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 a two-tiered screening program: Tier 1 screening assays (the Tier 1 battery) are intended to detect chemicals with the potential for endocrine activity (estrogen, androgen, or thyroid), and Tier 2 tests to determine whether such interactions adversely affect reproduction. The first phase of Tier I testing began at the end of 2009.

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 who are killed at the end of the experiment. 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 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 can be a completely 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. Unfortunately, the EPA has decided to use a method that uses an animal extract sourced from the prostates of rats. This 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 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 in which the ER is isolated from the uteruses of rats; again, this method is not used anywhere else in the world.

• Estrogen Receptor (hERa) Transcriptional Activation: The hERa transcriptional activation assay is a non-animal, cell-based method that measures transcriptional activation of the estrogen receptor in vitro 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. This assay uses a human cell line from human breast-cancer cells (H295R). The OECD validation is nearly complete; however, the EPA participated in the validation studies and has accepted this method as being validated.

• 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 uteruses 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. After several days of injections, the animals are killed and their uteruses are removed and 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 (the prostate and the seminal vesicle) 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 daily until the vaginal opening appears 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 30 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.

• 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 30 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 30 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.

• 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. In practice, at least 60 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. 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.

Tier 2 Tests

• 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.

• Avian 2-Generation: The EPA is developing a 2-Generation reproduction study using Japanese quail in order to evaluate and characterize potential adverse chemical effects on bird populations. 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 Lifecycle: The Fish Lifecycle assay involves the use of whole 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. 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.

• Mammalian 2-Generation: Mammalian 2-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 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 for each chemical tested.

Efforts are underway at the OECD to assess each of these methods, review them for relevance, and minimize the numbers of animals used. The OECD is also working to create a new mammalian reproductive test guideline that does not generate a second generation, saving the majority of animals used in the standard 2-generation assay.

What is the Cost?

Performing all the tests for a single chemical of the current Tier 1 battery would use a minimum of 536 to 554 animals and cost a minimum of $384,995 per chemical (conservatively estimated, with no range-finding or repeat experiments; Table 1). If all tests were performed for all 67 Phase I chemicals selected for Tier 1 screening, the first phase of the EDSP would likely use more than 35,000 animals and cost at least $25,794,665.

Table 1: Cost of Tier 1 Assays¹ and Number of Animals Used²

¹Low and high cost data was obtained from price lists for two contract research laboratories: Harlan (November 2009) and Smithers Viscient (January 2010).

²Estimated from relevant OECD Test Guidelines.