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Assays Under Consideration
EPA is currently developing and validating in vitro and in vivo assays to determine the potential for chemicals to cause endocrine disruption in humans or wildlife. EPA is using a two-tiered approach. The Tier 1 Screening battery, as proposed by EPA, is based on the Endocrine Disruptor Screening and Testing Advisory Committee’s (EDSTAC) recommendations and is intended to identify chemicals affecting the estrogen, androgen, or thyroid hormone systems through any of several recognized modes of action. Tier 2 Testing is intended to confirm, characterize, and quantify those effects for estrogen, androgen, and thyroid active substances. The assays are described below.
Visit Status of the Assays to view more details about each assay and for an update on the assay’s current status in the validation process.
Tier 1 Screening Assays
Amphibian (Frog) Metamorphosis - The Amphibian Metamorphosis assay involves the use of tadpoles to determine if chemicals affect the thyroid during metamorphosis and consequently result in developmental effects.
Receptor Binding in vitro Assays - Chemicals can affect the endocrine system by binding to hormone receptors to either mimic the action of the natural hormone or block access of the hormone to the site and thus block hormone controlled activity. The androgen receptor (AR) is involved in the development of male sexual characteristics and the estrogen receptor (ER) is involved in female maturation and reproductive function. Several receptor binding assays are being considered, including:
1) An AR binding assay that utilizes rat prostate cytosol to examine the ability of a test chemical to bind with androgen receptors;
2) An AR binding assay that utilizes a rat recombinant AR to examine the ability of a test chemical to bind with androgen receptors;
3) An ER binding assay that utilizes rat uterine cytosol to examine the ability of a test chemical to bind with estrogen receptors; and
4) An ER binding assay utilizes the alpha isoform of the human recombinant ER to examine the ability of a test chemical to bind with estrogen receptors.
Aromatase - Aromatase is an enzyme complex responsible for estrogen biosynthesis that converts androgens into estrogens, estradiol, and estrone. The Aromatase in vitro assay focuses on this portion of the steroidogenic pathway to detect substances that inhibit aromatase activity.
Fish Screen - The Fish Screen assay screens for estrogenic and androgenic effects. The assay examines abnormalities associated with survival, reproductive behavior, secondary sex characteristics, histopathology, and fecundity (i.e., number of spawns, number of eggs/spawn, fertility, and development of offspring) of fish exposed to test chemicals.
Hershberger - The Hershberger assay is designed to detect androgenic and anti-androgenic effects. In this in vivo assay, accessory sex gland weights, including several androgen-dependent tissues, are measured in castrated or immature male rats.
Pubertal Female - The Pubertal Female assay involves the use of rats to screen for estrogenic and thyroid activity in females during sexual maturation. This assay examines abnormalities associated with sex organs and puberty markers, as well as thyroid tissue.
Pubertal Male - The Pubertal Male assay involves the use of rats to screen for androgenic, anti-androgenic, and thyroid activity in males during sexual maturation. This assay examines abnormalities associated with sex organs and puberty markers, as well as thyroid tissue
Steroidogenesis - The Steroidogenesis in vitro assay detects interference with the body’s production of male and female steroid sex hormones. A version of the assay using sliced testis as a source of steroidogenic enzymes was optimized by EPA to detect chemicals that inhibit synthesis of steroid hormones, but continued concerns about being able to distinguish between compounds that inhibit steroid hormone synthesis and chemicals that kill the cells responsible for testosterone synthesis led to a halt in further work on validating this assay. This assay is being replaced by a cell-based assay using the H295R human adrenocortical carcinoma cell line. The H295R cell line also holds promise in being able to detect inducers of enzymes responsible for steroid synthesis in addition to chemicals that inhibit it.
Uterotrophic - The Uterotrophic assay involves the use of female rats to screen for estrogenic effects. In this in vivo assay, uterine weight changes are measured in ovariectomised or immature female rats.
15-day Adult Intact Male - The Adult Male assay involves the use of rats to screen primarily for anti-androgenic and thyroid activity. The assay will screen for abnormalities associated with primary and secondary sex organs, systemic hormone concentrations, and thyroid.
Tier 2 Testing Assays
Amphibian Development, Reproduction - The Amphibian Development, Reproduction assay involves the use of frogs to characterize dose-response characteristics and adverse reproductive and developmental effects.
Avian 2-Generation - The Avian 2-Generation assay involves the use of Japanese quail to characterize dose-response characteristics and adverse reproductive and developmental effects.
Fish Lifecycle - The Fish Lifecycle assay involves the use of fish to characterize dose-response characteristics and adverse reproductive and developmental effects.
Invertebrate Lifecycle - The Invertebrate Lifecycle assay involves the use of mysid shrimp to characterize dose-response characteristics and adverse reproductive and developmental effects.
Mammalian 2-Generation - The Mammalian 2-Generation assay involves the use of rats to characterize dose-response characteristics and adverse reproductive and developmental effects.
Tier to be determined
In Utero through Lactation - The In Utero through Lactation assay involves the use of pregnant rats to assess post-natal development of the neonate after in utero and lactational exposure.