Evaluating H295R steroidogenesis assay data for robust interpretation.

The in vitro H295R steroidogenesis assay (OECD TG 456) is used to determine a chemical's potential to interfere with steroid hormone synthesis/metabolism. As positive outcomes in this assay can trigger significant higher tiered testing, we compiled a stakeholder database of reference and test item H295R data to characterize assay outcomes. Information concerning whether a Level 5 reproductive toxicity study was triggered due to a positive outcome in the H295R assay was also included. Quality control acceptance criteria were not always achieved, suggesting this assay is challenging to conduct within the guideline specifications. Analysis of test item data demonstrated that pairwise significance testing to controls allowed for overly sensitive statistically significant positive outcomes, which likely contribute to the assay's high positive hit rate. Complementary interpretation criteria (e.g., 1.5-fold change threshold) markedly reduced the rate of equivocal and positive outcomes thus improving identification of robust positive effects in the assay. Finally, a case study (positive H295R outcome and no endocrine adversity in vivo) is presented, which suggests that stricter data interpretation criteria could refine necessary in vivo follow-up testing. Overall, the described additional criteria could improve H295R data interpretation and help inform on how to best leverage this assay for regulatory purposes.

Isoflucypram: Combining in vivo and NAMs data in a weight of evidence approach to demonstrate the human non-relevance of the mode of action leading to the subtle thyroid effects observed in the rat.

Isoflucypram (ISY) is a new cereal fungicide with an overall favorable toxicity profile. As the thyroid was identified as a target organ only in the rat, following repeat dosing; short term in vivo (rat) and in vitro mechanistic studies were conducted to substantiate the thyroid changes as being secondary to liver enzyme induction via PXR/CAR activation and to determine the human non-relevance of the thyroid effects. The in vivo studies established ISY as a weak prototypical hepatic PXR/CAR enzyme inducer (P450 and T4-UDP-glucuronosyltransferase (UDPGT-T4) activities), with the induction being associated with increased liver weight/hepatocellular hypertrophy/proliferation. Thyroid effects (minimal follicular cell hypertrophy/proliferation, slight, statistically significantly increased thyroid stimulating hormone) occurred at doses where liver stimulation was already established. Direct thyroid effects (in vitro thyroid peroxidase and sodium iodide symporter inhibition) were excluded. Marked quantitative species differences were identified when comparing rat and human hepatic enzyme activities in vitro, particularly for UDPGT-T4. Specifically, basal UDPGT-T4 was 4-fold lower in human compared to rat hepatocytes. In addition, UDPGT-T4 was induced in vitro in rat but not in human hepatocytes following ISY treatment. Overall, the weight of evidence supports a liver mediated mode of action for the isoflucypram-induced slight thyroid changes in the rat as well as the human non-relevance of these findings.

Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Low dose evaluation of the antiandrogen flutamide following a Mode of Action approach.

The dose-response characterization of endocrine mediated toxicity is an on-going debate which is controversial when exploring the nature of the dose-response curve and the effect at the low-end of the curve. To contribute to this debate we have assessed the effects of a wide range of dose levels of the antiandrogen flutamide (FLU) on 7-week male Wistar rats. FLU was administered by oral gavage at doses of 0, 0.001, 0.01, 0.1, 1 and 10mg/kg/day for 28 days. To evaluate the reproducibility, the study was performed 3 times. The molecular initiating event (MIE; AR antagonism), the key events (LH increase, Leydig cell proliferation and hyperplasia increases) and associated events involved in the mode of action (MOA) of FLU induced testicular toxicity were characterized to address the dose response concordance. Results showed no effects at low doses (<0.1mg/kg/day) for the different key events studied. The histopathological changes (Leydig cell hyperplasia) observed at 1 and 10mg/kg/day were associated with an increase in steroidogenesis gene expression in the testis from 1mg/kg/day, as well as an increase in testosterone blood level at 10mg/kg/day. Each key event dose-response was in good concordance with the MOA of FLU on the testis. From the available results, only monotonic dose-response curves were observed for the MIE, the key events, associated events and in effects observed in other sex related tissues. All the results, so far, show that the reference endocrine disruptor FLU induces threshold effects in a standard 28-day toxicity study on adult male rats.

Liver tumor formation in female rat induced by fluopyram is mediated by CAR/PXR nuclear receptor activation.

Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mold, botrytis). During the general toxicity evaluation of fluopyram in rodents, the liver was identified as a target organ (hepatomegaly and liver hypertrophy were observed in all studies). At the end of the guideline carcinogenicity study, an increased incidence of hepatocellular adenomas and carcinomas was observed in female Wistar rats following exposure to the highest fluopyram dose evaluated (1500ppm). Short-term mechanistic studies (3, 7 or 28days of exposure) were conducted in the female rat to identify the initial key events responsible for the tumor formation and to establish thresholds for each of the early hepatic changes. Increased expression of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) inducible genes was recorded after each exposure period. Further confirmation of CAR/PXR activation was provided by increased activity of specific Phase I enzymes (PROD/BROD respectively). Increased hepatocellular proliferation (measured by Ki67) was observed after each exposure period with the greatest proliferative response occurring after 3days of treatment. In these studies, dose responses and clear thresholds were established for gene expression, enzyme activity and cell proliferation. Furthermore, these early hepatic changes were shown to be reversible following compound withdrawal. Other modes of action for liver tumor formation such as DNA damage, cytotoxicity and peroxisome proliferation were excluded during the investigations. In conclusion, fluopyram is a threshold carcinogen and the resultant hepatocellular carcinomas in the female rat are due to hepatocellular proliferation mediated by CAR/PXR activation.

Thyroid tumor formation in the male mouse induced by fluopyram is mediated by activation of hepatic CAR/PXR nuclear receptors.

Fluopyram, a broad spectrum fungicide, caused an increased incidence of thyroid follicular cell (TFC) adenomas in males at the highest dose evaluated (750ppm equating to 105mg/kg/day) in the mouse oncogenicity study. A series of short-term mechanistic studies were conducted in the male mouse to characterize the mode of action (MOA) for the thyroid tumor formation and to determine if No Observed Effect Levels (NOELs) exist for each key event identified. The proposed MOA consists of an initial effect on the liver by activating the constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH). This change in TSH secretion results in an increase of TFC proliferation which leads to hyperplasia and eventually adenomas after chronic exposure. Car/Pxr nuclear receptors were shown to be activated as indicated by increased activity of specific Phase I enzymes (PROD and BROD, respectively). Furthermore, evidence of increased T4 metabolism was provided by the induction of phase II enzymes known to preferentially use T4 as a substrate. Additional support for the proposed MOA was provided by demonstrating increased Tsh ß transcripts in the pituitary gland. Finally, increased TFC proliferation was observed after 28days of treatment. In these dose-response studies, clear NOELs were established for phase 2 liver enzyme activities, TSH changes and TFC proliferation. Furthermore, compelling evidence for Car/Pxr activation being the molecular initiating event for these thyroid tumors was provided by the absence of the sequential key events responsible for the TCF tumors in Car/Pxr KO mice when exposed to fluopyram. In conclusion, fluopyram thyroid toxicity is mediated by activation of hepatic Car/Pxr receptors and shows a threshold dependent MOA.

The screening of everyday life chemicals in validated assays targeting the pituitary-gonadal axis.

Ten structurally diverse chemicals (vitamins C, B9, B6, B3, sucrose, caffeine, gingerol, xanthan gum, paracetamol, ibuprofen) deemed intrinsic to modern life but not considered as endocrine active, were tested in vitro using the human estrogen receptor transcriptional activation (hERTa) and the H295R steroidogenesis assays. All were inactive in the hERTa assay but paracetamol, gingerol, caffeine and vitamin C affected steroidogenesis in vitro from 250, 25, 500 and 750 µM respectively. One molecule, caffeine, was further tested in rat pubertal assays at the tumorigenic dose-level and at dose-levels relevant for human consumption. In females pubertal parameters (vaginal opening, estrus cycle), ovarian weight and Fsh and prolactin transcript levels were affected. In males, plasma progesterone levels and prostate and seminal vesicle weights were affected. Although the current regulatory focus is synthetic chemicals that can cause adverse effects on the hypothalamus-pituitary-gonadal axis, our data infer that the range of natural chemicals with the potential to affect this axis may be extensive and is probably overlooked. Thus, to avoid regulation of an overwhelming number of chemicals, a weight of evidence approach, combining hazard identification and characterization with exposure considerations, is needed to identify those chemicals of real regulatory concern.

Genome-wide expression screening in the cardiac embryonic stem cell test shows additional differentiation routes that are regulated by morpholines and piperidines.

The cardiac embryonic stem cell test (ESTc) is a well-studied non-animal alternative test method based on cardiac cell differentiation inhibition as a measure for developmental toxicity of tested chemicals. In the ESTc, a heterogenic cell population is generated besides cardiomyocytes. Using the full biological domain of ESTc may improve the sensitivity of the test system, possibly broadening the range of chemicals for which developmental effects can be detected in the test. In order to improve our knowledge of the biological and chemical applicability domains of the ESTc, we applied a hypothesis-generating data-driven approach on control samples as follows. A genome-wide expression screening was performed, using Next Generation Sequencing (NGS), to map the range of developmental pathways in the ESTc and to search for a predictive embryotoxicity biomarker profile, instead of the conventional read-out of beating cardiomyocytes. The detected developmental pathways included circulatory system development, skeletal system development, heart development, muscle and organ tissue development, and nervous system and cell development. Two pesticidal chemical classes, the morpholines and piperidines, were assessed for perturbation of differentiation in the ESTc using NGS. In addition to the anticipated impact on cardiomyocyte differentiation, the other developmental pathways were also regulated, in a concentration-response fashion. Despite the structural differences between the morpholine and piperidine pairs, their gene expression effect patterns were largely comparable. In addition, some chemical-specific gene regulation was also observed, which may help with future mechanistic understanding of specific effects with individual test compounds. These similar and unique regulations of gene expression profiles by the test compounds, adds to our knowledge of the chemical applicability domain, specificity and sensitivity of the ESTc. Knowledge of both the biological and chemical applicability domain contributes to the optimal placement of ESTc in test batteries and in Integrated Approaches to Testing and Assessment (IATA).

A novel method for measuring aromatase activity in tissue samples by determining estradiol concentrations.

Increasing scrutiny of endocrine disrupters has led to changes to European pesticide and biocide legislation and to the introduction of the Endocrine Disrupter Screening Program by the US EPA. One element of endocrine disrupter identification is to determine its effects on aromatase, but most available assays are limited as they depend on tritiated water production to indicate enzyme activity. Whilst acceptable for determining aromatase effects using a cell-free approach, this method is unreliable for cell or tissue-based investigations as other cytochrome P-450 isoenzyme activities can similarly produce tritiated water and consequently confound interpretation of the aromatase data. To address this lack of specificity an assay directly measuring the final estrogen product by incubating rat tissue protein with testosterone and measuring the resultant estradiol concentration was developed. Using this approach we demonstrated marked increases in enzyme activity in pregnant rat ovary samples and dose-related inhibitions when incubating non-pregnant rat ovary samples with known aromatase inhibitors. Hepatic aromatase activity was investigated using our method and by tritiated water production with microsomes from rats dosed with the antiandrogen 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane. Additional cytochrome P-450s were also measured. Treatment-related increased tritiated water production and general hepatic enzyme activity were recorded but estradiol was not increased, indicating that the increased tritiated water was due to general enzyme activity and not aromatase activity. A simple and specific method has been developed that can detect aromatase inhibition and induction, which when applied to tissue samples, provides a means of generating relevant animal data concerning chemical effects on the aromatase enzyme.

Molecular neural crest cell markers enable discrimination of organophosphates in the murine cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) originally used the differentiation of beating cardiomyocytes for embryotoxicity screenings of compounds. However, the ESTc consists of a heterogeneous cell population, including neural crest (NC) cells, which are important contributors to heart development in vivo. Molecular markers for NC cells were investigated to explore if this approach improved discrimination between structurally related chemicals, using the three organophosphates (OP): chlorpyrifos (CPF), malathion (MLT), and triphenyl phosphate (TPP). To decrease the test duration and to improve the objective quantification of the assay read-out, gene transcript biomarkers were measured on study day 4 instead of the traditional cardiomyocyte beating assessment at day 10. Gene expression profiling and immunocytochemistry were performed using markers for pluripotency, proliferation and cardiomyocyte and NC differentiation. Cell proliferation was also assessed by measurements of embryoid body (EB) size and total protein quantification (day 7). Exposure to the OPs resulted in similar patterns of inhibition of beating cardiomyocyte differentiation and of myosin protein expression on day 10. However, these three chemically related compounds induced distinctive effects on NC cell differentiation, indicated by changes in expression levels of the NC precursor (Msx2), NC marker (Ap2α), and epithelial to mesenchymal transition (EMT; Snai2) gene transcripts. This study shows that investigating NC markers can provide added value for ESTc outcome profiling and may enhance the applicability of this assay for the screening of structurally related test chemicals.

The effects of dose, route of administration, drug scheduling and MDR-1 gene transfer on the genotoxicity of etoposide in bone marrow.

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.

Uterotrophic activity of bisphenol A in the immature rat.

Bisphenol A (BPA) is active in immature AP rat uterotrophic assays when evaluated using either the oral or the subcutaneous (sc) injection routes of exposure (three daily administrations of 400-800 mg/kg BPA). Premature vaginal opening was seen for 8 of 14 animals exposed to 600 and 800 mg/kg BPA by sc injection. Vaginal opening was not produced by BPA in the gavage studies. These results are consistent with those of Dodds and Lawson [Nature 137:96 (1936)] who found that BPA induces persistent vaginal cornification in ovariectomized rats exposed to three twice-daily injections of 85 mg/kg BPA (total daily dose 170 mg/kg), but they conflict with the reported inactivity of BPA in the immature mouse uterotrophic assay. The uterotrophic activity of diethylstilbestrol in the rat is also established (0.04 mg/kg/day for three days).

Comparative activity of human carcinogens and NTP rodent carcinogens in the mouse bone marrow micronucleus assay: an integrative approach to genetic toxicity data assessment.

The mouse bone marrow micronucleus (MN) assay holds a key position in all schemes for detecting potential human carcinogens and mutagens. It was therefore of concern when Shelby et al. reported that only 5 of 25 rodent carcinogens defined by the U.S. NTP were positive in the assay. Further, each of these positive responses was weak and indistinguishable from the 4 positive responses observed among the 24 NTP noncarcinogens tested. To focus these findings, the activity in the MN assay of 26 human carcinogens, 6 reference rodent genotoxins, and the 9 NTP chemicals positive in the MN assay have been displayed in a common format. This involved plotting the minimum positive dose level (expressed as mumole/kilogram) and the maximum fold-increase in micronucleated polychromatic erythrocytes frequency observed at any dose level. By displaying the high sensitivity of the micronucleus assay to the reference human and rodent genotoxins, this analysis emphasizes the weakness in the MN assay responses given by the NTP carcinogens reported by Shelby et al. This, in turn, poses questions about the intrinsic hazard of this selection of NTP rodent carcinogens. Using fotemustine and vitamin C as models of a toxic and a nontoxic chemical known to be active in the MN assay, this analysis describes a method by which their relative potential human hazard can be distinguished (a synthetic, as opposed to an analytical approach to data assessment). The possibility that some weak responses observed in the MN assay at elevated dose levels may be stress induced is considered.

Arsenite as the probable active species in the human carcinogenicity of arsenic: mouse micronucleus assays on Na and K arsenite, orpiment, and Fowler's solution.

Sodium arsenite, potassium arsenite, and Fowler's solution (arsenic trioxide dissolved in potassium bicarbonate) are equally active in the mouse bone marrow micronucleus assay (approximately 10 mg/kg by IP injection). The natural ore orpiment (principally As2S3) was inactive despite blood levels of arsenic of 300 to 900 ng/mL in treated mice at 24 hr. Sodium arsenite was active in three strains of mice. It is suggested that the human lung cancer observed among arsenic ore smelters and the skin cancer among people exposed therapeutically to Fowler's solution, have, as their common origin, the genotoxic arsenite ion AsO2-. The difficulty experienced when attempting to demonstrate rodent carcinogenicity for derivatives of arsenic suggests that the bone marrow micronucleus assay may act as a useful assay for potentially carcinogenic arsenic derivatives.

Induction of hyperplasia and increased DNA content in the uterus of immature rats exposed to coumestrol.

Administration of the phytoestrogen coumestrol to ovariectomized rats leads to increases in both wet and dry uterine weights in the absence of an increase in uterine DNA content, as reported by Markaverich et al. [Effects of Coumestrol on Estrogen Receptor Function and Uterine Growth in Ovariectomized Rats. Environ Health Perspect 103:574-581 (1995)]. It was not possible to know if the observed atypical uterotrophic response of coumestrol was associated uniquely with the ovariectomized uterotrophic assay protocol. This question is answered in the present paper. Two experiments are described in which three daily oral gavage administrations of 60 mg/kg/day coumestrol to immature AP rats were followed by assessment of the reproductive tract on the fourth day. In both experiments coumestrol increased uterine fluid content and increased the weights of the uterus, cervix, and vagina. In addition, bromodeoxyuridine staining of uterine sections enabled confirmation of uterine hyperplasia for the coumestrol-treated animals. In the second experiment, total uterine DNA was determined; it doubled in the coumestrol-treated animals. Estradiol benzoate acted as the positive control agent for both of these experiments, and in each case it gave similar responses to those seen for coumestrol. We conclude that the uterotrophic activity of the phytoestrogen coumestrol in the immature intact rat is typical of the activity of the natural estrogen estradiol.

Estradiol-type activity of coumestrol in mature and immature ovariectomized rat uterotrophic assays.

Makaverich et al. [Environ Health Perspect 103:574-581 (1995)] reported that the uterotrophic activity of the phytoestrogen coumestrol in the immature ovariectomized rat was atypical in that it was not associated with increased uterine hyperplasia and DNA content. We previously reported that coumestrol gave a typical estradiol-type uterotrophic response in the immature intact rat, yielding increases in uterine epithelial cell height, glandular formation, cell labeling, and DNA content. These papers did not answer the question of whether there is a basic difference between the ovariectomized and the intact rat uterotrophic assays. In this paper, we report that coumestrol gives a typical estradiol-type uterotrophic response in uterotrophic assays using immature intact, immature ovariectomized, and mature ovariectomized rats. We concluded that the uterotrophic activity of coumestrol is typical of the natural estrogen estradiol.

Normal sexual development of two strains of rat exposed in utero to low doses of bisphenol A.

Pregnant Sprague-Dawley (SD) and Alderley Park (Wistar derived) rats were exposed by gavage during gestation days 6-21 to 20 microg/kg, 100 microg/kg, or 50 mg/kg body weight of BPA with ethinylestradiol (EE; 200 microg/kg) acting as a positive control agent. The sexual development of the derived pups was monitored until termination at postnatal day 90-98. The endpoints evaluated were litter size and weight, anogenital distance at birth, days of vaginal opening, first estrus and prepuce separation, weights of the liver, seminal vesicles, epididimydes, testes, ventral prostate, uterus, vagina, cervix and ovaries, and daily sperm production. Males were terminated at postnatal day 90 and females at postnatal day 98. The only statistically significant effects observed for any dose of BPA were a decrease in daily sperm production and an increase in the age of vaginal opening for the Alderley Park animals at the highest dose evaluated (50 mg/kg). The dose of EE evaluated proved to be maternally toxic in our laboratory, but provided gross evidence of endocrine disruption in the treated dams. These results diverge from those of Chahoud and his colleagues who indicated disturbances to the sexual development of both male and female SD rat pups administered the same 3 doses of BPA. This failure to confirm low dose endocrine effects for BPA is discussed within the context of similar divergent conclusions derived from other assessments of the endocrine toxicity of this agent to rats.

The effect on sperm production in adult Sprague-Dawley rats exposed by gavage to bisphenol A between postnatal days 91-97.

M. Sakaue et al. (2001,J. Occup. Health vol. 43, pp. 185-190) have described how oral exposure of sexually mature male rats to bisphenol A (BPA) between postnatal days (PND) 91-97 led to a reduction in daily sperm production (DSP) 5 weeks later (18 weeks of age). Activity was observed over the dose range 20 microgram/kg-200 mg/kg BPA, with an absence of activity over the dose range 2 ng/kg-2 microgram/kg BPA. There was no evidence of a dose response relationship over the active dose range (five orders of magnitude range). The observation of endocrine disruption (ED) effects for BPA at such low doses, and in sexually mature animals, was unexpected. It was therefore decided to mount an independent repeat of their study. A total of four independent studies were conducted according to the protocol used by Sakaue et al. Doses of 20 microgram/kg, 2 mg/kg, or 200 mg/kg BPA were administered to adult Sprague-Dawley (SD) rats over PND 91-97, and the studies were terminated when the rats reached the age of 18 weeks. Three different rodent diets were employed (RM3, Purina 5002, and CE2), the last of which had been used by Sakaue et al. BPA failed to give any evidence of ED activities, including the changes in DSP reported by Sakaue et al. 2001. During the course of these studies, the test protocol was adapted to coincide more precisely with that used by Sakaue et al.; this included restricting the number of animals per cage, removing bedding from the cages, and changing to the use of glass water bottles in the cages. The only thing of interest to emerge from our studies was the observation of a significant difference in DSP between the control groups of our first and second study. As the change in diet from RM3 to Purina 5002 was the major difference between those two studies, we conducted a repeat of the second study, but we were unable to confirm the differences seen between the first and second study. The probability that those differences arose either by chance, or as the result of intrinsic study-to-study variability, was strengthened by the absence of significant differences in the sperm parameters in a final (fifth) study where the sperm parameters for control animals maintained on the three different diets were compared under the conditions of the main experiments. No explanation for our failure to replicate the effects reported by Sakaue et al. is evident. A review of DSP values reported in the recent literature is provided and discussed, and it is concluded that use of the term DSP/g testis rather than DSP/testis could increase the sensitivity of DSP assessments.

Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning.

The objective of the study was to determine which period of exposure produces the most marked effects on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures. The endocrine active chemical, diethylstilbestrol (DES) was used as an agent known to affect many of the endpoints examined. Hitherto, such comparisons have been made between studies, rather than within a study. Our data will be helpful in the interpretation of future multigenerational assay data. In preliminary studies, DES was shown to be active in the immature rat uterotrophic assay with a lowest detected dose of 0.05 mg DES/kg body weight by sc injection and 10 mg DES/l (1.6 mg DES/kg body weight) by administration in drinking water. A dose of 60 microg DES/l drinking water ( approximately 6.5mg DES/kg body weight/day) was selected for the main study since this represented the midpoint of the drinking water uterotrophic dose response and produced no overt maternal toxicity. The study used 10 groups of concomitantly pregnant animals, including 2 control groups. The first comparison was between the effects of exposure to DES in utero, and exposure from conception to weaning. Another group of animals was exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational transfer of DES to pups. Two groups were exposed to DES neonatally, either from birth to postnatal day (PND) 10 (pups thus having only lactational exposure), or from birth until weaning (PND 21; pups thus having both lactational exposure and self-exposure via drinking water). In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, when the first phase of the study was terminated. Pups exposed to DES in utero and pups exposed from weaning to PND 100 were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. This last comparison was to determine the extent to which weanling rats could be used in endocrine toxicity studies to assess their potential to show activity in utero. The most sensitive period of exposure for inducing developmental effects in F1 animals was from weaning onwards. The neonatal to weaning period (PND 1-21) was the next most sensitive. Essentially no effects were induced in F1 animals exposed in utero. No effects of any kind were observed in animals only exposed over the early neonatal period of PND 1-10. The mean day of vaginal opening, testes descent, and prepuce separation was only altered in groups where postnatal exposure to DES continued beyond PND 10, or was started at weaning. No changes were observed in anogenital distance or caudal sperm counts. Some changes in organ weights were observed, but the interpretation of these was often confused by concomitant changes in body weight. In general, histopathological examination of tissues yielded no additional information. In breeding studies with animals exposed to DES in utero, or from weaning, reduced litter sizes and marginal advances in the day of vaginal opening were observed in the offspring, together with changes in organ weights. However, no unique sensitivity was noted for exposure in utero. Evaluation of the several exposure periods and the many markers monitored in this study may have individual strengths in individual cases, but when rigorously compared using the reference estrogen DES, many preconceptions regarding their absolute or relative value were not upheld. Further, each of these markers is subject to natural variability, as demonstrated by comparisons made among the 5 separate control groups available in parts of the present study. This variability increases the chance that small changes observed in endocrine toxicity studies employing small group sizes and a single control group, or no concomitant control group, may be artifactual. The most marked effects observed in this study were on the developmental landmarks in the F1 animals induced by exposures after PND 10. Some effects on developmental landmarks and organ weights were observed in F2 animals following exposure either in utero or postweaning. This study therefore does not establish a unique role for exposures in utero or during the early neonatal period.

Effect of rodent diets on the sexual development of the rat.

Five rodent diets have been evaluated for their possible effect on the sexual development of the rat. Groups of 12 pregnant Alpk rats were fed one of the following combinations of diets during pregnancy and postnatally: RM3/RM1, AIN-76A/AIN-76A, RM3/AIN-76A, Teklad Global 2016 (Global)/Global and Purina 5001/Purina 5001. AIN-76A is phytoestrogen-free while the other diets contained varying amounts of phytoestrogens. The phytoestrogens genistein and daidzein were determined in the diets studied, and the concentrations found agreed with earlier estimates. RM3/RM1 was selected as the control group, as this has been used routinely in this laboratory for the past decade. Determinations were made in offspring of the times of vaginal opening and first estrus among the females, and of prepuce separation and testes descent among the males. At postnatal day (PND) 26 the females from 6 of the 12 litters were terminated and tissue weights measured. Males from 6 of the 12 litters were similarly studied at PND 68. Animals from the remaining litters were transferred to RM1 diet at PND 70. Termination of the study was at PND 128 (males) and PND 140 (females) when body weights and tissue weights were determined. Marked differences in body weight, sexual development, and reproductive tissue weights were observed for rats maintained on AIN-76A or Purina 5001, with only minimal effects among rats maintained on the Global diet. These comparisons were against RM3/RM1 as the reference diet. However, using Purina 5001 as the reference diet reversed the direction of the differences seen when using RM3/RM1 as the reference diet. The differences observed when using RM3/RM1 as reference diet occurred mainly postnatally. In addition, the fact that similar differences were seen for the phytoestrogen-free diet, AIN-76A, and the phytoestrogen-rich diet, Purina 5001, indicate that these effects are more likely to be caused by nutritional differences between the diets that then have centrally mediated effects on rodent sexual development, rather than individual dietary components affecting peripheral estrogen receptors (ER). This proposal is supported by abolition of the uterotrophic activity of AIN-76A and Purina 5001 (relative to RM3/RM1) in the immature rat by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist ANTARELIX: The present data indicate that choice of diet may influence the timing of sexual development in the rat, and consequently, that when evaluating the potential endocrine toxicity of chemicals, the components of rodent diets used should be known, and as far as is possible, controlled.