Rodent Uterotrophic Bioassay Research Articles

The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay With Metabolic Competence.

The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances. A potential limitation to the use of in vitro assay data in regulatory decision-making is the lack of coverage for xenobiotic metabolic processes. Both hepatic- and peripheral-tissue metabolism can yield metabolites that exhibit greater activity than the parent compound (bioactivation) or are inactive (bioinactivation) for a given biological target. Interpretation of biological effect data for both putative endocrine active substances, as well as other chemicals, screened in HTS assays may benefit from the addition of xenobiotic metabolic capabilities to decrease the uncertainty in predicting potential hazards to human health. The objective of this study was to develop an approach to retrofit existing HTS assays with hepatic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform encapsulates hepatic S9 fractions in alginate microspheres attached to 96-well peg lids. Functional characterization across a panel of reference substrates for phase I cytochrome P450 enzymes revealed substrate depletion with expected metabolite accumulation. Performance of the AIME method in the VM7Luc estrogen receptor transactivation assay was evaluated across 15 reference chemicals and 48 test chemicals that yield metabolites previously identified as estrogen receptor active or inactive. The results demonstrate the utility of applying the AIME method for identification of false-positive and false-negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with the rodent uterotrophic bioassay. Integration of the AIME metabolism method may prove useful for future biochemical and cell-based HTS applications.

Evaluation and Optimization of Pharmacokinetic Models for in Vitro to in Vivo Extrapolation of Estrogenic Activity for Environmental Chemicals.

Background:To effectively incorporate in vitro data into regulatory use, confidence must be established in the quantitative extrapolation of in vitro activity to relevant end points in animals or humans.Objective:Our goal was to evaluate and optimize in vitro to in vivo extrapolation (IVIVE) approaches using in vitro estrogen receptor (ER) activity to predict estrogenic effects measured in rodent uterotrophic studies.Methods:We evaluated three pharmacokinetic (PK) models with varying complexities to extrapolate in vitro to in vivo dosimetry for a group of 29 ER agonists, using data from validated in vitro [U.S. Environmental Protection Agency (U.S. EPA) ToxCast™ ER model] and in vivo (uterotrophic) methods. In vitro activity values were adjusted using mass-balance equations to estimate intracellular exposure via an enrichment factor (EF), and steady-state model calculations were adjusted using fraction of unbound chemical in the plasma () to approximate bioavailability. Accuracy of each model-adjustment combination was assessed by comparing model predictions with lowest effect levels (LELs) from guideline uterotrophic studies.Results:We found little difference in model predictive performance based on complexity or route-specific modifications. Simple adjustments, applied to account for in vitro intracellular exposure (EF) or chemical bioavailability (), resulted in significant improvements in the predictive performance of all models.Conclusion:Computational IVIVE approaches accurately estimate chemical exposure levels that elicit positive responses in the rodent uterotrophic bioassay. The simplest model had the best overall performance for predicting both oral (PPK_EF) and injection (PPK_) LELs from guideline uterotrophic studies, is freely available, and can be parameterized entirely using freely available in silico tools. https://doi.org/10.1289/EHP1655

A Curated Database of Rodent Uterotrophic Bioactivity

Background:Novel in vitro methods are being developed to identify chemicals that may interfere with estrogen receptor (ER) signaling, but the results are difficult to put into biological context because of reliance on reference chemicals established using results from other in vitro assays and because of the lack of high-quality in vivo reference data. The Organisation for Economic Co-operation and Development (OECD)-validated rodent uterotrophic bioassay is considered the “gold standard” for identifying potential ER agonists.Objectives:We performed a comprehensive literature review to identify and evaluate data from uterotrophic studies and to analyze study variability.Methods:We reviewed 670 articles with results from 2,615 uterotrophic bioassays using 235 unique chemicals. Study descriptors, such as species/strain, route of administration, dosing regimen, lowest effect level, and test outcome, were captured in a database of uterotrophic results. Studies were assessed for adherence to six criteria that were based on uterotrophic regulatory test guidelines. Studies meeting all six criteria (458 bioassays on 118 unique chemicals) were considered guideline-like (GL) and were subsequently analyzed.Results:The immature rat model was used for 76% of the GL studies. Active outcomes were more prevalent across rat models (74% active) than across mouse models (36% active). Of the 70 chemicals with at least two GL studies, 18 (26%) had discordant outcomes and were classified as both active and inactive. Many discordant results were attributable to differences in study design (e.g., injection vs. oral dosing).Conclusions:This uterotrophic database provides a valuable resource for understanding in vivo outcome variability and for evaluating the performance of in vitro assays that measure estrogenic activity.Citation:Kleinstreuer NC, Ceger PC, Allen DG, Strickland J, Chang X, Hamm JT, Casey WM. 2016. A curated database of rodent uterotrophic bioactivity. Environ Health Perspect 124:556–562; http://dx.doi.org/10.1289/ehp.1510183

Bayesian nonparametric centered random effects models with variable selection

In a linear mixed effects model, it is common practice to assume that the random effects follow a parametric distribution such as a normal distribution with mean zero. However, in the case of variable selection, substantial violation of the normality assumption can potentially impact the subset selection and result in poor interpretation and even incorrect results. In nonparametric random effects models, the random effects generally have a nonzero mean, which causes an identifiability problem for the fixed effects that are paired with the random effects. In this article, we focus on a Bayesian method for variable selection. We characterize the subject-specific random effects nonparametrically with a Dirichlet process and resolve the bias simultaneously. In particular, we propose flexible modeling of the conditional distribution of the random effects with changes across the predictor space. The approach is implemented using a stochastic search Gibbs sampler to identify subsets of fixed effects and random effects to be included in the model. Simulations are provided to evaluate and compare the performance of our approach to the existing ones. We then apply the new approach to a real data example, cross-country and interlaboratory rodent uterotrophic bioassay.

Estrogen-Like Effects of Cadmium in Vivo Do Not Appear to be Mediated via the Classical Estrogen Receptor Transcriptional Pathway

BackgroundCadmium (Cd), a ubiquitous food contaminant, has been proposed to be an endocrine disruptor by inducing estrogenic responses in vivo. Several in vitro studies suggested that these effects are mediated via estrogen receptors (ERs).ObjectiveWe performed this study to clarify whether Cd-induced effects in vivo are mediated via classical ER signaling through estrogen responsive element (ERE)-regulated genes or if other signaling pathways are involved.MethodsWe investigated the estrogenic effects of cadmium chloride (CdCl2) exposure in vivo by applying the Organisation for Economic Co-operation and Development (OECD) rodent uterotrophic bioassay to transgenic ERE-luciferase reporter mice. Immature female mice were injected subcutaneously with CdCl2 (5, 50, or 500 μg/kg body weight) or with 17α-ethinylestradiol (EE2) on 3 consecutive days. We examined uterine weight and histology, vaginal opening, body and organ weights, Cd tissue retention, activation of mitogen-activated protein kinase (MAPK) pathways, and ERE-dependent luciferase expression.ResultsCdCl2 increased the height of the uterine luminal epithelium in a dose-dependent manner without increasing the uterine wet weight, altering the timing of vaginal opening, or affecting the luciferase activity in reproductive or nonreproductive organs. However, we observed changes in the phosphorylation of mouse double minute 2 oncoprotein (Mdm2) and extracellular signal-regulated kinase (Erk1/2) in the liver after CdCl2 exposure. As we expected, EE2 advanced vaginal opening and increased uterine epithelial height, uterine wet weight, and luciferase activity in various tissues.ConclusionOur data suggest that Cd exposure induces a limited spectrum of estrogenic responses in vivo and that, in certain targets, effects of Cd might not be mediated via classical ER signaling through ERE-regulated genes.

Estrogenic effects in vitro and in vivo of the fungicide fenarimol

The fungicide fenarimol has the potential to induce endocrine disrupting effects via several mechanisms since it possesses both estrogenic and antiandrogenic activity and inhibits aromatase activity in cell culture studies. Hence, the integrated response of fenarimol in vivo is not easy to predict. In this study, we demonstrate that fenarimol is also estrogenic in vivo, causing significantly increased uterine weight in ovariectomized female rats. In addition, mRNA levels of the estrogen responsive gene lactoferrin (LF) were decreased in uteri, serum FSH levels were increased, and T3 levels decreased in fenarimol-treated animals. To our knowledge, only two other pesticides ( o, p-DDT and methoxychlor) have previously been reported to induce an estrogenic response in the rodent uterotrophic bioassay. A pronounced xenoestrogenicity in serum samples from rats treated with fenarimol and estradiol benzoate (E2B) separately or in combination was observed, demonstrating the usefulness of this approach for estimating the integrated internal exposure to xenoestrogens. The MCF-7 cell proliferation assay was used to investigate further the dose–response curves for the estrogenic, antiestrogenic, and aromatase inhibiting properties of fenarimol in vitro. The results indicates that fenarimol exhibits a dual effect being aromatase inhibitor at low concentrations and estrogenic at higher concentrations.

Conflicts of Interest: Owens’ Response

To clarify comments from Goozner and the Center for Science in the Public Interest (CSPI), I would like to provide the pertinent facts on my compliance with EHP’s policy on competing financial interest disclosure. First, I was on loan or “seconded” from Procter & Gamble (PG the current manager, Andrew Wagner; and their manager, Robert Visser, as references for these points. Second, in this approximate period, I was a corresponding or contributing author on 11 relevant publications on assays that were either undergoing formal validation at the OECD or were being developed along similar lines for endocrine mechanisms (Ashby et al. 2002a, 2002b, 2003, 2004; Fang et al. 2003; Kanno et al. 2001, 2003a, 2003b; Owens and Ashby 2002; Owens and Koeter 2003; Owens et al. 2003; Yamasaki et al. 2003). In all cases but one, my P&G address was used. Third, the one exception involved the article questioned by Goozner (Yamasaki et al. 2003). Kanji Yamasaki of the Chemical Evaluation and Research Institute in Japan was the corresponding author in this case. Because work on this manuscript (Yamasaki et al. 2003) occurred while I was at the OECD, Yamasaki used the OECD affiliation when submitting the manuscript to EHP. I simply did not notice the affiliation. If I had, I would have changed it to use my P&G affiliation to be consistent with the other 10 publications during this period. Last, neither I nor P&G have any financial interest in the publications or their subject matter. Rather, this work was done to progress both development and validation work on safety tests that have potential for use to serve a diverse range of parties, including regulators, industry, and the public. Therefore, I do not see how allegations of a competing financial interest are valid in this case, even considering the oversight on the affiliation. I hope this fully clarifies the record on this matter and lays the CSPI allegations to rest.

The intact immature rodent uterotrophic bioassay: possible effects on assay sensitivity of vomeronasal signals from male rodents and strain differences.

The vomeronasal organ in rodents is an important social and sexual signaling pathway. We have investigated whether the housing of intact immature females in close proximity to mature males would interfere with the sensitivity of the immature rodent uterotrophic bioassay as the result of vomeronasal signals transmitted by male urinary proteins. The hypothesis was that the proximity of males might induce early puberty, thereby increasing mean uterine weight and reducing the responsiveness of the assay. The hypothesis was tested in both rats and mice by housing mature males above immature females, separated only by a wire screen, for 3 days and determining possible changes in uterine weight. The results were negative. Neither the mean uterine weight nor the group mean standard deviation of the uterine weights were changed in the uterotrophic bioassay. Given that the timing of sexual maturation may vary with the strain of mouse used, we also evaluated the sensitivity of the immature mouse uterotrophic assay to diethylstilbestrol (DES) using four strains of mice. Similar sensitivity was observed for the CD-1, C57Bl6, and Alpk strains, but B6CBF(1) mice were marginally less sensitive to DES than were the other strains. These findings add to earlier data indicating the robustness of the rodent uterotrophic assay protocol.

The OECD program to validate the rat uterotrophic bioassay: an overview.

The Organisation for Economic Co-operation and Development has undertaken an international validation program for the rodent uterotrophic bioassay. This validation program comprised two major parts. The first part was the development of a detailed background review document compiling the existing data on the bioassay's history, the molecular and physiologic basis for the bioassay's mechanistic relevance to detect estrogen agonists and antagonists, a review of important bioassay protocol parameters, and a review of the data generated by in vitro assays, previous uterotrophic bioassays, and developmental and reproductive assays to assess and support the overall predictivity of the uterotrophic bioassay. The second part was an extensive multiyear effort managed by a validation management group to demonstrate the operating characteristics of four protocols. The effort was conducted in two phases. The phase 1 results with the reference agonist ethinyl estradiol (EE) and antagonist ZM 189,154 has been published previously. This Environmental Health Perspectives mini-monograph is devoted to the phase 2 work using five weak estrogen agonists, bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT, as well as the negative substance dibutylphthalate. These data show that all protocols successfully detected increases in uterine weights when a sufficient dose level of the weak agonists was administered, whether the substances were known or provided as coded doses to the laboratory. The data with both the reference EE and all five weak agonists are reproducible over time and under a variety of different experimental conditions (e.g., animal strain, diet, housing, bedding, vehicle, animal age). In conclusion, all protocols now have sufficient data to support their validity.

The OECD program to validate the rat uterotrophic bioassay. Phase 2: dose-response studies.

The Organisation for Economic Co-operation and Development has completed phase 2 of an international validation program for the rodent uterotrophic bioassay. The purpose of the validation program was to demonstrate the performance of two versions of the uterotrophic bioassay, the immature female rat and the adult ovariectomized rat, in four standardized protocols. This article reports the dose-response studies of the validation program; the coded single-dose studies are reported in an accompanying paper. The dose-response study design used five selected weak estrogen agonists, bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT. These weak agonists were administered in a prescribed series of doses to measure the performance and reproducibility of the protocols among the participating laboratories. All protocols successfully detected increases in uterine weights when the weak agonists were administered. Within each protocol, there was good agreement and reproducibility of the dose response among laboratories with each substance. Substance-specific variations were observed in the influence of the route of administration on the uterine response, the potency as related to the dose producing the first statistically significant increase in uterine weights, and the maximum increase in uterine weight. Substantive performance differences were not observed between the uterotrophic bioassay versions or among the standardized protocols, and these were judged to be qualitatively equivalent. It is noteworthy that these results were reproducible under a variety of different experimental conditions (e.g., animal strain, diet, housing, bedding, vehicle, animal age), indicating that the bioassay's performance as a screen is robust. In conclusion, both the intact, immature, and adult OVX versions, and all protocols appear to be reproducible and transferable across laboratories and are able to detect weak estrogen agonists.

The OECD program to validate the rat uterotrophic bioassay. Phase 2: coded single-dose studies.

The Organisation for Economic Co-operation and Development has completed phase 2 of an international validation program for the rodent uterotrophic bioassay. This portion of phase 2 assessed the reproducibility of the assay with a battery of positive and negative test substances. Positive agonists of the estrogen receptor included the potent reference estrogen 17-ethinyl estradiol (EE), and the weak estrogen agonists bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT. The negative test substance or nonagonist was n-dibutylphthalate. The test substances were coded, and prescribed doses of each test substance were administered in 16 laboratories. Two versions of the uterotrophic assay, the intact immature and the adult ovariectomized female rat, were tested and compared using four standardized protocols covering both sc and po administration. Assay reproducibility was compared using a) EE doses identical to those used in phase 1 and in parallel dose-response studies, b) single doses of the weak agonists identical to one of five doses from the dose-response studies, and c) a single dose of the negative test substance. The results were reproducible and in agreement both within individual laboratories and across the participating laboratories for the same test substance and protocol. The few exceptions are examined in detail. The reproducibility was achieved despite a variety of different experimental conditions (e.g., variations in animal strain, diet, housing protocol, bedding, vehicle, animal age). In conclusion, both versions of the uterotrophic bioassay and all protocols appear robust, reproducible, and transferable across laboratories and able to detect weak estrogen agonists. These results will be submitted along with other data for independent peer review to provide support for the validation of the uterotrophic bioassay.

Critical Review and Evaluation of the Uterotrophic Bioassay for the Identification of Possible Estrogen Agonists and Antagonists: In Support of the Validation of the OECD Uterotrophic Protocols for the Laboratory Rodent

A current issue for regulatory agencies is endocrine-related modes of action such as those mediated by the estrogen, androgen, and thyroid nuclear receptors. At the national and international levels, the consensus recommendation for the assessment of such modes of action is a tiered series of in vitro and in vivo protocols. The tiered framework begins with screens for structural alerts and then moves to rapid, mechanistic in vitro screening assays, and then to in vivo screening bioassays. The objective of these screens is to identify substances that may warrant testing for endocrine-mediated adverse effects. The final framework tier as needed is to test these substances in long-term bioassays for adverse endocrine-mediated reproductive and/or developmental effects. The subject of this review, the rodent uterotrophic bioassay, is intended to be a rapid in vivo screening bioassay for possible estrogen agonists and antagonists, based on the response of the estrogen-sensitive uterus. The central metric of the bioassay is a statistically significant increase in the weight of the uterus after 3 consecutive days of test substance administration. The extensive background literature is summarized in this review on the mode of action underlying the bioassay and the uterine response to estrogens. The review includes the bioassay's history of development and how its employment has changed and evolved over time. The review describes two major uterotrophic bioassay versions, the intact, immature female and the mature, ovariectomized female, and the protocol factors likely to influence the relevance, reproducibility, and reliability of the bioassay. The emphasis of the review is the ability of the uterotrophic bioassay to identify the substances of current interest: weak estrogen agonists with binding affinities relative to the natural 17β-estradiol in the log 0 to log -3 range. Using selected model substances having RBAs in this target range, the bioassay's performance in a hierarchical, tiered approach is evaluated, including the predictive capability of the uterotrophic bioassay based on available reproductive and developmental testing data. The review concludes that the uterotrophic bioassay is reliable and can identify substances that may act via an estrogen-mode of action, supporting the validity of the uterotrophic bioassay and its regulatory use as an in vivo mechanistic screening bioassay for estrogen agonists and antagonists.

The OECD Program to Validate the Rat Uterotrophic Bioassay to Screen Compounds for in Vivo Estrogenic Responses: Phase 1

The Organisation for Economic Co-operation and Development has completed the first phase of an international validation program for the rodent uterotrophic bioassay. This uterotrophic bioassay is intended to identify the in vivo activity of compounds that are suspected agonists or antagonists of estrogen. This information could, for example, be used to help prioritize positive compounds for further testing. Using draft protocols, we tested and compared two model systems, the immature female rat and the adult ovariectomized rat. Data from 19 participating laboratories using a high-potency reference agonist, ethinyl estradiol (EE), and an antagonist, ZM 189,154, indicate no substantive performance differences between models. All laboratories and all protocols successfully detected increases in uterine weights using EE in phase 1. These significant uterine weight increases were achieved under a variety of experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). For each protocol, there was generally good agreement among laboratories with regard to the actual EE doses both in producing the first significant increase in uterine weights and achieving the maximum uterine response. Furthermore, the Hill equation appears to model the dose response satisfactorily and indicates general agreement based on calculated effective dose (ED)(10) and ED(50) within and among laboratories. The feasibility of an antagonist assay was also successfully demonstrated. Therefore, both models appear robust, reproducible, and transferable across laboratories for high-potency estrogen agonists such as EE. For the next phase of the OECD validation program, both models will be tested against a battery of weak, partial estrogen agonists.

The OECD program to validate the rat uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1.

The Organisation for Economic Co-operation and Development has completed the first phase of an international validation program for the rodent uterotrophic bioassay. This uterotrophic bioassay is intended to identify the in vivo activity of compounds that are suspected agonists or antagonists of estrogen. This information could, for example, be used to help prioritize positive compounds for further testing. Using draft protocols, we tested and compared two model systems, the immature female rat and the adult ovariectomized rat. Data from 19 participating laboratories using a high-potency reference agonist, ethinyl estradiol (EE), and an antagonist, ZM 189,154, indicate no substantive performance differences between models. All laboratories and all protocols successfully detected increases in uterine weights using EE in phase 1. These significant uterine weight increases were achieved under a variety of experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). For each protocol, there was generally good agreement among laboratories with regard to the actual EE doses both in producing the first significant increase in uterine weights and achieving the maximum uterine response. Furthermore, the Hill equation appears to model the dose response satisfactorily and indicates general agreement based on calculated effective dose (ED)(10) and ED(50) within and among laboratories. The feasibility of an antagonist assay was also successfully demonstrated. Therefore, both models appear robust, reproducible, and transferable across laboratories for high-potency estrogen agonists such as EE. For the next phase of the OECD validation program, both models will be tested against a battery of weak, partial estrogen agonists.

Uterotrophic Activity of a "Phytoestrogen-Free" Rat Diet

LetterOpen AccessUterotrophic activity of a "phytoestrogen-free" rat diet. J Ashby, H Tinwell, and J Odum J Ashby Search for more papers by this author , H Tinwell Search for more papers by this author , and J Odum Search for more papers by this author Published:1 January 2000https://doi.org/10.1289/ehp.108-a12cCited by:12AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit "Uterotrophic activity of a "phytoestrogen-free" rat diet.." Environmental Health Perspectives, 108(1), pp. A12–A13FiguresReferencesRelatedDetailsCited by Prongay K, Lewis A, Hurn P and Murphy S (2010) Dietary soy may not confound acute experimental stroke infarct volume outcomes in ovariectomized female rats, Laboratory Animals, 10.1258/la.2009.009031, 44:3, (238-246), Online publication date: 1-Jul-2010. Charles G, Gennings C, Tornesi B, Kan H, Zacharewski T, Bhaskar Gollapudi B and Carney E (2007) Analysis of the interaction of phytoestrogens and synthetic chemicals: An in vitro/in vivo comparison, Toxicology and Applied Pharmacology, 10.1016/j.taap.2006.11.029, 218:3, (280-288), Online publication date: 1-Feb-2007. Thigpen J, Setchell K, Padilla-Banks E, Haseman J, Saunders H, Caviness G, Kissling G, Grant M and Forsythe D (2007) Variations in Phytoestrogen Content between Different Mill Dates of the Same Diet Produces Significant Differences in the Time of Vaginal Opening in CD-1 Mice and F344 Rats but Not in CD Sprague-Dawley Rats, Environmental Health Perspectives, 115:12, (1717-1726), Online publication date: 1-Dec-2007. Masutomi N, Shibutani M, Takagi H, Uneyama C, Takahashi N and Hirose M (2003) Impact of dietary exposure to methoxychlor, genistein, or diisononyl phthalate during the perinatal period on the development of the rat endocrine/reproductive systems in later life, Toxicology, 10.1016/S0300-483X(03)00269-5, 192:2-3, (149-170), Online publication date: 1-Nov-2003. Wade M, Lee A, McMahon A, Cooke G and Curran I (2003) The influence of dietary isoflavone on the uterotrophic response in juvenile rats, Food and Chemical Toxicology, 10.1016/S0278-6915(03)00168-6, 41:11, (1517-1525), Online publication date: 1-Nov-2003. Owens W, Ashby J, Odum J and Onyon L (2003) The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses., Environmental Health Perspectives, 111:12, (1559-1567), Online publication date: 1-Sep-2003.Ashby J, Owens W, Odum J and Tinwell H (2003) The intact immature rodent uterotrophic bioassay: possible effects on assay sensitivity of vomeronasal signals from male rodents and strain differences., Environmental Health Perspectives, 111:12, (1568-1570), Online publication date: 1-Sep-2003. Degen G, Janning P, Diel P and Bolt H (2002) Estrogenic isoflavones in rodent diets, Toxicology Letters, 10.1016/S0378-4274(02)00009-7, 128:1-3, (145-157), Online publication date: 1-Mar-2002. Barlow S, Greig J, Bridges J, Carere A, Carpy A, Galli C, Kleiner J, Knudsen I, Koëter H, Levy L, Madsen C, Mayer S, Narbonne J, Pfannkuch F, Prodanchuk M, Smith M and Steinberg P (2002) Hazard identification by methods of animal-based toxicology, Food and Chemical Toxicology, 10.1016/S0278-6915(01)00117-X, 40:2-3, (145-191), Online publication date: 1-Feb-2002. Thigpen J, Haseman J, Saunders H, Locklear J, Caviness G, Grant M and Forsythe D (2002) Dietary factors affecting uterine weights of immature CD-1 mice used in uterotrophic bioassays, Cancer Detection and Prevention, 10.1016/S0361-090X(02)00122-8, 26:5, (381-393), Online publication date: 1-Nov-2002. Ashby J, Tinwell H and Odum J (2001) DNA adducts, estrogenicity and rodent diets, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 10.1016/S0027-5107(01)00224-X, 483:1-2, (105-106), Online publication date: 1-Nov-2001. Ashby J, Tinwell H, Odum J, Kimber I, Brooks A, Pate I and Boyle C (2000) Diet and the aetiology of temporal advances in human and rodent sexual development, Journal of Applied Toxicology, 10.1002/1099-1263(200009/10)20:5<343::AID-JAT719>3.0.CO;2-O, 20:5, (343-347), Online publication date: 1-Sep-2000. Vol. 108, No. 1 January 2000Metrics About Article Metrics Publication History Originally published1 January 2000Published in print1 January 2000 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.