Simple, rapid assays for conventional definite testing of endocrine disruptor hazard: Summary and recommendations*

Study protocols for the characterization of endocrine active compounds presented in Workshop 4 included the enhanced Organization for Economic Cooperation and Development (OECD) test guideline (TG) 407, the medium-term rat liver and rat multi-organ carcinogenicity assays, and an enhanced one-generation reproduction study. The outcome of rat studies on flutamide and ethinylestradiol indicated that these strongly active compounds can readily be detected even with a low animal number using the enhanced OECD TG 407. Both newly added (such as male accessory sex organ weights, histology of pituitary, vagina and male mammary gland) and already included parameters contributed to the detection of endocrine effects. Thorough evaluation of the results of 20 studies conducted with 10 compounds thought to interfere with the endocrine system by different mechanisms will identify the most appropriate enhancements to the current OECD TG 407. Medium-term rat liver and rat multi-organ carcinogenicity assays are well recognized in the International Conferences on Harmonization for Pharmaceutical Chemicals. They have been successfully used to detect carcinogenic and modifying potentials of new chemicals within a relatively short time and can be applied to endocrine active compounds. Dose–response studies on nonylphenol, bisphenol A, and styrene using the rat liver carcinogenicity assay did not reveal effects of any of these compounds on the development of preneoplastic lesions in rat liver. The enhanced one-generation reproduction study protocol included treatment of pregnant female rats from gestation day 0 through to lactation day 21, and examination of all offspring. Half of the animals were necropsied at weaning, the remaining animals were examined for vaginal opening, preputial separation, estrous cyclicity, and sperm characteristics and were necropsied at adulthood. In a pilot study ethinylestradiol inhibited maternal fertility at dose levels similar to those effective in the uterotrophic assay. It is recommended to rapidly evaluate the conducted enhanced OECD TG 407 studies and to enhance the current OECD TG 407 appropriately. Further compounds with different mechanisms of action should be studied in the one-generation reproduction study to further investigate the usefulness of this protocol. The established medium-term carcinogenicity assays can be used to study carcinogenic potential rapidly. Use of female animals and inclusion of carcinogens targeting at breast and uterus should be considered in order to explore further the predictibility of this model. *Report from a SCOPE/IUPAC project: Implication of Endocrine Active Substances for Human and Wildlife (J. Miyamoto and J. Burger, editors). Other reports are published in this issue, Pure Appl. Chem. 75, 1617–2615 (2003). ‡Corresponding author In the introduction to the workshop, Shoji Fukushima pointed to a number of areas that require particular emphasis when considering rapid assays for endocrine active substances (EASs) and the effects of these compounds on disease processes like reproductive lesions or neoplasia. First of all, and fundamental to the understanding of the mechanism of action of EASs, is the question of how an agent interacts with nuclear receptors or hormone synthesis, and which are the downstream events that then ensue. Related to this are metabolism of both EAS and endogenous hormones and the fate and potential activity of corresponding EAS metabolites and their environmental impact. In addition to studies on rodents, providing comprehensive information on a great diversity of endpoints, for human risk assessment due attention must be paid to any epidemiological evidence that may be available. It may also be helpful to take into account the effects of EASs in wildlife mammalian species should such information be available. Of particular importance are the dose response and the appropriate definition of threshold doses. The possibility of homeostasis playing a role also deserves special consideration. Clearly, appropriate choice of representative test compounds and screening is essential, and given the likelihood of species specificity mammals must be the animals of choice for testing. Furthermore, the complex influence of hormones and EASs, acting simultaneously on many organs and tissues, means that in vivo approaches must be given high priority if a comprehensive understanding is to be obtained. Attention must also be concentrated on the stage in life when sensitivity would be expected to be highest on the basis of mechanistic and developmental considerations. This underlies the selection of models for presentation in our workshop. In Workshop 4.1, by Freyberger et al., the enhanced OECD test guideline (TG) 407, was introduced. This subacute protocol for detecting endocrine modulation was discussed at the international level under the umbrella of the OECD, and the initial version was the basis for the reported feasibility study on flutamide (FLU). Furthermore, data on ethinyl estradiol (EE) using a modified protocol with fewer enhancements that is presently under investigation were presented for comparison. The enhancements considered were additional determination of: (a) thyroid-related hormones; (b) estrus cyclicity from vaginal smears starting in exposure week 4 to ensure necropsy of all females in the diestrus stage of the estrus cycle in week 5; (c) the number and morphology of cauda epididymal spermatozoa; (d) organ weights of ovaries, uterus, thyroid, male accessory reproductive organs, and pituitary; and (e) histopathological investigation of pituitary, male and female mammary gland, epididymes, pancreas. and vagina. For the study, groups of 5 male and 5 female rats were orally gavaged with FLU at 0, 1, 10, or 100 mg/kg body weight (b.w.)/day or with 0, 0.01, 0.05, or 0.2 mg/kg EE/day for at least 28 days. Two studies (A and B) were run in parallel to assess intra-laboratory variation and the potential increase in sensitivity with doubling of the animal numbers. Endocrine activity of both FLU and EE was readily demonstrated with the enhanced OECD TG 407 protocol. Relative weights of the male accessory sex organs (MASO) were consistently decreased by the highest doses of FLU and EE. Treatment-related increase of relative uterine weights in EE-treated animals was only observed with the combined data at the high dose, indicating that uterine weight in young adult animals is not a sensitive measure of estrogenicity. Consistent histopathological changes in studies A and B were atrophy of MASO and decreased tubular size of the epidiymus at 100 mg/kg FLU, along with increased numbers of PAS-positive cells from 10 mg/kg, hypertrophic basophilic cells at 100 mg/kg, and Leydig cell hypertrophy from 10 mg/kg, the latter indicating activation of the hypothalamic-pituitary-gonadal axis. Furthermore, microvesicular cytoplasmic vacuoles were observed in the zona fasciculata of the adrenals at the highest dose. With EE, similar atrophy of the MASO was recognized at the high dose, degeneration of the germinal epithelium and Leydig cell atrophy was apparent at 0.2 mg/kg, and feminization of the mammary gland was observed from 0.05 mg/kg. In contrast to the FLU study, reduced vacuolation in the zona fasciculata of the male adrenal at the high dose was seen with EE. In FLU-treated females, no endocrine-mediated histological changes were observed, while EE caused a striking discrepancy between the diagnosis of the stage of the female cycle by vaginal smear cytology (diestrus) and vaginal and uterS. FUKUSHIMA AND A. FREYBERGER © 2003 IUPAC, Pure and Applied Chemistry 75, 2479–2482 2480