In Vivo Assays for Endocrine Disruptor
Amphibian Metamorphosis Assay (21d) (OECD 231)
The Amphibian Metamorphosis Assay (AMA) is a screening assay intended to empirically identify whether test item may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. The AMA represents a generalized vertebrate model to the extent that it is based on the conserved structures and functions of the HPT axis.
Fish Short Term Reproduction Assay (21d) (OECD 229)
The purpose of the study is to assess the effects of test item on survival, reproductive behavior, secondary sexual characteristics, fecundity and fertility of the freshwater fish species Pimephales promelas (fathead minnow) exposed to various concentrations for a period of 21 days under flow-through conditions. At the end of the test, measurements will be made that are reflective of the status of the reproductive endocrine system, including male secondary sex characteristics, gonadal histopathology, gonado-somatic index and plasma concentrations of vitellogenin (VTG).
Rat Hershberger Assay (OECD 441, OPPTS 890.1400)
The Hershberger assay is a rat screening assay aimed at identifying substances with androgenic or antiandrogenic properties. Androgens are important for the development, maturation and maintenance of the reproductive system, accessory sex organs, and the development of secondary and tertiary sex characteristics at puberty. Orchidoepididyectomized (castrated) peripubertal male rats have low physiological levels of endogenous androgens. The animals are then exposed to potential androgen agonists, antagonists, and 5α-reductase inhibitors and changes in the weight of five androgen-dependent organs (ventral prostate, seminal vesicle (plus fluids and coagulating glands), LABC, Cowper’s glands, and the glans penis) are evaluated. Test substance that act as androgen agonists cause increases in the weight of these organs, and those that act as androgen antagonists cause a relative decrease in the organ weights, when co-administered with a potent androgen such as testosterone propionate.
Rat Uterotrophic Assay (OECD 440, OPPTS 890.1600)
The uterotrophic assay is a rat screening assay aimed at identifying substances with estrogenic properties, measured in terms of an increase in uterine weight i.e., uterotrophic response. Estradiol is important for the development and maturation of the reproductive system, and also for the cyclical growth and regression of the female reproductive tract (uterus, cervix, and vagina) during normal female cyclicity. The conversion of androgens to estradiol, primarily in the ovary, promotes cell proliferation and maturation of these organs. In the uterus, early changes include increased electrolytes and water imbibition and subsequently an increase in cell division leading to uterine growth. Both changes contribute to increases in uterine weight. Females with low physiological levels of endogenous estradiol (intact immature females between weaning and puberty, and ovariectomized young adult females) are exposed to the test compound. Low baseline uterine weights in these females allow for a maximum range of response to an administered estrogenic substance. Test substances that act as estrogen agonists cause an increase in wet and blotted uterine weight. The use of this assay for antagonist detection is far less common, and is expected to result in a relative decrease in uterine weight, when co-administered with a potent estrogen such as 17β-estradiol.
Female and Male Pubertal Assays (OPPTS 890.1450, OPPTS 890.1500)
The male and female pubertal assay is aimed at identifying substances that have the potential to interact with the endocrine system, by identifying effects on pubertal development and thyroid function in the intact juvenile/peripubertal rat. Weanling rats are exposed to the test substance daily from postnatal day 22 through postnatal day 42 (females) or 53 (males) to detect alterations in attainment of puberty (vaginal patency and balanopreputial separation), and any potential alterations in thyroid function. Wet and blotted uterine weights, age at first estrus, length and quality of the estrous cycle (females) and wet and blotted (as applicable) accessory sex organs and testosterone levels (males), as well as alterations in thyroid hormone levels are evaluated. Clinical chemistry and organ weights are collected before histopathological examination of selected tissues, with emphasis on evaluation of thyroid and reproductive organs. Data are evaluated based on performance criteria set forth in the published guideline for vehicle control animals.
The use of performance criteria assures the sensitivity of the endpoints relative to other potential confounding factors. A weight of evidence analysis is conducted to assess the potential of the substance to disrupt the estrogen, androgen or thyroid hormone systems.