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.

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.

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.

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.

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.

The effects of atrazine on the sexual maturation of female rats.

The mammalian hazard assessment of the herbicide atrazine (ATR) has focused on the induction of mammary tumors and accelerated reproductive aging of adult rats, and the relationship of these effects to the inhibition of leutinizing hormone (LH) release from the pituitary, an effect itself caused by inhibition of GnRH signaling by the adult rat hypothalamus. In earlier studies, Laws et al. (Toxicol. Sci., 58, 366-376, 2000) demonstrated a delay in female rat sexual maturation induced by ATR, effects that could equally have been caused by inhibition of hypothalamic GnRH release. The present studies were designed to compare the doses that interfere with GnRH signaling seen in previous studies in adult Sprague-Dawley (SD) rats (LH surge suppression) with doses that impair GnRH signaling in peripubertal rats, as indicated by delayed sexual maturation. The studies evaluated the effects of ATR treatment on the timing of uterine growth and vaginal opening (VO) in peripubertal female Wistar (Alderley Park, AP) and SD rats. Doses of 10, 30, and 100 mg/kg ATR were administered daily from postnatal day (pnd) 21 to up to pnd 46. Determinations of uterine weight were made at pnd 30, 33, 43 (AP), and 46 (SD) and the timing of VO was also assessed in the last two of these experiments. The centrally acting GnRH antagonist Antarelix (ANT) was used as a positive control agent as it has previously been shown to prevent uterine growth and to delay VO in peripubertal AP rats. Uterine growth and VO were completely prevented in AP rats exposed to ANT. Uterine growth was delayed at pnd 30 and 33 in AP rats exposed to 100 mg/kg ATR, but this growth inhibition had been overcome by pnd 43. VO was significantly delayed in AP rats for the 100 mg/kg ATR dose. By pnd 46, VO was significantly delayed in SD rats exposed to both 30 and 100 mg/kg ATR, but uterine weights were unaffected by that time (as for AP rats). It is concluded that the no-effect level for the effects of ATR on sexually immature rats (10 mg/kg in SD; 30 mg/kg AP) is approximately the same as reported previously by Laws et al. in peripubertal Wistar rats (25 mg/kg). However, the no-effect level in peripubertal female SD rats is nearly an order of magnitude greater than the no-observed effect level observed in female SD rats fed ATR for 6 months (1.8 mg/kg) where LH suppression was used as an indicator of effect on the pituitary/hypothalamic axis (USEPA, Atrazine-DACT Fourth Report of the Hazard Identification and Review Committee, April 5, 2002). These results support the conclusion that the pituitary/hypothalamic axis in peripubertal female SD rats is less sensitive than that in adult female SD rats.

Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay.

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.

The male rat carcinogens limonene and sodium saccharin are not mutagenic to male Big Blue rats.

Limonene and sodium saccharin are male rat specific carcinogens giving rise to renal and bladder tumours, respectively. Both compounds give negative results in genetic toxicity assays suggesting a non-genotoxic mode of action for their carcinogenicity. The alpha 2U-globulin accumulation theory has been invoked to explain the renal carcinogenicity of limonene: the accumulation of micro masses of calcium phosphate in the bladder, coupled with a high pH environment in the male rat bladder, has been suggested to be responsible for the bladder carcinogenicity of sodium saccharin. The implication of these proposed mechanisms is that limonene and sodium saccharin will not be mutagenic to the rat kidney and bladder, respectively. This proposal has been evaluated by assessing the mutagenic potential of the two chemicals to male lacI transgenic (Big Blue) rats. Male Big Blue rats were exposed for 10 consecutive days to either limonene in diet, at a dose level in excess of that used in the original National Toxicology Program gavage carcinogenicity bioassay, or to sodium saccharin in diet at the dose known to induce bladder tumours. The multi-site rat carcinogen 4-aminobiphenyl was used as a positive control for the experiment. Limonene failed to increase the mutant frequency in the liver or kidney of the rats, and sodium saccharin failed to increase the mutant frequency in the liver or bladder of the rats. 4-Aminobiphenyl was mutagenic to all three of these tissues. These results add further support to a non-genotoxic mechanism of carcinogenic action for both limonene and sodium saccharin.

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.

The application of the restriction site mutation assay to compare 1-ethyl-1-nitrosourea-induced mutations between the endogenous p53 gene and the transgenic LacZ gene in MutaMouse testes.

Transgenic mouse modelling has provided a new approach to study the various steps involved in spontaneous and induced mutagenesis in rodent somatic and germline tissues in vivo. However, the important question arises as to whether mutations occur at the same rate in transgenes as in endogenous genes. Here, the restriction site mutation (RSM) assay was used to study mutations induced in the endogenous p53 gene and LacZ transgene of MutaMouse testes treated with 1-ethyl-1-nitrosourea (ENU). The aim of these experiments was to compare mutation susceptibility between the endogenous p53 gene and the integrated LacZ gene in the transgenic mouse. ENU-treated and control testes were analysed 102 days after treatment; a total of 297 RSM analyses were performed on ENU-treated and untreated testis DNA. Ten mutational events were detected in the p53 gene (exon 5 and intron 8), two of which occurred in untreated animals and probably represent spontaneous events. Only a single mutation was detected in the LacZ gene of an ENU-treated animal by the RSM assay. Thus the RSM assay can readily detect ENU-induced mutations in the p53 gene, but not in the LacZ transgene. Comparison of the LacZ RSM mutation data with results from a previous study of identically dosed MutaMice in the transgenic selection assay [Ashby, J., Gorelick,N.J. and Shelby,M.D. (1997) Mutat. Res., 388, 111-122] showed that LacZ mutations were far more readily recovered with the MutaMouse transgenic selection assay than by RSM analysis. The reason for the relative inability of the RSM assay to detect LacZ mutations may be the smaller target size of the RSM analysis compared with the transgenic selection assay (16 bases compared with 3000 bases). Taking into account the different target sizes by calculating the mutation frequency per base allowed the RSM data regarding p53 and LacZ to be compared with previously published data from transgenic selection assays. These studies demonstrated that the p53 mutations were present at mutation frequencies (per base) 5- to 70-fold higher than the LacZ gene mutations. In addition, the LacZ mutation frequency per base found in the RSM was an order of magnitude higher than that found in the transgenic selection assay. The transgenic selection assay is more sensitive per locus (due to the larger target of the LacZ gene), as evidenced by ability to detect ENU-induced testes mutations readily.

The uterotrophic activity of nonylphenol in the rat is not mediated by aromatase enzyme induction.

p-Nonylphenol (NP) is weakly estrogenic to rodents and to some species of fish. All evidence to date has indicated that the estrogenic effects of NP are due to the interaction of NP with the estrogen receptor. Recent findings of increased plasma estradiol in fish exposed to NP have, however, led to the proposal of an alternative mechanism for NP-induced estrogenicity in this species, possibly via induction of aromatase enzymes. In the present studies, this hypothesis was investigated in rats using the aromatase inhibitor anastrozole. The results indicated that the uterotrophic action of NP, as with estradiol used as a positive control, is mediated directly by its interaction with uterine ER, rather than an indirect effect via aromatase enzyme induction. Circulating levels of estradiol were unchanged after NP treatment and the aromatase inhibitor anastrozole failed to inhibit NP-induced uterine growth. These results are consistent with previous published data on NP in rodents.

Lack of binding to isolated estrogen or androgen receptors, and inactivity in the immature rat uterotrophic assay, of the ultraviolet sunscreen filters Tinosorb M-active and Tinosorb S.

The presence of structurally diverse chemicals as contaminants in the environment has led to concerns regarding their possible endocrine disturbing effects. Recently, some ultraviolet absorbing components of sunscreen preparations have given positive responses in assays monitoring estrogen-like activity both in vitro and in vivo. Consequently, two recently developed sunscreen components, Tinosorb M-active and Tinosorb S, were evaluated using the in vitro estrogen and androgen receptor competitive binding assays. Neither compound gave a positive response in either of the assays, consistent with the large molecular dimensions of each chemical disfavoring binding to the hormone receptors. Both of the chemicals were inactive in immature rat uterotrophic assays conducted using the subcutaneous route of administration. It is concluded that neither of these agents possess intrinsic estrogenic/antiestrogenic or androgenic/antiandrogenic activity. The several positive control chemicals evaluated gave the expected positive responses in the assays used.

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 mouse.

Bisphenol A (BPA) has been evaluated in eight independent immature mouse uterotrophic assays using the subcutaneous route of administration, and in a single study employing oral gavage. The dose range covered was from 0.02 microg to 300 mg/kg BPA and some experiments were supplemented by assessments of uterine hypertrophy and hyperplasia. Pooling of the test data indicates no uterotrophic activity for the chemical. However, in a subset of the subcutaneous injection studies, where control uterine weights were relatively low, significant, but weak, uterotrophic activity was observed over a range of dose levels, but in the complete absence of a dose relationship. In the oral gavage study, no increases in uterine weight were seen, but there were increases in uterine labeling with bromodeoxyuridine at 200-300 mg/kg BPA. The present study illustrated that when a large number of observations are made, a certain level of chance observations may be made, and that surrogates for an increase in uterine weight do not necessarily enhance assay sensitivity, albeit such data may complement uterine weight data. The data indicate that reducing control uterine weights may enhance assay sensitivity, but that animal body weight is an imperfect indicator of control uterine weight. The data also show that it is possible for individual investigators to be unable to confirm their own observations. It is concluded that BPA may be weakly uterotrophic to the mouse under specific conditions of test, and in the complete absence of a dose-response relationship to this activity. However, overall, we have failed to define BPA as reproducibly active in the immature mouse uterotrophic assay, and in that sense, our data are broadly consistent with those reported earlier by Coldham et al. (Environ. Health Perspect. 105, 734-742, 1997) in 1997 using a similar assay.

Hepatic gene mutations induced in Big Blue rats by both the potent rat liver azo-carcinogen 6BT and its reported noncarcinogenic analogue 5BT.

The potent rat liver carcinogen 6-p-dimethylaminophenylazobenzthiazole (6BT) and its reported noncarcinogenic analogue 5-p-dimethylaminophenylazobenzthiazole (5BT; evaluated for carcinogenicity under the similar limited bioassay conditions used for 6BT) have been studied in order to seek an explanation for their different carcinogenic activities. Both compounds act as DNA-damaging agents to the rat liver, and both have now been shown to induce lacI (-) gene mutations in the liver of Big Blue(trade mark) transgenic rats. Both compounds were mutagenic following ten daily gavage doses or following administration in diet for 10 days. Neither chemical induced cell proliferation in the liver following repeat gavage administrations. In contrast, dietary administration of 6BT, and to a lesser extent of 5BT, induced hepatic cell proliferation. The carcinogen 6BT, but not the noncarcinogen 5BT, caused proliferation of oval stem cells in the livers by both routes of administration. It is possible that mutations induced in oval cells by 6BT are responsible for its potent carcinogenicity, and that the comparative absence of these cells in 5BT-treated livers may account for the carcinogenic inactivity of 5BT. Equally, the proliferation of the oval cells may reflect changes in liver homeostasis associated with the liver toxicity observed at the dose level of 6BT used (which was, nonetheless, the dose level used in the positive cancer bioassays). It is concluded that the new data presented cannot explain the differing carcinogenic activities of 5BT and 6BT, and that the reported noncarcinogen 5BT may also be carcinogenic when adequately assessed for this activity.

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.

Partial and weak oestrogenicity of the red wine constituent resveratrol: consideration of its superagonist activity in MCF-7 cells and its suggested cardiovascular protective effects.

It was recently reported that the red wine phytoestrogen resveratrol (RES) acts as a superagonist to oestrogen-responsive MCF-7 cells. This activity of RES was speculated to be relevant to the 'French paradox' in which moderate red wine consumption is reported to yield cardiovascular health benefits to humans. We report here that RES binds to oestrogen receptors (ER) isolated from rat uterus with an affinity approximately 5 orders of magnitude lower than does either the reference synthetic oestrogen diethylstilboestrol (DES) or oestradiol (E2). In comparison with E2 or DES, RES is only a weak and partial agonist in a yeast hER-alpha transcription assay and in cos-1 cell assays employing transient transfections of ER-alpha or ER-beta associated with two different ER-response elements. Resveratrol was also concluded to be inactive in immature rat uterotrophic assays conducted using three daily administrations of 0.03-120 mgkg(-1)/day(-1) RES (administered by either oral gavage or subcutaneous injection). These data weaken the suggestion that the oestrogenicity of RES may account for the reported cardiovascular protective effects of red wine consumption, and they raise questions regarding the extent to which oestrogenicity data derived for a chemical using MCF-7 cells (or any other single in vitro assay) can be used to predict the hormonal effects likely to occur in animals or humans.