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Screens & Tests

The EPA, in partnership with the chemical and pesticide industry and other world governments, is currently developing and validating approximately 14 new chemical screens and tests, the majority of which are animal-poisoning studies. The EPA has proposed to require chemical assessments in two separate phases, or tiers: Tier 1 screening is intended to detect chemical substances capable of interacting with the estrogen, androgen, or thyroid hormonal systems. Chemicals that interact with the above hormone systems may then be required to undergo Tier 2 testing to determine whether such interactions adversely affect reproduction.

Below is a summary of each of the screens and tests currently under development by the EPA and its counterparts in the 30-member-country Organization for Economic Cooperation and Development (OECD). Click here for detailed information about each of the assays and for up-to-date information on each assay’s status in the validation process.

Tier 1 Screens


• Uterotrophic – The Uterotrophic assay examines the weight and microscopic appearance of the uterus of female rats. An increase in uterine weight is considered to be an indicator of exposure to chemicals that mimic the hormone estrogen (which is associated with female sex characteristics). In this assay, either immature female rats or mature rats whose ovaries have been surgically removed are exposed to a test substance through force-feeding or injection beneath their skin or into their stomach. After several days of injections, the animals are killed and their uteri are weighed. In practice, at least 50 female rats are killed in this study per chemical tested; however, the OECD’s validation program alone is estimated to have killed 6,000 or more animals just to evaluate the assay’s performance. Moreover, the published results of this massive effort suggest that the Uterotrophic assay has failed the validation test (e.g., the amount of estrogen in the diet can skew the results, as can differences within and between laboratories in the manner in which the uterus is dissected and weighed). Click here to read a critique of the OECD’s validation and peer review program for the Uterotrophic assay.

• Hershberger – The Hershberger assay examines the weight of specific sex glands in male rats to screen for chemicals that suppress the activity of male sex hormones. Male rats are castrated, treated with the male sex hormone testosterone and exposed to a test chemical daily via oral gavages (in which a syringe or force-feeding tube is inserted into their stomachs). After several days, the animals are killed, and the weights of specific sex glands (prostate and seminal vesicle) are measured. At least 20 male rats are killed in this study per chemical tested.

• Enhanced Repeated-Dose – Studies in which animals are force-fed a substance daily for 28-days are a common requirement for the testing of pesticides and other chemicals. However, since conventional Repeated-Dose studies have not examined endocrine effects as a rule, the test is being “enhanced” and validated by the OECD and its member countries for use in screening chemicals for effects on endocrine organs (e.g., uterus, testes, thyroid, etc.). At least 40 rats are killed in this study per chemical tested.

• Pubertal Female – The Pubertal Female assay examines the age at which female rats reach “puberty” according to when their “vaginal opening” appears (which normally occurs in rats two weeks after weaning; thus premature vaginal opening is considered a possible sign of endocrine disruption). Young, unweaned female rats are force-fed a test chemical daily until the vaginal opening is attained in all females. When the average age at vaginal opening is determined, the animals are killed, and their thyroid gland and ovaries are removed for further study. At least 30 female weanling rats are killed in this study per chemical tested.

• Pubertal Male – The Pubertal Male assay examines the age at which male rats reach “puberty” and examines abnormalities associated with sex organs and secondary sexual characteristics. Young, unweaned male rats are force-fed a test chemical daily for one month, after which they are killed, and their reproductive and thyroid tissues are weighed and examined microscopically, and blood serum is collected for hormone analysis. At least 30 male weanling rats are killed in this study per chemical tested.

• Steroidogenesis – The Steroidogenesis assay is intended to detect interference with any steps leading to the production of male and female steroid sex hormones. Two approaches have been proposed to evaluate these effects: one using testes tissue from male rats (who are killed for this purpose) and another non-animal method using the same human cell-line as is being considered for the Aromatase assay outlined below.

• Aromatase – Aromatase is an enzyme responsible for the synthesis of the hormone estrogen. The Aromatase assay is a non-animal method that uses either human placental tissue or a human cell-line to detect substances that inhibit aromatase activity.

• Androgen Receptor (AR) Binding – 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 AR Binding assay is a non-animal, cell-based method that measures chemical binding to the androgen receptor in vitro using receptors isolated from human or rat cells.

• Estrogen Receptor (ER) Binding – The ER Binding assay is a non-animal, cell-based method that measures chemical binding to the estrogen receptor in vitro using receptors isolated from human or rat cells.

• Amphibian Metamorphosis – As frogs mature, they undergo a metamorphosis from their larval (tadpole) stage to adulthood, during which their tails gradually disappear back into the body (resorption). The Amphibian Metamorphosis assay measures the rate of tail resorption as a measure of thyroid action in the Xenopus frog. Over a two-week period, a test chemical is pumped into the water of tanks holding the tadpoles and the rate of tail resorption is measured by means of computer-aided video image processing. At least 30 tadpoles/frogs are killed in this study per chemical tested.

• Fish Screen – The Fish Screening assay is intended to examine abnormalities associated with survival, reproductive behavior, secondary sex character, and fecundity (i.e., number of spawns, number of eggs/spawn, fertility, and development of offspring). A test chemical is pumped into the water of tanks holding the fish, who are later killed and their bodies examined. In practice, at least 60 fish are killed in this study per chemical tested. However, because of “geo-political” differences between the U.S., Europe, and Japan in terms of the “preferred” species of fish for use in toxicity studies, the EPA and its partners are conducting parallel validation studies using three different species of fish (fathead minnow, zebrafish and Japanese medaka), which has tripled the animal body-count just to develop a validated Fish Screen.


Tier 2 Tests

• Mammalian 2-Generation – Mammalian 2-Generation reproductive toxicity studies in rats are common requirement for most pesticide chemicals for the purpose of characterizing and determining dose-response relationships for potential adverse effects on reproduction and development. As with the Repeated-Dose study outlined above, conventional 2-Generation studies have not routinely examined endocrine effects, so the study is being “enhanced” and validated by the EPA to serve as the so-called “definitive test” for adverse endocrine effects in humans. As many as 2,600 rats are killed in this study per chemical tested.

• Fish Lifecycle – The Fish Lifecycle assay involves the use of whole fish to characterize dose-response characteristics and adverse reproductive and developmental effects.

• Avian 2-Generation – For the purpose of evaluating and characterizing potential adverse chemical effects on populations of birds, the EPA is developing a 2-Generation reproduction study using Japanese quail. As many as 5,500 birds (Japanese quail) may be killed in this study per chemical tested. Click here to read PETA’s comments on this massive animal test.

• Amphibian 2-Generation – For the purpose of evaluating and characterizing potential adverse chemical effects on populations of amphibians, the EPA is developing a 2-Generation reproduction study using the Xenopus frog.

• Invertebrate Lifecycle – The Invertebrate Lifecycle assay involves the use of Mysid shrimp to characterize dose-response characteristics and adverse reproductive and developmental effects. Chemical exposure occurs via the water in which the shrimp are held. Exposure continues over the entire lifecycle, through development, maturation, reproduction, and early development of offspring.


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