The Ramazzini Institute 13-week study on glyphosate-based herbicides at human-equivalent dose in Sprague Dawley rats: study design and first in-life endpoints evaluation

The Ramazzini Institute 13-week study on glyphosate-based herbicides at human-equivalent dose in... Background: Glyphosate-based herbicides (GBHs) are the most widely used pesticides worldwide, and glyphosate is the active ingredient of such herbicides, including the formulation known as Roundup. The massive and increasing use of GBHs results in not only the global burden of occupational exposures, but also increased exposure to the general population. The current pilot study represents the first phase of a long-term investigation of GBHs that we are conducting over the next 5 years. In this paper, we present the study design, the first evaluation of in vivo parameters and the determination of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) in urine. Methods: We exposed Sprague-Dawley (SD) rats orally via drinking water to a dose of glyphosate equivalent to the United States Acceptable Daily Intake (US ADI) of 1.75 mg/kg bw/day, defined as the chronic Reference Dose (cRfD) determined by the US EPA, starting from prenatal life, i.e. gestational day (GD) 6 of their mothers. One cohort was continuously dosed until sexual maturity (6-week cohort) and another cohort was continuously dosed until adulthood (13-week cohort). Here we present data on general toxicity and urinary concentrations of glyphosate and its major metabolite AMPA. Results: Survival, body weight, food and water consumption of the animals were not affected by the treatment with either glyphosate or Roundup. The concentration of both glyphosate and AMPA detected in the urine of SD rats treated with glyphosate were comparable to that observed in animals treated with Roundup, with an increase in relation to the duration of treatment. The majority of glyphosate was excreted unchanged. Urinary levels of the parent compound, glyphosate, were around 100-fold higher than the level of its metabolite, AMPA. Conclusions: Glyphosate concentrations in urine showed that most part of the administered dose was excreted as unchanged parent compound upon glyphosate and Roundup exposure, with an increasing pattern of glyphosate excreted in urine in relation to the duration of treatment. The adjuvants and the other substances present in Roundup did not seem to exert a major effect on the absorption and excretion of glyphosate. Our results demonstrate that urinary glyphosate is a more relevant marker of exposure than AMPA in the rodent model. Keywords: Glyphosate, Roundup, 13-week, Sprague-Dawley rat, Glyphosate based herbicides, GBH * Correspondence: belpoggif@ramazzini.it Simona Panzacchi and Daniele Mandrioli contributed equally to this work. Cesare Maltoni Cancer Research Center (CMCRC), Ramazzini Institute (RI), Via Saliceto, 3, 40010 Bentivoglio, Bologna, Italy Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Panzacchi et al. Environmental Health (2018) 17:52 Page 2 of 13 Background the major microbial metabolite [22]. In humans, the main Glyphosate [IUPAC chemical name N-(phosphono- exposure routes to glyphosate are inhalation and dermal methyl)glycine] is the most widely applied pesticide exposure in the occupational setting and consumption of worldwide and it is an active ingredient of all water and food for the general population [22]. The results glyphosate-based herbicides (GBHs), including in the of oral studies with [ C] glyphosate in rats, rabbits and formulation “Roundup” [1, 2]. It is mainly marketed as a goats indicate that absorption from the gastrointestinal broad-spectrum systemic herbicide and crop desiccant tract is incomplete and amounts to up to 30% of the dose [3]. The Asia-Pacific region represents the largest sup- [23–25]. The most relevant routes of excretion following plier of glyphosate active ingredient worldwide in terms oral administration of glyphosate [ C] are feces (70–80%) of production.. In 2016, China contributed the largest and urine (20–30%) [26]. In rats, after a single oral share in the Asia Pacific, and is likely to remain a dom- administration of [ C] glyphosate, almost all radioactivity inant market for years to come. The United State trails was detected in urine and feces, and the radiolabeled behind the Asia-Pacific market in the production of detected chemical was present as the unchanged parent GBHs. Latin America, Middle East and Africa are ex- compound [27–29]. Elimination through exhaled air was pected to grow in terms of use at a significant rate dur- very low. AMPA was the only metabolite detected, ing 2017–2025 [4]. Production and use of glyphosate accounting for only 0.2–0.3% of the applied dose of [ C] have risen dramatically with the introduction in 1996 of glyphosate [30]. The limited data currently available on genetically modified (GM) glyphosate tolerant crop var- glyphosate pharmacokinetics in vertebrates are insufficient ieties. In the United States (US) glyphosate is contained to predict transport and fate of glyphosate in different in over 750 products, particularly herbicides used for in- mammalian tissues, organs and fluids in the body, and to tensive GM crops that have built-in tolerance to glypho- determine whether or where bioaccumulation occurs, sate, but also in other products used in agriculture, although animal metabolism studies indicate kidney and forestry, urban, and home applications [5]. In 2015, 89% liver as target tissues [1]. of corn, 94% of soybeans, and 89% of cotton cropped in The possible effects of GBHs on human health is the the US were genetically modified to be glyphosate- topic of intense public debate, for both its potential car- tolerant [6]. Only a few data on the use of individual cinogenic and non-carcinogenic effects, including endo- pesticides are available for certain countries in the Euro- crine disruption, neurotoxicity, developmental and pean Union (EU), making it difficult to find out how reproductive toxicity, which might occur even at doses much glyphosate is being used by farmers [7]. However, much lower than the ones considered for risk assess- surveys in individual countries give some indication. ment, in particular during sensitive periods of life (such Glyphosate is the top ranked herbicide in United King- as fetal development) [5, 12, 31, 32]. Glyphosate, as the dom arable crop production [8]. In Denmark, glyphosate pure active substance, and GBHs may not be quite the accounts for 35% of all pesticides used in agricultural same from the toxicological standpoint. Glyphosate for- production [9]. In Germany, it has been estimated that mulations contain a number of so-called ‘inert’ ingredi- glyphosate is used on 4.3 million hectares (39%) of agri- ents or adjuvants to facilitate the uptake by plants, most cultural land each year, with nearly two thirds applied to of which are patented and not publicly known (in many just 3 crops - oilseed rape, winter wheat and winter bar- countries the law does not require a full disclosure of ley [10]. The EU has a strict regulation regarding the pesticide ingredients). GBHs that contain surfactants planting of GM crops (Directive EU 2015/412) [11] and and adjuvants might act differently than glyphosate GBHs are mainly applied to cereals for post-harvest des- alone [33, 34]. In fact, adjuvants might potentiate the iccation purposes (wheat, rye, triticale, barley and oats), toxic effects of glyphosate [35–38]. oilseeds (rapeseed, mustard seed and linseed), orchards and vineyards [12]. The Ramazzini Institute 13-week pilot study: aims and The massive and increasing use of GBHs leads to a experimental design global burden of occupational exposures in manufactur- The present pilot study is the first phase of an integrated ing workers and GBH applicators (farmers), as well as long-term project on GBHs that we are conducting dur- increasing exposures in the general population, as demon- ing the next 5 years [39]. The initial focus of our pilot strated by environmental contamination from glyphosate study is to assess techniques and methods for glyphosate residues found in air [13], groundwater [14, 15], drinking- detection in different matrices (results presented here), water [16], crops [17, 18], food [19, 20] and animal feed then to evaluate target organ toxicity, genotoxicity and [21]. Microbial biodegradation of glyphosate occurs in soil, endocrine disrupting activities, together with omics and aquatic sediment and water. The main pathway of biodeg- microbiome alterations (not presented here). In our pilot radation of glyphosate appears to be by splitting the C–N study, we exposed Sprague-Dawley (SD) rats to either bond to produce aminomethylphosphonic acid (AMPA), glyphosate or Roundup, one of the most popular Panzacchi et al. Environmental Health (2018) 17:52 Page 3 of 13 branded GBHs, with a dosage considered to be The protocol of the pilot study commences with “safe”, the United States Acceptable Daily Intake (US exposure from gestation day (GD) 6 (implantation) ADI) of 1.75 mg/kg bw/day, defined as the chronic continuously through pregnancy and lactation. To satisfy Reference Dose (cRfD) determined by the US EPA the need to consider multiple effects across multiple life [40]. The design of the pilot study derives from the stages, at weaning the offspring were assigned to two 13-week cohort protocol of the National Toxicology testing cohorts at random, so as to have minimal differ- Program (NTP) guideline Modified One-Generation ences in body weight among groups (standard deviation study (MOG) [39, 41]. It incorporates exposure dur- < 10% of the average). The first cohort (6-week cohort) ing the perinatal period (i.e., gestation and lactation) was continuously dosed until full sexual maturity (Post and later for 13 weeks after the pups are weaned, Natal Day-PND 73 ± 2), then sacrificed. The second evaluating standard sub-chronic toxicity and func- cohort (13-week cohort) was continuously dosed until tional endpoints (e.g., sperm analysis, vaginal cy- adulthood (PND 125 ± 2), then sacrificed. Both co- tology, indices of puberty and sexual differentiation) horts were analyzed for post-natal developmental to investigatepossibleeffects on thereproductive landmarks, microbiome, target organs toxicity and and endocrine systems. In order to provide more in- clinical pathology. formation about specific modes of action, we further The design of the pilot study has been developed by integrated the 13-week cohort NTP MOG design the Ramazzini Institute in collaboration with all Institu- with transcriptome analyses of potential target tis- tions taking part in the overall Glyphosate Study. All of sues and gut microbiome evaluation at different the in vivo experimental phases of the study were per- time-points and life stages in both dams and their formed at the Ramazzini Institute, while the other col- offspring. The whole-transcriptome analysis can pro- laborating Institutions have independently assessed vide important mechanistic information and support different outcomes and endpoints of interest. In this the pathological evaluation of target organs and hor- paper, we present the study design, the first evaluation mone analysis. The gut microbiome evaluation is a of in vivo parameters and the determination of glypho- novel endpoint representingthe potentialroleofal- sate and its major metabolite AMPA in urine. tered balance in the gut microbiota that relate to several health disorders such as metabolic diseases, Methods hepatic, coronary and gastrointestinal diseases (e.g., Experimental model inflammatory bowel disease) [32]. The experimental The study was conducted following the rules established plan and the endpoints investigated in the study are by the Italian law regulating the use and humane presented in Table 1 and Table 2. treatment of animals for scientific purposes [Decreto Table 1 Experimental plan Breeders Offspring a b Group Animals Group Animals Treatment End of the experiment Sex No. N. Sex Cohort Compound Dose Age at Cohort start 6-week (No.) 13-week (No.) 6-week (PND) 13-week (PND) e f I F 8 I M 8 10 Control 0GD670 120 (drinking water) M8 F 8 10 F + M 16 M + F 16 20 e f II F 8 II M 8 10 Glyphosate US ADI GD 6 70 120 M8 F 8 10 F + M 16 M + F 16 20 e f III F 8 III F 8 10 Roundup US ADI Glyphosate GD 6 70 120 equivalent M8 M 8 10 F + M 16 F + M 16 20 Total M + F 48 M + F 48 60 No more than 2 sisters and 2 brothers per litter Test compounds are administered ad libitum in drinking water Doses are calculated considering the Glyphosate US ADI defined as the chronic Reference Dose (cRfD) determined by the US EPA (1.75 mg/kg bw/day) Solutions are admistered to dams starting from the 6th day of pregnancy Animals are treated until the landmarks of sexual development are acquired (PND 73 ± 2) Animals are treated from embryonic life (GD 6) indirectly from dams milk until PND 28 ± 2, then directly for 90 days after weaning (until PND 125 ± 2) Panzacchi et al. Environmental Health (2018) 17:52 Page 4 of 13 Table 2 Summary of the endpoints and relative monitoring time points evaluated in the study, in dams and offspring (6-week and 13-week cohorts) Endpoints Time points Dams Offspring 6- Offspring 13- week cohort week cohort Gestation length GD0-delivery ✓ –– AGD and body weight in male and female PND 1 – ✓✓ pups Litter size PND 1, 4, 7, 10, 13, 16, 19, 21, 25 – ✓✓ Live-birth index PND 1 – ✓✓ Survival index PND 4, 7, 10, 13, 16, 19, 21, 25 – ✓✓ Age and body weight at BPS in male pups PND 35 – ✓✓ Age and body weight at VO in female pups PND 28 – ✓✓ First estrous in female pups 3 days after VO – ✓ – Estrous cycle length and percentage of days PND 95 - PND 116 –– ✓ in each stage Estrous cycle prior to necropsy PND 125 ± 2 –– ✓ Serum hormone measures End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Clinical biochemistry PND 73 ± 2 and PND 125 ± 2 – ✓✓ Urinalysis PND 73 ± 2 and PND 125 ± 2 – ✓✓ Glyphosate and AMPA detection in urine End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Sperm counts PND 73 ± 2 and PND 125 ± 2 – ✓✓ Daily Sperm production PND 73 ± 2 and PND 125 ± 2 – ✓✓ Sperm transit time through the epididymis PND 73 ± 2 and PND 125 ± 2 – ✓✓ Sperm morphology PND 73 ± 2 and PND 125 ± 2 – ✓✓ Sperm aneuploidy PND 73 ± 2 and PND 125 ± 2 – ✓✓ Partial histopathology (reproductive organs, End of lactation (dams) ✓ –– brain, liver, kidney) Complete histopathology PND 73 ± 2 and PND 125 ± 2 – ✓✓ Organ weight End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Micronuclei test (bone marrow) PND 73 ± 2 and PND 125 ± 2 – ✓✓ Transcriptome on mammary glands End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Transcriptome on brain PND 125 ± 2 –– ✓ Transcriptome on liver End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Transcriptome on kidneys End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Microbiome analysis in dams Before mating, GD 5 (before treatment), GD 13, LD 7, LD 14 ✓ –– Microbiome analysis in offspring PND 7, PND 14, PND 31 (before puberty), PND 57 (after puberty), – ✓✓ PND 125 ± 2 (adulthood) GD gestation day, LD lactation day, PND postnatal day, AGD anogenital distance, VO vaginal opening, BPS balano preputial separation Legislativo (D.Lgs.) N. 26, 2014. Attuazione della diret- of the Ramazzini Institute (CMCRC/RI) for over 40 years. tiva n. 2010/63/UE in materia di protezione degli animali An animal disease screening program enforced by the utilizzati a fini scientifici. - G.U. Serie Generale, n. 61 del Italian Health Authority and Research Organization for 14 Marzo 2014]. Before starting, the protocol was exam- Animal Health is in place and ongoing on sentinel ani- ined by the Internal Ethical Committee for approval. mals belonging to the RI colony. The protocol of the experiment was also approved and Female breeders SD rats were placed individually in formally authorized by the ad hoc commission of the Polycarbonate cage (42x26x18cm; Tecniplast Buguggi- Italian Ministry of Health (ministerial approval n. 710/ ate, Varese, Italy) with a single unrelated male until evi- 2015-PR). The experiment was performed on both male dence of copulation was observed. After mating, and female SD rats, which belong to the colony used at matched females were housed separately during gesta- the Cesare Maltoni Cancer Research Center laboratories tion and delivery. Newborns were housed with their Panzacchi et al. Environmental Health (2018) 17:52 Page 5 of 13 mothers until weaning. Weaned offspring were housed, The opening and the use date of the different batches of by sex and treatment group, not more than 3 per each test substances were recorded in the raw data. An ali- cage. Cages were identified by a card indicating: study quot of each lot of the test article is maintained in the protocol code, experimental and pedigree numbers, dos- ventilated storage cabinet, until 5 years from the end of age group. A shallow layer of white fir wood shavings the main experiment. The solutions of glyphosate and served as bedding (supplier: Giuseppe Bordignon, Tre- Roundup were prepared by the addition of appropriate viso, Italy). Analysis of chemical characteristics (pH, volume of tap drinking water. ashes, dry weight, specific weight) and possible contam- ination (metals, aflatoxin, polychlorobiphenyls, organo- phosphorus and organochlorine pesticides) of the Experimental plan bedding was performed by CONSULAB Laboratories Each of twenty-four virgin female SD rats (17 weeks old, (Treviso, Italy). The cages were placed on racks, inside a 270-315 g) was cohabited outbred with one breeder male single room prepared for the experiment at 22 °C ± 3 °C rat of the same age and strain. Every day, the females temperature and 50 ± 20% relative humidity. Daily were examined for presence of sperm. Gestational day checks on temperature and humidity were performed. (GD) 0 was defined as the one in which the sperm was The light was artificial and a light/dark cycle of 12 h was found in vaginal smears. The day on which parturition maintained. was completed was designated as lactating day (LD) 0 During the experiment SD rats received ad libitum the for the dam and PND 0 for the offspring. Each dam and standard “Corticella” pellet feed supplied by Laboratorio delivered litter was co-housed in common nesting box Dottori Piccioni Srl (Piccioni Laboratory, Milan, Italy). during the postpartum period. Following the NTP MOG The constituents of the diet are: ground corn (23%), bar- design, on PND 28, thus 28 days after the last litter was ley milled (15%), soybean meal extract (20.6%), wheat delivered, the offspring were weaned and identified by middling (24%), wheat bran (2%), spray dried whey (2. ear punch according to the Jackson Laboratory system. 5%), di-calcium phosphate (2%), calcium carbonate (1. Sequentially, they were allocated in the same treatment 1%), chicken meal (6%), carob bean gum (3%), sodium group of their mother in order to have 18 males (8 for chloride (0.5%), mixed vitamins (0.3%). Every day, the the 6-week cohort and 10 for the 13-week cohort) and animals drank fresh municipal tap water from glass bot- 18 females (8 for the 6-week cohort and 10 for the 13- tles ad libitum. Both feed and water were periodically week cohort) for each dose group. No more than 2 analyzed to identify possible chemical or microbiological males and 2 females from the same litter were included contaminants or impurities; the analyses are included in in the same cohort/treatment group. Altogether, 108 SD the documentation of the experiment. The pelleted feed rats (54 males and 54 females) were enrolled in the post- was tested for possible glyphosate contamination in weaning treatment phase. The experimental plan of the compliance with Commission Regulation (EU) No 293/ pilot study is outlined in Table 1. A summary of the end- 2013 [maximum residue levels (MRLs) < 1 mg/kg]. Tap points and relative monitoring time points evaluated in drinking water was tested for possible glyphosate con- the pilot study, both in dams and in the offspring (6- tamination in compliance with Directive 2008/105/EC, week and 13-week cohorts) is presented in Table 2. D.Lgs. 152/2006, Directive2006/118/EC (active sub- Two groups of SD rats were treated with either glyphosate stances in pesticides, including their relevant metabo- or Roundup diluted in tap water administered ad libitum and lites, degradation and reaction products < 0.1 μg/l). onegroup receivedonlytap waterascontrol.Roundup was Active ingredient glyphosate (Pestanal™ analytical diluted in tap water in order to obtain an equivalent dose of standard, CAS number 1071–83-6, purity > 99,5%) was glyphosate of 1.75 mg/kg bw/day. During gestational and lac- supplied from Sigma-Aldrich (Milan, Italy). The com- tational periods, embryos and newborns (F1) received the test mercial formulation Roundup Bioflow (containing compounds mainly through their dams (F0). Glyphosate and 360 g/L of glyphosate acid in the form of 480 g/l isopro- Roundup water formulations during these periods were pylamine salts of glyphosate (41.5%), water (42.5%) and freshly prepared on a daily base depending on individual surfactant (16%; chemical name, CAS number and/or body weight and water consumption of dams as measured at exact percentage have been withheld as a trade secret) each scheduled time point (see below). After weaning, until was supplied from a local agricultural consortium (Con- the end of the experiment (PND 73 ± 2 or 125 ± 2), the test sorzio Agrario dell’Emilia, Bologna, Italy). The original substances were administered in tap water to F1 animals on containers/bottles of glyphosate and Roundup were the basis of the average body weight and average water con- stored in its original container and kept in a ventilated sumption per sex and per experimental group, as measured storage cabinet at room temperature (22 °C ± 3 °C) at each scheduled time point (see below). Males and females throughout the study. Purity data for each batch of gly- were considered separately because of their difference in phosate and Roundup were provided by the supplier. weight gain, body weight and water consumption. Panzacchi et al. Environmental Health (2018) 17:52 Page 6 of 13 At least every week, the exposure doses were recalcu- Neotron Laboratories (Modena, Italy), an officially accre- lated and registered. The actual levels of test compounds dited laboratory by Accredia (Lab. N. 0026) according to that reached the fetus during gestation or that were European regulation UNI CEI EN ISO/IEC 17025:2005. ingested postnatally by the offspring during the period The specification and results are maintained in the experi- of lactation were not estimated in the present study. mental documentation. The analytical method is based on Animals were monitored during the entire experimen- liquid chromatography tandem mass spectrometry (LC- tal period. The following procedures were performed: MS/MS) [42–45]. The limit of quantification (LQ) for gly- Health status control: from the start of the experiment, phosate and AMPA corresponded to 0.10 μg/l in water, animals were checked three times daily, except on Sun- 50 μg/kg in feed, and 1 μg/kg in urine. days and non-working days, when they were only checked twice. All observed variations from normal sta- Statistical analysis tus were recorded. Summary statistics, means ± standard deviations (sd), Clinical control: status, behavior and clinical observation were calculated for continuous variables. For body on the experimental animals were checked before the start weight, water and feed consumption over time further of the treatment, and at least every two days until the end analyses were performed using multilevel mixed-effect of the experiment. Any findings listed below were then re- linear regression models, to control for within subject corded: alterations of skin, hair, eyes and mucosa; modifi- correlation across time; moreover we have considered cation in production of secretions or excretions and in also the litter effect during the lactation period. Analysis autonomic activity; respiratory symptoms; postural of variance and Dunnett’s tests (when applicable) were changes or changes in walk; presence of tonic or clonic also performed to compare body weight gain in different contractions; unusual stereotypes and behavior. periods and consumption of food and water as mean Dams’ body weights were recorded on GD 0, 3, 6 and consumption in several periods. then daily during gestation until parturition. During lac- All tests were two tailed, with alpha set at 0.05. Statis- tation, dams’ body weights were recorded at LD 1, 4, 7, tical analyses were perfomed by using STATA version10 10, 13, 16, 19, 21 and 25 (last measurement before wean- (Stata Corporation, College StationTexas, USA). ing). Pups’ body weight by sex and litter was determined on PND 1, 4, 7, 10, 13, 16, 19, 21 and 25. After weaning, Results the body weight was measured twice a week, until PND In dams, during both gestation and lactation, body 73 ± 2, then weekly until PND 125 ± 2 and before ter- weight and weight gain were not statistically different minal sacrifices; the means of individual body weights among the different groups (Fig. 1 a-b). In both female were calculated for each group and sex. and male offspring, post weaning body weights were Dams’ feed and water consumption were recorded homogenous and no statistically significant differences twice weekly during gestation (GD 0, 3, 6, 9, 12, 15, 18, in body weight gain were observed among groups (Fig. 1 21), whereas during lactation were measured at LD 1, 4, c-f). All 24 dams and 108 SD rats from the 6-week (48/ 7, 10, 13, 16, 19, 21, 25 and 28. 48) and 13-week (60/60) cohorts survived until sacrifice. After weaning the daily water and feed consumption Water and feed consumption during gestation and lac- per cage were measured twice a week, until PND 73 ± 2, tation were no different across the groups (Fig. 2 a-b then weekly until PND 125 ± 2; the means of individual and Fig. 3 a-b). Litter sizes were fully comparable among consumptions were calculated for each group and sex. groups, with mean number of live pups: control group The day before the terminal sacrifices, all the animals 13.6 (range 10–16); glyphosate group 13.3 (range 11– were located individually in metabolic cages and starved for 17); Roundup group 13.9 (range 11–16). Post weaning around 16 h. During this time, the animals had free access water and feed consumption were not affected by the to water alone or to the programmed test compound solu- treatment (Fig. 2 c-d and Fig. 3 c-d). tions. The day after, in the morning, samples of at least No unexpected clinical signs or symptoms were ob- 5 ml of spontaneous urine from each animal were collected served in the experimental animals during the in vivo and put in separate labelled tubes. Urine samples for ana- phase. In particular, there was no clinical evidence of al- lysis of glyphosate and AMPA excretion were obtained terations in activity or behavior, reflexes, the eye or skin, from 3 dams/group and from 10 (5 males + 5 females) rats/ or the respiratory, gastrointestinal, genito-urinary and group belonging to the 6-week and 13-week cohorts. cardiovascular systems. The results of glyphosate and AMPA urinary concen- Glyphosate and aminomethylphosphonic acid (AMPA) trations are reported in Table 3 and Fig. 4. The urinary detection concentration of both glyphosate and AMPA of SD rats Analyses of glyphosate and its metabolite AMPA in treated with 1.75 mg/kg bw/day of glyphosate were com- drinking water, feed and urine were performed by parable to the ones observed in SD rats treated with Panzacchi et al. Environmental Health (2018) 17:52 Page 7 of 13 Fig. 1 Average body weight: dams during gestation (a), treatment starting at gestation day 6 (↓); dams (b), male (c) and female (d) offspring during lactation; male (e) and female (f) offspring after weaning. At week 6 after weaning 8 male and 8 female pups per group were sacrificed Roundup dose equivalent to 1.75 mg/kg bw/day, despite of quantitation (0.001 mg/kg). In the treated SD rats, the limited sample size and the large standard deviations. In majority of glyphosate was excreted unchanged (as par- the control group, as expected, the glyphosate and ent compound), with urinary levels about 100-fold AMPA urinary levels were all below or close to the limit higher than that of its metabolite AMPA. For example, Panzacchi et al. Environmental Health (2018) 17:52 Page 8 of 13 Fig. 2 Average water consumption: dams during gestation (a), treatment starting at gestation day 6 (↓); dams and litter (b) during lactation; male (c) and female (d) offspring after weaning. At week 6 after weaning 8 male and 8 female pups per group were sacrificed glyphosate and Roundup treated females in the 13-week cohort. In the Roundup treatment group, the increase was cohort presented mean urinary levels of glyphosate re- less steep, but the time-dependent pattern was still evi- spectively of 1.354 mg/kg and 1.524 mg/kg, while the dent. In glyphosate and Roundup treated SD rats, the AMPA levels were respectively 0.013 mg/kg and 0. levels of AMPA were comparable at the different time 021 mg/kg. In glyphosate and Roundup treated SD rats, points in both males and females. In these animals, large a time-dependent increase in the mean urinary concen- standard deviations of the values of AMPA concentrations tration of glyphosate was observed. In glyphosate and in urine have been observed, in particular for values close Roundup treated males, an approximate 2-fold in- to the limit of quantitation as in the control groups. crease of mean urinary concentration of glyphosate in the 13-week cohort (animals exposed prenatally until Discussion 125 ± 2 days after birth) compared to the 6-week cohort Survival, body weights, food and water consumption of (animals exposed prenatally until 73 ± 2 days after birth) SD rats were not affected by the treatment with glypho- was observed. In glyphosate treated females, the 6-week sate and Roundup. Clinical changes in the animals were cohort (animals exposed prenatally until 73 ± 2 days not observed in the various groups. Overall, both gly- after birth) showed a 2-fold higher value of mean urinary phosate and Roundup treatments seemed to be well tol- concentration of glyphosate than the dams after weaning erated, which is consistent with previous experiments (exposed for 49 ± 2 days), while the 13-week cohort (ani- performed by the US NTP [26]. mals exposed prenatally and 125 ± 2 days after birth) Glyphosate and Roundup exposure led to comparable showed a 1.5-fold increase compared to the 6-week concentrations of glyphosate and AMPA in urine, Panzacchi et al. Environmental Health (2018) 17:52 Page 9 of 13 Fig. 3 Average feed consumption: dams during gestation (a), treatment starting at gestation day 6 (↓); dams and litter (b) during lactation; male (c) and female (d) offspring after weaning. At week 6 after weaning 8 male and 8 female pups per group were sacrificed indicating that systemic exposure does occur at the se- major effect on the absorption and excretion of glypho- lected exposure level of 1.75 mg//kg bw/day, corre- sate, even though mean values of glyphosate seem to be sponding to the US ADI. The bioavailability of somewhat higher in the Roundup treated group. The glyphosate in our study is also supported by the evident levels in urine were also comparable between the two increase of glyphosate concentration in urine in relation sexes; however, a consistent inter-individual variability to the length of treatment. The adjuvants and the other was observed. In rats, glyphosate in urine appears to be substances present in Roundup did not seem to exert a the most accurate biomarker of exposure to GBHs. In Table 3 Glyphosate and AMPA concentration in urine. Results are reported as mean ± standard deviations Dams Offspring (6-week cohort) Offspring (13-week cohort) Treatment Glyphosate AMPA Glyphosate AMPA Glyphosate AMPA (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) Male Control 0.012 ± 0.010 0.003 ± 0.003 0.011 ± 0.010 0.006 ± 0.004 Glyphosate _ _ 0.938 ± 0.414 0.014 ± 0.007 1.684 ± 0.768 0.023 ± 0.012 Roundup 1.174 ± 0.439 0.011 ± 0.005 2.280 ± 1.520 0.027 ± 0.016 Female Control 0.009 ± 0.001 0.006 ± 0.002 0.013 ± 0.007 0.005 ± 0.001 0.008 ± 0.005 0.003 ± 0.005 Glyphosate 0.480 ± 0.010 0.024 ± 0.002 0.938 ± 0.377 0.016 ± 0.010 1.354 ± 0.359 0.013 ± 0.006 Roundup 0.700 ± 0.106 0.024 ± 0.001 0.910 ± 0.383 0.018 ± 0.007 1.524 ± 0.585 0.021 ± 0.007 Panzacchi et al. Environmental Health (2018) 17:52 Page 10 of 13 Fig. 4 Average urinary concentrations of glyphosate and AMPA, expressed in mg/kg, collected at terminal sacrifices. Dams glyphosate (a) and AMPA (b) excretion; 6-week cohort male and female offspring; glyphosate (c) and AMPA (d) excretion; 13-week cohort male and female pups Glyphosate (e) and AMPA (f) excretion fact, our results confirm previous evidence that in rodents administered dose [46]. Furthermore, with the level of ex- most of the administered dose of glyphosate (98%) is posure to glyphosate used in this pilot study, AMPA urin- excreted as unchanged parent compound, whereas the ary values of treated animals (0.011–0.027 mg/kg) were metabolite AMPA in urine is at around 0.2–0.3% of the already close to the chromatographic LQ (0.001 mg/kg) Panzacchi et al. Environmental Health (2018) 17:52 Page 11 of 13 and this might limit the reliability of the measures. On the confirm that, in rodents, glyphosate in urine is the much other hand, glyphosate concentration in urine of treated more relevant marker of exposure than AMPA in par- animals (0.480–2.280 mg/kg) resulted up to 100-fold ticular at doses that are equal or lower than the one used higher than the AMPA concentration and at least 500- in this pilot study (1.75 mg/kg bw/day). The evaluation fold higher than the chromatographic LQ (0.001 mg/kg). of different outcomes and endpoints of interest (i.e., Therefore, in order to assess exposure to glyphosate in pathology of target organs, molecular toxicity, genotoxi- rats, in particular at doses that are equal or lower than city, endocrine disrupting activities, microbiome, devel- the one used in this pilot study (1.75 mg/kg bw/day), opmental toxicity, etc.) is currently ongoing in the glyphosate appears to be the biomarker of choice. different partner laboratories of the project. The presence of negligible levels of glyphosate (0.003– Abbreviations 0.013 mg/kg), close to the chromatographic LQ (0.001 AMPA: Aminomethylphosphonic acid; CMCRC: Cesare Maltoni Cancer mg/kg), in some of the urine of the control groups might Research Center; EU: European Union; GBH: Glyphosate-based herbicides; GD: Gestational day; GM: Genetically modified; LC-MS/MS: Liquid reflect an ubiquitous environmental contamination at chromatography tandem mass spectrometry; LD: Lactating day; LQ: Limit of ultra-low doses of glyphosate, which is consistent with Quantification; MOG: Modified One-Generation study; NTP: National previous reports from other authors [21]. As the current Toxicology Program; PND: Post Natal Day; RI: Ramazzini Institute; SD: Sprague-Dawley; US ADI: United States Acceptable Daily Intake limit of quantitation of glyphosate in HPLC for pelleted animal feed is 0.050 mg/kg, this represents a technical Acknowledgements limiting factor for testing ultra-low doses of glyphosate. We thank the over 30,000 associates and volunteers of the Ramazzini Institute that made this pilot study possible through their commitment and As reported by a recent inter laboratory comparative study generosity. We thank the Municipality of Bologna, the Emilia-Romagna Re- on the quantitative determination of glyphosate at low gion, and the International Society of Doctors for Environment for organizing levels, caution should be taken when interpreting results if several events to promote this pilot study; “Coop Reno” and “Coopfond Fondo Mutualistico Legacoop” for supporting our research activity. the tested doses of glyphosate are close to the LQ of HPLC [47]. Funding It is noteworthy that the commercial formulation used This work was funded by Institution fund of the Ramazzini Institute, Bologna, Italy. in this study, Roundup Bioflow, was the representative formulated product recently evaluated for the renewal of Availability of data and materials the approval of glyphosate in EU and considered in the All raw data recorded and used during the current study are available from the corresponding author on reasonable request. European Food Safety Authority peer review (MON 52276) [48]. Authors’ contributions Our results seem particularly relevant in light of the All authors provided substantial contributions to the conception/design of the work, acquisition, analysis or interpretation of the data, revised the massive global burden of exposure to glyphosate, as shown manuscript critically, and approved the final version for submission. FM, SP, by the exponential increase in the last 20 years of the levels DM participated in the design of the study, performed the animal of glyphosate and AMPA measured in the urine of the experiments and sample collection, and drafted the manuscript. LB and LF performed the animal experiments and sample collection. FB supervised the general population in Germany [49]and in theUS[50]. study, participated in the design of the study and helped to draft the manuscript. MS, GG, GD, RM, AM, SL, JH, JC, MJP, PJL, helped to draft the Conclusion manuscript. All authors read and approved the final manuscript. We performed a pilot study on the health effects of gly- Ethics approval and consent to participate phosate and its formulation Roundup administered at N/A currently admitted doses (US ADI = 1.75 mg/kg bw/day) to SD rats. In this paper, we described the study design, Competing interests The authors declare that they have no competing interests. the first evaluation of in vivo parameters and the deter- mination of glyphosate and its major metabolite AMPA Publisher’sNote in urine. The treatment with either glyphosate or Springer Nature remains neutral with regard to jurisdictional claims in Roundup seemed to be overall well tolerated, consist- published maps and institutional affiliations. ently with previous experiments performed by the US Author details NTP [26]. Both glyphosate and Roundup exposure led Cesare Maltoni Cancer Research Center (CMCRC), Ramazzini Institute (RI), Via to comparable urinary concentrations of glyphosate and 2 Saliceto, 3, 40010 Bentivoglio, Bologna, Italy. Department of Agricultural AMPA with an increasing pattern of glyphosate excreted Sciences, University of Bologna, Viale Fanin 44, 40127 Bologna, Italy. Department of Veterinary Medical Sciences, University of Bologna, Via Tolara in urine in relation to the duration of treatment, indicat- di Sopra 50, 40064 Ozzano dell’Emilia, Bologna, Italy. Department of ing the systemic bioavailability of the active sub- Statistical Sciences, University of Bologna, Via Belle Arti 41, 40126 Bologna, stance and a possible mechanism of bioaccumulaton. Italy. Department of Food safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. The adjuvants and the other substances present in Department of Genetics and Genomic Sciences, Icahn School of Medicine at Roundup did not seem to exert a major effect on the ab- 7 Mount Sinai, 1425 Madison Ave, New York, NY 10029, USA. Department of sorption and excretion of glyphosate. 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The Ramazzini Institute 13-week study on glyphosate-based herbicides at human-equivalent dose in Sprague Dawley rats: study design and first in-life endpoints evaluation

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Abstract

Background: Glyphosate-based herbicides (GBHs) are the most widely used pesticides worldwide, and glyphosate is the active ingredient of such herbicides, including the formulation known as Roundup. The massive and increasing use of GBHs results in not only the global burden of occupational exposures, but also increased exposure to the general population. The current pilot study represents the first phase of a long-term investigation of GBHs that we are conducting over the next 5 years. In this paper, we present the study design, the first evaluation of in vivo parameters and the determination of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA) in urine. Methods: We exposed Sprague-Dawley (SD) rats orally via drinking water to a dose of glyphosate equivalent to the United States Acceptable Daily Intake (US ADI) of 1.75 mg/kg bw/day, defined as the chronic Reference Dose (cRfD) determined by the US EPA, starting from prenatal life, i.e. gestational day (GD) 6 of their mothers. One cohort was continuously dosed until sexual maturity (6-week cohort) and another cohort was continuously dosed until adulthood (13-week cohort). Here we present data on general toxicity and urinary concentrations of glyphosate and its major metabolite AMPA. Results: Survival, body weight, food and water consumption of the animals were not affected by the treatment with either glyphosate or Roundup. The concentration of both glyphosate and AMPA detected in the urine of SD rats treated with glyphosate were comparable to that observed in animals treated with Roundup, with an increase in relation to the duration of treatment. The majority of glyphosate was excreted unchanged. Urinary levels of the parent compound, glyphosate, were around 100-fold higher than the level of its metabolite, AMPA. Conclusions: Glyphosate concentrations in urine showed that most part of the administered dose was excreted as unchanged parent compound upon glyphosate and Roundup exposure, with an increasing pattern of glyphosate excreted in urine in relation to the duration of treatment. The adjuvants and the other substances present in Roundup did not seem to exert a major effect on the absorption and excretion of glyphosate. Our results demonstrate that urinary glyphosate is a more relevant marker of exposure than AMPA in the rodent model. Keywords: Glyphosate, Roundup, 13-week, Sprague-Dawley rat, Glyphosate based herbicides, GBH * Correspondence: belpoggif@ramazzini.it Simona Panzacchi and Daniele Mandrioli contributed equally to this work. Cesare Maltoni Cancer Research Center (CMCRC), Ramazzini Institute (RI), Via Saliceto, 3, 40010 Bentivoglio, Bologna, Italy Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Panzacchi et al. Environmental Health (2018) 17:52 Page 2 of 13 Background the major microbial metabolite [22]. In humans, the main Glyphosate [IUPAC chemical name N-(phosphono- exposure routes to glyphosate are inhalation and dermal methyl)glycine] is the most widely applied pesticide exposure in the occupational setting and consumption of worldwide and it is an active ingredient of all water and food for the general population [22]. The results glyphosate-based herbicides (GBHs), including in the of oral studies with [ C] glyphosate in rats, rabbits and formulation “Roundup” [1, 2]. It is mainly marketed as a goats indicate that absorption from the gastrointestinal broad-spectrum systemic herbicide and crop desiccant tract is incomplete and amounts to up to 30% of the dose [3]. The Asia-Pacific region represents the largest sup- [23–25]. The most relevant routes of excretion following plier of glyphosate active ingredient worldwide in terms oral administration of glyphosate [ C] are feces (70–80%) of production.. In 2016, China contributed the largest and urine (20–30%) [26]. In rats, after a single oral share in the Asia Pacific, and is likely to remain a dom- administration of [ C] glyphosate, almost all radioactivity inant market for years to come. The United State trails was detected in urine and feces, and the radiolabeled behind the Asia-Pacific market in the production of detected chemical was present as the unchanged parent GBHs. Latin America, Middle East and Africa are ex- compound [27–29]. Elimination through exhaled air was pected to grow in terms of use at a significant rate dur- very low. AMPA was the only metabolite detected, ing 2017–2025 [4]. Production and use of glyphosate accounting for only 0.2–0.3% of the applied dose of [ C] have risen dramatically with the introduction in 1996 of glyphosate [30]. The limited data currently available on genetically modified (GM) glyphosate tolerant crop var- glyphosate pharmacokinetics in vertebrates are insufficient ieties. In the United States (US) glyphosate is contained to predict transport and fate of glyphosate in different in over 750 products, particularly herbicides used for in- mammalian tissues, organs and fluids in the body, and to tensive GM crops that have built-in tolerance to glypho- determine whether or where bioaccumulation occurs, sate, but also in other products used in agriculture, although animal metabolism studies indicate kidney and forestry, urban, and home applications [5]. In 2015, 89% liver as target tissues [1]. of corn, 94% of soybeans, and 89% of cotton cropped in The possible effects of GBHs on human health is the the US were genetically modified to be glyphosate- topic of intense public debate, for both its potential car- tolerant [6]. Only a few data on the use of individual cinogenic and non-carcinogenic effects, including endo- pesticides are available for certain countries in the Euro- crine disruption, neurotoxicity, developmental and pean Union (EU), making it difficult to find out how reproductive toxicity, which might occur even at doses much glyphosate is being used by farmers [7]. However, much lower than the ones considered for risk assess- surveys in individual countries give some indication. ment, in particular during sensitive periods of life (such Glyphosate is the top ranked herbicide in United King- as fetal development) [5, 12, 31, 32]. Glyphosate, as the dom arable crop production [8]. In Denmark, glyphosate pure active substance, and GBHs may not be quite the accounts for 35% of all pesticides used in agricultural same from the toxicological standpoint. Glyphosate for- production [9]. In Germany, it has been estimated that mulations contain a number of so-called ‘inert’ ingredi- glyphosate is used on 4.3 million hectares (39%) of agri- ents or adjuvants to facilitate the uptake by plants, most cultural land each year, with nearly two thirds applied to of which are patented and not publicly known (in many just 3 crops - oilseed rape, winter wheat and winter bar- countries the law does not require a full disclosure of ley [10]. The EU has a strict regulation regarding the pesticide ingredients). GBHs that contain surfactants planting of GM crops (Directive EU 2015/412) [11] and and adjuvants might act differently than glyphosate GBHs are mainly applied to cereals for post-harvest des- alone [33, 34]. In fact, adjuvants might potentiate the iccation purposes (wheat, rye, triticale, barley and oats), toxic effects of glyphosate [35–38]. oilseeds (rapeseed, mustard seed and linseed), orchards and vineyards [12]. The Ramazzini Institute 13-week pilot study: aims and The massive and increasing use of GBHs leads to a experimental design global burden of occupational exposures in manufactur- The present pilot study is the first phase of an integrated ing workers and GBH applicators (farmers), as well as long-term project on GBHs that we are conducting dur- increasing exposures in the general population, as demon- ing the next 5 years [39]. The initial focus of our pilot strated by environmental contamination from glyphosate study is to assess techniques and methods for glyphosate residues found in air [13], groundwater [14, 15], drinking- detection in different matrices (results presented here), water [16], crops [17, 18], food [19, 20] and animal feed then to evaluate target organ toxicity, genotoxicity and [21]. Microbial biodegradation of glyphosate occurs in soil, endocrine disrupting activities, together with omics and aquatic sediment and water. The main pathway of biodeg- microbiome alterations (not presented here). In our pilot radation of glyphosate appears to be by splitting the C–N study, we exposed Sprague-Dawley (SD) rats to either bond to produce aminomethylphosphonic acid (AMPA), glyphosate or Roundup, one of the most popular Panzacchi et al. Environmental Health (2018) 17:52 Page 3 of 13 branded GBHs, with a dosage considered to be The protocol of the pilot study commences with “safe”, the United States Acceptable Daily Intake (US exposure from gestation day (GD) 6 (implantation) ADI) of 1.75 mg/kg bw/day, defined as the chronic continuously through pregnancy and lactation. To satisfy Reference Dose (cRfD) determined by the US EPA the need to consider multiple effects across multiple life [40]. The design of the pilot study derives from the stages, at weaning the offspring were assigned to two 13-week cohort protocol of the National Toxicology testing cohorts at random, so as to have minimal differ- Program (NTP) guideline Modified One-Generation ences in body weight among groups (standard deviation study (MOG) [39, 41]. It incorporates exposure dur- < 10% of the average). The first cohort (6-week cohort) ing the perinatal period (i.e., gestation and lactation) was continuously dosed until full sexual maturity (Post and later for 13 weeks after the pups are weaned, Natal Day-PND 73 ± 2), then sacrificed. The second evaluating standard sub-chronic toxicity and func- cohort (13-week cohort) was continuously dosed until tional endpoints (e.g., sperm analysis, vaginal cy- adulthood (PND 125 ± 2), then sacrificed. Both co- tology, indices of puberty and sexual differentiation) horts were analyzed for post-natal developmental to investigatepossibleeffects on thereproductive landmarks, microbiome, target organs toxicity and and endocrine systems. In order to provide more in- clinical pathology. formation about specific modes of action, we further The design of the pilot study has been developed by integrated the 13-week cohort NTP MOG design the Ramazzini Institute in collaboration with all Institu- with transcriptome analyses of potential target tis- tions taking part in the overall Glyphosate Study. All of sues and gut microbiome evaluation at different the in vivo experimental phases of the study were per- time-points and life stages in both dams and their formed at the Ramazzini Institute, while the other col- offspring. The whole-transcriptome analysis can pro- laborating Institutions have independently assessed vide important mechanistic information and support different outcomes and endpoints of interest. In this the pathological evaluation of target organs and hor- paper, we present the study design, the first evaluation mone analysis. The gut microbiome evaluation is a of in vivo parameters and the determination of glypho- novel endpoint representingthe potentialroleofal- sate and its major metabolite AMPA in urine. tered balance in the gut microbiota that relate to several health disorders such as metabolic diseases, Methods hepatic, coronary and gastrointestinal diseases (e.g., Experimental model inflammatory bowel disease) [32]. The experimental The study was conducted following the rules established plan and the endpoints investigated in the study are by the Italian law regulating the use and humane presented in Table 1 and Table 2. treatment of animals for scientific purposes [Decreto Table 1 Experimental plan Breeders Offspring a b Group Animals Group Animals Treatment End of the experiment Sex No. N. Sex Cohort Compound Dose Age at Cohort start 6-week (No.) 13-week (No.) 6-week (PND) 13-week (PND) e f I F 8 I M 8 10 Control 0GD670 120 (drinking water) M8 F 8 10 F + M 16 M + F 16 20 e f II F 8 II M 8 10 Glyphosate US ADI GD 6 70 120 M8 F 8 10 F + M 16 M + F 16 20 e f III F 8 III F 8 10 Roundup US ADI Glyphosate GD 6 70 120 equivalent M8 M 8 10 F + M 16 F + M 16 20 Total M + F 48 M + F 48 60 No more than 2 sisters and 2 brothers per litter Test compounds are administered ad libitum in drinking water Doses are calculated considering the Glyphosate US ADI defined as the chronic Reference Dose (cRfD) determined by the US EPA (1.75 mg/kg bw/day) Solutions are admistered to dams starting from the 6th day of pregnancy Animals are treated until the landmarks of sexual development are acquired (PND 73 ± 2) Animals are treated from embryonic life (GD 6) indirectly from dams milk until PND 28 ± 2, then directly for 90 days after weaning (until PND 125 ± 2) Panzacchi et al. Environmental Health (2018) 17:52 Page 4 of 13 Table 2 Summary of the endpoints and relative monitoring time points evaluated in the study, in dams and offspring (6-week and 13-week cohorts) Endpoints Time points Dams Offspring 6- Offspring 13- week cohort week cohort Gestation length GD0-delivery ✓ –– AGD and body weight in male and female PND 1 – ✓✓ pups Litter size PND 1, 4, 7, 10, 13, 16, 19, 21, 25 – ✓✓ Live-birth index PND 1 – ✓✓ Survival index PND 4, 7, 10, 13, 16, 19, 21, 25 – ✓✓ Age and body weight at BPS in male pups PND 35 – ✓✓ Age and body weight at VO in female pups PND 28 – ✓✓ First estrous in female pups 3 days after VO – ✓ – Estrous cycle length and percentage of days PND 95 - PND 116 –– ✓ in each stage Estrous cycle prior to necropsy PND 125 ± 2 –– ✓ Serum hormone measures End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Clinical biochemistry PND 73 ± 2 and PND 125 ± 2 – ✓✓ Urinalysis PND 73 ± 2 and PND 125 ± 2 – ✓✓ Glyphosate and AMPA detection in urine End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Sperm counts PND 73 ± 2 and PND 125 ± 2 – ✓✓ Daily Sperm production PND 73 ± 2 and PND 125 ± 2 – ✓✓ Sperm transit time through the epididymis PND 73 ± 2 and PND 125 ± 2 – ✓✓ Sperm morphology PND 73 ± 2 and PND 125 ± 2 – ✓✓ Sperm aneuploidy PND 73 ± 2 and PND 125 ± 2 – ✓✓ Partial histopathology (reproductive organs, End of lactation (dams) ✓ –– brain, liver, kidney) Complete histopathology PND 73 ± 2 and PND 125 ± 2 – ✓✓ Organ weight End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Micronuclei test (bone marrow) PND 73 ± 2 and PND 125 ± 2 – ✓✓ Transcriptome on mammary glands End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Transcriptome on brain PND 125 ± 2 –– ✓ Transcriptome on liver End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Transcriptome on kidneys End of lactation (dams), PND 73 ± 2 and PND 125 ± 2 ✓✓ ✓ Microbiome analysis in dams Before mating, GD 5 (before treatment), GD 13, LD 7, LD 14 ✓ –– Microbiome analysis in offspring PND 7, PND 14, PND 31 (before puberty), PND 57 (after puberty), – ✓✓ PND 125 ± 2 (adulthood) GD gestation day, LD lactation day, PND postnatal day, AGD anogenital distance, VO vaginal opening, BPS balano preputial separation Legislativo (D.Lgs.) N. 26, 2014. Attuazione della diret- of the Ramazzini Institute (CMCRC/RI) for over 40 years. tiva n. 2010/63/UE in materia di protezione degli animali An animal disease screening program enforced by the utilizzati a fini scientifici. - G.U. Serie Generale, n. 61 del Italian Health Authority and Research Organization for 14 Marzo 2014]. Before starting, the protocol was exam- Animal Health is in place and ongoing on sentinel ani- ined by the Internal Ethical Committee for approval. mals belonging to the RI colony. The protocol of the experiment was also approved and Female breeders SD rats were placed individually in formally authorized by the ad hoc commission of the Polycarbonate cage (42x26x18cm; Tecniplast Buguggi- Italian Ministry of Health (ministerial approval n. 710/ ate, Varese, Italy) with a single unrelated male until evi- 2015-PR). The experiment was performed on both male dence of copulation was observed. After mating, and female SD rats, which belong to the colony used at matched females were housed separately during gesta- the Cesare Maltoni Cancer Research Center laboratories tion and delivery. Newborns were housed with their Panzacchi et al. Environmental Health (2018) 17:52 Page 5 of 13 mothers until weaning. Weaned offspring were housed, The opening and the use date of the different batches of by sex and treatment group, not more than 3 per each test substances were recorded in the raw data. An ali- cage. Cages were identified by a card indicating: study quot of each lot of the test article is maintained in the protocol code, experimental and pedigree numbers, dos- ventilated storage cabinet, until 5 years from the end of age group. A shallow layer of white fir wood shavings the main experiment. The solutions of glyphosate and served as bedding (supplier: Giuseppe Bordignon, Tre- Roundup were prepared by the addition of appropriate viso, Italy). Analysis of chemical characteristics (pH, volume of tap drinking water. ashes, dry weight, specific weight) and possible contam- ination (metals, aflatoxin, polychlorobiphenyls, organo- phosphorus and organochlorine pesticides) of the Experimental plan bedding was performed by CONSULAB Laboratories Each of twenty-four virgin female SD rats (17 weeks old, (Treviso, Italy). The cages were placed on racks, inside a 270-315 g) was cohabited outbred with one breeder male single room prepared for the experiment at 22 °C ± 3 °C rat of the same age and strain. Every day, the females temperature and 50 ± 20% relative humidity. Daily were examined for presence of sperm. Gestational day checks on temperature and humidity were performed. (GD) 0 was defined as the one in which the sperm was The light was artificial and a light/dark cycle of 12 h was found in vaginal smears. The day on which parturition maintained. was completed was designated as lactating day (LD) 0 During the experiment SD rats received ad libitum the for the dam and PND 0 for the offspring. Each dam and standard “Corticella” pellet feed supplied by Laboratorio delivered litter was co-housed in common nesting box Dottori Piccioni Srl (Piccioni Laboratory, Milan, Italy). during the postpartum period. Following the NTP MOG The constituents of the diet are: ground corn (23%), bar- design, on PND 28, thus 28 days after the last litter was ley milled (15%), soybean meal extract (20.6%), wheat delivered, the offspring were weaned and identified by middling (24%), wheat bran (2%), spray dried whey (2. ear punch according to the Jackson Laboratory system. 5%), di-calcium phosphate (2%), calcium carbonate (1. Sequentially, they were allocated in the same treatment 1%), chicken meal (6%), carob bean gum (3%), sodium group of their mother in order to have 18 males (8 for chloride (0.5%), mixed vitamins (0.3%). Every day, the the 6-week cohort and 10 for the 13-week cohort) and animals drank fresh municipal tap water from glass bot- 18 females (8 for the 6-week cohort and 10 for the 13- tles ad libitum. Both feed and water were periodically week cohort) for each dose group. No more than 2 analyzed to identify possible chemical or microbiological males and 2 females from the same litter were included contaminants or impurities; the analyses are included in in the same cohort/treatment group. Altogether, 108 SD the documentation of the experiment. The pelleted feed rats (54 males and 54 females) were enrolled in the post- was tested for possible glyphosate contamination in weaning treatment phase. The experimental plan of the compliance with Commission Regulation (EU) No 293/ pilot study is outlined in Table 1. A summary of the end- 2013 [maximum residue levels (MRLs) < 1 mg/kg]. Tap points and relative monitoring time points evaluated in drinking water was tested for possible glyphosate con- the pilot study, both in dams and in the offspring (6- tamination in compliance with Directive 2008/105/EC, week and 13-week cohorts) is presented in Table 2. D.Lgs. 152/2006, Directive2006/118/EC (active sub- Two groups of SD rats were treated with either glyphosate stances in pesticides, including their relevant metabo- or Roundup diluted in tap water administered ad libitum and lites, degradation and reaction products < 0.1 μg/l). onegroup receivedonlytap waterascontrol.Roundup was Active ingredient glyphosate (Pestanal™ analytical diluted in tap water in order to obtain an equivalent dose of standard, CAS number 1071–83-6, purity > 99,5%) was glyphosate of 1.75 mg/kg bw/day. During gestational and lac- supplied from Sigma-Aldrich (Milan, Italy). The com- tational periods, embryos and newborns (F1) received the test mercial formulation Roundup Bioflow (containing compounds mainly through their dams (F0). Glyphosate and 360 g/L of glyphosate acid in the form of 480 g/l isopro- Roundup water formulations during these periods were pylamine salts of glyphosate (41.5%), water (42.5%) and freshly prepared on a daily base depending on individual surfactant (16%; chemical name, CAS number and/or body weight and water consumption of dams as measured at exact percentage have been withheld as a trade secret) each scheduled time point (see below). After weaning, until was supplied from a local agricultural consortium (Con- the end of the experiment (PND 73 ± 2 or 125 ± 2), the test sorzio Agrario dell’Emilia, Bologna, Italy). The original substances were administered in tap water to F1 animals on containers/bottles of glyphosate and Roundup were the basis of the average body weight and average water con- stored in its original container and kept in a ventilated sumption per sex and per experimental group, as measured storage cabinet at room temperature (22 °C ± 3 °C) at each scheduled time point (see below). Males and females throughout the study. Purity data for each batch of gly- were considered separately because of their difference in phosate and Roundup were provided by the supplier. weight gain, body weight and water consumption. Panzacchi et al. Environmental Health (2018) 17:52 Page 6 of 13 At least every week, the exposure doses were recalcu- Neotron Laboratories (Modena, Italy), an officially accre- lated and registered. The actual levels of test compounds dited laboratory by Accredia (Lab. N. 0026) according to that reached the fetus during gestation or that were European regulation UNI CEI EN ISO/IEC 17025:2005. ingested postnatally by the offspring during the period The specification and results are maintained in the experi- of lactation were not estimated in the present study. mental documentation. The analytical method is based on Animals were monitored during the entire experimen- liquid chromatography tandem mass spectrometry (LC- tal period. The following procedures were performed: MS/MS) [42–45]. The limit of quantification (LQ) for gly- Health status control: from the start of the experiment, phosate and AMPA corresponded to 0.10 μg/l in water, animals were checked three times daily, except on Sun- 50 μg/kg in feed, and 1 μg/kg in urine. days and non-working days, when they were only checked twice. All observed variations from normal sta- Statistical analysis tus were recorded. Summary statistics, means ± standard deviations (sd), Clinical control: status, behavior and clinical observation were calculated for continuous variables. For body on the experimental animals were checked before the start weight, water and feed consumption over time further of the treatment, and at least every two days until the end analyses were performed using multilevel mixed-effect of the experiment. Any findings listed below were then re- linear regression models, to control for within subject corded: alterations of skin, hair, eyes and mucosa; modifi- correlation across time; moreover we have considered cation in production of secretions or excretions and in also the litter effect during the lactation period. Analysis autonomic activity; respiratory symptoms; postural of variance and Dunnett’s tests (when applicable) were changes or changes in walk; presence of tonic or clonic also performed to compare body weight gain in different contractions; unusual stereotypes and behavior. periods and consumption of food and water as mean Dams’ body weights were recorded on GD 0, 3, 6 and consumption in several periods. then daily during gestation until parturition. During lac- All tests were two tailed, with alpha set at 0.05. Statis- tation, dams’ body weights were recorded at LD 1, 4, 7, tical analyses were perfomed by using STATA version10 10, 13, 16, 19, 21 and 25 (last measurement before wean- (Stata Corporation, College StationTexas, USA). ing). Pups’ body weight by sex and litter was determined on PND 1, 4, 7, 10, 13, 16, 19, 21 and 25. After weaning, Results the body weight was measured twice a week, until PND In dams, during both gestation and lactation, body 73 ± 2, then weekly until PND 125 ± 2 and before ter- weight and weight gain were not statistically different minal sacrifices; the means of individual body weights among the different groups (Fig. 1 a-b). In both female were calculated for each group and sex. and male offspring, post weaning body weights were Dams’ feed and water consumption were recorded homogenous and no statistically significant differences twice weekly during gestation (GD 0, 3, 6, 9, 12, 15, 18, in body weight gain were observed among groups (Fig. 1 21), whereas during lactation were measured at LD 1, 4, c-f). All 24 dams and 108 SD rats from the 6-week (48/ 7, 10, 13, 16, 19, 21, 25 and 28. 48) and 13-week (60/60) cohorts survived until sacrifice. After weaning the daily water and feed consumption Water and feed consumption during gestation and lac- per cage were measured twice a week, until PND 73 ± 2, tation were no different across the groups (Fig. 2 a-b then weekly until PND 125 ± 2; the means of individual and Fig. 3 a-b). Litter sizes were fully comparable among consumptions were calculated for each group and sex. groups, with mean number of live pups: control group The day before the terminal sacrifices, all the animals 13.6 (range 10–16); glyphosate group 13.3 (range 11– were located individually in metabolic cages and starved for 17); Roundup group 13.9 (range 11–16). Post weaning around 16 h. During this time, the animals had free access water and feed consumption were not affected by the to water alone or to the programmed test compound solu- treatment (Fig. 2 c-d and Fig. 3 c-d). tions. The day after, in the morning, samples of at least No unexpected clinical signs or symptoms were ob- 5 ml of spontaneous urine from each animal were collected served in the experimental animals during the in vivo and put in separate labelled tubes. Urine samples for ana- phase. In particular, there was no clinical evidence of al- lysis of glyphosate and AMPA excretion were obtained terations in activity or behavior, reflexes, the eye or skin, from 3 dams/group and from 10 (5 males + 5 females) rats/ or the respiratory, gastrointestinal, genito-urinary and group belonging to the 6-week and 13-week cohorts. cardiovascular systems. The results of glyphosate and AMPA urinary concen- Glyphosate and aminomethylphosphonic acid (AMPA) trations are reported in Table 3 and Fig. 4. The urinary detection concentration of both glyphosate and AMPA of SD rats Analyses of glyphosate and its metabolite AMPA in treated with 1.75 mg/kg bw/day of glyphosate were com- drinking water, feed and urine were performed by parable to the ones observed in SD rats treated with Panzacchi et al. Environmental Health (2018) 17:52 Page 7 of 13 Fig. 1 Average body weight: dams during gestation (a), treatment starting at gestation day 6 (↓); dams (b), male (c) and female (d) offspring during lactation; male (e) and female (f) offspring after weaning. At week 6 after weaning 8 male and 8 female pups per group were sacrificed Roundup dose equivalent to 1.75 mg/kg bw/day, despite of quantitation (0.001 mg/kg). In the treated SD rats, the limited sample size and the large standard deviations. In majority of glyphosate was excreted unchanged (as par- the control group, as expected, the glyphosate and ent compound), with urinary levels about 100-fold AMPA urinary levels were all below or close to the limit higher than that of its metabolite AMPA. For example, Panzacchi et al. Environmental Health (2018) 17:52 Page 8 of 13 Fig. 2 Average water consumption: dams during gestation (a), treatment starting at gestation day 6 (↓); dams and litter (b) during lactation; male (c) and female (d) offspring after weaning. At week 6 after weaning 8 male and 8 female pups per group were sacrificed glyphosate and Roundup treated females in the 13-week cohort. In the Roundup treatment group, the increase was cohort presented mean urinary levels of glyphosate re- less steep, but the time-dependent pattern was still evi- spectively of 1.354 mg/kg and 1.524 mg/kg, while the dent. In glyphosate and Roundup treated SD rats, the AMPA levels were respectively 0.013 mg/kg and 0. levels of AMPA were comparable at the different time 021 mg/kg. In glyphosate and Roundup treated SD rats, points in both males and females. In these animals, large a time-dependent increase in the mean urinary concen- standard deviations of the values of AMPA concentrations tration of glyphosate was observed. In glyphosate and in urine have been observed, in particular for values close Roundup treated males, an approximate 2-fold in- to the limit of quantitation as in the control groups. crease of mean urinary concentration of glyphosate in the 13-week cohort (animals exposed prenatally until Discussion 125 ± 2 days after birth) compared to the 6-week cohort Survival, body weights, food and water consumption of (animals exposed prenatally until 73 ± 2 days after birth) SD rats were not affected by the treatment with glypho- was observed. In glyphosate treated females, the 6-week sate and Roundup. Clinical changes in the animals were cohort (animals exposed prenatally until 73 ± 2 days not observed in the various groups. Overall, both gly- after birth) showed a 2-fold higher value of mean urinary phosate and Roundup treatments seemed to be well tol- concentration of glyphosate than the dams after weaning erated, which is consistent with previous experiments (exposed for 49 ± 2 days), while the 13-week cohort (ani- performed by the US NTP [26]. mals exposed prenatally and 125 ± 2 days after birth) Glyphosate and Roundup exposure led to comparable showed a 1.5-fold increase compared to the 6-week concentrations of glyphosate and AMPA in urine, Panzacchi et al. Environmental Health (2018) 17:52 Page 9 of 13 Fig. 3 Average feed consumption: dams during gestation (a), treatment starting at gestation day 6 (↓); dams and litter (b) during lactation; male (c) and female (d) offspring after weaning. At week 6 after weaning 8 male and 8 female pups per group were sacrificed indicating that systemic exposure does occur at the se- major effect on the absorption and excretion of glypho- lected exposure level of 1.75 mg//kg bw/day, corre- sate, even though mean values of glyphosate seem to be sponding to the US ADI. The bioavailability of somewhat higher in the Roundup treated group. The glyphosate in our study is also supported by the evident levels in urine were also comparable between the two increase of glyphosate concentration in urine in relation sexes; however, a consistent inter-individual variability to the length of treatment. The adjuvants and the other was observed. In rats, glyphosate in urine appears to be substances present in Roundup did not seem to exert a the most accurate biomarker of exposure to GBHs. In Table 3 Glyphosate and AMPA concentration in urine. Results are reported as mean ± standard deviations Dams Offspring (6-week cohort) Offspring (13-week cohort) Treatment Glyphosate AMPA Glyphosate AMPA Glyphosate AMPA (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) Male Control 0.012 ± 0.010 0.003 ± 0.003 0.011 ± 0.010 0.006 ± 0.004 Glyphosate _ _ 0.938 ± 0.414 0.014 ± 0.007 1.684 ± 0.768 0.023 ± 0.012 Roundup 1.174 ± 0.439 0.011 ± 0.005 2.280 ± 1.520 0.027 ± 0.016 Female Control 0.009 ± 0.001 0.006 ± 0.002 0.013 ± 0.007 0.005 ± 0.001 0.008 ± 0.005 0.003 ± 0.005 Glyphosate 0.480 ± 0.010 0.024 ± 0.002 0.938 ± 0.377 0.016 ± 0.010 1.354 ± 0.359 0.013 ± 0.006 Roundup 0.700 ± 0.106 0.024 ± 0.001 0.910 ± 0.383 0.018 ± 0.007 1.524 ± 0.585 0.021 ± 0.007 Panzacchi et al. Environmental Health (2018) 17:52 Page 10 of 13 Fig. 4 Average urinary concentrations of glyphosate and AMPA, expressed in mg/kg, collected at terminal sacrifices. Dams glyphosate (a) and AMPA (b) excretion; 6-week cohort male and female offspring; glyphosate (c) and AMPA (d) excretion; 13-week cohort male and female pups Glyphosate (e) and AMPA (f) excretion fact, our results confirm previous evidence that in rodents administered dose [46]. Furthermore, with the level of ex- most of the administered dose of glyphosate (98%) is posure to glyphosate used in this pilot study, AMPA urin- excreted as unchanged parent compound, whereas the ary values of treated animals (0.011–0.027 mg/kg) were metabolite AMPA in urine is at around 0.2–0.3% of the already close to the chromatographic LQ (0.001 mg/kg) Panzacchi et al. Environmental Health (2018) 17:52 Page 11 of 13 and this might limit the reliability of the measures. On the confirm that, in rodents, glyphosate in urine is the much other hand, glyphosate concentration in urine of treated more relevant marker of exposure than AMPA in par- animals (0.480–2.280 mg/kg) resulted up to 100-fold ticular at doses that are equal or lower than the one used higher than the AMPA concentration and at least 500- in this pilot study (1.75 mg/kg bw/day). The evaluation fold higher than the chromatographic LQ (0.001 mg/kg). of different outcomes and endpoints of interest (i.e., Therefore, in order to assess exposure to glyphosate in pathology of target organs, molecular toxicity, genotoxi- rats, in particular at doses that are equal or lower than city, endocrine disrupting activities, microbiome, devel- the one used in this pilot study (1.75 mg/kg bw/day), opmental toxicity, etc.) is currently ongoing in the glyphosate appears to be the biomarker of choice. different partner laboratories of the project. The presence of negligible levels of glyphosate (0.003– Abbreviations 0.013 mg/kg), close to the chromatographic LQ (0.001 AMPA: Aminomethylphosphonic acid; CMCRC: Cesare Maltoni Cancer mg/kg), in some of the urine of the control groups might Research Center; EU: European Union; GBH: Glyphosate-based herbicides; GD: Gestational day; GM: Genetically modified; LC-MS/MS: Liquid reflect an ubiquitous environmental contamination at chromatography tandem mass spectrometry; LD: Lactating day; LQ: Limit of ultra-low doses of glyphosate, which is consistent with Quantification; MOG: Modified One-Generation study; NTP: National previous reports from other authors [21]. As the current Toxicology Program; PND: Post Natal Day; RI: Ramazzini Institute; SD: Sprague-Dawley; US ADI: United States Acceptable Daily Intake limit of quantitation of glyphosate in HPLC for pelleted animal feed is 0.050 mg/kg, this represents a technical Acknowledgements limiting factor for testing ultra-low doses of glyphosate. We thank the over 30,000 associates and volunteers of the Ramazzini Institute that made this pilot study possible through their commitment and As reported by a recent inter laboratory comparative study generosity. We thank the Municipality of Bologna, the Emilia-Romagna Re- on the quantitative determination of glyphosate at low gion, and the International Society of Doctors for Environment for organizing levels, caution should be taken when interpreting results if several events to promote this pilot study; “Coop Reno” and “Coopfond Fondo Mutualistico Legacoop” for supporting our research activity. the tested doses of glyphosate are close to the LQ of HPLC [47]. Funding It is noteworthy that the commercial formulation used This work was funded by Institution fund of the Ramazzini Institute, Bologna, Italy. in this study, Roundup Bioflow, was the representative formulated product recently evaluated for the renewal of Availability of data and materials the approval of glyphosate in EU and considered in the All raw data recorded and used during the current study are available from the corresponding author on reasonable request. European Food Safety Authority peer review (MON 52276) [48]. Authors’ contributions Our results seem particularly relevant in light of the All authors provided substantial contributions to the conception/design of the work, acquisition, analysis or interpretation of the data, revised the massive global burden of exposure to glyphosate, as shown manuscript critically, and approved the final version for submission. FM, SP, by the exponential increase in the last 20 years of the levels DM participated in the design of the study, performed the animal of glyphosate and AMPA measured in the urine of the experiments and sample collection, and drafted the manuscript. LB and LF performed the animal experiments and sample collection. FB supervised the general population in Germany [49]and in theUS[50]. study, participated in the design of the study and helped to draft the manuscript. MS, GG, GD, RM, AM, SL, JH, JC, MJP, PJL, helped to draft the Conclusion manuscript. All authors read and approved the final manuscript. We performed a pilot study on the health effects of gly- Ethics approval and consent to participate phosate and its formulation Roundup administered at N/A currently admitted doses (US ADI = 1.75 mg/kg bw/day) to SD rats. In this paper, we described the study design, Competing interests The authors declare that they have no competing interests. the first evaluation of in vivo parameters and the deter- mination of glyphosate and its major metabolite AMPA Publisher’sNote in urine. The treatment with either glyphosate or Springer Nature remains neutral with regard to jurisdictional claims in Roundup seemed to be overall well tolerated, consist- published maps and institutional affiliations. ently with previous experiments performed by the US Author details NTP [26]. Both glyphosate and Roundup exposure led Cesare Maltoni Cancer Research Center (CMCRC), Ramazzini Institute (RI), Via to comparable urinary concentrations of glyphosate and 2 Saliceto, 3, 40010 Bentivoglio, Bologna, Italy. Department of Agricultural AMPA with an increasing pattern of glyphosate excreted Sciences, University of Bologna, Viale Fanin 44, 40127 Bologna, Italy. Department of Veterinary Medical Sciences, University of Bologna, Via Tolara in urine in relation to the duration of treatment, indicat- di Sopra 50, 40064 Ozzano dell’Emilia, Bologna, Italy. Department of ing the systemic bioavailability of the active sub- Statistical Sciences, University of Bologna, Via Belle Arti 41, 40126 Bologna, stance and a possible mechanism of bioaccumulaton. Italy. Department of Food safety, Nutrition and Veterinary Public Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. The adjuvants and the other substances present in Department of Genetics and Genomic Sciences, Icahn School of Medicine at Roundup did not seem to exert a major effect on the ab- 7 Mount Sinai, 1425 Madison Ave, New York, NY 10029, USA. Department of sorption and excretion of glyphosate. 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Environmental HealthSpringer Journals

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