A. Michael Kaplan

Evaluation of Tier I screening approaches for detecting endocrine-active compounds (EACs).

In 1996, Congress passed legislation requiring the U.S. Environmental Protection Agency (EPA) to implement screening/testing strategies for endocrine-active compounds (EACs). In response, EPA convened the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to advise the agency on a strategy to screen and test xenobiotics for endocrine disruption. EDSTAC completed their charter in 1998 by recommending a tiered screening and testing scheme to evaluate compounds for their potential to act as agonists or antagonists to the estrogen or androgen receptors, steroid biosynthesis inhibitors, or their ability to alter thyroid function. For Tier I, the EDSTAC-recommended screening battery comprised eight different assays, but EDSTAC also proposed two alternative batteries that were deemed worthy of further evaluation. The challenge currently confronting EPA is to choose among the Tier I screening options and then to standardize protocols, validate the assays, and determine the criteria for judging a compound as positive or negative in the battery. The purpose of the current review is to: (1) provide an overview of the three EDSTAC options, (2) evaluate the data currently available for the individual assays of the three EDSTAC options and discuss the strengths and limitations of each, and (3) provide a final recommendation for a Tier I screen based on the experiences of the authors who have used all of the individual assays under consideration by EDSTAC. The goal of this report is not to provide an exhaustive historical review of each assay, but rather to summarize some of the more relevant data from available published reports as it relates to current proposed study designs for those particular assays. Based on the current data, a Tier I screening battery consisting of in vitro receptor binding assays, a 3-day uterotrophic assay, and a 15-day intact male assay are recommended as the preferred approach on which future validation efforts should be focused. This screening approach is a mode-of-action screen that will identify specific types of endocrine activity. Because it utilizes many endpoints from the same test animals (i.e., it integrates), it is the most cost-effective and efficient option in terms of animal usage. The mode-of-action screening approach advances scientific understanding and is preferred over other options based on apical tests, as these essentially are reproductive effects screens that are not necessarily specific for endocrine activity. Because Tier II tests include the critical apical endpoints used in the pubertal models, a mode-of-action approach provides complementary rather than redundant data. By identifying the potential mode of action, critical endpoints can be included in Tier II studies that will be used to define dose-response curves and no observed adverse effect levels (NOAELs)/no observed effect levels (NOELs) for the compound.

Toxicity studies with methyl N-[[(methylamino)carbonyl]oxy]-ethanimidothioate.

Abstract Methomyl exhibited a low order of chronic oral toxicity even though it exhibited high acute oral toxicity. When applied dermally to rabbits, aqueous suspensions of methomyl exhibited low acute toxicity. Subacute oral tests in rats indicated no cumulative oral toxicity, and plasma cholinesterase activity for test rats was similar to that of the controls. Also, when methomyl was applied to intact skin of rabbits, 15 daily treatments produced no cumulative effects. Fish residue studies showed no build-up of methomyl in the tissues and no signs of a compound-related effect were noted in a 30-day feeding study in Japanese quail. Methomyl fed to pregnant New Zealand white rabbits on Days 8–16 of gestation at dietary levels of 0, 50, and 100 ppm was not teratogenic. In a 3-generation, 6-litter reproduction study with rats, at the same dietary levels, methomyl had no adverse effects upon reproduction and lactation performance; no pathologic changes were observed in weanling pups of the F3b generation. The oral administration of 28 mg/kg to adult hens did not result in demyelination of the sciatic nerve. Rats of both sexes were fed nutritionally complete diets containing 0, 10, 50, 125–500, and 250 ppm of methomyl in a 90-day feeding study and 0, 50, 100, 200, and 400 ppm of methomyl in a 22-month feeding study. The weight gain for the high-level males was significantly lower than that of the controls. No clinical, hematological, biochemical, urinary, or pathologic evidence of toxicity was observed at 90 days. However, in the 22-month study, decreased hemoglobin values were noted in the two higher level female test groups. A significantly higher testis/body weight ratio was observed in the high-level males. Histopathologic alterations were observed in the kidneys of male and female rats receiving 400 ppm and the spleens of the female rats receiving 200 and 400 ppm of methomyl. Beagle dogs of both sexes, fed nutritionally complete diets containing 0, 50, 100, and 400 ppm of methomyl in 90-day and 2-year feeding studies, showed no nutritional, clinical, urinary, or biochemical evidence of toxicity; in the latter study, an additional dietary level of 1000 ppm caused some clinical signs of toxicity and mortality. Similar to the findings in the 22-month feeding study in rats, histopathologic changes were observed after 2 years in the kidney and spleen as well as in the liver at the two higher feeding levels. In addition, dogs receiving the high-level diet showed a compound-related anemia. There was no evidence of carcinogenicity at any levels in either rats or dogs. Based on the results of the long-term studies, the “no-effect” level was determined for rats and dogs to be 100 ppm.

An enhanced tiered toxicity testing framework with triggers for assessing hazards and risks of commodity chemicals

This paper presents an enhanced integrated testing framework based on tiered testing and endpoint-specific decision triggers envisioned for application to commodity chemical safety assessments. The framework has two tiers in which exposure information can be integrated with hazard data at each Tier. Tier 1 tests are used to screen chemicals for major toxic effects (i.e., acute toxicity potential, target organs of repeat dose toxicity, genotoxicity potential, neurotoxicity potential, reproductive toxicity potential, immunotoxicity potential, and developmental toxicity potential), and to direct planning for more complex and targeted testing in Tier 2. The proposed decision triggers coupled with information on use and potential for exposure allow for scientifically-based decisions to be made about further testing in Tier 2, indicating which specific endpoints and tests warrant further evaluation, and which do not. The testing framework addresses risks to humans during all stages of development and provides data relevant to assessing hazards to sensitive subpopulations, such as infants and children. The REACH program in Europe and TSCA in the United States have led to an increased focus on development of hazard and risk information for chemicals used in industrial processes and consumer products. This framework and its toxicity decision triggers will allow for scientifically justified evaluation of chemicals that is comprehensive in terms of hazard screening, focuses resources on the specific complex tests that are most important for hazard characterization, and minimizes the use of animals.

Toxicity studies with 5-bromo-3-sec-butyl-6-methyluracil.

Abstract The herbicide, 5-bromo-3- sec -butyl-6-methyluracil (bromacil) exhibited a low order of acute toxicity as evaluated by oral, dermal, and inhalation studies in mammals and by fish and wildlife studies. Bromacil fed to pregnant New Zealand white rabbits on days 8–16 of gestation at dietary levels of 0, 50, and 250 ppm was not teratogenic. In a three-generation six-litter reproduction study with rats, a dietary level of 250 ppm had no adverse effects upon reproduction and lactation performance; no pathologic changes were observed in weanling pups of the F 3b generation. Rats of both sexes were fed nutritionally complete diets containing 0, 50, 250, and 1250 ppm bromacil for 2 yr. Except for a lower rate of weight gain, a slightly decreased food intake and a slightly lower food efficiency among the female rats that received 1250 ppm bromacil, there was no nutritional, clinical, hematologic, urinary, or biochemical evidence of toxicity, nor any evidence of carcinogenicity; there was, however, a suggestion of slight hyperplasia in the thyroids of animals given the highest dietary level. Beagle dogs of both sexes, 1–2 yr of age, fed nutritionally complete diets containing 0, 50, 250, and 1250 ppm bromacil for 2 yr, showed no nutritional, clinical, hematological, urinary, biochemical, or pathologic evidence of toxicity.

Results of the negative control chemical allyl alcohol in the 15-day intact adult male rat screening assay for endocrine activity.

Development, standardization, and validation of methods to assess the potential of chemicals to disrupt hormonal homeostasis have been the focus of considerable research efforts over the past 10 years. As part of our validation effort, we evaluated the specificity of the 15-day intact adult male rat assay, using a negative control chemical, allyl alcohol, a known hepatotoxicant that was not expected to induce endocrine effects. Male rats were dosed for 15 days via oral gavage with 0, 10, 30, 40, or 50 mg/kg/day allyl alcohol. The endpoints evaluated included final body and organ weights, serum hormone concentrations, and a limited histopathology assessment. No mortality or adverse clinical signs were observed. Mean final body weight for rats in the 50-mg/kg/day dose group was decreased to 90% of control. Mean relative liver weights were increased at 40 and 50 mg/kg/day (115% and 117% of control, respectively). Serum testosterone and DHT concentrations were statistically significantly decreased at 50 mg/kg/day (72% of control). Serum prolactin concentrations were statistically significantly decreased at 40 mg/kg/day (58% of control), but not at 50 mg/kg/day. There were no effects on the other endpoints evaluated. Consistent with previous guidance for interpreting the 15-day intact adult male rat assay, histological and weight changes of target organs were given a higher weight-of-evidence than changes in serum hormone concentrations alone. Therefore, with only minimal changes in serum hormone concentrations and no effects on organ weights or microscopic alterations, the results of allyl alcohol in the 15-day intact adult male rat assay were considered negative and consistent with the predicted results.

Chronic Toxicity, Reproductive, and Teratogenic Studies of Hexazinone

Hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine 2,4(1H,3H)-dione; CAS 51235-04-2] was tested for oral toxicity in rats (both 90-day and 2-year feeding studies), mice (8-week and 2-year feeding studies), and dogs (90-day feeding study). The teratogenic potential was evaluated in rabbits and rats and functional reproductive capacity was studied in rats. Ninety-day feeding of up to 1000 ppm produced no signs of a toxic response in rats. Rats fed 5000 ppm had growth curves slightly inferior to those of the controls as the only detectable difference. Extending the feeding period to 2 years produced decreased body weights in males fed 2500 ppm (top level tested) and in females fed either 1000 or 2500 ppm. All other indices of response, including the type and distribution of tumors, were similar in the test and control rats with the no-effect level being 200 ppm. Eight-week feeding of up to 10,000 ppm in mice produced increased liver weight only at the highest level without any other changes. Two-year feeding of either 200, 2500, or 10,000 ppm resulted in sloughing of the distal tip of the tail and increased liver weights among mice fed 10,000 ppm. Hypertrophy of centrilobular hepatocytes and hyperplasic nodules were increased in mice fed either 2500 or 10,000 ppm. No evidence of a tumorigenic response was evident. The no-effect level was 200 ppm. Dogs fed 5000 ppm for 90 days had decreased rate of body weight gain with clinical enzyme changes suggestive of liver damage. Microscopic examination of the liver failed to reveal any alterations and dogs fed either 200 or 1000 ppm were indistinguishable from controls. The no-effect level in the dog was 1000 ppm. No evidence of a teratogenic response was seen in either rats or rabbits and reproduction capacity in rats fed up to 2500 ppm for three generations was unaffected.

Challenges in using the ToxRefDB as a resource for toxicity prediction modeling

Developing and evaluating toxicity prediction models requires selection and use of datasets of known positive and negative agents for the endpoint(s) of interest. EPAs Toxicity Reference Database (ToxRefDB) is a publicly available dataset containing detailed study and effect information on more than 400 chemicals, and it has been used by EPA researchers to develop toxicity prediction models. During an initial evaluation of reproductive toxicity, however, limitations were uncovered in applying data from ToxRefDB that involved interpretation of toxicity effects and designation of toxicity endpoints, core attributes of the database that are critical to its use. These limitations for reproductive toxicity were found to be related, at least in part, to challenges faced in (1) evaluating the source of the original study data (EPA Data Evaluation Records (DERs)) for input into ToxRefDB and (2) interpretation of the biological significance of responses. These limitations of the ToxRefDB have important implications for the wider use of the database as it currently exists. Our results point to a need for improvements to the existing ToxRefDB and/or for researchers to independently evaluate, assign and verify positive or negative designations to data from ToxRefDB before use in development or validation of prediction models or testing frameworks.

Interlaboratory Study Comparison of the 15-Day Intact Adult Male Rat Screening Assay: Evaluation of an Antithyroid Chemical and a Negative Control Chemical

Validation of the 15-day intact adult male rat screening assay (IAMRSA), an endocrine activity screen, was extended beyond the 28 substances evaluated to date. Two independent laboratories evaluated specificity using allyl alcohol (AA), a putative negative control, and DE-71 (technical grade pentabromodiphenyl ether) for comparison with previous pubertal assays that demonstrated thyroid effects. Male rats (15/group) were gavaged daily with AA (0, 10, 30, or 40 mg/kg/day) or DE-71 (0, 3, 30, or 60 mg/kg/day) for 15 days. Body and organ weights and serum hormone concentrations were measured, and a limited histopathological assessment was conducted. AA results were considered negative at doses that did not exceed the maximum tolerated dose (MTD); effects reported were dose-related decreases in weight gain, increased liver weights and, although the pattern varied across studies, alterations in some androgen-sensitive endpoints in the high-dose where the maximum tolerated dose was exceeded. In the DE-71 studies, dose-dependent increases in liver weights (consistent with hepatic enzyme induction), decreases in tri-iodothyronine and thyroxine, concomitant thyroid stimulating hormone increases were observed and one laboratory reported histopathological thyroid changes in mid- and high-dose groups, and the other increased thyroid weights. For DE-71, the IAMRSA was comparable in sensitivity to the pubertal assays. Overall, the specificity and sensitivity of the IAMRSA for deployment in an endocrine screening battery are supported. However, differentiating primary endocrine-mediated effects from secondary effects caused by systemic toxicity will be challenging, emphasizing the need to utilize a battery of assays and a weight of evidence approach when evaluating the potential endocrine activity of chemicals.