How well can in vitro data predict in vivo effects of chemicals? Rodent carcinogenicity as a case study.

A recent research article by the National Center for Computational Toxicology (NCCT) (Kleinstreuer et al., 2013), indicated that high throughput screening (HTS) data from assays linked to hallmarks and presumed pathways of carcinogenesis could be used to predict classification of pesticides as either (a) possible, probable or likely rodent carcinogens; or (b) not likely carcinogens or evidence of non-carcinogenicity. Using independently developed software to validate the computational results, we replicated the majority of the results reported. We also found that the prediction model correlating cancer pathway bioactivity scores with in vivo carcinogenic effects in rodents was not robust. A change of classification of a single chemical in the test set was capable of changing the overall study conclusion about the statistical significance of the correlation. Furthermore, in the subset of pesticide compounds used in model validation, the accuracy of prediction was no better than chance for about three quarters of the chemicals (those with fewer than 7 positive outcomes in HTS assays representing the 11 histopathological endpoints used in model development), suggesting that the prediction model was not adequate to predict cancer hazard for most of these chemicals. Although the utility of the model for humans is also unclear because a number of the rodent responses modeled (e.g., mouse liver tumors, rat thyroid tumors, rat testicular tumors, etc.) are not considered biologically relevant to human responses, the data examined imply the need for further research with HTS assays and improved models, which might help to predict classifications of in vivo carcinogenic responses in rodents for the pesticide considered, and thus reduce the need for testing in laboratory animals.

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. EPA's 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.

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.

Non-coplanar 2,2',3,3',4,4',5,5',6,6'-decachlorobiphenyl (PCB 209) did not induce cytochrome P450 enzyme activities in primary cultured rat hepatocytes, was not genotoxic, and did not exhibit endocrine-modulating activities.

2,2',3,3',4,4',5,5',6,6'-Decachlorobiphenyl (PCB 209) is a fully chlorinated, non-coplanar biphenyl. To demonstrate that PCB 209 is not likely to exhibit human health hazards common to coplanar PCBs it was tested for cytochrome P450 (P450) enzyme induction potentials, genetic toxicity, and endocrine-modulating activity. PCB 209 (dose from 0.005 to 5000 ng/mL) did not significantly induce P450 CYP1A, 2A, 2B, 3A, or 4A enzyme activities in primary cultured rat hepatocytes. In contrast, Aroclor 1260, a PCB mixture that contains approximately 60% chlorine by weight, showed significant induction of P450 CYP1A, 2A, 2B, and 3A within the same dose range. PCB 209 (dose from 100 to 5000 microg/plate) was negative in the bacterial mutagenicity (Ames) test in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 or in Eschericia coli strain WP2uvrA. PCB 209 (dose from 25 to 150 microg/mL) was also negative for forward mutations at the thymidine kinase (TK+/-) locus of L5178Y mouse lymphoma cells. The Ames and the mouse lymphoma assays were both conducted in the absence and presence of rat liver S9 fraction. PCB 209 (dose from 500 to 2000 mg/kg by single dose oral gavage) did not induce an increase in the frequency of micronuclei in polychromatic erythrocytes in mouse bone marrow in vivo. PCB 209 did not induce estrogenic effects when administered by gavage to ovariectomized adult female rats at 500 and 1000 mg/kg for 4 days, nor did it produce alterations consistent with endocrine-modulating activity in adult intact male rats when administered by gavage at 500 and 1000 mg/kg for 15 consecutive days.

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.

Tiered toxicity testing: evaluation of toxicity-based decision triggers for human health hazard characterization.

A set of biologically-based toxicity testing decision triggers was developed and analyzed within a tiered testing and decision-making framework for evaluating potential human health hazards and risks associated with chemical exposures. The proposed three-tiered toxicity testing approach starts from a base set of toxicity studies (acute toxicity, in vitro genetic toxicity, in vitro cytogenetics, repeat dose/subchronic toxicity, developmental toxicity, reproductive toxicity) and then uses the toxicity triggers to identify which specific additional tests are needed to adequately characterize a substance's hazard potential. The toxicity triggers were initially evaluated using published information for eight chemicals, representing diverse classes. A retrospective validation study was then conducted using seven chemicals which had completed the USEPA's Voluntary Children's Chemical Evaluation Program (VCCEP). The toxicity triggers were shown to identify appropriate higher tier tests and to be reasonably predictive of the results expected in higher tiered tests. Employing these toxicity triggers within a tiered testing framework could lead to a reduction in the number of laboratory animals without diminishing the degree of scientific certainty necessary for hazard evaluations. The toxicity triggers appear to be suitable for identifying which specific endpoints and tests warrant further evaluation, and which do not, and for documenting the scientific basis for such decisions.

Mortality among workers exposed to acrylonitrile in fiber production: an update.

OBJECTIVE: The investigation updates the mortality experience through 2002 for a cohort of workers exposed to acrylonitrile (AN). METHODS: Standardized mortality ratios (SMR) were estimated based on two reference populations: the US population and a regional employee population. Exposure-response analyses were conducted using Cox regression models for cumulative and mean intensity exposure measures. RESULTS: In the cohort of 2548 workers, 839 deaths have occurred with 91 deaths due to respiratory system cancer. Most standardized mortality ratio estimates are at or near no-effects levels. Hazard ratio (HR) estimates indicate no increased mortality risk for respiratory system cancer (adjusted HR = 0.96, 95% confidence interval: 0.74, 1.25). CONCLUSIONS: In summary, no mortality outcome of a priori interest, principally respiratory system cancer, is associated with increased AN exposure among fiber production workers over five decades of follow-up.

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.