Fish differ in steroid profiles from mammals ( e. g., 11 ketotestosterone as opposed to testosterone is the most important androgen in fish). The estrogen receptor in fish appears to differ structurally and functionally from the mammalian estrogen receptor ( Petit et al., 1995; Gustafsson, 1996). Also, steroid receptors in eggs and hepatic vitellogenin production have no known analogous receptors in mammals, which would suggest sites of endocrine disruption unique to oviparous animals. Therefore, this assay is essential to address these known endocrine differences. EDSTAC Final Report Chapter Five August 1998 5  29 F. Alternative Assays for Possible Inclusion 1. Placental Aromatase Assay One critical enzyme present at very low levels in the testis, and at higher levels in the ovary, uterus, and placenta, is aromatase, which converts testosterone to estradiol and is another P450 isozyme. Human placental aromatase is commercially available and could be used in vitro to assess the effects of toxicants on this enzyme fairly easily.

2. Modified Rodent 3 Day Uterotrophic Assay ( Intraperitoneal)

This is an in vivo assay ( O'Conner et al., 1996) for estrogenic activity in ovariectomized female rats. It can detect certain antiestrogens with mixed activity, i. e., those with some agonistic activity ( e. g., tamoxifen). The rats are injected intraperitoneally with the test agent daily for three days. The ip injection method may enhance the sensitivity of the assay and is capable of detecting the estrogenic potential of methoxychlor, which has been cited as an example of a compound not detectable by the sc route. The females are necropsied either 6 hours or 24 hours after the final treatment, depending on the protocol employed by the laboratory. Vaginal cytology is evaluated by vaginal lavage to determine whether the epithelium has become cornified, indicative of estrus. Presence of fluid in the uterine lumen is noted and recorded, and the number of animals that have fluid in the uterus is reported. Fluid imbibition ( uptake) is indicative of estrogenic potential. The uterus is excised and weighed. It is then preserved in an appropriate fixative for subsequent histological evaluation, if needed.

Subsequent histological evaluation will be triggered by an equivocal uterine weight or uterine fluid response ( i. e., an increase that is not statistically significant). This evaluation will consist of a characterization of the appearance of the uterine epithelium, a measurement of uterine epithelial cell height, and epithelial mitotic index or proliferating cell nuclear antigen ( PCNA) immunohistochemistry. Uterine cell height and cell proliferation are sensitive indicators of estrogenic potential.

3. 14 Day Intact Adult Male Assay This in vivo assay is intended to detect effects on male reproductive organs that are sensitive to antiandrogens and agents that inhibit testosterone synthesis or inhibit 5 alpha reductase ( Cook et al., 1997). The duration of the assay is anticipated to be sufficient to detect effects on thyroid gland activity. The rats are anatomically intact and mature; therefore, they have an intact HPG axis, allowing an assessment of the higher order neuroendocrine control of male reproductive function and the thyroid.

Young adult male rats ( 70 90 days of age) are used in this assay. They are dosed daily with the test agent for 14 days. The recommended route of administration is ip, which may, in some cases, maximize the sensitivity of the assay. They are necropsied 24 hours after the final dose. Immediately after sacrifice one cauda epididymis is weighed and processed for evaluation of sperm motility and concentration. The following organs are weighed: testes, epididymides, EDSTAC Final Report Chapter Five August 1998 5  30 seminal vesicles, and prostate. The following are fixed and evaluated histologically: one testis and epididymis, and the thyroid. The following hormones are measured in blood plasma: T4, TSH, LH, testosterone, DHT, and estradiol.

Empirical assessment of this assay has shown it to be sensitive to agents that are directly antiandrogenic, inhibit 5 alpha reductase, inhibit testosterone synthesis, or affect thyroid function. The sensitivity of this assay, as defined as the ability to detect a hazard, may be comparable to other assays that have been recommended.

4. Rodent 20 Day Thyroid/ Pubertal Male Assay This assay detects androgens and antiandrogens in vivo in a single stage apical test. " Puberty" is measured in male rats by determining age at PPS ( preputial separation). Animals are dosed by gavage beginning one week before puberty ( which occurs at about 40 days of age) and PPS is measured. Androgens will accelerate and antiandrogens and estrogens will delay PPS. The assay takes about 3 weeks, and allows for comprehensive assessment of the entire endocrine system in one study ( 10 per group, selected for uniform body weights to reduce variance). The animals are dosed daily, seven days a week, and examined daily for PPS. Dosing continues until 53 days of age; the males are then necropsied. The body, heart ( thyroid), adrenal, testis, seminal vesicle plus coagulating glands ( with fluid), ventral prostate, and levator ani plus bulbocavernosus muscles ( as a unit) are weighed. The thyroid is retained for histopathology and serum is taken for T4, T3, and TSH. Testosterone, LH, prolactin, and dihydrotestosterone analyses are optional. These endpoints take several weeks to evaluate and are affected not only by estrogens but by environmental antiandrogens, drugs that affect the hypothalamic pituitary axis ( Hostetter and Piacsek, 1977; Ramaley and Phares, 1983), and by prenatal exposure to TCDD ( Gray et al., 1995a; Bjerke and Peterson, 1994) or dioxin like PCBs ( Gray et al., 1995b). In contrast to these other mechanisms, only peripubertal estrogen administration accelerates this process in the female and delays it in the male. Preputial separation in the male rodent is easy to measure and this is not a terminal measure ( Korenbrot et al., 1977).

Age and weight at puberty, reproductive organ weights, and serum hormone levels can also be measured. Delays in male puberty result from exposure to both estrogenic and antiandrogenic chemicals including methoxychlor ( Gray et al., 1989), vinclozolin ( Anderson et al., 1995b) and p, p' DDE ( Kelce et al., 1995). Exposing weanling male rats to the antiandrogenic pesticides p, p' DDE or vinclozolin delays pubertal development in weanling male rats as indicated by delayed preputial separation and increased body weight ( because they are older and larger) at puberty. In contrast to the delays associated with exposure to estrogenic substances, antiandrogens do not inhibit food consumption or retard growth ( Anderson et al., 1995). Antiandrogens cause a delay in preputial separation and affect a number of endocrine and morphological parameters including reduced seminal vesicle, ventral prostate, and epididymal weights. It is apparent that PPS is more sensitive than are organ weights in this assays. In addition, responses of the HPG are variable. In studies of vinclozolin, increases in serum LH were a sensitive response to this antiandrogen, whereas serum LH is not increased in males exposed to p, p' DDE during puberty ( Kelce et al., 1997). Furthermore, a systematic review of the literature indicates that the sex accessory glands of the immature intact male rat are consistently more affected than in the adult intact male rat. EDSTAC Final Report Chapter Five August 1998 5  31 In summary, preputial separation and sex accessory gland weights are sensitive endpoints. However, a delay in preputial separation is not pathognomonic for antiandrogens. Pubertal alterations result from chemicals that disrupt hypothalamic pituitary function ( Huhtaniemi et al., 1986), and, for this reason, additional in vivo and in vitro tests are needed to identify the mechanism of action responsible for the pubertal alterations. For example, alterations of prolactin, growth hormone, gonadotrophin ( LH and FSH) secretion, or hypothalamic lesions alter the rate of pubertal maturation in weanling rats.

As indicated above, the determination of the age at " puberty" in the male rat are endpoints that already have gained acceptance in the toxicology community. Preputial separation in the male is a required endpoint in the new EPA two generation reproductive toxicity test guideline. In this regard, this assay would be easy to implement because these endpoints have been standardized and validated and PPS data are currently being collected under GLP conditions in most toxicology laboratories. In addition, PPS data are reported in many recently published developmental and reproductive toxicity studies ( i. e., see studies from R. E. Peterson's, J. Ashby's, R. Chapin's and L. E. Gray's laboratories on dioxins, PCBs, antiandrogens, and xenoestrogens).

Sex accessory gland weights in intact adult male rats also can be affected directly or indirectly by toxicant exposure. The HPG axis in an intact animal is able to compensate for the action of antiandrogens by increasing hormone production, which counteracts the effect of the antiandrogen on the tract ( Raynoud et al., 1984; Edgren, 1994; Hershberger, 1953).

IV. General Principles in Evaluating Tier 1 and Tier 2 Results A. Introduction Apart from substances yielding negative results in all assays, it is likely that most substances will produce a unique array of results requiring a judgment as to whether the weight of evidence indicates the substance should or should not be judged a candidate for T2T ( after completing T1S), and, designated as an endocrine disruptor for EAT ( after completing T2T). A table consisting of 18 chemical types along with known or expected T1S results can be found in Appendix P, Examples of " Weight of Evidence" Determinations.

There are two senses in which a " weight of evidence" determination will need to be made. The first is with respect to the question of whether consistent results are being obtained across multiple assays. If the results are not consistent, it will be necessary to " weight" the conflicting results, allowing some to carry more weight than others. The second sense is with respect to the question of whether a particular body of evidence, even if it is fully consistent, is sufficient to justify a decision. In this sense, it is the " weight" of the entire body of evidence, relative to some minimal level established as being required for sound decisions, that is being judged.

Assessing the " weight of evidence," and using that assessment in forming judgments about a substance, can be done in a variety of ways. On one extreme are approaches based solely on expert judgment in which an individual reflects on the data and offers an informed, yet personal, EDSTAC Final Report Chapter Five August 1998 5  32 opinion. On the other extreme are more formal and mathematical procedures such as Bayesian analysis in which data are viewed sequentially and used to formulate a priori and a posteriori judgments. An intermediate approach is one in which a group debates the available data, presents alternative arguments, and collectively reaches a judgment.

All three of these possibilities are forms of " weight of evidence" assessments. The EDSTAC has agreed not to prescribe a particular " weight of evidence" approach, as these are controversial and a matter of science policy to be established by the Agency. Instead, the EDSTAC offers general guidelines for reasoning from the data produced in the two tiers, which conform to the outline provided in the NAS/ NRC report Science and Judgment in Risk Assessment ( National Academy Press, 1994). These guidelines provide a framework within which one may take into account multiple features of relevant screening and testing data in determining whether the substance should be a priority for T2T ( after T1S) and/ or is determined to have endocrine disrupting effects ( after T2T).

" Weight of evidence" considerations will arise at two places within the EDSTP. It first will arise in considering whether the evidence collected solely within a given tier ( T1S) warrants a particular conclusion ( e. g., that the substance may have endocrine activity for EAT). The second place where it will arise is in considering whether results from the previous tier ( T1S) should affect the conclusions drawn from the subsequent tier ( T2T). By this, the EDSTAC is not referring to the fact that T1S results may guide selection and/ or design of Tier 2 tests ( with the results of the Tier 2 tests then being interpreted without further reference to the T1S results). The EDSTAC is, instead, referring to the possibility that the T1S assay results may be " weighted into" the determination of whether a substance has passed or failed the Tier 2 tests.

A broad range of results may need to be weighted into a final judgment at either tier. Information routinely taken into consideration in determining the " weight of evidence" will include:

· the balance of assays/ tests that gave positive and negative results; · results of in vitro versus in vivo assays/ tests; · the nature of the biological effects induced; · the range of effects observed; · the slope and shape of the dose response curves; · the level, magnitude, or severity of the effects induced; and · the presence or absence of response in multiple taxa.

The " weight of evidence" approach makes explicit the assumption that results of some assays/ tests, in some taxa, at some level of severity, are intrinsically " worth" more than others and should, therefore, carry more weight in decisions following T1S and T2T. For example, positive results showing reproducible, high levels of effects at low doses ( near the doses produced by environmental or human exposures) are likely of greater weight than weak effects observed only at very high, perhaps excessively toxic, levels of exposure.

The EDSTAC has taken the approach here of providing guidance on the use of " weight ofevidence Any approach used must satisfy several broad criteria which are essential. The EDSTAC Final Report Chapter Five August 1998 5  33 weighting system should be transparent, allowing individuals to review the " weight of evidence" determination. It should be possible to understand the procedure before viewing the data, so individuals have a reasonable expectation of the final decision at the time when the data are presented. This does not mean the decision is fully determined by the data, removing the need for scientific judgment, but it does mean that any deviations from the expected decision should be supported by an explanation detailing the " weight of evidence" assigned. EDSTAC Final Report Chapter Five August 1998 5  34 B. False Negatives and False Positives Within the Context of T1S and T2T The guiding principle for the treatment of false positives and negatives should be one of valuing sensitivity more than specificity at the screening level unless this compromises the ability to sort chemical substances or mixtures into a subset most likely to be of concern. False positives and negatives can arise in at least three different ways in the screening and testing batteries ( see Figure 5.1):

· The false result may be due to the stochastic nature of screens and tests. A false result leading to an incorrect claim that the screen/ test is positive is a Type I ( false positive) error. A false result leading to an incorrect claim that the screen/ test is negative is a Type II ( false negative) error. The frequency of these types of errors is expressed by the p value2 for an assay, so the selection of a required p value to classify a result as positive will determine the frequency of Type I and Type II errors. The guiding principle above suggests that required p values should be chosen so Type II errors are minimized, while also ensuring that Type I errors do not become so frequent that chemical substances or mixtures can no longer be sorted meaningfully.

· False positives and negatives may arise due to unknown or unexpected limitations of the test or assays, such as anomalous activity of chemicals or classes in a particular assay or interference from assay procedures.

· The third source of error arises from a potential lack of predictivity of results in T1S for endocrine disruptive responses in T2T. This source of error is shown in Figure 5.1 by the bold arrow going from positive results in T1S to T2T. A negative result in T1S may simply mean the assay battery misses a mechanism of action that would have been active in a Tier 2 Test. This will result in a false classification of the substance as not having endocrine activity, an error that would have been caught in T2T had the chemical substance or mixture proceeded to that stage. For this reason, the T1S battery was designed to capture all known endocrine mechanisms for EAT and to minimize false negative results specifically as opposed to false positives. A positive result in T1S could be followed by negative results in T2T because the endpoints measured in T1S may not accurately predict adverse effects in longterm whole animal tests. This will result in unnecessary testing of some chemicals in T2T, a possibility considered more acceptable than missing potential endocrine disruptors for EAT.

In treating the frequency of Type I and Type II errors, it is important to consider both the frequency of these errors in each particular assay/ test and the number of assays/ tests in a battery. As the number of assays/ tests in a battery increases, the probability that at least one of the assays/ tests will show a false positive increases. This is shown in Figure 5.2, which displays the 2 " P value" is the statistical probability that two groups, e. g., control and treated animals, come from the same population ( i. e., p= 0.05 means that the probability is 5 out of 100 that the two groups, based on values for a given parameter, belong to the same population and conversely, that 95 out of 100 do not belong to the same population and therefore they are significantly different). EDSTAC Final Report Chapter Five August 1998 5  35 relationship between the probability of any one assay/ test showing a false positive ( the X axis), the number of assays/ tests in the battery ( the Y axis), and the probability that the battery shows at least one false positive result. In this figure, it is assumed that the chemical substance or mixture tested actually has no endocrine activity, but might yield a false positive result due primarily to stochastic variation. The goal of the Tier 1 or Tier 2 stages should be to minimize the probability of a battery producing a Type II error for a chemical substance or mixture, while not causing the probability of a Type I error from getting so large that the battery becomes ineffective at sorting chemical substances or mixtures. Figure 5.3 displays the analogue of Figure 5.2 ( i. e., the probability that a battery produces a false negative result if each assay/ test has a given false negative frequency, there are N assays/ tests in the battery, and the chemical substance or mixture truly is an endocrine disruptor for EAT).

The EDSTAC cautions that the statistical properties of actual assays/ tests in a battery will not be identical, so Figures 5.2 and 5.3 are simply illustrative and must be modified for any particular battery developed. What the figures indicate is that weighting a single positive result from a battery into the " weight of evidence" judgment should reflect a concern for both Type I and Type II errors. From these figures, it can be seen that a large battery ( e. g., with 10 assays/ tests), each with a false positive frequency of only 10%, can result in a very high probability of producing at least one assay/ test showing a false positive when applied to a substance that in reality has no endocrine disrupting properties. Such a battery would be essentially useless in sorting chemical substances or mixtures and focusing society's resources. The final advice here is that an effort should be made to characterize statistically the frequency of Type I and Type II errors associated with any selected battery, and to use this characterization in deciding the weight assigned to a single positive result from that battery.

C. Specific Principles for Evaluating T1S There are several specific criteria to be met by the decision process assuming appropriate dose and route of exposure as discussed previously in this chapter:

1. If functionally equivalent information is available ( e. g., from the sorting and prioritization phase), it may be appropriate that only those T1S assays which evaluate the endocrine activity of concern ( based on prior information) of a chemical substance or mixture would be performed ( i. e., only a subset of assays would be run). Similarly, the results of the T1S assays may require that only a subset of the Tier 2 tests be conducted.

2. If all assays are performed, and all assays are negative, then the chemical substance or mixture does not have endocrine activity for estrogen, androgen, or thyroid hormone at this time. EDSTAC Final Report Chapter Five August 1998 5  36 EDSTAC Final Report Chapter Five August 1998 5  37 EDSTAC Final Report Chapter Five August 1998 5  38 EDSTAC Final Report Chapter Five August 1998 5  39 3. In vitro assays cannot and will not be " gatekeepers;" they cannot constitute a " decision node;" they are useful as information for possible mechanisms ( or site of action) but not as " yes/ no" determinants to proceed to the in vivo screens or T2T because: a) in vitro assays mediated by receptor binding evaluate only one of many possible sites and modes of action; b) negative results may mean relatively little due to limitations of the assays, e. g., lack of metabolic capability, solubility, etc. ( i. e., false negatives); and c) positive results may be false positives.

4. Results from in vivo assays have more weight than results from in vitro assays since: a) in vitro assays will generate false negatives as well as false positives, based on differences in access to the target tissue, metabolism, etc. relative to in vivo assays; and b) in vivo results are considered to be more relevant.

5. Results from in vitro assays that assess endocrine activity with and without metabolic activation are worth more than results from in vitro assays without metabolic activation ( since the former can assess the activity of metabolites generated within the culture if the correct metabolic activation is used ( e. g., rat liver S9) and the latter can only assay the parent compound).

6. Results from apical in vivo assays are worth more than results from specific in vivo assays ( since they indirectly assay many more sites of action to get to the same endpoint; e. g., uterotrophic assay in ovariectomized adult females [ specific assay; chemical substance or mixture must act at level of uterus] versus in intact immature females [ apical assay; chemical substance or mixture can act at level of hypothalamus, pituitary, gonad, thyroid, and/ or uterus]). A positive specific assay provides mechanistic information but other mechanisms of action may also be present and go undetected; a negative specific assay is less informative.

7. Biologically plausible results are worth more than biologically implausible results ( obviously dependent on the state of current scientific knowledge).

8. Statistical significance is a useful tool, but must be interpreted within the context of biological significance. For example, an observed association which does not achieve statistical significance, but which is consistent with results from related assays suggesting a common mechanism of action, might be interpreted as biologically significant. This means the use of any particular criterion such as p equal to 0.05 should be carefully considered, and there may be no hard and fast rule for weighting by statistical significance.

9. A consistent pattern of positive ( or negative) results in various related assays is worth more than a single isolated positive ( or negative) result ( e. g., positive results for binding to ER and transcriptional activation in vitro and positive results in an apical or specific uterotrophic assay in vivo are worth more than a positive result for receptor binding and transcriptional activation, but no uterotrophic response) ( see additional comments in discussion of false negatives and false positives above). EDSTAC Final Report Chapter Five August 1998 5  40 10. The decision which will emerge from T1S is: a) The chemical substance or mixture does not require further testing for EAT activity at this time ( the chemical substance or mixture goes to the " hold box"); or b) The chemical substance or mixture should be tested further for EAT activity at this time, and i) proceed to T2T; or ii) proceed to Hazard Assessment/ Regulatory Action.

V. Tier 2 Testing Concepts and Design Parameters A. Introduction to T2T The purpose of T2T is to characterize the nature, likelihood, and dose response relationship of endocrine disruption of estrogen, androgen, and thyroid in humans and wildlife. T2T is a complement to T1S and T2T results supersede T1S results. As already discussed, T1S is composed of a battery of in vitro and in vivo assays designed to detect whether a substance may have EAT activity. The in vitro screening assays are highly sensitive and quite selective for a particular mode of action. They are, however, quite far removed from the biological complexity of an intact animal and may give false positive readings because, for instance, not all substances which bind to a receptor will cause an adverse biological effect; false negative readings may also result from the in vitro receptor binding or transcriptional assays because not all endocrine disruptors act via a receptor. In vivo assays encompass the metabolic and response capability of a whole organism but focus on such a short time frame that the full effects of exposure to a chemical substance may not be identified and characterized. Since there is considerable biological conservation in the endocrine system, it is not necessary to screen in every major taxonomic group. Screens based on mammalian cell lines or intact animals will determine whether a chemical substance or mixture may interact with the endocrine system, and if so, the effects of the chemical substance or mixture must be characterized in longer term studies in species representing a variety of taxa.

T2T is the final phase of the screening and testing program and is intended to provide more detailed information regarding endocrine disruption activity of a tested chemical substance or mixture. This tier should assess the concentrations which elicit endocrine disruption and the consequences of such disruption to inform hazard assessments. To fulfill this purpose, tests are longer term studies designed to encompass critical life stages and processes, a broad range of doses, and administration by a relevant route of exposure, so a more comprehensive profile of biological consequences of chemical exposure can be identified and related to the dose or exposure which caused them. Effects associated with endocrine disruption may be latent and not manifested until later in life or may not appear until the reproductive period is reached. Tests for endocrine disruption will usually encompass two generations including effects on fertility and mating, embryonic development, sensitive neonatal growth and development, and transformation from the juvenile life stage to sexual maturity unless a rationale exists to limit the mammalian test to one generation. EDSTAC Final Report Chapter Five August 1998 5  41 The outcome of T2T is designed to be conclusive in relation to the outcome of T1S, and any other prior information, in the sense that a negative outcome in T2T will supersede a positive outcome in T1S. Furthermore, each full test in T2T has been designed to include those endpoints that will allow one to reach a definitive conclusion as to whether or not the tested chemical substance or mixture is or is not an endocrine disruptor for EAT in that species/ taxa. Conducting all five tests in the T2T battery would provide a more comprehensive profile of the effects a chemical substance or mixture could induce via EAT endocrine disruption mode( s)/ mechanism( s) of action than would be the case if only a subset of tests were performed. The EDSTAC recommends that the " default" action, in the absence of any prior information, be to perform all tests in the T2T battery with all endpoints. However, performance of the entire battery with multiple generations may not always be necessary, as is discussed below.

The next section of this report provides guidance on the selection of the taxa to be subjected to T2T, and the use of alternative tests, focusing particularly on alternative mammalian tests. Conducting Tier 2 tests on less than the five recommended taxa and/ or the use of an alternative mammalian test will result in T2T outcomes that are, by definition and design, less comprehensive in comparison to the outcomes from T2T using the recommended two generation mammalian test and/ or the full set of recommended taxa. However, the EDSTAC believes the guidance set forth below will ensure that the information generated from tests conducted with less than the full set of recommended taxa and/ or using alternative Tier 2 tests will still be sufficient, along with prior information, for EPA to make a decision about whether the chemical substance or mixture should be placed in the " hold box" or forwarded to hazard assessment.

While two generation tests are designed to fully characterize potential effects of concern, there may be instances ( depending on available prior information) when a less comprehensive study design would provide adequate information on which to make decisions. In addition, there may be reasons why all of the non mammalian tests need not be conducted in the same time frame, or at all. Below is a discussion of how the decisions of which tests to perform should be informed.

In determining which mammalian and non mammalian tests to include in T2T, the EDSTAC chose tests which would require the least modification from existing standardized methods in order to minimize the time necessary to standardize and validate those tests. Thus, the Tier 2 tests for mammals ( rats), fish, birds, and invertebrates are based on existing test guidelines ( the recommended study design for amphibians has never been utilized in a testing scheme). The existing test guidelines were considered adequate for evaluating the most obvious consequences of EAT related endocrine disruption, but not adequate to pick up some of the more subtle or insidious effects. However, more modification of the non mammalian guidelines was required than for their mammalian counterparts and the elements described in the existing non mammalian guidelines, in general, have not been widely and routinely run, as have the elements described in the mammalian guidelines.

B. Outline of Recommended T2T Battery EDSTAC Final Report Chapter Five August 1998 5  42 The EDSTAC recommends that the T2T battery include a mammalian two generation reproductive toxicity study, or a less comprehensive mammalian test in accordance with guidelines outlined below, and tests addressing four additional taxonomic groups, including birds, amphibians, fish, and invertebrates as follows.

Mammalian Tests 1. Two Generation Mammalian Reproductive Toxicity Study; or 2. A Less Comprehensive Test: a) Alternative Mammalian Reproductive Test; or b) One Generation Test.

Multigeneration Tests in Other Taxa 1. Avian Reproduction ( with bobwhite quail and mallard) 2. Fish Life Cycle ( fathead minnow) 3. Mysid Life Cycle ( Americamysis) 4. Amphibian Development and Reproduction ( Xenopus)

C. Guidance for Selecting Tier 2 Tests The Conceptual Framework, found in Chapter Three, states that existing information on biological effects and exposure and the results of T1S should be used to inform decisions regarding the selection and design of Tier 2 tests. The EDSTAC believes that T1S information may be of use in determining whether to evaluate thyroid effects, for example, but may be of limited value in determining whether all five, or some subset of the recommended Tier 2 tests should be conducted since only a limited number of taxa are recommended for the in vivo assays and mammalian cell lines are used for in vitro assays in T1S. Therefore, the EDSTAC recommends that the choice of whether Tier 2 tests will be conducted on all five of the recommend taxa, or a more limited subset of the five taxa, should be based on the physicochemical characteristics and environmental release and exposure information of the chemical substance or mixture to be tested, together with biological data from T1S.

This section addresses the need for guidance in the selection of Tier 2 tests, focusing upon: ( 1) the determination of which of the five taxonomic groups should be included in the Tier 2 testing of a specific chemical substance or mixture; ( 2) the circumstances under which it may be appropriate to perform an alternative test, with a particular focus on the selection of alternative mammalian tests; ( 3) the selection of endpoints; ( 4) the special case of chemicals that bypass T1S and go directly to T2T; and ( 5) the potential need for supplemental information to complete T2T. EDSTAC Final Report Chapter Five August 1998 5  43 1. Determining Which Taxa Should be Included in Tier 2 Testing for a Specific Chemical In general, the number of studies underway at any one time is limited, in part, by laboratory capacity. In addition, the pace at which each study can be completed is controlled by the nature of the protocol for the study. Priority should be given to conducting tests for those species/ taxa to which exposure is known or expected to occur. The EDSTAC recommends that in the absence of information, as described below, the default assumption should be that chemical substances or mixtures should be subjected to the full set of five recommended tests with all endpoints, once they are fully standardized and validated. Building on these overarching principle, the EDSTAC recommends that EPA should use the following guidelines in determining which taxa/ tests should be included T2T for a specific chemical substance or mixture:

a) Where use, exposure, and release of a substance are well characterized, it may be possible to tailor T2T for particular exposure scenarios. Conversely, if they are poorly characterized, positive results in T1S would trigger the entire battery of Tier 2 tests unless other data clearly indicated that certain taxa would not be affected. As noted above, the EDSTAC recommends the default assumption, in the absence of information that certain taxa would not be affected, be to conduct all five of the recommended tests with all endpoints.

b) If sufficient exposure data are available, the owner of the chemical may conduct an exposure assessment to provide information that will help inform the selection of Tier 2 tests. Exposure assessments vary in scope and complexity, but, for this purpose, " sufficient data" at a minimum includes chemical identity, basic chemical properties ( water solubility, octanol: water partition coefficient, vapor pressure, Henry's Law constant), rates of significant transformation processes such as biodegradation, and use and release profiles, including accidental releases. Measured values are preferable for chemical properties and environmental fate, but estimation methods are often satisfactory for supplying missing data. Ideally, the chemical use and release profile should provide information on the distribution of releases ( if any) to air, water, soil, etc., and the amounts and frequency of such releases.

For example, freshwater aquatic exposure modeling often can be conducted using models which incorporate environmental degradation processes. One such model, the Probablistic Dilution Model, yields the frequency of exceedance of an ecological concern level preset by the modeler, and is useful not only when releases are from known point sources but also when only the category of use ( via Standard Industrialization Classification Codes) is known. At a higher level of complexity, even site specific models such as EXAMS II may be appropriate under some circumstances, for example, when there are only a few point sources and the site of release and downstream environments are well characterized.

Soil fate models such as SESOIL and PRZM exist but also have extensive parameterization requirements. Short of this, rough estimates of mobility in soil and thus likelihood of reaching groundwater or surface waters that are in hydrological contact with ground water can be EDSTAC Final Report Chapter Five August 1998 5  44 made using certain screening level tools routinely applied by EPA in, for example, PMN review.

For air releases numerous fate models such as ISCLT are available, and like the water models, they vary in complexity. They calculate concentrations of chemicals in air at assumed locations of human receptors ( e. g., at certain distances from stack releases), but such exposure data may also be used as input for aquatic or terrestrial ecological exposure assessments.

Models for calculating environmental concentrations of chemicals released to estuaries are less well developed in general, but the model ESTUARY contains extensive hydrologic data for several major estuaries in the U. S. and is potentially useful. It was designed for high volume consumer products that are widely used and dispersed in the environment ( i. e., surfactants).

c) If a chemical is released to or can be predicted to reach streams, rivers, or lakes, a fish life cycle test with freshwater species and invertebrate life cycle test should be conducted. If release is to an estuary, marine species should be substituted for freshwater species in toxicity testing. If release is to both types of environment, freshwater species are preferred. In a broad sense, freshwater fish species are often more sensitive than saltwater fish species in laboratory toxicity tests. The greater sensitivity is due, in part, to greater bioavailability in freshwater which has fewer dissolved solids than brackish or saltwater. Moreover, currently freshwater species are more widely available and somewhat easier to accommodate in laboratory settings than saltwater species. Species selection, independent of taxa, should be performance based, and as more appropriate species are developed for use and validated, the EDSTAC strongly encourages their use.

d) Pesticides with agricultural or other outdoor uses, and chemicals that would be expected to bioaccumulate and biomagnify through the food chain or that present a potential exposure to birds and aquatic organisms, should be tested in the appropriate tests. Alternatively, if the pesticide or other chemical is not likely to be used in a manner that results in exposure to birds and aquatic organisms, it does not need to be tested in either the avian or aquatic tests. For example, as per current regulatory requirements under FIFRA ( 40 CFR 158), this would be the case if a pesticide registrant is requesting the first use of a pesticide only for application to flowers grown only in greenhouses. Since a greenhouse is considered a confined use situation, the studies to assess the impact on birds or aquatic organisms would not be required. If, at a later time, the registrant requests additional uses, then additional studies might be required. In this example, assume the second use is for application to turf. Then, impacts on birds and aquatic organisms would be of concern, and therefore studies on those organisms would be necessary.

A similar situation exists for commercial chemicals under TSCA. A manufacturer may supply information to EPA that a substance is a site limited intermediate with no significant releases to the environment. In this case, the exposure of concern would be exposure to workers and the appropriate test would be the mammalian reproduction test. If new uses were developed for the chemical which could result in environmental exposure, the ecological tests could be EDSTAC Final Report Chapter Five August 1998 5  45 required. The rationale for testing should not be limited solely to use considerations but should also consider potential releases from manufacturing, storage, transportation, and disposal.

e) Finally, as noted in Chapter Three, if a chemical is placed in the " hold box" based on the results of Tier 2 tests that are conducted on less than the full set of the five recommended taxa/ tests, and the use and exposure patterns for the chemical substance subsequently change, EPA should determine whether the new use or uses warrant testing in one or more of the taxa that had previously not been included in Tier 2 testing of the chemical.

2. Determining When to Perform Alternative Tests In Chapter Three the EDSTAC sets forth a definition of " function equivalency." In presenting the guidelines for determining when it may be appropriate to perform alternative Tier 2 tests, the EDSTAC wishes to emphasize the difference between alternative tests that meet the definition of functional equivalency from those that do not. As stated in Chapter Three, an assay, test, or endpoint can be considered to be " functionally equivalent" to a T1S or T2T assay, test, or endpoint when it provides equivalent information for each endpoint being studied. Furthermore, the EDSTAC recommended assays, tests, and endpoints must be validated and standardized prior to EPA's use of functionally equivalent information.

The EDSTAC recommends that, as new tests are developed and proposed for inclusion in T2T, EPA should determine whether such tests are functionally equivalent to the recommended Tier 2 tests. If they are determined not to be functionally equivalent, the decision about whether and, if so, under what condition, any newly developed alternative tests should be used, should be subjected to the criteria listed below.

This section sets forth guidelines for determining when to conduct alternative tests, which are less comprehensive than the recommended tests, and therefore do not meet the definition of " functional equivalency."

The EDSTAC believes there are instances when a less comprehensive test for any of the five recommended taxa, when considered along with existing information, would provide sufficient information upon which to make a decision that meets the overall purpose of T2T ( i. e., whether to go to the hold box or hazard assessment). The EDSTAC identified two alternative study designs for mammalian species. The EDSTAC believes such alternative designs could be developed for all species currently included in T2T. When and if such alternative tests are developed for the other taxa, in making the decision to perform a less comprehensive test EPA should consider the same issues, and apply the same principles that are described below for the alternative mammalian tests. The EDSTAC recommends alternative study designs be developed for other species/ taxa only after their respective multi generation study protocols are validated and standardized.

The primary considerations for determining whether an alternative, less comprehensive test should be conducted include an understanding of the toxicity profile of the chemical substance or mixture EDSTAC Final Report Chapter Five August 1998 5  46 under study; its mechanisms of action; exposure scenarios; use patterns; populations at risk; and other prior information. The criteria for determining when an alternative test might be run include: ( 1) a full two generation reproductive toxicity study has been run in the past, but it either was conducted in accordance with the " old" guideline and/ or the results in the previous study require additional follow up that may be accomplished using one of the alternative protocols; ( 2) production volume and potential for exposure is low; or ( 3) there is low probability of at risk populations being exposed. In applying criteria 2 and 3, the alternative test may be used more as a preliminary evaluation and, therefore, not necessarily as the last evaluation of the potential for EAT effects in a reproductive toxicity study. Furthermore, the EDSTAC wishes to note that chemical substances determined to be a high priority for the early phases of T1S based primarily on exposure considerations are not likely to meet these criteria; whereas the chemicals permitted to bypass T1S due to existing statutory requirements ( i. e., food use pesticides) will more often than not meet the first of these three criteria.

Below are potential scenarios for a specific chemical substance or mixture that requires a decision on whether to perform the two generation mammalian test or one of the alternative tests. These examples pertain only to the mammalian reproduction tests as they are the only ones currently discussed in the report.

a) A chemical substance or mixture has not been through T1S, and thus, a weight of evidence evaluation cannot be performed. One may or may not have prior information which offers an understanding of the chemical substance or mixture's potential to show endocrine activity for the EAT hormone systems. In this case, the EDSTAC recommended two generation reproduction study would be conducted for each taxa/ species which has been identified as being exposed or likely to be exposed. Whether or not all four of the non mammalian tests would be performed should be determined in accordance with the principles described in Section V, C, 1 of this chapter.

b) A chemical substance or mixture has been through T1S and the weight of evidence evaluation concludes it does not have endocrine activity for EAT. The chemical substance or mixture has use patterns suggesting no or low exposure potential to populations of concern. In this case, no further screening or testing for EAT would be needed. However, a decision might be made to ask for a reproductive toxicity study to satisfy other regulatory requirements. One might argue that an alternative test protocol would provide sufficient information at this time since it is important to evaluate the potential for inducing reproductive effects of concern that may have other underlying mode( s)/ mechanism( s) of action. Obviously, depending upon the outcome of the study and later discovery of additional relevant information, additional, follow up studies could be required. EDSTAC Final Report Chapter Five August 1998 5  47 3. Determining Selection of Endpoints In addition to guidance regarding selection of tests, the EDSTAC believes it is necessary to offer some guidance related to selection of which endpoints to include in performance of the tests for each chemical substance or mixture. Below are some potential scenarios regarding selection of which particular endpoints should be evaluated.

a) A chemical substance or mixture has been through T1S and the " weight of evidence" evaluation concludes it may have endocrine activity in all three hormone systems ( E, A, and T). The chemical substance or mixture has use patterns suggesting exposure could possibly occur to populations of concern. In this case, the two generation test, including the thyroid related enhancements ( recommended later in the report), should be conducted.

b) A chemical substance or mixture has been through T1S and the " weight of evidence" evaluation concludes it may have endocrine activity for E and A, but not for T. The chemical substance or mixture has use patterns suggesting exposure could possibly occur to populations of concern. In this case, it would be appropriate to require the EDSTACrecommended mammalian two generation test, without the recommended enhancements for evaluating T. In other words, the study design would be tailored to provide results only for the endpoints of concern identified in T1S.

4. Chemicals That Bypass T1S and go Directly to T2T In Chapter Three, the EDSTAC makes a distinction between two different scenarios under which a chemical would be permitted to bypass T1S and go directly to T2T. The first includes chemicals that have previously been subjected to two generation mammalian and wildlife developmental and reproductive toxicity testing. The second includes chemicals for which the owner of the chemical has decided to voluntarily go to T2T without having completed the full T1S battery or any prior two generation reproductive toxicity testing. The EDSTAC recommends elsewhere that both categories of " bypass chemicals" should be required to complete the HTPS assays.

T1S assays, in the aggregate, provide preliminary information on the presence of endocrine activity for EAT, the mechanism of action, and the species and sex at risk. The EDSTAC believes the absence of such information in the case of chemicals that voluntarily bypass T1S without having completed the full T1S battery or any prior two generation reproductive toxicity testing provides sufficient justification for requiring such chemicals to complete all five tests in the T2T battery ( i. e., the two generation mammalian and non mammalian tests with all the recommended endpoints). However, the determination of whether all of the non mammalian tests would need to be conducted should be made consistent with the principles governing their selection, which were set forth in Section V, C, 1 of this chapter.

For chemicals that bypass T1S because they have previously been subjected to two generation mammalian and wildlife developmental and reproductive toxicity testing, as noted above, the EDSTAC Final Report Chapter Five August 1998 5  48 EDSTAC believes these chemicals are the primary candidates for meeting the criteria specified for possible use of alternative Tier 2 tests. Once again, the determination of whether all of the nonmammalian tests would need to be conducted should be made consistent with the principles governing their selection, which were set forth in Section V, C, 1 of this chapter.

The EDSTAC recognizes it may be necessary, after completion of T2T, to conduct a limited number of assays that are similar, if not identical, to those that would have been conducted during T1S for chemicals which are permitted to bypass the T1S battery. The purpose of conducting these assays as part of T2T is to gain knowledge about specific mechanisms of action that are necessary to complete the hazard assessment step and/ or to determine whether any adverse effects observed in T2T are in fact endocrine mediated.

5. Potential Need for Supplemental Information to Complete T2T The EDSTP focuses on identifying agents that act as reproductive or developmental toxicants through the EAT endocrine mediated mechanisms. Properly conducted, Tier 2 tests are intended to be the final arbiter of whether or not a substance is an endocrine disruptor for EAT. In other words, when the results of T2T are unambiguous, they provide a conclusive answer. Given the current definition of testing, both the expression of " endocrine mediated" and " adverse effects" are necessary conditions for designation as an endocrine disruptor for EAT. In some cases, particularly for those chemicals that have received a positive in T1S, it will be apparent that the type of effect seen in T2T is endocrine mediated. In other cases, particularly for those chemicals that bypass T1S, the results of T2T may not allow a judgment to be reached that the adverse effect is endocrine mediated. The EDSTAC recommends that such information, if determined to be necessary to achieve a " weight of evidence" judgment, should be generated through further study using more focused assays, some of which may be drawn from the T1S battery.

Furthermore, it is inevitable with toxicological testing that equivocal results will sometimes be obtained in T2T. When this occurs one must look at the possible reasons for the ambiguity and see if there are ways to resolve it. Conducting other tests, or running assays to investigate the mechanism of action ( if T1S was bypassed), may resolve the ambiguity and allow a more informed " weight of evidence" determination of the hazard potential of the chemical substance or mixture. Alternatively, repeating the study, perhaps at different exposure levels, may resolve the ambiguity.

D. Low Dose Considerations for T2T 1. Introduction to the Issue Issues have been raised regarding approaches to regulatory toxicity testing that employ dosing regimens up to maximally tolerated dosages in order to identify a hazard and extrapolate from these doses to estimate risk or safe levels in the range of environmentally relevant exposures. There are two principal issues: ( 1) whether or not a threshold dose exists for receptor mediated toxicity; and ( 2) whether the dose response curve is monotonic or non monotonic in nature. EDSTAC Final Report Chapter Five August 1998 5  49 With respect to the first issue, some believe that there is no threshold for effects of exogenous endocrine disruptors since there exist background levels of endogenous hormones that are already biologically active. Thus, any additional exposure constitutes an exceedance of the threshold ( e. g., McLachlan et al., 1982; Wibbels et al., 1991; Russo, 1996; Gray et al., 1997b).

The second issue relates to the shape of the dose response curve. Although monotonic curves may vary in slope, the slope of such curves is always in the same direction ( either positive or negative including zero) and therefore there are no local maximum or minimum points along the curve. With non monotonic dose response curves, local minima can exist such that an effect may be pronounced at low doses, then becomes statistically insignificant or disappears at intermediate doses, and finally reappears ( or a different effect appears) at higher doses. The issue raised by non monotonic curves is that " high dose" testing may fail to detect toxicity that occurs in the " low dose" region of the dose response curve. This is because the classical approach to finding the NOAEL by progressive reduction of the dose beginning at high doses will locate the nadir ( i. e., a local minimum at some intermediate dose) but will not locate the second region of increased effect at doses below the apparent NOAEL. This issue is further complicated by the possibility of different effects at low doses as compared to high doses. There are examples of this phenomenon for endogenous hormones. For example, it is well known that testosterone stimulates sperm production up to a point, but at excess levels inhibits it. There is evidence that the developing mouse prostate responds in a non monotonic manner to estrogens, in that prostate weight is increased initially, then decreased by higher maternal dosages of potent estrogens like DES and ethinyl estradiol. There is one report in the literature indicating that the weak estrogen bisphenol A has effects similar to these potent estrogens at low doses ( Nagel et al., 1997). There is intense scientific debate surrounding these issues that centers on two principal questions: first, are data implicating xenobiotics in such phenomena reproducible and broadly generalizable to endocrine endpoints and endocrine active chemicals? and second, is the low dose phenomenon indicative of adverse effects at the individual or population level? If low dose phenomena are reproducible, generalizable, and related to adverse effects, the implications for regulatory toxicity testing and risk assessment are profound. The EDSTAC recognizes there are divergent scientific opinions on the " low dose" issue at the present time and that more research is necessary to answer these questions.

The EDSTAC notes that, historically, testing has sometimes missed critical endpoints either by: ( 1) failing to dose during the most sensitive life stage ( Morrissey et al., 1987); ( 2) failing to test in a susceptible organism ( Chamberlin, 1979; Fraser, 1988); or ( 3) failing to examine subtle ( yet biologically important) endpoints. For example, early studies on the developmental effects of PCBs in rodents identified fetotoxicity as a critical endpoint, yet these studies failed to test at low enough doses or to measure subtle enough endpoints. As a result these early studies missed the neurotoxic effects of PCBs which occur at much lower doses ( Tilson et al., 1990).

These omissions in testing may lead either to missing a critical effect completely, or to identifying a NOAEL which is erroneously high. The EDSTAC has attempted to minimize the likelihood of these types of errors by requiring testing in a variety of organisms during sensitive life stages. A variety of endpoints which appear to be low dose sensitive have also been added to the EDSTP EDSTAC Final Report Chapter Five August 1998 5  50 testing protocols. In addition, a number of endocrine disruptor sensitive endpoints have already been added to EPA's reproductive toxicity testing guidelines ( OPPTS Public Draft, 1996; U. S. EPA TSCA finalized testing guidelines, 1997; U. S. EPA FIFRA finalized testing guidelines, 1998).

The Committee agrees, however, that dose selection in T2T must include special attention to setting the low dose. In particular, the low dose should not be selected by identifying the high dose and then dropping the dose by a fixed formula of a couple of orders of magnitude. Instead, a number of considerations need to go into selecting the low dose, including the results of prior information ( e. g., HTPS, toxicity testing, pharmacokinetic, and epidemiology data). Information about environmental exposure levels might also be used where appropriate. Finally, range finding studies are currently constructed so as to inform both the high dose and the low dose for the definitive testing, with inclusion of low dose sensitive endpoints in the range finding study. Range finding studies, with fewer animals per group and a more limited set of endpoints than in definitive tests, will not necessarily identify the appropriate low dose or a NOAEL but they will indicate whether effects are observed for low dose sensitive endpoints and therefore whether low doses should be included in the definitive tests. These precautions will minimize the likelihood that critical effects will be missed or that erroneously high NOAELs will be identified in T2T.

2. Recommended Project to Address Low Dose Issues As previously discussed, questions have been raised as to the adequacy of conventional toxicology study designs for assessment of endocrine active substances, particularly with regard to low dose selection and the identification of no observed adverse effect levels ( NOAELs). The EDSTAC believes a project is required to resolve the underlying uncertainties and controversy about these issues. The purpose of the project is to address the nature of the dose response curves for exogenous estrogenic substances in order to allow more informed judgments about appropriate toxicology study designs for substances that have hormonal activity. The recommended project focuses only on substances with estrogenic activity, since it has been reported that some estrogens can produce nonmonotonic dose response curves at environmentally relevant dosage levels. However, the results of the study could be more broadly applicable to substances with other types of hormonal activity ( e. g., those that have androgenic or antiandrogenic activity).

Several very important studies related to low dose issues are currently in progress. The exact design of the project ( e. g., chemicals to be tested, strains of animals, endpoints to be evaluated) will be based on a thorough evaluation of all relevant data that can be made available. The EDSTAC believes this evaluation should be completed within a reasonable timeframe, such as four to six months. Preliminary discussions to date have focused on a two phased project. Phase I ( Replication Studies) is intended to replicate previously reported low dose results in male and female mice with one test substance and a positive control substance ( e. g., DES). The replication studies would be done in at least 3 different laboratories using an identical, mutually agreed upon protocol. The Phase I studies will allow a systematic evaluation of inter laboratory variability in the assessment of various estrogen responsive endpoints in male mice. Depending on the timing for completing studies currently in progress, or results of studies, if they have been completed, EDSTAC Final Report Chapter Five August 1998 5  51 Phase I may not be required. If Phase I is required, it should be conducted simultaneously with Phase II ( described below).

Phase II ( Exploratory Studies) is intended to evaluate potential new endpoints and compare the effects in male and female rats and mice with approximately four to seven different substances, with varying degrees of estrogenic potency ( including estradiol and at least one natural estrogenic substance) using an identical protocol for each substance. The number of substances, as well as the number of laboratories, will depend on available resources. The Phase II studies will allow a systematic determination of any species or sex differences in sensitivity, as well as elucidation of any qualitative differences in the responses to the various test substances. Phase II will allow assessment of the complete time course for any observed effects, and a determination of whether or not observations early in life are predictive of any specific adverse outcomes during later life stages.

If the results of the project confirm there is a need to include additional low doses for Tier 2 testing of substances with estrogen, androgen, or thyroid activity, there are implications for additional testing of any substances that may already have been subjected to dosing in the EDSTAC recommended Tier 2 tests. For any substance that has previously been tested and found to be positive in T2T ( i. e., produces adverse effects on EAT sensitive endpoints), it will be necessary to retest that substance with additional low doses. This additional low dose testing would be done for the purpose of conclusively identifying the NOAEL.

If the results of the project demonstrate the need to include any additional EAT endpoints, there would be a need for retesting to assess those new endpoints for any substances previously tested using the EDSTAC recommended T2T protocols. Additional endpoints would be those that are plausibly linked to adverse effects and not covered in current tests, or those that would be more sensitive than those in current tests.

Given the current state of the science, the EDSTAC believes its recommended T2T protocols ( once standardized and validated) will be adequate to determine whether chemicals exhibit EATmediated adverse effects. If the results of the project confirm there is a need to include additional low doses, but there is not a need to include additional EAT endpoints, then it will be necessary to retest chemicals that have been found positive in the EDSTAC recommended T2T protocols to conclusively identify the NOAEL, if one exists. However, it will not be necessary to retest chemicals that have already been found negative, and placed in the " hold box," using the EDSTAC recommended T2T protocols. As indicated in Chapter Three, all chemicals placed in the " hold box," can be reconsidered for additional testing if " new information on the endocrine disrupting potential of the chemical substance or mixture becomes available and it is determined that this new information warrants additional testing."

If retesting is required, a test concentration that produced adverse effects in the EDSTACrecommended T2T protocol ( if any) would be required as the high dose of the repeat study to demonstrate reproducibility of the initial finding. EDSTAC Final Report Chapter Five August 1998 5  52 If the project demonstrates that conventional protocols are adequate for identifying the NOAEL of endocrine active substances, then the NOAEL derived from conventional T2T should not be contested.

The EDSTAC recommends that a collaborative group, involving government, industry, and appropriate individuals in academia, design the study protocols, be kept abreast of the conduct of the studies, evaluate results, and develop overall conclusions and recommendations.

E. Methods to Select the Target Doses for T2T For T2T in mammals, other vertebrates, and invertebrates, the EDSTAC recommends that the information to select doses used in the performance of these tests include:

1. existing information such as that available during the priority setting phase including results from the HTPS ( or its equivalent by bench level assays); 2. results from T1S ( including the range finding study results); 3. results from other assays or tests for pesticide registration, etc.; and 4. results from range finding studies.

Range finding studies specifically for T2T should be performed at multiple doses ( at least five) with a limited number of animals per dose, an abbreviated duration ( which must include exposures during gestation or egg development and lactation), and a limited number of relevant endpoints including low dose sensitive ones. If further research validates the low dose concern, the EDSTAC would recommend using the low dose sensitive endpoints in the range finding study to determine the need for inclusion of low doses in the definitive T2T. Range finding studies are already conducted for toxicity studies, and have the principal purpose of defining both the maximal level that can be dosed and, at the other end of the dose range, informing a dose that will have no observable effect on the most sensitive endpoint in the full scale study. Endpoints, identified in recent publications, which, at present, appear to be low dose sensitive include: prostate weight ( for mammals), epididymal sperm concentration ( for mammals), other accessory sex organ weights ( all vertebrates), thyroid weight ( all vertebrates), reproductive capability ( all T2T), and vaginal threads ( for mammals). All of these, except for vaginal threads, are included in the 1996 guidelines; however, vaginal threads would be identified during the examination of offspring females for vaginal patency. New and/ or different low dose sensitive endpoints may be identified as new data are generated.

Current toxicological test guidelines generally require testing at a minimum of three dose levels plus a control. These guidelines specify that the top dose level should be a maximally tolerated dose ( MTD), that is, a dose which by definition is toxic but which does not result in excessive mortality ( not to exceed 10%). In reproductive and developmental toxicity studies, the MTD is usually based on parental or maternal toxicity, which is expressed as depressed body weight gain, actual weight loss, reduced feed and/ or water consumption, treatment related clinical signs of toxicity, etc. The MTD is based on available toxicity information such as data from a range finding study. The next lower dose is ideally set at an intermediate toxic dose and the lowest dose EDSTAC Final Report Chapter Five August 1998 5  53 at a level at which no toxic effects are observed. If additional lower doses are included in the study because of identified concerns at low doses, the additional doses should be widely spaced ( perhaps in orders of magnitude) to identify the nature of the dose response curve in the low dose region. The toxicity upon which the MTD is based may or may not be related to the endpoints that are the object of the investigation ( e. g., cancer, neurotoxicity, reproductive, or developmental effects).

The rationale and results of range finding studies should be included in the submission of the T2T results for evaluation by the Agency.

F. Testing Antithyroid Activities in T2T Thyroid hormones are well known to play essential roles in vertebrate development ( Dussault and Ruel, 1987; Myant, 1971; Porterfield and Hendrich, 1993; Porterfield and Stein, 1994; Timiras and Nzekwe, 1989). Experimental work focused on the effects of thyroid hormone on brain development in the neonatal rat supports the concept of a " critical period," during which thyroid hormone must be present to avoid irreversible damage ( Timiras and Nzekwe, 1989). Though the duration of this critical period may be different for different thyroid hormone effects, the general view has developed that this is the period of maximal developmental sensitivity to thyroid hormone, and it occurs during the lactational period in the rat ( Oppenheimer et al., 1994; Timiras and Nzekwe, 1989). Although thyroid hormone receptors are expressed in fetal rat brains ( Bradley et al., 1989; Strait et al., 1990) and thyroid hormone can exert effects on the fetal brain ( Escobar et al., 1990; Escobar et al., 1987; Escobar et al., 1988; Porterfield, 1994; Porterfield and Hendrich, 1992; Porterfield and Hendrich, 1993; Porterfield and Stein, 1994), the lactational period represents a stage of rapid expansion of the central nervous system that coincides with a large increase in the expression of thyroid hormone receptors ( Perez Castillo et al., 1985) and an increase in the number of demonstrated effects of thyroid hormone on brain development.

In conducting thyroid related tests in Tier 2, the EDSTAC recommends using an approach in which dosing occurs during the fetal and lactational period. In addition, there are a variety of endpoints that would provide reliable markers of thyroid disruption. Brain weight offers a simple measure, though it is not thyroid specific. Characteristics of myelination, or of myelin basic protein expression ( either mRNA or protein), would provide a more selective measure ( Bhat et al., 1981; Bhat et al., 1979; Farsetti et al., 1991; Figueiredo et al., 1993; Rodriguez Pena et al., 1993; Shanker et al., 1987). In this regard, the expression of myelin basic protein and/ or neurogranin/ RC3 may offer the simplest and most specific endpoints of thyroid disruption during the perinatal period ( Farsetti et al., 1991; Iniguez et al., 1993). These mRNAs are both enormously abundant and robustly affected by thyroid hormone. However, their sensitivity to xenobiotics has not been studied. A list of existing endpoints for thyroid hormone function, and additional ones recommended by the EDSTAC for validation and inclusion, are found in Table 5.3. EDSTAC Final Report Chapter Five August 1998 5  54 VI. Recommended Tier 2 Testing Battery A. Outline of Recommended T2T Battery The EDSTAC recommends that the T2T battery include a mammalian two generation reproductive toxicity study, or a less comprehensive test in accordance with the guidelines outlined above, and tests addressing four additional taxonomic groups, including birds, amphibians, fish, and invertebrates as follows:

Mammalian Tests 1. Two Generation Mammalian Reproductive Toxicity Study; or 2. A Less Comprehensive Test: a) Alternative Mammalian Reproductive Test; or b) One Generation Test.

Multigeneration Tests in Other Taxa 1. Avian Reproduction ( with bobwhite quail and mallard) 2. Fish Life Cycle ( fathead minnow) 3. Mysid Life Cycle ( Americamysis) 4. Amphibian Development and Reproduction ( Xenopus)

B. Two Generation Mammalian Reproductive Toxicity Study The two generation reproductive toxicity study in rats ( TSCA 799.9380, August 15, 1997; OPPTS 870.3800, Public Draft, February, 1996; OECD no. 416, 1983; FIFRA, Subdivision F, Guidelines 83 4) is designed to comprehensively evaluate the effects of a chemical on gonadal function, estrous cycles, mating behavior, fertilization, implantation, pregnancy, parturition, lactation, weaning, and the offspring' ability to achieve adulthood and successfully reproduce, through two generations, one litter per generation. Administration is usually oral ( dosed feed, dosed water, or gavage) but other routes are acceptable with justification ( e. g., inhalation). In addition, the study also provides information about neonatal survival, growth, development, and preliminary data on possible teratogenesis. The experimental design for a two generation reproductive toxicity study is presented in Figure Q. 1, which is found in Appendix Q, Tier 2 Test Study Designs.

In the existing two generation reproductive toxicity test, a minimum of three treatment levels and a concurrent control group are required. At least 20 males and sufficient females to produce 20 pregnant females must be used in each group as prescribed in this current guideline. The highest dose must induce toxicity but not exceed 10% mortality. In this study, potential hormonal effects can be detected through behavioral changes, ability to become pregnant, duration of gestation, signs of difficult or prolonged parturition, apparent sex ratio ( as ascertained by anogenital distances) of the offspring, feminization or masculinization of offspring, number of pups, stillbirths, gross pathology and histopathology of the vagina, uterus, ovaries, testis, epididymis, EDSTAC Final Report Chapter Five August 1998 5  55 seminal vesicles, prostate, and any other identified target organs, and gynecological assesments ( evaluation of parental estrous cyclicity, onset of puberty, acquisition of VO).

Table 5.3 provides a summary of the endpoints evaluated within the framework of the experimental design of the updated two generation reproductive toxicity test ( and some recommended additional endpoints for validation and inclusion to cover EAT concerns). These endpoints are comprehensive and cover every phase of reproduction and development. Tests that measure only a single dimension or component of hormonal activity, ( e. g., in vitro or short term assays) provide supplementary and/ or mechanistic information, but cannot provide the breadth of information listed in Table 5.3, which is critical for risk assessment.

Additionally, in this study type, hormonally induced effects such as abortion, resorption, or premature delivery as well as abnormalities and anomalies such as masculinization of the female offspring or feminization of male offspring, can be detected. Substances such as the phytoestrogen, coumesterol, and the antiandrogen, cyproterone acetate, which possess the potential to alter normal sexual differentiation, were similarly detected in this study test system ( i. e., 1982 Guideline). The initial prebreed exposure period ( 10 weeks) of the two generation reproductive toxicity test also provides information on subchronic exposures which can be used for other regulatory purposes.

C. Alternative Approaches to Mammalian T2T The EDSTAC acknowledges that the developing organism may be uniquely sensitive to the effects of endocrine active agents. Therefore, any mammalian Tier 2 test should include a careful assessment of the consequences of in utero and lactational exposure on subsequent growth and development. Below are two alternative mammalian tests the EDSTAC recommends be included, once validated and standardized, as part of T2T. The determination on whether to perform the two generation test or one of the alternative tests should be made consistent with the criteria described in Chapter Five, Section V, C, 2.

Although EAT relevant endpoints are not optimally detected by developmental toxicity study designs, both of these alternative tests can be modified to allow assessment of term fetuses by adding additional F0 females to each dose group ( i. e., create satellite groups) and terminating these satellite females just prior to anticipated parturition ( i. e., on gd 20 21) and performing gestational and fetal structural evaluations ( i. e., ovarian corpora lutea, uterine implantation sites, EDSTAC Final Report Chapter Five August 1998 5  56 Table 5.3 Mammalian Tier 2 Test Endpoints Below are two types of lists. First, those endpoints required in current EPA test guidelines 1996. Second, additional endpoints recommended by EDSTAC for validation and inclusion in both the recommended two generation test, as well as the alternative mammalian tests discussed below. These additional endpoints will detect estrogen, androgen, and thyroid hormone perturbations.

As discussed above, in Chapter Five, Section V, C, the default assumption is that all of these endpoints would be evaluated unless the conditions which are set forth in the guidelines for determining the selection of endpoints are met.

Current Guideline Endpoints Sensitive to Estrogens/ Antiestrogens · sexual differentiation · gonad development ( size, morphology, weight) > accessory sex organ ( ASO) development · ASO weight ± fluid; histology · sexual development and maturation: acquisition of vaginal patency ( VP), preputial separation ( PPS) · fertility · fecundity · time to mating · mating and sexual behavior · ovulation · estrous cyclicity · gestation length · abortion · premature delivery · dystocia · spermatogenesis · epididymal sperm numbers and morphology; testicular spermatid head counts; daily sperm production ( DSP); efficiency of DSP · gross and histopathology of reproductive tissues · anomalies of the genital tract · viability of the conceptus in utero ( prenatal demise) · survival and growth of offspring · maternal lactational behaviors ( e. g., nursing, pup retrieval, etc.)

Recommended Additional Endpoints for Validation and Inclusion · accessory sex organ function ( secretory products) · sexual development and maturation ( nipple development and retention) · androgen and estrogen levels · LH and FSH levels · testis descent EDSTAC Final Report Chapter Five August 1998 5  57 Current Guideline Endpoints Sensitive to Androgens/ Antiandrogens · altered apparent sex ratio ( based on AGD) · malformations of the urogenital system · altered sexual behavior · changes in testis and accessory sex organ weights · effects on sperm numbers, morphology, etc. · retained nipples in male offspring · altered AGD ( now triggered from PPS/ VP) · reproductive development; PPS/ VP ( puberty) · male fertility · agenesis of prostate · changes in androgen dependent tissues in pups and adults ( not limited to sex accessory glands)

Current Guideline Endpoints Sensitive to Thyroid Hormone Agonists/ Antagonists ( general) · growth, body weight · food consumption, food efficiency · developmental abnormalities · perinatal mortality · testis size and DSP · VP; PPS Recommended Additional Endpoints for Validation and Inclusion · neurobehavioral deficits ( see developmental landmarks below) · TSH, T4, thyroid weight and histology ( e. g., goiter) · developmental landmarks: · prewean includes pinna detachment, surface righting reflex, eye opening, acquisition of auditory startle, negative geotaxis, mid air righting reflex, motor activity on PND 13, 21, etc. · postwean includes motor activity PND 21 and postpuberty ages ( sex difference); learning and memory PND 60  active avoidance/ water maze · brain weight ( absolute), whole and cerebellum · brain histology EDSTAC Final Report Chapter Five August 1998 5  58 total, resorbed, dead and live fetuses, live fetal number, sex, weight, external, visceral, and skeletal alterations), i. e., a " standard" developmental toxicity evaluation by OPPTS 1996 draft guidelines; USEPA TSCA guidelines 870.3700, 1997; FDA guidelines, 1993. The remaining females in each group would continue on study as described below.

1. Alternative Mammalian Reproduction Test A graphical representation of the study design ( Figure Q. 2) and additional descriptive text for the Alternative Mammalian Reproduction Test ( AMRT) are provided in Appendix Q, Tier 2 Test Study Designs. The objectives of this test are to describe the consequences of in utero and/ or lactational exposure on reproduction and development from compounds that displayed EAT activity in the T1S. If validated, this test may be used, under certain defined circumstances, instead of the recommended two generation reproductive toxicity test ( TSCA guidelines, 1997) in T2T. In this regard it will be conducted with at least three treatment groups plus a control and include endpoints sensitive to chemicals that alter development via EAT activities. As with the two generation mammalian reproductive toxicity study, the default assumption is that all of the endpoints in Table 5.3 would be evaluated in the AMRT, unless the conditions set forth in the guidelines for determining the selection of endpoints are met.

The AMRT involves exposure of maternal rats ( designated F0 generation) from gestational day 6 ( time of implantation), through parturition ( birth), and through the lactation period until weaning of offspring ( designated F1 generation) on postnatal day 21. F1 offspring ( both sexes) are retained after weaning with no exposures for 10 weeks and then mated within groups. F1 males are necropsied after the mating. F1 females and their litters ( designated the F2 generation) are retained until the F2 generation is weaned. F0 females ( and a subset of F1 weanlings) are necropsied with organ weights and possible histopathology. F1 animals are evaluated for reproductive development ( VP, PPS), estrous cyclicity, and, at necropsy, for organ weights, possible histopathology, andrological assessments, and T3/ T4 ( with TSH triggered). F2 weanlings are counted, sexed, weighed, examined externally, and discarded.

The AMRT differs from the " standard" two generation study design in that it:

a) does not include exposures prior to mating, during mating, or during the early preimplantation stage of pregnancy in the dams; b) does not include exposures to parental males; and c) does not include direct exposure to the postweanling offspring; potential exposure is limited to in utero transplacental and/ or lactational routes.

The AMRT differs from the one generation test ( see below) in that its study design provides for:

a) exposure to the F0 dam only from gd6, through weaning of the F1 offspring on pnd 21; b) no exposure to parental males; c) mating of the F1 animals ( who have not been directly exposed) to produce F2 offspring; and d) following the F2 offspring to weaning on pnd 21. EDSTAC Final Report Chapter Five August 1998 5  59 2. One Generation Test A second alternative to the standard two generation reproductive toxicity test is a one generation reproductive toxicity test. A graphical representation of the one generation test ( Figure Q. 3), and additional text, is provided in Appendix Q, Tier 2 Test Study Designs. It has been used in rats and mice. It has been used as a range finding study prior to performance of a guideline twogeneration ( or more) study for the last 10 years under EPA ( TSCA/ FIFRA) GLPs; the design is similar to that used by Sharpe et al., 1996. This is a shortened, scaled down version of the new draft OPPTS and Final TSCA guidelines for reproductive toxicity testing. As with the twogeneration mammalian reproductive toxicity study, the default assumption is that all of the endpoints in Table 5.3 would be evaluated in the one generation test, unless the conditions set forth in the guidelines for determining the selection of endpoints are met.

The one generation test is a less comprehensive evaluation of functional reproductive development than the AMRT ( since it does not follow F1 animals through production of F2 offspring), but it has the advantage of assessing postnatal development and adult reproductive capacity after in utero lactational and post lactational exposure. In the presence of continued exposure, the postnatal component of the test is extended to evaluate acquisition of vaginal patency, preputial separation, estrous cyclicity, and andrological assessments in the F1 offspring. Inappropriate retention of Mullerian duct derivations ( e. g., oviducts) in males and of Wolffian duct derivatives ( e. g., seminal vesicles, epididymides) in females can be identified in all three proposed tests ( with or without satellite F0 females and examination of term fetuses).

The one generation test involves a short prebreed exposure period for male and female rats of the initial parental generation ( designated F0), and exposure continues through mating, gestation, and lactation of F1 litters. F0 males are necropsied after F1 deliveries; F0 females are necropsied after F1 weaning. Postweanling F1 animals are directly exposed for a 10 week postwean period and are then necropsied. F1 animals are evaluated for reproductive development ( VP, PPS), estrous cyclicity and at necropsy for organ weights, possible histopathology, andrological assessments, and T3/ T4 ( TSH triggered). F0 animals will undergo the same necropsy assessments.

The one generation test differs from the " standard" two generation study design in that it:

a) is shorter ( basic design calls for two weeks, can be prolonged) than in the standard twogeneration study ( 10 weeks to encompass one full spermatogenic cycle in rats), though it does include a prebreed exposure period; and b) does not evaluate effects of in utero and/ or lactational exposure ( and beyond) on generation of F2 offspring though it does include direct exposure of F1 offspring after weaning, including exposure through puberty and sexual maturation. F1 male and female reproductive organs ( weight/ histology), estrous cyclicity, and andrological endpoints are assessed at scheduled necropsy on PND 90 ± 2. EDSTAC Final Report Chapter Five August 1998 5  60 The one generation test differs from the AMRT in that its study design provides for:

a) exposure to both male and female F0 parental animals prior to mating, during mating, and during gestation and lactation of F1 offspring ( F0 males are necropsied after F1 deliveries, F0 females are necropsied after F1 weaning); b) direct exposure of postweanling F1 offspring after lactation until termination; and c) no mating of F1 animals to produce F2 offspring.

D. Description of the Tests for Other Animal Taxa The EDSTAC agrees T2T should address at least four other animal taxonomic groups, including birds, amphibians, fish, and invertebrates. Each of the four basic non mammalian tests should be multigenerational, as is the basic mammalian reproduction study. It is recommended that the following standardized tests be used as a basis for a non mammalian battery:

1. Avian Reproduction ( with bobwhite quail and mallard) 2. Fish Life Cycle ( fathead minnow) 3. Mysid Life Cycle ( Americamysis) 4. Amphibian Development and Reproduction ( Xenopus)

Except for the amphibian study, these tests are routinely performed for chemicals with widespread outdoor exposures that are expected to affect reproduction. Modifications to each may be warranted to enhance the ability to detect endocrine related effects. The amphibian test, though not standardized, is considered warranted because of the extensive fundamental knowledge base on amphibian development and reproduction.

Just as for mammalian testing, there may be instances when less comprehensive study designs would be adequate. Considerations for determining whether the full battery of comprehensive non mammalian tests should be implemented include an understanding of mechanisms of action, environmental fate and transport, persistence, potential for bioaccumulation, and potential ecosystems exposed.

Production volume is also a consideration for less comprehensive approaches. Comprehensive assessments of environmental toxicity, including chronic toxicity assays in a variety of species, are already generated for pesticides and very high production volume chemicals (> 1,000 tons per annum) in Europe. The European Union explicitly requires less comprehensive assessments for lower production volume chemicals, with additional testing required as production increases. As with mammalian assessments, these moving triggers recognize that the potential for exposure is correlated with production volume. While there are no explicitly required data sets in the U. S. under TSCA, similar decisions are made on data adequacy based on other information, including production volume. EDSTAC Final Report Chapter Five August 1998 5  61 There are a number of alternative, less comprehensive assays that may be appropriate to consider for environmental toxicity assessments. These might include shorter term avian development tests, and the fish early life cycle test and Daphnia reproduction test, both of which are already established protocols.

1. Avian Reproduction Test While birds are not included as subjects in the T1S battery, it is important to evaluate the effects of exposure of birds to chemical substances or mixtures with endocrine activity. Furthermore, birds are fundamentally different from mammals in the control of sexual differentiation ( males are the homogametic sex) so results using mammalian subjects will not provide complete information relevant to birds.

Use of the EPA's Avian Reproduction Test guidelines ( OPPTS 850.2300) is recommended, modified to include the additional endpoints presented below to make the test more sensitive to chemical substances or mixtures with endocrine activity. Table 5.4 provides a summary of the endpoints evaluated within the framework of the Avian Reproduction Test ( and recommended additional endpoints for validation and inclusion to cover EAT concerns). Two important extensions of this guideline are recommended: ( 1) modification and standardization of the husbandry and dosing of the offspring from EPA's Avian Reproduction Test guidelines ( OPPTS 850.2300) to create a two generation avian reproduction test; and ( 2) using the procedures of the modified Avian Reproduction Test protocol, evaluate an additional exposure pathway ( i. e., direct topical exposure, which is common in the wild, by dipping eggs). The recommended extensions to the guideline are outlined in Appendix Q.

In the current Avian Reproduction Test guidelines, two species are commonly used, mallards and northern bobwhite. Exposure of adults begins prior to the onset of maturation and egg laying and continues through the egg laying period; their offspring are exposed, in early development, by material deposited into the egg yolk by the females. These offspring can be used efficiently to test for the effects of chemical substances or mixtures on avian development. There are several endpoints currently required ( see OPPTS 850.2300, c, 2) that are particularly relevant to disruption of endocrine activity, including: eggs laid, cracked eggs, eggshell thickness, viable embryos, and chicks surviving to 14 days. The guidelines should be extended with additional observations made for circulating steroid titers, thyroid hormones, major organ ( including brain) weights, gland weights, bone development, leg and wing bone lengths, ratios of organ weights to bone measurements, skeletal x ray, histopathology, functional tests, and reproductive capability of offspring ( Baxter et al., 1969; Bellabarba et al., 1988; Dahlgren and Linder, 1971; Emlen, 1963; Cruickhank and Sim, 1986; Fleming et al., 1985a; Fleming et al., 1985b; Fox, 1976; Fox et al., 1978; Freeman and Vince, 1974; Hoffman and Eastin, 1981; Hoffman and Albers, 1984; Hoffman, 1990; Hoffman et al., 1993; Hoffman et al., 1996; Jefferies and Parslow, 1976; Kubiak et al., 1989; Maguire and Williams, 1987; Martin, 1990; Martin and Solomon, 1991; McArthur et al., 1983; McNabb, 1988; Moccia et al., 1986; Rattner et al., 1982; Rattner et al., 1987; Summer et al., 1996; Tori and Mayer, 1981). Other avian assays were considered including the Japanese quail androgenic assay ( proctodeal gland), egg injection, draft OECD Japanese quail EDSTAC Final Report Chapter Five August 1998 5  62 reproduction, and two generation avian reproduction tests, but were not selected because the endpoints addressed were limited or there was a lack of accepted and standardized methods.

2. Fish Life Cycle Test The freshwater fathead minnow Pimephales promelas is the recommended species to be used and is continuously exposed from fertilization through development, maturation, reproduction, and early development of offspring with a test duration of up to 300 days. The fathead minnow is also the recommended species for use in the screening battery for the fish gonadal recrudescence assay, and as such, the relevance of any activity detected in the screening assay would be evaluated. However, EDSTAC recommends a performance based approach to species selection and, as more appropriate species are developed and validated, EDSTAC strongly encourages their use. For example, if exposure to a particular chemical substance or mixture is predominantly estuarine or marine, the estuarine sheepshead minnow Cyprinodon variegatus may be substituted since experience and an established method exist for this species.

Likewise, other species may offer more specificity for certain endpoints or geographic relevance and should be considered in a performance based approach. For example, the sex linked color gene in a medaka strain may afford early, non intrusive specificity for determining the genetic sex of test animals following estrogenic and androgenic screens or tests and anadromous salmon may have better value to examine thyroid function in a sea water challenge ( smoltification) assay.

Fish are the most diverse and least homologous to mammals of all vertebrates. Reproductive strategies extend from oviparity, to ovoviviparity, to true viviparity. The consequences of an endocrine disruptor may be quite different across the many families of fishes. As a first step though, only a fathead minnow, or in special cases the sheepshead minnow, life cycle test is suggested to confirm and quantify any effects detected by the Tier 1 battery. Subsequent tests with other species will then be a function of the risk assessment and nature of the hormones involved and effects expected/ obtained.

The fish life cycle test ( OPPTS 850.1500) follows procedures outlined in Benoit, 1981, for the fathead minnow, and Hansen et al., 1978, for the sheepshead minnow. In general, the test begins with 200 embryos distributed among eight incubation cups in each treatment group. When hatching is completed, the number of larvae are reduced to 25 individuals, if available, which are released to each of four replicate larval growth chambers. Four weeks following their release into the larval growth chambers, the number of juvenile fish are reduced again and 25 individuals, if available, are distributed to each of two replicate adult test chambers. When fish reach sexual maturity, fish are separated into spawning groups ( pairs or one male/ two females) with a minimum of eight breeding females. Remaining adults will be maintained in the tank but will be segregated from the spawning groups. Adults will be allowed to reproduce, at will, until the 300th day of exposure. Alternatively, the test may be continued past 300 days until one week passes in which no eggs from any group have been laid. The embryos and fish are exposed to a geometric series of at least five test concentrations, a negative ( dilution water) control, and, if necessary, a solvent control. EDSTAC Final Report Chapter Five August 1998 5  63 Table 5.4 Avian Reproduction Test Endpoints Current Guideline Endpoints Sensitive to Estrogens/ Antiestrogens, Androgens/ Antiandrogens, and/ or Hypothalamic Pituitary Gonadal Axis · egg production · eggs cracked · viable embryos ( fertility) · eggshell thickness · fertilization success · live 18 day embryos · hatchability · 14 day old survivors Recommended Additional Endpoints for Validation and Inclusion · sex ratio · major organ ( including brain) weights · gland weights · histopathology · plasma steroid concentrations · neurobehavioral test ( e. g., nest attentiveness)

Current Guideline Endpoints Sensitive to Thyroid Hormone Agonists/ Antagonists · body weight of adults · food consumption of adults · body weight of 14 day old survivors · developmental abnormalities Recommended Additional Endpoints for Validation and Inclusion · plasma T3/ T4 · thyroid histology · bone development ( skeletal x ray) · ratio of organ weights to bone measurements · neurobehavioral test ( cliff test) · cold stress test EDSTAC Final Report Chapter Five August 1998 5  64 Assessment of effects on offspring of the parental group ( first filial or F1 generation) will be made by collecting two groups of 50 embryos from each experimental group and incubating those embryos. When embryos hatch, the number of larvae hatched from each group will be impartially reduced to 25, if available, and released into the larval growth chambers. After four weeks of exposure, lengths and weights of surviving individuals will be made.

Observations are made of the effects of the test substance on embryo hatching success, larvaejuvenile adult survival, growth of parental and F1 generation, and reproduction of the adults. Table 5.5 provides a summary of the endpoints evaluated within the framework of the Fish Life Cycle Test ( and recommended additional endpoints for validation and inclusion to cover EAT concerns).

3. Mysid Life Cycle Test Invertebrates ( especially arthropods such as insects and crustaceans) constitute the vast majority of animal species on earth. Yet, relatively few invertebrate toxicity test protocols are routinely used in regulatory toxicity testing, and none have been designed with endocrine endpoints in mind. Nevertheless, invertebrate growth, reproduction, and development are under endocrine control. However, invertebrate endocrine systems and hormones are not directly analogous to those of vertebrates.

Two invertebrate life cycle toxicity tests are commonly used in chemical and pesticide testing, both using crustaceans. The opossum or mysid ( Order Mysidacea) shrimp is an estuarine species, whereas water fleas or daphnids ( Daphnia magna or Daphnia pulex) are freshwater species. The former is sexually dimorphic with males and females, whereas the latter undergoes parthenogenetic reproduction for the majority of its life cycle. Although sexual reproduction occurs in Daphnia, the standard test protocol ( U. S. EPA Public Draft OPPTS 850.1300 and OECD 202) is designed solely for the parthenogenetic reproductive phase. The chronic tests for both species are designed to provide No Observed Effect Levels ( NOEL) using apical effect endpoints for fecundity and growth.

Although neither chronic test was designed to examine endocrine specific endpoints, both species are crustaceans and therefore share common physiology. Ecdysone is a steroid hormone that regulates growth and molting in arthropods, and exhibits some functional and structural similarities to estrogen. The central role of ecdysone makes it an attractive candidate for examining endocrine effects in invertebrates; however, other possibilities also exist. Morphogenetic and reproductive development of arthropods is controlled in part by juvenile hormone ( JH). Methyl farnesoate is a JH like compound that may play a role in reproduction and development ( Borstet et al., 1987; Laufer et al., 1987a, b).

Invertebrate hormones are beyond the immediate scope of the EDSTAC which has focused on the vertebrate estrogen, androgen, and thyroid hormones. Nevertheless, invertebrate hormones that are functionally equivalent to estrogen, androgen, and thyroid need to be examined in more EDSTAC Final Report Chapter Five August 1998 5  65 Table 5.5 Fish Life Cycle Test Endpoints Current Guideline Endpoints Sensitive to Estrogens/ Antiestrogens, Androgens/ Antiandrogens, and/ or Hypothalamic Pituitary Gonadal Axis · viability of embryos · time to hatch · spawning frequency · egg production · fertilization success Recommended Additional Endpoints for Validation and Inclusion · sexual differentiation ( tubercle formation, gonadal histology)

· sex ratio · gonadosomatic index · gamete maturation ( production, final oocyte maturation, sperm motility test, etc.)

· vitellogenin · plasma steroid concentrations · in vitro gonadal steroidogenesis Current Guideline Endpoints Sensitive to Thyroid Hormone Agonists/ Antagonists · growth, length, and body weight · developmental abnormalities Recommended Additional Endpoints for Validation and Inclusion · plasma T3/ T4 · neurobehavioral tests ( e. g., activity level and swimming performance, nesting ( spawning) behavioral endpoints such as territory defense, courtship, and egg protection and care, or whether test fish retain the ability to avoid known deterrent chemicals after exposure to a test chemical)

· thyroid histology EDSTAC Final Report Chapter Five August 1998 5  66 depth. More importantly, chemicals that affect these vertebrate hormones may also affect invertebrate hormones resulting in altered reproduction, development, and growth.

Chemicals with estrogenic properties are reported to have altered normal function of ecdysone systems ( Mortimer, 1993; 1994; 1995a; 1995b; Chu et al., 1997). ( Satyanarayana et al., 1994) showed stimulation of vitellogenin in insect prepupae and pupae by methoprene, a JH mimic with retinoid properties. Whether vitellogenin production is controlled through an estrogen receptor or an alternative mechanism is not crucial for obtaining test results that show alteration occurs.

Therefore, the mysid shrimp chronic life cycle test ( OPPTS 850.1350) may be adapted to determine whether chemicals that affect hormonal activity in vertebrates also affect arthropods. Once adapted to include reproductive and developmental endpoints relevant to the EDSTP, the test could be a useful component in screening and testing. Although mysids would provide some indication of endocrine effects for arthropods, it is unlikely to identify vertebrate effects.

The other common invertebrate bioassay, one using the water flea, Daphnia, is used internationally ( OECD 202). It incorporates life cycle assessment and reproductive and developmental endpoints, albeit applied quite differently in this group of animals. Reproduction is usually parthenogenic in the laboratory in these animals, limiting the applicability to endpoints identified in this report. The particular aspect of this system is that the Daphnia is sensitive to estrogenic compounds ( Baldwin et al., 1995; Baldwin et al., 1997; Shurin and Dodson, 1997), and possesses receptors for testosterone, making the system sensitive to another vertebrate hormone. Again, this bioassay would have to be adapted for the endpoints and process of interest in the EDSTP as a protocol for including invertebrate species in the endpoints addressed by the EDSTP screening and testing battery. Other invertebrates, such as molluscs, crayfishes, and echinoderms, do have EA systems, but again relevant standardized tests for evaluating the consequences of interfering with these systems are not currently available. We simply do not know whether one ( mysid) or two ( mysid and daphnia) Tier 2 tests will provide sufficiently valid information for other invertebrate groups not tested. This is a source of uncertainty, potentially leading to Type II errors of unknown magnitude.

4. Amphibian Development and Reproduction A definitive amphibian test, which exposes larvae through metamorphosis and reproduction, is important to evaluate the consequences of endocrine disruption in a poikilothermic oviparous vertebrate distinct from fishes. A rich literature on metamorphosis, growth, and reproduction exists for frogs and promising methods are being developed. No established method has been identified which is suitably comprehensive to stand as a Tier 2 test at this time. The EDSTAC feels a test to address this taxonomic group and set of endpoints is needed in T2T and should be given a high priority for development and standardization.

VII. Validation, Standardization, Methods Development, and Research EDSTAC Final Report Chapter Five August 1998 5  67 A. Concept of Assay Validation and Standardization As stated earlier, the role of standardization and validation is to provide sufficient data to allow informed decisions about the relative merits of the recommended T1S battery component assays and alternative assays ( based on sensitivity, specificity, technical complexity, inter and intralaboratory variability, time, and cost).

Validation is the scientific process by which the reliability and relevance of an assay method are evaluated for the purpose of supporting a specific use ( ICCVAM, 1996). Relevance refers to the ability of the assay to measure the biological effect of interest. Measures of relevance can include sensitivity ( the ability to detect positive effects), specificity ( the ability to give negative results for chemicals that do not cause the effect of interest), statistically derived correlation coefficients, and determination of the mechanism of the assay response with the toxic effects of interest. Reliability is an objective measure of a method's intra and inter laboratory reproducibility. The process of validation includes standardization, that is, definition of conditions under which the assay is run ( species, strain, culture medium, dosing regimen, etc.). Standardization is critical to ensure reliability, that is, valid results from time to time and between laboratories. Even in those instances where there is currently some degree of de facto acceptance of a given screening method as valid, there is a need for such standardization.

B. Statutory Need for Validation The Food Quality Protection Act ( FQPA) requires EPA " to develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or other such endocrine effect as the Administrator shall designate" by August 1998 with implementation of the peer reviewed program by August 1999. In requiring the use of validated test systems the FQPA is merely mandating good science. There are numerous reasons for using only validated assays. These include: having confidence that the assay is detecting the effect it purports to be detecting, that the results of the assay are reproducible and comparable from laboratory to laboratory, and that the results permit a comparison of the toxicity of various chemicals. These factors are important in being able to interpret results to establish a relative priority among chemicals for progressing from screening to testing and ultimately to perform a hazard and risk assessment.

C. Addressing the Validation Issue The assays and tests recommended by the EDSTAC vary considerably in terms of their degree of development and validation. EPA ( Dr. Lynn Goldman, April 24, 1997, letter to EDSTAC) recognized that few screening assays have actually met the " gold standard" of validation and that other assays have been accepted on the basis of peer review and general use without formal validation. Because the number of validated off the shelf assays is so limited, EDSTAC was asked by EPA to extend its consideration to all existing assays. EDSTAC Final Report Chapter Five August 1998 5  68 Thus, although formal validation was not a prerequisite for assay selection by the EDSTAC, the ability or potential of an assay to be validated must be considered because assays must be capable of passing the " validation test" before the screening program is fully implemented. The following are a list of factors that EPA might consider in estimating the likelihood that a candidate assay will actually survive the validation process. If possible, it would be useful to devise a quantitative or semi quantitative scale for expressing these characteristics so that test methods reviewed by various people could more easily be compared.

1. Number of independent, peer reviewed publications reporting results from the assay. 2. Similarity of results from independent publications performing the assay. 3. Number of independent laboratories publishing results from the assay. 4. Consistency of the methods used across laboratories. 5. Consistency of results of the assay between labs ( to the extent results are available for the same chemicals). 6. Known variability of the assay within single laboratories ( may not preclude use as a valuable research tool, but may have important implications for use as a widespread screening tool). 7. Age of the assay  is it an " old standby" or a " new kid on the block." 8. The extent to which the assay relies upon calibrated equipment and calibrated standards. 9. The extent to which the assay depends upon the skill of the technician ( a professional opinion from those who know about the assay). 10. The extent to which the assay utilizes internal controls or standards. 11. Use of the assay to develop clinically effective drugs ( although not of itself proof of validity, success speaks well for itself). 12. Number of examples of false positives/ false negatives from the assay ( although we have a clear charge to eliminate false negatives at the screening stage, it is nonetheless important to consider the overall performance of an assay in order to estimate its likelihood of being validatable). 13. Any known species or strain specific sensitivities of the assay. For in vitro assays, any knowledge of critical sensitivity to cell characteristics such as passage number, plating density, doubling time, etc. or other specific sensitivities of the assay, such as receptor number, transfection technique, serum requirements, media composition, etc. ( trying to get another angle on how finicky the assay is).

Information related to validation status of the assays is summarized in Chapter Five, Section VII, E, and was among the factors considered in deciding among assays. These same considerations regarding validation and standardization apply to T2T.

D. Validation and Standardization Process While not all assays would necessarily need to be validated since they may have de facto acceptance as being valid in the scientific community due to their long history of use and EDSTAC Final Report Chapter Five August 1998 5  69 performance, others have little or no data that would allow judgments to be made regarding their validity.

The following is a description of elements of a such a validation process for endocrine disruptor screening assays.

1. Characterize Reference Substances/ Vehicles. · A reference substance for each hormone endpoint ( minimum) · Reference substances for each hormone endpoint: · positive control substances · negative control substances · Natural and man made substances · Composition and purity defined ( e. g., GC/ MS) · Stability verified · Coded · Centralized distribution ( using same batch number, lot number, etc.)

2. Develop a Standard Protocol for Each Assay Method. · Assay system to be used ( species, strain, sex, age, cell line, clone, gene construct, etc.) · Dose levels/ exposure concentrations · General criteria for selection · MTD ( whole animal systems; e. g., mortality, decreased body weight, etc.) · Cell viability ( in vitro systems): 2 methods · Solubility limitations · Specific for each reference substance · Dose/ exposure regimen · Number of doses · Duration · Guidance of mixtures · Concentrations of reference substances · Interpretation of data · Route of exposure ( whole animal systems) · Description of endpoint( s) to be measured · Materials ( equipment, media, vehicles, etc.) · Time( s) of measurements · Criteria for positive/ negative response · Statistical methods to used · Number of replications required ( depending on the study design)

3. Define Specialized Skills and Equipment Required for Each Assay Method 4. Conduct in a Variety of Laboratories. EDSTAC Final Report Chapter Five August 1998 5  70 5. Compile and Evaluate Data · Expert scientific oversight group · Inter laboratory/ intra laboratory variability · Positive versus negative response · Relative potency ( in comparison to reference substance) · Viability/ maintenance of assay system ( including passage number and growth curves) · Sensitivity of assay system ( e. g., minimal effective dose/ concentration) · Specificity of assay system ( positive versus negative controls)

E. Levels of Effort Necessary to Validate the Recommended Screens and Tests The EDSTAC believes validation and standardization of the recommended screens and tests are essential for implementation of the EDSTP. The EDSTAC also believes the validation and standardization program is of highest priority, and recommends that it proceed on an accelerated schedule. As indicated earlier, the EDSTAC recommends that the validation and standardization program be consistent with the principles articulated by the national ( ICCVAM, 1996; Zeiger, 1998) and international ( OECD, 1996) alternative methods validation groups. As mentioned throughout the chapter, each screen and test recommended for T1S or T2T needs some level of validation, standardization, methods development, or further research before being accepted as a regulatory toxicity screen or test for inclusion in the EDSTP. The level of effort needed to fully standardize and validate may be different for each individual screen or test ( including all recommended endpoints). The effort required for each screen or test will be defined by a variety of criteria including: period of time in use, existing level of general acceptance in the endocrine toxicology field, and existing understanding of relevancy and reliability. Regardless of the effort required, EDSTAC believes all the screens and tests recommended for T1S and T2T must be fully validated and standardized before being included in the EDSTP. The EDSTAC recommends that as individual screens and tests are validated and standardized, they can be utilized in the EDSTP without waiting for all screens and tests in the batteries to be validated. The EDSTAC further recommends that a multi stakeholder process, involving government, industry, and academics, be utilized in validating and standardizing the T1S and T2T batteries ( see Section VII, G, of this chapter, for more explanation).

As mentioned earlier, the screens and tests being recommended by the EDSTAC vary considerably in terms of the effort necessary to be fully validated and standardized. Each screen or test lies somewhere along a spectrum of validation from already fully validated and standardized to needing further research to determine their utility in the EDSTP. Outlined in the following few paragraphs, in some detail, is the spectrum of possible effort needed to validate and standardize the screens and tests. In addition, the Screening and Testing Work Group attempted to categorize the levels of validation needed of the recommended screens and tests and their preliminary efforts are found in Appendix R. It is possible that the status of these screens and tests may have changed as work has progressed in the interim. The EDSTAC recommends that EPA update this categorization scheme as part of their validation and standardization program. EDSTAC Final Report Chapter Five August 1998 5  71 The recommended screens and tests ( including all endpoints) will have to meet all the criteria of relevance and reliability for use in regulatory toxicity screening or testing for EAT in order to be considered fully validated and standardized ( ICCVAM, 1996; Zeiger, 1998). As screens and tests become fully validated and standardized, they will warrant inclusion in the EDSTP according to their specific and appropriate use. None of the screens, new tests, or enhancements to existing test guidelines included in T1S or T2T completely fulfill these criteria to date.

Some of the recommended screens and tests have been in use for a sufficient period of time and have therefore gained sufficient general acceptance within the field of endocrine toxicology to be considered de facto validated ( reliable and relevant). These assays measure relevant endpoints, are responsive to endocrine active compounds with a high degree of specificity, are sufficiently sensitive to identify all known active agents, and can reasonably be expected to give reproducible results from laboratory to laboratory, assuming a general level of competence and expertise. Nonetheless, variations in protocols for these screens and tests can produce disparate results. Therefore, before the recommended screens and tests are utilized in the EDSTP, a standard performance guideline should be developed that produces an acceptable level of consistency in results for each one.

Some of the recommended screens and tests have sufficiently broad use to be generally considered relevant OR reliable to either screening for endocrine activity ( Tier 1) or to testing for adverse endocrine mediated effects ( Tier 2). These screens and tests cannot, however, be generally considered to be both relevant and reliable. The level of performance that can be expected of these screens and tests with respect to identifying endocrine active agents or endocrine disruptive effects of chemicals must be clarified. Therefore, these assays should undergo further but focused validation and standardization to define their relevance and reliability for the task of endocrine disruptor screening or testing. The validation required may be focused to answer specific questions about relevance and to provide information regarding specificity and sensitivity.

Some of the recommended screens and tests may have relevance to the task of either screening for endocrine activity or testing for endocrine disruptive effects, but their performance in identifying endocrine active agents or endocrine disruptive effects has seen only limited testing. Questions as to whether these assays measure endpoints that are relevant to endocrine activity or endocrine disruptive effects, whether these assays respond with specificity and sensitivity to known endocrine active agents, or whether they identify endocrine disruptive effects cannot be addressed with information currently available. In addition, questions regarding the specific protocols and conditions under which the assays should be conducted must be answered before relevance and reliability can be assessed. Nonetheless, the EDSTAC feels these assays would have sufficient utility, if further developed and validated, to enhance or augment the screening and testing program. Therefore, the EDSTAC recommends that resources be made available to pursue methods development and validation and standardization of these assays.

F. Screens and Tests Recommended for Further Research EDSTAC Final Report Chapter Five August 1998 5  72 As discussed in Section III, A, 5, the EDSTAC recognizes the importance of evaluating postnatal consequences of in utero and in ovo exposures to chemicals with EAT activities. Such an assay has not been incorporated into T1S due, in part, to the lack of an appropriate, short term, costeffective assay. The EDSTAC, however, does recommend that EPA take the lead in designing, standardizing, and validating such an assay. The EDSTAC also identified other screens and tests which, if available, could have an important utility in the screening and testing program, and recommends that research be conducted to determine whether such assays can be developed, and if so, what purpose the assays could fulfill within the endocrine disruptor screening and testing program. The rationale for including each of the following assays and tests is found below.

· in utero developmental screening assay · in ovo developmental screening assay · avian androgenicity screening assay · invertebrate screening assays · amphibian development and reproduction test ( Xenopus) · reptilian reproduction test In Utero Developmental Screening Assay: The rationale for developing an in utero developmental screening assay was discussed earlier; however, the EDSTAC did consider one possible study design which is summarized here ( and further elaborated upon in Appendix O). In the study design, pregnant rats would be exposed to the tested chemical from the start of the embryonic period through weaning of the offspring three weeks after birth. Types of EATsensitive endpoints that could be measured in this assay might include apparent sex ratio ( based on anogenital distance), numbers of offspring per litter, anogenital distance, retention of nipples in males, precocious puberty in females, uterine and ovarian weight and various uterine histological and biochemical parameters in females, and reproductive tract anomalies in both sexes. Blood samples could be analyzed for estradiol, testosterone, T4, and TSH. Myelin basic protein in brains could be measured as an indicator of thyroid hormone activity.

How much effort such an assay would entail or what its cost might be is not currently known. There may be additional protocols that could be predictive of EAT in developing systems and which are more amenable to screening applications than the protocol included and therefore the EDSTAC encourages development of other assays to address this issue.

In Ovo Developmental Screening Assay: A major route of excretion of lipophilic contaminants for female birds is into the yolk of their eggs; therefore, their embryos can have high levels of exposure from the earliest stages of development. In addition, the endocrine control of sexual and reproductive development is fundamentally different in birds than in mammals. Hence, a shortterm screening assay for chemical substances or mixtures that alter avian development is highly desirable. There is a moderate amount of research on the effects of environmental contaminants injected into bird eggs that could be the basis for developing such an assay. The more general rationale for developing an in ovo developmental screening assay, as with an in utero one, is found earlier in the chapter. EDSTAC Final Report Chapter Five August 1998 5  73 Avian Androgenicity Screening Assay: This assay would be useful in the T1S battery to improve and extend our assessment of chemical substances or mixtures for androgenic and antiandrogenic activity in birds. Development of this screen will become important if data from T2T point to differences in the actions of chemical substances or mixtures in birds versus mammals.

Reptilian Reproduction Test: Several distinctive features of reptilian reproduction ( e. g., ovoviviparity, temperature dependent sex determination), and a generally long life span that allows high body burdens of environmental contaminants to accumulate in reptiles, underscore the importance of developing a practical reproductive test in this class of ecologically important vertebrates.

G. Stakeholder Involvement in the Validation Program The EDSTAC recommends that a multi stakeholder process involving government, industry, and academics be utilized in standardizing and validating the T1S and T2T batteries. One key step in instituting a validation program for T1S assays is the identification of a set of " standard test substances" for the individual assays as well as for the overall T1S battery. To the extent possible, the standard test substances will be chosen according to the following criteria:

1. known EAT positives which act via receptor binding; 2. known EAT positives that do not appear to act via receptor binding ( i. e., via some other mechanism such as alterations of hormone synthesis, degradation, transport, etc.); 3. known EAT negatives ( i. e., substances known not to have hormonal activity); 4. known EAT positives which are active as the parent compound; 5. known EAT positives which require metabolic activation; 6. substances that cover a wide range of EAT potencies; 7. substances with a wide range of physical properties ( e. g., pH, reactivity, volatility, etc.); and 8. substances with extensive in vivo databases with in vivo effects that have been well documented.

It may not be possible to satisfy every one of the above criteria ( e. g., there are currently no known examples of environmental thyroid or androgen receptor agonists), but every standard test substance selected should meet at least one of the criteria.

In addition, careful definition of the expected use of the set of chemicals is necessary to avoid inappropriate use. Such a set of chemicals, developed with the already mentioned criteria in mind, would be used in the validation program to assist in defining their relevance and reliability for the task of endocrine disruptor screening, ( i. e., to identify whether a specific chemical substance or mixture has endocrine activity, or can be placed in the " hold box").

Further, as was also stated earlier, it is critical to acknowledge state of the science in this area is evolving rapidly, and assays currently being developed, or ones developed in the future, may offer EDSTAC Final Report Chapter Five August 1998 5  74 distinct advantages over some included in the current options. As they are developed, validated, and standardized, the use of these new assays for screening is strongly encouraged.

H. Preliminary Cost Estimates for the T1S and T2T Batteries An EDSTAC member completed a survey of contract labs in an effort to estimate the cost of the recommended T1S and T2T batteries. The results of this survey are summarized in Tables 5.6 and 5.7 and Appendix S. A detailed description of the methodology used to conduct the survey is included in the EDSTAC Docket (# OPPTS 42189, TSCA Public Docket Office, U. S. EPA). The EDSTAC includes these estimates in its final report, but acknowledges they are both preliminary and uncertain given the inherent uncertainties regarding the outcome of the validation and standardization process. The EDSTAC also notes that these cost estimates should be viewed in the context of the near and long term public health and environmental protection benefits to society. EDSTAC Final Report Chapter Five August 1998 5  75 EDSTAC Final Report Chapter Five August 1998 5  76 EDSTAC Final Report Chapter Five August 1998 5  77 VIII. Compilation of Chapter Five Recommendations A. Tier 1 Screening 1. The EDSTAC recommends that any T1S battery designed to detect endocrine disruptors should meet five criteria. The battery should:

· maximize sensitivity to minimize false negatives while permitting an acceptable level of false positives; · include a range of organisms representing known or anticipated differences in metabolic activity; · detect all known modes of action for endocrine endpoints of concern; · include a sufficient range of taxonomic groups among the test organisms; and · incorporate sufficient diversity and complementarity among the endpoints and assays to reach conclusions based on weight of evidence considerations.

2. The EDSTAC recommends the following assays for inclusion in the T1S battery: