et al., 2014; Wan et al., 2012); ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG (Table 12). All tests with carbon nanotubes had at least one test result that was positive (Cancino et al., 2013; Chatterjee et al., 2014; Kermanizadeh et al., 2012, 2013; Lindberg et al., 2013; Ursini et al., 2012, 2014). Table 12. In Vitro Comet Assay: Positive Responses Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Open in new tab Table 12. In Vitro Comet Assay: Positive Responses Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Open in new tab One publication was identified that follows most recommendations: use of a positive control, verification of NM uptake, analysis of an acceptable number of cells per sample, and detailed characterization of the NM used (nanotubes; the characterization was shown in a separate publication and was done before the use of the material) (Lindberg et al., 2009). Though analysis of 100 cells per treatment was conducted in this study, a minimum of 150 cells per sample would have been preferred, as recommended by OECD TG 489. Although this study followed many recommendations, the use of a nonstandard cell line makes data interpretation somewhat difficult. In Vitro Mammalian Gene Mutation Assays Seventeen papers published from 2007 to 2017 were included in this review (Table 13) and nineteen NMs were evaluated (Table 1), including MWCNT (plain and carboxylated), SWCNT (plain and carboxylated), anatase TiO2, TiO2, carbon black, C60 fullerenes, Ag, Cd/Se quantum dots (carboxyl, hexadecylamine [HDA], and amine), WC-Co, SiO2, ultrafine quartz, ZnO and 2 poly(anhydride) NMs. Table 13. In Vitro Mammalian Gene Mutation Assay: Papers Evaluated Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Open in new tab Table 13. In Vitro Mammalian Gene Mutation Assay: Papers Evaluated Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Open in new tab There are publications using the standard assays (Tk in L5178Y cells [OECD TG 490], and Hprt (hypoxanthine-guanine phosphoribosyltransferase) in V79, CHO, CHL, L5178Y or TK6 cells [OECD TG 476]). In addition, publications were identified using nonstandard assays including mutation at the HPRT locus in MCL-5 and WIL2-NS cells (Manshian et al., 2013; Wang et al., 2007a,b,c); Aprt (Adenine Phosphoribosyltransferase) in 3C4ES cells (Zhu et al., 2007); transgenes (cII and/or lacZ) in FE1 cells (Jacobsen et al., 2007, 2008); and loss of CD59 in AL cells (Wang et al., 2015). There were 4 papers for the standard mouse lymphoma assay (MLA, OECD TG 490) (Iglesias et al., 2017; Kim et al., 2010; Mei et al., 2012; Moche et al., 2014) and 5 papers for the standard HPRT assay using cell lines that are recommended in OECD TG 476 (Asakura et al., 2010; Chen et al., 2014; Manshian et al., 2016; Mrakovcic et al., 2015; Wang et al., 2011). Four papers evaluated Hprt mutation in cell lines not included in OECD TG 476 (Manshian et al., 2013; Wang et al., 2007a,b,c), one each evaluated mutation at the Aprt or CD59 loci (Wang et al., 2015; Zhu et al., 2007), and 2 evaluated cII and/or lacZ in FE1 cells (Jacobsen et al., 2007, 2008). There is no OECD TG for the latter 3 assays. Only a few materials were evaluated in more than one study. Only Ag was evaluated in the same assay in 2 laboratories (Kim et al., 2010; Mei et al., 2012). None of the other materials (or similar materials) was evaluated in the same test system using the same endpoint in multiple studies or laboratories (although replicate experiments were sometimes reported and were reproducible). When the same or similar materials were evaluated in multiple laboratories or test systems, conflicting results were sometimes observed; however, not all of these studies were determined to be acceptable (see below for a more detailed discussion). Summarizing all of the studies (acceptable and not acceptable), Ag was reported to be positive in one MLA (Mei et al., 2012) and negative in a second study (Kim et al., 2010), but it was not clear that the same size particles were tested or that uptake was assured in the second study. MWCNT were reported positive in 1 of 3 studies (Asakura et al., 2010; Mrakovcic et al., 2015; Zhu et al., 2007); 2 samples of various SWCNTs were reported positive in 2 of 3 studies (Jacobsen et al., 2008; Manshian et al., 2013; Mrakovcic et al., 2015); TiO2 was reported positive in 2 of 3 studies (Chen et al., 2014; Wang et al., 2007a, 2015); and one study reported 20 nm ZnO negative, but 90–200 nm ZnO positive (Wang et al., 2015). In addition, for most materials assessed, where the authors reported positive responses, the maximal increases observed were usually only 2–3 times negative control values, or even less (Table 14). Table 14. In Vitro Mammalian Gene Mutation Assay: Unconfirmed Positive Responses Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Open in new tab Table 14. In Vitro Mammalian Gene Mutation Assay: Unconfirmed Positive Responses Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Open in new tab As with the other mammalian cell assays and summarized in Table 1, there was the substantial variation in cell lines, dose levels, treatment times, and endpoints analyzed. Such variation precluded any definitive conclusions. Due to the variability in the methods used, the protocols used in each study were evaluated and the following criteria were applied for excluding tests from the analysis: Reports with negative control mutant frequencies outside typical reported ranges, or those with no concurrent negative control. Concurrent positive controls failed to elicit a significant increase in mutant frequency (not all studies included positive controls, but this was not used as a specific exclusion criterion, especially if a positive response was observed for the NMs; it also would have reduced the number of papers available). Studies without a concurrent cytotoxicity evaluation, an inappropriate cytotoxicity parameter, or responses observed only at excessively cytotoxic dose levels (generally below 10%–20% survival by the appropriate parameter). Insufficient numbers of cells scored or an inappropriate scoring method was used (not all studies were specifically excluded due to low cell numbers, especially if a positive response was observed for the NMs). Missing details that prevented full evaluation of methods used. From this review, 15 of 17 studies characterized the test material and provided detailed dispersion protocols for the NM treatments (Table 15) and 2 did not (Kim et al., 2010; Zhu et al., 2007). In total 7 of 17 studies verified uptake of the NMs (Table 16), which was not determined or discussed in the remaining 10 papers. Only one study included S9 (Kim et al., 2010). In addition, 3 studies used extremely long treatment protocols of 24–60 days (Jacobsen et al., 2007, 2008; Wang et al., 2011); 3 studies had negative control mutant frequencies that appeared unusually high (Kim et al., 2010; Wang et al., 2007b,c). One study scored far too few cells to support a negative finding (Asakura et al., 2010); one study did not include concurrent negative controls, instead relying on comparison to historical control values (Wang et al., 2015); and one study had no statistical analyses, or any other valid criteria for judging the response, and also lacked historical control data for comparison (Zhu et al., 2007). One study used relative suspension growth rather than relative total growth to express cytotoxicity, so that it was impossible to know whether the doses were appropriate to conclude negative results (Iglesias et al., 2017). Table 15. In Vitro Mammalian Gene Mutation Assay: Studies With Adequate NM Characterization Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Open in new tab Table 15. In Vitro Mammalian Gene Mutation Assay: Studies With Adequate NM Characterization Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Open in new tab Table 16. In Vitro Mammalian Gene Mutation Assay: Uptake Studies Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Open in new tab Table 16. In Vitro Mammalian Gene Mutation Assay: Uptake Studies Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Open in new tab Based upon our exclusion criteria, which generally followed or were adapted from the appropriate OECD TG, 9 studies (53%) were classified as acceptable (Table 17) and 8 (47%) were considered to be unacceptable (Table 18). Of the acceptable studies, 6 individual NMs were considered positive by the authors, but the expert review only considered 5 of them to be clearly positive: 400–800 nm SWCNT, tungsten carbide cobalt nm, TiO2, Ag, carboxylated SWCNT (Chen et al., 2014; Manshian et al., 2013; Mei et al., 2012; Moche et al., 2014; Mrakovcic et al., 2015). Ultrafine TiO2 was deemed to be positive by the authors (Wang et al., 2007a), but equivocal by the review group. Interestingly, 2 of these studies had 3 additional, related NMs (carbon nanotubes) that were deemed to be negative by the authors and the review group (Manshian et al., 2013; Mrakovcic et al., 2015). Two additional studies, evaluating a total of 4 NMs, produced negative results with which the reviewers agreed. Table 17. In Vitro Mammalian Gene Mutation Assay: Studies Classified as Acceptable Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Open in new tab Table 17. In Vitro Mammalian Gene Mutation Assay: Studies Classified as Acceptable Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Open in new tab Table 18. In Vitro Mammalian Gene Mutation Assay: Studies Considered to Be Unacceptable Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Open in new tab Table 18. In Vitro Mammalian Gene Mutation Assay: Studies Considered to Be Unacceptable Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Open in new tab In the 8 studies judged to be unacceptable for the reasons described, the authors reported 7 NMs to be negative and 5 NMs to be positive. However, the increases in mutant frequencies observed for these latter “positive” NMs only reached 1.2- to 2.5-fold control values. For the MLA, Mei et al. (2012) reported Ag NM (5 nm) to be positive. In this study, the investigators fully characterized the test material and uptake into the cells, and also conducted the study according to OECD TG 490. In contrast, Kim et al. (2010) reported Ag NM (<100 nm) to be negative in the MLA. However, no information was provided regarding characterization of the Ag NM or its uptake into cells. Moche et al. (2014) reported WC-Co to be positive in the MLA using a 4-h treatment, but negative using a 24-h treatment, which is an unusual pattern of responses (Moore et al., 2007), and cellular uptake was not addressed. The review team felt that a reasonable study design and approach for the MLA/TK assay was as described by Mei et al. (2012). For the standard Hprt assay (OECD TG 476), there were 5 papers. Asakura et al. (2010) reported MWCNT to be negative in CHL cells. Although they did characterize the NM, they did not provide information concerning cellular uptake. Chen et al. (2014) reported anatase TiO2 to be positive in V79 cells. The test material was characterized, but there was no evaluation of cellular uptake. Manshian et al. (2016) reported 3 different types of Cd/SE quantum dots to be negative in TK6 cells. The test materials were fully characterized, and cellular uptake was confirmed. Mrakovcic et al. (2015) evaluated SWCNT and MWCNT (both plain and carboxylated) using V79 cells. Both NMs were fully characterized, and cellular uptake was verified. In this study, SWCNT was mutagenic while MWCNT was negative. Wang et al. (2011) reported TiO2 to be negative in CHO cells. The test material was fully characterized and cellular uptake was verified, but the assay was not conducted according to OECD TG 476 (treatment time and the measure of cytotoxicity were not according to the recommendations). The review team felt that a reasonable study design and approach for the HPRT assay was as described by Manshian et al. (2016) or Mrakovcic et al. (2015). For the publications using the HPRT locus in other cell lines, some studies fully characterized the test material including cellular uptake while others characterized the test material, but did not confirm cellular uptake. Three publications using WIL2-NS cells (Wang et al., 2007a,b,c) did not confirm cellular uptake of the test materials. Although the authors called the responses for all 3 positive, the review team disagreed and called SiO2 and UF quartz negative (maximum MFs were approximately 1.4- to 1.6-fold control values) and called TiO2 equivocal (with a dose-dependent increase to 2.5-fold control values). Manshian et al. (2013) used MCL-5 cells to evaluate different sizes of SWCNT. The NM was fully characterized, and cellular uptake was confirmed. One (400–800 nm) was positive, whereas the other two (1–3 and 5–30 µm) were negative. Two papers by Jacobsen et al. (2007, 2008) used transgenic loci (cII and/or lacZ). Although they did characterize the 4 NMs evaluated, cellular uptake was not confirmed. The authors reported quartz (1.59 µm), SWCNT and C60 fullerenes to be negative and carbon black to be positive. However, the review team judged all 4 to be negative (all were using repeated exposures for 24 days, and the largest increases seen for carbon black were only approximately 1.2- to 1.4-fold control values). Wang et al. (2015) reported ZnO (90–200 nm) to be positive, but ZnO (20 nm) and TiO2 (15 nm) to be negative, for inducing mutation at the CD59 locus in AL hybrid cells. They did characterize the test material and confirmed cellular uptake. However, the review team called all 3 responses negative (overturning the authors’ positive call on the basis of the magnitude of the increases seen). The one publication using the Aprt locus in mouse 3C4 ES cells (Zhu et al., 2007) had insufficient detail to adequately evaluate the reported positive call for MWCNT. In total, the review team disagreed with 6 of the 11 positive calls made by the authors, deeming 4 to be outright negative, 1 to be equivocal, and 1 to be uninterpretable. Overall, there was insufficient information in the published studies to draw any firm conclusions concerning the mutagenicity of NMs in mammalian cells in culture, or in the ability of the various assays to detect potential effects. For the standard MLA and Hprt assays there were 2 and 6 papers, respectively, not all of which were conducted appropriately. In particular, not all of the publications in which negative responses were reported showed confirmation that the test material was taken up by the cells. For the entire set of papers for in vitro mammalian gene mutation, approximately half of the publications used cell lines and/or mutation endpoints for which there is very little published data for standard chemicals. Furthermore, given the wide variety of NMs included in the small number of publications, it is not possible to make any conclusions concerning the relative sensitivity of the various reporter genes to the potential mutagenicity of NMs. It should, however, be noted that the MLA using the Tk locus detects a wider spectrum of genetic damage, including large scale chromosomal effects (Applegate et al., 1990; Wang et al., 2009), while the Hprt locus detects primarily single base pair changes or other small scale DNA sequence alterations (Moore et al., 1989). Thus, some chemicals are positive at the Tk locus, negative at the Hprt locus, and positive for chromosome aberrations, micronuclei, or comet (Doerr et al., 1989; Moore et al., 1989). It would be expected that the same would be true for NMs. In Vivo Bone Marrow MN Assay and Chromosomal Aberration Assay In total 18 papers were assessed for the in vivo MN endpoint (Table 19; results summarized in Table 2); from 2012 to 2016, 8 were from prior to 2012 and included in the COM (2012) analysis. In general, the NM characterization, particle size distribution and genotoxicity test methods were better conducted in the in vivo studies than in the in vitro studies. Approximately half of the studies were in mice and half were in rats. There are few separate papers on chromosome aberrations in vivo; however, there were chromosome aberration results along with in vivo MN results in 5 out of 18 papers (Balasubramanyam et al., 2009; Ghosh et al., 2012; Landsiedel et al., 2010; Schulz et al., 2012; Singh et al., 2013) and these results have been included in this analysis. All papers were assessed for additional information than was summarized in the COM (2012) analysis. No papers were excluded from the analysis but the interpretation of 2 papers is debatable, due to the low magnitude of induction of micronuclei in NM test groups—barely a doubling over control (Dobrzyńska et al., 2014; Downs et al., 2012) which may put into question the biological relevance of the findings. In cases where both CA and MN were assessed in the same study, the results were generally the same for both assays, either positive or negative. Table 19. In Vivo MN Assay and Chromosomal Aberration (CA) Assay References Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Open in new tab Table 19. In Vivo MN Assay and Chromosomal Aberration (CA) Assay References Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Open in new tab When different results were seen for the same NMs (eg, TiO2 and Ag) it could be attributed to exposure methods and different dose levels. 6 papers for 5 different NMs (Ag, TiO2, Al2O3, MnO, and CrO2) showed clearly positive results in in vivo micronuclei or chromosome aberrations assays (Balasubramanyam et al., 2009; Chen et al., 2014; Dobrzyńska et al., 2014; Ghosh et al., 2012; Singh et al., 2013; Trouiller et al., 2009). Two equivocal or weak positives were determined as approximately a doubling or less above control. The positive with MnO gave 2- to 3-fold increases following dosing at 1000 mg/day for 28 days (Schulz et al., 2012), and the positive with Al2O3 (Doerr et al., 1989) was seen following dosing of up to 2000 mg/kg as a single dose. TiO2, Ag and ZnO papers had dose response studies. Of the 5 papers with silver NMs (Dobrzyńska et al., 2014; Ghosh et al., 2012; Jiang et al., 2013; Landsiedel et al., 2010; Wang et al., 2015), 3 were positive (Dobrzyńska et al., 2014; Landsiedel et al., 2010), one of these following intraperitoneal (i.p.) exposure of a single dose with groups of animals given between 10 and 80 mg/kg Ag NPs (Ghosh et al., 2012). A second Ag NP study dosed intravenously (i.v.) gave increases in micronuclei of 3-fold at 24 h following exposure to 5 or 10 mg/kg of Ag NP (Dobrzyńska et al., 2014). The positive response persisted for up to 1 week with a 2-fold increase at the 10 mg/kg dose. However, a study dosed up to 1000 mg/kg via the oral route for 28 days with Ag NPs of a similar size (52–71 nm) was negative in bone marrow (Kim et al., 2008); thus it appears that the route of exposure needed to be i.v. or i.p to get a positive with Ag NPs. With TiO2, 2 papers described positive results via an oral route (Chen et al., 2014; Trouiller et al., 2009) while one was negative Donner et al. (2016); one was negative via inhalation (Lindberg et al., 2012). A fourth paper with TiO2 is equivocal with exposure via an i.v. route (Dobrzyńska et al., 2014); therefore, again the differences seen might be attributed to routes of administration. A further paper on silica had MN increases considered equivocal but were accompanied by significant increased γH2AX and 8 Oxo-G signals, indicating the signal might be due to inflammatory responses (Downs et al., 2012). The study with europium was performed at approximately one-tenth the doses of most of the in vivo studies, and the negative result was a desirable one from the perspective of potential therapeutic use of the product. However, the optimum choice of dosing for in vivo safety assessment is still an unresolved issue. One paper that could be considered a model for the in vivo MN assay is Donner et al. (2016). It was conducted according to OECD guidelines, with good characterization of the TiO2 test articles and methods for the MN assay. Dosimetry included high test doses up to 2000 mg/kg. This dose is high, but appropriate for the goal of this test, which was to demonstrate a negative. This study lacked an analysis of uptake into the target tissue, but the negative results were discussed in the context of likely poor target tissue (bone marrow) exposure following oral administration of the NM, due to poor systemic distribution from gastrointestinal exposure. A well-run assay for both micronuclei and chromosome aberrations in vivo is by Balasubramanyam et al. (2009) where both assays were conducted according to OECD guidelines, the aluminum oxide NPs are well described, and the authors monitored uptake of the NPs into a variety of tissues in the rats. The genotoxicity assays showed similar, positive dose-response results for 30 and 40 nm particles, with a significant, possibly exponential increase within a defined linear dose range, up to 2000 mg/kg dosed orally. Such high doses might be questionable from a risk assessment context, but the lowest dose tested, 500 mg/kg, was also significantly positive although this dose may still be considered high for a particulate material. These 2 papers demonstrated divergent results from oral administration of different nanomaterials, one positive and one negative. Overall, the data reviewed show that the in vivo MN assay (OECD TG 474) and in vivo chromosome aberration assay (OECD TG 475) can be used in the standard form for NP evaluation, but attention should be paid to mechanistic aspects such as relevant tissue exposure and potential particle overload effects. In Vivo Comet Assay The dataset considered 17 papers from 2009 up to the end of 2014 (Table 20), addressing genotoxicity of about 20 different NPs, including those that were also reviewed by COM (2012). The findings are summarized in Table 2. The studies did encompass a wide range of NMs, including carbon nanotubes, carbon black NP’s, silica and fullerenes, the majority of data being generated on metal oxides. For the NMs investigated, a positive result in the comet assay was claimed by the authors in >50% of these studies. Effects were mostly small, 1.3-fold up to approximately 2-fold increases in tail DNA. Some of these calls may be considered questionable, in light of the OECD guideline requirements for a positive call for this assay (OECD TG 489) which requires a clear positive call to be statistically significant, dose-related and one data point to exceed the distribution of the historical negative control of the performing laboratory. However, no clear pattern was observed in terms of the responses for different types (classes) of NMs investigated. Regarding MoA, there was one positive study that was directly associated by the authors with the release of genotoxic metal ions (Tiwari et al., 2011). Several other studies associated the positive response with signs of tissue inflammation as indication of an inflammation-driven, oxidative MoA (Bourdon et al., 2012; Downs et al., 2012; Saber et al., 2012b; Sharma et al., 2012; Totsuka et al., 2009). Although the data reviewed seem to indicate that the in vivo comet assay (OECD TG 489) can be used without modification for NP assessment, attention should be paid to potential artifacts when residual particulates remain during the electrophoresis of the DNA (Ferraro et al., 2016; Karlsson et al., 2015). Table 20. In Vivo Comet Assay References Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Open in new tab Table 20. In Vivo Comet Assay References Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Open in new tab If maximal assay sensitivity for detection of oxidative DNA damage (eg, inflammation-induced) is desired, the use of a modified version may be beneficial (addition of OGG1 or FPG glycosylases for recognition of particular oxidative DNA adducts). Bourdon et al. (2012) have shown that an FPG protocol did boost the comet effects, but the standard protocol had already given a positive response. Sharma et al. (2012) found a positive response when using the FPG protocol but no data were generated in parallel using the standard protocol. Taken together, there is not enough data available to develop a firm recommendation on the need to modify the standard protocol. Another key aspect associated with the observed responses is the selection of a maximum dose, as well as route of exposure. Most of the studies associated with a positive response were using very high particle loads, and maximized tissue exposure by, eg, intratracheal instillation (Saber et al., 2012b) or i.v. injection (Downs et al., 2012). The effects may therefore be indication of a “hazard” that may not be associated with a relevant human exposure scenario and reflect particle overload, triggering inflammatory responses (Downs et al., 2012). In Vivo Gene Mutation Assays in Transgenic Rodents In total 8 papers were reviewed that were published from 1997 to 2015 (Driscoll et al., 1997; Kato et al., 2013; Kovvuru et al., 2015; Louro et al., 2014; Shvedova et al., 2008, 2014; Totsuka et al., 2009, 2014); these described mutagenic responses to NM exposure in both generic and transgenic rats and mice. Although the extensive COM (2012) analysis covered genotoxicity data from NM exposure in vitro and in vivo, there is no analysis of in vivo mutation data on endogenous reporter genes or transgenes using Transgenic Rodent (TGR) models. Therefore, our analysis of 11 NMs ranging from 1.2 to 180 nm in size using different mutation reporter systems, reviewed in Table 2, is an extension of the COM (2012) analysis. The NMs tested included the following: C60 fullerenes (Totsuka et al., 2009), carbon black (Driscoll et al., 1997; Totsuka et al., 2009), Kaolin (Totsuka et al., 2009), MWCNT (Kato et al., 2013), anatase TiO2 (Driscoll et al., 1997; Louro et al., 2014), α-quartz (Driscoll et al., 1997), SWCNT (Shvedova et al., 2008), carbon nanofiber (CNF) (Shvedova et al., 2008), asbestos (Shvedova et al., 2014), magnetite NPs (in the form of Fe3O4) (Totsuka et al., 2014), and PVP-coated Ag NPs (Kovvuru et al., 2015). The mutation reporter systems included gpt-delta, lacZ and myh−/− mice; F344 and C57BL/6 rats with K-RAS codons 8 and 12. The tested NMs were well characterized in most studies and the investigators followed OECD guidelines for treatment and sacrifice. Titanium, carbon black and asbestos were all negative in the TGR systems whereas most other NMs showed positive responses in one or the other mutational reporter systems. Most of the studies used multiple doses and 6 of them, C60 fullerenes (Totsuka et al., 2009), α-quartz (Driscoll et al., 1997), CNF (Shvedova et al., 2008), SWCNT of 2 different sizes (Shvedova et al., 2008), magnetite NPs (Shvedova et al., 2014) showed a positive dose-response. However, MWCNT (Kato et al., 2013), anatase TiO2 (Driscoll et al., 1997) and carbon black (Driscoll et al., 1997) tested positive only at the highest dose, while one NM (asbestos) (Shvedova et al., 2014) was negative in both mice and rats at all doses. The positive mutagenic responses (mutant frequencies) were 2- to 3-fold higher than the background response and in some studies which evaluated the mutational spectra, showed significant shifts in the NP-induced spectra compared with untreated control or background spectra. In general, the mutagenic responses induced by NMs appear to be substantially lower than that of standard mutagenic agents such as ENU and DMBA. Some of the NMs-induced mutagenic responses comprised of G→A (K-ras) and G→T (gpt) mutations; the G→T mutations are likely to be a hallmark of oxidative damage, while the mutagenic effects by other NMs may be secondary as a consequence of inflammation and release of ROS or free radicals. However, more studies are needed to evaluate the MoA of these NMs. Most of the studies performed uptake analysis of NMs in the target tissues, and the majority showed the presence of NMs in the target tissues, suggesting the mutagenic response was associated with NM exposure. However, it is notable that when the same NM was tested using different species of animals, different mutagenic responses (mutant frequencies) were observed. For example, carbon black was positive in F344 rats but negative in gpt delta mice (Driscoll et al., 1997; Totsuka et al., 2009), whereas TiO2 was positive in F344 rats (lung epithelial hprt assay) but negative in lacZ mice (Driscoll et al., 1997; Louro et al., 2014). There may be some correlation between positive mutagenic response and NM uptake because all NMs except asbestos produced positive responses and most of the NMs were taken up in the target tissues. However, this does not hold for asbestos, which was not mutagenic even though there was tissue accumulation or uptake. These results suggest the importance of animal species or test endpoint in addition to character of the NM. Interestingly, one study on long-term effects of carbon-containing engineered NMs such as SWCNT and CNF showed the presence of NMs in the target tissue even after one year following an acute exposure. What is also notable is that both SWCNT and CNF induced significant increases in the rate of K-ras mutations in codons 12 and 8, one year after exposure in mice lungs. However, the exposed mice did not have any tumors in the lungs at the time examined (Shvedova et al., 2014). DISCUSSION The major genotoxicity assays utilized for assessment of chemicals have been reviewed here as they have been applied to the evaluation of NMs. The goal was to recommend approaches appropriate for hazard identification and risk assessment of NMs. This review is anchored on and adds to the EMGS 2010 Workshop (Pfuhler et al., 2013), the UK COM (2012) report, and the reviews and work of the authors and others, but it is also focused on a robust and critical evaluation of the current nanogenotoxicity testing literature as a basis for specific recommendations on test assays and test methods. Understanding the Test Material The physical characterization of the NM for size, shape, and properties is of utmost importance (Bouwmeester et al., 2011; Boverhof and David, 2010; Love et al., 2012). This information has been provided in most of the more recent papers, as this is now generally recognized and required by most journals. However, of equal or greater importance is an understanding of the fate of NMs when added to the biological test systems. In many cases, NMs are characterized under one set of conditions and then encounter a different set of conditions in the test system. Thus, there may be uncertainty regarding the actual nature of the NM to which the test system is being exposed, and how this relates to human exposure. Many papers have noted that certain standard assays and test conditions may not suffice for assessment of NMs, eg, due to lack of particle uptake and a requirement for assay method modification (Doak et al., 2009, 2012; EFSA Scientific Committee, 2011; Landsiedel et al., 2009; Warheit and Donner, 2010). Several studies have explored the effects of media, pH, surface charge, coatings, and proteins on fate, action and toxic outcomes of NMs (Dutta et al., 2007; French et al., 2009; Jiang et al., 2009; Ju et al., 2013; Li et al., 2013; Pagnout et al., 2012; Pathakoti et al., 2013; Pele et al., 2015; Rivera Gil et al., 2010). Other important issues affecting NM toxicity measurements include experimental handling and interference with endpoints (Akabori and Nagle, 2014; Cullen et al., 2011; French et al., 2009; Jiang et al., 2009; Lindberg et al., 2009). It is recommended that NMs should be characterized for size distribution and properties under the test conditions in the genotoxicity assay, and consideration should be given to the properties of the NM for intended human exposure/use. Understanding Exposure Although genotoxicity testing is usually carried out without regard to a human exposure scenario, the route of exposure of NMs is a critical feature of human risk. Much of the focus on the toxicity assessment of NMs is related to inhalation exposure in humans; however, the effects of inhalation exposure may not be extrapolated to other routes. Dermal exposure is another common route of exposure to NMs, but many studies have indicated the lack of penetration of NMs into dermal tissues (reviewed in Warheit and Donner, 2015). However, dermal penetration may be NM and/or species specific (Hirai et al., 2012). Another major consideration is the possibility that the Absorption, Distribution, Metabolism and Elimination (ADME) of NMs may be different from that of the non-nano material of the same composition. A classic review of “Principles for characterizing the potential human health effects from exposure to nanomaterials” by different routes of exposure (Oberdorster et al., 2005) still seems relevant. However, for genotoxicity testing, exposure consideration is generally related to assurance that the test agent reaches the cells in the test system. Consideration of human exposure may be a factor, but generally the genotoxicity tests need to be performed in standard available systems in order that the results may be interpretable. Thus, in addition to characterizing the physico-chemical form of the NM in the conditions of the test system, there is a need to characterize how NMs behave in the test system; specifically, whether the NM gets into the cell/nucleus. If a NM does not get into a cell, genotoxicity is not expected unless there is release of ions or other genotoxic moieties that have the capacity to penetrate the cell. The selection of an “appropriate” dose for testing remains a difficult and unresolved issue. Exposure is typically elevated beyond actual human exposures in toxicology testing designed for hazard identification, as recommended in the current OECD TGs for the various tests. Elevated doses compensate for sensitive populations, statistically small samples, and extended time of exposure. However, high dose testing may lead to effects that would not occur at human use exposures, including artifacts caused by particle “overload”, which would be specific for the testing of NMs. There is not enough supporting evidence for a recommendation of a cut-off for maximum NM exposures at present, in vitro or in vivo. Rather than a particular cut-off value, it is likely more useful to monitor for potential overload effects of particular NMs in the relevant experimental system under study. In the case of bacteria, information to date indicates that NMs do not traverse the bacterial cell wall and the results are almost always negative. In in vivo systems, NMs may not be distributed systemically to the tissues evaluated in the assay, which is required for a valid test, per OECD testing guidelines. There is evidence that high dose artifacts (eg, related to particle effects or sequestration in tissues) in in vivo genotoxicity tests can be induced that would not occur under normal human exposures, such as demonstrated by Downs et al. (2012), who administered the NMs via the i.v. route and noted that this was a “worst case” exposure scenario that will rarely have a human correlate. Currently, technological limitations prevent these exposure issues from being fully addressed in each assay. However, this represents an important gap in our understanding and advances in the field will assist with understanding the biological and toxicological impact of exposure to NMs moving forward. At this time, it is recommended that genotoxicity tests for NMs be conducted within the guidelines (eg, OECD) as for testing of other agents, with the exception of specific methods adaptations required for NMs (as described further in the “Recommendations on method standardization and assay modifications” section below. Dose responses over a range of doses would be most informative and would aid risk assessment. Understanding MoAs/Mechanisms of NMs Several papers have addressed potential toxic and genotoxic mechanisms of action of NMs (Liu et al., 2016; Pati et al., 2016; Saptarshi et al., 2015; Zijno et al., 2015). The diversity of genotoxicity systems, NMs, and results found in this review are not conducive to clarifying MoAs of NMs. However, some useful conclusions can be noted. Nanomaterials, when positive in a genotoxicity assay, do not generally induce the large increases in genotoxic responses that are characteristic of many classical DNA damaging agents. This might not be surprising, since NMs typically do not interact directly with DNA (ie, do not involve covalent interactions such as alkylation, or intercalation). The observations in this analysis are consistent with the concept that the genotoxicity of most NMs is likely to be indirect, eg, via generation of oxidative species or indirect consequences of inflammation (Landsiedel et al., 2009; Xia et al., 2013; and many others). Another possible mechanism involves direct physical interaction with the spindle apparatus during cell division (Sargent et al., 2010; Siegrist et al., 2014). Based on this, it appears appropriate to apply the principles of indirect versus direct genotoxic effects of NMs in risk assessments. It is important to note that to date, most focus has been on the oxidative stress MoA, but there has been limited evaluation of other mechanisms and thus it is not possible to rule out other DNA damage mechanisms including perturbation of systems, eg, DNA repair or DNA synthesis. The comet assay may be particularly useful in studies on oxidative MoAs and its sensitivity for this type of lesion can be further improved by using OGG1 or FPG glycosylases for recognition of oxidative DNA adducts. However, the in vitro comet assay, as currently practiced, is subject to variable responses resulting from the use of diverse methods and cell systems, which may vary in metabolic and DNA repair capability. Standardized methods for the in vitro comet assay, including the use of well-characterized glycosylases, have not been established, and are a notable gap in the genotoxicity test battery. This is therefore another area that requires further clarification, which can impact the scope of the testing framework to take forward. If indirect effects represent the major mode of genotoxicity of NMs, genotoxicity assessment of NM may be better integrated into a broader context of systems toxicology. Attempts to categorize NMs (Godwin et al., 2015) may be a useful approach to understanding MoA as well as facilitating the analysis of large numbers of material agents. The UCLA Center for Nano Biology and Predictive Toxicology has adopted a Tox21 high content approach targeting defined pathways of toxicity involving pulmonary inflammation, ROS and membrane effects, as related to the physical and chemical properties of NMs (Godwin et al., 2015; Nel, 2013). Arts et al. (2015, 2016) have furthered the idea and developed a decision-making framework (DF4) for the grouping and testing of NMs which considers intrinsic material and system-dependent properties, biopersistence, uptake and biodistribution as well as cellular and apical toxic effects which are derived from in vitro studies. This concept can be helpful for MoA understanding but can be useful also for selection of appropriate assays or protocols since the categories they provide, ie, (1) soluble NMs, (2) biopersistent high aspect ratio NMs, (3) passive NMs, and (4) active NMs, provide initial clues about the behavior of a NM in a test system, and whether testing is relevant within a particular context, eg, human exposure. These approaches and “read across” (Oomen et al., 2015) may be most useful for minimizing testing where there is no human relevance, but any utility in risk assessment remains to be demonstrated. Recommendations on Method Standardization and Assay Modifications General Notes on Standardization of Approaches (Relevant for All Assays) If NMs are used by a particular route of administration in humans, this might be considered in genotoxicity testing if feasible (however, see below on testing within the context of cells and systems recommended in OECD TGs). Consideration should be given to the potential release and solubilization of NMs or components of complex NMs in cell culture medium or other vehicle, to aid understanding of potential ion versus particle effects. Examples would be the metal oxides, which release ions. Preliminary studies with the NM test article should provide a rationale for type of media, dispersion method, surfactant use, duration of treatment, test system and dosimetry. The presence of serum or proteins in the medium can impact the extent of particle internalized in the cells. Characterization of NMs should occur in the test media (as well as in other media, as appropriate). Consideration should be given to potential artifacts occurring with NMs in the test media (eg, agglomeration). Per the OECD TGs, toxicity should be measured in parallel with genotoxicity, not in separate trials. The toxicity parameters used in all genotoxicity studies should be those recommended for each assay in the relevant OECD guidelines (eg, relative population doubling, cloning efficiency or relative total growth). OECD guidelines have been updated, and previous methods, such as confluence estimation or dye exclusion, are no longer considered adequate. Uptake of the NMs into the cells of the test system should be documented and the location of particles determined within the cells if feasible (ie, nucleus or cytoplasm); evidence should be provided that the NM reached the target tissue in any in vivo assays. In some cases, results may be positive in the absence of uptake (eg, for bacteria), which could reflect breakdown of the material in the test environment and/or the release of diffusible genotoxins. Cell lines and test systems should be limited to those recommended in the OECD guidelines and for which methods are well characterized. Lorge et al. (2016) provide resources and information on some preferred cell lines. Cell lines for mammalian in vitro cytogenetic studies include V79, CHL, L5178Y, CHO, TK6 cells, and human lymphocytes (although it is now recognized that rodent cell lines may be more sensitive to cytotoxicity and genotoxicity than the human and/or p53 competent cells when one is conducting cytogenetic assays (Hashimoto et al., 2011; Honma and Hayashi, 2011). Nonetheless, it is important to note that the type of cell considered may have an impact on the outcome of the assay, as different cells may have different internalization capacity (Manshian et al., 2015), different DNA repair capability, or different metabolic capability. It may be critical to characterize the capability of the cells to take up model particles prior to the choice of a cell type. Other cell lines and systems should be used only if they are justified as particularly related to human use. These cell systems should however be compared with the standard systems and methods to aid data interpretation; they should have a sufficiently stable genetic background to support genotoxicity assessment, as demonstrated through response to appropriate positive and negative controls. They also must be validated and the same recommendations applied as are in the OECD TGs. Appropriate attention should be given to study design and statistical power, including the choice of doses from preliminary tests to be within an acceptable range based on toxicity; adequate number of doses spaced for maximum information relative to a biological effect; and statistical validity of the test results, eg, adequate cell numbers and replicates within a countable range. These parameters are fully described in the appropriate OECD TG. Positive and negative controls for each assay should be included and the results should be within the acceptable/expected range of the assay (as described in the OECD TGs). Although controls for the assays are recommended, NM-specific controls have rarely been demonstrated, and are not necessary to show the performance of the assay. Recommendations on Modification, If Needed, of Each Assay for Use With Nanomaterials Bacterial assays Since S. typhimurium and E. coli tester strains appear not to take up or respond to NMs, the recommendation is for an in vitro mammalian mutagenicity assay instead of a bacterial mutation test. Results from negative bacterial assays are not definitive as a test result for NMs. However, the bacterial assays may be appropriate to assess soluble genotoxic or toxic agents released from NMs. In vitro MN assay The in vitro MN assay is recommended as a component of a test battery for assessment of NMs. However, a modification of the assay is needed because CB treatment can inhibit the uptake of NPs by endocytosis or pinocytosis. Thus CB, if used, should be applied after NM exposure, allowing sufficient time for NP uptake (Doak et al., 2009, 2012; Haynes et al., 1996). Appropriate cytotoxicity evaluation must also be carried out in parallel as described earlier. Other outstanding questions surround the ideal exposure time for this assay and the exact choice of cell type. Regarding exposure time, it is important that the cells be allowed to complete more than one cell cycle in the presence of the NM so that NMs taken up by the cells may come into direct contact with the DNA when the nuclear membrane breaks down during mitosis (Doak et al., 2012; Nelson et al., 2017). Standard cell lines with suitably low background MN frequencies and stable genetic backgrounds are recommended (Doak et al., 2012; Lorge et al., 2016). In vitro chromosomal aberration assay Cytogenetic damage is an important genotoxicity endpoint; thus the chromosome aberration assay would be a recommended assay in a test battery. However, it takes a significant level of expertise to score chromosomal aberrations. If the chromosomal aberration assay is performed, aberrations should be characterized according to typical categories (eg, chromatid breaks), especially to ensure that chromatid and chromosome gaps are noted separately from aberrations. Test methods do not require modification for assessment of NMs, but confirmation of particle uptake should be included. In vitro comet assay There is no standard method (eg, OECD TG) for the in vitro comet assay. Thus, the methods used are more likely to vary in ways that may not be validated. The comet assay generally shows positive results with NMs, but there are major questions about the reliability or validity of these results that were not discernible in the methods review undertaken here. There is no consistent evidence that the use of glycosylases targeting oxidative damage lesions enhances sensitivity. Is this a result that truly reflects variability among NMs? Or is it related to different enzymes, incubation times, cell type or handling method. Several papers have postulated (Karlsson et al., 2015) or demonstrated sources of artefactual positive results with the comet assay (Ferraro et al., 2016). A standard protocol for the alkaline comet assay post treatment is found in the in OECD TG 489 for the in vivo comet assay. This includes technical details on slide prep, lysis, electrophoresis and analysis and may be useful in performance of the in vitro assay. If maximal assay sensitivity for detection of oxidative DNA damage is desired, the use of a modified version (use of DNA glycosylases) may be beneficial. Reduced effects when NMs are removed prior to the posttreatment stage are important indicators that NMs can affect the electrophoretic mobilities of DNA. Therefore, NM assessments using the comet assay should consider a rinsing step to remove residual NMs after a defined treatment stage. Comparison of results with and without posttreatment rinsing would be informative. In addition, the DNA strand breaks measured in the comet assay are not a fixed genetic endpoint. They are intermediates that can change during the process of measurement and thus require strict conditions for assessment. Because of the lack of standard methods and uncertainty over the meaning of results, the comet assay is not recommended as a screening assay for NM genotoxicity assessment. However, careful experimentation with attention to the potential generation of artefacts can be useful in understanding NM effects. In vitro mammalian gene mutation assays Since it is suggested to waive the bacterial assays for evaluating NMs, a general recommendation would be to include an in vitro mammalian cell gene mutation assay in the test battery for NMs. Assays for NMs should be conducted based on OECD TG 490 (for the Tk locus) or TG 476 (for Hprt). All of the recommendations in these TGs are relevant to the testing of NMs and should be followed. The literature review did not reveal any assay modifications from the standard assay designs and performance that would be required for the conduct of these assays for evaluating NMs (other than the general recommendations affecting all assays such as NM characterization, evaluation of uptake, cytotoxicity measurements, etc). However, consideration should be given to the adequacy of NM clearance from suspension cultures after exposure, or whether the NM is present during mutation fixation and development. In vivo genotoxicity assays There is generally much less data on NM effects in vivo than in vitro; however, because of ADME issues, effects in a living organism are considered more important for safety assessment of NMs than for other test agents. Recommendations on in vivo assays for genotoxicity assessment of NM are summarized below: Dosing should include appropriate dose escalation for toxicological assessment, but avoid particle overload that could lead to artifacts. The in vivo assays generally do not need modifications and may be performed according to OECD TGs: 474 (Mammalian Erythrocyte MN Test), 475 (Mammalian Bone Marrow Chromosomal Aberration Test), 488 (TGR Somatic and Germ Cell Gene Mutation Assay), 489 (In Vivo Mammalian Alkaline Comet Assay). If detection of oxidative damage is a goal, the comet assay modification that includes DNA glycosylases (Bourdon et al., 2012; Sharma et al., 2012) may provide maximal sensitivity (TG 489). The in vivo assays should be considered and chosen within the context of the need for in vivo-specific information, eg, distribution and sequestration of NMs in target tissues or organs, or to model human exposure. Recommended Approach for Assessing Genotoxicity of Nanomaterials 1. Scoping assessment A scoping assessment is recommended to evaluate available data on physico-chemical characterization and potential systemic distribution of NMs (consistent with the model of Next Gen genotoxicity assessment (Dearfield et al., 2017). This can be based on human exposure or sub-chronic animal experiments, if available as part of a standard safety assessment (data are not available in certain contexts, eg, for the review of cosmetics in the EU). Information on NM distribution, tissue or organ accumulation, or sequestration would be considered in the development of testing strategies. If systemic availability or tissue targeting effects did not occur, in vitro but not in vivo genotoxicity testing would be recommended. In the case where systemic effects were noted, in vivo genotoxicity testing would be considered. The selection of tests would be defined based on systemic/tissue targeting observed. 2. Recommended test battery Recommended test battery includes in vitro mammalian assays that detect the 2 major classes of genetic damage: gene mutation and chromosomal damage with a choice of assay from each group: A. In vitro mammalian mutagenicity assay (replaces bacterial mutation assays) (choose one) Mouse Lymphoma (L5178Y) TK±Assay (MLA) (OECD TG 490) HPRT gene mutation assay (HPRT) (OECD TG 476) Rationale: These forward mutation assays detect the same types of small scale genetic events as bacterial assays, including single base pair changes and frameshifts. In addition, the MLA detects a broad spectrum of genetic damage including chromosome rearrangements, deletions (both small and large) and mitotic recombination. The MLA and the HPRT assays thus detect a different spectrum of genetic damage and there can be situations where one is preferred. That is, if the detection of only small scale events is desired, it may be preferable to use the HPRT assay. For hazard identification it is often desirable to use the MLA to detect a broader array of events. The bacterial assays are not included in this recommended test battery because of substantial evidence that the bacteria used for standard genotoxicity testing (E. coli and S. typhimurium) have limited uptake of NMs. They also lack the capability for mammalian-specific responses, which may be the more important reason for their lack of response to NMs. Results in bacteria have been generally negative and are considered uninformative. B. Chromosomal damage assays In vitro Chromosomal aberration assay (OECD TG 473) In vitro MN assay with assay modification as described earlier (OECD TG 487) In vitro MLA (OECD TG 490) Rationale: These assays detect large scale genetic damage affecting chromosomes, particularly breaks, rearrangements, or whole chromosome loss. The in vitro MN assay is more commonly used than the chromosomal aberration assay, because it is less subjective and requires less skill in reading the endpoint. In addition, the MN assay can be automated for the assessment of a large number of cells, enhancing statistical validity. However, consistent with international strategies for chemical testing, there are 3 equally acceptable options for assessing the ability of NMs to cause chromosomal effects (MN assay, Chromosomal aberration assay, and the MLA). Although the MLA detects both large and small scale genomic damage, it is recommended that 2 in vitro assays be chosen for assessment of nanomaterials. Because NMs generally do not require metabolic activation, there is no compelling reason to perform an in vitro assay with S9, and no reason for default testing in vivo. Thus, NMs are generally tested in the absence of S9, unless composed of organic materials or agents likely to be affected by mammalian metabolism. 3. Additional tests for consideration A. The in vitro comet assay, a DNA strand break assay, is sometimes considered, especially in the context of assessment of oxidative damaging effects. However, the assay lacks an OECD guideline, defined protocols, and a mechanistic understanding based on positive and negative responses to a defined set of mutagens. Handling issues may be particularly important because of ongoing DNA repair processes that affect the quantitative endpoint. TK6(TK±) (human lymphoblastoid) assay (OED 490) has not undergone validation for hazard identification, but may be considered, especially if genetic variants (p53 or DNA repair deficiencies) would contribute to mechanistic understanding of a positive result (http://www.nihs.go.jp/dgm/tk6.html) In vivo assay if targeting/sequestering of NM to a specific tissue is demonstrated, or for additional information relative to in vivo risk Comet assay (OECD TG 489) Rodent transgenic mutagenicity assay (OECD TG 488). The in vivo MN assay (OECD TG 474) is part of some international testing strategies and is valid only when NMs are systemically available or when exposure of target tissues has been demonstrated. CONCLUSION A great diversity of test systems and methods has been used to assess the genotoxicity of nanomaterials, with almost a similar level of diversity of results. Thus few conclusions on NM genotoxicity can be made, despite a substantial body of work. This review sought to critique the published literature, with a view of providing recommendations on validated methods and systems for genotoxicity assessment of NMs. A multitude of issues were documented from this analysis, including a wide variation in physical and chemical properties of NMs, inconsistent NM characterization in the test medium, diversity of test systems often failing to meet OECD standards, difficulty of applying NMs to biological systems (including uptake), interference of NM with the test endpoint, potential variation in systemic distribution in vivo, and lack of a definitive MoA. Based on current data, it appears that NM genotoxicity responses are smaller than observed from classical DNA damaging agents, consistent with genotoxicity induced via a secondary effect rather than a result of direct DNA interaction. As a way forward, we recommend: (1) the use of carefully defined NMs, including characterization in the test medium; (2) assessment of uptake and distribution within cells and in vivo systems; (3) dose ranges carefully chosen to avoid artefacts related to system overload; (4) a modified test battery that includes genotoxicity testing in in vitro mammalian mutagenicity and chromosomal damage assays, coupled to assay modifications as described within; (5) adherence to tests and cell systems described in the OECD TG and in Lorge et al. (2016); (6) and a greater effort on understanding mechanisms. ACKNOWLEDGMENTS The authors wish to thank the HESI Genetic Toxicology Technical Committee for intellectual and financial support. The authors also acknowledge the assistance by Ms Teyent Getaneh, Ms Lauren Peel, Ms Christina West, and Dr Stanley Parish of HESI for administrative support. REFERENCES Aardema M. J. , Barnett B. 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Concentration-dependent effects of fullerenol on cultured hippocampal neuron viability . Int. J. Nanomed. 7 , 3099 – 3109 . Google Scholar OpenURL Placeholder Text WorldCat Zhu L. , Chang D. W., Dai L., Hong Y. ( 2007 ). DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells . Nano Lett . 7 , 3592 – 3597 . Google Scholar Crossref Search ADS PubMed WorldCat Zijno A. , De Angelis I., De Berardis B., Andreoli C., Russo M. T., Pietraforte D., Scorza G., Degan P., Ponti J., Rossi F., et al. . ( 2015 ). Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells . Toxicol. In Vitro 29 , 1503 – 1512 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Present address: Toward Safer LLC, Washington, DC 20009. Disclaimer: This article has been reviewed by the agencies and organizations of the authors and approved for publication. The views expressed in the manuscript do not necessarily reflect the policy of these agencies and organizations. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. Published by Oxford University Press on behalf of the Society of Toxicology 2018. This work is written by US Government employees and is in the public domain in the US. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) Published by Oxford University Press on behalf of the Society of Toxicology 2018. This work is written by US Government employees and is in the public domain in the US. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

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Copyright © 2022 Society of Toxicology
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1096-6080
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10.1093/toxsci/kfy100
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Abstract

Abstract Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute’s Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing. genotoxicity, genetic toxicology, nanoparticles, test battery, mutagenicity, clastogenicity, testing strategy Nanomaterials (NMs) generally refer to nano-objects and particles with one or more dimensions in the nanometer size range [a diversity of definitions exists]. NMs present challenges in safety evaluation owing to small size, relatively large surface area, and unknown disposition in biological systems. A working group of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC) has been addressing the genotoxicity assessment of NMs. The group’s evaluation is anchored by a critical review of published primary data that evaluates potential genotoxic effects of NMs in the standard genotoxicity assays. The assays include the bacterial reverse mutation (Ames) assay; in vitro mammalian assays for mutations, chromosomal damage, micronucleus (MN) induction, or DNA strand breaks; and in vivo assays for genetic damage in various target tissues (MN, comet and transgenic mutation assays). This review follows and extends the UK’s Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM)’s review of genotoxicity assessment of NMs and experimental considerations in publications identified between 1992 and early 2012 (COM, 2012). It also builds on the Environmental Mutagenesis and Genomics Society (EMGS) 2010 workshop report (Pfuhler et al., 2013) in which issues related to assay utility and mechanistic endpoints were also addressed. Since our review began, other authors have come to similar assessments of the issues related to genotoxicity testing, using different analyses (Golbamaki et al., 2015; Magdolenova et al., 2014; Swedish Chemicals Agency, 2016). Our work provides a summary and an extensive critique of genotoxicity test data, methods validity, and potential conclusions about mechanisms. From this context we make recommendations for future testing of NMs (summarized in the Roadmap, Figure 1). Figure 1. Open in new tabDownload slide Nanomaterial genotoxicity testing roadmap. Figure 1. Open in new tabDownload slide Nanomaterial genotoxicity testing roadmap. MATERIALS AND METHODS For each genotoxicity assay, the published literature on NMs was analyzed by a team of genetic toxicologists experienced in that assay, who are the coauthors of this article. At a minimum, the teams conducted exhaustive literature searches to identify papers published after COM (2012) and through the end of 2014, using search terms designed to identify papers in diverse biological, physical, and nanotechnology journals. The combination of multiple assays without definition other than “genotoxicity” in many papers made searching difficult, particularly in the more recent literature. For the assays with fewer citations (except in vitro MN), earlier and more recent papers were often included in the analysis; each assay section below explains the range considered and any additional criteria that were part of the analysis. The following issues were considered in the literature evaluation: NM evaluated Type and size Characterization and sample preparation Test system uptake Test systems/assays used Standard methods or other Organization for Economic Co-operation and Development (OECD) Test Guidelines (TG) compliance Metabolic activation Positive controls for the assay Toxicity measurements Test validity (see Tables 1 and 2) Table 1. Summary of Literature Using in Vitro Genotoxicity Tests In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant a Including (as appropriate) S9, CB, No. cells treated, No. replicates/repeats, No. cells scored. b S. typhimurium strains TA1535, TA 97, TA98, TA100, TA102. c For example no significant increase in MN with positive control or data not provided. d CB = cytochalasin B Open in new tab Table 1. Summary of Literature Using in Vitro Genotoxicity Tests In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant In Vitro Test . No. Papers Evaluated . No. of NMs . Test System . % with Cytotoxicity Assessment . % With Positive Control . % That Evaluated NM Uptake Into Cells . Results . % Papers Meeting Acceptable Criteria . Cell Types . Treatment Time . Standard Test Systema . Bacterial Ames Assay 26 32 S. typh.b and/or E. coli WP2 uvrA (Standard assay) Preincubation: 20 min to 20 h 65% Standard agar plate assay (4) (1 other method) Not performed with this assay 100% 19% (5/26) 31% were called positive (revertants ≥ 2× background) Few if any positive results were judged valid due to unacceptable methods, errors, or credibility issues. No experiments with positive results were repeated. In vitro MN 79 27 32 different cell lines 15 different treatment times (2h-80 days). 54%: 24h CBd coexposure: 11%. CB postexposure: 59% Mononuclear MN: 22% Unclear: 8%Insufficient cells scored: 23% None: 5% Standard: 45% Colorimetric/fluorometric dyes: 38% Other: 12% Yes: 62% No: 25% Inappropriatec: 13% 46% Almost all NP showed a (usually) weak positive response. For the most widely studied materials (eg SWCNT, silica, Ag, TiO2) at least one negative study also exists. 46% (36/79) meet set criteria, including those with nonstandard cytotoxicity evaluation; If studies not conducting approved cytotoxicity tests are removed, 24% remain (19/79). In vitro Chromosomal Aberration (CA) 11 7 5 different cell lines 3 different treatment times 73% (8/11) were 3 and 24 h Standard use of short and long exposure conditions: 90% (10/11) None: 9% Standard: 55% Colorimetric / fluorometric dyes: 36% 73% 81% Results in 3 CA papers were considered positive, involving tests of ZnO, carbon nanotubes and Ag. 55% meet acceptable criteria; 45% do not, mainly due to a lack of assessment of cytotoxicity In vitro Comet 22 33 23 different cell lines 8 different treatment times (1–72 h) (1, 2, 3, 4, 6, 24, 28, and 72) 17/29 used 24 h treatment Standard in vitro alkaline comet assay: 79% FPG-modified comet assay: 21% None: 5% Colorimetric/fluorometric dyes: 95% Other: 16% (in addition to colorimetric/fluorometric) 73% 45% 3 NM were considered positive: ZnO2, carbon nanotubes and Ag 55% meet acceptable criteria In Vitro Mammalian Cell Gene Mutation 17 19 10 different cell lines Various, from 3–4 h to 24 days 53% used standard (OECD) cell systems; only one used S9 Cloning efficiency, biochemical markers: not always performed or concurrent 65% 37% Inconclusive; conflicting results for some NM; review group did not always agree with author conclusions 53% studies used standard cell lines, but not all would be considered OECD TG compliant a Including (as appropriate) S9, CB, No. cells treated, No. replicates/repeats, No. cells scored. b S. typhimurium strains TA1535, TA 97, TA98, TA100, TA102. c For example no significant increase in MN with positive control or data not provided. d CB = cytochalasin B Open in new tab Table 2. Summary of Literature Using In Vivo Genotoxicity Tests In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. Open in new tab Table 2. Summary of Literature Using In Vivo Genotoxicity Tests In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. In Vivo Test . No. Papers Evaluated . No. of NMs . Test System . % with Organ Toxicity Assessment . % with Positive Control1 . % that Evaluated NM Uptake into Cells . . % Papers Meeting Acceptable Criteria . Animal Model . Treatment Time . Exposure Route . Standard Test System . Results . In vivo MN And/or Chromosomal Aberration (CA) 18 17 10 rats 8 mice mostly 1 sex only (males) 15 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 8 oral 1 intra-tracheal instillation 1 inhalation 5 i.v. injection 4 i.p. injection Standard short term and 28 day studies; also single dose studies, with recovery time Hematology: 6% 58% 17% of studies evaluated cellular and tissue uptake TiO2 positive in 2/3 studies; Ag positive in 2 studies; MgO2 positive in both MN and CA; AlO2 positive in both MN and CA 65% of studies lack proper characterization of NMs In vivo Comet 17 20 9 rats 8 mice mostly 1 sex only (males) 16 different treatment times (single treatment, 1 h sacrifice up to 28 day treatment) 5 oral 4 intra-tracheal instillation 2 inhalation 4 i.v. injection 3 i.p. injection 4 FPG or OGG1 modified comet Histopathology findings: 28% Blood haematology: 6% (1/18) 41% 6% of studies evaluated cellular /nuclear uptake 50% positive results across all studies, albeit some were questionable. No clear pattern of damage was evident. 41% acceptable. Main issues: lack of positive control, adequate protocols, characterization of NMs Mammalian Transgenic Mutation 8 11 Gpt-delta mice, MutaMice, C57BL6 mice, F344 rats, male and female Myh-/- mice From single treatment to multiple treatments spanning 28 days; sampling time ranged from weeks to one year in one case i.p., oral (gavage, drinking water) Intratracheal instillation, Lateral tail vein injection (i.v.), Whole body inhalation and pharyngeal aspiration, Assays detecting mutations in Gpt, Spi-lacZ or Hprt, K-ras codons 8, 12; Pun reversion/ deletion Histopathology of lungs for accumulation of macrophages and neutrophils, granulomas, epithelial hyperplasia bronchoalveolar lavage: 73% 33% 73% Not evaluated: PVP-coated Ag NPs, quartz and carbon black 91% tested positive in one of the mutation assay variants; the exception was asbestos 75% of the studies meet acceptance criteria; studies that used K-Ras and Myh -/- mice lack established OECD TG for conducting these assays. Open in new tab Appropriateness of the system for assessment of NMs Need for methods alteration for NM assessment Results (positive or negative) Critique of result based on acceptance criteria and comparison with a concurrent positive and negative control If positive, dynamic range of effect Consistency of results with particular NMs in diverse published papers Insight into potential modes of action (MoAs) In the analysis, experiments were evaluated for adherence to established guidelines/guidance, including the OECD TG, an approach that is critical for interpreting results in a regulatory context. We recognized that sample characterization is of major importance for the assays, particularly analysis of the sample in vehicle and in the test system. However, other than noting whether samples were characterized, comments have not been made on the adequacy of sample preparation or characterization. RESULTS Literature evaluation and methods recommendations for genotoxicity assays are summarized in Tables 1 and 2 (for in vitro and in vivo genotoxicity tests, respectively) and presented by assay in the following sections. Bacterial (Ames) Genotoxicity Assays For the bacterial (Ames) genotoxicity assessment, 26 papers published from 2009 to 2016 (including COM, 2012) were considered (Table 3), and findings summarized in Table 1. Nanomaterials tested included metal oxides (Al-, Cu-, Ti-, Zn-, Ce-, In- [indium], IN tin, Dy- [dysprosium], W- [tungsten]), Fe- (magnetic), TiSi-, elemental metals (Mo [molybdenum], Ag, Cu, gold nanorods) as well as carbon-based (single-walled carbon nanotubes [SWCNTs] and multi-walled carbon nanotube[MWCNTs]), quantum dots, and complex or combination NMs (Au-PMA-ATTO, coated Zn oxides, asbestos, diesel exhaust, polymeric nanocapsules, phospholipids), and organic NMs. Most tests included assessment with and without an exogenous metabolic activation system (S9). Although S9 would usually not be expected to have an effect, the proteins in S9 could affect uptake of the nanoparticles. Some changes in results were reported in the presence of S9 (Gomaa et al., 2013; Hasegawa et al., 2012; Kumar et al., 2011; Liu et al., 2014; Lopes et al., 2012), but these were not consistent among NMs, within a lab, or within a discernible context, eg, metal oxides. Table 3. Bacterial (Ames) Genotoxicity Assay: Papers Evaluated Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Open in new tab Table 3. Bacterial (Ames) Genotoxicity Assay: Papers Evaluated Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Description . References . 26 papers evaluated Akyıl et al. (2016), Aye et al. (2013), Butler et al. (2014), Clift et al. (2013), Di Sotto et al. (2009), Ema et al. (2012), Errico et al. (2012), Gomaa et al. (2013), Hasegawa et al. (2012), Hu et al. (2013), Jomini et al. (2012), Kim et al. (2013, 2015), Kumar et al. (2011), Kwon et al. (2014), Li et al. (2012), Liu et al. (2014), Lopes et al. (2012), Maenosono et al. (2009), Naya et al. (2011), Pan et al. (2010), Sadiq et al. (2015), Shinohara et al. (2009), Warheit et al. (2007), Wirnitzer et al. (2009), Woodruff et al. (2012) 8 papers reported positive bacterial mutagenicity results Clift et al. (2013), Gomaa et al. (2013), Jomini et al. (2012), Kumar et al. (2011), Liu et al. (2014), Lopes et al. (2012), Pan et al. (2010), Sadiq et al. (2015) Open in new tab Papers were not subjected to acceptance criteria, but were analyzed individually as described in the “Methods” section. Most studies used the standard set of Salmonella typhimurium and/or Escherichia coli tester strains or a subset consisting of TA98 and TA100. All papers incorporated a positive control, but positive and negative controls were often not adequate to determine the impact of methods variations. Most papers utilized a preincubation method, but this varied between 20 min and 20 h. Each paper was analyzed for validity individually, based on bacterial-specific methods required for mutation development, and on response outcomes typical for mutagens. Of major importance for these assays was the question of whether any studies with positive results seem valid. In total 5 out of 26 papers measured uptake of the particles into S. typhimurium standard tester strains (Butler et al., 2014, 2015; Clift et al., 2013; Kumar et al., 2011; Woodruff et al., 2012). The positive results in Kumar et al. (2011) with TiO2 were not replicated in Butler et al. (2014). Photographic evidence for uptake may not be definitive. Eight papers reported positive bacterial mutagenicity results (Clift et al., 2013; Gomaa et al., 2013; Jomini et al., 2012; Kumar et al., 2011; Liu et al., 2014; Lopes et al., 2012; Pan et al., 2010; Sadiq et al., 2015); however, this call was variously based on a 2-fold increase in revertant colonies over the control, a lesser increase that might be called marginal, or a greater increase under unacceptable conditions, ie, starvation (Jomini et al., 2012) or treatment using agar at 55° (Liu et al., 2014), likely to induce heat shock. The analysis provided here finds a lack of convincing validity for most if not all of the reported positives. For example, both Pan et al. (2010) and Kumar et al. (2011) report positive results that were borderline (approximately 2-fold over the control). More concerning is the pattern of response—a constant revertant colony number over a dose range encompassing 3 orders of magnitude, ie, an elevation without a dose response. Lopes et al. (2012) reported background colony counts with TA98 minus S9 as 10 times higher than the values plus S9 (200 vs approximately 20). Clearly there was a systematic error here with the minus S9 cultures. Gomaa et al. (2013) and Sadiq et al. (2015) respectively found a borderline positive only at the highest dose or doses, a limited but more credible result. Jomini et al. (2012) observed positive results with a fluctuation assay after preincubation up to 20 h in saline, a nonnutritive medium designed to minimize conditions impacting agglomeration and enhance association of particles with bacteria. There might be an important concept developed in this article regarding the ionic interactions of particles and cells. However, positive and negative controls did not appear to be subjected to the long preincubation time and thus the effect on the bacteria of a significant departure from standard methods, including starvation conditions, cannot be evaluated (discussed further below). Most concerning is a rationale for all 5 S. typhimurium tester strains testing positive with 1 agent (Dy2O3, in Hasegawa et al., 2012; iron oxide in Liu et al., 2014). Liu et al. (2014) found positive results with 10 nm polyethylene glycol (PEG)-coated iron oxide particles in all 5 strains of S. typhimurium (TA1535, TA97, TA98, TA100, and TA102) plus or minus S9, or with the 30 nm PEG-coated NM in the bacteria only with S9. This is problematic because the strains are designed to respond to different types of DNA interactions leading to different DNA sequence changes; there is little precedent for all of the tester strains responding similarly to one agent. These results indicate a systematic origin of positive responses that is unexplained. There are other issues in the papers reporting positive responses, eg, background mutant colony numbers that are not acceptable (eg, >200 for E. coli WP2) (Pan et al., 2010) and for TA98 as noted above (Lopes et al., 2012), and the lack of a rationale for positives in TA98 but not TA100 (Kumar et al., 2011), ie, results indicating sequence-specific alterations. Few of these studies appeared to confirm the results in a second experiment, a very important requirement for borderline or questionable positive results. When these results were reported, even though they are outside the norms of expectations, they were not discussed as such in the papers. It is possible that a few positives, eg, Cu metallic nanoparticles that showed a borderline dose response at high concentrations (Sadiq et al., 2015) perturb DNA replication, which could result in DNA sequence alterations. Our conclusion is that none of the studies reviewed were entirely credible with regard to induction of mutations in bacteria, and most were clearly not credible. As the scientific community has recognized that the standard bacterial Ames assay testing approach may not be adequate as a component of a test battery for NMs, method variations, including preincubation, expanded exposure times, and concentrated exposure of bacteria in small volumes, have been tried but did not change the negative outcome, eg, Butler et al. (2015). Some methods alterations, such as preincubation for a day in saline, ie, nongrowth medium at 37°, gave positive responses (Jomini et al., 2012), but the method alteration (without informative controls) compromised the validity of the test. A starvation condition induces stress responses and other responses designed to save the population from a nonviable condition. These conditions may lead to mutation generation that is not a result of classical DNA damage (Foster and Cairns, 1992; Wright, 2004). One paper used a nonstandard preincubation method in microtiter plates with generally negative results except for diesel exhaust particulates, which have tested positive in previous studies (Clift et al., 2013), likely due to the leaching of genotoxic polycyclic aromatic hydrocarbons. As noted previously by others (Doak et al., 2012; EFSA Scientific Committee, 2011; Landsiedel et al., 2009; Warheit and Donner, 2010), the gram-negative strains of bacteria used in the standard assays do not appear to have the capability for nanoparticle uptake, lacking mammalian mechanisms of endocytosis, pinocytosis, and phagocytosis. The lack of uptake is considered to be the reason for generally negative outcomes following nanoparticle exposure to bacteria. However, there is also the possibility that bacteria are not capable of the type of response that leads to positive effects with NMs in mammalian cells (eg, an indirect response to oxidative stress involving mitochondria). The negative result of AgNO3 in experiments with Ag nanoparticles indicated that the negative results were not necessarily due to the lack of uptake (Butler et al., 2015). Thus, the current standard bacterial mutagenicity test (Ames Assay) is not recognized as an informative component of a genotoxicity test battery for assessment of NMs. This conclusion is consistent with other reviews (COM, 2012; Vandebriel and De Jong, 2012) and discussions (Doak et al., 2012; Gonzalez et al., 2008; Landsiedel et al., 2009; Oesch and Landsiedel, 2012; Pfuhler et al., 2013), but we have provided an extensive analysis that goes well beyond previous work. In Vitro MN Assay The in vitro MN assay is a standard test system in the genotoxicity battery; however, it has been widely recognized over recent years that simply applying this test according to the OECD test guideline 487 is not wholly appropriate for nanomaterials. Thus, papers applying the in vitro MN assay to assess NMs from 1997 to 2014 (including the COM [2012] analysis) were carefully evaluated for their methodological approaches, as summarized in Table 1. Seventy-nine papers were identified (Table 4), in which 27 NMs had been evaluated consisting of metal oxides (eg, Al-, Cu-, Ce-, Fe-, Si-, Ti-, Y-, and Zn-oxides); metals (Au, Ag); carbon based (fullerenes, SWCNTs, MWCNTs), imogolite); and other combination materials (such as quantum dots, and WC). Table 4. In Vitro MN Assay: Papers Evaluated Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Open in new tab Table 4. In Vitro MN Assay: Papers Evaluated Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Description . References . 79 papers evaluated Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Bhattacharya et al. (2008), Choi et al. (2012), Cicchetti et al. (2011), Colognato et al. (2008), Conde et al. (2014), Cveticanin et al. (2009), Demir et al. (2014), Di Bucchianico et al. (2014), Di Virgilio et al. (2010), Downs et al. (2012), Ema et al. (2012), Falck et al. (2009), Gandin et al. (2013), Gonzalez et al. (2010, 2014), Guichard et al. (2012), Guidi et al. (2013), Gümüş et al. (2014), Gurr et al. (2005), Haroun et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kang et al. (2008), Kato et al. (2013), Kawata et al. (2009), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Lindberg et al. (2009, 2013), Linnainmaa et al. (1997), Manshian et al. (2013), Merhi et al. (2012), Migliore et al. (2010), Moche et al. (2014), Mrdanovic et al. (2009, 2012), Muller et al. (2008), Niwa and Iwai (2006), Nymark et al. (2013), Papageorgiou et al. (2007), Park et al. (2011), Patil et al. (2012), Pelka et al. (2013), Perreault et al. (2012), Pfaller et al. (2010), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Selvaraj et al. (2014), Shi et al. (2009), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Tavares et al. (2014), Tsaousi et al. (2010), Turkez et al. (2014), Uboldi et al. (2012), Vecchio et al. (2014), Wahab et al. (2011), Wang et al. (2007a,b,c), Xu et al. (2012), Yin et al. (2010) Open in new tab For the in vitro MN assay, there were few papers that had evaluated a similar material, making it difficult to draw general conclusions about the ability of NMs to reproducibly induce chromosomal damage in vitro. Both TiO2 and silver Nanoparticles (NPs) were most often reported to show significant increases in micronuclei, but these findings were not consistent and comparisons were difficult since dose ranges, cells used and exposure durations were too variable to reach a definitive conclusion. For most materials assessed, where reports indicated a significant increase in genotoxicity, this was usually only approximately 2- to 3-fold times over negative control values. An inherent problem with trying to reach conclusions regarding NM effects in the in vitro MN assay was the substantial variation in the methodologies applied. As seen in Table 1, numerous cell lines, variations in methods involving cytochalasin B (CB) and cytotoxicity measurements, treatment times, and endpoint analyses were used with the in vitro MN assay. Given the substantial variation in the methodology applied, the protocols used in each paper were critically evaluated and the following criteria were applied for excluding tests from this analysis: CB treatment: studies were excluded if simultaneous cotreatment with the test NM and CB was the only treatment regimen as this is known to hinder cellular uptake (Doak et al., 2009, 2012; Haynes et al., 1996). Reports with negative controls that had MN frequencies above 2%, or those where background MN frequency was not provided. Inappropriate positive controls where no increase in MN frequency was observed with the positive control, or where positive controls were included but data was not provided to allow evaluation of approach (not all studies included positive controls, but this was not used as a specific exclusion criterion as it would have substantially reduced the remaining papers making evaluation of test approaches very limited). Studies using excessively high doses (>500 μg/ml). [The issue of dose selection is considered in the Discussion; the acceptability of dose limits was not universally applied in the data review]. Studies without concurrent toxicity evaluation, an inappropriate toxicity test, or genotoxicity assessed at toxic doses ie, >50% cytotoxicity. Cell number scored was too low or an inappropriate scoring methodology applied. Missing experimental information that prevented full evaluation of methodology applied. From this evaluation, 36 studies were classified as acceptable (46%) (Table 5). However, this includes studies that conducted an evaluation of cytotoxicity using nonstandard approaches; if those studies that did not conduct approved cytotoxicity tests for the in vitro MN assay were removed from consideration, this number would be reduced to 19 acceptable studies (24%) (Table 6). Table 5. In Vitro MN Assay: Acceptable Studies Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Open in new tab Table 5. In Vitro MN Assay: Acceptable Studies Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Description . References . 36 studies classified as acceptable Asakura et al. (2010), Aye et al. (2013), Bhattacharya et al. (2008), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Guidi et al. (2013), Jiang et al. (2013), Kang et al. (2008), Kim et al. (2011), Kisin et al. (2007), Könczöl et al. (2011, 2012), Kühnel et al. (2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Nymark et al. (2013), Patil et al. (2012), Perreault et al. (2012), Prasad et al. (2014), Selvaraj et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Turkez et al. (2014), Uboldi et al. (2012), Wang et al. (2007a,b,c) Open in new tab Table 6. In Vitro MN Assay: Acceptable Studies With Approved Cytotoxicity Tests Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Open in new tab Table 6. In Vitro MN Assay: Acceptable Studies With Approved Cytotoxicity Tests Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Description . References . 19 acceptable studies with approved cytotoxicity tests Asakura et al. (2010), Colognato et al. (2008), Di Bucchianico et al. (2014), Downs et al. (2012), Gonzalez et al. (2010), Könczöl et al. (2011, 2012), Kumari et al. (2014), Landsiedel et al. (2010), Li et al. (2012), Manshian et al. (2013), Migliore et al. (2010), Mrdanovic et al. (2012), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Wang et al. (2007b,c) Open in new tab When assessing the number of studies that evaluated uptake of the NM into the cell, 34 studies out of the total 79 had considered whether the NMs assessed had been internalized within the test cells (Table 7). However, only 9 of the 19 studies that had conducted an “acceptable” in vitro MN had evaluated uptake of the test NM (Table 8). Some examples of studies following an appropriate study design included Könczöl et al. (2012), Singh et al. (2012), and Migliore et al. (2010). Even with the exclusion criteria applied, there were very few studies that evaluated similar NMs, with the exception of silica and silver. Silica nanoparticles (Downs et al., 2012; Gonzalez et al., 2010; Guidi et al., 2013; Uboldi et al., 2012; Wang et al., 2007b,c) were largely negative (4 out of 6 studies) (Downs et al., 2012; Gonzalez et al., 2010; Guidi et al., 2013; Uboldi et al., 2012), while silver (Jiang et al., 2013; Kim et al., 2011; Li et al., 2012; Nymark et al., 2013) was positive for MN induction (3 out of 4 studies) (Jiang et al., 2013; Kim et al., 2011; Li et al., 2012). Thus, these studies were not unanimous in their findings and no 2 studies were conducted using the same cell line; 32 different cell lines were applied in the 79 papers reviewed. The tremendous variation in approach made it almost impossible to detect trends in the capacity for groups of NMs to induce genotoxicity. It was therefore apparent that standardization in the approaches applied to evaluate NMs is urgently needed. Table 7. In Vitro MN References: Studies That Considered NM Uptake Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Open in new tab Table 7. In Vitro MN References: Studies That Considered NM Uptake Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Description . References . 34 studies considered NM internalization within the test cells Asakura et al. (2010), AshaRani et al. (2008), Auffan et al. (2009), Aye et al. (2013), Choi et al. (2012), Colognato et al. (2008), Conde et al. (2014), Di Virgilio et al. (2010), Gandin et al. (2013), Guichard et al. (2012), Guidi et al. (2013), Jiang et al. (2013), Jugan et al. (2012), Kim et al. (2011), Könczöl et al. (2011, 2012), Kruszewski et al. (2013), Kühnel et al. (2012), Lindberg et al. (2013), Manshian et al. (2013), Merhi et al. (2012), Papageorgiou et al. (2007), Park et al. (2011), Ponti et al. (2009, 2013), Prasad et al. (2013, 2014), Rahman et al. (2002), Rotoli et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012), Srivastava et al. (2013), Uboldi et al. (2012) Open in new tab Table 8. In Vitro MN Assay: Acceptable Studies That Evaluated Uptake Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Open in new tab Table 8. In Vitro MN Assay: Acceptable Studies That Evaluated Uptake Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Description . References . 9 acceptable studies that evaluated uptake Asakura et al. (2010), Colognato et al. (2008), Konczol et al. (2011 (2012), Manshian et al. (2013), Prasad et al. (2014), Shukla et al. (2011, 2013), Singh et al. (2012) Open in new tab A relatively new development is the 3D human reconstructed skin MN assay (Aardema et al., 2010). This test is growing in importance as it allows evaluation of topically applied compounds on a reconstructed human 3D skin model. It is proving to be particularly useful for assessing the genotoxic potential of cosmetics products that can no longer be tested in vivo (Pfuhler et al., 2014; Wills et al., 2015). In Vitro Chromosomal Aberration Assay A total of 11 papers were considered (Table 9), recapped in Table 1, including 3 published from 2012 to 2014 and 8 references previously reviewed by COM (2012). Nanomaterials tested included TiO2, ZnO, SWCNT and MWCNT, fullerene, Ag, hydroxyapatite, and FePt capped with 2-aminoethanethiol. Chinese hamster lung (CHL) or Chinese hamster ovary (CHO) cell lines were most commonly used (8 out of 11 publications: Dufour et al., 2006; Honma et al., 2012; Kwon et al., 2014; Maenosono et al., 2009; Mrđanović et al., 2009; Shinohara et al., 2009; Theogaraj et al., 2007; Warheit et al., 2007). Human lymphocytes were also used (Turkez et al., 2014). In addition, there were single papers describing chromosomal aberrations in a mouse macrophage cell line (Di Giorgio et al., 2011) and a human mesenchymal stem cell line (Hackenberg et al., 2011). One study was excluded that did not have any cytotoxicity measure and where excessive dose levels, ie, >500 μg/ml, were used (Maenosono et al., 2009). Table 9. In Vitro Chromosomal Aberration Assay (CA): Papers Evaluated Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Open in new tab Table 9. In Vitro Chromosomal Aberration Assay (CA): Papers Evaluated Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Description . References . 11 papers were evaluated Di Giorgio et al. (2011), Dufour et al. (2006), Hackenberg et al. (2011), Honma et al. (2012), Kwon et al. (2014), Maenosono et al. (2009), Mrdanovic et al. (2009), Shinohara et al. (2009), Theogaraj et al. (2007), Turkez et al. (2014), Warheit et al. (2007) Open in new tab Results in 3 chromosomal aberration papers were considered positive, involving tests of ZnO, carbon nanotubes, and Ag (Di Giorgio et al., 2011; Dufour et al., 2006; Hackenberg et al., 2011). ZnO had a maximum increase of total aberrations to 16% (Dufour et al., 2006). Carbon nanotubes generated 25% and 40% acentric fragments at 48 and 72 h, respectively, and were accompanied by detection of reactive oxygen species (ROS) by dichlorofluorescein in a mouse macrophage cell line (Di Giorgio et al., 2011). Ag NMs induced a relatively weak positive response (up to 10% aberrant cells), but this result was based only on 50 cells scored (Hackenberg et al., 2011). Positive controls were included in 9 out of 11 papers reviewed (Di Giorgio et al., 2011; Dufour et al., 2006; Hackenberg et al., 2011; Kwon et al., 2014; Maenosono et al., 2009; Mrđanović et al., 2009; Shinohara et al., 2009; Theogaraj et al., 2007; Turkez et al., 2014). The duration of treatment varied between 24 and 72 h. S9 was included in 5 out of 11 papers (Honma et al., 2012; Kwon et al., 2014; Maenosono et al., 2009; Shinohara et al., 2009; Warheit et al., 2007); no studies indicated that S9 affected the results. The way the chromosome aberrations results were expressed varied greatly throughout the papers, from a summary % of total chromosome aberrations to the detailed analysis of chromosome and chromatid breaks, acentric fragments and centromeric fusions. A paper that followed an OECD-compliant method for the standard chromosome aberration tests was that of Shinohara et al. (2009). The methods described a standard chromosome aberration test in a well-known cell line according to OECD TG 473. The results were well displayed to ensure that the types of aberrations scored were clear, indicating a knowledge of the assay. The NPs were characterized for primary and secondary size, but nuclear or cellular uptake was not measured; these data would have made this paper more informative. Two out of 11 papers did show nuclear or cellular uptake (Di Giorgio et al., 2011; Hackenberg et al., 2011), using electron microscopy and TEM respectively. Both of these papers showed positive results with carbon nanotubes and silver respectively. In Vitro Comet Assay Twenty-two papers on tests of metal oxides and carbon nanotubes in the in vitro comet assay published from 2012 to 2014 (postCOM, 2012) were considered (Table 10). Findings are summarized in Table 1. Naturally occurring and “soft” NMs, those that contain components such as polymers, gels, and biomaterials, were not considered. No papers were excluded from the analysis, but acceptability criteria were applied: the presence of a positive control, assessment of cytotoxicity, and evaluation of material uptake. There were 2 major methods used in these studies—the standard alkaline comet and the comet with the addition of formamidopyrimidine-DNA glycosylase (FPG) in order to cause strand breaks at sites of oxidative damage in the DNA, thereby providing evidence of the presence of oxidative lesions. Some studies compared both methods, thereby in principle providing information on the proportion of strand breaks caused by oxidative damage lesions. Table 10. In Vitro Comet Assay: Papers Evaluated Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab Table 10. In Vitro Comet Assay: Papers Evaluated Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 22 papers were evaluated Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Cancino et al. (2013), Chatterjee et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Lindberg et al. (2013), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab A diversity of cell lines was used in the studies, as follows: FPG method: A549 human lung alveolar carcinoma (2 papers) (Ursini et al., 2012, 2014); other cells used included BEAS 2B normal human bronchial epithelial cells, C3A human hepatoblastoma cell line, and HK-2 human renal proximal tubule epithelial cells (Kermanizadeh et al., 2012, 2013; Ursini et al., 2014). Standard method: BEAS 2B normal human bronchial epithelial (Chatterjee et al., 2014; Lindberg et al., 2013), HK-2 human renal proximal tubule epithelial cells (Kermanizadeh et al., 2013), MeT-5A human mesothelial (Lindberg et al., 2013), C2C12 -mouse myoblast (Cancino et al., 2013), A549 human lung alveolar carcinoma cells (De Marzi et al., 2013; Mu et al., 2012;, V79 hamster fibroblast cells (Chen et al., 2014), HepG2 (Alarifi et al., 2013; De Marzi et al., 2013; Kermanizadeh et al., 2012), Caco2 (De Marzi et al., 2013), Hacat (Mu et al., 2012), AGS (human gastric epithelial cancer cells) (Botelho et al., 2014), primary human lymphocytes (Battal et al., 2015; Moche et al., 2014), RAW 264.7 macrophages (Wilhelmi et al., 2013), HEK293 cells (Demir et al., 2014), NIH/3T3 cells (Demir et al., 2014; Ould-Moussa et al., 2014), IMR-90 human fibroblasts (Lim et al., 2012), M059K human glioblastoma cells and their PKC deficient counterpart (M059J cells) (Lim et al., 2012), CHO AA8 and their PKC deficient counterpart (CHO V33 cells) (Lim et al., 2012), mouse oocytes (Courbiere et al., 2013), and rat neurons (Zha et al., 2012). The type of cell used may have an impact on the outcome of the assay as different cells may have different internalization capacity, as well as differences in DNA repair and metabolic capability. Seventeen papers (Table 11) dealt with metal oxides, 2 with FPG (Kermanizadeh et al., 2012, 2013), and the remaining with the standard method. Seven papers tested carbon NMs, 3 with the FPG-modified method (Kermanizadeh et al., 2012; Ursini et al., 2012, 2014), 4 with the standard method (Cancino et al., 2013; Chatterjee et al., 2014; Kermanizadeh et al., 2013; Lindberg et al., 2013). Responses ranged from 2- to 7-fold increase in % tail DNA over the negative control level, with most in the 2- to 3-fold range. The 7-fold increase was seen for carbon nanotubes using the FPG-modified method (Ursini et al., 2012); it should be noted that the standard method was tested simultaneously and a negative response was observed. Table 11. In Vitro Comet Assay References: Papers With Metal Oxides Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab Table 11. In Vitro Comet Assay References: Papers With Metal Oxides Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Description . References . 17 papers with metal oxides Alarifi et al. (2013), Battal et al. (2015), Botelho et al. (2014), Chen et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lim et al. (2012), Moche et al. (2014), Mu et al. (2012), Ould-Moussa et al. (2014), Sarkar et al. (2014), Wan et al. (2012), Wilhelmi et al. (2013), Zha et al. (2012) Open in new tab Few studies measured uptake of the NPs: 3 of 7 total studies for carbon nanotubes (Lindberg et al., 2013; Ursini et al., 2012, 2014); 2 of 3 with FPG (Ursini et al., 2012, 2014); 1 of 4 for the standard method (Lindberg et al., 2013); and 6 of 17 studies for metal oxides (Alarifi et al., 2013; Courbiere et al., 2013; Mu et al., 2012; Ould-Moussa et al., 2014; Wan et al., 2012; Wilhelmi et al., 2013), all using the standard method. There were considerable variations in methods in the studies reviewed. The doses ranged from 0.01 to 760 µg/ml (Cancino et al., 2013; Chatterjee et al., 2014; Kermanizadeh et al., 2012, 2013; Lindberg et al., 2013; Ursini et al., 2012, 2014) for carbon nanotubes and from 0.001 to 1000 µg/ml for metal oxides (Table 11). Treatment ranged from 2 to 72 h, with a 24-h treatment being most common. Most positive responses were seen at 24 h. Different sample sizes ranging from 50 to 300 cells were analyzed, and not all studies used a positive control. From the studies reviewed, it proves difficult to draw conclusions on the genotoxic profile of any specific NM using the in vitro comet assay. Most of the studies on metal oxides and carbon nanotubes tended to yield positive results using either the FPG method or the standard method (Kermanizadeh et al., 2012, 2013; Ursini et al., 2012, 2014). Although TiO2 and WC-Co generated mixed results (Botelho et al., 2014; Chen et al., 2014; Kermanizadeh et al., 2012; Moche et al., 2014; Wan et al., 2012); ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG (Table 12). All tests with carbon nanotubes had at least one test result that was positive (Cancino et al., 2013; Chatterjee et al., 2014; Kermanizadeh et al., 2012, 2013; Lindberg et al., 2013; Ursini et al., 2012, 2014). Table 12. In Vitro Comet Assay: Positive Responses Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Open in new tab Table 12. In Vitro Comet Assay: Positive Responses Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Description . References . 7 Tests with carbon nanotubes Cancino et al. (2013), Chatterjee et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ursini et al. (2012, 2014) ZnO, Ag, CeCO2, amorphous silica, MWCNT, and SWCNT tended to yield a positive genotoxic response with or without FPG Cancino et al. (2013), Chatterjee et al. (2014), Courbiere et al. (2013), De Marzi et al. (2013), Demir et al. (2014), Kermanizadeh et al. (2012, 2013), Lindberg et al. (2013), Ould-Moussa et al. (2014), Sarkar et al. (2014), Ursini et al. (2012, 2014), Wilhelmi et al. (2013) Open in new tab One publication was identified that follows most recommendations: use of a positive control, verification of NM uptake, analysis of an acceptable number of cells per sample, and detailed characterization of the NM used (nanotubes; the characterization was shown in a separate publication and was done before the use of the material) (Lindberg et al., 2009). Though analysis of 100 cells per treatment was conducted in this study, a minimum of 150 cells per sample would have been preferred, as recommended by OECD TG 489. Although this study followed many recommendations, the use of a nonstandard cell line makes data interpretation somewhat difficult. In Vitro Mammalian Gene Mutation Assays Seventeen papers published from 2007 to 2017 were included in this review (Table 13) and nineteen NMs were evaluated (Table 1), including MWCNT (plain and carboxylated), SWCNT (plain and carboxylated), anatase TiO2, TiO2, carbon black, C60 fullerenes, Ag, Cd/Se quantum dots (carboxyl, hexadecylamine [HDA], and amine), WC-Co, SiO2, ultrafine quartz, ZnO and 2 poly(anhydride) NMs. Table 13. In Vitro Mammalian Gene Mutation Assay: Papers Evaluated Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Open in new tab Table 13. In Vitro Mammalian Gene Mutation Assay: Papers Evaluated Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Description . References . 17 papers were evaluated Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Kim et al. (2010), Manshian et al. (2013), Manshian et al. (2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c , 2011, 2015), Zhu et al. (2007) Open in new tab There are publications using the standard assays (Tk in L5178Y cells [OECD TG 490], and Hprt (hypoxanthine-guanine phosphoribosyltransferase) in V79, CHO, CHL, L5178Y or TK6 cells [OECD TG 476]). In addition, publications were identified using nonstandard assays including mutation at the HPRT locus in MCL-5 and WIL2-NS cells (Manshian et al., 2013; Wang et al., 2007a,b,c); Aprt (Adenine Phosphoribosyltransferase) in 3C4ES cells (Zhu et al., 2007); transgenes (cII and/or lacZ) in FE1 cells (Jacobsen et al., 2007, 2008); and loss of CD59 in AL cells (Wang et al., 2015). There were 4 papers for the standard mouse lymphoma assay (MLA, OECD TG 490) (Iglesias et al., 2017; Kim et al., 2010; Mei et al., 2012; Moche et al., 2014) and 5 papers for the standard HPRT assay using cell lines that are recommended in OECD TG 476 (Asakura et al., 2010; Chen et al., 2014; Manshian et al., 2016; Mrakovcic et al., 2015; Wang et al., 2011). Four papers evaluated Hprt mutation in cell lines not included in OECD TG 476 (Manshian et al., 2013; Wang et al., 2007a,b,c), one each evaluated mutation at the Aprt or CD59 loci (Wang et al., 2015; Zhu et al., 2007), and 2 evaluated cII and/or lacZ in FE1 cells (Jacobsen et al., 2007, 2008). There is no OECD TG for the latter 3 assays. Only a few materials were evaluated in more than one study. Only Ag was evaluated in the same assay in 2 laboratories (Kim et al., 2010; Mei et al., 2012). None of the other materials (or similar materials) was evaluated in the same test system using the same endpoint in multiple studies or laboratories (although replicate experiments were sometimes reported and were reproducible). When the same or similar materials were evaluated in multiple laboratories or test systems, conflicting results were sometimes observed; however, not all of these studies were determined to be acceptable (see below for a more detailed discussion). Summarizing all of the studies (acceptable and not acceptable), Ag was reported to be positive in one MLA (Mei et al., 2012) and negative in a second study (Kim et al., 2010), but it was not clear that the same size particles were tested or that uptake was assured in the second study. MWCNT were reported positive in 1 of 3 studies (Asakura et al., 2010; Mrakovcic et al., 2015; Zhu et al., 2007); 2 samples of various SWCNTs were reported positive in 2 of 3 studies (Jacobsen et al., 2008; Manshian et al., 2013; Mrakovcic et al., 2015); TiO2 was reported positive in 2 of 3 studies (Chen et al., 2014; Wang et al., 2007a, 2015); and one study reported 20 nm ZnO negative, but 90–200 nm ZnO positive (Wang et al., 2015). In addition, for most materials assessed, where the authors reported positive responses, the maximal increases observed were usually only 2–3 times negative control values, or even less (Table 14). Table 14. In Vitro Mammalian Gene Mutation Assay: Unconfirmed Positive Responses Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Open in new tab Table 14. In Vitro Mammalian Gene Mutation Assay: Unconfirmed Positive Responses Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Description . References . Positives with minimal responses Chen et al. (2014), Jacobsen et al. (2007), Moche et al. (2014), Wang et al. (2007a,b,c, 2015), Zhu et al. (2007) Open in new tab As with the other mammalian cell assays and summarized in Table 1, there was the substantial variation in cell lines, dose levels, treatment times, and endpoints analyzed. Such variation precluded any definitive conclusions. Due to the variability in the methods used, the protocols used in each study were evaluated and the following criteria were applied for excluding tests from the analysis: Reports with negative control mutant frequencies outside typical reported ranges, or those with no concurrent negative control. Concurrent positive controls failed to elicit a significant increase in mutant frequency (not all studies included positive controls, but this was not used as a specific exclusion criterion, especially if a positive response was observed for the NMs; it also would have reduced the number of papers available). Studies without a concurrent cytotoxicity evaluation, an inappropriate cytotoxicity parameter, or responses observed only at excessively cytotoxic dose levels (generally below 10%–20% survival by the appropriate parameter). Insufficient numbers of cells scored or an inappropriate scoring method was used (not all studies were specifically excluded due to low cell numbers, especially if a positive response was observed for the NMs). Missing details that prevented full evaluation of methods used. From this review, 15 of 17 studies characterized the test material and provided detailed dispersion protocols for the NM treatments (Table 15) and 2 did not (Kim et al., 2010; Zhu et al., 2007). In total 7 of 17 studies verified uptake of the NMs (Table 16), which was not determined or discussed in the remaining 10 papers. Only one study included S9 (Kim et al., 2010). In addition, 3 studies used extremely long treatment protocols of 24–60 days (Jacobsen et al., 2007, 2008; Wang et al., 2011); 3 studies had negative control mutant frequencies that appeared unusually high (Kim et al., 2010; Wang et al., 2007b,c). One study scored far too few cells to support a negative finding (Asakura et al., 2010); one study did not include concurrent negative controls, instead relying on comparison to historical control values (Wang et al., 2015); and one study had no statistical analyses, or any other valid criteria for judging the response, and also lacked historical control data for comparison (Zhu et al., 2007). One study used relative suspension growth rather than relative total growth to express cytotoxicity, so that it was impossible to know whether the doses were appropriate to conclude negative results (Iglesias et al., 2017). Table 15. In Vitro Mammalian Gene Mutation Assay: Studies With Adequate NM Characterization Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Open in new tab Table 15. In Vitro Mammalian Gene Mutation Assay: Studies With Adequate NM Characterization Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Description . References . 15 studies characterized the test material and provided detailed dispersion protocols Asakura et al. (2010), Chen et al. (2014), Iglesias et al. (2017), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a,b,c, 2011, 2015) Open in new tab Table 16. In Vitro Mammalian Gene Mutation Assay: Uptake Studies Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Open in new tab Table 16. In Vitro Mammalian Gene Mutation Assay: Uptake Studies Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Description . References . 7 studies verified uptake of the NMs Manshian et al. (2013, 2016), Mei et al. (2012), Mrakovcic et al. (2015), Wang et al. (2011, 2015), Zhu et al. (2007) Open in new tab Based upon our exclusion criteria, which generally followed or were adapted from the appropriate OECD TG, 9 studies (53%) were classified as acceptable (Table 17) and 8 (47%) were considered to be unacceptable (Table 18). Of the acceptable studies, 6 individual NMs were considered positive by the authors, but the expert review only considered 5 of them to be clearly positive: 400–800 nm SWCNT, tungsten carbide cobalt nm, TiO2, Ag, carboxylated SWCNT (Chen et al., 2014; Manshian et al., 2013; Mei et al., 2012; Moche et al., 2014; Mrakovcic et al., 2015). Ultrafine TiO2 was deemed to be positive by the authors (Wang et al., 2007a), but equivocal by the review group. Interestingly, 2 of these studies had 3 additional, related NMs (carbon nanotubes) that were deemed to be negative by the authors and the review group (Manshian et al., 2013; Mrakovcic et al., 2015). Two additional studies, evaluating a total of 4 NMs, produced negative results with which the reviewers agreed. Table 17. In Vitro Mammalian Gene Mutation Assay: Studies Classified as Acceptable Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Open in new tab Table 17. In Vitro Mammalian Gene Mutation Assay: Studies Classified as Acceptable Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Description . References . 9 studies classified as acceptable Chen et al. (2014), Jacobsen et al. (2007, 2008), Manshian et al. (2013, 2016), Mei et al. (2012), Moche et al. (2014), Mrakovcic et al. (2015), Wang et al. (2007a) Open in new tab Table 18. In Vitro Mammalian Gene Mutation Assay: Studies Considered to Be Unacceptable Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Open in new tab Table 18. In Vitro Mammalian Gene Mutation Assay: Studies Considered to Be Unacceptable Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Description . References . 8 studies considered to be unacceptable Asakura et al. (2010), Iglesias et al. (2017), Kim et al. (2010), Wang et al. (2007b,c, 2011, 2015), Zhu et al. (2007) Open in new tab In the 8 studies judged to be unacceptable for the reasons described, the authors reported 7 NMs to be negative and 5 NMs to be positive. However, the increases in mutant frequencies observed for these latter “positive” NMs only reached 1.2- to 2.5-fold control values. For the MLA, Mei et al. (2012) reported Ag NM (5 nm) to be positive. In this study, the investigators fully characterized the test material and uptake into the cells, and also conducted the study according to OECD TG 490. In contrast, Kim et al. (2010) reported Ag NM (<100 nm) to be negative in the MLA. However, no information was provided regarding characterization of the Ag NM or its uptake into cells. Moche et al. (2014) reported WC-Co to be positive in the MLA using a 4-h treatment, but negative using a 24-h treatment, which is an unusual pattern of responses (Moore et al., 2007), and cellular uptake was not addressed. The review team felt that a reasonable study design and approach for the MLA/TK assay was as described by Mei et al. (2012). For the standard Hprt assay (OECD TG 476), there were 5 papers. Asakura et al. (2010) reported MWCNT to be negative in CHL cells. Although they did characterize the NM, they did not provide information concerning cellular uptake. Chen et al. (2014) reported anatase TiO2 to be positive in V79 cells. The test material was characterized, but there was no evaluation of cellular uptake. Manshian et al. (2016) reported 3 different types of Cd/SE quantum dots to be negative in TK6 cells. The test materials were fully characterized, and cellular uptake was confirmed. Mrakovcic et al. (2015) evaluated SWCNT and MWCNT (both plain and carboxylated) using V79 cells. Both NMs were fully characterized, and cellular uptake was verified. In this study, SWCNT was mutagenic while MWCNT was negative. Wang et al. (2011) reported TiO2 to be negative in CHO cells. The test material was fully characterized and cellular uptake was verified, but the assay was not conducted according to OECD TG 476 (treatment time and the measure of cytotoxicity were not according to the recommendations). The review team felt that a reasonable study design and approach for the HPRT assay was as described by Manshian et al. (2016) or Mrakovcic et al. (2015). For the publications using the HPRT locus in other cell lines, some studies fully characterized the test material including cellular uptake while others characterized the test material, but did not confirm cellular uptake. Three publications using WIL2-NS cells (Wang et al., 2007a,b,c) did not confirm cellular uptake of the test materials. Although the authors called the responses for all 3 positive, the review team disagreed and called SiO2 and UF quartz negative (maximum MFs were approximately 1.4- to 1.6-fold control values) and called TiO2 equivocal (with a dose-dependent increase to 2.5-fold control values). Manshian et al. (2013) used MCL-5 cells to evaluate different sizes of SWCNT. The NM was fully characterized, and cellular uptake was confirmed. One (400–800 nm) was positive, whereas the other two (1–3 and 5–30 µm) were negative. Two papers by Jacobsen et al. (2007, 2008) used transgenic loci (cII and/or lacZ). Although they did characterize the 4 NMs evaluated, cellular uptake was not confirmed. The authors reported quartz (1.59 µm), SWCNT and C60 fullerenes to be negative and carbon black to be positive. However, the review team judged all 4 to be negative (all were using repeated exposures for 24 days, and the largest increases seen for carbon black were only approximately 1.2- to 1.4-fold control values). Wang et al. (2015) reported ZnO (90–200 nm) to be positive, but ZnO (20 nm) and TiO2 (15 nm) to be negative, for inducing mutation at the CD59 locus in AL hybrid cells. They did characterize the test material and confirmed cellular uptake. However, the review team called all 3 responses negative (overturning the authors’ positive call on the basis of the magnitude of the increases seen). The one publication using the Aprt locus in mouse 3C4 ES cells (Zhu et al., 2007) had insufficient detail to adequately evaluate the reported positive call for MWCNT. In total, the review team disagreed with 6 of the 11 positive calls made by the authors, deeming 4 to be outright negative, 1 to be equivocal, and 1 to be uninterpretable. Overall, there was insufficient information in the published studies to draw any firm conclusions concerning the mutagenicity of NMs in mammalian cells in culture, or in the ability of the various assays to detect potential effects. For the standard MLA and Hprt assays there were 2 and 6 papers, respectively, not all of which were conducted appropriately. In particular, not all of the publications in which negative responses were reported showed confirmation that the test material was taken up by the cells. For the entire set of papers for in vitro mammalian gene mutation, approximately half of the publications used cell lines and/or mutation endpoints for which there is very little published data for standard chemicals. Furthermore, given the wide variety of NMs included in the small number of publications, it is not possible to make any conclusions concerning the relative sensitivity of the various reporter genes to the potential mutagenicity of NMs. It should, however, be noted that the MLA using the Tk locus detects a wider spectrum of genetic damage, including large scale chromosomal effects (Applegate et al., 1990; Wang et al., 2009), while the Hprt locus detects primarily single base pair changes or other small scale DNA sequence alterations (Moore et al., 1989). Thus, some chemicals are positive at the Tk locus, negative at the Hprt locus, and positive for chromosome aberrations, micronuclei, or comet (Doerr et al., 1989; Moore et al., 1989). It would be expected that the same would be true for NMs. In Vivo Bone Marrow MN Assay and Chromosomal Aberration Assay In total 18 papers were assessed for the in vivo MN endpoint (Table 19; results summarized in Table 2); from 2012 to 2016, 8 were from prior to 2012 and included in the COM (2012) analysis. In general, the NM characterization, particle size distribution and genotoxicity test methods were better conducted in the in vivo studies than in the in vitro studies. Approximately half of the studies were in mice and half were in rats. There are few separate papers on chromosome aberrations in vivo; however, there were chromosome aberration results along with in vivo MN results in 5 out of 18 papers (Balasubramanyam et al., 2009; Ghosh et al., 2012; Landsiedel et al., 2010; Schulz et al., 2012; Singh et al., 2013) and these results have been included in this analysis. All papers were assessed for additional information than was summarized in the COM (2012) analysis. No papers were excluded from the analysis but the interpretation of 2 papers is debatable, due to the low magnitude of induction of micronuclei in NM test groups—barely a doubling over control (Dobrzyńska et al., 2014; Downs et al., 2012) which may put into question the biological relevance of the findings. In cases where both CA and MN were assessed in the same study, the results were generally the same for both assays, either positive or negative. Table 19. In Vivo MN Assay and Chromosomal Aberration (CA) Assay References Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Open in new tab Table 19. In Vivo MN Assay and Chromosomal Aberration (CA) Assay References Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Description . References . 18 papers were evaluated Balasubramanyam et al. (2009), Bollu et al. (2016), Chen et al. (2014), Dobrzyńska et al. (2014), Donner et al. (2016), Downs et al. (2012), Durnev et al. (2010), Ghosh et al. (2012), Kim et al. (2008), Kwon et al. (2014), Landsiedel et al. (2010), Li et al. (2014), Lindberg et al. (2012), Schulz et al. (2012), Shinohara et al. (2009), Singh et al. (2013, 2016), Trouiller et al. (2009) Open in new tab When different results were seen for the same NMs (eg, TiO2 and Ag) it could be attributed to exposure methods and different dose levels. 6 papers for 5 different NMs (Ag, TiO2, Al2O3, MnO, and CrO2) showed clearly positive results in in vivo micronuclei or chromosome aberrations assays (Balasubramanyam et al., 2009; Chen et al., 2014; Dobrzyńska et al., 2014; Ghosh et al., 2012; Singh et al., 2013; Trouiller et al., 2009). Two equivocal or weak positives were determined as approximately a doubling or less above control. The positive with MnO gave 2- to 3-fold increases following dosing at 1000 mg/day for 28 days (Schulz et al., 2012), and the positive with Al2O3 (Doerr et al., 1989) was seen following dosing of up to 2000 mg/kg as a single dose. TiO2, Ag and ZnO papers had dose response studies. Of the 5 papers with silver NMs (Dobrzyńska et al., 2014; Ghosh et al., 2012; Jiang et al., 2013; Landsiedel et al., 2010; Wang et al., 2015), 3 were positive (Dobrzyńska et al., 2014; Landsiedel et al., 2010), one of these following intraperitoneal (i.p.) exposure of a single dose with groups of animals given between 10 and 80 mg/kg Ag NPs (Ghosh et al., 2012). A second Ag NP study dosed intravenously (i.v.) gave increases in micronuclei of 3-fold at 24 h following exposure to 5 or 10 mg/kg of Ag NP (Dobrzyńska et al., 2014). The positive response persisted for up to 1 week with a 2-fold increase at the 10 mg/kg dose. However, a study dosed up to 1000 mg/kg via the oral route for 28 days with Ag NPs of a similar size (52–71 nm) was negative in bone marrow (Kim et al., 2008); thus it appears that the route of exposure needed to be i.v. or i.p to get a positive with Ag NPs. With TiO2, 2 papers described positive results via an oral route (Chen et al., 2014; Trouiller et al., 2009) while one was negative Donner et al. (2016); one was negative via inhalation (Lindberg et al., 2012). A fourth paper with TiO2 is equivocal with exposure via an i.v. route (Dobrzyńska et al., 2014); therefore, again the differences seen might be attributed to routes of administration. A further paper on silica had MN increases considered equivocal but were accompanied by significant increased γH2AX and 8 Oxo-G signals, indicating the signal might be due to inflammatory responses (Downs et al., 2012). The study with europium was performed at approximately one-tenth the doses of most of the in vivo studies, and the negative result was a desirable one from the perspective of potential therapeutic use of the product. However, the optimum choice of dosing for in vivo safety assessment is still an unresolved issue. One paper that could be considered a model for the in vivo MN assay is Donner et al. (2016). It was conducted according to OECD guidelines, with good characterization of the TiO2 test articles and methods for the MN assay. Dosimetry included high test doses up to 2000 mg/kg. This dose is high, but appropriate for the goal of this test, which was to demonstrate a negative. This study lacked an analysis of uptake into the target tissue, but the negative results were discussed in the context of likely poor target tissue (bone marrow) exposure following oral administration of the NM, due to poor systemic distribution from gastrointestinal exposure. A well-run assay for both micronuclei and chromosome aberrations in vivo is by Balasubramanyam et al. (2009) where both assays were conducted according to OECD guidelines, the aluminum oxide NPs are well described, and the authors monitored uptake of the NPs into a variety of tissues in the rats. The genotoxicity assays showed similar, positive dose-response results for 30 and 40 nm particles, with a significant, possibly exponential increase within a defined linear dose range, up to 2000 mg/kg dosed orally. Such high doses might be questionable from a risk assessment context, but the lowest dose tested, 500 mg/kg, was also significantly positive although this dose may still be considered high for a particulate material. These 2 papers demonstrated divergent results from oral administration of different nanomaterials, one positive and one negative. Overall, the data reviewed show that the in vivo MN assay (OECD TG 474) and in vivo chromosome aberration assay (OECD TG 475) can be used in the standard form for NP evaluation, but attention should be paid to mechanistic aspects such as relevant tissue exposure and potential particle overload effects. In Vivo Comet Assay The dataset considered 17 papers from 2009 up to the end of 2014 (Table 20), addressing genotoxicity of about 20 different NPs, including those that were also reviewed by COM (2012). The findings are summarized in Table 2. The studies did encompass a wide range of NMs, including carbon nanotubes, carbon black NP’s, silica and fullerenes, the majority of data being generated on metal oxides. For the NMs investigated, a positive result in the comet assay was claimed by the authors in >50% of these studies. Effects were mostly small, 1.3-fold up to approximately 2-fold increases in tail DNA. Some of these calls may be considered questionable, in light of the OECD guideline requirements for a positive call for this assay (OECD TG 489) which requires a clear positive call to be statistically significant, dose-related and one data point to exceed the distribution of the historical negative control of the performing laboratory. However, no clear pattern was observed in terms of the responses for different types (classes) of NMs investigated. Regarding MoA, there was one positive study that was directly associated by the authors with the release of genotoxic metal ions (Tiwari et al., 2011). Several other studies associated the positive response with signs of tissue inflammation as indication of an inflammation-driven, oxidative MoA (Bourdon et al., 2012; Downs et al., 2012; Saber et al., 2012b; Sharma et al., 2012; Totsuka et al., 2009). Although the data reviewed seem to indicate that the in vivo comet assay (OECD TG 489) can be used without modification for NP assessment, attention should be paid to potential artifacts when residual particulates remain during the electrophoresis of the DNA (Ferraro et al., 2016; Karlsson et al., 2015). Table 20. In Vivo Comet Assay References Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Open in new tab Table 20. In Vivo Comet Assay References Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Description . References . 17 papers were evaluated Bourdon et al. (2012), Dandekar et al. (2010), Dobrzyńska et al. (2014), Downs et al. (2012), Durnev et al. (2010), Jacobsen et al. (2009), Kwon et al. (2014), Landsiedel et al. (2010), Lindberg et al. (2012), Saber et al. (2012a), Schulz et al. (2012), Sharma et al. (2012), Sycheva et al. (2011), Tiwari et al. (2011), Totsuka et al. (2009), Trouiller et al. (2009), Wessels et al. (2011) Open in new tab If maximal assay sensitivity for detection of oxidative DNA damage (eg, inflammation-induced) is desired, the use of a modified version may be beneficial (addition of OGG1 or FPG glycosylases for recognition of particular oxidative DNA adducts). Bourdon et al. (2012) have shown that an FPG protocol did boost the comet effects, but the standard protocol had already given a positive response. Sharma et al. (2012) found a positive response when using the FPG protocol but no data were generated in parallel using the standard protocol. Taken together, there is not enough data available to develop a firm recommendation on the need to modify the standard protocol. Another key aspect associated with the observed responses is the selection of a maximum dose, as well as route of exposure. Most of the studies associated with a positive response were using very high particle loads, and maximized tissue exposure by, eg, intratracheal instillation (Saber et al., 2012b) or i.v. injection (Downs et al., 2012). The effects may therefore be indication of a “hazard” that may not be associated with a relevant human exposure scenario and reflect particle overload, triggering inflammatory responses (Downs et al., 2012). In Vivo Gene Mutation Assays in Transgenic Rodents In total 8 papers were reviewed that were published from 1997 to 2015 (Driscoll et al., 1997; Kato et al., 2013; Kovvuru et al., 2015; Louro et al., 2014; Shvedova et al., 2008, 2014; Totsuka et al., 2009, 2014); these described mutagenic responses to NM exposure in both generic and transgenic rats and mice. Although the extensive COM (2012) analysis covered genotoxicity data from NM exposure in vitro and in vivo, there is no analysis of in vivo mutation data on endogenous reporter genes or transgenes using Transgenic Rodent (TGR) models. Therefore, our analysis of 11 NMs ranging from 1.2 to 180 nm in size using different mutation reporter systems, reviewed in Table 2, is an extension of the COM (2012) analysis. The NMs tested included the following: C60 fullerenes (Totsuka et al., 2009), carbon black (Driscoll et al., 1997; Totsuka et al., 2009), Kaolin (Totsuka et al., 2009), MWCNT (Kato et al., 2013), anatase TiO2 (Driscoll et al., 1997; Louro et al., 2014), α-quartz (Driscoll et al., 1997), SWCNT (Shvedova et al., 2008), carbon nanofiber (CNF) (Shvedova et al., 2008), asbestos (Shvedova et al., 2014), magnetite NPs (in the form of Fe3O4) (Totsuka et al., 2014), and PVP-coated Ag NPs (Kovvuru et al., 2015). The mutation reporter systems included gpt-delta, lacZ and myh−/− mice; F344 and C57BL/6 rats with K-RAS codons 8 and 12. The tested NMs were well characterized in most studies and the investigators followed OECD guidelines for treatment and sacrifice. Titanium, carbon black and asbestos were all negative in the TGR systems whereas most other NMs showed positive responses in one or the other mutational reporter systems. Most of the studies used multiple doses and 6 of them, C60 fullerenes (Totsuka et al., 2009), α-quartz (Driscoll et al., 1997), CNF (Shvedova et al., 2008), SWCNT of 2 different sizes (Shvedova et al., 2008), magnetite NPs (Shvedova et al., 2014) showed a positive dose-response. However, MWCNT (Kato et al., 2013), anatase TiO2 (Driscoll et al., 1997) and carbon black (Driscoll et al., 1997) tested positive only at the highest dose, while one NM (asbestos) (Shvedova et al., 2014) was negative in both mice and rats at all doses. The positive mutagenic responses (mutant frequencies) were 2- to 3-fold higher than the background response and in some studies which evaluated the mutational spectra, showed significant shifts in the NP-induced spectra compared with untreated control or background spectra. In general, the mutagenic responses induced by NMs appear to be substantially lower than that of standard mutagenic agents such as ENU and DMBA. Some of the NMs-induced mutagenic responses comprised of G→A (K-ras) and G→T (gpt) mutations; the G→T mutations are likely to be a hallmark of oxidative damage, while the mutagenic effects by other NMs may be secondary as a consequence of inflammation and release of ROS or free radicals. However, more studies are needed to evaluate the MoA of these NMs. Most of the studies performed uptake analysis of NMs in the target tissues, and the majority showed the presence of NMs in the target tissues, suggesting the mutagenic response was associated with NM exposure. However, it is notable that when the same NM was tested using different species of animals, different mutagenic responses (mutant frequencies) were observed. For example, carbon black was positive in F344 rats but negative in gpt delta mice (Driscoll et al., 1997; Totsuka et al., 2009), whereas TiO2 was positive in F344 rats (lung epithelial hprt assay) but negative in lacZ mice (Driscoll et al., 1997; Louro et al., 2014). There may be some correlation between positive mutagenic response and NM uptake because all NMs except asbestos produced positive responses and most of the NMs were taken up in the target tissues. However, this does not hold for asbestos, which was not mutagenic even though there was tissue accumulation or uptake. These results suggest the importance of animal species or test endpoint in addition to character of the NM. Interestingly, one study on long-term effects of carbon-containing engineered NMs such as SWCNT and CNF showed the presence of NMs in the target tissue even after one year following an acute exposure. What is also notable is that both SWCNT and CNF induced significant increases in the rate of K-ras mutations in codons 12 and 8, one year after exposure in mice lungs. However, the exposed mice did not have any tumors in the lungs at the time examined (Shvedova et al., 2014). DISCUSSION The major genotoxicity assays utilized for assessment of chemicals have been reviewed here as they have been applied to the evaluation of NMs. The goal was to recommend approaches appropriate for hazard identification and risk assessment of NMs. This review is anchored on and adds to the EMGS 2010 Workshop (Pfuhler et al., 2013), the UK COM (2012) report, and the reviews and work of the authors and others, but it is also focused on a robust and critical evaluation of the current nanogenotoxicity testing literature as a basis for specific recommendations on test assays and test methods. Understanding the Test Material The physical characterization of the NM for size, shape, and properties is of utmost importance (Bouwmeester et al., 2011; Boverhof and David, 2010; Love et al., 2012). This information has been provided in most of the more recent papers, as this is now generally recognized and required by most journals. However, of equal or greater importance is an understanding of the fate of NMs when added to the biological test systems. In many cases, NMs are characterized under one set of conditions and then encounter a different set of conditions in the test system. Thus, there may be uncertainty regarding the actual nature of the NM to which the test system is being exposed, and how this relates to human exposure. Many papers have noted that certain standard assays and test conditions may not suffice for assessment of NMs, eg, due to lack of particle uptake and a requirement for assay method modification (Doak et al., 2009, 2012; EFSA Scientific Committee, 2011; Landsiedel et al., 2009; Warheit and Donner, 2010). Several studies have explored the effects of media, pH, surface charge, coatings, and proteins on fate, action and toxic outcomes of NMs (Dutta et al., 2007; French et al., 2009; Jiang et al., 2009; Ju et al., 2013; Li et al., 2013; Pagnout et al., 2012; Pathakoti et al., 2013; Pele et al., 2015; Rivera Gil et al., 2010). Other important issues affecting NM toxicity measurements include experimental handling and interference with endpoints (Akabori and Nagle, 2014; Cullen et al., 2011; French et al., 2009; Jiang et al., 2009; Lindberg et al., 2009). It is recommended that NMs should be characterized for size distribution and properties under the test conditions in the genotoxicity assay, and consideration should be given to the properties of the NM for intended human exposure/use. Understanding Exposure Although genotoxicity testing is usually carried out without regard to a human exposure scenario, the route of exposure of NMs is a critical feature of human risk. Much of the focus on the toxicity assessment of NMs is related to inhalation exposure in humans; however, the effects of inhalation exposure may not be extrapolated to other routes. Dermal exposure is another common route of exposure to NMs, but many studies have indicated the lack of penetration of NMs into dermal tissues (reviewed in Warheit and Donner, 2015). However, dermal penetration may be NM and/or species specific (Hirai et al., 2012). Another major consideration is the possibility that the Absorption, Distribution, Metabolism and Elimination (ADME) of NMs may be different from that of the non-nano material of the same composition. A classic review of “Principles for characterizing the potential human health effects from exposure to nanomaterials” by different routes of exposure (Oberdorster et al., 2005) still seems relevant. However, for genotoxicity testing, exposure consideration is generally related to assurance that the test agent reaches the cells in the test system. Consideration of human exposure may be a factor, but generally the genotoxicity tests need to be performed in standard available systems in order that the results may be interpretable. Thus, in addition to characterizing the physico-chemical form of the NM in the conditions of the test system, there is a need to characterize how NMs behave in the test system; specifically, whether the NM gets into the cell/nucleus. If a NM does not get into a cell, genotoxicity is not expected unless there is release of ions or other genotoxic moieties that have the capacity to penetrate the cell. The selection of an “appropriate” dose for testing remains a difficult and unresolved issue. Exposure is typically elevated beyond actual human exposures in toxicology testing designed for hazard identification, as recommended in the current OECD TGs for the various tests. Elevated doses compensate for sensitive populations, statistically small samples, and extended time of exposure. However, high dose testing may lead to effects that would not occur at human use exposures, including artifacts caused by particle “overload”, which would be specific for the testing of NMs. There is not enough supporting evidence for a recommendation of a cut-off for maximum NM exposures at present, in vitro or in vivo. Rather than a particular cut-off value, it is likely more useful to monitor for potential overload effects of particular NMs in the relevant experimental system under study. In the case of bacteria, information to date indicates that NMs do not traverse the bacterial cell wall and the results are almost always negative. In in vivo systems, NMs may not be distributed systemically to the tissues evaluated in the assay, which is required for a valid test, per OECD testing guidelines. There is evidence that high dose artifacts (eg, related to particle effects or sequestration in tissues) in in vivo genotoxicity tests can be induced that would not occur under normal human exposures, such as demonstrated by Downs et al. (2012), who administered the NMs via the i.v. route and noted that this was a “worst case” exposure scenario that will rarely have a human correlate. Currently, technological limitations prevent these exposure issues from being fully addressed in each assay. However, this represents an important gap in our understanding and advances in the field will assist with understanding the biological and toxicological impact of exposure to NMs moving forward. At this time, it is recommended that genotoxicity tests for NMs be conducted within the guidelines (eg, OECD) as for testing of other agents, with the exception of specific methods adaptations required for NMs (as described further in the “Recommendations on method standardization and assay modifications” section below. Dose responses over a range of doses would be most informative and would aid risk assessment. Understanding MoAs/Mechanisms of NMs Several papers have addressed potential toxic and genotoxic mechanisms of action of NMs (Liu et al., 2016; Pati et al., 2016; Saptarshi et al., 2015; Zijno et al., 2015). The diversity of genotoxicity systems, NMs, and results found in this review are not conducive to clarifying MoAs of NMs. However, some useful conclusions can be noted. Nanomaterials, when positive in a genotoxicity assay, do not generally induce the large increases in genotoxic responses that are characteristic of many classical DNA damaging agents. This might not be surprising, since NMs typically do not interact directly with DNA (ie, do not involve covalent interactions such as alkylation, or intercalation). The observations in this analysis are consistent with the concept that the genotoxicity of most NMs is likely to be indirect, eg, via generation of oxidative species or indirect consequences of inflammation (Landsiedel et al., 2009; Xia et al., 2013; and many others). Another possible mechanism involves direct physical interaction with the spindle apparatus during cell division (Sargent et al., 2010; Siegrist et al., 2014). Based on this, it appears appropriate to apply the principles of indirect versus direct genotoxic effects of NMs in risk assessments. It is important to note that to date, most focus has been on the oxidative stress MoA, but there has been limited evaluation of other mechanisms and thus it is not possible to rule out other DNA damage mechanisms including perturbation of systems, eg, DNA repair or DNA synthesis. The comet assay may be particularly useful in studies on oxidative MoAs and its sensitivity for this type of lesion can be further improved by using OGG1 or FPG glycosylases for recognition of oxidative DNA adducts. However, the in vitro comet assay, as currently practiced, is subject to variable responses resulting from the use of diverse methods and cell systems, which may vary in metabolic and DNA repair capability. Standardized methods for the in vitro comet assay, including the use of well-characterized glycosylases, have not been established, and are a notable gap in the genotoxicity test battery. This is therefore another area that requires further clarification, which can impact the scope of the testing framework to take forward. If indirect effects represent the major mode of genotoxicity of NMs, genotoxicity assessment of NM may be better integrated into a broader context of systems toxicology. Attempts to categorize NMs (Godwin et al., 2015) may be a useful approach to understanding MoA as well as facilitating the analysis of large numbers of material agents. The UCLA Center for Nano Biology and Predictive Toxicology has adopted a Tox21 high content approach targeting defined pathways of toxicity involving pulmonary inflammation, ROS and membrane effects, as related to the physical and chemical properties of NMs (Godwin et al., 2015; Nel, 2013). Arts et al. (2015, 2016) have furthered the idea and developed a decision-making framework (DF4) for the grouping and testing of NMs which considers intrinsic material and system-dependent properties, biopersistence, uptake and biodistribution as well as cellular and apical toxic effects which are derived from in vitro studies. This concept can be helpful for MoA understanding but can be useful also for selection of appropriate assays or protocols since the categories they provide, ie, (1) soluble NMs, (2) biopersistent high aspect ratio NMs, (3) passive NMs, and (4) active NMs, provide initial clues about the behavior of a NM in a test system, and whether testing is relevant within a particular context, eg, human exposure. These approaches and “read across” (Oomen et al., 2015) may be most useful for minimizing testing where there is no human relevance, but any utility in risk assessment remains to be demonstrated. Recommendations on Method Standardization and Assay Modifications General Notes on Standardization of Approaches (Relevant for All Assays) If NMs are used by a particular route of administration in humans, this might be considered in genotoxicity testing if feasible (however, see below on testing within the context of cells and systems recommended in OECD TGs). Consideration should be given to the potential release and solubilization of NMs or components of complex NMs in cell culture medium or other vehicle, to aid understanding of potential ion versus particle effects. Examples would be the metal oxides, which release ions. Preliminary studies with the NM test article should provide a rationale for type of media, dispersion method, surfactant use, duration of treatment, test system and dosimetry. The presence of serum or proteins in the medium can impact the extent of particle internalized in the cells. Characterization of NMs should occur in the test media (as well as in other media, as appropriate). Consideration should be given to potential artifacts occurring with NMs in the test media (eg, agglomeration). Per the OECD TGs, toxicity should be measured in parallel with genotoxicity, not in separate trials. The toxicity parameters used in all genotoxicity studies should be those recommended for each assay in the relevant OECD guidelines (eg, relative population doubling, cloning efficiency or relative total growth). OECD guidelines have been updated, and previous methods, such as confluence estimation or dye exclusion, are no longer considered adequate. Uptake of the NMs into the cells of the test system should be documented and the location of particles determined within the cells if feasible (ie, nucleus or cytoplasm); evidence should be provided that the NM reached the target tissue in any in vivo assays. In some cases, results may be positive in the absence of uptake (eg, for bacteria), which could reflect breakdown of the material in the test environment and/or the release of diffusible genotoxins. Cell lines and test systems should be limited to those recommended in the OECD guidelines and for which methods are well characterized. Lorge et al. (2016) provide resources and information on some preferred cell lines. Cell lines for mammalian in vitro cytogenetic studies include V79, CHL, L5178Y, CHO, TK6 cells, and human lymphocytes (although it is now recognized that rodent cell lines may be more sensitive to cytotoxicity and genotoxicity than the human and/or p53 competent cells when one is conducting cytogenetic assays (Hashimoto et al., 2011; Honma and Hayashi, 2011). Nonetheless, it is important to note that the type of cell considered may have an impact on the outcome of the assay, as different cells may have different internalization capacity (Manshian et al., 2015), different DNA repair capability, or different metabolic capability. It may be critical to characterize the capability of the cells to take up model particles prior to the choice of a cell type. Other cell lines and systems should be used only if they are justified as particularly related to human use. These cell systems should however be compared with the standard systems and methods to aid data interpretation; they should have a sufficiently stable genetic background to support genotoxicity assessment, as demonstrated through response to appropriate positive and negative controls. They also must be validated and the same recommendations applied as are in the OECD TGs. Appropriate attention should be given to study design and statistical power, including the choice of doses from preliminary tests to be within an acceptable range based on toxicity; adequate number of doses spaced for maximum information relative to a biological effect; and statistical validity of the test results, eg, adequate cell numbers and replicates within a countable range. These parameters are fully described in the appropriate OECD TG. Positive and negative controls for each assay should be included and the results should be within the acceptable/expected range of the assay (as described in the OECD TGs). Although controls for the assays are recommended, NM-specific controls have rarely been demonstrated, and are not necessary to show the performance of the assay. Recommendations on Modification, If Needed, of Each Assay for Use With Nanomaterials Bacterial assays Since S. typhimurium and E. coli tester strains appear not to take up or respond to NMs, the recommendation is for an in vitro mammalian mutagenicity assay instead of a bacterial mutation test. Results from negative bacterial assays are not definitive as a test result for NMs. However, the bacterial assays may be appropriate to assess soluble genotoxic or toxic agents released from NMs. In vitro MN assay The in vitro MN assay is recommended as a component of a test battery for assessment of NMs. However, a modification of the assay is needed because CB treatment can inhibit the uptake of NPs by endocytosis or pinocytosis. Thus CB, if used, should be applied after NM exposure, allowing sufficient time for NP uptake (Doak et al., 2009, 2012; Haynes et al., 1996). Appropriate cytotoxicity evaluation must also be carried out in parallel as described earlier. Other outstanding questions surround the ideal exposure time for this assay and the exact choice of cell type. Regarding exposure time, it is important that the cells be allowed to complete more than one cell cycle in the presence of the NM so that NMs taken up by the cells may come into direct contact with the DNA when the nuclear membrane breaks down during mitosis (Doak et al., 2012; Nelson et al., 2017). Standard cell lines with suitably low background MN frequencies and stable genetic backgrounds are recommended (Doak et al., 2012; Lorge et al., 2016). In vitro chromosomal aberration assay Cytogenetic damage is an important genotoxicity endpoint; thus the chromosome aberration assay would be a recommended assay in a test battery. However, it takes a significant level of expertise to score chromosomal aberrations. If the chromosomal aberration assay is performed, aberrations should be characterized according to typical categories (eg, chromatid breaks), especially to ensure that chromatid and chromosome gaps are noted separately from aberrations. Test methods do not require modification for assessment of NMs, but confirmation of particle uptake should be included. In vitro comet assay There is no standard method (eg, OECD TG) for the in vitro comet assay. Thus, the methods used are more likely to vary in ways that may not be validated. The comet assay generally shows positive results with NMs, but there are major questions about the reliability or validity of these results that were not discernible in the methods review undertaken here. There is no consistent evidence that the use of glycosylases targeting oxidative damage lesions enhances sensitivity. Is this a result that truly reflects variability among NMs? Or is it related to different enzymes, incubation times, cell type or handling method. Several papers have postulated (Karlsson et al., 2015) or demonstrated sources of artefactual positive results with the comet assay (Ferraro et al., 2016). A standard protocol for the alkaline comet assay post treatment is found in the in OECD TG 489 for the in vivo comet assay. This includes technical details on slide prep, lysis, electrophoresis and analysis and may be useful in performance of the in vitro assay. If maximal assay sensitivity for detection of oxidative DNA damage is desired, the use of a modified version (use of DNA glycosylases) may be beneficial. Reduced effects when NMs are removed prior to the posttreatment stage are important indicators that NMs can affect the electrophoretic mobilities of DNA. Therefore, NM assessments using the comet assay should consider a rinsing step to remove residual NMs after a defined treatment stage. Comparison of results with and without posttreatment rinsing would be informative. In addition, the DNA strand breaks measured in the comet assay are not a fixed genetic endpoint. They are intermediates that can change during the process of measurement and thus require strict conditions for assessment. Because of the lack of standard methods and uncertainty over the meaning of results, the comet assay is not recommended as a screening assay for NM genotoxicity assessment. However, careful experimentation with attention to the potential generation of artefacts can be useful in understanding NM effects. In vitro mammalian gene mutation assays Since it is suggested to waive the bacterial assays for evaluating NMs, a general recommendation would be to include an in vitro mammalian cell gene mutation assay in the test battery for NMs. Assays for NMs should be conducted based on OECD TG 490 (for the Tk locus) or TG 476 (for Hprt). All of the recommendations in these TGs are relevant to the testing of NMs and should be followed. The literature review did not reveal any assay modifications from the standard assay designs and performance that would be required for the conduct of these assays for evaluating NMs (other than the general recommendations affecting all assays such as NM characterization, evaluation of uptake, cytotoxicity measurements, etc). However, consideration should be given to the adequacy of NM clearance from suspension cultures after exposure, or whether the NM is present during mutation fixation and development. In vivo genotoxicity assays There is generally much less data on NM effects in vivo than in vitro; however, because of ADME issues, effects in a living organism are considered more important for safety assessment of NMs than for other test agents. Recommendations on in vivo assays for genotoxicity assessment of NM are summarized below: Dosing should include appropriate dose escalation for toxicological assessment, but avoid particle overload that could lead to artifacts. The in vivo assays generally do not need modifications and may be performed according to OECD TGs: 474 (Mammalian Erythrocyte MN Test), 475 (Mammalian Bone Marrow Chromosomal Aberration Test), 488 (TGR Somatic and Germ Cell Gene Mutation Assay), 489 (In Vivo Mammalian Alkaline Comet Assay). If detection of oxidative damage is a goal, the comet assay modification that includes DNA glycosylases (Bourdon et al., 2012; Sharma et al., 2012) may provide maximal sensitivity (TG 489). The in vivo assays should be considered and chosen within the context of the need for in vivo-specific information, eg, distribution and sequestration of NMs in target tissues or organs, or to model human exposure. Recommended Approach for Assessing Genotoxicity of Nanomaterials 1. Scoping assessment A scoping assessment is recommended to evaluate available data on physico-chemical characterization and potential systemic distribution of NMs (consistent with the model of Next Gen genotoxicity assessment (Dearfield et al., 2017). This can be based on human exposure or sub-chronic animal experiments, if available as part of a standard safety assessment (data are not available in certain contexts, eg, for the review of cosmetics in the EU). Information on NM distribution, tissue or organ accumulation, or sequestration would be considered in the development of testing strategies. If systemic availability or tissue targeting effects did not occur, in vitro but not in vivo genotoxicity testing would be recommended. In the case where systemic effects were noted, in vivo genotoxicity testing would be considered. The selection of tests would be defined based on systemic/tissue targeting observed. 2. Recommended test battery Recommended test battery includes in vitro mammalian assays that detect the 2 major classes of genetic damage: gene mutation and chromosomal damage with a choice of assay from each group: A. In vitro mammalian mutagenicity assay (replaces bacterial mutation assays) (choose one) Mouse Lymphoma (L5178Y) TK±Assay (MLA) (OECD TG 490) HPRT gene mutation assay (HPRT) (OECD TG 476) Rationale: These forward mutation assays detect the same types of small scale genetic events as bacterial assays, including single base pair changes and frameshifts. In addition, the MLA detects a broad spectrum of genetic damage including chromosome rearrangements, deletions (both small and large) and mitotic recombination. The MLA and the HPRT assays thus detect a different spectrum of genetic damage and there can be situations where one is preferred. That is, if the detection of only small scale events is desired, it may be preferable to use the HPRT assay. For hazard identification it is often desirable to use the MLA to detect a broader array of events. The bacterial assays are not included in this recommended test battery because of substantial evidence that the bacteria used for standard genotoxicity testing (E. coli and S. typhimurium) have limited uptake of NMs. They also lack the capability for mammalian-specific responses, which may be the more important reason for their lack of response to NMs. Results in bacteria have been generally negative and are considered uninformative. B. Chromosomal damage assays In vitro Chromosomal aberration assay (OECD TG 473) In vitro MN assay with assay modification as described earlier (OECD TG 487) In vitro MLA (OECD TG 490) Rationale: These assays detect large scale genetic damage affecting chromosomes, particularly breaks, rearrangements, or whole chromosome loss. The in vitro MN assay is more commonly used than the chromosomal aberration assay, because it is less subjective and requires less skill in reading the endpoint. In addition, the MN assay can be automated for the assessment of a large number of cells, enhancing statistical validity. However, consistent with international strategies for chemical testing, there are 3 equally acceptable options for assessing the ability of NMs to cause chromosomal effects (MN assay, Chromosomal aberration assay, and the MLA). Although the MLA detects both large and small scale genomic damage, it is recommended that 2 in vitro assays be chosen for assessment of nanomaterials. Because NMs generally do not require metabolic activation, there is no compelling reason to perform an in vitro assay with S9, and no reason for default testing in vivo. Thus, NMs are generally tested in the absence of S9, unless composed of organic materials or agents likely to be affected by mammalian metabolism. 3. Additional tests for consideration A. The in vitro comet assay, a DNA strand break assay, is sometimes considered, especially in the context of assessment of oxidative damaging effects. However, the assay lacks an OECD guideline, defined protocols, and a mechanistic understanding based on positive and negative responses to a defined set of mutagens. Handling issues may be particularly important because of ongoing DNA repair processes that affect the quantitative endpoint. TK6(TK±) (human lymphoblastoid) assay (OED 490) has not undergone validation for hazard identification, but may be considered, especially if genetic variants (p53 or DNA repair deficiencies) would contribute to mechanistic understanding of a positive result (http://www.nihs.go.jp/dgm/tk6.html) In vivo assay if targeting/sequestering of NM to a specific tissue is demonstrated, or for additional information relative to in vivo risk Comet assay (OECD TG 489) Rodent transgenic mutagenicity assay (OECD TG 488). The in vivo MN assay (OECD TG 474) is part of some international testing strategies and is valid only when NMs are systemically available or when exposure of target tissues has been demonstrated. CONCLUSION A great diversity of test systems and methods has been used to assess the genotoxicity of nanomaterials, with almost a similar level of diversity of results. Thus few conclusions on NM genotoxicity can be made, despite a substantial body of work. This review sought to critique the published literature, with a view of providing recommendations on validated methods and systems for genotoxicity assessment of NMs. A multitude of issues were documented from this analysis, including a wide variation in physical and chemical properties of NMs, inconsistent NM characterization in the test medium, diversity of test systems often failing to meet OECD standards, difficulty of applying NMs to biological systems (including uptake), interference of NM with the test endpoint, potential variation in systemic distribution in vivo, and lack of a definitive MoA. Based on current data, it appears that NM genotoxicity responses are smaller than observed from classical DNA damaging agents, consistent with genotoxicity induced via a secondary effect rather than a result of direct DNA interaction. As a way forward, we recommend: (1) the use of carefully defined NMs, including characterization in the test medium; (2) assessment of uptake and distribution within cells and in vivo systems; (3) dose ranges carefully chosen to avoid artefacts related to system overload; (4) a modified test battery that includes genotoxicity testing in in vitro mammalian mutagenicity and chromosomal damage assays, coupled to assay modifications as described within; (5) adherence to tests and cell systems described in the OECD TG and in Lorge et al. (2016); (6) and a greater effort on understanding mechanisms. ACKNOWLEDGMENTS The authors wish to thank the HESI Genetic Toxicology Technical Committee for intellectual and financial support. The authors also acknowledge the assistance by Ms Teyent Getaneh, Ms Lauren Peel, Ms Christina West, and Dr Stanley Parish of HESI for administrative support. REFERENCES Aardema M. J. , Barnett B. 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The views expressed in the manuscript do not necessarily reflect the policy of these agencies and organizations. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any agency determination or policy. The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services. Published by Oxford University Press on behalf of the Society of Toxicology 2018. This work is written by US Government employees and is in the public domain in the US. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) Published by Oxford University Press on behalf of the Society of Toxicology 2018. This work is written by US Government employees and is in the public domain in the US.

Journal

Toxicological SciencesOxford University Press

Published: Aug 1, 2018

Keywords: vasovagal syncope; chromosome abnormality; mutation; neuroleptic malignant syndrome; batteries; mutagenic effect; nanostructures; best practice; organisation for economic co-operation and development; mammals; animals, transgenic; comet assay; micronucleus

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