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Interim Report To The EDSTAC
Submitted By The Screening And Testing Work Group
October 2, 1997
A. Purpose and Scope
The purpose of this Interim Report is to provide a description of the Screening and Testing Work Group (STWG) progress to date and identify areas requiring further discussion and work. The STWG members attending the September 9-11 work group meeting reached consensus on the general categories of assays to be included in Tier 1 Screening (T1S) which, if implemented, will detect all known estrogen, androgen, and thyroid disruptors in mammals.(1) The STWG has not reached this level of certainty for other vertebrate classes.
Overviews of the component assays will be provided, and their relation to the criteria required of the T1S battery will be discussed. This battery does not currently include an in utero assay; however, the STWG has identified that there are both benefits and costs of including such an assay which impinge on the overall effectiveness of T1S. Additional STWG work on this issue will be needed. Assays for endocrine disruptors using subjects from all vertebrate classes have been identified using well-documented endocrine endpoints. For both mammals and other vertebrates, the STWG formally considered many more assays than those presented here; the following background is included to provide a framework for inclusion of assays. While group members have discussed the possible role of invertebrate assays in accomplishing the goals of the battery, as of now, none are being considered (for additional discussion see Attachment B).
Tier 1 Screening (T1S) is intended to act as a "gatekeeper." After the T1S battery is applied, a chemical substance or mixture (CSM) will be designated as having either: 1) potential for estrogen, androgen, or thyroid hormone disruption and therefore will go to Tier 2 Testing (T2T); or 2) low or no potential for endocrine disruption for estrogen, androgen, or thyroid and therefore will go to the "hold unless ..." box. Criteria for this designation have not yet been agreed upon by the STWG members.
The STWG has interpreted the nature of the decision, based on T1S results, to mean T1S must therefore: 1) maximize sensitivity (if need be, over specificity) to minimize false negatives, while permitting an as-of-yet undetermined, but acceptable, level of false positives; 2) encompass a sufficient range of metabolic activity; 3) capture all known areas of activity for estrogen, androgen, and thyroid; 4) capture a sufficient range of taxonomic groups; and 5) incorporate sufficient diversity among the assays to reach conclusions based on weight of evidence. The T1S battery must therefore be structured such that, at the completion of the assays, the STWG and EDSTAC (during the T1S development) and the EPA and other stakeholders (during its implementation) will be scientifically and ethically comfortable in assigning CSMs as either having 1) low or no potential for estrogen, androgen, or thyroid endocrine disruption, or 2) as having such potential. This comfort level in moving a CSM to the "hold unless ..." box requires that T1S "covers the waterfront," (i.e., that T1S includes a sufficient diversity of screening assays, endpoints, and taxa). At the same time, the T1S must be relatively fast and economical while meeting the criteria described below. T1S is not, and should not be, designed to produce definitive answers; that is the goal of T2T.
T1S results should also inform T2T, in terms of guidance on which tests to perform, which endpoints to include, and which organisms to assess to satisfy the goals of T2T. These goals include identifying: at what doses (dose-response), at which life stage(s) (most sensitive), and in which organism(s) (most appropriate, most sensitive, most at risk, etc.) do the adverse effects occur. Based on these concerns and goals, the STWG is considering a tailored approach to T2T, with a necessarily more global approach to T1S.
The STWG has, therefore, spent a great deal of time evaluating existing and potential assays for inclusion in the T1S Battery. The component assays included in this paper are being evaluated further for what they add and which other assays they can replace. The goal is to create a T1S battery with the necessary breadth, depth, and rigor to allow assignment of CSMs to the "hold unless ..." box, to inform subsequent T2T, and to provide a comprehensive detection of hazard, but be as economical as possible (in terms of time and costs) to maximize throughput.
B. The STWG Working Model of Endocrine Disruption and the Implications for the Design of T1S
Xenobiotics can alter endocrine function by affecting the availability of a hormone to the target tissue, and/or affecting the cellular response to the hormone. Mechanisms regulating hormone availability to a responsive cell are complex, including hormone synthesis, serum binding, metabolism, cellular uptake (thyroid), and neuroendocrine mechanisms that control overall function of the endocrine axis. Mechanisms regulating cellular responses to hormones are likewise complex and are tissue-specific. Because the role of receptors is crucial to cellular responsiveness, specific nuclear receptor binding assays are included. In addition, tissue responses that are particularly sensitive and specific to a hormone are included as end-points for Tier 1 screens.
C. Criteria for Tier 1 Screening
The STWG has identified five criteria for constructing a screening battery and used these criteria in discussions of which specific assays to recommend. These criteria were presented to the EDSTAC during the Chicago meeting and derive from the Conceptual Framework. They are presented here again with brief discussion:
- 1. Does the T1S battery minimize false negatives while permitting an as-of-yet undetermined, but acceptable, level of false positives? [this criterion expresses the need to 'cast the screening net widely' and not miss potential endocrine disruptors]
- 2. Does the T1S battery capture an adequate range of metabolic activity? [the battery should include assays from representative vertebrate classes to reduce the likelihood that important pathways for metabolic activation of parent CSMs, or other events, are not overlooked]
- 3. Does the T1S battery have the potential to capture known endocrine endpoints? [all known chemicals affecting the action of estrogen, androgen, or thyroid hormones should be detected]
- 4. Does the T1S battery capture a sufficient range of taxonomic groups? [there are known differences at the organismal level (e.g., in detoxification) among taxa that may affect endocrine activity of CSMs]
- 5. Does the T1S battery incorporate sufficient diversity among the assays to reach conclusions based on weight of evidence? [we suspect many decisions on further testing of CSMs will require weighing the evidence of several assays]
II. Tier 1 Screening Battery
The assays being considered by the STWG for inclusion in the T1S battery are listed below, followed by a rationale for each one. They are arranged in categories that reflect the sources of subjects or material, the kinds of assays performed, and the amount of further development needed to make them ready for screening. Unresolved issues within the STWG, related to each category of assays, follow the information given about the assays. The STWG recognized these assays require development, standardization, and validation.
A. Mammalian Subjects/Material - in vitro Assays
The STWG members attending the September 9-11 work group meeting reached tentative consensus on the following screens to be further considered for inclusion in the final T1S recommendation. In vitro assays are specific for known endocrine mechanisms and sensitive to small doses. These assays were chosen because many xenobiotics are known to act by these mechanisms.
It is critical to acknowledge that the state-of-the-science in this area is evolving rapidly and assays are currently being developed that may offer distinct advantages over some of those discussed below. As they are developed and validated, the use of new assays for screening is strongly encouraged.
1. Cell-free Receptor Binding
Receptor binding assays (RBAs)are long-standing and relatively simple in vitro assays that detect specific mechanism of endocrine activity. This is important because several xenobiotics display affinity for the estrogen and/or androgen receptors (thyroid hormone receptors exist are structurally and functionally similar to receptors for estrogens and androgens, but there is no information on CSMs binding to these proteins). Binding assays identify, but do not discriminate between, agonists and antagonists. The apical nature of these assays can be an advantage rather than a limitation because either activity can produce adverse endocrine effects. These assays typically are less sensitive than the functional assays, and they lack metabolic activity, which is an advantage if one wishes to identify the specific compound with endocrine activity. However, the lack of metabolic activation is also a limitation because some xenobiotics require metabolic activation. With this limitation in mind, the STWG along with some members of the PSWG, have suggested that in vitro screening assays should screen a metabolically activated fraction, as well as the parent material. For the whole cell assays, discussed below, one could employ two cell lines, one that can and one that cannot metabolize toxicants. In vitro assays conducted in a high-throughput mode, with and without metabolites, could provide an extremely powerful tool for potential receptor binding activity of large numbers of CSMs. Importantly, because thyroid hormone binding is not a known mode of endocrine disruption, activity of chemicals through mechanisms altering thyroid hormone synthesis or removal must be detected with high confidence.
2. Functional Assay (Transcriptional Activation or Cell Proliferation)
In addition to RBAs, the STWG recommends the use of whole-cell transcriptional activation to detect endocrine disruptor activity. The work group will have further discussions to determine which, if any, of these assays should be recommended over others in the future. These assays can also be conducted in a manner that allows them to distinguish between receptor agonists and antagonists.
Although these functional assays often provide information similar to the above binding assays, this is not always the case. There are occasionally well-founded biological reasons for a chemical to display positive results in either the binding or the transcriptional activation assays but not both. Hence, the use of both of these would reduce the incidence of false negatives. At present, the STWG feels both sets of assays should be considered. However, due to a higher degree of difficulty, the STWG is concerned that proper execution of whole-cell assays requires a level of skill and training that may not currently exist in the toxicology community. If so, these assays might be more difficult to validate, standardize, and implement than the binding assays, some of which have been used for decades and are less complex.
The information obtained from RBAs is a subset of that obtained from a transcriptional activation assay; thus, transcriptional activation assays would be an acceptable alternative to cell-free RBAs and may even be preferable if such assays can be standardized and validated, and if patent restriction questions can be resolved. Functional assays are more sensitive than cell-free receptor binding assays; however, there are many versions of functional assays that may not be acceptable; specific assays have been identified and defined and we are discussing their relative merits and ability to replace RBAs.
a. Cellular Assays Detecting Estrogen Receptor Agonists and Antagonists: Assays like the MVLN cell assay for ER activity, an MCF-7 cell line stably transfected with a promoter and luciferase reporter gene, are recommended over transiently transfected cells. If the stably transfected cells are unavailable, the transiently transfected cells can be used to assess estrogen and anti-estrogen action. In addition to the MVLN, other stably transfected cell lines have been or are being developed. The MCF-7 cell proliferation assay is not specifically recommended because the proliferation response is less specific, being indirect, and because the assay response varies, dependent upon unspecified serum factors, cell strain, and passage number. The assay would, therefore, be more difficult to standardize, especially for a high-throughput mode (a question as to whether this assay will be more difficult to standardize has been raised). However, if one wishes to use this assay, the data are acceptable and would obviate the necessity of running one of the other transcriptional activation assays mentioned above. Another possibility is to run each of these assay types through a validation process to determine which type best meets the goals of T1S. In contrast, the Yeast Estrogen Screen (YES) assay is not considered acceptable because of its inability to detect the estrogenic activity of chlorinated chemicals (positive YES data are acceptable and useful, but negative data are not). In addition, since the YES assay, when it works, does not distinguish between agonists and antagonists, the results are more equivalent to a binding assay than other transcriptional activation assays.
b. Cellular Assays Detecting Androgen Receptor Agonists and Antagonists: For AR-mediated activity, stably transfected cell lines are under development, but not yet available. Assays like the CV-1 cell line transiently co-transfected with hAR and a promoter construct with a luciferinase reporter are recommended. It is noteworthy, that as compared to MCF-7 cells the CV-1 has considerable metabolic capability. Here again, the Yeast Androgen Screen (YAS) is not acceptable as it is unable to detect the AR-mediated activity of chlorinated pesticides, however, positive YAS data are acceptable.
3. Steroidogenesis Using Minced Testis Assay
Anti-androgens and anti-estrogens act via a number of direct mechanisms in addition to those which directly involve the steroid hormone receptors. One prominent mechanism of anti-hormonal activity is inhibition of hormone synthesis by inhibiting the activity of P450 enzymes in the steroid pathway. Such activity could be detected in a fairly simple in vitro procedure with minced testicular tissue obtained from adult male rats. Although for many of the pesticides known to alter this pathway the parent material is active, testing a "metabolically activated" fraction would enhance the utility of this assay. Further development and validation of this assay is required. Leydig cell cultures could be used in place of the minced testis culture; the results are more precise, but the technique is more difficult.
4. Additional Enzyme Assays (e.g., placental aromatase and thyroid peroxidase)(2)
One critical enzyme present at very low levels in the testis is aromatase, which converts testosterone to estradiol and is another P450 enzyme. It was proposed because human placental aromatase is commercially available and could be used in vitro to assess the effects of toxicants on this enzyme fairly easily. An additional enzyme under consideration, and commercially available, is thyroid peroxidase; one can assess effects of toxicants on this enzyme as well. These enzyme assays are still under consideration by the STWG.
B. Mammalian Subjects - in vivo Assays
In vivo assays are more apical; that is, they incorporate endocrine-specific endpoints but disruption of a number of hormone regulation/delivery mechanisms can be evaluated at once. In addition, the in vivo assays allow evaluation of mechanisms of hormonal action that cannot be isolated in in vitro assays.
1. Female: Uterotrophic Assay
A 3-day Uterotrophic Assay for estrogenicity or anti-estrogenicity in immature (18-21 day old) or adult ovariectomized female rat. Females are injected sc or ip with the chemical for 3 days and necropsied 6 hours after the last dose. This assay, which is one of several used for over 50 years, has been used for thousands of estrogenic chemicals, including hundreds of xenoestrogens. Some of the xenobiotics of current concern, e.g., bisphenol A, were identified in such assays in 1938. The assay detects estrogens and anti-estrogens and should incorporate weighing and histological examination, including the percent of endometrial luminal epithelial cells in the proliferative cycle, of the uterus (the examination could be accomplished in the Developmental Thyroid/Uterotrophic Assay described below).
2. Male: Hershberger Assay
This assay has been used for over 40 years in the pharmaceutical industry and for several xenoantiandrogens as well. An in vivo assay is required to screen chemicals for androgenic and anti-androgenic activity. The STWG prefers the standard Hershberger et al. (1953) assay over a longer, more apical "pubertal" assay using juvenile males (or adult male rates). In the standard Hershberger, weanling male rats are castrated and for five consecutive days are given either: 1) vehicle, 2) testosterone, 3) the xenobiotic (detects androgens as compared to group 1), or 4) the xenobiotic plus testosterone (detects anti-androgens as compared to group 2). In conducting the assay one should necropsy male and weigh seminal vesicle (SV), ventral prostate (VP), and levator ani plus bulbocavernosus muscles (LA), which are all androgen-dependent (T or DHT) tissues. This assay could be extended beyond 5 days to 14 days (or longer) for assessment of thyroid function. However, in the absence of a group of intact males (with and without treatment), this assay would provide no assessment of the hypothalamic-pituitary-gonadal axis.
3. An Assay to Assess Thyroid, EROD, and Other Activities(3)
Peripheral effects of thyroid hormones in the adult and the developing fetus are complex and require considerable time to manifest. Therefore, the STWG proposes the measurement of circulating thyroxin (T4), thyrotropin (TSH) and thyroid histopathology in an in vivo assay in one sex. Measurement of T4 is recommended because many xenobiotics affect circulating levels of thyroxin by increasing T4 clearance rate and/or by displacing thyroxin from carrier proteins in the blood. Thyrotropin level is the single endpoint used clinically to evaluate thyroid hormone action. Changes in circulating TSH can compensate for changes in T4; thus, both T4 and TSH must be measured. Additionally, changes in the biopotency of TSH that are not detected by RIA will be evaluated by histological examination of the thyroid gland. Finally, the STWG feels that 14 days of dosing are required for manifestation of these effects.
One of the following three assays would be needed in order to detect altered thyroid hormone status; a 14-day dosing assay reported to be able to detect agonists and antagonists which may detect alterations of the hypothalamic-pituitary-gonadal axis, a 14-day dosing assay reported to detect Ah-receptor agonists, estrogens, anti-estrogens, and altered hypothalamic-pituitary-gonadal maturation; or a 30-day thyroid/pubertal male assay. The potential of these assays for screening could be determined in validation studies. The current approach is to recommend performance of the female Uterotrophic assay and the male Hershberger assay and one of the following three assays. Alternatively, 3a or 3b might replace the female Uterotrophic assay and/or assay 3c might replace the male Hershberger Assay. Further discussion by the group is required.
a. 14-day Developmental Thyroid/Uterotrophic Assay. Intact female rats are dosed daily from postnatal day 9 to 22. Endpoints include uterine weight and uterine gland height. Uteri are cross-sectioned and the number of uterine glands and the height of the luminal and glandular epithelia are measured. Altered uterine weight may suggest complete agonist activity (large weight gain) or partial agonist/antagonist activity (smaller weight gain). Inhibition of gland appearance is an irreversible developmental effect (i.e., not dependent on the continued presence of the estrogen) and unlike uterine weight, is completely responsive to chemicals from either pharmacological class. Epithelial hypertrophy, measured as cell height, occurs at all ages and is reversible, as is uterine weight gain, but like inhibition of gland appearance, is completely responsive to chemicals in both pharmacological categories. Glandular epithelial hypertrophy only responds to mixed agonists/antagonists. Furthermore, the ovary makes estrogens starting on day 10 and treatment with a pure anti-estrogen or removal of the ovary lowers uterine weight. Thus, lowered uterine weight may indicate action as a steroidogenesis inhibitor or as an inhibitor of estrogen production via the hypothalamic-pituitary-ovarian axis. Finally, increased estrogen production via the same route would increase uterine weight. This developmental assay defines patterns of activity for chemicals that act via the receptor and additionally detects those acting via the hypothalamic-pituitary-ovarian axis or other mechanisms that cannot be detected in an ovariectomized animal or using in vitro screens. Effects in thyroid hormone (as discussed above) can also be detected in this assay.
b. 14-day Thyroid/Pubertal Assay. Using the immature female rat, oral dosing is initiated at 21 days of age and continued for two weeks to day 35. Oral dosing allows for the detection of chemicals not active in the 3-day uterotrophic assay due to the fact that they are not effectively absorbed via sc injection (i.e., methoxychlor). Estrogens accelerate vaginal opening, induce vaginal cornification, and alter estrous cyclicity (triggered measures from accelerated VO). Anti-estrogens delay VO. In addition, as puberty is the landmark of hypothalamic-pituitary-gonadal maturation culminating in vaginal opening and ovulation, alterations of these functions can be detected as well. The VO assay has been used for about 80 years and is a part of current Agency test guidelines. Hence, test labs have standard operating procedures (SOPs) and experience in collecting pubertal endpoints in female rats. From these studies it is clear that VO is more sensitive to estrogens than traditional multi-generational endpoints. Effects in thyroid hormone (as discussed above) can also be detected in this assay.
c. 30-day Thyroid/Pubertal Male Assay. This "pubertal" assay takes longer, is more apical and is not likely necessary as long as one of the above assays using female rats provides for sufficient assessment of the thyroid and hypothalamic-pituitary-gonadal axis. In conducting the assay one should: initiate oral dosing of weanling male rats at 25 days of age (10 per group, selected for uniform body weights at 24 days of age to reduce variance); dose daily, seven days a week and examine daily for PPS; continue dosing until 55 days of age and necropsy males; optional endpoints include weighing body, liver (Ah and etc.), heart (thyroid), adrenal, testis (sperm counts), portions of epididymis (do sperm counts), seminal vesicle plus coagulating glands (with fluid), ventral prostate, levator ani plus bulbocavernosus muscles (as a unit); save thyroid for histopathology; save liver for EROD; take serum for T4, T3, testosterone and dihydrotestosterone analyses; optional biochemical and gene activation measurements could be taken as well; if T4 or T3 is altered, then measure TSH; if liver weight is increased, run EROD; if EROD is elevated, run toxicant and metabolites in in vitro Ah-receptor binding assay (could use stably transfected cell line); and run minced testis culture, ex vivo. One advantage of using the pubertal assay in the male rather than the female is that both anti-androgens and estrogens delay puberty in the male, while the female is unresponsive to anti-androgens. The length of this assay was considered a drawback.
4. Assays With in utero Exposure
There is consensus among STWG members that the T1S battery (including the mammalian assays and the other in vivo vertebrate assays discussed later), when validated, will detect all known disruptors of estrogen, androgen, and thyroid action. However, some work group members believe mechanisms may exist by which unknown disruptors can effect the embryo/fetus with out showing positive on any of the above assays. Therefore, these work group members have indicated an in utero developmental assay needs to be included in the T1S battery as well. All work group members agreed such assays should be presented, discussed, and considered. At the last STWG meeting four developmental assays were presented. These assays differed in dosing length and number of endpoints assessed in the offspring. The shortest of these involves dosing the dams from gestational day 14 to postnatal day 14, with most assessments being carried out prior to weaning, while the longest of these takes several months to conduct.
Several of the assays/protocols under discussion are hypothetical as they have never been used for any chemicals. As these assays have yet to be executed as proposed, analysis of their ability to detect certain endocrine activities and their sensitivity, as compared to more standard assays, is premature.
C. Other Vertebrate Subjects/Material - in vivo Assays(4)
As mentioned earlier, there are known differences at the organismal level (e.g., in detoxification) among taxa that may affect the endocrine activity of chemical substances and mixtures (CSMs), but comparative physiology is not extensive enough to predict which taxa are the most sensitive. Hence, we have evaluated in vivo assays using representatives of selected vertebrate classes. All the assays in this category have been used previously to investigate basic mechanisms of development or reproduction and the influence of hormones on these processes. The assays vary in the amount of data extant relevant to the action of CSMs. Each assay needs at least some of the following: more data to demonstrate their efficacy with CSMs; validation and standardization relative to estrogen, androgen, or thyroid hormone; and/or modification to improve its suitability for screening. The STWG believes the taxonomic range represented by these procedures is a vital characteristic of the battery, and that, further, the evaluation, necessary modifications, standardization, and validation for each assay should be accomplished as soon as possible.
Although several short-term in vitro and in vivo non-mammalian assays of EAT were identified, the STWG did not feel it was necessary to have a separate comprehensive battery like that presented for mammals. The group generally felt estrogens in mammalian assays would also be estrogenic in most non-mammalian systems as well. Rather than unnecessarily duplicate the assays in the screening process, it was our objective to include non-mammalian assays which could complement, rather than duplicate, the mammalian assays. For example, it is unlikely that in vitro binding assays of avian ER would yield results different from those obtained with mammalian ER. In contrast, the proposed amphibian metamorphosis assay provides an example where a simple developmental process, one which has no mammalian equivalent, can facilitate identification of chemicals that display (anti)thyroid activity. No simple mammalian assay of (anti)thyroid activity exists that is as specific as the amphibian metamorphosis assay.
A different strategy was adopted for the development of a fish EAT assay. This vertebrate class is farthest removed phylogenetically from mammals than the other vertebrate classes. The committee felt if significant differences existed between mammals and any other vertebrate class with respect to EAT activities then they would be reflected in fish. For example, fish ER differs from mammalian ER more than the ER of other classes. In addition, fish have some unique androgens and possibly different hormone receptors. Not only is there less conservation of structure but the function of the hormones often differs greatly between these two vertebrate classes. Part of the difficulty in designing a screening battery specifically for EAT in fish lies with the fact that this class is very diverse and in many cases, little is known about the role of these hormones. With this in mind, the STWG adopted the screening approach recommended by the EPA/KC Workshop on this issue, which was to develop a simple, yet comprehensive apical test, that would detect EAT in selected species. This assay includes exposure by injection and evaluation of gonadosomatic index (GSI), histology of the reproductive tract, and induction of vitellogenin in males.
The committee is also considering (anti)androgen assays in avian species to complement the mammalian assays. Two short-term assays for androgenicity in birds have been considered, including crop growth in chickens and proctodeal gland size in Japanese Quail. Several additional assays for non-mammalian vertebrates have been discussed by the STWG, but their role in screening versus testing has not been finalized. In avian and reptilian species, oviductal weight has been used to screen chemicals for estrogenicity in the same manner that the uterotrophic assay is used in rodents. This is a short-term assay that can detect estrogenicity and could be modified to detect anti-estrogens as well. Two fairly long-term developmental assays that are responsive to estrogens and aromatase inhibitors are being discussed. One involves in ovo injection in the Japanese quail, while the other examines the effects of xenobiotics on turtle temperature-dependent sex differentiation. In contrast to mammalian sex differentiation where androgens predominate, in these species estrogens and the aromatization of androgens normally plays a key role in the development of the female phenotype. Another consideration is to extend the length of exposure in the amphibian metamorphosis assay which would enable the detection of (anti)androgens and estrogens in a single assay.
Frog Metamorphosis: Tail resorption in frogs is under thyroid control with selective cell death specifically controlled by thyroxin. Thus, monitoring patterns of tail resorption, as well as the rate at which tail resorption occurs is a suitable indicator of thyroid hormone function. The South African clawed frog (Xenopus laevis) has been utilized in a short term (14-day) assay to evaluate the rate and behavior of tail resorption. This assay provides a comparatively simple procedure for evaluating thyroid active CSMs. Preliminary trials demonstrate this assay detects thyroid and anti-thyroid active compounds and, as a logical extension of the FETAX assay, is suitable for standardization. Modifications of this assay to include other morphological endpoints such as hind limb growth and eye migration, histopathology, and deiodinase activity are also under consideration. This assay could be extended to detect anti-androgens as well.
Fish In Vivo Screening Assay: Adult male and female fish are exposed to test chemicals in the water for 3-4 weeks at the beginning of gonadal recrudescence. The following primary measures will be taken 1) gonadosomatic index (GSI); 2) secondary sexual characteristics; 3) final oocyte maturation (FOM)/ovulation/ spermiation; 4) plasma sex steroids; and plasma vitellogenin. These endpoints were selected on the basis of their broad applicability and ease of routine measurement. This assay will respond to a broad range of EDCs including estrogen, anti-estrogen, anti-androgen, and anti-progestin. It is expected that this assay should detect endocrine disruption at any site of chemical interference on the hypothalamus-pituitary-gonadal-liver axis, including neuroendocrine effects.
Androgen Screen: Rationale: This procedure exploits the activational action of androgens on male externally visible morphology and behavior in seasonally reproductive Japanese quail. Reproductive behavior (crowing and copulation) as well as proctodeal gland hypertrophy can be stimulated when supplemental testosterone is given to adult male quail, which have been made non-reproductive by housing them under short day conditions. Three responses measured can together distinguish which pathway for androgenic action is stimulated by a chemical being screened: proctodeal gland hypertrophy alone indicates a DHT agonist, copulation alone indicates an estradiol agonist, and both (along with crowing) indicates a testosterone agonist, (but only if the test chemical can be metabolized to DHT and estradiol). Extensive study of maturing male Japanese quail is consistent with a dose-response relationship between serum testosterone concentrations and these responses. This methodology has been widely used to investigate basic processes of avian endocrinology, but has not been applied to the purpose of identifying CSMs with endocrine activity. This is a procedure with excellent potential as a screen, but the specific data needed to evaluate its sensitivity to EDCs that might have androgenic activity are lacking. It has the advantage of being an in vivo procedure and hence will capture mechanisms that might not be assayed with in vitro screens, it uses avian subjects, which increases the taxonomic range of the screening battery, and it targets androgen activity. These qualities are important features that lend confidence to the comprehensive coverage of endocrine activity of the battery.
Sex Determination/Egg Exposure: This assay measures the ability of estrogens to induce female sex determination under temperature conditions normally producing males. Chemicals are applied to the eggshell and sex is scored at hatching. This assay is slightly more sensitive than the rat uterotrophic assay, has a different steroid specificity, and appears to be the most sensitive of the wildlife assays. The assay also detects steroidogenesis inhibitors. Eggs are readily available in season, and studies can be conducted at a large scale for relatively low cost.
D. Invertebrate Assays
No invertebrate assays have been evaluated for use in a screening battery for detecting estrogen, androgen, or thyroid hormone disruption. It is recommended that a workshop of invertebrate endocrinologists and toxicologists be convened to address first, the suitability of invertebrate assays for estrogen and androgen (not thyroid) for use in a screening battery, and second, future improvements to the broader consideration of endocrine disruption in the environment and the utility of invertebrates as surrogate test organisms.
There are two aspects to considering endocrine disruption for invertebrates; one is relevance to the health of invertebrate organisms themselves and the other is relevance of invertebrates as surrogates for investigating vertebrate-related phenomena. Conventional risk assessment of toxic chemicals such as outdoor-use pesticides and high volume industrial chemicals generally include a crustacean reproduction or life cycle test in the data set used in the assessment. Although specific endocrine system endpoints are not considered, the apical nature of these tests may be adequate to detect the adverse consequences of an endocrine disrupting chemical in crustacean arthropods. Additional information is needed to determine what is most useful beyond these conventional tests for the wider invertebrate taxa. As surrogates, more information on the correlation of endocrine phenomena between invertebrates and vertebrates would be helpful. For instance, to what degree does a substance which disrupts ecdysteroid metabolism in crustacea disrupt sex steroid metabolism in vertebrates? Perhaps good correlations may be found, but more comparative information is needed before recommendations of specific invertebrate tests useful for evaluating potential endocrine disrupting activity relevant to vertebrates can be made. Please see the attached document, Endocrine Disruption and Invertebrates, for further discussion of invertebrate issues.
1 Throughout this document, recommendations and consensus pertains only to those work group members who attended the September 9-11 work group meeting. All work group members have participated in the evolution of this paper and many of them have commented on the substance. Subsequent work group meetings will enable all work group members to further discuss the issues raised in this paper as well as other issues.
2 The need for the placental aromatase and thyroid peroxidase assays is unclear until T1S in vivo screens have been fully defined.
3Thyroid endpoints may be incorporated into a uterotrophic assay or the Hershberger assays if they can be extended for 14 days as described below.
4The relative sensitivities of various hormone responsive endpoints, within each species, needs to be established, and the relative sensitivities of each species needs to be assessed to determine which, if any, are required for T1S assays.