REVISED DRAFT DETAILED REVIEW PAPER FOR AVIAN TWO GENERATION TOXICITY TEST EPA CONTRACT NUMBER 68 W 01 023 WORK ASSIGNMENT 2 16 April 23, 2003 Prepared for LESLIE TOUART, PH. D. WORK ASSIGNMENT MANAGER U. S. ENVIRONMENTAL PROTECTION AGENCY ENDOCRINE DISRUPTOR SCREENING PROGRAM WASHINGTON, D. C.

BATTELLE 505 King Avenue Columbus, Ohio 43201 Battelle Draft ii April 23, 2003 TABLE OF CONTENTS 1.0 EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2.0 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.1 Developing and Implementing the Endocrine Disruptor Screening Program ( EDSP) . . . . . 4 2.2 The Validation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.3 Purpose of the Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.4 Objectives of the Avian Two Generation Toxicity Study . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.5 Methods Used in this Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.6 Acronyms and Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.0 OVERVIEW AND SCIENTIFIC BASIS OF AVIAN TWO GENERATION TESTS . . . . . . . . 10 3.1 Avian Endocrinology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2 Differences in Avian and Mammalian Endocrine Systems . . . . . . . . . . . . . . . . . . . . . . . . 13 3.3 Avian Two Generation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.0 CANDIDATE TEST SPECIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 4.1 Japanese Quail ( Coturnix japonica) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 4.1.1 Natural History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 4.1.2 Availability, Culture, Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 4.1.3 Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.2 Bobwhite ( Colinus virginianus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4.2.1 Natural History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4.2.2 Availability, Culture, Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 4.2.3 Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4.3 Strengths and Weaknesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 5.0 EXPERIMENTAL DESIGN CONSIDERATIONS FOR TWO GENERATION AVIAN TESTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 5.1 Exposure Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 5.1.1 Exposure of the Parental ( P1) Generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 5.1.2 Exposure of the Offspring ( F1) of the Parents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 5.1.3 Combined Exposure Scenarios for P1 and F1 Generations . . . . . . . . . . . . . . . . . . . 34 5.1.4 Selection of Egg Cohort for F1 Breeding Pairs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 5.1.5 Selection of P1 and F1 Birds for Pairing and Breeding . . . . . . . . . . . . . . . . . . . . . . 37 5.1.6 Comparison of One Generation and Two Generation Exposure Regimens . . . . . . 37 5.2 Route of Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 5.2.1 Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 5.2.2 Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 5.2.3 Bolus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 5.3 Dose Selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 5.3.1 Dose Adjustment for Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 5.3.2 Dose Adjustment by Life Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5.4 Statistical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Battelle Draft iii April 23, 2003 TABLE OF CONTENTS ( Contd)

5.4.1 Sample Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 5.4.2 Hypothesis Testing or Regression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 6. ASSAY ENDPOINTS: FITNESS ENDPOINTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 6.1 Growth Rate, Food Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 6.2 Measures of Reproductive Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 6.2.1 Fecundity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 6.2.2 Gamete Viability and Fertilization Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 6.2.3 Changes in Breeding Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 6.3 Neurological/ Central Nervous System Impairment Tests . . . . . . . . . . . . . . . . . . . . . . . . . 71 7.0 ASSAY ENDPOINTS: PHYSIOLOGICAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 7.1 Organ Growth and Morphological Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 7.1.1 Development of Gonadal and Accessory Structures . . . . . . . . . . . . . . . . . . . . . . . . 73 7.1.2 Histopathology in Juveniles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 7.1.3 Histopathology in Sexually Mature Individuals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 7.1.4 Histopathology and Organ Weights of Nonreproductive Tissues . . . . . . . . . . . . . . 78 7.1.5 Japanese Quail Male Cloacal Gland Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 7.2 Sexual Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 7.3 Secondary Sex Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 7.4 Sex Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 7.5 Biochemical Measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 7.5.1 Vitellogenin and Other Biomarkers of Hepatic Metabolic Changes . . . . . . . . . . . . 85 7.5.2 Plasma and Renal/ Urate Hormone Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . 89 8.0 RESPONSE TO ESTROGEN AGONISTS AND ANTAGONISTS . . . . . . . . . . . . . . . . . . . . . . 92 8.1 Sexually Mature Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 8.1.1 Sensitivity to 17ß estradiol or Synthetic Estrogen Exposure . . . . . . . . . . . . . . . . . . 93 8.1.2 Antiestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 8.1.3 Relevance, Sensitivity, Use History, Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . 94 8.2 Juvenile Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 8.2.1 Sensitivity to 17ß estradiol or Synthetic Estrogen Exposure . . . . . . . . . . . . . . . . . . 94 8.2.2 Antiestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 8.2.3 Relevance, Sensitivity, Use History, Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . 96 9.0 RESPONSE TO ANDROGEN AGONISTS AND ANTAGONISTS . . . . . . . . . . . . . . . . . . . . . . 97 9.1 Sexually Mature Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 9.1.1 Sensitivity to Androgenic Steroid Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 9.1.2 Antiandrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 9.1.3 Relevance, Sensitivity, Use History, Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . 99 9.2 Juvenile Life Stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 9.2.1 Sensitivity to Androgenic Steroid Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 9.2.2 Antiandrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 9.2.3 Relevance, Sensitivity, Use History, Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . 100 Battelle Draft iv April 23, 2003 TABLE OF CONTENTS ( Contd )

10.0 RESPONSE TO THYROID AGONISTS AND ANTAGONISTS . . . . . . . . . . . . . . . . . . . . . . 101 10.1 Sensitivity to Thyroid Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 10.2 Inhibition of Thyroid Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 10.3 Relevance, Sensitivity, Use History, Uncertainty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 11.0 CANDIDATE PROTOCOLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 11.1 One Generation Reproduction Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 11.1.1 OPPTS 850.2300 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 11.1.2 OECD 206 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 11.1.3 ASTM E1062 86 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 11.1.4 OECD Revised Draft Proposal ( April 2000) for a New Test Guideline, Avian Reproduction Toxicity Test in the Japanese Quail or Northern Bobwhite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 11.2 Two Generation Life Cycle Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 11.2.1 Proven Breeder Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 11.2.2 Pre Egg Laying Exposure Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 12.0 RECOMMENDED PROTOCOL AND ADDITIONAL DATA NEEDS . . . . . . . . . . . . . . . . . 122 12.1 Preferred Test Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 12.2 Exposure Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 12.3 Appropriateness of Reproductive Endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 12.4 Preferred Methods for Quantification of Biochemical Endpoints . . . . . . . . . . . . . . . . . . 126 12.5 Significant Data Gaps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 13.0 IMPLEMENTATION CONSIDERATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 14.0 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 APPENDIX A   Literature Search ( Will be added) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 1 APPENDIX B   Interviews ( Will be added) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B 1 FIGURES 5 1 Calculated Dose at Weekly Intervals of Growing Japanese Quail Consuming a Diet Amended with 100 or 167 ppm of Test Substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 5 2 Power of a One Sided Independent Samples T Test as a Function of the Percentage Reduction Detected Between the Test and Reference Means, with 16 Replicates per Treatment . . . . . . . . . 50 TABLES 2 1 Acronyms and Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 4 1 Stocks of Random Bred or Wild Type Japanese Quail Maintained in North America and Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 4 2 Body Weight and Reproductive Parameters of Egg Producing and Meat Producing Strains of Japanese Quail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Battelle Draft v April 23, 2003 TABLES ( Contd)

4 3 Major Strengths and Weaknesses of Japanese Quail and Northern Bobwhite Related to Use in Avian Two Generation Reproduction Toxicity Tests . . . . . . . . . . . . . . . . . . . 28 4 4 Typical Values for Reproductive Parameters in Japanese Quail and Northern Bobwhite . . . . . . . 29 4 5 Comparison of Development Phases in Japanese Quail and the Northern Bobwhite . . . . . . . . . . 30 5 1 Required Attributes of Parental and Offspring Exposure Regimens . . . . . . . . . . . . . . . . . . . . . . . 35 5 2 Control Data Coefficient of Variation for the Raw and Transformed Endpoints Measured from an Interlaboratory Comparison Study on Japanese Quail Exposed to Bis( tri butyltin) oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 6 1 Fitness Endpoints Specific to Endocrine Active Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 7 1 Physiological Endpoints Specific to Endocrine Active Substances . . . . . . . . . . . . . . . . . . . . . . . . 72 7 2 Gross Landmarks of Sexual Differentiation in Japanese Quail and Age of Appearance . . . . . . . . 75 11 1 Comparison of Avian Reproductive Toxicity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Battelle Draft vi April 23, 2003 SPECIAL NOTE A substantial technical contribution to the creation of this document was provided by Dr. Ann Fairbrother. Written text included the author's own published work, either as a lead or coauthor During this time, Dr. Fairbrother was working for a private company. She has since taken a position with the EPA. Battelle Draft 1 April 23, 2003 1.0 EXECUTIVE SUMMARY Endocrine disruptors are any chemicals that are known or suspected to cause adverse endocrine effects in organisms or their progeny. Such chemicals have received increased attention over the past decade because of the potential harm they can do to wild and domestic animals and ultimately to humans. Therefore, Congress authorized the United States Environmental Protection Agency ( EPA) to develop a program to screen a wide array of chemicals found in drinking water sources and food to determine whether they possess estrogenic or other endocrine activity that could have disruptive endocrine effects in humans. The aim of this program is to develop a two tiered approach: that is, a combination of in vitro and in vivo mammalian and ecotoxicological screens ( Tier 1), and a set of in vivo tests ( Tier 2) for identifying and characterizing endocrine effects of pesticides, industrial chemicals, and environmental contaminants. The organisms used in the screening and testing will represent a variety of taxonomic groups, such as fish, birds, and mammals, for example.

The present detailed review paper fulfills one of EPA's objectives in its validation process, namely, to summarize, explain, and document the relevant principles, methods, and techniques for a Tier 2 multigenerational test to determine the effects of potential endocrine disrupting chemicals on birds. Although avian species are not part of the Tier 1 screening battery for assessment of the effects of endocrine disrupting chemicals, they are included in Tier 2. Birds are fundamentally different from mammals in the control of their sexual differentiation, and therefore, mammalian tests provide little predictive value for assessing a chemical's potential impact to birds; separate testing is required.

The present report contains a review of the current literature, and a recommendation of an initial Tier 2 protocol and of an organism that will best meet the needs for testing. It identifies issues that could require prevalidation studies. The recommended avian test protocol is designed to accomplish the following:

° determine whether effects are a primary or secondary disturbance of endocrine function ° establish exposure, concentrations, timing, and effects relationships ° be sensitive and specific ° assess relevant endpoints ° include a dose range for full characterization of effects ° be conducted in accordance with good laboratory practices ( GLP) ° be validated.

Two quail species, the northern bobwhite, Colinus virginianus, and the Japanese quail Coturnix japonica, were considered for the test species, because they are commercially cultured and available year round, are among the few avian species that breed successfully under laboratory conditions, and unlike most birds, are able to produce eggs almost indefinitely under long day photoperiods. Also, both species are accepted models for assessing acute and reproductive effects of pesticides and other chemicals in birds. Although the Japanese quail is not indigenous Battelle Draft 2 April 23, 2003 to the United States and has undergone extensive domestication, and therefore may be less representative of wild species, it is recommended here as the preferred test species because of its small size, high fecundity, well characterized reproductive biology, and in particular, its very rapid incubation and maturation stages. Pragmatically, the completion of a mutigenerational test using Japanese quail can be completed in less than half the time of a test using the bobwhite.

The experimental design of avian reproduction tests has undergone considerable discussion. The tests can be designed to define concentrations of environmental chemicals at which no observable adverse effects occur ( NOAECs), or they can be designed to develop dose response relationships for endpoints of concern ( ECx). Two principal exposure protocols have also been considered for the parental ( P1) generation in the avian two generation reproduction test; a " proven breeder design," in which reproduction is monitored pre exposure, and a " pre egg laying exposure design," in which effects on maturation are included. Several first generation ( F1) exposures have also been considered, the two most likely of which are treatment from hatch through egg laying and no treatment. Currently, there are insufficient data to determine the combination of exposure protocols for the P1 and F1 generations that is the most robust for documenting changes in ecologically important fitness endpoints, and that at the same time is most effective in determining mechanism of action. A side by side performance evaluation in prevalidation of the prebreeding and proven breeder exposure regimens and the F1 treatment options is recommended to evaluate the sensitivity and cost benefit of these exposure protocols.

Because of the variety of responses that test substances can induce, a broad set of reproductive fitness and physiological endpoints was selected to evaluate reproductive impact and endocrine activity in an avian two generation reproduction toxicity test. Emphasis was given to endpoints recommended by the Endocrine Disruptor Screening and Testing Advisory Committee and the Organization for Economic Cooperation and Development ( Bennett, et al., 2001). Additional endpoints were added from the literature review. Fitness endpoints are egg production and viability, hatching success, survival of chicks to 14 days of age, genetic sex, onset of sexual maturation, cloacal gland area, body weight, and male copulatory behavior. The physiological endpoints are gross morphology and histology of specific organs, levels of sex hormones, corticosterone, and thyroid hormones by specific methods. Because of the effects of handling on plasma concentrations of many of these hormones, it is preferable that hormone status be evaluated via the noninvasive sampling of fecal/ urate matter. Monitoring of T4/ T3 values over time would particularly benefit from sampling of feces; however, methods for fecal analysis have not been developed for these hormones, although they are excreted in the bile. Measuring the sperm: egg interaction is recommended as an inexpensive, but direct means of assessing gender specific effects on fertility during egg production and for establishing relative fertility of proven breeder males prior to treatment. Some modification of these endpoints is recommended to reduce redundancy, increase the cost effectiveness of the test, and provide higher quality data.

To further optimize two generation testing with Japanese quail, several research areas have been identified: Battelle Draft 3 April 23, 2003 ° Many strains of Japanese quail have been developed, largely along egg production or body mass lines. The impact of strain selection on the ability of the test to detect endocrine activity and reproductive deficit needs to be evaluated. ° Japanese quail are recommended over the northern bobwhite largely for pragmatic reasons. Information that is not known at this time that could greatly influence this selection is the relative sensitivity of these two species to reproductive and endocrine effects of environmental chemicals. ° The potential impact of natural phytoestrogens in the feed on the outcome the test needs to be delineated so that limits of contamination can be set. ° If ANOVA methods continue to be applied to avian reproduction toxicity tests, a statistical approach for delayed effects must be investigated. ° Though seemingly a minor data gap, the lack of specific information on husbandry requirements of the Japanese quail that will result in consistent results in laboratory toxicity tests is important. ° The number of replicates ( pens) needed for dose response testing is not known. The replicate needs for sufficient power to detect an effect in the various physiological endpoints using ANOVA approaches have not been established. ° The most appropriate treatment regimen needs to be defined for the P1 and F1 generations to best characterize effects of potential endocrine disruptors on fitness endpoints with consideration of endocrine and other toxic effects. ° Baseline data on the variance, sensitivity, and range of responses of many of the selected and potential physiological endpoints ( steroid and thyroid hormones, behavior, vitellogenin, very low density lipoproteins, aromatase) are needed. ° The source of the in ovo concentrations of test substances has impact on decisions regarding exposure duration and interpretation of endpoint responses. However, chemical transfer to the egg has been variably described from tissue or dietary sources. Elucidation of source transfer is needed. ° Further study of estrogenic xenobiotics that require metabolic activation is needed to determine whether or not this occurs in the embryo and to understand its relationship to this sensitive developmental period.

Considerations for a recommended protocol are discussed in Section 12 of this DRP using the Japanese Quail as the test system. Battelle Draft 4 April 23, 2003 2.0 INTRODUCTION 2.1 DEVELOPING AND IMPLEMENTING THE ENDOCRINE DISRUPTOR SCREENING PROGRAM ( EDSP)

Chemicals that are known or suspected of being endocrine disruptors ( Kavlock et al. 1996), also referred to as hormonally active agents ( NRC 1999), have received increased attention over the past decade. In 1996, the passage of the two laws, the Food Quality Protection Act ( FQPA) and Amendments to the Safe Drinking Water Act ( SDWA) mandated the United States Environmental Protection Agency ( EPA) to screen substances found in drinking water sources or food to determine whether they possess estrogenic or other endocrine activity ( Federal Register 1998a, 1998b). Pursuant to this goal, the EPA is required 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..." ( FQPA 1996).

In 21 U. S. C. § 346a( p)( 3), the FQPA also states that in carrying out its screening program, the EPA ( A) shall provide for the testing of all pesticide chemicals and ( B) may provide for the testing of any other substance that may have an effect that is cumulative to an effect of a pesticide chemical if the Administrator determines that a substantial population may be exposed to such a substance.

Additionally, Congress amended the Safe Drinking Water Act ( SDWA) ( 42 U. S. C. § 300j 17), authorizing the EPA to provide for the testing, under the FFDCA Screening Program . . . any other substance that may be found in sources of drinking water if the Administrator determines that a substantial population may be exposed to such substance.

Prior to the passage of the FQPA and the SDWA, the EPA initiated several endocrine disruptor investigations, including the development of a special report and effects assessment ( EPA 1997a); a series of endocrine disruptor methods workshops funded by the World Wildlife Fund, Chemical Manufacturers Association ( later known as the American Chemistry Council), and the EPA ( Gray et al. 1997; EPA 1997b; Ankley et al. 1998); and co sponsorship ( with the National Institute of Environmental Health Sciences [ NIEHS] and the Department of the Interior) of an independent critical literature analysis of hormone active toxicants in the environment by the National Academy of Sciences ( NRC 1999).

The EPA established the Endocrine Disruptor Screening and Testing Advisory Committee ( EDSTAC), to provide recommendations regarding a strategy for developing a testing paradigm for compounds that may have activities similar to naturally occurring hormones. Following the recommendations made by EDSTAC in its final report ( EDSTAC 1998), the EPA established the Battelle Draft 5 April 23, 2003 Endocrine Disruptor Screening Program ( EDSP). The program's aim is to develop a two tiered approach, e. g. a combination of in vitro and in vivo mammalian and ecotoxicological screens ( Tier 1) and a set of in vivo tests ( Tier 2) for identifying and characterizing endocrine effects of pesticides, industrial substances, and environmental contaminants ( Federal Register 1998a, 1998b).

To date, the EPA has implemented the program on two fronts: ( 1) the development of the Endocrine Disruptor Priority Setting Database, and the approach that will be used to establish priorities for screening compounds, and ( 2) prevalidation and validation studies of some of the Tier 1 and Tier 2 assays that are likely to be included in the testing battery. The Endocrine Disruptor Methods Validation Subcommittee ( EDMVS) has been set up to advise and review new and ongoing work in the validation of these assays.

The EDSP's proposed statement of policy, including public comments, was reviewed by a joint panel of the Federal Insecticide, Fungicide, and Rodenticide Act ( FIFRA) Scientific Advisory Panel ( SAP) and the EPA Science Advisory Board ( SAB) in May 1999. Gray et al. ( 1997), EDSTAC ( 1998), and the National Research Council ( NRC 1999) concluded that a tiered approach relying on a combination of in vivo and in vitro screens for Tier 1 was scientifically reasonable. This conclusion was based upon each group's assessment of the current state of the science on the evaluation of agents affecting the endocrine system. Another consistent conclusion was the need to validate the individual screens and tests in the EDSP. Validation and peer review are prerequisites to the development and approval of test guidelines for regulatory use. Many of the documents cited above and other EPA EDSP related information may be found at http:\\ www. epa. gov/ scipoly/ oscpendo.

In addition to the EPA's domestic EDSP validation program, a separate effort to validate certain screening assays and tests for international use is being conducted by the Organization for Economic Cooperation and Development ( OECD) Test Guidelines Program. The EPA actively participates as a member of the OECD test guidelines program and its Endocrine Disruptor Testing and Assessment Task Force. The EPA is relying on the OECD effort to serve as the mechanism for validation of some of the components of its EDSP. Separate domestic and international activities are necessary in that laws and regulatory procedures differ in various countries. Although international activities are distinct from domestic activities, overlapping membership on various committees ensures appropriate liaison and communication, eliminates duplication of effort, and facilitates international harmonization.

2.2 THE VALIDATION PROCESS The EPA ( and EDMVS) chose to follow the validation process established by the Interagency Coordinating Committee on the Validation of Alternative Methods ( ICCVAM), of which the EPA was a charter member, for validation of the EDSP screening and testing methods. ICCVAM was established by the National Institute of Environmental Health Sciences ( NIEHS) as a standing interagency committee to aid in the validation, acceptance, and harmonization of test methods designed to reduce animal use, refine procedures involving the use of animals so Battelle Draft 6 April 23, 2003 that they would experience less stress, and to replace animal tests whenever appropriate ( ICCVAM 2000). To this end, ICCVAM defined a flexible, adaptable framework for test method validation that was applicable to conventional and alternate methods, and could be applied to the needs of different agencies and regulatory processes.

The purpose of the validation is to establish the reliability and relevance of a test method with respect to a specific use. The process is science driven, and addresses the scientific principles of objectivity and experimental design ( NIEHS 1997). In addition, as stated in the ICCVAM report, " A test is considered validated when its performance characteristics, advantages, and limitations have been adequately determined for a specific purpose." ( NIEHS 1997).

The validation process consists of four discrete phases: ( 1) initial protocol development, ( 2) prevalidation studies, ( 3) validation studies, and ( 4) external scientific peer review. The initial protocol, developed from existing information and experience ( past and current research), serves as the starting point for initiating the validation process. Prevalidation studies consist of further development and optimization of specific initial protocols through targeted investigations. Either before or during prevalidation, a detailed review paper ( DRP) addressing all critical areas outlined in Validation and Regulatory Acceptance of Toxicological Test Methods ( NIEHS 1997) is prepared for each method to summarize, explain, and document decisions regarding the relevant principles, methods, and techniques recommended for the initial protocol. Targeted prevalidation investigations are designed to address questions necessary for completing an optimized, transferrable protocol suitable for interlaboratory validation studies. Validation studies consist of comparative interlaboratory studies to establish the reliability and relevance of the protocols developed in the prevalidation stage. Validation requires the development of a detailed review paper to document what is known about the assay system proposed for validation.

A test is considered validated when its performance characteristics, advantages, and limitations have been adequately determined for a specific purpose. The measurement of a test's reliability and relevance are independent stages in the validation of a test method, and both are required. Reliability is an objective measure of a method's intra and interlaboratory reproducibility. If the test is not sufficiently reliable, it cannot be used for its intended purpose. Alternatively, if the test is not relevant, of questionable relevance to the biological effect of interest, or if it is not an appropriate measure of the effect, its reliability is academic. The relevance of a test may be linked to the mechanism of the toxic effect it measures and to its proposed uses ( NIEHS 1997). The studies conducted will be used to develop, standardize, and validate methods, prepare appropriate documents for peer review of the methods, and develop technical guidance and test guidelines in support of the EDSP.

Following the validation studies, results of an external scientific peer review of the study and the optimized protocols will be used to develop the EPA test guidelines. Battelle Draft 7 April 23, 2003 2.3 Purpose of the Review The purpose of this detailed review paper ( DRP), prepared as part of Work Assignment 2 16, is to define the basis and purpose of the proposed avian two generation reproductive and developmental toxicity study for evaluating effects of potential endocrine disrupting chemicals. The DRP will summarize, explain, and document the relevant principles, methods, and techniques; it will recommend an initial Tier 2 protocol that will best meet the needs for testing; and it will identify issues that could require prevalidation studies.

2.4 Objectives of the Avian Two Generation Toxicity Study Tier 2 is the final phase of the screening and testing program and therefore should provide more detailed information regarding the endocrine disruption activity of a tested chemical or mixture. To fulfill this purpose, tests are often longer term studies designed to encompass critical life states and processes, a broad range of doses, and administration by relevant route of exposure. In addition, the effects associated with endocrine disrupting chemicals ( EDCs) can be latent and not manifested until later in life or may not be apparent until reproductive processes occur in an organism's life history. Thus, tests for endocrine disruption often encompass two generations to address effects on fertility and mating, embryonic development, sensitive neonatal growth and development, and transformation from the juvenile life state to sexual maturity. The results form the Tier 2 testing should be conclusive in documenting a discernable cause and effect relationship of chemical exposure to measurable manifestation in the test organisms.

The avian test protocol that will be recommended in this report ( Section 11.0) will be designed to be capable of the following:

° to determine whether effects are a primary or secondary disturbance of endocrine function ° to establish exposure, concentrations, timing, and effects relationships ° to be sensitive and specific ° to assess relevant endpoints ° to include a dose range for full characterization of effects to be conducted in accordance with good laboratory practices ( GLP) ° to be validated.

Although avian species are not part of the Tier 1 screening battery, they are included in Tier 2 and serve an important role in that birds are fundamentally different from mammals in the control of sexual differentiation, and results from mammalian tests provide little predictive value for assessing a chemical's potential impact on birds. The following sections describe the methods used and results obtained from conducting this DRP on avian multigenerational test methods. Battelle Draft 8 April 23, 2003 2.5 Methods Used in This Analysis In Appendix A, a detailed description of the methods employed for the literature search, such as key words, databases, and results, is provided. After key papers were identified, retrieved, and read for content, pertinent information was synthesized to create this DRP. At the back of this report is a compact disk read only memory ( CD ROM) containing the Reference Manager Database of all documents reviewed. This database includes the reference citation and abstract.

2.6 Acronyms and Abbreviations Table 2 1 lists the acronyms and abbreviations used in the DRP, with the exception of commonly used units, such as h for hour or L for liter. Each of the acronyms and abbreviations also is introduced at first use in the text.

Table 2 1. Acronyms and Abbreviations ACTH adrenocorticotropic hormone ANOVA analysis of variance A& M ( Texas) Agricultural and Mechanical ( University) ASTM American Society for Testing and Materials apo apolipoprotein BSA bovine serum albumin BST bed nucleus of the stria terminalis, part of the quail brain CNS central nervous system CV coefficient of variation CyA cyproterone acetate DDE dichlorodiphenylethylene DDT dichlorodiphenyltricloroethane DES diethylstilbestrol DEHP diethylhexylphthalate DHT dihydrotestosterone DNA deoxyribonucleic acid DRP detailed review paper EB estradiol benzoate EC50 median effective concentration EDx effective dose ( x = percentage of effect, from 0 to 100) EDC endocrine disrupting chemical EDMVS Endocrine Disruptor Methods Validation Subcommittee EDSP Endocrine Disruptor Screening Program EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee EDSTP Endocrine Disruptor Screening and Testing Protocols EE2 ethinyletradiol ELISA enzyme linked immunosorbent assay EPA U. S. Environmental Protection Agency EROD ethoxyresorufin O deethylase FFDCA Federal Food, Drug, and Cosmetics Act FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FQPA Food Quality Protection Act FSH follicle stimulating hormone GLP good laboratory practice GnRH gonadotropin releasing hormone Battelle Draft 9 April 23, 2003 Table 2 1. Acronyms and Abbreviations ( Continued)

HAH halogenated aromatic hydrocarbon HCP highly carboxylated porphyrine hCG human chorionic gonadotropin HPG hypothalmic pituitary gonadal HPV high production volume ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods in vitro outside the body, in an artificial environment in ovo in the egg in vivo within the body IPVL inner perivitelline layer LC50 median lethal concentration LH luteinizing hormone LOAEL lowest observable adverse effect levels MTD minimum tolerated dose NIEHS National Institute of Environmental Health Sciences NOAEL No observable adverse effect level NRC National Research Council OC organochlorine OECD Organization for Economic Cooperation and Development OP organophosphorus OPPT Office of Pollution Prevention and Toxics, EPA OPVL outer perivitelline layer PAH polycyclic aromatic hydrocarbon PCB polychlorinated biphenyl PCR polymerase chain reaction P4 progesterone PMSG pregnant mares' serum gonadotropin POM medial preoptic nucleus, part of the quail brain Prl prolactin Random bred Random bred lines are relatively large populations of birds (> 100) in which minimal selection of breeding stock is done by the curator ( Pisenti et al. 1998) RH relative humidity RIA radioimmunoassay RMD Reference Manager Database RNA ribonucleic acid RTI Research Triangle Institute SAB Science Advisory Board SAP Scientific Advisory Panel SDS PAGE sodium dodecyl sulfate polyacrylamide electrophoresis SDWA Safe Drinking Water Act SHBG sex hormone binding globulin T testosterone T1S Tier 1 Screening T3 triiodothyronine T4 thyroxin TCDD tetrachlorodibenzodioxin TSH thyroid stimulating hormone UBC University of British Columbia USC United States Code VTG vitellogenin Battelle Draft 10 April 23, 2003 3.0 OVERVIEW AND SCIENTIFIC BASIS OF AVIAN TWO GENERATION TESTS Effects of chemicals on avian reproduction, development, and survival have been studied for several decades, and standardized protocols have been in place for screening potential environmental contaminants for such effects. Although these methods have some relevance to the discrimination of endocrine disruptors from among the category of all substances that causes adverse effects, they are not sufficiently robust to determine mode of action. Because endocrine disruptors are categorized on the bases of how they exert adverse effects in the animal, specific endpoints must be built into the studies to differentiate them from other " reproductive or developmental toxicants" ( DeFur et al. 1999). Furthermore, current test protocols are not designed to determine long term effects of in ovo exposure, which may be the most relevant responses to endocrine disrupting compounds in birds ( Fry 1995). Therefore, a two generation avian reproduction study is being developed to provide the necessary protocol to determine endocrine disruption potential and ecologically relevant effects of environmental chemicals.

Endocrine disruptors are " exogenous agent[ s] that interfere with the production, release, transport, metabolism, binding, action, or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes" ( Kavlock et al. 1996). Such effects must result in adverse health effects in an organism or its progeny, to be ecologically relevant ( DeFur 1999). It is important to note, however, that endocrine disruptors are defined on the basis of their mechanism of action. Many physiological processes that are hormonally regulated, including reproduction and development, may be affected by chemicals through other toxicological mechanisms. Hoffman ( 1990) reviewed the data on embryo toxicity and teratogenicity of environmental chemicals to bird eggs. Many chemicals have shown such effects, either through direct application or via maternal transfer, including organochlorines ( OCs), polycyclic aromatic hydrocarbons ( PAHs), organophosphorus compounds ( OPs), some herbicides, and fungicides. However, there are multiple causes of such effects, not all of which are endocrine related.

For example, selenium is a well known avian embryotoxicant, causing severe embryonic developmental effects following in ovo exposure. Such effects are due to interference with glutathione peroxidase and subsequent oxidation of cell membranes ( Ohlendorf 1996). Thus, selenium would be classified as a reproductive and developmental toxicant but not as an endocrine disruptor. Endocrine disruptors need to interact with the hormone system, not simply alter a process that is under normal endocrine control ( DeFur 1999).

The concept that environmental chemicals have the potential to disrupt hormonally regulated processes in birds is not new. Such mechanisms have been studied since the late 1960s, and it was first discussed as a generalized class of chemicals at a NIEHS symposium, " Estrogens in the Environment," held in Eugene, Oregon, in 1979. Chemicals such as OCs, PAHs, diethystilbesterol ( DES), and others ( e. g., nonylphenols) were identified as having estrogenic properties due to competitive binding with cellular estrogen receptors ( McLachlan 1997). Potential endocrine effects in birds exposed to environmental pollutants were reviewed further by Rattner et al. ( 1984), who introduced the concept of relative potency to the discussion of Battelle Draft 11 April 23, 2003 environmental relevance. For example, whereas dichlorodiphenyltrichloroethane ( DDT) and its derivatives have been shown to bind to nuclear estrogen receptors in the magnum and shell gland regions of the oviduct, their relative potency is only 1/ 1000 of that of the natural estrogen, 17$ estradiol. This further underscores the necessity of conducting studies in a dose response fashion and correlating measured hormonal changes with ecologically relevant fitness endpoints, such as successful reproduction, growth, and maturation. Rattner et al. ( 1984) also broadened the discussion to include the potential for chemicals such as the OCs to result in thyroid dysfunction. The current definition of endocrine disrupting chemicals now includes those that act or have the potential to act on estrogens, androgens, and thyroid hormone systems.

3.1 Avian Endocrinology " The endocrine system is responsible for regulating most of the body's essential functions. It controls reproduction and secondary sex characteristics, molting and metamorphosis, fluid balances, growth rates, salt balance, and response to stress, among others. These functions are accomplished by a diverse set of small organs located throughout the body that communicate with each other and their target organs by releasing small amounts of very potent substances known as hormones. The primary endocrine organs are the adrenal glands, pituitary, thyroid, parathyroid, ovaries/ testes, and pancreas. The kidney produces hormones in addition to filtering the blood. In mammals, the placenta also functions as an important endocrine organ Although many of the processes and hormones involved with reproduction, development, and homeostasis are well conserved across species, there are sufficient differences between mammalian and avian systems to warrant the development of avian specific tests ( Dawson 2000).

Control of the endocrine system is by the neurosecretory neurons within the hypothalamus. These receive input from internal and external cues and also control the rate at which the hypothalamus synthesizes and secretes various peptides or glycoproteins known as releasing hormones ( e. g., gonadotrophic releasing hormone [ GnRH]). These hormones then stimulate the pituitary to synthesize and secrete other peptide hormones ( e. g., follicle stimulating hormone [ FSH]; thyroid stimulating hormone [ TSH]), which pass into the circulation and go to the target endocrine glands. Glands such as the ovaries, testes, and oviduct are stimulated to secrete the active steroid sex hormones, including estrogens and androgens. " Estrogen, testosterone, and other steroid hormones are distributed around the body bound to another protein, the sex hormone binding globulin ( SHBG). The relative affinity of the hormones to bind to SHBG versus to cell receptors influences the potency of hormones within and across species. Interaction of chemicals with the SHBG hormone binding may be a cause of species specific differences in hormone mediated effects of xenobiotics. There are a number of interactions among the various endocrine axes and between the endocrine and nervous systems. In the case of the reproductive, hypothalamic pituitary gonadal ( HPG) axis, steroid hormones produced by the gonads feed back to the hypothalamus and pituitary gland to alter HPG axis function. This is termed a negative feedback loop and is a mechanism by which many of the endocrine axes are regulated. Blood calcium levels are regulated in a manner similar by the parathyroid gland. Estrogen interacts with parathormone, which is produced by the parathyroid gland, regulating calcium levels by causing an increased deposition of calcium in the long bones. Hormones Battelle Draft 12 April 23, 2003 produced by the kidney also participate in the regulation of calcium as well as in the maintenance of blood pressure and proper balance in the body of ions such as potassium and sodium. The adrenal glands, located just above the kidneys, produce corticosteroids, which are important under conditions of chronic stress" ( Bennett, et al., 2001).

" The corticosteroids also influence the immune system and estrogen production. Less well known is their production of hormones that interact with the kidney to maintain ionic balance" ( Bennett, et al., 2001).

" In birds, sex steroid hormones are responsible for a variety of reproductive functions. These include development of secondary sexual characteristics such as plumage coloration and mating songs, phenotypic gender determination including oviduct development and maturation, shell gland function, oviduct development and maturation, and cornification of the cloacal epithelium. Progesterone causes development of the brood patch, which is further vascularized under the influence of estrogen. Progesterone also is responsible for broody behavior, such as nest attentiveness during incubation and during posthatch in altricial species. Estrogen also regulates calcium deposition and release from bones, as well as brain development. In birds and other egg laying animals, estrogen is responsible for the stimulation of production of vitellogenin ( VTG) by the liver. VTG is the precursor for the primary proteins that are incorporated into the egg for nourishment of the embryo during prehatch development" ( Bennett, et al., 2001).

The steroid sex hormones are synthesized from cholesterol through the hydrolysis of side chains by cytochrome P450 enzymes in families 11, 17, 19, 21, and 27, to form progesterone. These enzymes are not inducible by xenobiotics, but they can be inhibited. Progesterone is further modified to form testosterone and its active derivative, androstenedione. Another P450 enzyme, aromatase, takes these androgens to estrogens, estradiol and estrone, respectively ( Fairbrother 2000). Cytochrome P450 enzymes also play a role in the degradative metabolism of the steroid hormones. Cytochrome P450 enzymes in families 1 through 4 metabolize natural steroids to more polar forms for excretion in the urine and are inducible by a variety of chemicals ( Dawson 2000).

" The thyroid gland is another major endocrine organ. It is a butterfly shaped gland located at the base of the neck of most vertebrates and depends upon an adequate supply of iodine for normal functioning" ( Bennett, et al., 2001). Thyroid hormones are peptide hormones and are synthesized in the follicles of the thyroid gland; this synthesis produces a large globular glycoprotein called thyroglobulin. This contains iodinated tyrosine residues, which are coupled to form iodinated thyronine, the major form of which contains four iodides and is known as thyroxin, also known as tetraiodothyronine ( T4). T4 is the major circulating hormone, but is not the active form. T4 is converted into tri iodothyronine ( T3), which is the physiologically active thyroid hormone. Conversion occurs in the liver and is controlled by the enzyme Type I deiodinase. Only about 30% to 40% of the circulating T4 is converted to T3; another 15% to 20% is deaminated to form tetraiodothyroacetate, which is rapidly excreted. Other glucuronides also are formed and excreted. Battelle Draft 13 April 23, 2003 " Production of thyroid hormone is regulated very closely by the hypothalamus and pituitary, through a negative feedback cycle. The corticosteroids, testosterone, and estrogen can depress production of thyroid hormone, and some of the soluble mediators of immune system function ( interferon and interleukins) have slight effects as well. T3 is responsible for controlling the metabolic rate of an animal, regulating body temperature, interacting with growth hormone to determine body size, and playing an integral role in molting, metamorphosis, and smoltification of various species of invertebrates, amphibians, and fish" ( Bennett, et al., 2001).

3.2 Differences in Avian and Mammalian Endocrine Systems " There are many similarities among birds, mammals, and other vertebrate classes regarding endocrine systems. These vertebrate classes share similar hormones and hormone receptors, and fundamental feedback mechanisms are similar. However, important differences between birds and mammals and other vertebrate classes do exist. Birds offer unique morphological, physiological, and behavioral adaptations that are not widely found in other vertebrates, including flight, which requires adaptations for high metabolic rate and reduced body mass, oviparity with hard shelled eggs, a different physiological basis for gender development, and complex mate attraction behaviors, especially in songbirds" ( Bennett et al. 2001). These differences were reviewed by Fry ( 1995) and Fairbrother ( 2000), and they are summarized here.

Sex determination and control of differentiation is linked to the heterogametic sex. In mammals, this is the male ( XY); in birds, it is the female ( ZW). The so called default sex, which is the phenotype to which the embryo will develop in the absence of sex specific hormones, is the homogametic sex. Mammals will develop as female ( XX) and birds as male ( ZZ) if no sex differentiation hormones are present. Therefore, birds require that estradiol be synthesized to cause differentiation of the gonad into an ovary. Lack of estrogen, regardless of androgen levels, will result in development of phenotypic males. In mammals, the reverse is true: embryos will develop into phenotypic females unless sufficient levels of androgens are present to induce gonadal differentiation into testicular tissue. In mammals, normal maternal estrogens are sequestered by fetoprotein, which has a high estrogen binding affinity and protects fetal tissues from estrogen exposure. Exogenous estrogenic substances also may bind to this protein, reducing the potential for effect on the developing embryo. Birds do not have this protein, and embryos are exposed in ovo to the same level of estrogen as was in maternal circulation during egg formation.

Because of the differences in steroid hormonal control of sexual differentiation, xenobiotic estrogens have different effects in birds and mammals during embryonic development. In male birds, excess estrogens will stimulate the primordial germ cells to become localized in the cortex of the gonad as well as in the more normal location in the medulla in a dose dependent manner. These cortical cells differentiate into primordial follicles, and the gonad begins to resemble an ovary. The seminiferous tubules are retained, but the number of primordial germ cells will be lower than normal, resulting in low to no spermatogenesis. Genetic males also may develop an oviduct. Estrogenization of female birds does not change the ovary, but does change the oviduct and causes retention of the right oviduct, which in birds normally regresses prior to maturation. Battelle Draft 14 April 23, 2003 Some of the OC pesticides and other xenobiotics need to be metabolized into active products before the estrogenizing effects are expressed. Most of the metabolic products of OCs are hydroxylated forms that are water soluble and readily excreted in the urine/ urates. However, in bird embryos, the metabolized products cannot be excreted ( i. e., they remain in the egg), and so the embryo can be exposed throughout the length of gestation. In mammals, maternal excretion can reduce the effective dose and/ or completely eliminate the product before the end of gestation. Because these products have 100 to 1000 times less potency or binding efficiency than natural estrogens, direct reproductive effects on adults are expected to be negligible. However, avian embryos are at significant risk because of retention in the egg and estrogen dependency of sex expression.

3.3 Avian Two Generation Test The proposed avian two generation reproductive study for determination of toxicants with endocrine disrupting effects is based on a premise similar to that of the mammalian study ( OECD 1999). The test must be sufficiently robust to document changes in fitness endpoints, such as reproductive output, developmental adequacy, and appropriate behaviors, but equally specific to determine mechanism of action. It is essential that a two generation test be able to assess the impact of endocrine disrupting chemicals on endocrine mediated processes as systems organize during embryonic development and as they are activated in adult birds. There are four critical life stages of birds during which endocrine mediated processes take place and that therefore could be sensitive to endocrine disruption: 1) in ovo, 2) offspring ( F1) generation chick growth, 3) parental ( P1) generation and F1 sexual maturation, and 4) P1 and F1 egg laying. Therefore, the study must include two egg laying cycles to assess effects on ecologically relevant fitness endpoints of endocrine dysfunction at each of these stages.

In brief, the test is designed to expose birds to environmental chemicals suspected as having endocrine disrupting effects from examination of structure activity relationships or test data from other species or in vitro studies. The P1generation is exposed, and reproductive endpoints such as egg production are measured, along with appropriate measures of estrogen, testosterone, and thyroid hormone synthesis, metabolism, and activity. The F1 generation is hatched and evaluated to 2 weeks of age, at which time a subset is selected for pairing and further evaluation of reproduction, development, and endocrine function. This generation may be exposed to the chemical throughout its lifetime, or a subset may be exposed while another cohort is not. The F2 generation also is hatched and observed to 2 weeks of age but is not exposed to the chemical. Test species will be either Japanese quail ( Coturnix japonica) or bobwhite quail ( Colinus virginianus) because of their small size, short time to sexual maturation, ease of handling, and an existing large database of information regarding reproductive effects of environmental contaminants.

The identification of endocrine mediated effects on avian reproduction can occur at the test individual or test population level of analysis. This needs to be considered in determining the ultimate objective of the test. If the emphasis in a two generation study is to determine the impact of a potential endocrine disruptor at the test population level, then an integrative endpoint Battelle Draft 15 April 23, 2003 that expresses the outcome of the study in terms of effect on overall productivity of a population must be identified. This type of study requires determination of dose response curves for the integrative endpoint. Other, specific endocrine related endpoints would then be evaluated alongside the primary productivity endpoint to identify the chemical as an endocrine disruptor rather than simply a reproductive toxicant, and to identify sensitive life stages. Endocrine specific endpoints also may identify exposure levels at which endocrine mediated effects occur in individual birds but not in the treatment group as a whole, or cause measurable hormonal changes without resulting in gross physiological differences. For example, because only 30% to 40% of the naturally circulating levels of T4 is converted into the active T3 form, a change in production of T4 may not result in significant effects to T3 levels. Thus, incorporation of sensitive measures of endocrine related endpoints will allow a hazard assessment   that is, a determination of whether chemicals are potentially endocrine disrupting   without resulting in ecologically relevant risk estimates ( Bennett et al. 2001).

Treatment of the P1 generation can begin either prior to sexual maturation or after the onset of egg laying. The advantages of starting dietary treatment prior to sexual maturation are ". . . the ability to measure changes in the onset of laying and effects resulting from inhibition or delay of gonadal development. The disadvantages are that birds that are incompatible or infertile for reasons other than the test substance cannot be removed, which may reduce the statistical power of the test and the ability to detect treatment effects if they exist. The advantages of starting treatment during egg laying include the ability to remove nonlaying and infertile pairs prior to the initiation of treatment and the option to use pretreatment measurements as covariates in statistical analyses to remove nontreatment sources of variation. Starting treatment during egg laying also provides information on rapidity with which reproductive effects can be observed, which can be useful in risk assessments of test substances that degrade rapidly in the environment. Because treatment effects on reproductive endpoints may increase during the course of the treatment period, statistical analyses need to be sensitive to the potential temporal patterns in the endpoint measurements" ( Bennett, et al., 2001). Disadvantages include the inability to measure changes in the onset of laying and effects resulting from inhibition or delay of gonadal development. These two options are discussed in greater detail in Section 5.1.1.

Treatment of the F1 chicks ensures that endocrine mediated effects occurring during growth and development of chicks as a result of direct exposure as well as from in ovo exposure will be assessed. Treatment of the F1 chicks also is considered to more closely represent a continuous exposure scenario ( Bennett, et al., 2001). " However, analysis and interpretation of the results will have to consider how effects occurring at various life stages may be confounded" ( Bennett, et al., 2001). Additionally, effects from such full life cycle exposure scenarios need to be interpreted with caution for those test substances that degrade rapidly in the environment that could be present at only certain life stages. " Ending treatment of the F1 generation prior to egg laying removes some of the potentially confounding effects of endocrine mediated effects on F1 reproductive potential occurring from in ovo and/ or early life exposures. It can be argued that the effects of exposure during egg laying can be observed in the P1 generation and may not need to be repeated in the F1 generation. However, by ending the treatment period just prior to egg laying, there may be a temporal pattern of effects observed in the production of the F2 Battelle Draft 16 April 23, 2003 generation, depending on the clearance rate of the test substance and related deposition rate into eggs, and the rate of recovery of the F1 birds from any residual toxic effects" ( Bennett, et al., 2001). Several exposure options have been proposed to cover these various arguments. They are discussed in more detail in Section 5.1.2.

The experimental design of avian reproduction tests has undergone considerable discussion. The tests can be designed to define concentrations of environmental chemicals at which no observable adverse effects occur, or they can be designed to develop dose response relationships for endpoints of concern. Determination of no observable adverse effects levels ( NOAELs) requires use of analysis of variance ( ANOVA) designs, whereas development of dose response curves uses regression techniques ( Bennett et al. 2001). The issue of appropriate statistical design is examined in further detail in Section 5.4.2.

4.0 CANDIDATE TEST SPECIES Two quail species, the northern bobwhite and the Japanese quail, are the most likely candidates for use in a two generation reproductive screening assay and are the focus of this review. Because of their terrestrial habit, both species are considered to be representative of terrestrial birds and are accepted models for assessing both acute and reproductive effects of pesticides and other chemicals in wild birds ( Office of Pollution Prevention and Toxics [ OPPT], EPA, OPPT Guideline 850.230 [ Federal Register 1978]; OECD Guideline 206 [ OECD 1984]). Although both species, but particularly the Japanese quail, have undergone domestication and therefore may be less representative of wild birds, they are used in reproductive assays, because few wild species adapt well to laboratory conditions and breed successfully in the laboratory. Also, unlike most birds, the breeding cycle of quail is not restricted with a photorefractory period, so they are able to produce eggs almost indefinitely under photoperiods longer than about 12 h. In addition, as precocial species, they are likely to be more sensitive to changes in steroid concentrations during all life stages.

4.1 Japanese Quail ( Coturnix japonica)

This Old World quail has been intensively domesticated for more than 800 years in Japan. Since the beginning of the twentieth century, breeding stock has been selected for body size and egg production throughout the world ( Cooper 1976). Use of Japanese quail as a laboratory animal began in the late 1950s, and because of its adaptability to battery breeding cages, small size, and high fecundity, it has been used extensively in research. As a result, numerous strains of Japanese quail have been developed and conserved in government and academic institutions and by commercial suppliers. Among these strains, body size characteristics, rates of sexual maturation, and reproductive capabilities can differ significantly. The Japanese quail has been used extensively in reproductive toxicity testing in the European community and to a lesser extent in the United States. Some testing guidelines include detailed information on their laboratory husbandry ( OECD Guideline 206; OPPT Guideline 850.2300; ASTM Method E1062 86). Battelle Draft 17 April 23, 2003 4.1.1 Natural History The Japanese quail, once classified as a race of the common quail ( Coturnix coturnix), is now considered to form a superspecies with C. coturnix and possibly also with C. pectoralis ( Cheng and Kimura 1990). It belongs to the largest family ( Phasianidae) of the galliformes, and based on deoxyribonucleic acid ( DNA) DNA hybridization evidence is only distantly related to New World quail, such as the northern bobwhite ( Hoyo et al. 1994). The Japanese quail is a migratory species widely distributed throughout eastern Asia. Feral populations of Japanese quail have been established in the Hawaiian Islands and in Great Britain, but three attempts to introduce the species as a game bird into the United States have been unsuccessful ( Cooper 1976). Little habitat information is available for the Japanese quail; however, this ground dwelling species has been observed using a wide range of open habitats, including cultivated areas. It feeds on a variety of plant materials and terrestrial invertebrates and nests in grassland areas. Clutch sizes vary from 5 to 8 eggs in Japan to 9 to 10 eggs in Russia. Incubation is typically 18 days in the wild ( Hoyo et al. 1994). Siblings form coveys through their first winter and disperse the following spring. In the wild, their average body weight is about 90 g ( Hoyo et al. 1994).

4.1.2 Availability, Culture, Handling Several academic and governmental institutions in the United States and Canada maintain random bred stocks of Japanese quail ( Pisenti et al. 1999). The largest quail collection in North America is maintained at the Quail Genetic Stock Center at the University of British Columbia ( UBC), Vancouver, British Columbia, through support by the Natural Sciences and Engineering Research Council of Canada. Among its conserved strains of Japanese quail are several outbred stocks including a wild type from Japan. In the United States, some of the oldest established quail lines for research are at the Universities of California, Georgia, and Maryland ( Table 4 1). In addition, some commercial sources maintain large colonies of Japanese quail to supply research and toxicity testing organizations. Most of the stocks available in North America and Japan are derived from stocks originally selected for egg production and are smaller, faster maturing, less docile strains than those commonly used in the European community for reproductive toxicity testing. However, as the quail meat industry grows, more strains of the higher body weight birds are being developed in the United States. As addressed in Section 4.1.3, the growth and reproductive differences between the strains can be substantial.

The care and handling of Japanese quail for laboratory use have been well documented. ( NRC 1969; Cooper 1976; CCAC 1984; Ottinger and Rattner 1999), and testing guidelines include detailed information on their husbandry for reproductive toxicity tests ( OPPT Guideline 850.230; OECD 206). Coturnix grow very rapidly; females reach sexual maturity in about 6 weeks, depending on strain. Day old quail weigh about 7 g, and at maturity, they weigh between 120 g to more than 200 g. As a consequence, provision of adequate space per bird during this short growth period is crucial. During the growth period, temperature requirements that are 38 º C at hatch decrease by about 3 º C per week until the young reach 4 weeks of age. Young are very susceptible to drafts during this period. At 4 weeks of age, they are fully feathered and often are Battelle Draft 18 April 23, 2003 moved to breeder cages. Pairing birds before sexual maturity aids in reducing aggressive behavior between pairs ( NRC 1969). Early pairing can be accomplished, because gender is discernable by plumage color and pattern as early as 3 weeks of age. The cloacal gland, a bulbous structure located at the upper edge of the cloaca in males, also can be used to discriminate between sexes, but it is affected by photoperiod ( Sachs 1967). Japanese quail are very hardy once the brooding stage is past.

Wild type hens and hens from strains selected for egg production will lay about 1 egg per day and will produce about 300 eggs per year. Heavier strains lay fewer eggs ( Table 4 2). The average weight of eggs laid by mature Japanese quail is about 10 g. However, older birds usually produce larger eggs, larger embryos, and larger chicks ( Cooper 1976). A mating ratio of one male to one female results in the highest fertility rate. Duration of fertility after removal of the male is about 6 days. After remating the pair, fertile eggs are produced on the third day ( Woodard and Abplanalp 1967). Full egg production is reached about 2 to 3 weeks after egg laying begins. Optimal reproduction typically spans 5 to 6 months ( CCAC 1993); maximum hatchability occurs when parents are between 8 and 20 weeks of age ( CCAC 1984). Fertility in Japanese quail progressively decreases after about 56 weeks of age under stimulatory photoperiod ( 16L: 8 D). By this time, courtship and mating behavior are greatly reduced and plasma testosterone levels also are decreased ( Ottinger et al. 1983). Also, eggshells of older birds are usually thinner and result in a lower hatch ( Cooper 1976).

Table 4 1. Stocks of Random Bred or Wild Type Japanese Quail Maintained in North America and Europe( a)

Stock Name and Description History of Origin Number of Birds Curator Quail Random bred, Arkansas RBS random bred control from Eastern Shore random bred Acquired from Eastern Shore as random bred control, 1990 36M/ 36F Anthony Athens control quail random bred control w/ white eggshell mutation in gene pool, propagated by random pair matings Kept as closed flock since 1963 120M/ 120F Burke Louisiana random bred quail unselected, random bred population, some color mutations ( tuxedo, redhead, white egg) Kept at Louisiana State as closed flock for > 20 years 60M/ 120F Satterlee Ohio R1 propagated using 36 pairs/ generation Derived from cross of Athens random bred, Athens white egg, and Wisconsin stock; closed flock 38 generations 36M/ 36F Nestor UBC A random bred flock Derived from cross of UCD randombred quail and quail stock from Korea; closed flock 70 generations 48M/ 96F Cheng UBC B exceptionally nervous random bred; spade homozygotes have defective feathers Acquired from U Alberta 1977, combined with UBC SP ( spade mutation, affects feathers) by 1998 34M/ 48F Cheng Stock Name and Description History of Origin Number of Birds Curator Battelle Draft 19 April 23, 2003 UBC M rough textured homozygotes have feathers that appear matted and rough; females produce fewer viable embryos ( RT mutation not yet reported in literature); have extended brown allele; slightly heavier than UBC A UBC M from commercial ( Marsh Farms) strain in 1975; combined with UBC RT in 1989 24M/ 48F Cheng UBC N very docile random bred Acquired from U Nagoya ( Japan), 1988 24M/ 48F Cheng UBC NC random bred, sensitive to changes in photoperiod Acquired from NCSU random bred quail, 1990 34M/ 48F Cheng UBC S Acquired from U Saskatchewan, 1983 24M/ 48F Cheng UBC WILD Foundation stock was 12 feral birds caught in Hawaii, 1985 48M/ 96F Cheng UCD Random bred quail wild type feather color pattern, unselected, randomly grouped in colony cages ( 2M/ 4F); reproduce every 6 to 8 months Derived from stock imported from Japan and Taiwan ( 1950s and 1972) 200+ Wilson U Maryland random bred quail Acquired from U Wisconsin/ US Department of Agriculture, 1970s; kept as closed flock for ~ 20 years 100 Ottinger UNL Wild type Coturnix Acquired from U Georgia Athens 30 60 pairs Beck Purdue Coturnix KGB   selected behavioral trait selected 18 generations for nonaggressive behavior, starting 1988 Derived from Athens control quail ( U Georgia Athens) 10,000 birds Muir Arkansas H10   selected growth trait selected 18 generations for high 10 day body weight Derived from Arkansas RBC 36M/ 36F Anthony Arkansas H17   selected growth trait selected 18 generations for high 17 day body weight Derived from Arkansas RBC 36M/ 36F Anthony Arkansas H28   selected growth trait selected 18 generations for high 28 day body weight Derived from Arkansas RBC 36M/ 36F Anthony Arkansas H40   selected growth trait selected 18 generations for high 40 day body weight Derived from Arkansas RBC 36M/ 36F Anthony Arkansas HL   selected growth trait selected for high early body weight gain ( 10 17 days), low late body weight gain ( 17 28 days) 36M/ 36F Anthony Arkansas LH  selected growth trait selected for low early body weight gain ( 10 17 days), high late body weight gain ( 17 28 days) 36M/ 36F Anthony Athens 52 high body weight selected Derived from cross Athens 51 and 53 ( both selected 38 generations for high 4 week body weight) 36M/ 36F Burke Stock Name and Description History of Origin Number of Birds Curator Battelle Draft 20 April 23, 2003 Athens 54 low body weight selected Derived from cross of two stocks, both selected 38 generations for low 4 week body weight 36M/ 36F Burke Athens 56   selected growth trait Intermediate body weight stock, cross of longterm selected high and low body weight stocks Derived from cross of Athens P, T, and S lines 36M/ 36F Burke Athens P line selected 100 generations for high 4 week body weight, 28% protein diet; at 70 generations, adult size was > 150% above that of controlled standard population Derived from Athens control quail 60M/ 60F Burke Athens T line selected 100 generations for high 4 week body weight, low protein, thioruacil stress diet; resists growth depression on diets with up to 0.2% thiouracil Derived from Athens control quail 60M/ 60F Burke Ohio HW inbreeding coefficient ( F) = 0.417; selected 30 generations generations for increased 4 week body weight Derived from Ohio R1 48M/ 48F Nestor Ohio HW HP inbreeding coefficient ( F)= 0.375; selected for male increased 4 week body weight, for female increased plasma phosphorus ( indicator of yolk precursors) Derived from Ohio HW 36M/ 36F Nestor Ohio HW LP inbreeding coefficient ( F)= 0.357; selected for male increased 4 week body weight, for female decreased plasma phosphorus 2 weeks after start of lay Derived from Ohio HW 36M/ 36F Nestor Ohio LW inbreeding coefficient ( F)= 0.357; selected 30 generations for decreased 4 week body weight Derived from Ohio R1 48M/ 48F Nestor UBC G QM selected for average female body weight 280g at 6 weeks UBC G derived from a commercial strain ( Marsh Farms) combined with UBC QM in 1992 24M/ 48F Cheng UBC QF selected for average female body weight 280g at 6 weeks Acquired from Deschambault in 1990 48M/ 96F Cheng UBC QM selected for average male body weight 280g at 6 weeks Acquired from Deschambault in 1990, combined with UBC G in 1992 see UBC G QM Cheng Bobwhite NIU bobwhites, blood type   gene pool various erythrocyte alloantigens in quail Acquired from Mississippi State U in 1992 50+ Briles a) Adapted from data in Pisenti et al. ( 1999). Battelle Draft 21 April 23, 2003 Table 4 2. Body Weight and Reproductive Parameters of Egg Producing and Meat Production Strains of Japanese Quail Parameter UBC Wild Type Northwest Game Birds UBC QO Quail Internt Georgia, USA Texas A& M Body weight ( g) Male Female 144 170 232 297 218 243 312 331 Age at sexual maturity ( days) 32 28 35 63 49 70 42 53 49 56 Number eggs/ year 288 300 183 230 234 Number eggs/ hen/ day 0.79 0.82 0.5 0.64 Percent Fertility 87 90 Percent Hatchability 90 85 Reproduction is greatly affected by inbreeding in Japanese quail, and mating systems need to be selected to avoid pairing of closely related individuals. Sittmann et al. ( 1966) demonstrated a 66% decrease in hatch from one generation of full sibling mating and nearly complete loss of hatch by the third generation. Fertility and hatchability decline by about 1% for each 1% additional inbreeding. Brooder mortality also increases substantially with each increase in inbreeding. Bateson ( 1983) demonstrated a mating preference for unfamiliar first cousins among Japanese quail; therefore, single enclosure mating systems may be less successful than the more controlled mating systems in preventing inbreeding. To prevent inbreeding depression in resource stocks bred in single enclosure systems where all males have access to all females, a minimum of 100 birds are needed. Japanese quail stocks are often maintained by random pairing or grouping in cages, in which case 25 pairs but usually more than 150 birds, are needed to keep inbreeding at a minimum ( Pisenti et al. 1999). Typically, random bred stocks are kept as closed populations; however, new bloodlines are often introduced to the flock if inbreeding depression is observed.

Japanese quail lay large eggs relative to their body size   7% to 8% of their body weight   with large yolk volume, providing a relatively large potential depot for xenobiotic transfer from hen to developing embryo. As is typical for nidifugous young, a reserve of yolk is gradually discharged into the blood plasma for several days post hatch. Most strains of Japanese quail produce colored, often mottled eggshells that can make detection of cracks difficult. However, some strains produce light colored or white shells ( Poole 1964). Incubating eggs require a temperature of 37.5 ± 0.3 º C and relative humidity ( RH) of at least 60%. Egg incubations systems are available to automatically rotate the eggs every 2 to 4 h. Pedigree baskets are routinely used to track parentage of the young at hatch. Fertility of eggs stored at 14 ± 0.3 º C and 70% RH is high for the first 7 days, after which it falls off dramatically. Rapid decline in hatchability occurs if the eggs are held longer than 7 days. Storage can be extended to 10 to 14 days, if the Battelle Draft 22 April 23, 2003 eggs are covered in plastic to prevent desiccation ( NRC 1969; Ottinger and Rattner 1999). The incubation period for Japanese quail is 14 to 18 days depending on strain. Humidity requirements during hatch are higher ( 70% RH minimum), and eggs can be sprayed with distilled water to help prevent embryonic membranes from drying after pipping ( Olsen 1992; Ottinger and Rattner 1999). Hatchlings can be sexed by cloacal examination ( Homma et al. 1966).

Japanese quail respond to sudden disturbance by flushing, and injury can result during capture, if the bird strikes against the ceiling of the cage. Cage height is therefore kept at about 25.5 cm to reduce the potential for injury. Some species, such as the feral Hawaiian strain, are considered flighty, whereas larger strains appear to be more docile. In all, they are relatively easy to handle, although manual restraint can occasionally induce tonic immobility, an instinctive fear response characterized by motor inhibition and loss of righting response for a period of time ( Satterlee et al. 1993). Disturbing laying hens may cause a pause in egg laying. Moving layers to new caging may result in a cessation of egg production for 2 to 3 weeks ( NRC 1969). Interruption of egg laying will have to be considered in selection of endpoints and/ or sampling schedule.

The threshold for reproductive system stimulation in Japanese quail is 11 h of light and 13 h of darkness per day ( 11L: 13D). Maximal photoresponse is obtained at 14L: 10D and maintained at maximum up to 18L: 6D. However, captive quail populations often display marked photoperiodic drift and large variability in reproductive response. This appears to be a result of the increasing population of individuals in stocks of Coturnix that reach sexual maturity on shorter day lengths. Dobson et al. ( 1992) reported up to 10% of a population may breed on 8L: 16D photoperiods if selective breeding against early photoresponses is not maintained. Others have reported even greater percentages of test populations that enter into reproduction when raised under the recommended photoperiod for gonadal quiescence in avian reproduction tests ( OECD Guideline 206). For example, Yamamoto et al. ( 1996) found that 60% of the Japanese quail used for a reproductive toxicity study entered into reproduction when maintained under the recommended short day length of 7 h to 8 h of light. When the day length was shortened to 6 h of light, 20% of the birds became reproductive. Decreasing day length to 5 h of light reduced the incidence of reproduction to less than 5% of the population. However, the reduced day length ( 6L: 18D) resulted in lower food intake, and thus, reduced uptake of the test substance ( fenthion), which in turn resulted in a much reduced response to the chemical exposure than was previously observed in their laboratory. Some strains of Japanese quail have been developed that do not require photostimulation of reproduction ( Dobson et al. 1992). Photoperiodic drift within a strain of quail could compromise attempts to measure reproductive endpoints associated with sexual maturation ( gonadal development, cloacal gland size, time to first egg laid, level of circulating hormones). Therefore, careful consideration should be given to strain selection prior to use in avian reproduction tests.

Light intensity is also important in maintaining quail. Light intensity that is too high will encourage aggressive behavior. The quail should be exposed to about 10 lux artificial light, measured at the level of the feeder, which approximates the daylight visual spectrum to initiate and maintain reproduction. However, to reduce aggression or maintain birds in nonreproductive Battelle Draft 23 April 23, 2003 condition, lower levels of light may need to be provided. To ensure a stimulatory photoperiod as well as a long feeding period to maximize consumption of the test substance, a photoperiod of 16 h or 17 h of light and 7 h or 8 h of dark is commonly used. If the breeding colony is maintained on this light cycle throughout the year, the hens may experience premature aging from the calcium drain associated with constant egg laying ( Ottinger and Rattner 1999).

Coturnix can be maintained from hatchling through reproduction on a diet containing 26% to 28% protein ( Howes 1965) and an energy content of 2000 productive energy calories per kilogram. High energy diets should be avoided, because they cause steatosis ( Howes and Fitzgerald 1966). Supplementary calcium ( 3.0%) is required for egg laying and should be supplied just prior to maturity ( Nelson et al. 1964). Commercial game bird and turkey starter diets are available and are commonly used for Japanese quail production. Most commercial and custom diets are formulated with soy and corn, which contain phytoestrogens in widely varying amounts. The impact of soy and corn diet on endocrine disruptor testing has not been studied. However, commercial turkey starter amended with extract from forage plants containing elevated levels of phytoestrogens ( biochenin A and genistein) was shown to cause a delay in the onset of egg production in California qual ( Lophortyx californicus) and to reduce egg production overall in this species ( Leopold et al. 1977). Unfortunately, only relative concentrations   high, medium, and low   of the amended phytoestrogens were reported. A later study by Lien et al. ( 1987) showed that bobwhite would have to consume in excess of 1 mg/ bird/ day of biochenin A to adversely impact reproduction.

Stainless steel construction is recommended for caging used in avian toxicity tests, although galvanized construction is acceptable. Quail cages and brooders are commercially available in galvanized steel from production factories. However, stainless steel caging must be customconstructed There is an increasing interest in cages constructed of metal wire coated with perflourocarbon plastics to provide thermoneutral surfaces, ease of cleaning, and reduced cost. Some also feel that the coatings reduce egg breakage and may provide a more comfortable flooring for the birds. However, use of plastic water providers and food containers may contain plasticizers that should be avoided in tests measuring endocrine endpoints. Wire pens with slanting floors and egg catchers or other measures to prevent breakage of eggs are recommended for adults.

4.1.3 Strains A review of available documentation on genetic stocks of the Japanese quail sponsored by the University of California Genetic Resources Conservation Program indicated that all the strains of Japanese quail in North America and the European community developed through selective breeding for food production and/ or biological research are descended from C. japonica ( NRC 1985; Pisenti et al. 1999). Small wild populations of C. japonica still are found in Japan, and a random bred population of the wild type is maintained at the Quail Genetic Resource Centre at UBC. A strain originating from feral quail captured on the island of Hawaii also is maintained at the center. Battelle Draft 24 April 23, 2003 Many specialized genetic stocks of morphological and physiological mutations have been developed by universities and government research organizations ( Pisenti et al. 1999). Likewise, research institutions and some commercial suppliers have developed and continue to conserve through randomly breeding populations a number of strains in which individual birds have many traits in common ( Table 4 1). These random bred strains are the sources of test organisms for current avian endocrine and reproductive toxicity studies conducted with Japanese quail ( Pisenti et al. 1999). In the United States and Japan, quail currently used for toxicity testing and endocrine studies are derived from strains selected for their egg producing traits. European studies often make use of strains of Japanese quail that have been selected for meat production. These strains may differ not only in growth rate and body size, but also in carcass composition ( Barbato et al. 1984), sexual maturation, and egg production ( Table 4 2). Increased lipid deposition in the form of body fat and yolk material is often observed in meat production strains fed ad libitum ( Reddy and Siegel 1976; Barbato et al. 1984). Increased food consumption and lipid deposition in yolk could result in greater exposure of heavier birds to test substance in the feed and in ovo. To determine whether there are significant differences in growth and reproductive characteristics for the various strains of Japanese quail currently in use, researchers and commercial quail facilities in the United States, Japan, and the European community were canvassed to obtain data on strain maturation rate, productivity, length of peak production period, eggshell properties, aggression between members of a pair, reliance on photostimulation to induce reproduction, and sexual aggressiveness. This information is reported in Table 4 2.

In general, the smaller, egg producing strains appear to mature more rapidly, have higher productivity, and retain peak fertility and productivity for a longer period than the larger, meatproduction strains. However, there is some evidence in the literature that strains selected for rapid growth have more ovarian follicles in rapid development during the egg laying cycle than do random bred lines ( Ye et al. 1999). This apparent contradiction may be explained by the higher incidence of follicles lost into the body cavity, follicular atresion, and production of abnormal eggs in strains selected for increased growth rate ( Bacon et al. 1973; Nestor et al. 1982). Egg production of Japanese quail lines selected for high body weight at 4 weeks of age declined over generations ( Nestor et al. 1996). Notably, in a random bred line maintained using a paired mating system, nine reproduction traits declined over 30 generations ( Nestor 1977). Many of these traits are related in the Japanese quail; therefore, changes in a single trait over the generations could be correlated with other traits ( Nestor et al. 1995). The declining traits were egg production, hatch of eggs set, egg weight, albumen weight, shell weight, yolk weight, body weight at end of lay, and change in body weight during laying, and percentage liver dry matter. Selection for high body weight also appears to reduce the minimal requirements for males to advance to sexual maturity ( Anthony et al. 1993). Selection is also made for docility, both for its effects on poultry welfare and on egg quality ( Jones 1996). These quail tend to have reduced corticosterone responses ( Satterlee and Johnson 1988).

How the various types of strains differ in sensitivity to EDCs is not known and should be evaluated to determine whether the differences would affect interlaboratory comparisons and hazard evaluation. An option currently being considered in Japan to address this issue of comparable test birds is to restrict test populations to those arising from a specific Battelle Draft 25 April 23, 2003 egg production strain and source for all endocrine disruptor tests ( Ito, Yoshihiko, Research Institute for Animal Science in Biochemistry and Toxicology, Kanagawa, Japan, currently Chair of the Endocrine Disruptor Task Force for Japanese Environmental Protection Agency, personal communication, 2002). However, criteria for selecting a standard strain or standard type strains must be carefully established and should take into consideration limits on body size, eggshell quality, fertility, photoperiod drift, behavior, and EDC sensitivity.

4.2 Bobwhite ( Colinus virginianus)

The northern bobwhite is a New World quail only distantly related to quail of Old World origins ( Hoyo et al. 1994). As a native species of the United States adaptable to laboratory conditions, the northern bobwhite has been used extensively under EPA and American Society for Testing and Materials ( ASTM) test guidelines as a representative species for toxicological evaluation of pesticides and other environmental contaminants. Although there are many natural strains of bobwhite, few domesticated strains have been intentionally developed. Guidelines specifically call for bobwhite used on test to be phenotypically indistinguishable from the wild type to retain capacity to represent wildlife responses to chemicals. The life stages of the bobwhite are significantly longer than those of the Japanese quail, particularly the period from growth to reproductive maturity.

4.2.1 Natural History The bobwhite is distributed from southeastern Ontario ( Canada) to Guatemala and in Cuba but reaches its highest density in the eastern United States and Mexico. The species has been widely transplanted from pen raised and wild trapped birds in the United States and is well established in areas beyond its natural range, with disjunct populations found in Washington, Oregon, and Idaho. Northern bobwhite has been introduced also to British Columbia ( Canada), Puerto Rico, Hawaii, and New Zealand ( Hoyo et al. 1994). It is a ground dwelling species with forest and grassland affinities. It also has adapted to intensive agricultural areas in the United States and to xeric ecosystems of Mexico and Central America. Bobwhite nests in the spring with clutch sizes of 10 to 15 eggs. The incubation period is 23 days, and nest success is usually between 20% to 40%. After the breeding season, family coveys of about 12 in its southern range and 15 in northern United States are formed. Body size also increases from south to north in its native range with birds in Chiapas, Mexico, averaging 129 g, whereas those in the eastern United States weigh about 172 g. The northern bobwhite is a sedentary species with a home range of 10 ha to 31 ha. It is an opportunistic feeder, consuming mainly seeds and invertebrates.

4.2.2 Availability, Culture, Handling Numerous commercial and game farm sources of northern bobwhite are available. Most sources supply phenotypical wild type birds for hunting and dog training. These or similar wild type strains are the stocks also used or maintained by testing and research laboratories. Bobwhite husbandry is similar to that of the Japanese quail. Methods of incubation, brooding, maintenance of juveniles, and breeder support are generally the same as for the smaller Japanese quail. However, space requirements are greater, 400 to 900 cm2 per adult bobwhite ( Olsen Battelle Draft 26 April 23, 2003 1993), and humidity requirement during incubation is higher ( 85% RH) for bobwhite ( Ottinger and Rattner 1999). Similar to Coturnix, the northern bobwhite has a low photoperiodic threshold ( 10L: 14D) for stimulation of the reproductive system ( Kirkpatrick 1955), and also lacks the photorefractoriness of most other birds.

All life phases of the bobwhite are longer than those of Japanese quail. The incubation period is 23 to 24 days, development of sexually distinguishable plumage occurs at about 12 weeks of age, and sexual maturity is reached at 24 weeks of age ( Ballard et al. 1994). Peak production is reached at about 6 weeks after the onset of lay. The northern bobwhite is bred to retain its flightiness, and excessive handling may result in mortality. Inbreeding depression will occur in bobwhite stocks under 200 pairs. Therefore, unrelated breeder stock is introduced into the resource stocks of bobwhite at least every 3 years to reduce inbreeding ( International Hatchery Practice 1989).

4.2.3 Strains There are 22 recognized subspecies of bobwhite ( Hoyo et al. 1994). Five of the subspecies, C. v. marilandicus, C. v. virginianus, C. v. floridanus, C. v. texanus, and C. v. taylori, are native to the United States ( Rosene 1969). The northern bobwhite is commonly bred commercially or by hobbyists as wild stock. Very little deliberate selective breeding of northern bobwhite has been practiced, with the exception of the bobwhite blood type variants maintained at Northern Illinois University. However, a few strains, such as Eastern, for example, are selected for body weight for meat production. They are phenotypically similar to the wild type except for body weight and are generally not used in avian toxicity tests. However, because most suppliers raise bobwhite in game farm situations, there is some use of these meat strains by commercial testing laboratories when production of eggs and juveniles has decreased in winter. These larger strains are usually more docile and lay fewer eggs ( International Hatchery Practice 1989).

4.3 Strengths and Weaknesses Table 4 3 summarizes major strengths and weaknesses of the two species for two generation tests. The major advantages of using the Japanese quail over bobwhite in a two generation test guideline with endocrine endpoints are related to the very rapid incubation and maturation ( Table 4 2) and high rate of egg production of the Japanese quail. These traits allow for the completion of a multigenerational test within a relatively short time, about 30 weeks, compared with 70 weeks for a bobwhite test. Further, they allow a large number of eggs per hen from which to sample for egg quality and chemical residue analyses. The cloacal gland of the Japanese quail provides an indirect measure of gonadal development, indicating reproductive fitness, and sexual maturation of the males. Because of the extensive use of Coturnix in biochemical and steroid research, endocrine and behavioral patterns in this species are well characterized ( Hutchinson et al. 2000; Ottinger and Brinkley 1978, 1979a, 1979b). Spermatogenesis is most fully characterized in the Japanese quail ( Lin and Jones 1992), which provides the ability to quantify histopathological evaluations of testicular function and to determine length of exposure required before spermatogonial damage could be detected Battelle Draft 27 April 23, 2003 extragonadally ( Section 5.1.1). No information is available on the seminiferous cycles of bobwhite.

Major disadvantages associated with the use of the Japanese quail are related to its extensive history of domestication. Stock populations are very sensitive to inbreeding and loss of fertility. Consequently, heed must be given to appropriate mating protocols in source flocks and to the parental and offspring pairing in a multigenerational test protocol. Also, many strains of Japanese quail are being used around the world for reproduction testing. These strains differ in body size, maturation rates, egg production, and lipid deposition in yolk. The impact of different strain selection on the outcome of a two generation study is not known.

In contrast, the northern bobwhite is far less domesticated and thus faces less inbreeding pressure and presumably is more representative of wildlife responses. However, the comparative sensitivity of the two species to environmental chemicals, particularly those with potential endocrine effects, is not known. A study by Solecki et al. ( 1996) compared the reproductive effects of an OP pesticide on Japanese quail with those published by Bennett et al. ( 1990), but most of the reproductive impacts observed could be attributed to parental toxicity in both studies. Although no sensitivity comparisons in chronic reproductive toxicity tests have been conducted, comparison of the relative sensitivity of the Japanese quail and bobwhite to acute dietary exposure ( Romijin et al. 1995) and to acute oral dose with pesticides ( Romijin et al. 1995; Baril et al. 1994) have been made using data from the open literature and from studies in support of registration of pesticides. Both studies showed that the northern bobwhite tended to be more sensitive to more compounds than the Japanese quail, but the difference was not statistically significant. In the Romijin et al. study, the differences in sensitivity between the two quail species did not exceed a factor of 5. Sensitivity to some dietary mycotoxins was also greater in bobwhite chicks than in Japanese quail ( Ruff et al. 1992). Aflatoxin, toxic metabolites produced by the mold, Aspergillus flavus, and T 2 toxin resulted in 40% and 23% mortality, respectively, in 2 and 3 week old bobwhite. No mortality was observed in Japanese quail chicks fed the same contaminated diets. Body weight loss, feed conversion ratio, and mouth lesions were all less severe in Japanese quail ( Ruff et al. 1992). Currently, a study directly comparing the relative reproductive sensitivity of the Japanese quail and northern bobwhite to known EDCs is in progress at the University of Maryland for the EPA. Until these results are available, existing data indicate that the sensitivity of the two species to environmental chemicals is comparable. Battelle Draft 28 April 23, 2003 Table 4 3. Major Strengths and Weaknesses of Japanese Quail and Northern Bobwhite Related to Use in Avian Two Generation Reproduction Toxicity Tests Japanese Quail Northern Bobwhite ADVANTAGES ADVANTAGES ° Endocrine, behavioral pattern characterized ° Small bird; occupies 230 cm2 per bird ° Reach sexual maturity by 6 weeks ° Prolific layer, close to 1 egg per day ° Early maturity ( 36 days males, 42 days females) ° Short incubation period ( 16  17 days) ° Males are aggressive breeders ° Males maintain high fertility ( 90%) ° Adapts well to breeder cages ° Biological, physiological, biochemical data available ° Produce a large egg ( 8% of body weight) ° Naturally hardy in the laboratory ° Highly photosensitive ° Physiological aging is rapid and lifespan short ° Dimorphism of plumage color and pattern makes it possible to identify sex at 3 weeks of age ° Cloacal gland of the male may be used as measure of maturity ° History of use in toxicity testing ° Spermatogenesis is well characterized ° Less domesticated, wild type ° Small bird; occupies 400 900 cm2 per bird ° Prolific layer ( somewhat less than Japanese quail) ° Males are aggressive breeders ° Males maintain high fertility ( 95%) ° Adapts well to laboratory ° Produces a relatively large egg ( 8% 105% of body weight) ° Hardy in the laboratory ° Highly photosensitive ° Populations not prone to photoperiodic drift. ° Dimorphism of plumage color ° History of use in toxicity testing ° More yolk per egg ( 39.8% by weight) compared with Japanese quail ( 31.9%)

DISADVANTAGES DISADVANTAGES ° Inbreeding not tolerated, ( leads to impaired fertility) ° Strains differ in body weight, maturation rate, egg production, lipid deposition in body and egg. ° Populations can show marked photoperiodic drift with large variability in reproductive response ° Food wastage making food consumption measurement difficult ° Most strains have colored eggshells that are difficult to handle. Mottling makes detection of cracks in shell difficult ( some white egg strains are available) ° Less yolk per egg ( 31.9% by weight) compared with bobwhite ( 39.8%) ° Long incubation period ° Long maturation period ( 24 months) ° Food wastage making food consumption measurement difficult ° Sex cannot be distinguished by plumage until 12 weeks of age ° Lack cloacal gland ° Spermatogenesis is not characterized Battelle Draft 29 April 23, 2003 Also comparable are baseline ranges of reproductive parameters of the two quail species. Tables 4 4 and 4 5 show typical reproductive values for the Japanese quail and northern bobwhite compiled in a revised draft guideline for avian reproduction toxicity testing ( OECD 2001). For additional comparison are shown the baseline values reported by Piccirillo and Orlando ( 1985) for evaluation of control data for one generation reproduction studies using the bobwhite. Ranges for the Japanese quail will likely vary, depending on strain selection. To evaluate which of the quail species would be the better choice for increasing the power of an avian reproduction test to detect effects, by displaying less background variation in reproductive endpoints, Springer and Collins ( 1999) compared the results of two simulation studies. Using historical control data of reproductive parameters for Japanese quail ( Baus et al. 1999) and northern bobwhite ( Springer and Collins 1999), the two studies employed simulation methods to compare power of the respective test to detect a 20% change in the mean value of a treated group relative to controls. Power estimates for both species were high (> 0.80) for ratio type endpoints, such as eggs hatched / fertile eggs, but lower for count type endpoints, such as eggs laid/ hen. No similar power comparisons are available for endocrine endpoints.

Table 4 4. Typical Values for Reproductive Parameters in Japanese Quail and Northern Bobwhite Parameter Japanese Quail Northern Bobwhite OECD Piccirilo and Orlando 1981 No. eggs laid/ hen/ day 0.66 to 0.89 0.40 to 0.81 0.6 ± 0.004 Percentage cracked or broken eggs 0% to 10% 0% to 6% 8.2% ± 7.2%

Viability ( percentage of fertile eggs that develop live embryos at 2/ 3 through incubation) 85% to 96% 72% to 98% 73.6% ± 19.9%

Hatchability ( percentage of viable eggs that hatch) 70% to 80% 70% to 98% 82.0% ± 12.8%

Percentage hatchlings that survive to 14 days 85% to 97% 69% to 98% 76.3% ± 5.0%

Mean number 14 dayold survivors/ hen/ day 0.34 to 0.71 0.24 to 0.38 Eggshell thickness ( mm) 0.19 to 0.22 0.20 to 0.25 0.31 ± 7.2% Battelle Draft 30 April 23, 2003 ( a) Data adapted from OECD ( 2000). Table 4 5. Comparison of Development Phases in Japanese Quail and the Northern Bobwhite( a)

Species Candling for fertility and viability ( days) Incubation ( days) Hatch ( days) Dimorphism of Plumage ( weeks) Sexual Maturity ( weeks) Peak Egg Production after Onset of Lay Japanese quail 8 15 to 16 17 to 18 3 6 3 Northern bobwhite 11 20 to 21 24 to 25 12 24 6 5.0 EXPERIMENTAL DESIGN CONSIDERATIONS FOR TWO GENERATION AVIAN TESTS 5.1 Exposure Duration The exposure duration should be long enough for the test substance to reach equilibrium in the tissues and all possible effects on the reproductive processes to be expressed in the selected endpoints and provide exposure to all life stages.

5.1.1 Exposure of the Parental ( P1) Generation ( Pre or Post Egg Laying)

Current EPA and OECD guidelines were originally designed to detect reproductive deficits resulting from chronic exposure to bioaccumulating substances. Therefore, birds are exposed under these guidelines well in advance of egg laying so that the compound reaches equilibrium in the tissues and presumably a maximum exposure level in eggs. Newer pesticides are much less persistent, and significant reproductive effects have been detected with treatment periods of only 1 to 3 weeks ( Bennett and Bennett 1990; Bennett et al. 1990; Rattner et al. 1982; Stromborg 1981, 1986). This led several authors to review the statistical and physiological advantages and disadvantages of the pre egg laying and post egg laying exposure regimens for one generation avian reproductive toxicity tests ( e. g., Collins 1994; Mineau et al. 1994; Bennett et al. 1990; Bennett and Ganio 1991; David Farrar, personal communication to T. Maciorowski, D. Urban, D. McLane, and D. Balluff, Statistical aspects of the evaluation of avian reproductive effects: accomplishments and objectives, 1995) and for the design of multiple generation tests that would include endocrine endpoints ( Hart et al. 1999; Baus et al. 1999; Springer and Collins 1999; Bennett et al. 2001). These authors pointed out that exposure following proven breeding would allow for the removal of incompatible or infertile birds prior to exposure, which would remove a nontreatment related source of variation and could increase the power of the test. Pretreatment measurements could also be used as covariates in the statistical analysis, again potentially increasing the power of the statistical test. Battelle Draft 31 April 23, 2003 Conversely, exposure before pairs are proven to be breeders risks a loss of replication and corresponding statistical power due to infertility or incompatibility. Using control data from a variety of historical data sets, several investigators ( Baus et al. 1999; Springer and Collins 1999; David Farrar, personal communication to T. Maciorowski, D. Urban, D. McLane, and D. Balluff, Statistical aspects of the evaluation of avian reproductive effects: accomplishments and objectives, 1995) studied the power of the proposed ( OECD 2000) design of the avian reproduction test through modeled simulations of reproductive effects. These investigators showed that use of pretreatment measures as covariates in simulation tests resulted an increase in the power of the test for number of eggs laid and the number of eggs incubated that hatched for both the Japanese quail and bobwhite ( Springer and Collins 1999). Although the simulation studies also demonstrated that significantly increased power can be obtained for all count variables by replacing nonproducing pairs of bobwhite ( Springer and Collins 1999), replacing pairs in tests using Japanese quail appeared to be ineffective ( Baus et al. 1999) or only slightly effective ( Springer and Collins 1999) in increasing the power of the test. These simulation comparisons, however, were based on study designs that included only post egg laying treatment.

No simulation studies have been conducted that provide information for selection of treatment period based on power comparisons of pre egg laying and post initiation of egg laying exposure scenarios. However, Collin ( 1994) showed that the power of the FIFRA avian reproduction test ( EPA 1982) wherein treatment is started prior to egg laying is low. He determined that to have 80% power to detect a 20% decline in the number of hatchlings per eggs incubated required a sample size of 27 pens for hatchlings exposed to a test substance for 10 weeks prior to egg laying and for 8 weeks after the start of egg laying . To detect a reduction in the number of eggs laid in the pre egg laying exposure tests would require 74 pens. In contrast, Springer and Collins ( 1999) determined that only 16 pens would be required to detect a 20% reduction in these same parameters when treatment is started after the initiation of egg laying and that only 12 pens would be required if the post initiation of egg laying test included a covariate. This comparison, however, is potentially confounded by differences in the assumptions, and the simulation and statistical methods between the studies. In particular, Collins ( 1994) used reproduction data from the entire study period rather than limiting it to the peak laying period of 5 to10 weeks used in the simulation reviewed by Springer and Collins ( 1999). Restricting the simulations to a period of high egg production when the number of hens in production and their rate of production are most stable appears to greatly enhance the statistical power of the test. Improved statistical power from this high production period is probably the result of the increased median value of the endpoints ( David Farrar, personal communication to T. Maciorowski, D. Urban, D. McLane, and D. Balluff, Statistical aspects of the evaluation of avian reproductive effects: accomplishments and objectives, 1995). Even so, the power values obtained in investigations that used only data from the peak production period in bobwhite varied greatly ( Springer and Collins 1999).

When compared with the power increases obtained from screening out nonproductive pairs or using pretreatment data as covariates, using data from the period of high egg production appears to be more influential in increasing the power to detect effects in avian reproduction studies. Battelle Draft 32 April 23, 2003 Springer and Collins ( 1999) reported a doubling of the power to detect effects in data from Weeks 3 to 8 after the onset of laying, as compared with Weeks 1 to 6 from onset of laying in bobwhite. Smaller increases of 24% to 32% were seen when nonlayers were removed or when a covariable was included in the analysis from simulations based on data from birds treated prior to egg laying. Therefore, it is possible to increase the power of a test to detect effects in a study where the birds are exposed prior to maturation by analyzing data during peak egg production, that is, by excluding the low, variable egg production interval from statistical analysis. A proven layer exposure also benefits from this approach, but in addition, has the ability to further improve the power to detect effects through screening out nonlayers and using pretreatment data as covariates.

Only one study was found in the literature that directly compared the results of a pre egg and a post egg laying exposure regimen in reproductive toxicity studies ( Bennett et al. 1990). Bobwhite in the pre egg laying test received methyl parathion, an OP insecticide, in their diet for a total of 25 weeks. Birds receiving dietary exposure of the insecticide after the onset of egg laying were treated for 3 weeks during the peak egg laying period. All dose related reproductive effects that were found in the pre egg laying exposure study, including decreased food consumption and egg production, were also seen in the post egg laying study. Unfortunately, the advantage of using proven breeders to reduce variability in the measured endpoints was compromised by the abnormally large number of injured and nonproducing birds, a total of 40%, in the post egg laying study. The elevated number of injured birds was attributed to the higher rate of aggression in the post egg laying birds that was apparently a result of changing abruptly from the short to the long day length light cycle. However, comparison of the two exposure regimens did demonstrate that initiating exposure to a test substance after the onset of laying significantly improved the dose response relationship of certain variables by using pretreatment means as covariates for each hen. Variables that demonstrated an improved dose response were those with lower within pen variation than between pen variations such as eggshell quality ( Hunt et al. 1977; Thompson et al. 1983).

From the available experimental and simulation data, it is clear that there is insufficient information to determine whether a post initiation of egg laying exposure will provide significant improvements over pre egg laying exposure in increasing the power of statistical tests to detect reproductive effects.

Besides the potential statistical advantages to be gained from the ability to remove nonproductive birds from the test before exposure begins and/ or to use pretreatment data during egg laying for each bird as a control, a proven breeder exposure can also provide information on the rapidity with which many reproductive effects are manifested in exposed birds. Information of this type is particularly useful in risk assessments of chemicals that undergo rapid degradation in the environment or have use patterns that preclude long term exposure during the breeding season. Another advantage is the savings in time and cost that is attained with the reduction in exposure period from the 20 weeks of the prematuration exposure regimen to less than 10 weeks of the proven breeder regimen. However, it must be remembered that the reduced exposure period may also not be long enough for histological or biochemical lesions to develop. Battelle Draft 33 April 23, 2003 There are potential reproductive effects, however, that cannot be detected in the P1 generation by exposing birds only after reproductive maturity. Of these effects, the age at which sexual maturation is attained is of particular importance to reproductive success and maintenance in wild bird populations. Altered rate of maturation is emerging as one of the more significant endpoints of endocrine disruption in birds ( Yoshimura et al. 2000). In a study by Edens et al. ( 1976), age at sexual maturity was among the most sensitive measures of impaired reproduction in the female Japanese quail exposed to dietary lead from hatch through 12 weeks of age. Delayed sexual maturation was detected at dietary concentrations that caused no body weight loss or overt signs of toxicity. Onset of egg laying was delayed by up to 2 weeks in treated birds. In turn, peak egg production was also delayed and in most of the treatment groups was not attained before termination of the test at 12 weeks of age. Such a delay in peak production of treated birds could confound a direct comparison not only of fecundity but also body weight, because female quail typically increase in body weight by 20% to 40% during sexual maturation. Exposure duration may need to be lengthened under these circumstances to characterize the delayed response in egg production. Conversely, chronic treatment with tamoxifen, an estrogen antagonist, resulted in precocious puberty and accelerated spermatogenesis in chickens ( Rozenboim et al. 1986), similarly offering the potential to confound direct comparison with reproductive parameters in control and treated birds. A regression approach would capture the time course of such events.

As pointed out in Bennett et al. ( 2001), the effects of delayed or accelerated maturation do not determine the ability of the F1 generation to reproduce successfully, because they are not transgenerational effects. Maturation information could be better attained in separate tests. Bennet et al. ( 2001) also noted that it is possible to obtain data to evaluate the effects of pre egg laying exposure from the F2 chicks, assuming the F1 chicks are treated prior to sexual maturation. Another alternative would be to add relatively inexpensive sexual maturation endpoints, such as cloacal gland size/ function and day of first egg, to the avian one generation test. The additional observations would also provide information on exposure duration to aid in identifying a delayed peak egg production period. However, if Japanese quail are used in the one generation tests, strain selection becomes an important factor in assuring the pre egg laying exposure is adequate. As noted earlier, many strains of Coturnix exhibit photoperiod drift, resulting in the maturation of significant proportions of the population under regimens of < 8 h light. Yamamoto et al. ( 1996) found that the day length had to be reduced severely to prevent the birds from entering into reproduction prior to the 10 week prereproduction period. The reduced day length resulted in reduced food consumption, and thus reduced uptake of the test substance in the birds.

Selection of an exposure regimen for the P1 generation of the avian two generation test must also take into account the propensity of the test substance to bioaccumulate. For compounds that require a long time to reach equilibrium in tissues, an exposure period that begins prior to egg laying may be necessary in order to reach a maximum exposure level in testicular tissue ( Section 5.1.6) and in the egg. It is not clear, however, to what extent maternal transfer of contaminants to the egg is derived from stored lipid. Although residues of OCs in eggs and Battelle Draft 34 April 23, 2003 tissues of females have been found to be highly correlated ( Mineau 1982; Custer et al. 1990; Loncore and Stendell 1977; Bogan and Newton 1977), other studies indicated that dietary, not adipose lipid may be the primary source of fat soluble contaminants in eggs of exposed birds ( Norstrom et al. 1986; Roudybush et al. 1979). Until the source of contaminants in the egg is resolved under the conditions of the test, the potential for a delayed response in the F1 endpoints for bioaccumulating chemicals should be presumed and the exposure duration selected to accommodate the required build up in tissues.

5.1.2 Exposure of the Offspring ( F1) of the Parents ( No Exposure vs. Exposure from Hatch through Egg Laying)

Bennett et al. ( 2001) reviewed the various potential exposure scenarios of the F1 generation. The two major exposure regimens involve 1) a worst case scenario, wherein the chicks are exposed from hatch through egg laying to the same environmental concentrations as their parents, or 2) a nonexposure scenario wherein the chicks do not receive additional exposure other than in ovo. The former scenario is similar to the exposure regimen of classical mammalian reproductive toxicity studies ( EPA 1982, 1985; OECD 1981) allowing observation of reproductive, developmental, and endocrine mediated effects at all susceptible life stages, not just those expressed in the P1 generation or resulting from in ovo exposure. There is a real potential, however, that exposure to the test substance as the chicks mature will result in nonendocrine related effects and potentially high juvenile mortality. This confounding influence of direct toxicity could mask the detection of endocrine mediated effects. Limiting the F1 exposure to in ovo exposure ( e. g., sexual differentiation) of the developing embryo would eliminate these potential interference and interpretative problems, and focus the test on the reproductive success of the F1 chicks. However, it is likely that for many chemicals, chicks will be exposed in the wild. Elimination of exposure during vulnerable growth and maturation stages may overlook impacts to reproductive success. Currently, there is no information available that would help to resolve the relative importance of treating the F1 chicks. It is important to the design of a two generation reproduction test to determine whether endocrine disruption can be detected amid the responses induced by direct toxicity of compounds such as OP insecticides.

Because the potential exposure patterns will differ for different chemicals, effects from both the in ovo and continuous exposure regimens must be interpreted with caution, if the environmental exposures are likely to differ from the exposure regimen of the test. Continuous exposure is used for chemicals that degrade rapidly in the environment or in cases in which F1 chicks are not exposed to chemicals to which birds in the wild would be exposed throughout their life cycle.

5.1.3 Combined Exposure Scenarios for P1 and F1 Generations The exposure scenarios for each generation should be combined in such a manner that the maximum number of reproductive processes and vulnerable life stages are exposed over the period of the study. In addition, the exposures should not mask endocrine related effects or confound interpretation of results. The selection of exposures should also maximize time and cost effectiveness of the test relative to the information obtained. From the discussions above, a list of desirable attributes were obtained and matched to the various exposure regimens ( Table Battelle Draft 35 April 23, 2003 5 1) regardless of the generation ( P1, F1, or F2). In all exposure scenarios, the F2 generation is not exposed to the test substance. The combination of exposures that provides the most comprehensive number of the attributes with minimum disadvantages was determined ( Table 5 1): post egg laying production exposure of the P1 generation; exposure of the F1 chicks through growth and egg laying; no exposure of the F2 chicks. The resulting exposure combination provides a different exposure history for each generation such that the P1 generation receives a subchronic exposure during the reproductive adult stage; the F1 offspring are exposed continuously from in ovo embryogenesis through egg laying; and the F2 generation is exposed in ovo only. By contrasting the P1 proven breeders and F1, the reproductive impact from full life cycle exposure from in ovo exposure by combined parental transfer and exposure of F1 birds through growth and breeding can be obtained. Contrasting the F1 with the F2 exposures allows evaluation of the contribution of in ovo exposure alone to the reproductive and endocrine mediated effects of the test substance. Although effects on sexual maturation are included in the F1 exposure, it is not separated from effects induced in ovo. If this information were needed, a separate study could be performed, or maturation endpoints added to existing one generation tests. A combination that employs the pre egg laying exposure of the P1generation would provide similar information as that previously described, but it would add direct maturation information. However, acquisition of these data would be at considerable time and labor cost and with potential loss of statistical power in ANOVA based comparisons. Preegg laying exposure could affect the F1 and consequently the F2 generation by influencing the transport of any of a number of essential maternal substances that are needed in the egg for normal development. In addition, if egg laying were delayed or accelerated by prebreeding exposure, comparison of the periods of peak egg production between treated and control birds would have to be made with caution.

Table 5 1. Required Attributes of Parental and Offspring Exposure Regimens TREATMENT Parameter P1 F1 F2 Pre egg Post egg None Chicks( a) None Increased power of test ( b) X

Detect altered maturation X   X

Detect effects of in ovo exposure   X Confound X Detect effects of chick exposure Partial   X( c)

Worst case environmental exposure Partial   X  Battelle Draft 36 April 23, 2003 Exposure with advantages that cannot be replaced by other exposure scenario without consideration of cost  X  X ( X)( d)

Relative time/ cost savings  High Moderate

Exposure with advantages that cannot be replaced by other exposure scenario with consideration of cost  X  X X a) exposed from hatch through egg laying. b)  no effect. c) Possible confounding of endocrine effects from direct toxicity. d) Could be replaced by F1 no exposure.

5.1.4 Selection of Egg Cohort for F1 Breeding Pairs The egg cohort from which the F1 breeding pairs will be obtained should be selected at a time during egg production when the full effect of the test substance on the production and quality of the eggs and the viability of the young have been attained. In tests where an exposure initiated after egg laying has begun, effects in eggs may not appear for some time after the initiation of the exposure. For example, yolk is recruited by ovarian follicles up to 9 days before the egg is laid ( Bacon et al. 1973) possibly delaying potential developmental effects of the test substance on embryos after the initiation of treatment. If the test substance has not reached equilibrium in the tissues, the period of maximum deposition of the chemical into eggs may be further delayed and maximum effects observed even later. As noted in Section 5.1.1, the source of chemical deposited in egg may be from the diet rather than fat tissue, in which case little extra delay would be observed. Another source of delayed response in birds exposed during breeding is the time required ( 17   21 days) for effects on early spermatogenic processes to be observed in eggs ( see Section 5.1.6). Because of these and other potential delays in response after the onset of exposure, selection of the egg cohort for the next breeding population ( F1) should be obtained as late as possible in the treatment period. Therefore, the last weekly egg batch at the end of the parental treatment period should be used for the F1 breeding pairs.