Toxicology and Industrial Health

Lazarow, Paul B.

doi: 10.1177/074823378700300202pmid: 3303444

Barber, Eugene D. ;Astill, Bernard D. ;Moran, Elizabeth J. ;Schneider, Bernard F. ;Gray, Tim J.B. ;Lake, Brian G. ;Evans, John G.

doi: 10.1177/074823378700300203pmid: 3617071

Seven phthalate esters, representing a variety of chain lengths and degrees of branching in the alcohol moiety, were tested for their ability to produce peroxisome proliferation in the Fischer 344 rat. Di(2-ethylhexyl)adipate (DEHA) was tested using the same protocol and di(2-ethylhexyl)phthalate (DEHP) was run with each study as an internal control. Each ester was administered in the feed for a period of 21 days at levels of 2.5%, 1.2% and either 0.6% or 0.3%. DEHP and DEHA were also fed at levels of 0.1% and 0.01%. The animals were sacrificed and samples of liver were prepared for both light and electron microscopy. Serum samples were assayed for both triglyceride and cholesterol. The remaining portion of the liver was homogenized and assayed for cyanide-insensitive palmitoyl-CoA oxidation, lauric acid 11-hydroxylase and lauric acid 12-hydroxylase. The results show that there is approximately a ten-fold difference between the weakest and strongest esters in terms of their potency to induce changes in relative liver weight and in several of the biochemical parameters. In general, the longer chain esters were more potent than the shorter chain ones, and branched chain esters seemed more potent than straight. Several statistical analyses of the dataset have been performed and all render similar conehcsions. The results of one of these evaluations are presented elsewhere in this volume (Lin, 1987).

Lin, Lawrence I-Kuei

doi: 10.1177/074823378700300204pmid: 3617068

This paper contains statistical evaluations of the effect of 9 different plasticizers on rat hepatic peroxisome proliferation. There were 9 21-day feeding studies with one plasticizer in each study. The plasticizers are: di(2-ethylhexyl)phthalate (DEHP), butyl benzyl phthalate (BBP), 711 phthalate (711P), di(n-butyl)phthalate (DBP), di(undecyl)phthalate (DUP), di(isodecyl)phthalate (DIDP), di(isononyl)phthalate (DINP), 610 phthalate (610P), and di(ethylhexyl)adipate (DEHA). For each plasticizer, several dosage (as % of diet) groups, plus a negative control group (0%) and a reference control group (1.2% DEHP) were included. There were 5 males and 5 females, approximately 5 weeks old, per group per plasticizer, for a total of 470 animals. For each animal, body weight and food intake ivere measured prior to treatment and twice a week during the treatment period. Animals were necropsied at the end of the 21-day treatment period. Selected organs and blood samples of each animal were collected for measuring hepatic enzymes, proteins, fat stains, organ weights and serum fats. The endpoints included electron microscopy examination of liver peroxisome proliferation and liver histological abnormalities. The statistical evaluation utilized a multivariate approach that condensed a cornplex phenornenon into certain meaningful and simple endpoints for quantitative comparisons. The dose of each plasticizer that would protect 99% and 99.9% of rats against peroxisome proliferation (xx% statistically predicted dose, or xx% SPD) was estimated. Marked differences were seen in the ability of the 9 test conipoziilds to cause hepatic peroxisome proliferation in rats. The graphic ranking of overall potency is, in order from most potent to least potent: DEHP, DIDP, DINP, DBP, DEHA, DUP, BBP, 711P and 610P. The ranking in terms of 99.9% SPD is: DEHP, DINP, DIDP, DBP, 610P, DUP, DBP, 711P and DEHA. It was also found that the histological findings of reduced basophilia or increased eosinophilia were highly correlated with peroxisome proliferation (r = 0.94). The other histological findings were incidental observations for specific plasticizers.

Hodgson, John R.

doi: 10.1177/074823378700300205pmid: 3617069

The results of these studies demonstrate that Tris(2-ethylhexyl)trimellitate (TOTM) produces the same spectrum of morphological and biochemical changes in the rat liver as di(2-ethylhexyl)phthalate (DEHP). TOTM, however, was much less potent in its action, witlt a dietary level of 2.0%, causing less peroxisome proliferation and enzyme induction than 0.67% DEHP. Also, these studies show that the level of peroxisome induction in rats given TOTM is less than in those receiving a metabolically equivalent dose of 2-ethylhexanol (2-EH). Furthermore, on a molar basis, effects were lower than with DEHP. A "monoester effect" of the kind attributed to MEHP, a metabolite of DEHP, was not seen with TOTM.

Topping, Douglas C. ;Ford, Graham P. ;Evans, John G. ;Lake, Brian G. ;O'Donoghue, John L. ;JR, Haines B. Lockhart,

doi: 10.1177/074823378700300206pmid: 3617070

Groups of five male and five female rats were fed diets containing from 0% to 2.5% di(2-ethylhexyl)terephthalate (DEHT) or 1.2% di(2-ethylhexyl)phthalate (DEHP) for 21 days. Feed consumption and body weight gains were collected and, at study termination, animals were examined for alterations in body weight, differences in serum lipids, changes in the activities of certain enzymes associated with fat metabolism, and proliferatiort of hepatic peroxisomes. Feed consumption and weight gain were greatly decreased in DEHT-fed animals only at 2.5%. No biologically significant alterations in absolute liver weight occurred with DEHT. Relative liver weights were increased at 2.5% in both sexes and at 1.0% and 1.2% in females. The alterations were due wholly to decreased terminal body weights. Serum triglyceride and cholesterol levels were not found useful in interpreting the effects of DEHT Cyanide-insensitive palmitoyl CoA oxidation and lauric acid 11- and 12-hydroxylation were increased in animals cortsuming 2.5%, but no lower levels of DEHT. Induction of hepatic peroxisomes did not occur at 1.2% DEHT. Interpretation of minimal peroxisomal effects with 2.5% DEHT ivas confounded by reduced feed consumption. Slight decreases in weight gain occurred in males consuming the 1.2% DEHP diet, but differences were minor relative to effects observed at 2.5% DEHT. Results with DEHP contrasted with those obtained with DEHT. Absolute and relative liver weights, activities of enzymes of lipid metabolism, and peroxisorne content were all significantly increased at 1.2% DEHP. Reduction of feed intake was implicated in the effects observed at 2.5% DEHT, since the amount of DEHT consumed by 2.5% animals ivas only 1.4 times as much as by 1.2% animals. A possible explanation for the observed differences between DEHP and DEHT was related to the results of a metabolic fate study on DEHT. Metabolism of DEHT by the rat appears to occur via rapid hydrolysis of both ester linkages to give two moles of 2-ethylhexanol and one mole of terephthalic acid. Although 2-ethylhexanol has been shown to induce peroxisome proliferation, it appears to be less active in this respect than the monoester of DEHP. The relatively smaller amounts of monoester produced during the metabolism of DEHT may explain the differences seen in these experiments.

Hammond, Bruce G. ;Levinskas, George J. ;Robinson, Ellen C. ;Johannsen, Frederick R.

doi: 10.1177/074823378700300207pmid: 3617072

This review compares the subchronic toxicity of butyl benzyl phthalate (BBP) across several species. Data from the published literature as well as previously unpublished studies sponsored by Monsanto are presented. BBP-induced toxicity occurs only at relatively high levels of exposure and is dependent on the species, age and strain of test animals used. These factors should be considered in extrapolating findings from animal toxicology studies to humans when assessing the safety of BBP.

Melnick, Ronald L. ;Morrissey, Richard E. ;Tomaszewski, Konrad E.

doi: 10.1177/074823378700300208pmid: 3617073

In a 2-year feed study previously reported by the National Toxicology Program (NTP), the plasticizer di(2-ethylhexyl)phthalate (DEHP) was found to produce increased incidences of hepatocellular neoplasms in both sexes of Fischer 344 (F344) rats and B6C3F1 mice. Further studies by the NTP on this chemical have investigated its genotoxicity, dermal absorption, reproductive and developmental toxicity, and biochemical mechanism of action. DEHP was not mutagenic in Salmonella typhimurium (strains TA98, TA100, TA1535 or TA1537), in L5178Y mouse lymphoma cells, or in Drosophila melanogaster. DEHP did not induce chromosomal aberrations, but did cause a marginal dose-related increase in sister chromatid exchanges in CHO cells. In a dermal absorption study, DEHP was not absorbed well through the skin of F344 rats. In a fertility assessment study, DEHP was shown to be a reproductive toxicant in both male and female CD-1 mice. The teratogenic potential of DEHP was evaluated in F344 rats and CD-1 mice. In the rat study, there were no significant differences in percent fetuses malformed between control and treatment groups, even at dose levels (1.0, 1.5 and 2.0%) which produced significant maternal and fetal toxicity. In the mouse study, the incidence of fetuses with malformations was significantly increased at dose levels which produced maternal and/or fetal toxicity (0.10 and 0.15%), and at a dose level (0.05%) which did not cause maternal or fetal toxicity. The no-observed effect level for developmental toxicity in mice was 0.025% DEHP. Kinetic data on the rates of formation of H2O2 by peroxisomal palmitoyl CoA oxidase, and of degradation of H2O2 by catalase, was used to estimate in vitro steady-state H2O2 concentrations during peroxisofnal oxidation of palmitoyl CoA. Increases in steady-state H 2O2 in liver homogenates of rats treated with DEHP, di(2-ethylhexyl)adipate, or nafenopin, a hypolipidemic drug, correlated well with the carcinogenic potential of these cheniicals determined in previous carcinogenicity studies, and are consistent with but not definitive evidence for the involvement of peroxisome proliferation in the hepatocarcinogenesis of these compounds.

Michalopoulos, George K. ;Eckl, Peter M. ;Cruise, Jennifer L. ;Novicki, Deborra L. ;Jirtle, Randy L.

doi: 10.1177/074823378700300209pmid: 3303445

Stimulation of DNA synthesis by peroxisonte proliferators, including DEHP, should be viewed differently from the stimulation of DNA synthesis by xenobiotic chemicals which stimulate restorative hyperplasia after hepatic necrosis induced by the toxicity of the chemical. The emerging picture of the control mechanisms for hepatocyte proliferation suggests that rather few and distinct factors are involved. The stimulation of DNA synthesis by peroxisome proliferators should be examined in the context of the effects of these factors. Comparisons with other xenobiotics show that induction of DNA synthesis at rates comparable to those of peroxisome proliferators is not sufficient to explain the rates of carcinogenicity associated with peroxisonie proliferators. These considerations lead to the conclusion that although DNA synthesis enhances the incidence of neoplasia, it should not be viewed as a complete carcinogen, nor should it be considered as resulting in initiation at rates that can explain the carcinogenic potency of compounds such as peroxisome proliferators.

Butterworth, Byron E. ;Loury, David J. ;Smith-Oliver, Tracey ;Cattley, Russell C.

doi: 10.1177/074823378700300210pmid: 3303446

Di(2-ethylhexyl)phthalate (DEHP) is a widely used plasticizing agent resulting in substantial human exposure and environmental contamination. In a chronic bioassay, high doses of DEHP induced hepatocellular carcinomas in female Fischer-344 rats and male and female B6C3F1 mice. Thus, there is considerable concern as to the species specificity, mechanism of action, and human risk assessment of DEHP. DEHP belongs to a class of agents described as hypolipidemic hepatocarcinogens. These chemicals share the ability to induce hepatic peroxisomal proliferation and range from very weak to very potent hepatocarcinogens. Unlike most identified carcinogens, the hypolipidemic carcinogens lack DNA reactivity in sensitive cell culture systems such as the Ames test. It has been proposed that active oxygen radicals, produced as a result of peroxisomal proliferation, induce DNA damage. While this is an attractive hypothesis, no genotoxic activity has been observed in hepatocytes with peroxisomal proliferation in treated animals. Another biological activity shared by this class of compounds is their ability to stimulate liver growth or hyperplasia. This additive hyperplasia results from direct mitogenic stimulation rather than regenerative growth following liver toxicity. This hyperplasia can be dramatic, with liver to body weight ratios from treated animals reaching two to three times normal. The degree of induced hyperplasia correlates well with the carcinogenic potency of these agents, whereas genotoxicity does not correlate at all. Increased cellular growth may result in spontaneous mutational events or proniotional effects. While some feedback mechanism eventually inhibits liver growth, it is possible that key genes related to the regulation of cellular growth and cancer remain stimulated during continued administration of the chemical. Thus, determination of hyperplastic activity represents an attractive first-step approach to the short-term detection and study of the mode of action of nongenotoxic carcinogens.

Popp, James A. ;Garvey, Linda K. ;Cattley, Russell C.

doi: 10.1177/074823378700300211pmid: 3303447

In a study sponsored by the National Toxicology Program, di(2-ethylhexyl)phthalate (DEHP) fed in the diet at 1.2% significantly increased the incidence of female rats with hepatocellular carcinomas. Extensive evaluation of DEHP for carcinogenicity has yielded negative results. The present investigations were designed to elucidate the mechanism of DEHP hepatocarcinogenesis under the conditions of the original bioassay. Short-term studies designed to evaluate the promoting capability of DEHP, when administered after initiation, were negative when livers of female Fischer-344 rats were evaluated using multiple histochemical stains to identify foci of cellular alteration. Two different protocols were used to evaluate the initiating potential of DEHP in the liver using histochemically defined foci as the endpoint. In both experiments the results were negative. Chronic exposure to DEHP at 1.2% in the diet for 2 years resulted in elevation of hepatic peroxisomal enzymes while DNA replication, an indication of cell proliferation, was not affected in hepatocytes. The number of foci was not elevated in the DEHP group compared to the controls, even though a low incidence of rats with liver tumors occurred in the treated group. The results of this series, as well as other published results, suggest that DEHP and other peroxisomal proliferating chemicals have unique effects on the development of hepatic neoplasms. The absence of altered foci after chronic administration or in initiation-promotion protocols distinguishes DEHP and perhaps other peroxisomal proliferating chemicals from both classic liver carcinogens and promoters.