Reliability of the AR-CALUX®In Vitro Method Used to Detect Chemicals with (Anti)Androgen Activity: Results of an International Ring Trial.

The AR-CALUX® in vitro method is a reporter gene-based transactivation method where endocrine active chemicals with androgenic or anti-androgenic potential can be detected. Its primary purpose is for screening chemicals for further prioritization and providing mechanistic (endocrine mode of action) information, as defined by the Organisation of Economic Cooperation and Development (OECD) conceptual framework for the testing and assessment of endocrine-disrupting chemicals. This article describes the conduct and results of an international ring trial with 3 EU-NETVAL laboratories and the test method developer. It was organized by EURL ECVAM to validate the method by testing 46 chemicals. A very good reproducibility within and between laboratories was concluded (94.7-100% and 100% concordance of classification) with low within and between laboratory variability (less than 2.5% CV on EC50 values). Moreover, the variability is within the range of other validated, mechanistically similar methods. In comparison to the AR-reference list compiled by ICCVAM, an almost 100% concordance of classifications was obtained. This method allows the detection of the agonist and antagonist properties of a chemical. A specificity control test was developed during the validation study and added to the antagonist assay rendering the assay more specific. A comparison is made with the mechanistically similar methods AR-EcoScreen™ and 22Rv1/MMTV GR-KO TA. The AR-CALUX® method was approved for inclusion in the recently updated OECD test guideline TG458 which incorporates all 3 methods.

An analysis of the setting of the acute reference dose (ARfD) for pesticides in Europe.

To protect human health, acute reference values have been established for pesticides which have the potential to cause a toxic effect after acute human exposure. These values are used to identify exposure levels below which there is no appreciable risk. Comprehensive reference documents, including OECD criteria, are available to aid identification of relevant toxicological endpoints. Within Europe, there is a concern that the identification process is inconsistent and unnecessarily conservative such that safe products with no established human health risk are being restricted. For this reason, the basis for the setting of an acute reference dose (ARfD) has been investigated for 130 pesticides to better understand how the toxicological endpoints are selected. The investigation has shown that most ARfDs are derived from repeat dose studies and that there is an over-representation of prenatal developmental toxicity studies. There is clear evidence that ARfDs derived from rabbit developmental toxicity studies are set over conservatively with regard to acute maternal effects and often inappropriately. To facilitate an improved system, refinements to the existing process are recommended, the use of maternal data in the rabbit as the basis for deriving an ARfD is critically evaluated and a new, more pragmatic approach to ARfD derivation is proposed.

Recommended approaches to the scientific evaluation of ecotoxicological hazards and risks of endocrine-active substances.

A SETAC Pellston Workshop® "Environmental Hazard and Risk Assessment Approaches for Endocrine-Active Substances (EHRA)" was held in February 2016 in Pensacola, Florida, USA. The primary objective of the workshop was to provide advice, based on current scientific understanding, to regulators and policy makers; the aim being to make considered, informed decisions on whether to select an ecotoxicological hazard- or a risk-based approach for regulating a given endocrine-disrupting substance (EDS) under review. The workshop additionally considered recent developments in the identification of EDS. Case studies were undertaken on 6 endocrine-active substances (EAS-not necessarily proven EDS, but substances known to interact directly with the endocrine system) that are representative of a range of perturbations of the endocrine system and considered to be data rich in relevant information at multiple biological levels of organization for 1 or more ecologically relevant taxa. The substances selected were 17α-ethinylestradiol, perchlorate, propiconazole, 17ß-trenbolone, tributyltin, and vinclozolin. The 6 case studies were not comprehensive safety evaluations but provided foundations for clarifying key issues and procedures that should be considered when assessing the ecotoxicological hazards and risks of EAS and EDS. The workshop also highlighted areas of scientific uncertainty, and made specific recommendations for research and methods-development to resolve some of the identified issues. The present paper provides broad guidance for scientists in regulatory authorities, industry, and academia on issues likely to arise during the ecotoxicological hazard and risk assessment of EAS and EDS. The primary conclusion of this paper, and of the SETAC Pellston Workshop on which it is based, is that if data on environmental exposure, effects on sensitive species and life-stages, delayed effects, and effects at low concentrations are robust, initiating environmental risk assessment of EDS is scientifically sound and sufficiently reliable and protective of the environment. In the absence of such data, assessment on the basis of hazard is scientifically justified until such time as relevant new information is available. Integr Environ Assess Manag 2017;13:267-279. © 2017 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

In vitro metabolism and bioavailability tests for endocrine active substances: what is needed next for regulatory purposes?

Legislation and prospective legislative proposals internationally (may) require that chemicals be tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive in vitro are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs) in vivo. While there is a growing body of international in vitro test guidelines addressing EAS mechanisms and modes of action, to date there are still few or no standardized methods to incorporate metabolic and toxicokinetic aspects into these in vitro tests for EAS. In vitro assays for EAS should incorporate metabolic enzyme systems to better address the relevance of EAS tests to in vivo adverse outcome pathways, and a previous OECD review paper indicated how this could be done. This paper revisits those recommendations, addressing where research and funding efforts are needed to expedite the development of suitable in vitro metabolism systems to improve the accuracy of in vitro assays for identifying EAS and EDs. Recommendations are made for projects to support short, medium, and long-term goals. The complexity of in vivo metabolism presents major challenges for the development of predictive models suitable for the extrapolation of data from in silico/in vitro approaches to models that can occur in vivo. Therefore, the long-term recommendations are intended to foster an international harmonization of databases, delineation of metabolic pathways, and development of predictive tools that will provide a fundamental understanding of the processes by which metabolism occurs, increasing the predictive accuracy of in silico/in vitro methods.

The reported in vitro anti-estrogen pentachloronitrobenzene enhances the estrogenic activity of estradiol in vivo in the rat.

Pentachloronitrobenzene (PCNB) has been shown to inhibit foci-formation for MCF-7 cells in vitro (Zou, E., Hatakeyama, M., Matsumra, F., 2002. Foci-formation of MCF-7 cells as an in vitro screening method for estrogenic chemicals. Environ. Toxicol. Pharmacol. 11, 71) This effect was referred to as representing an anti-estrogenic property of PCNB. However, we have found no evidence that PCNB acts as either an estrogen or an anti-estrogen, either in vitro or in vivo. The assays conducted were binding to human and rat estrogen receptors (ER), a hER yeast trans-activation assay, the immature rat uterotrophic assay and a pubertal female rat assay. Nonetheless, when PCNB was evaluated as a possible anti-estrogen against estradiol in the immature rat uterotrophic assay, it enhanced, rather than reduced the activity of estradiol. Absence of an effect by PCNB on the uterotrophic activity of diethylstilbestrol suggests that the effect with estradiol was related to alteration of its metabolism. However, PCNB was not hepatotoxic and failed to inhibit cytochrome P450 or estradiol sulphotransferase. Pentachlorophenol, a major metabolite of PCNB, was inactive as an estrogen and failed to enhance the uterotrophic activity of estradiol.

Cumulative dietary energy intake determines the onset of puberty in female rats.

Laboratory animal diets for studies to determine the endocrine-disrupting potential of chemicals are under scrutiny because they can affect both assay control values and assay sensitivity. Although phytoestrogen content is important, we have previously shown that a phytoestrogen-rich diet and a phytoestrogen-free diet were equally uterotrophic to rats and advanced vaginal opening (VO) when compared with the standard diet RM1. Abolition of the effects by the gonadotrophin-releasing hormone antagonist Antarelix indicated that these effects were mediated through the hypothalamus-pituitary-reproductive organ axis. In the present study, we investigated the relationship between cumulative energy intake and sexual maturation in female rats. Infant formula (IF) at different concentrations and synthetic diets, with a wide range of metabolizable energy (ME) values, were used to modulate energy intake. Increasing energy intake was associated with an increase in uterine weight (absolute and adjusted for body weight) for both IF and the synthetic diets. In both cases, the increased uterine weight was directly proportional to energy intake. Body weight was unaffected by IF consumption but, in the case of the diets, was increased proportionally with energy consumption. Antarelix abolished the uterine weight increases with both formula and the diets, whereas body weight was unaffected. The mean day of VO was also advanced by high-ME diets and IF, whereas body weight at VO was unaffected. VO occurred at an energy intake of approximately 2,300 kJ/rat determined by measuring total food intake from weaning to VO, indicating that this cumulative energy intake was the trigger for puberty. ME is therefore a critical factor in the choice of diets for endocrine disruption studies.

Natural variability and the influence of concurrent control values on the detection and interpretation of low-dose or weak endocrine toxicities.

While defining the no effect level for the 5 alpha-reductase inhibitor finasteride in the Hershberger assay, we encountered an inverted-U low-dose trophic effect on the prostate gland of the rat. Two attempts to confirm this observation were unsuccessful, and we concluded that the positive effect initially observed was associated with normal biologic variability. During the same period we attempted, unsuccessfully, to repeat our own observation of weak uterotrophic activity in the rat for the sunscreen 3-(4-methylbenzylidene)camphor (4MBC). Further evaluation led us to conclude that 4MBC is uterotrophic only when the control uterine weights are at the low end of their normally encountered range. This led us to reevaluate our earlier mouse uterotrophic assay data for bisphenol A (BPA). Originally we had concluded that BPA gave irreproducible evidence of weak uterotrophic activity, but upon ordering the eight experiments we had conducted, according to decreasing control uterine weight, we confirmed reproducible weak uterotrophic activity for BPA when the control uteri were at the low end of their normal range. In this article, we describe these observations, together with a reanalysis of the data associated with several reported instances of weak or low-dose endocrine effects that have proven difficult to confirm in independent laboratories. These include the activity of BPA on the CF1 mouse prostate; the activities of BPA, octylphenol, and nonylphenol on the rat testis; and the effect of polycarbonate caging on control mouse uterine weight. In all of these cases, variability among controls provides a major obstacle to data interpretation and confirmation. Our recommendations on experimental design are also presented, with a view to ending the current impasse on the reality, or otherwise, of low-dose or weak endocrine toxicities.

The OECD program to validate the rat uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses.

Many commercial laboratory diets have detectable levels of isoflavones (e.g., phytoestrogens such as genistein [GN]) that have weak estrogenic activity both in vitro and in vivo. During validation studies of the uterotrophic bioassay, diet samples from 20 participating laboratories were collected and analyzed for three major phytoestrogens: GN, daidzein (DN), and coumestrol (CM). Soy phytoestrogens GN and DN were found at total phytoestrogen levels from 100 to 540 microg/g laboratory diet; a forage phytoestrogen, CM, ranged from nondetectable to 4 microg/g laboratory diet. The phytoestrogen levels were compared with both baseline uterine weights of the control groups and with the relative uterine weight increase of groups administered two weak estrogen agonists: bisphenol A (BPA) and nonylphenol (NP). The comparison uses a working assumption of additivity among the phytoestrogens, despite several significant qualifications to this assumption, to estimate total genistein equivalents (TGE). Some evidence was found that phytoestrogen levels in the diet > 325-350 microg/g TGE could diminish the responsiveness of the uterotrophic bioassay to weak agonists. This was especially true for the case of the intact, immature female version of the uterotrophic bioassay, where higher food consumption relative to body weight leads to higher intakes of dietary phytoestrogens versus ovariectomized adults. This dietary level is sufficient in the immature female to approach a biological lowest observable effect level for GN of 40-50 mg/kg/day. These same data, however, show that low to moderate levels of dietary phytoestrogens do not substantially affect the responsiveness of the assay with weak estrogen receptor agonists such as NP and BPA. Therefore, laboratories conducting the uterotrophic bioassay for either research or regulatory purposes may routinely use diets containing levels of phytoestrogens < 325-350 microg/g TGE without impairing the responsiveness of the bioassay.

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.

Confirmation of uterotrophic activity of 3-(4-methylbenzylidine)camphor in the immature rat.

In this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage. These data confirm earlier reports of uterotrophic activity for this agent when administered to immature rats in the diet or by whole-body immersion; however, they are in contrast to negative unpublished immature rat uterotrophic assay results. Data also indicate that 4MBC binds to isolated rat uterine estrogen receptors and shows activity in a human estrogen receptor yeast transactivation assay; however, we considered both of these effects equivocal. In this study, we confirmed the original observation that 4MBC was active as a mitogen to MCF-7 breast cancer cells. We evaluated and discounted the possibility that the estrogenic activity of 4MBC is related to its bulky camphor group, which is of similar molecular dimensions to that of the weak estrogen kepone. Uncertainty remains regarding the mechanism of the uterotrophic activity of 4MBC.

Prediction of rodent nongenotoxic carcinogenesis: evaluation of biochemical and tissue changes in rodents following exposure to nine nongenotoxic NTP carcinogens.

We studied nine presumed nongenotoxic rodent carcinogens, as defined by the U.S. National Toxicology Program (NTP), to determine their ability to induce acute or subacute biochemical and tissue changes that may act as useful predictors of nongenotoxic rodent carcinogenesis. The chemicals selected included six liver carcinogens (two of which are peroxisome proliferators), three thyroid gland carcinogens, and four kidney carcinogens. We administered the chemicals (diethylhexyl phthalate, cinnamyl anthranilate, chlorendic acid, 1,4-dichlorobenzene, monuron, ethylene thiourea, diethyl thiourea, trimethyl thiourea, and d-limonene to the same strains of mice and rats used in the original NTP bioassays (nine chemicals to rats and seven to mice). Selected tissues (liver, thyroid gland, and kidney) were collected from groups of animals at 7, 28, and 90 days for evaluation. Tissue changes selected for study were monitored for all of the test groups, irrespective of the specificity of the carcinogenic responses observed in those tissues. This allowed us to assess both the carcinogen specificity and the carcinogen sensitivity of the events being monitored. We studied relative weight, cell labeling indices, and pathologic changes such as hypertrophy in all tissues; a range of cytochrome P450 enzymes and palmitoyl coenzyme A oxidase in the liver; changes in the levels of plasma total triiodothyronine, total thyroxine, and thyroid-stimulating hormone (TSH) as markers of thyroid gland function; and hyaline droplet formation, tubular basophilia, and the formation of granular casts in the kidney. There were no single measurements that alerted specifically to the carcinogenicity of the agents to the rodent liver, thyroid gland, or kidney. However, in the majority of cases, the chemical induction of cancer in a tissue was preceded by a range of biochemical/morphologic changes, most of which were moderately specific for a carcinogenic outcome, and some of which were highly specific for it (e.g., increases in TSH in the thyroid gland and increases in relative liver weight in the mouse). The only measurements that failed to correlate usefully with carcinogenicity were the induction of liver enzymes (with the exception of the enzymes associated with peroxisome proliferation). Most of the useful markers were evident at the early times studied (7 days and 28 days), but no overall best time for the measurement of all markers was identified. The judicious choice of markers and evaluation times can aid the detection of potential nongenotoxic rodent carcinogens.