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Screens and Tests

The Environmental Protection Agency (EPA) has developed a battery of screens and tests to evaluate endocrine activity, the majority of which are animal-poisoning studies. The Endocrine Disrupter Screening Program (EDSP) is organized into two tiers: Tier 1 screening assays (the Tier 1 battery) that are intended to detect chemicals with the potential to interact with the estrogen, androgen, or thyroid hormone systems, and Tier 2 tests that are intended to determine whether such interactions result in adverse effects and establish a dose-response relationship. Test orders were issued at the end of 2009 on the first list of chemicals, and testing is now complete. The EPA is evaluating these results and plans to report on them in the summer of 2013.

Below is a summary of each of the screens and tests. Methods included in the Tier 1 battery have been finalized, and the EPA is in the process of developing and validating the Tier 2 tests. Click here for detailed information about each of the assays and for up-to-date information on the status of each Tier 2 assay in the validation process.

Tier 1 Screens

Tier 1 consists of 11 assays, five in vitro (non-animal) tests, and six in vivo (animal) tests. In vitro tests use proteins, cell lines, or tissues to examine biological activity on a microscopic level. In vivo tests use rats, mice, frogs, and fish, all of whom are killed at the end of experiments. In vitro studies save time and resources and use many fewer animals (for cells or tissue samples) or no animals at all.

In Vitro

  • Androgen Receptor (AR) Binding: Chemicals can affect the endocrine system by binding to hormone receptors, either to mimic the action of the natural hormone or block the access of the hormone to the site and thus block hormone-controlled activity. The current Tier 1 AR binding assay depends on the collection of a substance called cytosol from rats’ prostate glands. One lab conducting the assay reported killing about 10 rats per compound tested to collect sufficient cytosol. Evaluation of AR binding can be done with a non-animal, cell-based method—in fact, methods routinely used by contract laboratories and in laboratories outside the U.S. use a recombinant human AR that does not rely on animals at all. The current method is fraught with reproducibility problems that do not occur in the non-animal methods, and the EPA is the only agency or other entity worldwide that uses it.
  • Estrogen Receptor (ER) Binding: Like the AR assay, the ER binding assay uses animals—in this case, uterine tissue from female rats. About 13 rats per compound tested were used by one lab reporting its results. This Tier 1 screen could also be a completely non-animal, cell-based method that measures chemical binding to the estrogen receptor in vitro using recombinant human receptors. Instead, the EPA has chosen to use a method not used anywhere else in the world.
  • Estrogen Receptor (hERα) Transcriptional Activation (ERTA): The ERTA assay is a non-animal, cell-based method that measures transcriptional activation of the estrogen receptor in vitro by using a human-derived cell line (HeLa-9903). The method was developed and validated in Japan and approved by the Organisation for Economic Co-operation and Development (OECD) in 2009.
  • Steroidogenesis: The steroidogenesis assay is intended to detect interference with any steps leading to the production of male and female steroid sex hormones. It uses a cell line from human adreno-carcinoma cells (H295R). The OECD validation was completed in 2011. However, the EPA participated in the validation studies and accepted this method as being validated prior to this date.
  • Aromatase: Aromatase is an enzyme responsible for the synthesis of the hormone estrogen. The aromatase assay is a non-animal method that uses human recombinant microsomes to detect substances that inhibit aromatase activity. The EPA carried out its own validation studies of this method.

In Vivo

  • Uterotrophic: The uterotrophic assay examines the weight and microscopic appearance of the uteri 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 under the skin or into the stomach. Minimally, there are two dose groups and one control group, with six rats to a group. After three days of exposure to the test substance, the animals are killed and their uteri are removed and weighed. In practice, 50 female rats may be killed in this study per chemical tested because of additional tests to determine the maximum tolerable dose (MTD) or to test additional dose levels. 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 phytoestrogens in the diet or bedding 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 weights of several androgen-dependent tissues 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 at different dose levels for 10 days via oral gavages (in which a syringe or force-feeding tube is inserted into their stomachs). The animals are killed, and the weights of specific tissues are measured. At least 20 male rats are killed in this study for each chemical tested. This assay was validated through efforts of the Validation Management Group—Mammalian at the OECD.
  • 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 development of the vaginal opening is considered a possible sign of endocrine disruption). Young, unweaned female rats are force-fed a test chemical for 21 days when presumably the vaginal opening has appeared in all females. When the average age at vaginal opening is determined, the animals are killed, and their thyroid glands and ovaries are removed for further study. At least 45 female weanling rats are killed in this study for each chemical tested. This method was “validated” through EPA-sponsored work; however, the peer review of the validation studies was contentious: Serious concerns were raised about the specificity and reproducibility of this assay. Chemicals fed to rats must begin before post-natal day 22, or the results can be significantly skewed.
  • 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. Their reproductive and thyroid tissues are removed, weighed, and examined microscopically, and blood serum is collected for hormone analysis. At least 45 male weanling rats are killed in this study for each chemical tested. The EPA sponsored the validation exercise for this method.
  • Amphibian Metamorphosis: As frogs mature, they undergo a metamorphosis from their larval (tadpole) stage to adulthood, during which time their tails gradually disappear and are absorbed 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 320 tadpoles/frogs are killed in this study for each chemical tested. The EPA sponsored the validation exercises for this method; however, the peer review raised serious concerns regarding its variability and specificity. No chemical was negative in this assay, including the negative “control” chemicals. In addition, for chemicals with certain properties, it is difficult to maintain stable test concentrations in the tank water, possibly further skewing results.
  • Fish Screen: The fish screen assay is intended to examine abnormalities associated with survival, reproductive behavior, secondary sex characteristics, and fecundity (i.e., the number of spawns, the number of eggs/spawn, fertility, and the development of offspring). A test chemical is pumped into the water of tanks holding the fish, who are later killed and their bodies examined. At least 96 fish are killed in this study per chemical tested. The Validation Management Group—Ecotox at the OECD has been reviewing this method along with another closely related method. The OECD did not agree with the U.S. that the histopathology and fecundity endpoints were relevant and reproducible; however, the EPA insists on including these endpoints. Labs conducting this test on the first list of chemicals reported that changes in fecundity may not be specific to an endocrine mode of action but could be a result of stress, bacterial infections, and other factors. Again, the EPA sponsored the validation exercises for this method, but serious concerns were raised by the peer review regarding its variability and specificity. No chemical was negative in this assay, including the negative “control” chemicals. As with the amphibian assay, labs conducting the assay reported difficulties in maintaining stable test-chemical concentrations in tank water for some chemicals, with possible further skewing of results.

Tier 2 Tests

  • Larval Amphibian Growth and Development Assay: This assay is a long-term (130 days), chronic toxicity test using the Xenopus frog species that considers growth and development from fertilization through the early juvenile period and includes partial reproductive maturation. Endpoints evaluated during the course of the exposure include those indicative of generalized toxicity, mortality, abnormal behavior, and growth determinations (length and weight) as well as endpoints designed to characterize endocrine-specific modes of action targeting estrogen-, androgen-, or thyroid-mediated physiological processes. The EPA has nearly completed its validation of this assay.
  • Avian Multigeneration: The EPA is in the process of validating a multigeneration reproduction study using Japanese quail in order to evaluate and characterize potential adverse chemical effects on bird populations. The testing protocol contains each primary phase in the life cycle with isolated exposure at that phase as well as cumulative exposure over the multiple life stages. Initial exposure in the testing protocol occurs in proven breeders followed by continuous exposure to F1 offspring to evaluation of their F2 offspring as the final step in the testing protocol. As many as 5,500 birds may be killed in this study for each chemical tested. Click here to read PETA’s comments on this massive animal test.
  • Fish Multigeneration Test: The medaka multigeneration assay (MMT) involves the use of whole fish to characterize dose-response characteristics and adverse reproductive and developmental effects. The protocol currently being validated uses the medaka, a small freshwater fish, to evaluate reproductive fitness as an integrated measure of toxicant effects across 2½ consecutive generations starting with pairs of adult medaka. It also measures a suite of other endpoints for diagnostic and quantitative evaluation of endocrine disruptors or other types of reproductive toxicants. The MMT is a relatively long-term assay (29 weeks of in-life exposure) that assesses hatch, growth, survival, gonadal development, and reproduction in 2½ generations (F0, F1, and part of F2).  Several thousand fish will be killed in each experiment.
  • Invertebrate Lifecycle: The mysid two-generation toxicity test (MTTT) and harpacticoid copepod development and reproduction assay (HCDRT) involves the use of mysid shrimp and harpacticoid copepods to characterize dose-response characteristics and adverse reproductive and developmental effects. The MTTT protocol describes a two-generation toxicity test with an estuarine mysid that evaluates reproductive fitness in parents and offspring as an integrated measure of toxicant exposure. The HCDRT measures the (sub)chronic effects of chemicals on an estuarine/marine harpacticoid copepod species under semi-static conditions. Exposure occurs over the complete life-cycle, approximately 25 to 36 days, which is sufficient to observe any developmental-rate changes induced by the test.  These two invertebrate tests are also currently in the process of validation.
  • Mammalian Two-Generation: Mammalian two-generation reproductive toxicity studies in rats are common requirements for most pesticide chemicals for the purpose of characterizing and determining dose-response relationships for potential adverse effects on reproduction and development. Conventional two-generation studies have not routinely examined endocrine effects, so the study has been “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 for each chemical tested. As an alternate that uses fewer animals, the OECD-validated extended one-generation reproduction toxicity screen (EOGRTS) can be used instead.

What Is the Cost?

Performing all the Tier 1 battery tests for a single chemical uses 595 animals. Although some assays were waived for some chemicals based on existing data, the total number of animals killed in testing the first list of chemicals is calculated to have been 27,731. Most likely, more animals were killed in studies to determine MTDs in dose range-finding studies and in tests repeated when the first attempt did not meet performance criteria. Recent estimates have put the cost of Tier 1 testing and associated work at between $750,000 and $1 million per chemical if all the assays are required.

Click here for more information about animal testing in the EPA’s EDSP program.

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