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Validation of the Japanese Quail Two-Generation Test:

Integrated Summary Report

U.S. Environmental Protection Agency

Endocrine Disruptor Screening Program

Washington, D.C.

Prepared by

Battelle

Columbus, Ohio

Mary Ann Ottinger

College Park, Maryland

Johanna Rochester

Paonia, Colorado

Contract No. EP-W-11-063

Task Order 5

28 May 2013
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1 Contents

2 Page

3 EXECUTIVE SUMMARY .......................................................................................................... 1

4 OBJECTIVE ................................................................................................................................. 3

5 1 INTRODUCTION............................................................................................................. 3

6 1.1 Endocrine Disruptor Screening Program................................................................ 4

7 1.2 Validation................................................................................................................ 5

8 2 OVERVIEW OF THE JAPANESE QUAIL TWO-GENERATION TEST ............... 8

9 2.1 Background on Endocrine Disruption in Avian Species ........................................ 8

10 2.2 Relevance of the Japanese Quail Two-generation Test ........................................ 10

11 2.3 Purpose of the Japanese Quail Two-generation Test............................................ 11

12 2.4 Overview of Japanese Quail Biology & Reproductive Endocrinology................ 11

13 2.4.1 Altricial versus precocial birds: Development Patterns and 

14 Vulnerable Stages ..................................................................................... 11

15 2.4.2 Post Hatch Growth and Maturation .......................................................... 16

16 2.5 Rationale for the Assay......................................................................................... 17

17 3 PROTOCOL DEVELOPMENT ................................................................................... 19

18 3.1 Summary of the Japanese Quail Two-generation Test Protocol........................... 19

19 3.2 Study Design, Test Chemicals and Dose Selection .............................................. 19

20 3.3 Preparation and Monitoring of the Test Diet ........................................................ 20

21 3.4 Equipment and Supplies (Husbandry, Endpoint Measurements, Analyses)......... 21

22 3.5 Feeding.................................................................................................................. 22

23 3.6 Animals (Species, Sex, Age, Weight, Number).................................................... 23

24 3.7 Experimental Design............................................................................................. 23

25 3.8 Offspring – Eggs................................................................................................... 23

26 3.9 Egg Parameters ..................................................................................................... 25

27 3.10 Chicks ................................................................................................................... 26

28 3.11 Pathology .............................................................................................................. 26

29 3.12 Biochemical/endocrine Measurements and Observations.................................... 27

30 3.13 Behavioral Measurements..................................................................................... 27

31 4 BACKGROUND AND RATIONALE FOR SELECTION OF ENDPOINTS.......... 29

32 4.1 Endpoints.............................................................................................................. 29

33 4.1.1 Survival..................................................................................................... 29

34 4.1.2 Food Consumption.................................................................................... 30

35 4.1.3 Body Weight ............................................................................................. 30

36 4.1.4 Secondary Sex Characteristics (plumage, cloacal gland area/foam 

37 production)................................................................................................ 31

38 4.1.5 Sexual Maturation and Puberty................................................................. 31

39 4.1.6 Behavior (activity, lacrimation, reproductive behaviors, etc.).................. 32

40 4.1.7 Fecundity (egg production)....................................................................... 32

41 4.1.8 Fertilization Success ................................................................................. 33

42 4.1.9 Embryo viability ....................................................................................... 33

43 4.1.10 Eggshell parameters (abnormalities, eggshell strength/thickness, 

44 eggshell color)........................................................................................... 33
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1 4.1.11 Hatchability (ED15, hatch)....................................................................... 35

2 4.1.12 Genotypic Sex Determination................................................................... 35

3 4.1.13 Histopathology.......................................................................................... 35

4 4.1.14 Neuroendocrine Systems Regulating Reproduction, Metabolism, 

5 and Stress.................................................................................................. 38

6 4.1.15 Key Neuroendocrine Measures for Endocrine Disruption........................ 39

7 5 PROTOCOL OPTIMIZATION AND DEMONSTRATION..................................... 42

8 5.1 Comparability with the Avian Reproduction Test Currently in Use .................... 42

9 5.2 Egg Injection Studies............................................................................................ 42

10 5.2.1 Atrazine (Experimental Pathology Laboratories 2006)............................ 43

11 5.2.2 Trenbolone ................................................................................................ 43

12 5.2.3 Estradiol .................................................................................................... 45

13 5.2.4 Vinclozolin (McGary et al. 2001)............................................................. 46

14 5.2.5 Other Egg Injection Treatments................................................................ 46

15 5.3 Reproduction and Two-Generation Dietary Studies............................................. 46

16 5.3.1 Perchlorate (Stafford 2011)....................................................................... 46

17 5.3.2 Bisphenol A (Frey et al. 2011).................................................................. 47

18 5.3.3 Trenbolone Reproduction Dietary Study (Frey et al. 2012)-Dietary 

19 Study #1 .................................................................................................... 48

20 5.3.4 Methoxychlor (dietary study with Japanese quail and bobwhite 

21 quail comparisons).................................................................................... 48

22 5.3.5 Two-Generation Dietary 17-Beta Estradiol (E2) Dosing Study............... 49

23 5.3.6 Two-Generation Endosulfan Dietary Test (Quinn et al. 2010)................. 51

24 5.3.7 Two-Generation Trenbolone Dietary Study (Quinn et al. 2011).............. 51

25 5.3.8 Two-Generation 17β Trenbolone Dietary Study (Karouna-Renier 

26 and Henry, 2013) ...................................................................................... 52

27 5.4 Transferability of the Avian Reproduction Test to an Avian 

28 Multi-Generation Test........................................................................................... 52

29 5.5 Lessons Learned from Optimization Studies........................................................ 53

30 6 INTERLABORATORY EVALUATION..................................................................... 56

31 6.1 Vinclozolin—Bayer Crop Science Study ............................................................. 56

32 6.1.1 Exposure Levels and Protocol .................................................................. 56

33 6.1.2 Sensitive Endpoints and Overall Conclusions.......................................... 56

34 6.2 Vinclozolin – Wildlife International Study........................................................... 58

35 6.2.1 Exposure Levels and Protocol .................................................................. 58

36 6.2.2 Sensitive Endpoints and Overall Conclusions.......................................... 58

37 6.3 Vinclozolin – Smithers Viscient Carolina Research Center Study....................... 59

38 6.3.1 Exposure Levels and Protocol .................................................................. 59

39 6.3.2 Sensitive Endpoints and Overall Conclusion............................................ 59

40 6.3.3 General Advantages of Japanese Quail Two-generation Test 

41 Reproducibility of the assay...................................................................... 60

42 6.3.4 Data Interpretation .................................................................................... 61

43 6.3.5 Other Considerations for Multi-Generation Tests .................................... 64

44 7 CONCLUSIONS ............................................................................................................. 66

45 7.1 General Advantages of Japanese Quail Two-generation Test .............................. 66
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1 7.2 Limitations of the Japanese Quail Two-generation Test ...................................... 66

2 7.3 Value-added of Multi-generation Exposure.......................................................... 66

3 7.4 Complementary Technologies to the Japanese Quail Two-generation Test......... 67

4 8 REFERENCES................................................................................................................ 68

5 9 LIST OF APPENDICES ................................................................................................ 89

6

7 APPENDIX A. VALIDATION OF SCREENING AND TESTING ASSAYS 

8 PROPOSED FOR THE EDSP (JULY 2007)................................................................ 89

9 APPENDIX B. DETAILED REVIEW PAPER FOR AVIAN TWO-GENERATION 

10 TOXICITY TEST (OECD NO. 74, AUGUST 2007) ................................................... 89

11 APPENDIX C. REPORTS ON THE INTER-LABORATORY TRIALS FOR THE 

12 JQTT................................................................................................................................ 89

13 APPENDIX D. AVIAN TWO-GENERATION TOXICITY TEST IN THE 

14 JAPANESE QUAIL, OECD GUIDELINE FOR TESTING OF CHEMICALS, 

15 DRAFT, SEPTEMBER 2011 ......................................................................................... 89

16 APPENDIX E. SUPPORTING TWO-GENERATION DEMONSTRATION 

17 STUDIES ......................................................................................................................... 89

18
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Tables

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Avian Two-Generation ISR v May 28, 2013

1 Table 1-1. Assays recommended for consideration for the Tier 1 screening battery............... 5

2 Table 3-1. Housing Conditions. ............................................................................................. 22

3 Table 3-2. Recommended Nutritrional Values....................................................................... 22

4 Table 3-3. Conditions for Egg Storage, Incubation, and Hatching. ....................................... 24

5 Table 3-4. Typical Values for Reproductive Parameters in Japanese Quail. ......................... 26

6 Table 4-1. Summary of Significant Effects (p<0.05) of Trenbolone Acetate and p,p7 DDE on Immune, Reproductive, and Behavioral Measures................................. 38

8 Table 5-1. Summary of Responses to Estradiol or Trenbolone with Day of Egg 

9 Injection from the Studies Reviewed.................................................................... 45

10 Table 6-1. Summary of Statistical Analyses Yielding Effects – Bayer. ................................ 57

11 Table 6-2. Summary of Statistical Analyses Yielding Effects – Wildlife International. ....... 58

12 Table 6-3. Summary of Statistical Analyses Yielding Effects – Smithers Viscient. ............. 60

13 Table 6-4. Ranges of CV% Across Laboratories (by Category)............................................ 62

14 Table 6-5. Ranges of Percent Deviations from Control Group Means for Detections 

15 with 80% Power and the Ratios Between Generations (by Category). ................ 63

16

17 Figures

18 Figure 2-1. Stages in Embryonic Development. Diagram shows timing of 

19 developmental events during embryogenesis with indications of endogenous 

20 endocrine change and potential periods of vulnerability ...................................... 13

21 Figure 2-2. Yolk androgens during the first half of embryonic development in Japanese 

22 Quail...................................................................................................................... 14

23 Figure 2-3. Yolk estradiol during the first half of embryonic development in Japanese 

24 Quail...................................................................................................................... 14

25 Figure 2-4. Maternal deposition of the phytoestrogen, genistein by the quail hen into 

26 eggs....................................................................................................................... 15

27 Figure 2-5. Diagram of the reproductive axis in Japanese Quail ............................................ 16

28

29

30
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1 EXECUTIVE SUMMARY

2 This Integrated Summary Report (ISR) provides a validation record of the Endocrine 

3 Disruptor Screening Program (EDSP) Tier 2 Japanese Quail (Coturnix japonica) Two-generation

4 Test aimed at characterizing potential disruption of the endocrine system by putative Endocrine 

5 Disrupting Chemicals (EDCs). Pertinent literature was previously summarized in an in-depth 

6 review completed by the Organisation for Economic Co-Operation and Development (OECD) in 

7 2007, and recent literature is reviewed to provide background on avian biology, and findings 

8 from laboratory and field studies have been discussed in the context of observed actions of EDCs 

9 in birds. Finally, relevant studies are presented that helped inform the ultimate design of the 

10 avian two-generation testing protocol. Studies include egg injection studies in addition to dietary 

11 studies of different generational duration (see Table of Contents for complete list). Egg injection 

12 studies take advantage of avian embryonic development in the egg, independent of parental input. 

13 As such, egg injection studies allow shorter duration tests that mimic maternal deposition of 

14 chemicals, providing an opportunity to dose the embryo with known concentrations of 

15 compound and track effects throughout ontogeny. These studies provide insight into the 

16 mechanisms of action, toxicity, and physiological systems that would be targeted by these 

17 compounds. 

18 Findings from the interlaboratory comparison were informative in evaluating the 

19 performance criteria for the two-generation testing protocol. More importantly, the 

20 interlaboratory comparison demonstrated consistency in the responses of control birds and to 

21 specific doses in dietary vinclozolin across laboratories. Control birds show similar patterns in 

22 maturation and reproduction. Most of the variability observed in controls appears to be due to 

23 strain differences; this indicates the importance of using a consistent strain, preferably closest to 

24 wild type in order to optimize detection of EDC effects. Further, the interlaboratory comparison 

25 provides valuable information relative to unique characteristics of birds in that the weak response 

26 to vinclozolin illustrates differences in the biological basis of sexual differentiation in this 

27 taxonomic group. As such, it should be recognized that it is critical to include an avian 

28 multigenerational test for characterizing potential adverse endocrine-mediated effects and to 

29 encompass these unique biological characteristics, resulting in differential sensitivities to EDCs 

30 compared to other vertebrate classes and as observed in the response to other compounds such as 

31 endosulfan and trenbolone.

32 To summarize, there are some key differences in avian species that provide strong 

33 rationale for the avian multigeneration test in Tier 2 of the EDC testing schedule. These unique 

34 characteristics include:

35  Metabolic systems

36 o High body temperature (105oF) with high metabolic rate

37 o Associated accelerated toxicokinetics

38 o Migratory associated energy drain and mobilization of lipid reserves

39 o Thyroid system function in precocial and altricial birds.

40  Reproductive system

41 o Female has one functional ovary

42 o Altered gonadal differentiation results in ovotestes

43  Hormones and behavior

44 o Males adversely impacted by xenoestrogens and androgenic compounds
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1 o Female behavior (receptivity) less sensitive to xenoestrogens

2  Sexual differentiation

3 o Males are the homogametic sex having ZZ; females are ZW

4 o HPG axis relies on relative exposure to estradiol and testosterone

5 o HPG axis and song system differ in precocial and altricial birds

6  Growth and migration

7 o High metabolism requires sufficient nutrient utilization

8 o Rapid growth rate especially for migratory species

9 o Thyroid system critical for pre-migratory fattening

10  Lifespan

11 o Long-lived birds produce few offspring annually over many years.

12 Analyzing core endpoints of survival, growth, and reproduction across generations will 

13 reveal potential impacts on reproductive, metabolic/thyroid systems, and adrenal/stress axes as 

14 well as general toxicity. The P0 (parent generation) provides maturation and adult responses to 

15 exposure. Although these phases of the life cycle are potentially less vulnerable, it is important to 

16 assess effects on adults, especially relative to adverse effects on reproductive and metabolic 

17 endocrine function. Moreover, because the Japanese quail has the same neuroendocrine circuitry 

18 regulating reproduction as other avian species, any impact may be extrapolated to field birds. 

19 Behavioral impacts observed in Japanese quail would likely indicate more serious impacts on 

20 songbirds because the song control system is steroid dependent as are the neural systems that 

21 modulate singing behavior. Because the F1 (first generation) birds are exposed both via maternal 

22 deposition and from the diet (same treatments as their parents), they would be affected by 

23 endocrine disruption during embryonic development and sexual differentiation as well as 

24 experiencing any impacts due to endocrine disruption during reproductive maturation and in 

25 adults. Some chemicals may exert epigenetic effects that would have long-term impacts on wild 

26 populations over multiple generations, even without continued EDC exposure. Because the F2 

27 (second generation) is exposed to maternally deposited EDCs, this generation reveals potential 

28 transgenerational effects and isolates embryonic effects of EDCs. In summary, each portion of 

29 the multi-generation test protocol provides a unique treatment in that the P0 has maturation and 

30 adult exposure, the F1 has embryonic and life long exposure, and the F2 tests solely embryonic 

31 exposure. Together, all of the salient and vulnerable life stages are considered, thereby allowing 

32 expression of potential adverse effects.

33
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1 OBJECTIVE

2 The objective of this integrated summary report is to provide a detailed account of the 

3 validation process for the Japanese Quail Two-generation Test (JQTT) so that Federal Insecticide, 

4 Fungicide and Rodenticide Act (FIFRA) Scientific Advisory Panel members may address the 

5 accompanying charge questions.

6 1 INTRODUCTION

7 Section 408(p) of the Federal Food Drug and Cosmetic Act (FFDCA or FD&C Act) 

8 requires the USEPA to:

9

10 develop a screening program, using appropriate validated test systems and other

11 scientifically relevant information, to determine whether certain substances may 

12 have an effect in humans that is similar to an effect produced by a naturally 

13 occurring estrogen, or other such endocrine effect as the Administrator may 

14 designate [21 U.S.C. 346a(p)].

15

16 Subsequent to passage of the Food Quality Protection Act in 1996, which amended 

17 FFDCA and FIFRA, the EPA formed the Endocrine Disruptor Screening and Testing Advisory 

18 Committee (EDSTAC), a committee of scientists and stakeholders that was charged by the EPA 

19 to provide recommendations on how to implement its Endocrine Disruptor Screening Program 

20 (EDSP). The EDSP is described in detail at the following website: 

21 http://www.epa.gov/scipoly/oscpendo/. Upon recommendations from the EDSTAC (EDSTAC 

22 1998), the EPA expanded the EDSP using the Administrator’s discretionary authority to include 

23 the androgen and thyroid hormonal systems as well as the reproductive endocrine systems of 

24 wildlife. Following broader international concerns and the creation of similar programs in other 

25 countries, the Organisation for Economic Co-operation and Development (OECD) established 

26 the Endocrine Disrupters Testing and Assessment (EDTA) Task Force in 1998 within its Test 

27 Guidelines Programme. EDTA is charged with developing an internationally harmonized testing 

28 strategy for the screening and testing of endocrine disrupting chemicals, taking into account the 

29 consequences of such a testing strategy on the development and validation of Test Guidelines, 

30 and on existing regulatory systems for new and existing substances.

31 As described below, EPSP involves Tier 1 screening and Tier 2 testing. The JQTT is one 

32 of the assays proposed as part of the EDSP Tier 2 and is the subject of this report. This report 

33 complements the attached supporting materials and is meant to provide the peer reviewers with 

34 the necessary information, and/or direction to necessary information, to address the peer review 

35 charge questions. It introduces the purpose of the JQTT and how it fits into the EDSP, the 

36 scientific rationale for the assay, and an historical account of the development and optimization 

37 of the assay protocol. It also synthesizes the information gained during the validation process and 

38 addresses the advantages and limitations of this avian test method based on its strengths and 

39 weaknesses, practicality, reproducibility, reliability, and protocol transferability.
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1 1.1 Endocrine Disruptor Screening Program 

2 To comply with its mandate, the EPA chartered a Federal advisory committee (EDSTAC) 

3 to provide advice and guidance on the development of a screening and testing program. In 1998, 

4 this committee recommended to the EPA a conceptual two-tiered approach that involved 

5 screening and testing chemical compounds for effects on the estrogen, androgen, and thyroid 

6 (EAT) hormone axes. The ultimate goal of these assays is to provide input into hazard 

7 identification to assess risk of adverse consequences to humans and wildlife (EDSTAC 1998).

8 The EPA submitted a proposal of the EDSP for public review and comment (FRN 1998) 

9 as well as peer review by a joint subcommittee of the EPA Science Advisory Board and FIFRA 

10 Scientific Advisory Panel (SAB, SAP 1999). A complete description of the program proposal 

11 can be found in the Federal Register Notice (FRN 1998). Briefly, the EDSP proposed by 

12 EDSTAC allows for: 1) initial sorting and prioritization of chemical compounds; 2) 

13 identification of chemicals for further testing using a Tier 1 screening battery that includes in 

14 vitro and in vivo mammalian, amphibian and fish assays; and 3) characterization of adverse 

15 consequences resulting from possible endocrine disruption and establishment of dose-response 

16 relationships for hazard identification using Tier 2 testing.

17 In comparison to the more refined, detailed, and definitive tests in Tier 2, the EDSTAC 

18 indicated that the in vitro and in vivo screening assays in the Tier 1 battery should:

19  Be relatively fast and efficient

20  Be standardized and validated

21  Be more sensitive than specific to minimize false negatives without an unreasonable rate 

22 of false positives

23  Be comprised of multiple endpoints that reflect as many modes of endocrine action as 

24 possible 

25  Have a sufficient range of taxonomic groups among test organisms represented 

26  Yield data that can be interpreted as either negative or positive for determining the

27 necessity and manner in which to conduct Tier 2 tests.

28 Together, the suite of Tier 1 assays form a battery in which some endocrine axis 

29 redundancy is incorporated (e.g., two different assays may cover some similar aspects of the 

30 estrogenic response). This redundancy allows for a weight-of-evidence (WOE) approach, as 

31 recommended by EDSTAC, to determine whether a chemical shall undergo further, more 

32 definitive, testing.

33 The assays listed in Table 1-1, recommended by EDSTAC, have undergone validation 

34 and have been adopted as the EDSP Tier 1 screening battery. These assays are meant to detect 

35 chemicals that may affect the estrogen, androgen, and thyroid (EAT) hormone axes through any 

36 known modes of action.

37
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1 Table 1-1. Assays recommended for consideration for the Tier 1 screening battery.

Assay Reason for Inclusion

Estrogen receptor binding or transcriptional 

activation assay

An in vitro test to detect chemicals that may affect the endocrine 

system by binding to the estrogen receptor.

Estrogen receptor transcriptional activation 

assay

An in vitro test to detect chemicals that may affect the endocrine 

system by binding to the estrogen receptor.

Androgen receptor binding assay An in vitro test to detect chemicals that may affect the endocrine 

system by binding to the androgen receptor.

In vitro steroidogenesis assay An in vitro test to detect chemicals that interfere with the 

synthesis of the sex steroid hormones

Placental Aromatase Assay An assay to detect interference with aromatase.

Uterotrophic Assay An in vivo assay to detect estrogenic chemicals. 

Hershberger Assay An in vivo assay to detect androgenic and antiandrogenic 

chemicals. 

Pubertal Male An in vivo assay to detect chemicals that act on androgen or 

through the hypothalamic-pituitary-gonadal (HPG) axis that 

controls the estrogen and androgen hormone systems. It is also 

enhanced to detect chemicals that interfere with the thyroid 

system. This assay could in part substitute for the female 

pubertal assay.

Pubertal female assay An in vivo assay to detect chemicals that act on estrogen or 

through the HPG axis that controls the estrogen and androgen 

hormone systems. It is also enhanced to detect chemicals that 

interfere with the thyroid system.

Amphibian metamorphosis assay An in vivo assay for detection of chemicals that interfere with the 

thyroid hormone system. 

Fish screening assay An in vivo assay for detection chemicals that interfere with the 

HPG axes.

2

3 A WOE review of the results of the Tier 1 screening battery and other scientifically 

4 relevant information (OSRI) will be made to ascertain if a chemical is deemed sufficiently 

5 endocrine active to prompt requirements for Tier 2 testing. The following tests, as generally 

6 recommended by EDSTAC, are being considered as part of the EDSP Tier 2:

7  Two-generation Rat Reproduction Test

8  Medaka Multi-generation Test

9  Japanese Quail Two-generation Test

10  Mysid Two-generation Toxicity Test

11  Larval Amphibian Growth and Development Test.

12 The Two-generation Rat Reproduction Test (OCSPP 870.3800) has already been 

13 adopted. The remaining four Tier 2 tests are the subject of the FIFRA Scientific Advisory Panel 

14 for which the JQTT is reviewed in this report.

15 1.2 Validation

16 Validation has been defined as “the process by which the reliability and relevance of a 

17 test method are evaluated for a particular use” (NIEHS 1997; OECD 1996).

18 Reliability is defined as the reproducibility of results from an assay within and between 

19 laboratories. Relevance describes whether a test is meaningful and useful for a particular purpose 

20 (OECD 1996). For Tier I EDSP assays, relevance can be defined as the ability of an assay to 

21 detect the effects of chemicals with the potential to interact with the endocrine system.
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1 Validation is generally recognized as necessary for the regulatory acceptance of new and 

2 revised test methods, and is now an integral component of the international development and 

3 acceptance of these methods (OECD 2005a). The criteria used to guide the validation process for 

4 the JQTT were based on the principles of validation developed by the U.S. Interagency 

5 Coordinating Committee for the Validation of Alternative Methods (ICCVAM) (NIEHS 1997)

6 and the OECD (OECD 2005a). These criteria, as stated by ICCVAM (NIEHS 1997), are as 

7 follows:

8 1. The scientific and regulatory rationale for the test method, including a clear statement of 

9 its proposed use, should be available.

10 2. The relationship of the endpoints determined by the test method to the in vivo biologic 

11 effect and toxicity of interest must be addressed.

12 3. A formal detailed protocol must be provided and must be available in the public domain. 

13 It should be sufficiently detailed to enable the user to adhere to it and should include data 

14 analysis and decision criteria.

15 4. Within-test, intra-laboratory and inter-laboratory variability and how these parameters 

16 vary with time should have been evaluated.

17 5. The test method’s performance must have been demonstrated using a series of reference 

18 chemicals preferably coded to exclude bias.

19 6. Sufficient data should be provided to permit a comparison of the performance of a 

20 proposed substitute test to that of the test it is designed to replace.

21 7. The limitations of the test method must be described (e.g., metabolic capability).

22 8. The data should be obtained in accordance with Good Laboratory Practices (GLPs).

23 9. All data supporting the assessment of the validity of the test methods including the full 

24 data set collected during the validation studies must be publicly available and, preferably, 

25 published in an independent, peer-reviewed publication. 

26 The EPA has adopted these various validation criteria for the EDSP as described in 

27 Appendix A (EDSP 2007), which were considered to have been met in the review of the Tier 1 

28 assays. Validation of these assays according to the criteria proved a challenge because these are 

29 not replacement assays (Validation Criterion No. 6). Many of them are novel assays; 

30 consequently, large databases do not exist as a reference to establish their predictive capacity 

31 (e.g., determination of false positive and false negative rates).

32 In general, the EPA uses a five-part or stage validation process outlined by ICCVAM 

33 (NIEHS 1997). The EPA also believes it is important to recognize that this process was 

34 originally developed for alternative assays intended to replace or refine existing assays. A 

35 rudimentary problem confronting the EPA is how to adapt and work with this process for rodent 

36 and ecological in vivo assays in Tiers 1 and 2 that are considered the standards and not as 

37 alternatives to other assays. Nonetheless, the stages of the process outlined by the ICCVAM are 

38 as follows:

39 The first stage of the process was test development, an applied research function, which 

40 culminated in an initial protocol. As part of this phase, EPA drafted a Detailed Review Paper 

41 (DRP) to explain the purpose of the test method, the context in which it will be used, and the 

42 scientific bases upon which the assay’s protocol, endpoints, and relevance rest (Appendix B).

43 The DRP reviewed the scientific literature for candidate protocols and evaluated them with 

44 respect to a number of considerations, such as whether candidate protocols meet the assay’s 

45 intended purpose, costs, and other practical considerations. The DRP also identified the 
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1 developmental status and questions related to each protocol; the information needed to answer 

2 the questions; and, when possible, recommended an initial protocol for the initiation of the 

3 second stage of validation, standardization and optimization. During standardization and 

4 optimization, studies were performed that were geared toward refining, optimizing, and 

5 standardizing the protocol, and initially assessing protocol transferability and performance. In 

6 inter-laboratory validation, studies were conducted in several independent laboratories with the 

7 refined protocol. The results of these studies were used to determine inter-laboratory variability 

8 and to set or cross-check performance criteria. The reports on the inter-laboratory trials for the 

9 JQTT are provided in Appendix C, and supporting two-generation demonstration studies are 

10 provided in Appendix E. Inter-laboratory validation is followed by peer review, i.e., an 

11 independent scientific review by qualified experts, and by regulatory acceptance, i.e., adoption 

12 for regulatory use by an agency. EPA has developed extensive guidance on the conduct of peer 

13 reviews because the Agency believes that peer review is an important step in ensuring the quality 

14 of science that underlies its regulatory decisions (US EPA 2006).

15 The purpose of this ISR is to provide a historical summary of the development and 

16 validation of a standardized protocol for the JQTT (Appendix D) proposed as an in vivo assay for 

17 the Tier-1 screening battery. The reasoning and judgments leading to the various studies, and 

18 conclusions concerning the strengths and weaknesses of the assay in its current form, are 

19 presented.

20
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1 2 OVERVIEW OF THE JAPANESE QUAIL TWO-GENERATION TEST

2 2.1 Background on Endocrine Disruption in Avian Species

3 Many environmental chemicals have potential activity in avian systems. This section will 

4 discuss some of the challenges and background that has informed the development of the testing 

5 protocol. One of the primary challenges, given the diversity of chemicals considered potential 

6 EDCs, is to ascertain risk across vertebrate classes. Within the context of EDSTAC 

7 recommendations, it is critical to consider the unique characteristics of birds that make them 

8 particularly vulnerable to certain EDCs. Specifically, birds have a broad range of life histories as 

9 well as developmental characteristics resulting in differential sensitivities and changing lifetime 

10 vulnerability. Further, migratory birds have enormous metabolic demands, requiring highly 

11 functional metabolic and thyroid endocrine systems. As such, EDCs that impact metabolic and in 

12 particular the thyroid systems pose risks to migratory avian species. Furthermore, sexual 

13 differentiation of reproductive and other endocrine systems is unique to birds, being driven 

14 primarily by estradiol-mediated mechanisms. Developmentally, songbirds and other passerine

15 species are altricial, while ducks, galliforms, and ground dwelling species are precocial. This 

16 developmental difference appears to relate to the extent and timing of sensitivity to EDCs 

17 because precocial species are fully functional and relatively independent at hatch. As such, they 

18 undergo sexual differentiation in ovo; similarly, other physiological systems are also well 

19 developed at hatch as compared to altricial birds, which require intensive parental care and hatch 

20 in a relatively immature state. Finally, the potential for adverse effects of EDCs on long- or 

21 short-lived birds is unknown especially in the context that long-lived birds tend to have small 

22 clutches and raise fewer chicks per year compared to short-lived birds (Ottinger et al. 2003; 

23 Stearns 1979).

24 Workshops, including the Wingspread Meetings and research publications, have 

25 documented impacts of EDCs and toxicants in laboratory studies and in wild populations in 

26 avian species (Berg et al. 1999; Bowerman et al. 2000; Brunstrom and Halldin 1998; Cohen27 Barnhouse et al. 2011; Corbitt et al. 2007; CM Custer et al. 2010a; TW Custer et al. 2010a; 

28 Custer et al. 2002; Golden et al. 2003; Hotchkiss et al. 2008; Kavlock et al. 1996; Rattner et al. 

29 2004). In general terms, the potential for deleterious results have been supported by observations 

30 of endocrine-related effects on organisms in areas in which dioxin, PCB, or other chemicals 

31 occurred. Furthermore, the exposure of wildlife to environmentally relevant levels is often at 

32 sublethal concentrations, making the determination of effects of EDCs a challenge, especially 

33 when attempting to separate other interacting factors in the animal’s environment. Additional 

34 variables may include environmental conditions, food availability, disease, or confounds such as 

35 simultaneous exposure to several chemicals. It is therefore important to develop reliable and 

36 sensitive measures that are appropriate for the endocrine or organ system that are the targets of 

37 the EDCs.

38 A further challenge is developing reliable and sensitive measurement endpoints that can 

39 serve as indices of exposure. Inherent in the selection of endpoints to be used as bioindicators is 

40 having an understanding of the association of an adverse impact on the individual and risk to 

41 populations as a whole. Steroid hormones and EDCs disrupt embryonic development including 

42 organ development, sexual differentiation, reproduction and immune function, reproductive 

43 behavior, and metabolic processes (Berg et al. 1999; Best et al. 2010; Darnerud 2003; Fernie et 

44 al. 2005a; Fox and Grasman 1999; Franceschini et al. 2008; Ottinger et al. 2005; Quinn et al. 
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Avian Two-Generation ISR 9 May 28, 2013

1 2009; Quinn et al. 2008; Rissman et al. 1984; Rochester et al. 2010; Rochester et al. 2008). 

2 Biomedical and epidemiological data reveal clear linkages between EDC exposure and effects on 

3 early embryonic development, later disease, and potential epigenetic effects. In addition to 

4 biomedical data, field studies have correlated EDC exposure to immunosuppression in a number 

5 of taxa demonstrating that the effects of these compounds have conserved mechanisms and 

6 impact across a range of species, including invertebrates.

7 Laboratory studies have been successful in establishing clear effects of various classes of 

8 EDCs on a number of physiological systems (Berg et al. 1998; Gee et al. 2004; Halldin 2005). 

9 Because many early EDCs of concern such as methoxychlor, dioxin, and some polychlorinated 

10 biphenyls (PCBs) have estrogenic activity, attention has focused on reproductive impact of these 

11 compounds in comparison to estradiol. However, at any one time, wildlife species are actually 

12 exposed to many different types of EDCs (e.g., androgenic, thyroid active) as well as other 

13 toxicants, often with potentially different mechanisms of action (Golden et al. 2003; Hotchkiss et 

14 al. 2008). As a result, it has been difficult to develop comprehensive risk assessment models. 

15 Furthermore as mentioned above, some EDCs, most notably DDE and vinclozolin are 

16 biologically active in androgenic circuits by acting as androgen receptor (AR) blockers, whereas

17 other compounds such as trenbolone have frank androgenic activity (Panzica et al. 2007; Quinn 

18 Jr et al. 2007; Quinn et al. 2008; Quinn et al. 2007). This is in contrast to the commonly held 

19 belief that most of the EDCs are xenoestrogenic, acting primarily via estrogen responsive targets.

20 It is clear that EDCs act via a variety of pathways, including not only steroid sensitive 

21 pathways, but also pathways involving thyroid, metabolic, stress, and immune systems 

22 (Brunstrom and Halldin 1998; Cohen-Barnhouse et al. 2011; Fry 1995; Grasman and Fox 2001; 

23 Head et al. 2008; Ishihara et al. 2003; Kavlock et al. 1996; Lavoie and Grasman 2007; McNabb 

24 and Fox 2003). Therefore, an emerging approach for capturing the activity of a compound is 

25 developing Adverse Outcomes Pathways [AOP; (Hotchkiss et al. 2008)]. This approach 

26 promises to put more structure into characterizing the range of effects of EDCs while considering 

27 fundamental actions and molecular targets underpinning these impacts.

28 Studies in birds include documented cases of exposures in wildlife associated with higher 

29 concentrations of toxicants originating from chemical applications and industrial spills. Often 

30 these occurrences are associated with high mortality, thereby prompting federal and regional 

31 responses aimed at protecting human and wildlife health. However, less is known about non32 lethal impacts of EDCs on wild birds and the potential for adverse effects translating even to the 

33 level of a risk to the population. There are extensively documented cases, such as incidences of 

34 avian impacts from the Great Lakes, PCBs released into the Housatonic and Hudson Rivers and 

35 high concentrations of munitions and other contaminants that indicate impacts on birds (Best et 

36 al. 2010; Bowerman et al. 2000; CM Custer et al. 2010a; TW Custer et al. 2010a; Custer et al. 

37 2002; Darnerud 2003; Franceschini et al. 2008).

38 Some extraordinary datasets also exist or are emerging from long-term monitoring and 

39 research programs, including the Great Lakes region; many of these studies have considered 

40 physiological and reproductive impacts of exposure to chemicals found in those areas. 

41 Laboratory studies have provided additional insights into potential mechanisms of action of 

42 EDCs as well as periods of sensitivity throughout the life cycle. Information from many of these 

43 studies will be discussed below in order to put the proposed two-generation test into the context 

44 of testing in avian species. Furthermore, it is important to consider unique characteristics of 

45 avian physiology with emphasis on sexual differentiation, hormonal modulation of endocrine and 
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1 behavioral components of reproduction, and functional impacts of EDCs on neuroendocrine 

2 regulatory systems, especially considering potential adverse outcomes.

3 2.2 Relevance of the Japanese Quail Two-generation Test

4 The JQTT provides a multigenerational test that captures critical life cycle stages and 

5 assesses the impacts of EDCs on all these life stages, both separately and as a continuum. 

6 Japanese quail provide an excellent avian model for laboratory trials of EDC actions with a great 

7 deal of experimental literature available for comparison for both toxicological and EDC studies 

8 (Adkins-Regan and Watson 1990; Ottinger et al. 2008; Ottinger et al. 2009b; Watson et al. 1990). 

9 Hypothalamic systems that direct endocrine and behavioral components of reproduction are 

10 responsive to EDCs and have been investigated in detail in Japanese quail (Adkins-Regan and 

11 Watson 1990; Halldin 2005; Halldin et al. 2001; Halldin et al. 1999; Halldin et al. 2003; Ishihara 

12 et al. 2003; Oshima et al. 2012; Ottinger et al. 2009a; Panzica et al. 2007; Quinn Jr et al. 2007; 

13 Quinn et al. 2009; Quinn et al. 2008; Thompson and Adkins-Regan 1992; Wilhelms et al. 2006).

14 Nonetheless, it is important to point out that EDCs act on a background of endogenous 

15 steroids, which play a critical role in sexual differentiation, maturation, and maintenance of adult 

16 breeding. It is against this backdrop that exogenous EDCs act, with variations in specific 

17 mechanism(s) of action and targets for a particular compound. Although much of the emphasis 

18 of EDC studies has been on the reproductive axis, it is clear that the thyroid, immune, and stress 

19 axes are also potential targets of endocrine disruption. Toxicological effects are also observed 

20 with many EDCs; measures including activation of ethoxyresorufin-O-dealkylase (EROD) are 

21 indicative of the liver detoxification processes and serve as excellent indicators of exposure. 

22 Elegant studies have also shown that subtle differences in the sequence of the aryl hydrocarbon 

23 receptor contribute to observed variations in species sensitivities to many of these compounds 

24 (Cohen-Barnhouse et al. 2011; Farmahin et al. 2012a; Head et al. 2008). Behavior appears to be 

25 a sensitive measurement endpoint; embryonic exposure by egg injection to known concentrations 

26 of EDCs or steroid hormones can reduce or eliminate male sexual behavior in male quail as well 

27 as impair other physiological responses. Behavioral measures also provide useful indicators of 

28 effects, including sensory deficits, stress responses, and impaired reproductive function (Sefton 

29 and Siegel 1973; Benoff and Siegel 1976; Cunningham et al. 1977; Cohen-Parsons et al. 1973).

30 A great deal of research has been conducted on sexual differentiation, with studies of 

31 hormone and EDC manipulations on development and the reproductive axis (Adkins-Regan et al. 

32 1994; Balthazart et al. 2009; Cohen-Barnhouse et al. 2011; Ottinger and Dean 2011; Schweitzer 

33 et al. 2013). These studies have shown that the male Japanese quail is exquisitely sensitive to 

34 exogenous steroids, with both estradiol and androgen treatments resulting in impaired male 

35 reproductive behavior (Adkins-Regan et al. 1982; Balthazart et al. 2009; Halldin et al. 1999; 

36 Schweitzer et al. 2012). As such, estradiol is very useful as a positive control and as a means of 

37 comparison for relative activity when considering endocrine, neural, and behavioral components 

38 of reproduction (Abdelnabi et al. 2000; Berg et al. 1998; Halldin 2005; Halldin et al. 1999; 

39 Panzica et al. 2007; Watson et al. 1990; Yamashita et al. 2011). There is a range of effects of 

40 embryonic exposure to estradiol within the context of examining EDC impacts, especially when 

41 directly compared to trenbolone (androgen active EDC) at specific points in embryonic 

42 development (Quinn Jr et al. 2007; Quinn et al. 2008; Quinn et al. 2007). It is crucial to have an 

43 avian model to assess potential adverse effects of EDC exposure to birds. An avian 

44 multigenerational test will provide some assurance for potential impacts to birds.
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1 2.3 Purpose of the Japanese Quail Two-generation Test

2 The JQTT is designed to assess potential risk posed by EDCs to birds. As such, an avian 

3 model must be responsive to EDCs, exhibiting sufficient sensitivity especially in characteristics 

4 unique to birds. The JQTT provides a multi-generation testing protocol that contains each 

5 primary phase in the life cycle with isolated exposure at that phase as well as cumulative 

6 exposure over the multiple life stages. Initial exposure in the current testing protocol occurs in 

7 proven breeders followed by continuous exposure to F1 offspring to evaluation of their F2 

8 offspring as the final step in the testing protocol.

9 The OECD Avian Two-generation Toxicity Test Draft Guideline (Appendix D, 2011)

10 includes a range of measurements intended to detect general toxicological endpoints including 

11 lethality, gross morphology, growth and body weight, organ weights, and additional measures 

12 indicative of endocrine disruption. The general list of endpoints includes: measures of health and 

13 overt toxicity (food consumption, body weights, secondary sex characteristics, activity, chick 

14 health, growth and maturation, lethargy, morbidity or other clinical signs of toxicity, mortality); 

15 reproductive measures (sexual differentiation, sexual maturation, egg production, egg quality, 

16 fertility, embryo death, hatchability, chick survival and condition, sex ratio, reproductive 

17 behavior and reproductive hormones, testes histology and sperm counts, cloacal gland size and 

18 foam); endocrine and physiological endpoints (weight and histology of liver, testes, thyroid, 

19 adrenals, gonads, and brain, fecal steroid hormones, serum hormones and vitellogenin, glandular 

20 thyroid hormone). In the avian multigeneration test, evidence of endocrine disruption also 

21 informs us with critical insights into impacts of EDCs for birds at different stages in the life 

22 history.

23 2.4 Overview of Japanese Quail Biology & Reproductive Endocrinology

24 2.4.1 Altricial versus precocial birds: Development Patterns and Vulnerable Stages

25 Two developmental strategies occur in birds: altricial chicks require extensive parental 

26 care whereas precocial chicks are well developed at hatch. The song system is steroid dependent 

27 with elements that are sexually differentiated primarily post-hatchand remains plastic throughout 

28 the life cycle, showing seasonal regression and hormone induced seasonal neurogenesis (Adkins29 Regan et al. 1990; Adkins-Regan and Ascenzi 1990; Adkins-Regan et al. 1994; Smith et al. 1997; 

30 Fusani and Gahr 2006; Wade and Arnold 2004; Ball and Balthazart 2010). This neuroplasticity 

31 contributes to an apparent lifelong vulnerability yet potential resilience to EDC exposure 

32 (Iwaniuk et al. 2006; Markman et al. 2008; Quaglino et al. 2002). Some studies in passerines 

33 have considered the song control nuclei as a measure of potential impacts of steroids and EDCs 

34 on singing behavior (Adkins-Regan and Ascenzi 1990; Adkins-Regan et al. 1994; Corbitt et al. 

35 2007; Iwaniuk et al. 2006; Rochester et al. 2010; Rochester and Millam 2009). Adverse effects 

36 of EDCs have also been observed in finches and the dark-eyed junco (Hoogesteijn et al. 2008; 

37 Iwaniuk et al. 2006; Rochester et al. 2010; Rochester and Millam 2009; Satre et al. 2009; Touart 

38 2004). The JQTT would provide information about impacts in an avian model; positive effects 

39 would indicate potential risk to songbirds that may be more sensitive as adults.

40 Avian species rely on genetic based sexual differentiation with well characterized 

41 developmental events occurring during ontogeny for both precocial and altricial birds. Moreover, 

42 the genetic basis for gender is reversed from that of mammals in that the male is the 

43 homozygotic sex (ZZ) and the female is the heterozygotic (ZW). Current research provides more 
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Avian Two-Generation ISR 12 May 28, 2013

1 insight into the actions of these gender-determining genes and provides further indication that 

2 avian species have unique adaptations governing sexual differentiation (Itoh et al. 2010). 

3 Interestingly, birds appear to use this sex determining system slightly differently than mammals, 

4 with relatively higher expression of the Z gene in the male, but less dose related adjustments of 

5 expression between males and female (Smith et al. 2009). This would be translated into making 

6 male/female gender differences less marked than in mammals and in terms of EDC effects, the 

7 impacts of EDCs may be more global across both males and females than in mammals, at least 

8 relative to gender specific effects. A further consideration is the sexual differentiation of the song 

9 system and reproductive axis in songbirds, which adds to the complexity of interpreting EDC 

10 effects. As will be discussed below, songbirds hatch in a more immature state and appear to 

11 complete sexual differentiation post hatch. They also appear to retain greater neuroplasticity 

12 throughout their life with seasonal recrudescence of reproductive and song systems. Precocial 

13 birds ultimately appear to be more vulnerable during embryonic development because they hatch 

14 in a more mature state, having undergone sexual differentiation and having more functional 

15 endocrine systems. Consequently, precocial birds appear to be more vulnerable to EDC impacts 

16 if exposed during embryonic development, with potential long-term effects over their lifespan.

17 As such, precocial birds provide an excellent avian model that will detect EDC effects on sexual 

18 differentiation. Moreover, effects of steroid hormones and a number of EDCs have been well 

19 characterized in quail.

20 Precocial Birds – Galliforms and in particular Japanese Quail

21 Precocial birds have often been the subject of toxicological studies and regulatory testing 

22 (Touart 2004). The domestic chicken is a known sensitive model commensurate with the 

23 structure of the aryl hydryl carbon receptor characteristic of species that are very responsive to 

24 dioxin like compounds (AhR; Cohen-Barnhouse et al. 2011; Farmahin et al. 2012a; Farmahin et 

25 al. 2012b; Head et al. 2008). The EPA Avian Reproduction Test (OSCPP 850.2300; US EPA 

26 2012) primarily tests for toxicological effects and overall impacts on reproduction. With the 

27 recognition of the potential for endocrine disruption to pose a hazard to avian species, attention 

28 has turned to evaluating physiological impacts and vulnerability of birds to suspected EDCs. 

29 Precocial birds such as Japanese quail (Coturnix japonica) are well developed at hatch, with 

30 sexual differentiation of the reproductive endocrine and functional competency of other 

31 physiological systems already relatively complete. Consequently, if precocial embryos are 

32 exposed to EDCs during the more limited sensitive period during sexual differentiation, there 

33 may be both short- and long-term impacts on the individual (Ottinger et al. 2002; Panzica et al.

34 2007). As discussed in the following section, the hormones deposited by the hen as well as 

35 embryonically produced hormones direct sexual differentiation. 

36 During development, each level of the reproductive (hypothalamic-pitutitary-gonadal, or

37 HPG) axis develops and begins to function as early as embryonic day 5 (gonadal differentiation), 

38 embryonic days 12 to 14 (hypothalamus and pituitary gland), and later in embryonic 

39 development (accessory structures). The HPG axis initiates function midway in embryonic 

40 development in both chickens and quail. Observations by Woods and colleagues provided a 

41 detailed profile of developmental events in the domestic chicken. They found that plasma steroid 

42 hormones, including estradiol (E2) and androgen (A), follow sexually dimorphic patterns in the 

43 domestic chicken with circulating androgen levels peaking at 13.5 days of incubation in males, 

44 whereas estradiol levels slowly increase in females until hatching (Woods and Brazzill 1981; 

45 Woods et al. 1989; Woods et al. 1995; Woods and Thommes 1984). The relative concentrations 
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1 of E2 were higher in females, whereas males generally had higher androgen levels relative to 

2 estradiol (Woods and Brazzill 1981). Based on hormonal patterns, the HPG axis became 

3 functional between 11.5 and 14.5 days of embryonic age (Woods et al. 1989).

4 Similar patterns in circulating steroid hormones occur during embryonic development in 

5 Japanese quail (Figures 2-1 to 2-3). Estradiol is a critical element in sexual differentiation in 

6 quail. During embryonic development, females have relatively high concentrations of 

7 estradiol/androgen, whereas males have a relatively low estradiol/androgen ratio. Moreover, 

8 there are sex related differences in the patterns of plasma steroid levels during embryonic 

9 development. In female embryos, plasma E2 rose until hatch and decreased, post-hatch (Ottinger 

10 and Abdelnabi 1997; Ottinger et al. 2001). In males, plasma androgen peaked at embryonic days 

11 14 to17 (ED14-ED17), during the 17-day incubation period, and declined post hatch (Ottinger 

12 and Bakst 1981). In addition, yolk steroid hormone content reflects embryonic steroid hormone 

13 levels. These steroid hormones are present and available to the embryo during sexual

14 differentiation of multiple organ and endocrine systems throughout incubation. Once the 

15 embryonic gonads and adrenal glands begin to produce steroid hormones, circulating 

16 concentrations rise to make the gender-related pattern in changes more evident, especially during 

17 the last half of embryonic development (Ottinger et al. 2002).

18

19 Figure 2-1. Stages in Embryonic Development. Diagram shows timing of developmental

20 events during embryogenesis with indications of endogenous endocrine change 

21 and potential periods of vulnerability

22

 E2 & androgen 

Embryonic

Day 1 3 5 8 10 12 15 17 Hatch 

Event cell gonadal GnRH-I cells behavior HPG axis piping

 differentiation development migrate differentiation function

 

Effects of (alter gonadal (affect GnRH-I (sexual differentiation

Treatments differentiation) system) of hypothalamic neural systems)
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1

Figure 2-2. Yolk androgens during 

the first half of 

embryonic development 

in Japanese Quail

Figure 2-3. Yolk estradiol during the 

first half of embryonic 

development in 

Japanese Quail

2

3 As discussed above, the pattern of changing embryonic steroid hormones differs in males 

4 and females; both genders experience increased steroid hormones after embryonic days 10 to 12 

5 in males and embryonic days 10 to 16 in females. Sexual differentiation of endocrine and 

6 behavioral components of reproduction occur during this time, organizing the HPG axis and 

7 gender specific behaviors (Adkins 1975; Adkins-Regan and Watson, 1990; Ottinger et al 2005). 

8 There is a large literature that links steroid hormones, brain regions that modulate reproductive 

9 behavior, onset of reproduction during maturation, and adult reproductive function (Ottinger and 

10 Brinkley 1978; 1979a,b; Halldin et al 2003; Ottinger et al 2003; 2005). The primary 

11 hypothalamic hormone that regulates the HPG axis is gonadotropin releasing hormone-I (GnRH12 I), which is produced by the cell bodies located in the pre-optic septal region of the 

13 hypothalamus. Hypothalamic GnRH-I levels rise between embryonic days 10 and 15, a sharp 

14 drop that appears to be associated with activiation of the function of the HPG axis and feedback 

15 regulation of GnRH-I (Li et al. 1991). Both the endocrine and behavioral components of 

16 reproduction in Japanese quail are sexually differentiated during embryonic development (see 

17 details below).

18 In concert with the changing steroid hormones during embryonic development, the 

19 adrenal axis and the thyroid axis develop and achieve function. Circulating concentrations of 

20 adrenal and thyroid hormones rise during embryogenesis to peak at about the time of hatching in 

21 precocial birds, with a slight delay in this pattern for altricial birds. Additionally, the steroid 

22 hormones circulating during embryonic development also impact the immune system, with 

23 circulating testosterone in males inducing regression in the bursal tissue in males (Fernie et al. 

24 2005a; Grasman 2010; Grasman et al. 2013). Clear effects of EDCs have been observed for the 

25 immune system, especially for the response of the bursa, which is exquisitely sensitive to steroid 

26 hormones during embryonic development. Additionally, the thyroid axis has been well 
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1 documented as a primary target for many EDCs, including PCBs, flame retardants, and other 

2 compounds. As will be discussed below, some of these effects are exerted during embryonic 

3 development, and the thyroid axis is involved in hatching as well as in overall metabolic function 

4 (McNabb and Fox 2003).

5 Maternal Deposition of Steroids and EDCs into the Egg is a primary mode of exposure 

6 to the avian embryo. There is increasing recognition of the important role of maternally 

7 deposited steroid hormones, as well as corticosterone and thyroid hormones (Almasi et al. 2012; 

8 Hayward and Wingfield 2004). Mounting data point to both direct and epigenetic effects of 

9 maternally deposited hormones, which vary according to the condition of the female, health, 

10 reproductive status, and stressors. These steroids can affect the viability as well as developmental 

11 characteristics of the chicks, as discussed above, and there is an extensive literature pertinent to 

12 this topic (Lipar et al. 1999; Schwabl 1993, 1996a, b). Deposition of EDCs is layered on top of 

13 these endogenously produced hormones from the hen. Moreover, most maternally transferred 

14 compounds and EDCs are lipid soluble, thereby deposited primarily into the egg yolk, whereas, 

15 water soluble compounds such as atrazine may be more evenly distributed throughout the egg 

16 compartments. There is significant deposition of EDCs into the eggs of field birds (CM Custer et 

17 al. 2010a; CM Custer et al. 2010b; TW Custer et al. 2010a, b). Similarly, laboratory studies have 

18 demonstrated that EDCs, including exogenous estradiol, methoxychlor, and soy phytoestrogens 

19 (Figure 2-4), all readily transfer from the hen into the egg and partition in the egg compartment 

20 according to lipid solubility (Adkins-Regan et al. 2005; Lin et al. 2004; Thompson et al. 1967).

21

22

23 Figure 2-4. Maternal deposition of the phytoestrogen, genistein by the quail hen into eggs

μg genistein/egg yolk

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

supplementation

post supplementation

day

Genistein supplementation

Genistin supplementation

Control (placebo)

Concentrations of Genistein in Japanese Quail egg yolks supplemented with genistein (aglycone), 

genistin (glucoside) or placebo capsules. Data points represent average of 4 replicates for treatment

groups and 2 replicates for the control group. Modified from Lin et al, 2004. 

μg genistein/egg yolk

0

0.5

1

1.5

2

2.5

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

supplementation

post supplementation

day

Genistein supplementation

Genistin supplementation

Control (placebo)

Genistein supplementation

Genistin supplementation

Control (placebo)

Concentrations of Genistein in Japanese Quail egg yolks supplemented with genistein (aglycone), 

genistin (glucoside) or placebo capsules. Data points represent average of 4 replicates for treatment

groups and 2 replicates for the control group. Modified from Lin et al, 2004. 
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1 2.4.2 Post Hatch Growth and Maturation

2 Steroid hormones continue to decline from the late embryonic and post hatch levels 

3 associated with sexual differentiation to remain at low quiescent concentrations until the onset of 

4 sexual maturation. During sexual maturation, reproductive endocrine and behavioral responses 

5 are initiated earlier in males so that they mature by 6 to 8 weeks of age (Ottinger and Brinkley 

6 1978, 1979a, b). Females begin to mature slightly later than males with onset of egg production 

7 by 8 to10 weeks of age. This dimorphism is reflected in hypothalamic GnRH-I levels, which 

8 increase in males earlier than in females (Li and Ottinger 1988). Organizational effects of 

9 steroids in quail are also reflected morphologically in the sexually dimorphic preoptic region of 

10 the quail, which is larger in males and testosterone sensitive in the adult; high levels of 

11 aromatase metabolize testosterone to estradiol, which is essential for sexual behavior (Panzica et 

12 al. 1996; Watson and Adkins-Regan 1989a, b). Because this area in known to be important in 

13 modulating courtship and mating behavior, a number of studies have focused on the neural 

14 systems contained in this area in Japanese quail; this area is potentially vulnerable to the effects 

15 of EDCs (Balthazart et al. 2003; Brunstrom et al. 2009; Ottinger et al. 2008). In addition, the

16 preoptic-septal region (POA-SL) contains many of the gonadotropin releasing hormone (GnRH) 

17 cell bodies; these cells project to the median eminence of the hypothalamus, making the POA-SL 

18 critical in the regulation of both endocrine and behavioral components of reproduction. Not 

19 included in the diagram is the newly discovered gonadotropin inhibiting hormone, which may 

20 prove to be a potent regulator of reproductive function, especially in seasonally breeding birds 

21 (Tsutsui et al. 2013). All these characteristics of the HPG axis (Figure 2-5) and sexually 

22 dimorphic neuroendocrine systems are vulnerable to EDC exposure, especially during 

23 organization of the reproductive axis during embryonic sexual differentiation.

24

25 Figure 2-5. Diagram of the reproductive axis in Japanese Quail

26

Hypothalamus

 GnRH-I

Pituitary Gland

 LH, FSH

 Gonads

androgens, estrogens,

 progestins

The Hypothalamic-Pituitary-

 Gonadal Axis

+

+
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1 Steroids or EDC treatment during embryonic development reveals a number of long-term 

2 impacts on the timing of sexual maturation, male sexual behavior, female ovulatory cycle, and 

3 metabolic endocrine systems. Specifically, early steroid exposure or treatment with fadrozole or 

4 tamoxifen also affects gonadal development, producing defeminization of the ovary and 

5 accesssory structures (Elbrecht and Smith 1992; Perrin et al. 1995; Rissman et al. 1984; Stoll et 

6 al. 1993). Some long-term studies of embryos treated with steroids or EDCs have examined 

7 mechanisms of the observed impacts (Schweitzer et al. 2012). These studies have shown that 

8 treatments altered neural systems, specifically those that regulate reproductive, immune, and 

9 potentially metabolic function (Ottinger and Dean 2011; Quinn Jr. and Ottinger 2006).

10 2.5 Rationale for the Assay

11 The potential impact of environmental chemicals on avian species was discussed at a 

12 National Institute of Environmental Health Sciences symposium, “Estrogens in the Environment” 

13 held in Eugene Oregon in 1979. This was followed by publication of potential endocrine effects 

14 on reproductive as well as thyroid systems (Rattner et al. 1986; Rattner et al. 1984). Subsequent 

15 meetings have provided detailed documentation of substantial risk to avian populations from the 

16 challenges of environmental chemicals with thorough review of existing information and 

17 progress in characterizing potential impacts (Ankley et al. 1998; Kavlock et al. 1996; Hotchkiss 

18 et al. 2008).

19 There are compelling reasons for having an avian test in Tier 2 of the EDSP. The 

20 Japanese quail provides an advantageous model compared to other avian models; this species is a 

21 well-characterized laboratory model that matures rapidly, shows moderate sensitivity to EDCs.

22 Wild type strains retain photoperiodicity, and are responsive to EDCs that impact reproductive, 

23 thyroid, and adrenal axes. Birds have unique characteristics (see Damstra et al. 2002; Scanes and 

24 McNabb 2003), including the following:

25  Metabolic systems

26 o High body temperature (105oF) with high metabolic rate

27 o Associated accelerated toxicokinetics

28 o Migratory-associated energy drain and mobilization of lipid reserves

29 o Thyroid system function in precocial and altricial birds

30  Reproductive system

31 o Female has one functional ovary

32 o Altered gonadal differentiation results in ovotestes

33  Hormones and behavior

34 o Males adversely impacted by xenoestrogens and androgenic compounds

35 o Female behavior (receptivity) less sensitive to xenoestrogens

36  Sexual differentiation

37 o Males are the homogametic sex having ZZ; females are ZW

38 o HPG axis relies on relative exposure to estradiol and testosterone

39  Males―primary exposure to testosterone

40  Females―primary exposure to estradiol

41 o HPG axis and song system differ in precocial and altricial birds

42  Growth and migration

43 o High metabolism requires sufficient nutrient utilization
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Avian Two-Generation ISR 18 May 28, 2013

1 o Rapid growth rate especially for migratory species

2 o Thyroid system critical for pre-migratory fattening

3  Lifespan

4 o Long-lived birds produce few offspring annually over many years.

5 The EPA Avian Reproduction Test (OSCPP 850.2300; US EPA 2012) provided guidance 

6 for determining potential impacts on birds. This standard evaluation procedure (SEP) focused on 

7 basic reproductive characteristics in birds approaching their first breeding season with birds in 

8 harem groups in pens. Test measurements included egg production, eggshell quality, fertility, 

9 hatchability, 14-day viability, mortality, and gross morphology. The previous Reproductive 

10 Toxicity Test often used the mallard duck (Anas platyrhynchos) and northern bobwhite quail 

11 (Colinus virginianus) as test species. However, both species require at least 6 months to mature, 

12 thereby requiring an extended period of time for a multigeneration test.

13 Subsequent to this test, the OECD Avian Toxicology Expert Group produced a white 

14 paper that summarized key issues for assessing potential adverse effects of EDCs in birds 

15 (Bennett et al. 2001). In addition to articulating the unique characteristics of avian species, 

16 concern was expressed that the Tier 1 screening assays would not detect compounds hazardous 

17 to birds. Although the wide span of vertebrate classes in Tier 1 are intended to detect the 

18 potential of chemicals to interact with the endocrine system across species, a Tier 2 avian test 

19 was proposed as a critical test to protect birds and ensure detection of EDCs that particularly 

20 target vulnerabilities in birds. An OECD draft Guideline for Testing of Chemicals—Avian Two21 generation Toxicity Test in the Japanese Quail was revised in September 2011 and continues to 

22 be the latest working document for harmonizing a multi-generational avian toxicity test. The 

23 discussion will encompass the strengths and weaknesses of this test; specific recommendations 

24 will also be made for suggested changes to the protocol that would increase the reliability and 

25 sensitivity of the test.

26 The JQTT includes four key life stages that are potentially vulnerable to endocrine 

27 disruption. These stages include embryonic development, post hatch growth and development, 

28 sexual maturation, and adult. This test protocol excludes lifetime impacts, which are beyond the 

29 scope of a practical testing paradigm due to time constraints. In the future, models of adverse 

30 effects of EDCs to wildlife populations should also consider the factor of lifetime reproduction 

31 as one aspect of overall fitness, both for the individual and the population as a whole.

32 The unique characteristics of birds include the genetic basis of sexual differentiation, 

33 precocial and altricial developmental patterns, sexual differentiation of the song system versus 

34 the HPG axis, high metabolic rate accompanied by higher body temperature, long lifespan in 

35 many species, and a broad range of reproductive strategies. It is critical to determine if EDCs 

36 have adverse effects in birds by testing these compounds in a model avian species. Japanese 

37 quail represents an advantageous laboratory species because there is a great deal of available 

38 literature on the biology and endocrinology of this species. There are many strains; the outbred, 

39 random colonies retain most characteristics of the wild type population and maintain 

40 responsiveness to photoperiodic regulation of reproduction and a high level of productivity when 

41 on stimulatory environmental conditions. Further, this species does well in captivity and is easily 

42 studied. These characteristics are important to optimize detection of the adverse effects of 

43 potential EDCs.
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1 3 PROTOCOL DEVELOPMENT

2 The sections below have been taken from the OECD Guideline for Testing of Chemicals3 Avian Two-generation Toxicity Test in the Japanese Quail (Draft 2011, Appendix D).

4 3.1 Summary of the Japanese Quail Two-generation Test Protocol

5 The purpose of this guideline is to describe a laboratory testing procedure for evaluating 

6 the impact of pesticides and other chemicals upon avian health and reproduction. Data collected 

7 from such tests may be used in the assessment of risks to birds exposed to pesticides and other 

8 chemicals under field conditions. These data from a two-generation test are principally intended 

9 to evaluate the adverse consequence of exposure to a possible endocrine disruptor to the 

10 reproductive viability of F1 birds, but may also provide information to confirm a chemical as an 

11 endocrine disruptor.

12 The test is designed for dietary exposure, but also may include exposure through drinking 

13 water if more appropriate for the chemical. This guideline describes a two-generation test. The 

14 test monitors health and reproductive parameters in Japanese quail (Coturnix japonica) adults 

15 and their progeny, including viability of second generation offspring.

16 The number and condition of second generation (F2) survivors per F1 generation hen is a 

17 measure of impact to primary. oocytes and follicles produced by F1 females, who have been 

18 exposed during embryonic development and through dietary exposure. Because F2 birds are only 

19 on control diet, their viability, morphological, endocrine, and behavioral measures reflect

20 embryonic exposure.

21 3.2 Study Design, Test Chemicals and Dose Selection

22 Birds in the parent (F0) generation are paired at 4 weeks of age, and acclimated for 2 

23 weeks prior beginning chemically treated diet. Dosing begins in the F1 generation at hatch. 

24 Parameters for adult health (F0 and F1), egg production (F0 and F1), and offspring health (F1 

25 and F2) are evaluated by making statistical comparisons between treated groups and the control 

26 group. The no observed effect concentration (NOEC) and the lowest observed effect 

27 concentration (LOEC) are determined for health, fitness, and endocrine parameters.

28 Care should be taken to ensure that birds are drinking water as they transition from group 

29 pens to the breeder pens. An acclimation period of at least 2 weeks is required for the F0 

30 generation prior to the start of the test. The test begins with the administration of treated diet (i.e.,

31 when birds are 6 weeks old, at the earliest). Breeding pairs should be maintained in a 16:8 or 

32 17:7 light:dark photoperiod for the duration of the study. Onset of egg laying can take place 

33 during the acclimation period.

34 Prior to the start of treatment, individual F0 generation birds are weighed, sexed 

35 (preferably, using genetic techniques), and impartially allocated to pens and treatment groups. 

36 Birds are paired by known lineage such that males from one line are paired with females from 

37 another line. Pairs should be observed for signs of excessive aggression, and if present the sexes 

38 separated except for periodic conjugal visits. A translucent separator wall may be used for this 

39 purpose in each pen. Otherwise, the pairs will be housed together in the same pen. When 

40 together, signs of excessive aggression should be monitored and the males and females separated 

41 if injury is imminent. The number of pairs allocated to each group (for example 16) should be 
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1 sufficient to ensure that there are 12 breeding pairs in the F0 control group that have successfully 

2 produced eggs at the end of the treatment period. Eggs are collected from F0 post-treatment 

week 5 (and possibly, week 8 and/or 9) to examine fertility. Broods from the post-treatment 6th 3

and 7th 4 week are used to establish the first generation offspring (F1). Optimally, at least 6 eggs 

5 are set for each parental pair, with a minimum of 40 eggs must be set for each test level. If during 

the 6th and 7th 6 post-treatment weeks, a treatment level does not produce at least 40 eggs, that 

7 level is dropped from further analysis in the test. At approximately 2 weeks of age F1 control and 

8 treatment chicks are genetically sexed. At 4 weeks of age, F1 control and treatment group birds 

9 are weighed and randomly allocated to pens by pairs with male offspring from odd numbered 

10 parental pens being paired with females from even numbered parental pens and even numbered 

11 pen males being paired with odd numbered females. (The process should be a stratified random 

12 process to assure, to the extent possible, that each of the F0 pairs are contributing genetically to 

13 the F1 breeding population in order to represent individuals that may vary in sensitivity to 

14 chemical exposure.) The F1 generation breeding birds are maintained until 6 to 7 weeks post15 fertility. Eggs collected from F1 pairs during weeks 1 through 5 will be used to evaluate fertility. 

16 Eggs collected from weeks 6 and 7 post-pairing will be used to establish the F2 generation;

17 optimally 6 eggs will be collected from each F1 breeding pair. F2 offspring survival will be 

18 recorded on day 14, and if there has been evidence of endocrine disruption in the F1 generation 

19 or F2 hatchlings, then the F2 offspring observations will be extended through sexual maturation.

20 Dietary concentrations

21 The concentration of the test substance in the diet is expressed as weight of the test 

22 substance per unit weight of diet with known water content: mg/kg of diet (or weight of test 

23 substance per weight of water when birds are exposed through drinking water). The 

24 concentration should also be expressed as mg/kg body weight per bird per day.

25 Dietary concentrations of the test substance should be chosen on the basis of 

26 toxicological data from a range-finding study or other preceding avian tests. Information may 

27 also be gained from tests with rodents or other mammals. The highest concentration should be a 

28 level that is expected to reveal significant effects on adult health or reproductive parameters. If 

29 no significant effects are expected, then the highest concentration to be tested should be the 

30 expected concentration of the chemical in the environment with the addition of a safety factor 

31 (e.g., 5X). However, the highest dose administered should not cause mortality or other severe 

32 signs of parental toxicity that will preclude the evaluation of reproductive parameters. The lowest 

33 concentration tested should be chosen so that no effects are seen on adult health or reproductive 

34 parameters. Intermediate concentrations should be geometrically spaced between the highest and 

35 lowest doses. A minimum of four concentrations and a control are required for the definitive test.

36 A range finding test can be performed before initiating the main study to aid in 

37 establishing test concentrations and to evaluate any potential avoidance effects.

38 3.3 Preparation and Monitoring of the Test Diet

39 To prepare the test diets, the appropriate amounts of test substance are mixed into the diet. 

40 The mixing method should be developed so as to obtain a homogeneous distribution of test 

41 substance in the diet. The use of a premix is advisable.

42 If necessary, a vehicle of negligible toxicity (e.g., food grade corn oil, water, etc.) may be 

43 used to ensure a uniform distribution. Acetone may be used to dissolve a test substance, provided 
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1 it is allowed to evaporate before feeding the diet to the birds. The amount of vehicle used should 

2 be as low as possible and should not exceed 2% by weight of the diet. When used, a constant 

3 amount of vehicle should be added to each test group and the control group diet, in order to keep 

4 the caloric value of the diet equal between dosage groups.

5 The frequency of diet preparation should be chosen so that degradation or volatilization 

6 of the test substance in the diet does not allow the actual concentration to fall below 80% relative 

7 to initial concentration. The frequency of diet renewal should not be more than once a day and 

8 not less than once a week. Residual feed should be visibly inspected for mold and if acceptable, 

9 weighed and mixed with fresh feed supplied to the birds. It may be desirable to keep all feed 

10 frozen until use.

11 Stability of the chemical in feed must be analyzed under typical test conditions prior to 

12 the start of the test or in parallel with the range-finding study. Stability data from previous 

13 (mammalian) feeding studies may be used as guidance, if available. Stability should be verified 

14 during the definitive reproduction test. In order to check stability of the test substance under test 

15 conditions, feed should be sampled and analyzed from the feed hoppers at the end of the first 

16 feeding period, i.e., before diet in the feeding bowls is renewed, and again at the end of the last 

17 feeding period. A sufficient number of samples should be taken into account for variability.

18 Homogeneity of the test substance in the diet may be evaluated prior to the test or 

19 samples for homogeneity measurements may be taken at the first mix prepared for the study.

20 To verify test concentrations, samples of diets fed to the birds should be taken every time 

21 new diet is mixed during the treatment period to allow measurement of the actual concentration 

22 of the test substance.

23 Both F0 and F1 generations are maintained on treated diet until test termination. All F2 

24 chicks are maintained on untreated diet.

25 3.4 Equipment and Supplies (Husbandry, Endpoint Measurements, Analyses)

26 Temperature, ventilation, and light controlled facilities are needed throughout the test. 

27 Recommended ventilation is about 8 to 15 air changes per hour. Sound and vibrations or other 

28 disturbances in the rooms should be kept to a minimum to minimize stress.

29 Artificial lighting should approximate the daylight visual spectrum and be automatically 

30 controlled. The photoperiod for adult birds, from the start of acclimation onwards, and the chicks 

31 is 16 or 17 hours light and 7 or 8 hours of darkness. Birds should be exposed to light intensity of 

32 at least 10 lux, measured at the level of the feeder. The light intensity is dictated by the amount 

33 required for daily observations. Light intensity that is too high will encourage aggressive 

34 behavior

35 Incubators and hatchers, preferably with automatic temperature and humidity controls 

36 and an egg-turning device, are necessary. In addition, suitable equipment is required to maintain 

37 stored eggs within the temperature and humidity ranges specified..

38 Suitable pens of appropriate size for adults and for chicks are required. Wire pens with 

39 slanting floors and egg-catchers or other measures to prevent breakage of eggs, are 

40 recommended for adults. Pens for both adults and chicks should preferably be of stainless or 

41 galvanized steel or other inert materials. When using wire meshing for the floors, the wire mesh 

42 should be of a size sufficient to prevent foot injury (trauma or pododermatitis) but large enough 
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1 to allow excreta to drop through. Measures should be taken to minimize spillage of diet (e.g., 

2 covering the food troughs with a wire grid).

3 Birds should be placed in breeder pens at 4 weeks of age, and then housed in opposite sex 

4 pairs (one male/one female). The parental cages should be distributed in such a way that cross5 contamination and positional effects are avoided. Pen mate aggression can be a problem and 

6 measures must be taken to minimize injuries, stress, and mortality resulting from such aggression. 

7 Pairs should be reintroduced daily to maintain fertility.

8 For rearing chicks, the use of rearing pens with thermostatically controlled warm 

9 compartments or cages, which are free of drafts and have a radiator (e.g., ceramics), is 

10 recommended. Sufficient space for feeding and drinking must be provided, especially during the 

11 first week after hatching, to promote the weaker animals accessing food and water resources. 

12 Chicks should be identified individually or by pen of origin. They may be housed in groups of 

13 approximately equal number, by treatment group. The optimal number of chicks per pen depends 

14 on pen size. Birds must be separated into pairs (one male/one female) when 4 weeks old after sex 

15 has been determined.

16 Guidance for housing conditions is provided in Table 3-1.

17 Table 3-1. Housing Conditions.

Age

(week)

Temperature

(°C)

Relative Humidity

(%)

Minimum Floor Area

(cm2/bird)

1 35-38 40-80 50

2 30-35 40-80 75

3-4 23-27 40-80 100

>4 16-27 40-80 625

Note: The acceptability of housing conditions of adults will be evaluated on the basis of results of reproductive 

performance.

18

19 3.5 Feeding

20 Diet and drinking water are provided ad libitum. The diet should be described and should 

21 meet the specific nutrient requirements of Japanese quail. The caloric contents of the diet must 

22 be reported. If the same diet is used for chicks and adults, extra calcium should be added for the 

23 adult diet. During the treatment period, birds are fed basal diet mixed with the test substance at 

24 specified concentrations.

25 Guidance for recommended nutritional values is provided in Table 3-2.

26 Table 3-2. Recommended Nutritrional Values.

Nutrient

Adults (> 4 weeks)

Recommended Range (%)

Chicks

Recommended Range (%)

Crude protein 27 to 29 27 to 30

Crude fibre 3.5 to 5.0 3.0 to 6.0

Crude fat 2.5 to 7.0 5.5 to 7.5

Calcium 2.6 to 3.6 0.75 to 1.2

Phosphorous 0.9 to 1.1 0.6 to 1.0

27
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1 3.6 Animals (Species, Sex, Age, Weight, Number)

2 The test species is the Japanese quail (Coturnix japonica).

3 Japanese quail generally start laying eggs at or later than 6 weeks of age. Once egg laying 

4 has begun it will take about 2 to 3 weeks for birds to reach full egg production. Depending on the 

5 strain, eggs will be fertile when birds are about 8 to 10 weeks of age.

6 Birds used in the test should appear healthy and be free of abnormalities or injuries that 

7 may affect test results. Birds should not receive any dietary medications beginning one week 

8 prior to the start of the test and continuing until the test is terminated. All F0 birds used in a test 

9 should be from the same hatch.

10 Birds in the parent (F0) generation are paired at 4 weeks of age, and acclimated for 2 

11 weeks prior beginning chemically treated diet. Dosing begins in the F1 generation at hatch. 

12 Parameters for adult (F0 and F1), egg production (F0 and F1), and offspring health (F1 and F2) 

13 are evaluated by making statistical comparisons between treated groups and the control group. 

14 The no observed effect concentration (NOEC) and the LOEC are determined for health, fitness,

15 and endocrine parameters.

16 3.7 Experimental Design

17 The test species is the Japanese quail (Coturnix japonica).

18 During the treatment period, the parental birds are observed daily to detect any overt 

19 signs of toxicity or other clinical signs. If there is an injury due to adult pairings, this should be 

20 recorded and the breeding pair(s) should be re-established as needed if possible. The following 

21 observations are to be performed on adults during the study:

22  Clinical signs of toxicity and health conditions should be evaluated at least once daily, 

23 during the acclimation and treatment periods. Observations should include mortality 

24 and clinical sign of toxicity such as lethargy, depression, wing droop, ruffled feathers, 

25 lacrimation, runny feces, etc. Any injuries sustained and subsequent treatment should 

26 also be recorded. Clearly moribund individuals or those otherwise in severe distress 

27 should be immediately euthanized.

28  Food consumption (per pair) should be recorded at least weekly as often as food is 

29 replaced in the feeders. Any apparent food spillage should be noted.

30  Body weights should be determined at least at the start of treatment and at the end of 

31 the treatment period. There should be no significant difference in mean body weight 

32 between test groups at the start of the treatment period. If there is a significant 

33 difference between test groups, birds should be again randomized.

34 Observations of secondary sex characteristics, e.g., male/rusty or female/mottled plumage, 

production of the first egg or first foam), cloacal length, should be recorded beginning on the 4th 35

36 week and every week after until distinctly dimorphic traits are expressed (F0 and F1 generations). 

37 If plumage is not distinctly “male” or “female,” note the plumage as “ambiguous.”

38 3.8 Offspring – Eggs

39 Eggs are collected at least once daily, numbered according to pen of origin and stored; 

40 broken eggs are numbered, recorded, and then discarded. The total number of eggs laid per pen 

41 should be accounted for at the end of the study.
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1 The eggs are stored in a cold storage facility, for a maximum of 2 weeks, prior to setting 

2 in the incubator (see Table 3-3). Cool storage prevents embryo development and aids in 

3 synchronizing development of embryos of eggs. Before placing the eggs in the incubator, they 

4 are candled to check for abnormalities and fine cracks that can be identified only by candling. 

5 After removing all cracked and abnormal eggs, the remaining eggs are equilibrated to room 

6 temperature and set in the incubator. All cracked and abnormal eggs should be recorded.

7 Table 3-3. Conditions for Egg Storage, Incubation, and Hatching.

Temperature

(°C)

Relative Humidity

(%) Turning

Storage 13-16 55-75 Optional

Incubation 37.5-37.8 50-70 Yes

Hatching 37-37.5 70-75 No

8

Eggs from F0 and F1 pairs will be collected during the 5th, 6th, and 7th 9 post-pairing weeks. 

Eggs collected during the 5th 10 week will be candled to estimate fertility and embryo viability on 

11 embryonic days (ED) 8 and 15, respectively. On ED 15, eggs will be opened and embryo weight 

12 and sex will be recorded. Tissues will be collected from two ED 15 chicks per pen. In addition, 

13 trunk blood and thyroid glands will be collected for biochemical analyses from two ED 15 chicks 

per pen. If egg production from the 5th 14 week is limited (i.e., < 4 eggs with viable ED15 

embryos/pen are produced), the priority is as follows: 1st embryo for histology, 2nd 15 embryo for 

biochemistry, 3rd embryo for histology, 4th 16 embryo for biochemistry. If the eggs produced during 

17 week 5 are not sufficient (e.g., < 3/pair) to estimate fertility, additional eggs can be collected 

18 from breeding pairs during weeks 8 and 9. F1 birds are maintained on treated diet until test 

19 termination. Eggs collected from weeks 6 and 7 will be set, artificially incubated, and allowed to 

20 hatch to establish the F1 generation. All F2 offspring will be maintained on untreated diet and 

21 survival through day 14 is recorded.

22 The onset of sexual maturity (egg laying or foam production) or absence thereof can be 

23 recorded for F2 hatchlings. The timing of sexual maturity will be considered egg or foam 

24 production in 90% of the control (i.e., birds maintained on an untreated diet) and appropriate 

25 behavioral responses among paired birds. Observations will extend for an additional week 

26 beyond sexual maturity of the control groups to estimate potential pubertal delays among treated 

27 birds.

28 During the study, one egg per pen is collected from odd numbered pens in odd numbered 

29 weeks and from even numbered pens in even numbered weeks to evaluate eggshell strength and 

thickness. If egg production is limited during the 5th (<5 eggs), 6th, and 7th 30 weeks (<7 eggs total 

31 for both weeks), forgo egg collection for shell measurements. Eggshell strength is measured in 

32 Newtons using a strength tester. The egg is placed on its side on the test stand so the 

33 compression head will contact the egg at the equator. To determine eggshell thickness, each egg 

34 will be cut open around the equator and washed out; subsequently the shells are left to dry with 

35 the membrane intact for at least 48 hours at room temperature. Shell thickness is measured at a 

36 minimum of four points around the girth using a calibrated micrometer. Eggshells are measured 

37 to at least 0.01 mm and the mean value calculated per egg. Eggs other than those collected for 

38 production of the next generation (post-treatment weeks 6 and 7), embryonic survival and 

39 fertility (post-treatment week 5, and if necessary post-treatments weeks 8 and 9), and those 

40 collected for eggshell thickness and strength (one egg/pen/2 weeks), can be discarded after 
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1 enumeration and examination for cracks or abnormalities. Egg yolks from eggs broken during 

2 shell measurements can be used for biochemical analyses.

Eggs collected from the F0 and F1 generation during the 5th, 6th, and 7th 3 post-pairing 

weeks will be incubated to assess embryo viability (5th 4 post-pairing week) and establish the next 

generation (6th and 7th 5 post-pairing weeks). Eggs collected from the F1 generation during post6 pairing weeks 1 to 4 will also be incubated through embryonic day 8 to evaluate fertility. 

7 Incubation is best performed when eggs are set in an incubator with temperature and humidity 

8 control and an automatic turning device. From the second day of incubation onwards, eggs 

9 should be turned at least three times per day. If turned by hand, eggs should be turned an odd 

10 number of times a day.

11 Fertility and embryo viability in all incubated eggs are determined by candling the eggs 

12 after 8 days of incubation (ED 8) and 2 days before hatching at embryonic day 15 (ED 15). Eggs 

13 will be transferred to a hatcher. During candling, eggs should not be allowed to cool to room 

14 temperature, since this may delay embryonic development. After hatching, chicks should be dry 

15 before they are taken out of the hatcher. Those chicks that have not hatched within 

16 approximately 24 hours of the majority of chicks hatching should be considered unhatched. No 

17 assistance should be given to chicks during hatching.

18 3.9 Egg Parameters

19 Prior to start of the treatment period, egg production of all F0 generation pairs of birds 

20 available for the study (including potential replacements) should be recorded. These data will be 

21 used to exclude pairs that do not lay eggs from further study. Eggs will be discarded after 

22 candling unless otherwise indicated.

23 During the treatment periods, the following information is recorded:

24  Eggs laid

25  Eggs cracked

26  Eggs broken

27  Description of all egg abnormalities

28  Eggs set

29  Eggshell strength

30  Eggshell thickness

31  Infertile eggs

32  Early viability (ED8)

33  Late viability (ED15)

34  Eggs that hatch

35  Eggs that do not hatch

36  Fertile eggs.

37 If a pair is removed during the study due to death, injury, etc., data from all preceding 

38 weeks are evaluated. If there is an injury due to adult pairings, this should be recorded and the 

39 breeding pair(s) should be re-established as needed if possible. If data from a pen in which 

40 mortality occurred or a bird was injured are used in study results, this should be justified.

41 Typical control values for reproductive parameters in Japanese quail are provided in 

42 Table 3-4. If reproductive parameters of control birds do not meet these values, the test 
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1 procedure and husbandry conditions should be checked for potential problems and this 

2 information should be recorded.

3 Table 3-4. Typical Values for Reproductive Parameters in Japanese Quail.

Parameter Value

Number of eggs laid per hen per day 0.66 to 0.89

Percentage of cracked/broken eggs 0 to 10%

Viability (percentage fertile, live embryos of eggs set) 85 to 96%

Hatchability (percentage hatching of fertile eggs) 70 to 98%

Percentage hatchlings that survive 14 days 85 to 97%

Mean number of 14-day old survivors per hen per day 0.34 to 0.71

Eggshell thickness (mm) 0.19 to 0.22

4 Source: OECD

5 3.10 Chicks

6 The following observations on F1 and F2 chicks are recorded during the treatment 

7 periods:

8  Day of hatch

9  Hatching mortality

10  Hatchling morphology and/or abnormalities

11  Chick body weight at hatching

12  Clinical signs of toxicity/disease

13  Number of 14-day-old surviving chicks

14  Chick body weight at 14 days after hatching

15  Time to reproductive maturation (F2 optional)

16  Phenotypic sex

17  Genetic sex (F0, F1, F2)

18  Mating behavior (F2 optional).

19 3.11 Pathology

20 The following observations on F1 and F2 chicks are recorded during the treatment 

21 periods:

22 Adults - All adults that survive to the end of the treatment period are euthanized with

23 gross pathology assessment. Adult birds that die, or are killed during the course of the treatment 

24 period, will be subjected to the same procedures. Trunk blood will be collected for biochemical 

25 analyses. The left thyroid gland is to be stored for glandular T4 content. Organs including brain 

26 (POM, pituitary, pineal gland), kidneys, liver, adrenal glands, reproductive organs and associated 

27 structures, cloacal gland, bursa, and right thyroid gland should preserved in situ using 

28 Davidson’s fixative for further histopathology. Any morphologic abnormality should be recorded 

29 at the time of necropsy. Fixed organ (or group of organs) weights will be collected when organs 

30 are removed for histopathology.

31 Embryos – For each mating pair, two late-stage embryos (ED 15, 2 days prior to hatch) 

that are not allowed to hatch (i.e., from 5th 32 week post-treatment eggs) are to undergo necropsy 

33 and tissues preserved for histopathology as described above for adults. Trunk blood and thyroid 

34 glands will be collected from two late-stage embryos per pair for biochemical analyses.
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1 Birds (adults or chicks) found in severe distress will be euthanized. If one member of a 

2 pair dies or is euthanized during the treatment period, the other member of the pair is euthanized 

3 as well.

4 3.12 Biochemical/endocrine Measurements and Observations

5 Thyroid hormone measurements – Measurements of thyroid function include thyroid 

6 gland weight, glandular thyroxine (T4) content, and thyroid histology/histopathology. Samples 

7 will be collected from embryonic and adult life stages. Eggs collected from post-treatment week 

8 5 incubated to ED 15, can be used for embryonic measurements. After assessing late embryo 

9 viability 2 days prior to hatch, the embryo is removed from the shell and necropsied. Thyroid 

10 glands are dissected. Fixed thyroid glands for histology will be collected from two embryos and 

11 unfixed thyroid glands for biochemistry will be collected from two embryos for each male12 female pair. In addition, during necropsy of the adult Japanese quail, thyroid glands are dissected 

13 and the left thyroid gland will be used for glandular T4 analysis and right gland used for 

14 histological assessment as above.e following observations on F1 and F2 chicks are recorded 

15 during the treatment periods:

16 Steroid hormone measurements - Non-invasive procedures are preferable for periodic 

17 measurements of estradiol and testosterone in developing birds, adults, and eggs. One approach 

18 for measuring steroid hormones in the developing birds and adults is through fecal/urate material. 

19 Steroids are to be measured from fecal/urate material using radioimmunoassay (RIA) starting 

20 Day 1 of adult pairing, and weekly thereafter. Adults are housed in pairs, and the fecal/urate 

21 material shall be linked to the pen. Steroid hormones shall be analyzed in trunk blood collected 

22 from two late-stage embryos per male-female pair, from blood samples collected from all 

23 animals at necropsy, and from egg yolks resulting from eggshell thickness/strength measures.

24 Vitellogenin measurements – Vitellogenin is a protein that is generally controlled by 

25 estrogen. It is present in females of oviparous species during egg development. It is not 

26 expressed in normal males due to lack of estrogen; however in males that have been exposed to 

27 estrogenic contaminants, it is expected that vitellogenesis will be stimulated. Vitellogenin is to 

28 be measured from trunk blood of the late embryos used for steroid and thyroid hormone analyses 

29 and from adults at necropsy, when appropriate, to evaluate estrogenic potential of a test chemical.

30 3.13 Behavioral Measurements

31 Males – Initiation of normal male mating behavior is important for successful 

32 reproduction. Mating behavioral observations will be conducted, preferably beginning at the 

33 onset of maturation, on males of the F1 (F2, optional) generation. Trials shall be performed to 

34 assess mating behaviors including time to mount attempts, number of mount attempts, and 

35 number of successful cloacal contacts. Details on the behaviors and the background of the testing 

36 protocol can be found in Adkins (1972) and in Ottinger and Brinkley (1978). Behavioral 

37 observations shall be conducted on naive birds raised singly or in same-sex pairs. At sexual 

38 maturity (i.e., egg laying in females and foam production in males), 7 naive male-female pairs 

39 will be introduced and behavior will be observed during three-minute trials for three consecutive 

40 days. Trials may be truncated after successful mating or if bird aggression becomes an issue. At 

41 the conclusion of the trial, birds will be returned to their single or same-sex housing. The same 

42 individuals shall be re-introduced for three consecutive days or until successful mating is 
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1 observed. If behavioral observations of the F1 generation suggest a treatment-related effect, 

2 observations may be extended to the F2 generation.

3 Handling-related disturbance during introduction and separation of birds involved in 

4 behavior observation trials may perturb egg production, and therefore, observational data should 

5 be collected from distinct pairs not used in collection of other data endpoints (e.g., egg 

6 production, fertility, etc.).
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1 4 BACKGROUND AND RATIONALE FOR SELECTION OF ENDPOINTS

2 4.1 Endpoints

3 The selection of endpoints depends on the dosing regimen (i.e., amount of compound 

4 administered and the timing of administration), projected dose-response, the window of exposure 

5 (i.e., life-stage), projected meachnisms of action, potential endocrine activity or induction of 

6 vitellogenin or other biochemical responses, and mode of exposure, all of which are critical 

7 variables in evaluating potential EDCs. As more information on EDC actions becomes available, 

8 it will be possible to use the structure proposed in the framework offered by the Adverse 

9 Outcomes Pathways to help elucidate both the targeted physiological systems vulnerable to a

10 chemical/chemical class and the potential for significant impacts to avian species. This approach 

11 is not currently integrated into the testing paradigm, but may prove useful for providing structure 

12 to interpreting the observations and findings. For example, the developing embryo is much more 

13 sensitive to the effects of EDCs than the adult, and developmental defects (i.e., permanent central 

14 and peripheral disruption) often persist into maturation and adult life stages. Conversely, adult 

15 exposure does not generally have as much impact, or effects appear to be more transient, 

16 especially in precocial species (Ottinger et al. 2008). Advances in our understanding of 

17 molecular mechanisms have revealed that EDCs often exert epigenetic effects. This has changed 

18 our understanding of both the activational and organizational effects of EDCs, allowing for the 

19 possibility that exposure of the F0 (or the developing F1) generation can cause lasting effects in

20 multiple generations (Skinner et al. 2011). Therefore, it is extremely important to understand the 

21 physiology of the model (i.e., Japanese quail) and the species-specific endocrine mechanisms 

22 behind the selection of endpoints.

23 4.1.1 Survival

24 Survival is a basic endpoint for acute and chronic toxicity testing of compounds. 

25 Determining the median lethal dose that will kill 50% of the population being tested (LD50) is 

26 important for traditional toxicology studies. The OECD Avian Acute Toxicity Test (OECD; 

27 Appendix F) is intended to ascertain the relative toxicity of a compound over a dose-response 

28 curve, using a minimal number of animals that are exposed as adults. The LD50 or LD20 of a 

29 compound provides information for risk assessors to determine the potential hazard from that 

30 compound, which then can be utilized for regulatory purposes. In dietary studies, survival must 

31 be monitored in order to assess health effects and other impacts that may occur over the time of 

32 the study. If there are any deaths of adults or offspring during the experiment, it is important to 

33 determine an apparent cause of death. If unexplained deaths occur during an experiment in the 

34 treated groups, especially in a dose-response fashion, it is possible that the test compound has 

35 acute toxic effects at the doses tested. However, in the context of endocrine disruption studies, it 

36 is unlikely that the doses administered will approach lethal concentrations. Indeed, the nature of 

37 endocrine disruption studies is to determine the sublethal effects of compounds on the endocrine 

38 systems of organisms (Clotfelter et al. 2004). Because only minuscule concentrations of 

39 endogenous hormones (pg-ng/kg amounts; Vandenberg et al. 2012) are required for normal 

40 endocrine function, even small perturbations in the system can cause sublethal effects that can 

41 reduce reproductive function, alter behavior, and/or lead to adult morbidity (Ottinger et al. 2008; 

42 Vandenberg et al. 2012). For example, 50 ug/kg/day of BPA has been determined to be a safe 

43 exposure by classic toxicology testing (Vogel 2009). However doses 100 to 1000 times lower 
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Avian Two-Generation ISR 30 May 28, 2013

1 than that have been shown to induce feminization of the gonads in Japanese quail (with a single 

2 injection into the egg), with no changes in survivability among groups (Oshima et al. 2012). It is 

3 possible that changes in behavior due to EDC exposure as adults or during development could 

4 lead to increased mortality in treated groups, for example, by increasing aggression (Laskey and 

5 Edens 1985; Schlinger and Callard 1989). Thus, it is important to examine causes of death 

6 carefully. Moreover, it is critical to examine impacts on survivors, especially if a dose-response 

7 relationship is observed, in order to reveal potential short- and long-term impacts. Although 

8 survival may not be a sensitive endpoint in terms of EDCs, it should be included in the JQTT as 

9 a basic endpoint of toxic effects. The toxic effects of compounds will vary between avian species 

10 (Head et al. 2008; Meyers et al. 1992) and according to the mode of exposure (Hill and 

11 Soares 1987).

12 4.1.2 Food Consumption

13 Food consumption is a basic measure of the general health; changes in food intake can 

14 indicate acute or chronic toxic effects of a compound. Additionally, if the route of chemical 

15 exposure is through the feed, it is important to monitor the consumption carefully to determine 

16 the exact exposure. Feeders can be weighed (or the amount of food measured) before and after 

17 filling on a daily or weekly basis to determine the amount of food consumed (see above; OECD 

18 Two-Generation Toxicity Test). It is critical to have measurements on the stability of any 

19 treatment compound; this stability may dictate the timing for feeding and changing the feed 

20 being offered to the test subjects. Steps should be taken to minimize mass lost through spillage

21 (via feeder/cage design), and to estimate the amount spilled in order to detect reduced food 

22 consumption. Reduced food consumption is often exhibited in animals in classic toxicological 

23 studies and may be indicative of chemical toxicity. In terms of endocrine disruption, appetite is 

24 regulated by hormonal signaling in humans and animals; chemical signals from the adipose 

25 tissue, gut, and other organs (i.e., leptin, gherilin, insulin) act in brain centers to regulate feeding 

26 (Coll et al. 2007). Food intake regulation in birds appears to be similar to that in mammals

27 (Denbow 1985). Estrogen has been shown to inhibit food uptake (Hirschberg 2012) and 

28 progesterone promotes food intake (Wade 1975). Further, a growing body of evidence points to 

29 EDCs as potential obesogens, indicating that food consumption may be altered by exposure to 

30 certain EDCs (Grun and Blumberg 2009). Reduced food consumption has been associated with 

31 EDC exposure in Japanese quail (Yamashita et al. 2011) and in rodents (Trentacoste et al. 2001). 

32 However, reduced feed intake may not occur until EDC doses are relatively high (Vandenberg et 

33 al. 2012), so the effects on food consumption may have been due to general toxic effects, rather 

34 than endocrine disruption. Because monitoring food intake is relatively easy, and changes in 

35 food consumption (especially in the lower dose groups) could indicate novel actions of EDCs as 

36 disrupting the metabolic endocrine system, this endpoint should be included in the JQTT 

37 protocol. Food intake may vary between strains, diets, and laboratory environments, so these 

38 aspects should be standardized as much as possible, with adequate controls.

39 4.1.3 Body Weight

40 Body weight is closely linked with food intake and overall health, and can be indicative 

41 of toxicological effects. This endpoint is easily measured in Japanese quail, using a weigh box or 

42 cage. Along with general toxic effects, changes in body weight over time can relate to metabolic 

43 system disruption, thyroid axis disruption, and altered growth, all of which may be linked to 

44 endocrine actions (Boas et al. 2009; Park et al. 2006; Swedenborg et al. 2009). For example, 
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1 perchlorate, a thyroid hormone system disruptor, significantly reduced the growth (measured in 

2 body mass) of zebra finches dosed for the first 2 weeks of life (Rainwater et al. 2008). These 

3 birds also exhibited altered begging and fledging behaviors, indicating that other endpoints may 

4 be linked to altered body weight, depending on the mechanism of disruption (Rainwater et al. 

5 2008). Further, altered body weight during hatch/development could indicate eventual adult 

6 morbidity (Siegel 1999), which is an important measurement endpoint in EDC studies. Therefore, 

7 body weight measurements are an important measure in the JQTT protocol, especially at the start 

8 and end of the experiment for each generation tested. In particular, the growth of chicks to 

9 adulthood should be closely monitored, preferably daily, to determine changes in growth rate; 

10 this can be accomplished by measurement of body weight gain. Because the average normal 

11 increase in body weight has been recorded over a variety of strains of Japanese quail, body 

12 weight becomes a reliable indicator of growth and health.

13 4.1.4 Secondary Sex Characteristics (plumage, cloacal gland area/foam production)

14 Males have several sex-specific characteristics that occur in adults and reflect sufficient 

15 circulating levels of testosterone. Japanese quail have a cloacal gland that is androgen dependent; 

16 that is, sufficient circulating testosterone must be present to stimulate the gland to grow larger 

17 through both hyperplasia and hypertrophy, as well as to produce a glycoproteinaceous secretion, 

18 termed cloacal gland foam (Ottinger and Brinkley 1978, 1979b; Seiwert and Adkins-Regan 

19 1998). Measurements of the cloacal gland area have provided a reliable correlated measure for 

20 the sexual maturation and reproductive status of an individual male (Ottinger and Brinkley 

21 1979b). An estimate of the cloacal gland area is roughly approximated through the product of the 

22 length times the width of the gland (Siopes and Wilson 1975). The foam produced can be 

23 estimated on a scale of 1 to 5 or a similar ordinal subjective measurement scale. The function of 

24 the foam is for marking the male’s territory; the foam has been called the ”topping on the 

25 dropping” (Schleidt and Shalter 1972). Since Japanese quail are ground dwellers and males 

26 establish territories during breeding season, cloacal gland foam provides a marker delineating 

27 their territory (Schleidt and Shalter 1972). Further, the male Japanese quail does not have a 

28 penile organ for transfer of the sperm to the female’s reproductive tract. Therefore, cloacal gland 

29 foam serves as a vehicle to accomplish transfer between the male to the female’s cloacal vent 

30 area.

31 4.1.5 Sexual Maturation and Puberty

32 Because Japanese quail mature rapidly, this species is ideal for monitoring the effects of 

33 potential endocrine disruptors on the initiation of sexual maturation and onset of full 

34 reproductive function, including initiation of egg production. Starting at 5 weeks of age, birds 

35 should be monitored for presence of cloacal gland foam in males as an initial indicator of 

36 testicular activity and production of androgens. Exposure to EDCs may either accelerate or slow 

37 sexual maturation. Further, depending on an EDC’s action(s), there may be differential effects in 

38 males and females. Briefly, males mature slightly earlier than females, showing the first evidence 

39 of sexually dimorphic plumage as early as 3 weeks of age. By 5 to 6 weeks of age, testicular 

40 activity initiates, beginning to produce increasing concentrations of testosterone leading to 

41 increasing expression of reproductive endocrine function, courtship, and mating behavior 

42 (Ottinger and Brinkley 1978, 1979b).
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1 4.1.6 Behavior (activity, lacrimation, reproductive behaviors, etc.)

2 Although the Japanese quail female exhibits a range of sex-specific behaviors, these 

3 behaviors are not readily quantifiable. Female specific behaviors include vocalizations at the 

4 time of egg laying, separation from her mate, and contact calls to the chicks (Mills et al. 1997). 

5 Males and females both exhibit aggressive behavior, which can be quantified (Ramenofsky 1985; 

6 Schlinger et al. 1987). Aggressive behavior can confound the behavioral testing paradigm if 

7 either the male or female is excessively aggressive, leading to damage of the other bird. If there 

8 is aggression with damage to one of the pair, the pair must be separated.

9 Male courtship and mating behavior provides an excellent bioindicator of reproductive 

10 competency. Sexual behaviors are steroid hormone dependent, meaning that there must be 

11 sufficient circulating concentrations of androgens to enable the expression of male sexual 

12 behavior. Moreover, male sexual behavior is an exquisitely sensitive endpoint for embryonic 

13 exposure to estrogenic and highly active androgen active compounds. As such, it is worthy of 

14 further refinement as a useful responsive measurement. Birds should be housed in individual 

15 cages at 5 weeks of age (prior to full sexual maturation). When most males (~90%) are 

16 producing cloacal gland foam, a 3-minute test is conducted in which a receptive female (control 

17 diet female that is from the same population; non-experimental) is introduced into the male’s 

18 home cage. The timing and behavioral measures in this testing paradigm have been validated and 

19 this method is in use by a number of laboratories (Ottinger and Brinkley 1978). Latency to 

20 mount, mount attempts, and cloacal contacts by the male are recorded. An individual may be 

21 tested on 3 successive days if it is important to determine the effects of experience on behavioral 

22 responses.

23 Interpretation of the outcomes of behavioral testing relates to the potential mechanisms of 

24 action of compounds under testing. More specifically, the female quail requires exposure to 

25 estradiol during embryonic development and is masculinized by exposure to androgens between 

26 embryonic days 12 and 18. Conversely, males are demasculinized as evidenced by impaired 

27 male sexual behavior as adults, if they have been exposed to either estrogens or androgens during 

28 this critical period (Adkins 1975, 1979). As such, the male Japanese quail is exquisitely sensitive 

29 to exposure to EDCs during sexual differentiation (Ottinger et al. 2008).

30 4.1.7 Fecundity (egg production)

31 Egg production and onset of laying are important measures of reproductive function in 

32 birds, and are easily monitored by checking cages daily and marking eggs. Avian EDC studies 

33 have shown reduced egg production (Scanes and McNabb 2003) as well as delayed onset of egg 

34 production (Rattner et al. 1984). Although some of these studies found reductions in fecundity 

35 with adult exposure, indicating direct actions of EDCs on gonadal or HPG axis function, most 

36 disruption occurs with either in ovo or maturation related exposures. Exposure during sensitive 

37 developmental periods, even to low doses of EDCs, can permanently disrupt the oviduct, as well 

38 as central reproductive control (i.e., the reproductive brain), and eventual fecundity of the hen as 

39 an adult. Therefore, it is important to monitor egg production in both the parent generation and 

40 the F1 generation, to assess both the activational and organizational effects of the EDCs being 

41 tested.
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1 4.1.8 Fertilization Success

2 EDCs may act directly on the gonads, gametes, or sperm-host glands, potentially 

3 resulting in decreased fertilization success. This outcome would be observed in the number of 

4 viable embryos at first candling. Although it is possible to observe the early development of 

5 blood vessels and see by candling the embryo as early as ED 3 to 5 in white-shelled eggs, most 

6 laboratories wait about a week (ED 7 to 8) to make a determination of fertility and embryonic 

7 viability. In the case of the lack of embryonic development, the egg is opened to determine if 

8 there was a germinal disc, which is indicative of initial fertilization and early embryonic death. 

9 This is important to discern early lethality due to frank toxicity of some compounds or doses. As 

10 such, decreased reproductive success in response to EDCs could be an indication of male or 

11 female gamete disruption, or both, and assessing gender-specific gamete disruption requires 

12 specialized fertility tests (OECD 2007). Therefore, measurement of fertility by observing 

13 viability of eggs produced by EDC exposed pairs and comparison to control animals provides an 

14 indication of potential sperm or ova disruption.

15 4.1.9 Embryo viability

16 Set eggs should be candled daily in order to monitor embryo viability. The timing of 

17 EDC effects will depend on the targeted mechanism of action for that compound. In the case of 

18 toxicity, the embryo may not survive beyond the first few hours or days of development. During 

19 this time, the uptake of yolk (white and yellow yolk) is minimal and as such reflects primarily a 

20 toxic effect of a compound or specific effects impairing the function of homeobox genes and 

21 disruption of cellular lineages contributing to the formation of organ systems (Morrison et al. 

22 2002; van de Lavoir et al. 2006, 2012; Carro et al. 2013a; Carro et al. 2013b). Later embryonic 

23 effects and lethality are more likely to relate to disruption of essential physiological systems, 

24 such as impacting the thyroid endocrine axis and other systems essential for the development and 

25 synchronization of endocrine and other physiological systems.

26 It has also been shown that a combination of early and late embryo mortality is a stronger 

27 indicator of toxicity that either alone (OECD 2007). Further, the embryo seems to be highly 

28 dependent on diet (i.e., the ratio of n-6/n-3 fatty acids), so care should be taken to standardize the 

29 diet (OECD 2007). Therefore, it is important to monitor embryo viability throughout 

30 development by regular candling of eggs. Moreover, there are distinct strain differences in 

31 Japanese quail. An extensive and excellent literature deals with a variety of physiological 

32 responses in Japanese quail that have been selected for multiple generations (Blohowaik et al.

33 1984; 1985; Marks et al. 1990; 1991; Hammad et al. 1996; Yang et al. 1999; Aggrey and Marks 

34 2002). In some cases, these highly selected strains mimic the Japanese quail strain found in 

35 Europe, which has been selected for high body weight. This heavier strain of Japanese quail 

36 tends to have slower maturation and lower production characteristics via reduced fertility, 

37 embryo viability, or egg production, similar to the heavier broiler breeder strain of domesticated 

38 chickens. Further, eggs produced by some strains have more coloration, thereby making it more 

39 difficult to visualize the early embryo by candling.

40 4.1.10 Eggshell parameters (abnormalities, eggshell strength/thickness, eggshell color)

41 Eggshell thickness, strength, and other abnormalities can be both quantitatively and 

42 quantitatively assessed, although some methods require more specialized equipment and training. 
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1 Gross abnormalities (such as apex abnormalities, calcium deposits, ridges) can be visually 

2 assessed. Eggshell thickness is often measured using calipers or a micrometer. The egg contents 

3 are removed, the shell is rinsed and air-dried, and then the shell thickness is measured, preferably 

4 at the middle/widest portion of the eggshell. Scanning electron microscope (SEM) analysis has 

5 been used to determine species-specific differences in the eggshell matrix; these studies have 

6 revealed species specific differences in the characteristics of the shell that are adaptive for the 

7 habitat or life history of that species or population (Igic et al. 2011; Morales et al. 2012). 

8 However species-specific morphological differences in the shell only become of importance if 

9 birds inhabit areas that are sprayed with insecticides or other agents; in that case, the shell 

10 permeability may alter embryonic exposure.

11 There is a long history of eggshell thinning as an endpoint for chemical exposure in birds. 

12 As early as the 1960s, exposure to DDT and DDE was linked to eggshell thinning and population 

13 decline in many wild bird species (Rattner 2009). This was thought to be an activational effect of 

14 pesticide exposure, and the endocrine disruptive effects of DDT/DDE were not known. More 

15 recently, it has been shown that exposure to EDCs during development can result in eggshell 

16 thinning in the eggs of adult birds (Berg et al. 2004; Holm et al. 2006). Nestling female zebra 

17 finches treated orally with estradiol benzoate had an increased incidence of cracked and broken 

18 eggs as adults (Millam et al. 2001; Rochester et al. 2008). In chickens, eggshell thinning has 

19 been observed in females exposed in ovo to estrogenic EDCs (including DDT) (Berg et al. 2004; 

20 Holm et al. 2006). The authors of these studies also proposed that, because of reduced frequency 

21 of shell gland capillaries and reduced carbonic anhydrase activity in the uteri of these birds, 

22 eggshell thinning may be due to organizational effects on the developing shell gland during 

23 development (Berg et al. 2004; Holm et al. 2006). Significant eggshell thinning was also found, 

24 however, in adult Japanese quail exposed to perchlorate, indicating that EDCs can also have 

25 activational effects on this parameter. Although the eggshells were significantly thinner, the 

26 authors state that they were not out of the ‘normal’ range for Japanese quail (Stafford 2011). It is 

27 unclear, however, if birds from different laboratories have different eggshell parameter variances, 

28 so any significant deviations from the control groups should be examined for adverse effects. 

29 Indeed, it has been shown that egg quality can vary between birds, depending on the husbandry 

30 methods of the laboratory (OECD 2007). Additionally, this study did not monitor the eggs of the 

31 offspring (exposed during development) to see if there were more substantial effects in the F1 

32 generation (Stafford 2011). Eggshell parameters are important endpoints for inclusion in the 

33 JQTT.

34 Historically, it is well documented that there are species differences in susceptibility to 

35 eggshell thinning from EDC exposures (Lundholm 1997), and there may be strain differences 

36 within species (OECD 2007). It would be useful to determine the extent to which strain 

37 differences occur, particularly for body weight and growth that often are negatively correlated 

38 with reproduction. If one species is selected (e.g., Japanese quail) there should be as little 

39 variation of strain and husbandry methods between laboratories as possible, in order to minimize 

40 intra-laboratory variation of eggshell parameters. Again, there are insights from studies in which 

41 a number of generations in Japanese quail have been selected for growth, reproductive, 

42 behavioral, and production characteristics (Blohowaik et al. 1984; 1985; Marks et al. 1990; 

43 Hammad et al. 1996; Yang et al. 1999; Aggrey and Marks 2002).
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1 4.1.11 Hatchability (ED15, hatch)

2 The Japanese quail chick begins to pip at ED15 in preparation for hatching at ED17. This 

3 process begins with the chick breaking through the egg membrane into the air cell. Once pipped, 

4 the chick is able to vocalize and often there is synchronizing in hatching of chicks in a nest or 

5 incubator. Pipping is followed by the chick pecking a hole, similar to an escape hatch, which 

6 requires a great deal of energy. During this time, thyroid hormones peak; corticosterone levels 

7 also increase (Tona et al. 2003). The hatching success of chicks has been shown to be highly 

8 sensitive to chemical perturbation, and thus hatchability is an important endpoint in EDC studies. 

9 Again, husbandry plays an important role, as sub-optimal handling, nutrition, incubation 

10 conditions, etc., can greatly affect hatchability (OECD 2007).

11 4.1.12 Genotypic Sex Determination

12 Genotypic sex is important when examining embryonic or early development (i.e.,

13 feminization or masculinization of reproductive organs), when the genetic sex of the bird may be 

14 unclear, either because of an early developmental stage or masculinization/feminization of the 

15 embryo (Berg et al. 1999). Since the rusty-red plumage of the male is not apparent until 3 weeks 

16 of age, it may be necessary to determine genetic sex prior (Tilmann and Capel 2002). In 

17 mammals, males are heterogametic (XY) and females are homogametic (XX). The testes18 determining factor (the SRY gene) located on the Y chromosome initiates differentiation of the 

19 testes and thus the steroidal milieu that promotes male development. In the absence of SRY, the 

20 embryo will differentiate into a female (Sekido and Lovell-Badge 2013). In birds, the female is 

21 the heterogemetic sex (ZW), and the male is homogametic (ZZ), with no SRY. Sexual 

22 differentiation in birds, therefore, is not as straightforward and is less well characterized. Either 

23 there is an equivalent ”ovary-determining factor” on the W chromosome for female development, 

24 or the”double dose” of the Z chromosome initiates male development, or perhaps both are at play 

25 (Smith et al. 2009). It is thought that a double dose of DMRT1, located on the Z chromosome, 

26 initiates male differentiation (Smith et al. 1997). Several W-specific genes are also implicated in 

27 ovarian development, and thus may play a role in female sexual differentiation (Ayers et al. 

28 2013). Genotype can be determined by polymerase chain reaction (PCR) (Berg et al. 1999; 

29 Morinha et al. 2012).

30 4.1.13 Histopathology

31 Many organ systems are highly sensitive to hormonal perturbations, particularly if 

32 exposed during development. Further, organs are relatively easy to collect and prepare for 

33 histological examination, and should be included in the analysis of EDC effects on the F0 and 

34 F1/F2 generations. Reproductive organ (i.e., testes/ovaries/oviduct) weights and gross disruption 

35 of organ morphology should be recorded, and the reproductive organs should be examined 

36 histologically for microscopic disruption (i.e., disrupted follicular function or spermatogenesis) 

37 (OECD 2007). In addition, thyroid glands, liver, and other organs should be examined, 

38 depending on the projected target(s) for the EDC (see below for more discussion). Detailed 

39 explanations of organ collection, preparation, and analysis for EDC endpoints are described in 

40 the OECD 2011 DRP and Histopathology Guidance Document for the Avian 2-Generation Test, 

41 2012. The organ systems that have been the focus of histological analyses have been those 

42 considered likely targets for endocrine active and/or toxicological impacts of EDCs. They 

43 include the organs and organ systems listed below.
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1 Reproductive organs (gonads) are often subjected to histological analyses for evidence of 

2 pathology. For both males and females, initial wet weight provides some information about 

3 potential abnormalities, specifically the retention of both male and female gonadal tissue as well 

4 as other potential pathologies. The sexual differentiation of the avian female involves regression 

5 of one of the two primordial ovaries with its accompanying oviduct (Sturkie 1991). As such, a 

6 critical observation for female birds is the presence/regression of the right ovary and oviduct; this 

7 provides initial information about appropriate sexual differentiation of the female reproductive 

8 axis (Van Tienhoven 1968; Muller et al. 1980; Sturkie 1991). Because gonadal sexual 

9 differentiation in birds relies on the differentiation of the female by genetic and then hormonal 

10 drivers that promote ovarian development and sexual differentiation of the hypothalamus and 

11 accessory sex structures, the female is vulnerable to impacts by EDCs. Some of the impacts 

12 noted have been for impaired sexual maturation with altered onset of egg production as well as 

13 reduced egg production and potentially reduced fertility (Rissman et al. 1984). There are sperm14 host glands in the oviduct of the avian female, in which sperm may reside for some weeks (Bakst 

15 1998), allowing continued fertility for ova produced as the female sequentially lays the eggs 

16 making up a complete clutch. However, there are no data available for a potential impact of 

17 EDCs on these glands; altered function of these glands would contribute to impaired fertility of 

18 the female in addition to any other direct effects on the gonads or on the HPG axis.

19 The ovary in the mature avian female has multiple follicles in varied stages of 

20 recruitment and maturation, often likened to a cluster of grapes (Van Tienhoven 1968). The 

21 stages in follicular development and maturation mirror those observed in mammals; however, the 

22 hormonal production by the follicle and hypothalamic endocrine support for ovulation differs 

23 from mammals and other vertebrate classes. Environmental signals stimulate the hypothalamic 

24 GnRH system that stimulates pituitary gland gonadotropin production (luteinizing hormone; LH 

25 and follicle stimulating hormone; FSH), which in turn stimulate ovarian function. Follicular 

26 recruitment generates a sequential developmental pattern in follicular maturation. The small 

27 white follicles produce primarily estradiol; as the follicle grows to secondary and then tertiary 

28 follicles, steroidogenic cells in the thecal layer continue producing estradiol as well as secreting 

29 increasing concentrations of progesterone (Bahr et al. 1983; Johnson and van Tienhoven 1980; 

30 Johnson et al. 1987). Progesterone produced by the tertiary follicle provides the stimulations for 

31 the pre-ovulatory GnRH surge that promotes the LH peaks critical for ovulation. Birds do not 

32 have a luteal phase in their ovulatory cycle; as such, there is no corpus luteum as in mammals. 

33 Histological analyses will provide information as to the status of ovarian function. Linking 

34 ovarian morphology to these components of ovarian function provides insight into potential EDC 

35 targets and actions.

36 The avian male is also vulnerable to embryonic exposure to EDCs. Early exposure to 

37 EDCs also can impact gonadal development in a genetic male; however, there is less information 

38 about these types of long-term impacts. In some studies, the gonadal histology attributed to 

39 effects of steroid or EDC exposure appears to be ovotestes, which are found in the hatchling but 

40 generally do not persist into the adult. Gonadal histology for adult functional males includes 

41 assessment of mass, with staining of the interstitial Leydig cells that produce testosterone and 5-

42 α dihydrotestosterone (5-αDHT) and the seminiferous tubules including staining of viable 

43 spermatocytes, numbers of cells in various stages of the spermatogenic cycle, and overall 

44 morphology of the gonad. Conversely, there are some neural markers of EDC exposure 

45 described further below.
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1 Histology of other organs has provided additional insights into the actions and impacts of 

2 EDCs. Liver histology can provide information on any abnormalities associated with toxic 

3 effects. In addition, the activation of detoxification enzymes are excellent markers for initiation 

4 of these enzyme systems; conduct of the ethoxyresorufin-O-deethylase (EROD) assay on liver 

5 subsamples can provide supporting evidence for histopathology findings. 

6 Previous studies have shown a link between embryonic exposure to polychlorinated 

7 biphenyls (PCBs) and cardiac malformations including decreased heart indices (heart weight as a 

8 percent of body mass), thinning of the ventricular walls, and abnormal heart apexes (DeWitt et al. 

9 2006; Walker and Catron 2000; Walker et al. 1997). Domestic chick embryos exposed to dioxin10 like PCBs had increased ventricular-septal defects, ventricular dilation, and higher overall heart 

11 weight by embryonic day (ED) 10 (Carro et al. 2013a; Walker and Catron 2000). 

12 Kidney histology can provide information about toxicological impacts of EDCs due to 

13 the critical role of the kidneys in detoxification and elimination of water soluble toxicants. Some 

14 protocols have been used to determine kidney pathologies associated with endocrine disruption; 

15 these observations support the use of kidney histopathology as a responsive endpoint for some 

16 classes of EDCs (Scanes and McNabb 2003; Shibuya et al. 2005). 

17 Pathophysiological measurements of the bursa provide a powerful tool to assess 

18 endocrine disruption affecting the immune system. There is evidence for cross-talk between the 

19 neuroendocrine and immune systems. Further, the bursal size differs in males and females, with 

20 smaller organ size in hatchling males. The bursa of Fabricius is exquisitely sensitive to steroid 

21 hormones, especially during development with testosterone having an immunosuppressive effect 

22 (al-Afaleq and Homeida 1998; Glick 1980; Marsh and Scanes 1994; Quinn Jr. and Ottinger 

23 2006). Embryonic exposure to trenbolone acetate resulted in persistent effects on bursal 

24 morphology with fewer, smaller follicles (Quinn et al. 2007; Table 4-1). As may be seen in Table 

25 5, consequences for the immune system from EDC exposure also relate to effects on the 

26 reproductive axis. Conversely, estradiol treatment resulted in larger bursal size; however, there 

27 was evidence of histopathology in treated individuals (Quinn et al. 2009). Many of these studies 

28 have also found a consistent relationship between bursal morphology and immune response. As 

29 such, bursal histopathology appears to be a reliable measure of impact to the immune system, 

30 especially assessing impacts during embryonic treatment and the persistence of these effects into 

31 adulthood. Moreover, immunosuppression in response to EDC exposure remains a critical 

32 responsive endpoint that has been shown to be sensitive to endocrine disruption 

33 (Grasman et al. 1996).

34
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1 Table 4-1. Summary of Significant Effects (p<0.05) of Trenbolone Acetate and p,p-DDE on 

2 Immune, Reproductive, and Behavioral Measures.

Endpoint 

Group Endpoint

Trenbolone Acetate 

(Androgen)

P,p-DDE

(Anti-androgen)

Immune 

Measures

Bursa-body weight index Decreased Increased

Bursal follicle number Decreased in hatchlings 

and adults

Decreased in chicks

Bursal follicle size Increased at 0.05 μmg/g, 

decreased at 50 μg/g

NA

Spleen-body weight index NA NA

Humoral response to chukar red blood cells NA NA

Cell-mediated response to phytohemagglutinin NA NA

Hatchling plasma immunoglobulin G Increased at only 0.05 and 

0.5 μg/g

NA

Hatchling total leukocyte counts NA Increased in chicks

Hatchling differential leukocyte counts Increased heterophil : 

lymphocyte ratio

NA

Physiological 

Reproductive 

Measures

Gonad-body weight indices NA NA

Testes morphology NA NA

Ovarian follicle count NA NA

Proctodeal foam gland weight Decreased NA

Onset of puberty Prolonged in males Shortened in females

Sperm penetration of perivitelline layer NA NA

Male 

Copulatory 

Behavior

Number of attempts to mount female Decreased Decreased

Number of successful copulations Decreased NA

Time to initial mount attempt NA NA

Time to achieve first successful copulation NA Increased

3 “NA” denotes measures that were not affected by treatments.

4 Adapted from Quinn and Ottinger (2006). Reprinted with permission, The Journal of Poultry Science Vol 43: 1-11, 

5 Japan Poultry Science Association, Tsukuba, Ibaraki, Japan.

6 4.1.14 Neuroendocrine Systems Regulating Reproduction, Metabolism, and Stress

7 A great deal is known about sexual differentiation of male sexual behavior in Japanese 

8 quail, which has proven to be sensitive to exogenous steroid exposure during embryonic 

9 development (Adkins 1979; Grasman et al. 1996; Halldin 2005; Halldin et al. 2005; Panzica et al. 

10 2005; Panzica et al. 1996; Panzica et al. 2007; Schweitzer et al. 2013). Embryonic exposures to 

11 steroids or compounds that are estrogen or androgen active result in impaired behavior in male 

12 adults (Cornil et al. 2006; Ottinger et al. 2008; Panzica et al. 2007; Quinn et al. 2008). Further, it 

13 has been shown that specific neural systems, including neurotransmitters (norepinephrine, 

14 dopamine, serotonergic) and neuropeptides (vasotocinergic) are modified in response to 

15 embryonic exposure to steroid hormones and to EDCs (Cornil et al. 2006; Ottinger et al. 2008; 

16 Panzica et al. 2005). Assessing the effects of embryonic exposure to EDCs on neurotransmitters 

17 and neuropeptides can provide information about direct effects of these compounds on regulatory 

18 systems that modulate reproductive, thyroid, and adrenal endocrine axes.

19 In addition, metabolic systems have been investigated in detail in birds, especially for 

20 domestic poultry. The thyroid system in birds has proven vulnerable to specific types of 

21 endocrine disruption in which this endocrine system is a target. Specifically, PCBs, flame 

22 retardants, and other classes of EDCs have been implicated in having thyroid system actions that 

23 impact birds, both in the laboratory and in field birds (Chen et al. 2009; Chen et al. 2008; Fernie 

24 et al. 2005b; McNabb and Fox 2003; McNabb 2006; Wada et al. 2009; Webb and McNabb 2008). 

25 The mechanisms of action exerted by these compounds in birds are not as clear; data from the 
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Avian Two-Generation ISR 39 May 28, 2013

1 McNabb laboratory and others suggest that altered enzyme activity, specifically deiodinases,

2 may accompany EDC exposure, thereby reducing availability of bioactive thyroid hormones to 

3 target tissues (Chen et al. 2009; Chen et al. 2008; Fernie et al. 2005b). It is not clear if 

4 histological methods provide a sensitive method for revealing these EDC effects; however, 

5 impaired production and release of thyroid hormones would be observed in abnormal thyroid 

6 gland follicles size and distribution. Presumably, these abnormalities would also be reflected in 

7 differences in circulating thyroxine (T4) and the more bioactive form, triiodothyronine (T3). 

8 Moreover, there are changes in thyroid responsive genes with EDC exposure in cultured avian

9 neural cells (Vongphachan et al. 2011). Finally, the hypothalamic-pituitary-adrenal axis (HPA) is 

10 another endocrine system potentially responsive to EDC challenges (Jönsson et al. 1994). In the 

11 case of EDCs that also have toxicological effects, the HPA axis is likely to be activated in 

12 response to both EDC and toxic actions, especially if there is sustained stress to the organism. A 

13 newer method of measuring corticosterone in feathers has recently been validated (Lattin et al. 

14 2011). This may be an interesting technique to determine if long-term stress has occurred; a 

15 method that may be especially useful to assess stress level in field birds over time.

16 Seasonality and migration also complicate responses by birds to EDCs; both affect the 

17 histology of EDC responsive organs and physiological systems. Birds often exhibit seasonal 

18 patterns in reproduction, and species that migrate often show complete regression of the 

19 reproductive axis with cessation of function followed by restimulation of the reproductive system, 

20 or seasonal recrudescence for the next breeding cycle. Initial stimulation of reproductive function 

21 in birds relies on environmental triggers, termed zeitgebers. In the case of temperate zone species,

22 this environmental cue is often photoperiod. Birds are generally long day breeders due to their 

23 relatively short incubation and relatively quick maturation times for their offspring. As such, 

24 most photoperiodic birds become reproductive with day length longer than 12 hours. Some 

25 species are termed relatively photoperiodic, meaning that they respond to changes in photoperiod 

26 length to act as their cue. There is a very large literature on this subject that deals with the neural 

27 mechanisms, response via the eyes and pineal gland, and the hypothalamic response to regulate 

28 reproductive function; review of this literature is beyond the scope of this ISR, but it is important 

29 to recognize seasonal variations in energy demands due to reproduction, migration, and 

30 environmental challenges especially as EDCs impact critical endocrine systems involved in these 

31 seasonal and adaptive responses.

32 4.1.15 Key Neuroendocrine Measures for Endocrine Disruption

33 Hypothalamic Gonadotropin Releasing Hormone-I (GnRH-I)

34 Gonadotropin releasing hormone (GnRH) is the primary hypothalamic hormone that 

35 directs the reproductive or HPG axis. The form of GnRH that is a primary form in avian species 

36 is termed GnRH-I. This form of the peptide has been shown to regulate reproductive function in 

37 birds (Li and Ottinger 1988; Li et al. 1991). In birds, there are approximately 1000 GnRH-1 

38 neurons that are located in the pre-optic/lateral septal region of the hypothalamus (Kuenzel and 

39 Masson 1988; McGary et al. 2001; Ottinger et al. 2002; Ottinger et al. 2008; Ottinger et al. 

40 2009b). These neurons send axonal projections to the median eminence, where the GnRH is 

41 released to the portal vessels that carry hypothalamic hormones to gonadotropin producing cells 

42 in the anterior pituitary gland. Synthesis and secretion of GnRH is modulated by 

43 neurotransmitter and neuropeptide systems that respond to environmental signals and to internal 

44 physiological modulators (see Figure 2-5 above for a diagram of the HPG axis).
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1 Neurotransmitters, Neuropeptides, and Enzymes

2 Impacts to neurotransmitter systems have been a subject of interest for many years. In the 

3 case of toxicity assessments, brain acetylcholine esterase (ACHase) has been used as a measure 

4 of toxicant exposure (Rattner et al. 1986). However, it is not clear how responsive the 

5 acetylcholine system is to endocrine disruption. There is mounting evidence that 

6 neurodegenerative diseases in humans may be linked to chemical exposures in mammals, and it 

7 would be expected that similar deleterious impacts would occur in avian neural systems. 

8 However, additional information is needed to understand if this type of long-term impact 

9 provides a bioindicator of adverse effects in birds. Monoamines are key neurotransmitters that 

10 regulate the reproductive and metabolic endocrine axes as well as modulate stress responses. 

11 Analytical methods include High Pressure Liquid Chromatography – Electrochemical Detection 

12 or by ELISA. Specific monoamines of interest include: serotonin, 5-hydroxyindolamine 

13 (5HIAA), norepinephrine (NE), and dopamine (DA; Abdelnabi and Ottinger 2003). Monoamines 

14 are critical regulators of neuroendocrine function. A number of studies in both mammals and 

15 birds have shown that alterations in these neuroregulatory systems result in diminished 

16 reproductive or metabolic endocrine function. As mentioned earlier, steroids and EDCs impact 

17 arginine vasotocin (AVT) and aromatase enzyme in brain regions that modulate reproduction and 

18 sexual behavior (Panzica et al. 2005; Panzica et al. 2009; Panzica et al. 1996; 

19 Panzica et al. 2007).

20 Behavioral Modulation by Critical Brain Regions – Effects of EDCs

21 As summarized earlier, behavior can serve as a sensitive measure of EDC exposure, 

22 especially in precocial species. The brain regions that modulate reproductive behavior in male 

23 Japanese quail have been thoroughly characterized; these regions depend on androgen 

24 stimulation (Adkins-Regan et al. 1994; Watson et al. 1990; Watson and Adkins-Regan 1989b). 

25 Songbirds and other altricial birds also show impacts of EDC exposure on avian brain 

26 morphology (Iwaniuk et al. 2006; Quaglino et al. 2002). Further, there is a large literature about 

27 the impacts on steroid hormones on the sexual differentiation and function of the nuclei that 

28 direct and modulate song control in songbirds (Quaglino et al. 2002; Wade and Arnold 2004). 

29 These species show maturation and seasonal changes in reproductive behavior; these changes are 

30 associated with recrudescence of neuroanatomical areas modulating singing behavior. It is the 

31 song control nuclei that exhibit with greater neuroplasticity (Hoogesteijn et al. 2008), thereby 

32 allowing songbirds greater adaptability to changing conditions and environmental challenges. 

33 Because of the neuroplasticity in songbirds, EDC effects may be more transient in songbirds; 

34 however, there are insufficient data to ascertain if this is the case. Nonetheless, EDC effects on 

35 brain regions that modulate singing and other behaviors that are steroid dependent in both 

36 precocial and altricial birds have been clearly documented.

37 Other Biochemical Endpoints Potentially Reflective of Endocrine Disruption

38 Vitellogenin is a yolk protein that is produced in the liver in egg-laying animals in 

39 response to estrogen. In sexually mature females, it is produced in response to endogenous 

40 estrogen. However, in males and pre-pubertal animals, vitellogenin production can indicate 

41 exposure to exogenous estrogens (Heppell et al. 1995). Tests of vitellogenin response have not 

42 shown this to be a sensitive measure in birds, either relative to increased plasma vitellogenin in 

43 males or in pre-pubertal animals.
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1 Oxidative damage has been linked to adverse effects of xenobiotics; however, few data 

2 are available for birds to support selection as a global indicator of endocrine disruption in birds. 

3 Similarly, DNA damage could provide important information as an indicator of adverse effects 

4 of toxicants (Dix et al. 2012). However, similar to oxidative damage, more information is needed 

5 to ascertain the reliability of these measures as bioindicators of effects. Finally, the receptor 

6 based assays have provided additional information on potential mechanisms of action of EDCs 

7 as well as important insights into the comparative sensitivity across avian species to Ah receptor 

8 activated compounds (Farmahin et al. 2013; Farmahin et al. 2012b; Herve et al. 2010; Inoue et al. 

9 2009; Lattin et al. 2012; Lee et al. 2011a; Lee et al. 2011b; Manning et al. 2013; 

10 Tazelaar et al. 2013; Yang et al. 2010).
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Avian Two-Generation ISR 42 May 28, 2013

1 5 PROTOCOL OPTIMIZATION AND DEMONSTRATION

2 5.1 Comparability with the Avian Reproduction Test Currently in Use

3 The EPA Avian Reproduction Test (OSCPP 850.2300; US EPA 2012) was designed 

4 primarily as a reproductive toxicological test to determine if a test compound has adverse effects 

5 on classic reproductive parameters. These include measures of reproductive function as well as 

6 toxicological endpoints. Further, most are largely measures of effects on adults. This testing 

7 paradigm has the potential to detect more extreme adverse effects of EDCs, including some 

8 measures of fitness such as number of offspring produced and thus may reveal endocrine 

9 disruption. However, the Avian Reproduction Test may not detect endocrine disruption or low10 dose effects on developing embryos. The studies reviewed below include egg injection studies as 

11 well as one- and two-generation dietary tests. Statistically significant differences are discussed 

12 below with the exception of trends, as noted in the text.

13 5.2 Egg Injection Studies

14 A series of studies performed in a number of laboratories provide background 

15 information about the effects, mechanisms of action, and relative impacts of various classes of 

16 EDCs in birds. Studies described below are for the most part those conducted under controlled 

17 laboratory conditions. These experiments were designed to simulate maternal deposition of 

18 EDCs into eggs prior to oviposition by the female. There are complexities to consider with the 

19 use of egg injection as a method of administering known concentrations of a compound. First, 

20 since the avian embryo develops outside constant maternal input as occurs in mammals, it is 

21 assumed that the egg is a relatively closed system in which a known amount of compound is 

22 inserted. This is advantageous, especially with administration of the test compound prior to 

23 incubation, because there is opportunity for enzymes in the albumin and embryonic organs to 

24 metabolize the compound into secondary substances similar to what would occur in wildlife 

25 populations. A second consideration is the method of administration; specifically the vehicle, site 

26 of administration, and movement of the compound through the egg compartments may vary with 

27 the chemical characteristics of the test substance. Third, it is critical to test field relevant 

28 concentrations as part of a dose response experiment to ascertain bioactivity as well as determine 

29 potential mechanisms of action. Finally, egg injection methods have improved to optimize 

30 vehicles, injection volumes and sites, and timing of injection to minimize treatment effects. The 

31 egg injection studies below provide information on the response of avian embryos to a dose 

32 range of selected EDCs.

33 It is instructive to compare results with the avian embryo assay. The avian embryo assay 

34 involves exposing the embryo via injection of the compound into the egg. While this method 

35 may be less relevant to real world exposure than dietary exposure in the Avian Reproduction 

36 Test (OCSPP 850.2300, EPA 2012) and maternal transfer in the Two-Generation Test, it has an 

37 advantage over the Avian Reproduction Test in that the embryo is exposed to the potential EDC; 

38 thus developmental disruption can be examined. Because there is maternal transfer of 

39 environmental chemicals to the yolk (Adkins-Regan et al. 2005; Lin et al. 2004; Thompson et al. 

40 1967), egg injection mimics this maternal transfer, although it is difficult to determine relevant 

41 doses, without maternal transfer studies. Further, egg injection bypasses the metabolism of the 

42 compound in the hen, and the possible actions of metabolites. The Avian Embryo Assay is a 

43 bioassay that can involve dosing the egg at different times and comparing the results during 
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Avian Two-Generation ISR 43 May 28, 2013

1 critical periods in development. However, there are concerns about how closely egg injections 

2 mimic maternal deposition and with the selection of vehicles that do not interfere with discerning 

3 impacts of test compound(s). There are a number of studies in the literature that address these 

4 issues, and provide guidance on vehicles, injection sites and volumes, and relative uptake of 

5 treatments (McKernan et al. 2009).

6 5.2.1 Atrazine (Experimental Pathology Laboratories 2006)

7 Exposure Levels and Protocol

8 On day 0 before incubation, eggs were injected at doses of 0, 0.05, 0.5, 5.0 g/egg, and 

9 untreated. Endpoints included embryonic viability, hatching success, growth/weight gain, timing 

10 of maturation, egg production, male reproductive behavior, genetic sexing of hatchlings and 

11 adults, hypothalamic dopamine/GnRH-I, and brain aromatase (days 1 and 50), fecal steroids 

12 (days 1 and 50), plasma steroids (day 50), histopathology of abnormal bursa (days 1 and 50), 

13 aromatase activity or histopathology of gonads (days 1 and 50), thyroid weight, plasma and 

14 gland thyroxine (T4) content (days 1 and 50), plasma T4 (days 1 and 50), cloacal gland area and 

15 foam production (day 50), initiation of egg production, hypothalmic/gonadal aromatase in half of 

16 the controls, low, and high dose chicks (when available) and adults (half of the gonads sent to 

17 EPL and half used for frozen tissue), and plasma vitellogenin in chicks and adults.

18 Sensitive Endpoints

19 Liver mass was reduced in hatchlings in all treatment groups; gonad weight was lower in 

20 5 ug/egg group. Dopamine was decreased in the males at 0.5 g/egg and 5.0 g/egg doses. 

21 Circulating estradiol was decreased in dosed females.

22 In adults, exposed males showed greater latency to mount with high dose. Low dose 

23 males also had fewer mating attempts, while the medium dose had the most mount attempts, but 

24 not necessarily the greatest mating success. There was a trend of lower dopamine in the medium 

25 and high dose groups. Aromatase activity was higher in the medium dose group. Females in the 

26 low dose group had lower estradiol than the other groups. Males in the low dose group had 

27 higher estradiol than the other groups. Androgen was reduced in all treatment groups. Some 

28 sample sizes were too small for statistical analyses.

29 Overall Conclusions

30 Vinclozolin administered prior to incubation affected organ weights, with some effects on 

31 dopamine and circulating estradiol levels. Adult males had impaired sexual behavior, with some 

32 effects noted for neuroendocrine systems and circulating steroids.

33 5.2.2 Trenbolone

34 Exposure Levels and Protocol

35 There were two trials for trenbolone. With Trial 1: 0.05, 0.5, 5, 50, and 125 μg/egg; and 

36 Trial 2: 0.05, 0.5, 5, 50 g/egg injected at embryonic day 4 (i.e., ED 4 or ED 11, respectively. 

37 Adult tissues sampled included blood, brain (frozen), gonads and bursa (weighed), testes (left 

38 frozen and right fixed in Bouin’s). Hatchling tissues were blood, brain (frozen), bursa, and

39 gonads (gross morphological deformities). Other endpoints included chick behavior (motor 

40 activity), onset of puberty (monitored starting at 5 wks for cloacal gland hypertrophy and foam 
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1 production in males and oviposition in females), egg production, embryo development and 

2 fertility, male sexual behavior, steroid hormones (estradiol and androgens), aromatase enzyme 

3 activity (brain), monoamines (brain), GnRH (brain), and tissue histology.

4 Sensitive Endpoints

5 There was increased embryo mortality at 50 and 125 g/egg (100% mortality in 125 

6 g/egg); surviving chicks showed delayed puberty, decreased bursa size at 5 and 50 g/egg (high 

7 doses) with fewer bursal follicles, and a trend for reduced circulating androgens (see Table 5-1

8 for summary). Males in all treatment groups had significantly fewer successful copulations than 

9 controls; treatment effects were observed in latency to mount and number of mating attempts. 

10 There was a trend of an increase in aromatase activity. Males in the low dose group had higher 

11 estradiol than the other groups. Androgen was reduced in all treatment groups. Females in the 

12 low dose group had lower estradiol than the other groups.

13 Treatment at ED11 also resulted in effects, many of which did not follow a dose-response 

14 relationship. There was increased mortality in the lowest dose, increased bursal index decreased 

15 circulating steroid hormones, reduced ovarian weight and follicle number; with these and other 

16 effects occurring in a non-dose-dependent pattern. Altered neurotransmitter levels were observed 

17 with specific doses in serotonin metabolite (5-HIAA) as well as a dose related increase in 

18 norepinephrine. Often, there have been non-linear responses observed in these endpoints; 

19 however, this is sometime due to the number of individuals examined and could be assessed 

20 through a power analysis to determine the vigor of that particular endpoint. Both are indicative of

21 long-term effects on the neural systems that modulate both endocrine and behavioral components 

22 of reproduction. Ovary weight was reduced in the 0.5 and in the 5.0 g/egg treatments. There 

23 was a trend of reduction in follicles in the 0.5 and 5.0 g/egg groups and increased left testis 

24 mass in 0.05 g/egg group. Statistics were conducted to compare groups with sufficient sample 

25 size. A summary of findings is shown in Table 5-1.

26 Overall Conclusions

27 Trenbolone had both toxicological and endocrine disrupting effects that were not dose28 related, suggesting that there may be a survivor effect. This would suggest that the more 

29 sensitive birds responded and did not survive, thereby leaving less sensitive birds to be sampled 

30 in the higher doses. Trenbolone administered during development (egg injection) resulted in 

31 disrupted development of the immune system (smaller bursa; Quinn et al. 2007) as well as 

32 disrupted reproductive parameters (delayed puberty, reduced cloacal glad development, reduced 

33 reproductive behaviors) in males (Quinn et al. 2007).
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1 Table 5-1. Summary of Responses to Estradiol or Trenbolone with Day of Egg Injection from 

2 the Studies Reviewed.

Endpoint

Hatchline Effects Summary*

Estradiol ED4 Estradiol ED11 Trenbolone ED4 Trenbolone ED11

Embryo Mortality  -  

Hatchability  -  

Body Mass - - - -

Male--Androgen -  () 

Female--Estradiol -  () 

Bursa Mass  -  

Aromatase ()  - -

Norepinephrine - () - -

Dopamine -  - -

Serotonin - - - -

5 HT - - - -

GnRH-I - - - -

* () or () means effect/trend was non-significant, () means a non-linear dose response was observed.

3 5.2.3 Estradiol

4 Effects of exogenous steroids on sexual differentiation in Japanese quail have been 

5 examined in a number of studies. These studies have investigated the effects of exogenous 

6 estradiol treatment on sexual differentiation in Japanese quail. These studies have shown that

7 neural systems are sensitive to the effects of estradiol; however, few have examined 

8 toxicological effects or studied the endocrine disrupting effects of exogenous estradiol in a dose 

9 response manner. Nonetheless, this literature is valuable because it does reveal some impacts of 

10 excess estradiol beyond that maternally deposited or produced by the embryonic gonads and 

11 adrenal glands.

12 Exposure Levels and Protocol 

13 The dose response study included 0, 0.5, 50, or 500 g per egg (20 l vehicle). Injections 

14 were conducted at either ED4 or ED11, to determine early effects compared to impacts on the 

15 HPG axis and other gender specific characteristics. Studies in the older literature had higher 

16 doses, often with more toxicity than endocrine disruption as a result. 

17 Sensitive Endpoints 

18 Response to the treatment differed with the timing of exposure. Early embryonic 

19 treatment impaired male sexual behavior and decreased bursa size. Females had reduced egg 

20 production. Treatment at ED11 also reduced egg production in females, with altered circulating 

21 steroid hormone levels. Brain catecholamines were altered by treatments. A summary of findings 

22 is shown in Table 5-1 above.

23 Conclusions 

24 Estradiol is particularly potent early in embryonic development. Effects in females may 

25 be due to direct effects on the ovary (Rissman et al. 1984). Male sexual behavior and associated 

26 neural systems are exquisitely sensitive to exogenous estradiol treatment during embryonic 

27 development. This study supports findings from other studies demonstrating that estradiol can 

28 act as a potent endocrine disruptor, acting on neural systems that regulate endocrine and 

29 behavioral components of reproduction, as well as impacting immune-related endpoints such as 

30 the bursa. Further, it is clear that early exposure has greater overall impacts, which would be 
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Avian Two-Generation ISR 46 May 28, 2013

1 similar to maternal deposition with embryonic exposure occurring through the entire 

2 developmental process. 

3 5.2.4 Vinclozolin (McGary et al. 2001)

4 Exposure Levels and Protocol 

5 Eggs injected on Day 4 of incubation at doses of 0, 25, 50, or 100 ppm (calculated for a 10 g 

6 egg), and untreated. Endpoints in hatchlings were steroid hormones and hypothalamic GnRH-I. 

7 Adult males were measured for cloacal gland, male sexual behavior, hypothalamic GnRH-I, and 

8 steroid concentrations (estradiol and androgens).

9 Sensitive Endpoints 

10 Results showed that exposure to VIN significantly (p<0.05) altered GnRH-I in male hatchlings, 

11 whereas GnRH-I levels in females remained unaffected. Although steroid levels were unaltered 

12 by any VIN treatment, maturation appeared affected, with male the onset of reproductive 

13 behavior delayed, with lower number of mounts and cloacal contacts being lower (p<0.05) in 

14 VIN-treated males at higher doses.

15 Overall Conclusions

16 Vinclozolin treatments impacted males at higher doses, indicating that the antiandrogenic actions 

17 adversely affected male sexual differentiation. However, responses were significant primarily at 

18 relatively high doses.

19 5.2.5 Other Egg Injection Treatments

20 Many other studies in birds have utilized egg injections as the method to administer 

21 treatments. Although these studies have not been detailed, there are numerous examples of both 

22 short- and long-term impacts of endocrine active compounds that are environmentally available. 

23 Original papers and reviews may be found in the additional information provided in support of 

24 this ISR.

25 5.3 Reproduction and Two-Generation Dietary Studies

26 The effects of EDCs have been studied in a number of dietary studies in quail, including 

27 reproduction and multigenerational tests. Selected studies are described below to demonstrate 

28 response to specific EDCs and to provide information on responsive endpoints.

29 5.3.1 Perchlorate (Stafford 2011)

30 Exposure Levels and Protocol

31 Adult Japanese quail were exposed to perchlorate in the diet for ~10 weeks at doses of 0, 

32 13.8, 36.4, 64.7 and 144.1 mg a.i./kg body weight/day. Birds were photostimulated for 19 days, 

33 and eggs were collected for 10 weeks and incubated. Adult and 14-day juvenile tissues collected 

34 included blood, heart, liver, spleen, head (with the occipital plate of the cranium removed), 

35 cervical and thoracic cavity (including the glands extending along the neck to above the heart), 

36 and caudodorsal cavity (including kidney, adrenals, and reproductive organs). Endpoints 

37 included overall adult male/female body weight change, overall average adult feed consumption 

38 expressed as grams per bird per day, total number of eggs laid, cracked eggs, non-cracked eggs, 
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Avian Two-Generation ISR 47 May 28, 2013

1 eggs set into the incubator, eggs set of total eggs, viable embryos (and of total eggs set), live 

2 embryos (and of total viable embryos), successful hatchlings (and of eggs laid set, of total eggs 

3 laid, and of total live embryos), total number of 14-day offspring survivors (and of total eggs set, 

4 and of total successful hatchlings), mean hatchling body weight, mean 14-day od offspring 

5 survivor body weight, and mean eggshell thickness per hen.

6 Sensitive Endpoints

7 A trend of increased aggression was observed, male weight decreased with dose, and 

8 there was a dose-related reduction in egg shell thickness at 250, 500, and 1000 ppm. A reduction 

9 in number of viable embryos, and viable embryos of eggs set was observed with significant 

10 reduction at 100 ppm. Offspring data were not reported. Endpoints including those for thyroid 

11 system impact were not measured in adults (i.e., T3 and T4 levels, and mRNA).

12 Conclusions

13 Perchlorate is expected to primarily affect the thyroid axis, not necessarily the 

14 reproductive axis. However, if the perchlorate is toxic in addition to having thyroid system 

15 effects, then additional effects may be associated with toxicity. Therefore, the response to 

16 perchlorate relative to egg quality indicates a capacity for the compound to affect the 

17 reproductive system as well as the thyroid system. More sensitive thyroid endpoints/behavioral 

18 endpoints were not tested in offspring. Perchlorate has been shown to alter behavior in birds 

19 exposed during development (Rainwater et al. 2008), and change embryonic thyroid levels (Chen 

20 et al. 2008).

21 5.3.2 Bisphenol A (Frey et al. 2011)

22 Exposure Levels and Protocol

23 Adult Japanese quail were treated with BPA (0, 1, 30, 300, and 1000 ppm) during a 4 

24 week acclimation period, then photostimulated. After 1 week, an 8-week egg laying period was 

25 initiated. Body weight, feed consumption, egg quality and production, fertility, hatchability, and 

26 14-day survival were measured. Adults were euthanized after an 8-week egg-laying period, eggs 

27 incubated and hatchlings reared to 14 days. Adult and offspring tissues collected included blood, 

28 head, heart, liver, spleen, kidneys, thyroid glands, bursa of Fabricius, adrenal glands, gonads and 

29 associated structures (oviduct, shell gland, epididymis, ductus deferens), and cloacal gland area.

30 Sensitive Endpoints

31 No adverse effects were detected in this study.

32 Overall Conclusions

33 This was a classic reproduction study conducted in adult reproductive Japanese quail with 

34 additional measurements to assess effects on immune, reproductive, thyroid, and stress axes, as 

35 well as collection of additional measures of impacts to the reproductive system. Organ weights 

36 and gross morphology were used to check for possible effects on the liver, spleen thyroid, bursa, 

37 and adrenal glands. These additional measures would reveal significant impacts, but may not be 

38 the most sensitive measures. Other endpoints, such as behavioral or neural measures, may reveal 

39 endocrine disruption (Halldin et al. 2005; Panzica et al. 2005; Yigit and Daglioglu 2010).
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1 5.3.3 Trenbolone Reproduction Dietary Study (Frey et al. 2012)-Dietary Study #1

2 Exposure Levels and Protocol

3 Adult quail were exposed to trenbolone at doses of 0, 0.1, 0.4, 1.6, and 6.4 ppm for 9

4 weeks. Birds were acclimated for 4 wks, then photostimulated for 1 week followed by an 8-

5 week egg-laying/collection period. Eggs were incubated and hatchlings were reared to 14 days. 

6 Body weight, feed consumption, egg quality and production, fertility, hatchability, and 14-day 

7 viability were measured. Adult and offspring tissues collected included blood, head, heart, liver, 

8 spleen, kidneys, thyroid glands, bursa of Fabricius, adrenal glands, gonads and associated 

9 structures (oviduct, shell gland, epididymis, ductus deferens), and cloacal gland area.

10 Sensitive Endpoints

11 There was no evidence of endocrine disruption using this one-generation reproductive 

12 test. Organ weights were reported and tissues appear to have been preserved, but there is no 

13 mention of additional analyses.

14 Overall Conclusions

15 Test doses were determined in a range finding test using adults. Based on the results, 

16 doses were selected and administered to adult Japanese quail. The age and reproductive status of 

17 the birds at the initiation of the study are unclear. In other protocols from this laboratory, the 

18 photostimulation just denotes lengthening the photoperiod from 15 to 18 hours of light/day. As 

19 such, the birds should be sexually mature and functional at the initiation of the treatment. The 

20 biochemistry for the chemical analysis is very thorough and verifies the efficacy of the test 

21 compound. Dietary trenbolone in adults did not result in endocrine disruption. This may be due 

22 to lower sensitivity of adults as well as a lack of impact of androgenic compound. In adult males, 

23 additional androgen would not impair the male system unless the concentrations and activity of 

24 the compound was sufficient to shut down the reproductive axis via negative feedback to inhibit 

25 hypothalamic GnRH. Similarly, females would not be affected by trenbolone unless high levels 

26 are sufficient to interfere with the preovulatory LH surge.

27 5.3.4 Methoxychlor (dietary study with Japanese quail and bobwhite quail 

28 comparisons)

29 Exposure Levels and Protocol

30 Two studies were conducted to compare Japanese quail and northern bobwhite quail 

31 exposed to dietary methoxychlor at 0, 5, and 10 ppm according to the OECD two-generation 

32 protocol. Japanese and northern bobwhite quail (parental generation; P) were raised under short 

33 photoperiod until somatic maturity, then placed on diet containing low concentrations of 

34 methoxychlor (MXC; 0, 5, and 10 ppm) and simultaneously photostimulated. A range of 

35 measures were monitored including feed intake, egg production, egg quality, fertility, and 

36 offspring viability. In addition, other measures were selected for their potential to detect 

37 endocrine disruption. Chicks (F1) were raised on the same diet as their parents, and paired as 

38 adults. Sexual maturation, reproductive behavior, and endocrine measures were examined, 

39 similar to the parent generation. F2 chicks were raised on control feed and assessed for the same 

40 endpoints as the F1 birds.
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Avian Two-Generation ISR 49 May 28, 2013

1 Sensitive Endpoints

2 There were some inherent species differences, including slower maturation and lower 

3 productivity in bobwhite quail. These differences were observed consistently over all generations 

4 in both species. No treatment effects were observed in either species for body weight, feed intake, 

5 egg quality, or viability. Effects of 5 ppm MXC were similar in both species, and no treatment 

6 effects were observed for egg production, shell quality, fertility, and chick growth. However, 

7 both species showed delayed sexual maturation with treatment. This was more pronounced in the 

8 10 ppm MXC treatment, with significant (p<0.05) reduction in the number of birds achieving 

9 optimal reproductive performance. Sexual behavior was significantly (p<0.05) impaired by MXC 

10 exposure in both F1 and F2 Japanese quail males; behavioral assessment was not conducted in 

11 bobwhite males due to species differences in courtship and mating behavior. A modified open 

12 field test was developed and pilot data was collected to assess potential impairment of motor and 

13 behavioral responses in young chicks. This testing method has potential for Japanese quail, but 

14 not for bobwhite quail, again due to species differences in behavioral responses.

15 Although variable, plasma and fecal steroid hormones were significantly (p<0.05) 

16 different with treatment. In the 5 ppm MXC treatment, there was an apparent bimodal response, 

17 especially in maturing birds. This is potentially an important marker because gonadal steroids 

18 typically are variable among individuals during sexual maturation whereas treated birds showed 

19 reduced and uniformly lower plasma steroid hormones. This variability in gonadal steroids may 

20 provide an index of EDC adverse effects on maturation, which would be important for field 

21 populations where timing of reproduction is likely to be a critical factor. This maturation related 

22 relationship between reproductive status and steroid hormones was weaker in bobwhite quail 

23 even though MXC exposure was associated with delayed sexual maturation, a similar effect was 

24 observed in Japanese quail. Hypothalamic neurotransmitters and GnRH-I reflected stage in 

25 maturation and reproductive capability; however these measures were variable and warrant

26 further characterization to link individual responses.

27 Overall Conclusions

28 A number of effects were observed in this multigeneration study, especially for endpoints 

29 that revealed reproductive impacts. These effects were more apparent in the Japanese quail, 

30 which also included male reproductive behavior. One purpose of the study was to directly 

31 compare Japanese quail and northern bobwhite quail in the same testing protocol with the same 

32 dietary treatments. Measures not conducted in this study included morphological, histological 

33 (gonad, bursa), immune, and hormonal (thyroid, vitellogenin, gonadotropins) measures. Overall, 

34 the two species had similar responses to the low levels of dietary MXC treatments. Japanese 

35 quail offer a fast maturing precocial avian model that is practical relative to time for completion 

36 of a two-generation dietary regimen.

37 5.3.5 Two-Generation Dietary 17-Beta Estradiol (E2) Dosing Study

38 Exposure Levels and Protocol

39 Japanese quail in the P1 generation were either given the treatment diet prior to sexual 

40 maturation (P1A, 3 weeks old; 10 weeks of egg laying), or after proven egg-laying ability (P1, 5 

41 weeks; 10 weeks of egg laying). For each group, the F1s were either exposed to the treated diet 

42 from hatch to egg-laying (F1a) or not exposed to the dietary treatment (F1b). F2 birds did not 

43 receive the treatment. Controls were used for P1A and P1B, and F1a controls were used as the 
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Avian Two-Generation ISR 50 May 28, 2013

1 control group for the other F1 treated groups. E2 doses were 0, 0.078, 0.31, 1.25, and 5.0 ppm in 

2 the diet.

3 Sensitive Endpoints

4 The P1 generation had elevated E2 levels in both P1A and P1B males and females. There 

5 was a trend of decreased testosterone in males with elevated E2. P1B females had significantly 

6 greater numbers of right oviducts than P1A females. Thyroid and adrenal gland weights were 

7 greater in the P1B groupwith diffuse hypertrophy in the adrenal. P1A had fewer eggs, thinner 

8 eggshell thickness, and higher yolk E2 concentration. P1A males had degenerative changes of 

9 the testis and reduced sperm number. P1B males had testicular lesions in the high dose group. 

10 P1A males had earlier sexual maturation and P1A males had increased non-male plumage with 

11 E2 treatment.

12 In the F1 generation, the P1A F1 females had lower hatch and adult body weights. F1a

13 females had increased thyroid gland weights and decreased pancreas and ovary weights. P1A14 F1a females had more active oocytes and increased oviduct weight. F1a females had later sexual 

15 maturation. F1a birds had increased Day 15 viability of eggs and hatchling production. An 

16 increase in the proportion of cracked shells was associated with P1B parental exposure. F1a hens 

17 also had increased eggshell thickness. F1 males (P1A-F1a) had increased terminal body weight, 

18 tibiotarsus weight, and pancreas, liver, and testis weights and weight indices. Cloacal gland 

19 weight was reduced in the high E2 diet, and smaller in the P1A-F1a males, although the area was 

20 greater in these males.

21 F2 generation females (P1A-F1a) from E2 treated birds had more surviving F2 offspring. 

22 F1a birds exposed to E2, P1A-F1a F2 chicks had significantly or nearly significantly reduced 

23 measures of growth (tibiotarsus diameter and tarsometatsus length) ovary-to-body weight ratios 

24 and increased gross and body weight-adjusted pancreas weights of the F1a 5 ppm treatment 

25 groups. F1A F2 chicks had greater thyroid gland and spleen weight. P1B-F1a F2 female chicks 

26 had increased adrenal gland mass. P1B-F1b chicks had increased liver weights. F2 chicks with 

27 P1A exposure had significantly lower ovary weights. F2 chicks with P1B exposures had 

28 increased incidence of limb malformations. The F1a-F2 male chicks had a lower body weight, 

29 and less bone growth than male offspring of F1b parents. These P1A-F1a F2 males also had 

30 increased thyroid, pancreas, liver and cloacal gland weights, and decreased testis weight. F1a F2 

31 males had significantly decreased brain weight.

32 Overall Conclusions

33 Even though the P1A dosing regime did not have a large impact on the P1 generation, the 

34 P1A offspring (F1 and F2 generations) had effects on many important endpoints, suggesting that 

35 the P1A protocol (i.e., dosing the P1 generation from 3 to 4 weeks of age) would be the most 

36 appropriate protocol for assessing reproductive and other impact of estrogen active compounds. 

37 Further,exposure of the F1 generation is important to observe combined effects of embryonic 

38 exposure followed by lifetime dietary exposure. The F2 birds provide insight into the impacts on 

39 F1 females’ reproductive systems including maternal deposition into eggs produced by these 

40 females. Moreover, these potential sources of endocrine disruption are isolated from direct 

41 dietary exposure in the F2 birds because they are fed only control diet.
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1 5.3.6 Two-Generation Endosulfan Dietary Test (Quinn et al. 2010)

2 Exposure Levels and Protocol

3 Dietary endosulfan was administered at 0, 30, 60, 125, and 250 ppm. The two-generation 

4 experimental protocol was followed, with measurement of the range of endpoints described 

5 above in the protocol.

6 Sensitive Endpoints

7 There were differences in organ weights; however, many of the responses were non8 linear relative to dose.

9 Overall Conclusions

10 There was clear maternal transfer of endosulfan into the eggs, which resulted in 

11 significant effects at specific doses, primarily in the F1 generation. Observed effects included 

12 body weight, brain weight, kidney weight, and thyroid weight. There were some gender related 

13 differences in response, with exposed females in the 60ppm treatment having smaller ovaries 

14 (p=0.003) associated with fewer follicles (p=0.057).

15 5.3.7 Two-Generation Trenbolone Dietary Study (Quinn et al. 2011)

16 Exposure Levels and Protocol

17 Dietary exposure was 0, 0.5, 5, 10, and 20 ppm, beginning at 4 weeks of age in the 

18 parental generation, followed by a F1 generation maintaining treatment groups and an untreated 

19 F2 generation. Measurement endpoints were conducted according to the two-generation protocol, 

20 with some additional endpoints for assessing reproduction. The two highest doses are beyond 

21 those used in the one-generation dietary test described above.

22 Sensitive Endpoints

23 Females showed impacts from the two high doses, with reduced egg production in all 

24 generations to the extent that there was no F2 generation in the 20ppm dose level. Additionally, 

25 females showed delayed sexual maturation in the F0 and F1 generations.

26 Males showed effects of the higher doses of trenbolone. Although the timing of sexual 

27 maturation was unaffected, courtship and copulatory behaviors were impacted. There were minor 

28 effects on sperm motion; otherwise no other sperm characteristics were affected. Similarly, 

29 cloacal gland area differed only in the 0.5 ppm treatment, where there was reduced size. Organ 

30 weights were largely unaffected, with some bursal response noted.

31 Overall Conclusions

32 In males, sperm characteristics and quality were not the most sensitive measures of effect. 

33 Cloacal gland area reflected androgen stimulation; the low dose reduction may reflect inhibition 

34 of the HPG axis with insufficient combined trenbolone (androgen active EDC) and endogenous 

35 androgen stimulation. Male sexual behavior was affected; however, the results are somewhat 

36 complex and this testing paradigm will need further refinement to develop a clear testing 

37 protocol for multiple users. The author compares these findings to injection of 

38 dichlorodiphenyldicholoroethylene (p, p’-DDE), which is an anti-androgen, concluding that both 

39 inhibited male sexual behavior. The bursa remains a potential bioindicator of immune function 
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1 impacts. Female reproductive function was a responsive endpoint, culminating in the lack of F2 

2 offspring in the highest dose group. In total, trenbolone exerted endocrine disrupting effects, 

3 especially on the F1 generation.

4 5.3.8 Two-Generation 17β Trenbolone Dietary Study (Karouna-Renier and 

5 Henry, 2013)

6 Exposure Levels and Protocol

7 Effects of continuous exposure to 17β trenbolone (17βT) were assessed in three 

8 successive generations of Japanese quail. For the F0 generation, 6 week-old Japanese quail were 

9 randomly assigned to one of six doses of 17ß-trenbolone: 0, 1, 5, 10, 20, and 40 mg/kg feed 

10 (n=16 pairs per dose). Eggs were collected from F0 and F1over weeks 7 to 8 to generate pairs for 

11 the next generation. F1 birds were assigned to the same dose as their parents and received dosed 

12 feed from day 1 of hatch. The F2 birds were exposed solely in ovo and were provided only 

13 untreated feed. The 40 ppm dose was dropped after F0 since the pairs exposed to this dose were 

14 unable to produce sufficient numbers of eggs to populate the F1 generation. F0, F1 and F2 

15 parents were euthanized at 16 weeks of age to harvest tissues for genomic and biochemical 

16 analysis.

17 Sensitive Endpoints

18 Significant changes were observed in liver vitellogenin (VTG) mRNA, which decreased 

19 with dose in F0 females and increased in F1 females and F0 males. Plasma testosterone levels 

20 were decreased in F0 females with dose and were higher in the F0 (10, 20 and 40 ppm) and F2 

21 females (20 ppm). Plasma estradiol significantly increased in F0 and F1 males with dose; 

22 females showed isolated differences in estradiol levels with generation and dose. Fecal steroids 

23 mirrored differences in plasma steroids with showed treatment related differences; whereas, 

24 thyroid hormones did not differ with treatment. Bursa weights were lower across all treatments. 

25 Behavioral trials indicated differences in latency to mount and mounting success among the 

26 males in the 40ppm group. Behavior and some other endpoints are undergoing further analysis.

27 5.4 Transferability of the Avian Reproduction Test to an Avian 

28 Multi-Generation Test

29 The previous Reproduction Test accurately detects gross morphological and overall 

30 reproductive impairment with dietary test chemical exposure. These measurement endpoints are 

31 useful in detecting effects, but do not discern endocrine disruption from frank toxicological 

32 activity. Additionally, the endpoints measured throughout the multi-generational study and 

33 especially in the F1 generation, (body weight, potentially pre-pubertal organ histology) provide 

34 assessment of both toxicological effects and endocrine disruption. It is important for more 

35 sensitive endpoints, such as fertility, behavior, histolopathology of the gonads and bursa, 

36 hormone concentrations, and neuroendocrine measures, to be included to ascertain potential 

37 short and long-term endocrine disruption. In summary, the Avian Reproduction Test is not 

38 appropriate to discern endocrine disruption from toxicological effects and the test is relatively 

39 insensitive to more subtle EDC effects of environmental chemicals, especially at lower (i.e. 

40 environmentally relevant) exposures. A series of specific points addressing these items follows 

41 in the next section.
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1 5.5 Lessons Learned from Optimization Studies

2  Avian physiology and sexual differentiation must underpin measurement endpoints 

3 chosen for the testing paradigm

4  Need consistency in test design (photoperiod, timing of initiation of both photoperiod 

5 and dosing, dosing period, timing of collections, measures, and genetic strain)

6  Need to consider “low” and “environmentally relevant” doses

7  Standard reproductive measures are relatively insensitive, especially in dietary studies

8  The two-generation test provides a means of dosing various life stages and focusing 

9 on potential vulnerable periods

10  Egg injection studies tend to have clearer effects because the dose received may be 

11 more consistent or there is no chemical breakdown through the digestive tract; it is 

12 also possible that the primary route of exposure has not been delineated

13  Behavioral assessments were variable so it was difficult to show clear dose related 

14 differences; same for cloacal gland measurements

15  Male sexual behavior may be one of the most sensitive endpoints to estrogenic 

16 chemicals; however, the protocol must be standardized

17  Species differences in behavioral measures

18 

19 Sampling Points and Schedule

20  Egg injection studies showed that the embryo is the most vulnerable stage for 

21 precocial birds

22  In a diet test, the F1 is expected to be the most affected because both embryonic and 

23 dietary exposure occurs throughout the lifespan

24  Sampling times:

25 o At hatch to ascertain embryonic/developmental effects,

26 o During maturation to detect any acceleration or delay 

27 o Monitor growth and achieving adult status

28 o Adult onset of reproduction

29 o Productivity during adult reproduction, once full reproductive function has 

30 been attained (about 10 weeks of age)

31 o Sustained reproductive function and any evidence of health issues for at 

32 least 6 weeks of production

33 Histopathological Analyses

34  Histology is revealing if the suspected target has significant chronic effects

35  May be more revealing for toxic actions of the compounds

36  Altered thyroid system function does not usually result in easily discernable 

37 histological effects except if there is a goiter (collection of hormone precursors in the 

38 glandular follicles)

39 Secondary Sex Characteristics 

40  Male cloacal gland area is testosterone dependent

41  Plumage characteristic of male and female

42  Body size and weight

43  Relative body fat
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Avian Two-Generation ISR 54 May 28, 2013

1 Vitellogenin.

2  Has not been readily detectable in hatchlings

3  Not clearly a responsive endpoint

4  More characterization is needed to delineate when this would be a responsive 

5 endpoint and in what situations; specifically does vitellogenin change in females and 

6 how much do males respond? 

7 Sex Steroid Measurements

8  Plasma measurements may be more variable, but revealing with smaller volumes 

9 from hatchlings

10  Fecal samples reflect more consistent effects

11  Responsive endpoint if the reproductive axis is affected

12 Thyroid hormone measurements

13  Appropriate measurement endpoint for PCBs and PBDEs as well as other thyroid 

14 active compounds

15  Gland content reflects long term impacts; response can be increased (lack of secretion) 

16 or decreased (impaired hormone synthesis)

17  Plasma T3 and T4 reflect alterations in glandular function, axis function, as well as 

18 altered enzymes (deiodinases) activity which are important in the conversion of T4 to 

19 T3 or rT3 (reverse T3). More information is needed to develop a predictive model for 

20 chemical classes with predicted diagnostic responses 

21 Exposure Analyses

22  Analytical data are very useful in determining the amount of chemical that elicits 

23 adverse effects 

24  Establish threshold of impact and relative sensitivity of species and/or life stage at the 

25 time(s) of exposure

26  Studies indicate that embryonic exposures are at much lower dose than previously 

27 believed; meaning that the effective concentration of EDCs that result in adverse 

28 effects are much lower when bioavailability and embryonic uptake are considered

29 o More data are needed to accurately predict effects—suggest embryonic 

30 bioassay as a range finder and initial assessment for potential mechanism

31 Statistical Analyses

32  Need to consider and allow for non-linear data analysis

33  Analysis of many endpoints may dilute detection of effects

34 o Primary responsive endpoints must be identified for assessment

35 Parametric tests 

36  Preferred, but many of the data do not meet statistical assumptions

37  Depending on the measurement endpoint, the statistical analyses must be pre-planned 

38 to accommodate a range of issues that are inherent in the types of data to be collected

39  Assessment should include some acknowledgement of variability as an indicator of 

40 effect; specifically there may be increased (due to individual differences in sensitivity) 

41 or decreased variability (high mortality and survivor effect or delayed maturation) 

42 associated with endocrine disruption 
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1 Non-parametric tests 

2  Appropriate for behavioral tests and for ordinal data (cloacal gland area) 

3  Standardized methods should be prescribed so that there is consistency in analyses.

4

5

6

7
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1 6 INTERLABORATORY EVALUATION

2 The following discussion summarizes the laboratory results and statistical analysis from 

3 various studies intended to evaluate the protocol on both an intra-laboratory (within a single 

4 laboratory) and inter-laboratory (across all laboratories) basis. The reports documenting these 

5 studies and analyses are presented in Appendix C. Supporting demonstration studies were also 

6 sponsored by USEPA and performed by the U.S. Geological Survey and the U.S. Army, the 

7 results of which are given in Appendix E.

8 6.1 Vinclozolin—Bayer Crop Science Study

9 6.1.1 Exposure Levels and Protocol

10 Four treatment groups, each initially containing 18 pairs of Japanese quail, were fed diets 

11 containing the test substance at 64, 160, 400 & 1000 ppm a.i. through two generations (F0 and 

12 F1). A control group, also with 18 pairs initially, was maintained concurrently. A third 

13 generation (F2) was reared without exposure to the test substance through their diet. The F0

14 Japanese quail were genetically sexed after hatching and were 6 weeks old at test initiation. The 

15 F0 generation was exposed to treated diets from week 6 of age through an egg laying cycle of 

seven weeks. Selected embryos (Hatch E, 5th 16 group of eggs set from F0 hens) at embryo day15 

17 (ED15) were sampled for possible analysis of endocrine and histological impacts. Hatched F1

chicks (Hatch F & G, 6th & 7th 18 group of eggs set from F0 birds) were exposed to treated diets 

19 from hatch through rearing to 14 days of age. At one week of age surviving chicks were 

20 genetically sexed. Selected F1 offspring (Hatch F & G) maintained on treated diets for grow-out 

21 to four weeks of age. With exposure continuing, at three or four weeks of age the F1 birds were 

22 paired and monitored through a seven-week reproduction cycle. Selected F1 embryos (Hatch E) 

23 at embryo development day 15 (ED15) were sampled for possible analysis of endocrine and 

24 histological impact. Surviving F2 chicks were genetically sexed at one week of age and reared to 

25 14-days of age on untreated diet. Tissues of selected F2 14-day old survivors (Hatch F & G) 

26 were sampled for possible analysis of histological impact. Additional F2 birds were selected and 

27 reared to sexual maturation for mating behavior observations.

28 For each phase, effects of exposure to the test substance on bird health, body weight and 

29 feed consumption were evaluated. The effects on the number of eggs laid, viability of the 

30 embryos, normal development of embryos, percent hatchability, offspring survival and body 

31 weight, and eggshell thickness for both the F0 and F1 generations were evaluated. Selected tissue 

32 samples were collected from embryos, 14-day old chicks and adults for evaluation of 

33 biochemical endpoints such as serum steroid concentration. In addition, observations were 

34 conducted on subsets of F1 and F2 birds to evaluate possible effects of exposure on mating 

35 behavior. 

36 6.1.2 Sensitive Endpoints and Overall Conclusions

37 The summary of statistical analyses provided by Dr. Tim Springer (see Appendix C for 

38 details) included 106 variables, some of which were subjected to multiple comparisons. He 

39 conducted two types of analyses for each variable-by-generation cell. First, response to each test 

40 concentration was compared to the controls using Dunnett’s multiple comparison procedure. 

41 These responses were subjected to a Jonkheere-Terpstra trend test to provide an indication a 

42 concentration-related trend. When effects in one direction (i.e., increase or decrease) were clearly 
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Avian Two-Generation ISR 57 May 28, 2013

1 deleterious, and not deleterious in the other direction, a one-sided Dunnett’s test was used. 

2 Otherwise, a two-sided test was performed. The one-sided or two-sided test as well as the 

3 direction of the test if one-sided, is indicated in Table 6-1. In either case, the cell-wise type I 

4 error rate was controlled at α = 0.05. The Jonkheere-Terpstra trend test was performed 156 times 

5 and showed that 7 of the significant trends were likely to reflect a false discovery rate. Similarly, 

6 the Benjamini-Hochberg approach was applied to the results of the 624 p-values from the 

7 Dunnett’s test and revealed 13 false discoveries.

8 Most variables did not reveal significant differences due to treatment (Table 6-1); body 

9 weight and feed consumption showed some differences. The only other variables with p-values 

10 less than 0.05 for both Dunnett’s test and trend were Day14fE2 (day 14 female E2 concentration) 

11 and AmT3 (adult male T3). As such, there were few significant effects of the vinclozolin at the 

12 dietary doses in this study.

13 Table 6-1. Summary of Statistical Analyses Yielding Effects – Bayer.

Obs Variable Name Generation N

a

of Dunnett p < 0.05 N

b

of JT p < 0.05 Bothc

Body Weight

1 bwF0 (body wt) F0 0 1 N

2 bwF0 F1 2 1 Y

11 offbw14 (chk body wt) F1 0 1 N

Feed Consumption

13 AdultFC1 (feed consumption) F0 1 1 Y

14 AdultFC1 F1 1 1 Y

15 AdultFC2 F0 0 1 N

17 AdultFC3 F0 0 1 N

20 AdultFC4 F1 3 1 Y

21 AdultFC5 F0 0 1 N

22 AdultFC5 F1 0 1 N

23 AdultFC6 F0 2 1 Y

24 AdultFC6 F1 0 1 N

25 AdultFC7 F0 0 1 N

26 AdultFC7 F1 2 1 Y

Reproduction

71 FG14Day_Hatch F0 1 0 N

First Foam First Egg Cloacal Size

95 CloacaF6 F1 2 0 N

Mating Behavior

103 CrowFluff1 F1 1 0 N

106 ChangeCF F1 1 0 N

Thyroid Weight

122 ThyAdult Female F1 1 0 N

Bio Chem

127 Day14fE2 F2 1 1 Y

132 Day14mT F2 1 0 N

143 AmE2 F0 1 0 N

147 AmT3 F0 1 1 Y

149 AmT4 F0 1 0 N

a Number of p-values from Dunnett’s test that were < 0.05

a

Value is 1 if the Johkheere-Terprstra trend test p-value was < 0.05

c

Both = Y if there was at least one Dunnett p-value < 0.05, and the Johnkheere-Terpstra trend test p-value 

 was also < 0.05 (see Springer report for more detail)

14

15
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1 6.2 Vinclozolin – Wildlife International Study

2 6.2.1 Exposure Levels and Protocol

3 This trial was conducted according to the same protocol as described above. The primary 

4 difference was that the Japanese quail used in this trial were from the Wildlife International quail 

5 colony as compared to birds purchased from breeders by the other entities performing the inter6 laboratory comparison two-generation trials.

7 6.2.2 Sensitive Endpoints and Overall Conclusions

8 Summary of statistical analyses (provided by Dr. Tim Springer; see Appendix C for 

9 details) showed evidence of few effects due to dietary vinclozolin exposure for the 106 variables 

10 analyzed (Table 6-2). The analysis was identical to that performed for the Bayer Crop Science 

11 multi-generation study. Seven variables gave Dunnett’s test p-values of less than 0.05 for at least 

12 one treatment group, and trend tests for nine variables suggested a concentration response trend 

13 (i.e. p < 0.05). Variables with Dunnett p-values < 0.05 were CloacaM4, CloacaF6, CloacaM6, 

and CloacaM7 (male or female cloacal area (mm214 ) at 4, 6, and 7weeks) in the F1 generation. The 

15 variables ThyF214DayM (Thyroid weight of day 14 birds in the F2 generation, AfE2 (adult 

16 female E2) in the F0 generation and AfT (adult female testosterone) in the F1 generation also 

17 gave Dunnett p-values < 0.05. Table 6-2 shows variables with at least one p value less than 0.05 

18 according to Dunnett’s test or p value for trend of less than 0.05 according to the Jonkheere19 Terpstra trend test. The nine variables with Jonkheere-Terpstra p values less 0.05 are shown.

20 Table 6-2. Summary of Statistical Analyses Yielding Effects – Wildlife International.

Obs Variable Name Generation N

a

of Dunnett p < 0.05 N

b

of JT p < 0.05 Bothc

Feed Consumption

13 AdultFC1 F0 0 1 N

Reproduction

53 FGViable F0 0 1 N

Thyroid Weight

95 ThyF214DayM F2 1 0 N

156 ThyWtED15 F2 0 1 N

First Foam First Egg Cloacal Size

101 CloacaF6 F1 1 1 Y

105 CloacaM4 F1 1 1 Y

107 CloacaM6 F1 1 1 Y

108 CloacaM7 F1 1 0 N

109 CloacaM8 F1 0 1 N

110 CloacaM9 F1 0 1 N

Mating Behavior

122 CrowFluff2 F2 0 1 N

Bio Chem

167 AfE2 F0 1 0 N

170 AfT F1 1 1 Y

182 AmT4 F1 0 1 N

a Number of p-values from Dunnett’s test that were < 0.05

a

Value is 1 if the Johkheere-Terprstra trend test p-value was < 0.05

c

Both = Y if there was at least one Dunnett p-value < 0.05, and the Johnkheere-Terpstra trend test p-value 

 was also < 0.05. Otherwise Both = N.

21
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1 When both the trend test and Dunnett’s tests have low p-values, there is evidence for both 

2 a relatively large effect and a concentration response trend. Observing low probabilities for both 

3 tests is stronger evidence for a meaningful response than detecting a large change without 

4 concentration response, or slight concentration related trend where response is small. Only three 

variables, CloacaM4 (male cloacal area (mm2) at 4 weeks), CloacaM6 (male cloacal area (mm25 ) 

6 at 6 weeks) and AfT (adult female testosterone) for F1 generation birds, had at least one 

7 Dunnett’s p-value of less than 0.05, and also showed a concentration response trend with p < 

8 0.05.

9 6.3 Vinclozolin – Smithers Viscient Carolina Research Center Study

10 6.3.1 Exposure Levels and Protocol

11 Four treatment groups, each initially containing 18 pairs of Japanese quail, were fed diets 

12 containing the test substance at 64, 160, 400 & 1000 ppm a.i. through two generations (F0 and 

13 F1). A control group, also with 18 pairs initially was maintained concurrently. A third generation 

14 (F2) was reared without exposure to the test substance through their diet. The approximate 

15 duration of the study was eight to nine months. Primary phases of the study included: production 

16 of test birds (hatching, rearing and DNA sexing) - 4 weeks, acclimation to reproduction test pens 

17 - 2 weeks, F0 egg production - 7 weeks, F0 post egg production (embryo sampling, adult 

18 termination and sampling, and final incubation) - 2 weeks, F1 hatching and rearing to sexual 

19 maturation (DNA sexing and selection of birds for second phase of reproduction and mating 

20 behavior observations) - 6 weeks, F1 egg production - 7 weeks, F1 post egg production (embryo 

21 sampling, adult termination and sampling, and final incubation) - 2 weeks, F2 hatching and 

22 rearing (DNA sexing and 14-day old offspring sampling) - 2 weeks, F2 rearing to sexual 

23 maturation (for mating behavior observations) – 6 weeks. Behavioral assessments were made for 

24 males only in the control and high treatment groups for a subset of 7 males in each. Blood 

25 samples were drawn by cardiac puncture, including samples taken from ED 15 embryos. Data 

26 were collected for the following endpoints: adult body weight, adult feed consumption, eggs laid 

27 per pen per day (eggs laid/maximum laid) - Days counted from test initiation for F0 and from the 

28 first egg layed by any bird for F1 and potentially F2, eggs cracked of eggs laid, fertile eggs of eggs 

29 incubated, viable embryos of eggs incubated, live 2-week embryos of viable embryos, hatchability 

30 of 2-week embryos, 14-day old survivors of hatchlings, hatchlings of eggs incubated, hatchlings of 

31 fertile eggs, hatchlings per pen per day (hatchlings/maximum set), 14-day old survivors of eggs 

32 incubated, 14-day old survivors per pen per day (14-day olds/maximum set), hatchling body 

33 weights, 14-day old survivor body weights, egg shell thickness, reproductive behavior, and 

34 secondary sex characteristics/phenotype.

35 6.3.2 Sensitive Endpoints and Overall Conclusion

36 There were no treatment related effects noted. No effects were observed in any 

37 generation related to mating behavior or reproductive parameters. As such, the NOEC was 

38 determined to be 1000 a.i./kg feed, which was the highest concentration.

39 Statistical analyses of 106 variables (provided by Dr. Tim Springer; see Appendix C) 

40 revealed that 21 variables gave Dunnett’s test p-values of less than 0.05 (Table 6-3), and the 

41 trend tests for 9 variables suggested that a concentration response trend was present (i.e. p < 

42 0.05). These data are shown below. However, the Benjamini-Hochberg procedure indicated that 
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Avian Two-Generation ISR 60 May 28, 2013

1 a number of these differences might be false discoveries; similarly there is reduced confidence in 

2 differences when The Jonkheere-Terpstra Trend Test was applied.

3 Table 6-3. Summary of Statistical Analyses Yielding Effects – Smithers Viscient.

Obs Variable Name Generation N

a

of Dunnett p 

< 0.05

N

b

of JT p < 

0.05

Bothc

Feed Consumption

29 AdultFC9 F0 1 1 1

Reproduction

33 Laid_Hen_Day F0 1 0 0

66 FG2Wk_Via F1 1 0 0

70 EFG2Wk_Via F1 1 0 0

Mating Behavior

109 CloCon3 F1 1 0 0

113 CrowFluff1 F1 3 1 1

119 ChangeCF F1 1 1 1

121 Mount1 F1 0 1 0

131 Attempts2 F1 1 0 0

135 ChangeAt F1 1 0 0

Thyroid Weight

145 ThyF214DayM F2 1 0 0

146 ThyF214DayF F2 1 0 0

147 ThyAdultMale F0 1 0 0

148 ThyAdultMale F1 1 1 1

Bio Chem

155 Day14fE2 F2 1 0 0

161 Day14mT3 F2 1 0 0

168 AfT3 F1 1 0 0

169 AfT4 F0 0 0 0

170 AfT4 F1 0 1 0

171 AmE2 F0 1 0 0

174 AmT F1 1 1 1

176 AmT3 F1 0 1 0

179 YolkE2 F0 1 0 0

180 YolkE2 F1 0 1 0

a Number of p-values from Dunnett’s test that were < 0.05

a

Value is 1 if the Johkheere-Terprstra trend test p-value was < 0.05

c

Both = Y if there was at least one Dunnett p-value < 0.05, and the Johnkheere-Terpstra trend test p-value 

 was also < 0.05. Otherwise Both = N.

4

5 6.3.3 General Advantages of Japanese Quail Two-generation Test Reproducibility of 

6 the assay

7 Interlaboratory comparisons were conducted to determine if there were adverse 

8 consequences of exposure to putative endocrine active substances in a two-generation testing 

9 paradigm and to ascertain reliability of the testing protocol. The interlaboratory tests were 

10 sponsored by EPA and conducted in three laboratories: Smithers Viscient LLC (SV), Bayer 

11 CropScience Inc. (later renamed SynTech, or ST), and Wildlife International (WLI). All these 

12 laboratories carried out tests with vincolozlin at 64, 160, 400, and 1000 ppm concentration levels. 

13 The intra-laboratory analyses were performed for each laboratory’s data by WLI and have been 

14 summarized above.
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1 The interlaboratory analysis compared first, control group results among the three 

2 laboratories to assess the range of variation within laboratory and among laboratories and the 

3 power analyses based on the control groups to detect alternatives from the control groups within 

4 a laboratory that can be detected with high power, based on average within laboratory variability. 

5 Second, the treatment and control group results were compared across laboratories. Third, 

6 generations were compared across laboratories.

7 Comparison of the data from the three laboratories shows evidence for the following:

8  There are clear differences in strains of Japanese quail in terms of body weight, and 

9 related variables that contribute to somewhat large differences and ranges in 

10 measurement endpoints

11  Strain differences may also be a factor in rate of maturation and reproductive 

12 characteristics, 

13  Some variables also show intra-assay variability; it is not clear if this is indicative of an 

14 unreliable measurement endpoint or if the method for measurement requires refinement. 

15 o Behavioral measures may be improved with more precise testing methods, 

16 specifically consistency in the number and treatments subjected to testing and 

17 potentially use of one testing day rather than three successive days

18  The primary measures indicative of endocrine disruption require a multi-generation test 

19 protocol in order to discern vulnerability on specific life stages such as embryos as 

20 opposed to adult stages of the life cycle.

21 6.3.4 Data Interpretation

22 The inter-laboratory comparison provides insights on sources of variation due to the 

23 testing paradigm and other factors. Vinclozolin did not exert strong effects in any of the inter24 laboratory trials. Comparison of control birds across the three laboratories revealed that strain 

25 differences are critical and in fact the Japanese quail strain that is closest to wild type tends to be 

26 smaller, matures more rapidly, and is generally more vigorous. More highly selected strains may 

27 also lose their photoresponsiveness and may show altered reproductive system function that 

28 differs from wild birds. In addition, comparison of control birds revealed variability in some of 

29 the selected measurement endpoints, which could be indicative of the level of power needed to 

30 discern endocrine disruption. Conversely, an EDC given in a dose response venue would be 

31 expected to overcome individual variation in response.

32 Vinclozolin, which was chosen for this inter-laboratory comparison, did not exhibit a 

33 substantial response in Japanese quail. Based on the biological basis of sexual differentiation, 

34 this is not a completely surprising outcome. Although effects were observed with egg injection

35 experiments, the levels achievable by this type of direct treatment were effectively higher than 

36 those associated with maternal deposition. Further, the mechanism of action of vinclozolin is as 

37 an anti-androgen. Sexual differentiation in avian species is genomic with females being the 

38 heterogametic (ZW) sex and males being the homogametic (WW) sex. The heterogametic female 

39 relies on estradiol exposure to differentiate; whereas, the male is often considered more similar 

40 to the mammalian female as the homogametic sex. It should be pointed out that similar to 

41 mammals, both genders do produce steroid hormones necessary for development of accessory 

42 sex structures. However, an anti-androgen would not be predicted to alter sexual differentiation 

43 of the female hypothalamus and unlikely to impact the male except at high levels. Conversely, 
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1 estrogen active and strong androgen active compounds did have adverse effects in both males 

2 and females in the optimization studies. Furthermore, thyroid active compounds also impact 

3 avian species.

4 Table 6-4 summarizes the coefficients of variability (CV) across laboratories by category. 

5 Table 6-5 summarizes and the ranges of percent deviations from control group means for 

6 detections with 80% power. For each parameter the CV across labs and the associated p-value 

7 for significance of the laboratory random variation (i.e., differences among laboratories) for each 

8 generation were summarized. The ranges of CVs were compared across categories. The 

9 following are some findings:

10  The median of F1 CVs exceeds that of the F0 CVs for 27 of 57 (47%) comparsions. This 

11 is nearly 50%, so there is no systematic generation difference with respect to CVs. Some 

12 of the highest CVs are in the F1 generation, particularly for mating behavior.

13  The median of F2 CVs exceeds that of the F1 CVs in 15 of 24 (62.5%) comparisons. This 

14 possibly suggests some degradation in sensitivity in F2 compared to F1. 

15  The smaller CVs for body weight in the F2 generation may be due to thefact that adult 

16 body weights were not measured for the F2 generation.

17 The ranges of percent deviations to attain high power were compared across categories. 

18 F1 was compared to F0 for six of nine categories. The F1 percent deviation exceeded the F0 

19 percent deviation for 34 of 57 comparisons (60%). This possibly suggests some degradation in 

20 sensitivity in F1 compared to F0. The F2 percent deviation exceeded the F1 percent deviation for 

21 14 of 24 comparisons (58%). This again possibly suggests some degradation in sensitivity in F2 

22 compared to F1.

23 Table 6-4. Ranges of CV% Across Laboratories (by Category).

F0 Generation F1 Generation F2 Generation # F1 > 

F0

# F2 > 

Category CV Range Median CV Range Median CV Range Median F1

Body Weight 27.3 – 29.6 28.1 17.2 – 31.1 28.3 16.8 – 24.8 16.8 3/4 0/2

Feed Consumption 16.9 – 25.9 19.0 2.5 – 26.2 18.7 NA NA 5/8 NA

Reproduction 0.54 –103.3 10.7 0.59 - 120.8 9.1 NA NA 12/29 NA

Egg Shell and Weight 7.6 – 19.6 10.8 6.7 – 20.5 11.2 NA NA 2/4 NA

First Foam First Egg 

Cloacal Size NA NA 5.8 – 81.9 18.0 NA NA NA NA

Mating Behavior NA NA 12.4 – 272.5 57.7 31.8 – 208.8 79.3 NA 13/20

Thyroid Weight 30.7 – 49.8 40.2 30.3 – 42.3 40.3 14.5 – 53.5 50.6 1/2 1/1

Sex Ratio NA NA 9.1 – 9.1 9.1 13.1 – 13.1 13.1 NA 1/1

Bio Chem 17.0 – 57.5 34.2 12.5 – 67.8 23.8 18.1 – 81.5 49.6 4/10 NA

Total 0.54 – 103.3 17.4 0.59 – 272.3 23.1 13.1 – 208.8 63.6 27/57 15/24

24

25
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1 Table 6-5. Ranges of Percent Deviations from Control Group Means for Detections with 80% Power and the Ratios Between 

2 Generations (by Category).

Category

Percent Distances from Control

Ratio between Generation in Percent 

Distances from Control

# F1 > 

F0

# F2 > 

F1

F0 Generation F1 Generation F2 Generation F1/F0 F2/F1

Range Median Range Median Range Median Range Median Range Median

Body Weight 7.282-10.463 8.687 6.870-10.835 8.636 8.043-8.450 8.246 0.983-1.226 1.079 1.012-1.171 1.092 3/4 0/2

Feed 

Consumption 8.446-14.503 11.583 9.101-14.263 12.560 NA NA 0.628-1.689 0.987 NA NA 3/8 NA

Reproduction 1.834-265.997 17.079 3.962-192.231 21.977 NA NA 0.402-2.161 1.208 NA NA 22/29 NA

Egg Shell and 

Weight 6.470-14.692 13.411 6.166-11.911 10.318 NA NA 0.722-0.953 0.816 NA NA 0/4 NA

First Foam 

First Egg 

Cloacal Size

NA NA 11.818-70.697 35.149 NA NA NA NA NA NA NA NA

Mating 

Behavior NA NA 80.840-732.628 153.293 96.549-3361.243 294.521 NA NA 0.462-9.463 1.035 NA 11/20

Thyroid Weight 40.219-53.943 47.081 38.273-48.355 45.910 39.774-57.242 45.884 0.851-1.202 1.027 1.199-1.199 1.199 1/2 1/1

Sex Ratio NA NA 40.136-40.136 40.136 34.590-34.590 34.590 NA NA 0.862-0.862 0.862 NA 1/1

Bio Chem 43.085-273.307 97.290 44.612-258.880 91.890 43.803-198.495 135.169 0.617-2.681 0.993 NA NA 5/10 NA

Total 1.834-273.307 16.726 3.962-732.628 31.371 8.043-3361.243 138.156 0.402-2.681 1.076 0.462-9.463 1.035 34/57 14/24

3

4
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1 6.3.5 Other Considerations for Multi-Generation Tests

2 Low Doses, Non-Linear Dose Responses, and Windows of Exposure

3 Determining endocrine disruptive effects of environmental chemicals requires a 

4 drastically different approach than the approach to determining classic toxicological effects such 

5 as lethality, weight changes, organ disruption, and teratogenic mutagenic actions. In order to 

6 understand the full extent of the endocrine disruptive effects of compounds, the design and 

7 interpretation of the EDC studies need to be firmly based in the principles of endocrinology:

8 1) Windows of exposure—the timing of exposure to EDCs is extremely important. Data 

9 indicate that precocial birds are primarily vulnerable to EDC exposure during embryonic 

10 development, likely due to sexual differentiation during embryonic and early post hatch 

11 phases of development. Effects of embryonic exposure to EDCs are manifest during 

12 sexual maturation and in adults.

13

14 2) Concentration Response—endocrine responsive tissues and systems may be affected 

15 differently than understood from classic toxicology studies. Even modest exposure 

16 concentrations of EDCs can interact with endogenous hormones that are often at pg17 ng/ml concentrations. Moreover, responses can be drastically different at different points 

18 along a concentration response curve. Results should be studied for trends and 

19 appropriate statistical models will be needed. For example, there was a non-linear 

20 response to vinclozolin in Japanese quail in a study by McGary et al. (2001). However, 

21 the increase in GnRH peptide concentrations occurred at mid-doses (inverted “U” shaped 

22 curve) and there was a “U” shaped response in copulatory behavior. Because these data 

23 were not analyzed to determine non-linear responses, there may have been less ability to 

24 detect statistical significance. While it is important not to use statistical analysis to extract 

25 significance when there is none, it is also important to take into account physiological 

26 relevance (i.e., the non-linear action of hormones) when deciding on the correct statistical 

27 model.

28

29 3) Strain/Species Variability— Bennett et al. (2001) state that “There are data that show 

30 strains of Japanese quail may be quite variable in reproductive and growth characteristics. 

31 For example, many of the strains common to Europe have been selected for rapid growth 

32 and higher body weight. They may weigh 250 to 300 gm. In comparison, most the 

33 random bred strains in the USA and Japan tend to have lower body weight (100 to 

34 150 gm). Some comparisons between strains exist for endocrine characteristics, which 

35 have shown that the heavier strains tend to be slower to mature, have lower productivity, 

36 have shorter periods of peak production, and some strains have lower fertility (Van Krey 

37 et al. 1983; Zelenka et al. 1984; Anthony et al. 1991; Lilja and Marks 1991; Marks and 

38 Washburn 1991a, 1991b; Peebles and Marks 1991; Yang et al. 1998; Aggrey et al. 2003). 

39 Conversely, some of the smaller birds, especially the Hawaiian feral colony may be more 

40 flighty. The smaller strains are preferable for long-term tests as the fertility and 

41 productivity tend to stay at peak for a longer period of time than the larger strains. 

42 However, even these birds show an age-related decline in reproductive performance. It is 

43 not clear which strains are more aggressive. Some of the larger strains may be more 

44 sedentary, but coincidentally may be less sexually aggressive as well.
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1

2 4) Eggshells of Japanese quail tend to have mottled coloration. Some strains have primarily 

3 light colored or white shells. This is desirable for easy candling of eggs, but is not a 

4 critical factor to consider. Current data suggest there are no differences in the shell 

5 strength across strains.

6

7 5) Birds have altricial and precocial reproductive and developmental strategies. Altricial 

8 species include song birds have post-hatch differentiation of the song system and 

9 copulatory behavior. Often, both males and female share incubation and taking care of 

10 the young. Altricial birds appear to be less sensitive to effects of EDCs during embryonic 

11 development, but may remain more vulnerable to EDC impacts as adults, with impaired 

12 singing, mating, and parental behaviors. Altricial birds studied in the laboratory include 

13 zebra finches, ring doves, and some other species; a number of studies have contributed 

14 to a substantial literature. Precocial birds that have been studied in the laboratory include 

15 Japanese quail, mallard ducks, and domestic chickens. The advantage of precocial birds 

16 as a model species for EDC studies is that these species are established models with a 

17 great deal of information. They are easily managed in the laboratory and mature rapidly.

18

19 6) The process of sexual differentiation in birds is a unique process; thereby making it 

20 critical to test an avian model for effects of EDCs.

21
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1 7 CONCLUSIONS

2 7.1 General Advantages of Japanese Quail Two-generation Test

3  Precocial avian model that matures rapidly

4  Well characterized with a great deal of information available

5  Multi-generation test can be completed in 12 months for the in life portion of the 

6 test

7  Methods and assays have been validated for this species

8  As more information becomes available, it is possible to develop straightforward 

9 assays and tests that elucidate mechanisms 

10  Japanese quail appear moderately sensitive to the effects of EDCs, thereby 

11 providing an indication that is more broadly applicable across avian species

12  Quail are precocial; chicks can be treated via dietary administration 

13  The wild type laboratory strain retains photoperiodicity and other characteristics 

14 of wild populations

15  The immune, adrenal, and thyroid systems have been well characterized.

16 7.2 Limitations of the Japanese Quail Two-generation Test

17  There are ontogenetic differences in the pattern and timing of hormone changes 

18 during development for the reproduction and thyroid endocrine systems for 

19 precocial and altricial birds 

20  Japanese quail may vary across strains, with birds selected for heavy body weight 

21 maturing more slowly and having less robust reproductive function

22  Japanese quail provide a model species that does not completely emulate all birds, 

23 especially very long lived species.

24 7.3 Value-added of Multi-generation Exposure

25  Data indicate that adult birds are generally less vulnerable to adverse effects of 

26 EDCs

27  It is critical to have the F1 generation and then to observe the capability of the F1 

28 to successfully reproduce viable offspring

29  Some chemicals may exert epigenetic effects that would have long term impacts 

30 on wild population over multiple generations, even without continued EDC 

31 exposure. The F2 generation provides a test for this type of adverse effect

32  Each portion of the multi-generation test protocol provides a unique treatment in 

33 that the P0 has maturation and adult exposure, the F1 has embryonic and life long 

34 exposure, and the F2 tests solely embryonic exposure. Together, all the salient 

35 and vulnerable life stages are tested.

36  It is important to incorporate both endocrine and behavioral tests to discern 

37 endocrine disruption at various targets, including neural and direct organ system 

38 effects.
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1 7.4 Complementary Technologies to the Japanese Quail Two-generation 

2 Test

3 Other kinds of testing paradigms may be considered, but at this point, the two-generation 

4 Japanese quail test is recommended for detection of endocrine disruption. Moreover, current 

5 technologies available for fish and mammals are now becoming available for avian species.

6 These technologies, including ELISAs and epigenetic endpoints, are expected to provide 

7 sensitive and precise detection of more subtle alterations due to endocrine disruption.
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1 9 List of Appendices

2

3 APPENDIX A. VALIDATION OF SCREENING AND TESTING ASSAYS 

4 PROPOSED FOR THE EDSP (JULY 2007)

5 APPENDIX B. DETAILED REVIEW PAPER FOR AVIAN TWO-GENERATION 

6 TOXICITY TEST (OECD NO. 74, AUGUST 2007)

7 APPENDIX C. REPORTS ON THE INTER-LABORATORY TRIALS FOR THE 

8 JQTT

9 C.1 Inter-laboratory Statistical Analysis, 2013

10

11 C.2 Vinclozolin Study (TO35) by SynTech (Bayer CropScience), 2013

12

13 C.3 Vinclozolin Study (185-133) by Wildlife International, 2013

14

15 C.4 Vinclozolin Study (13888.4106) by Smithers Viscient, 2013

16

17 C.5 Statistical Analysis for Vinclozolin Study Performed at Bayer (185-133-1), prepared by 

18 Wildlife International, 2013

19

20 C.6 Statistical Analysis for Vinclozolin Study Performed at Wildlife International (185-133-

21 3), prepared by Wildlife International, 2013

22

23 C.7 Statistical Analysis for Vinclozolin Study Performed at Smithers Viscient (185-133-2), 

24 prepared by Wildlife International, 2013

25

26 APPENDIX D. AVIAN TWO-GENERATION TOXICITY TEST IN THE 

27 JAPANESE QUAIL, OECD GUIDELINE FOR TESTING OF CHEMICALS, DRAFT, 

28 SEPTEMBER 2011

29 APPENDIX E. SUPPORTING TWO-GENERATION DEMONSTRATION 

30 STUDIES

31 E.1 DRAFT Trenbolone Quail Study Performed by U.S. Geological Survey, 2013

32

33 E.2 DRAFT Endosulfan Quail Study (87-XE-08JG-10) Performed by U.S. Army Public 

34 Health Command, 2010.

35

36 E.3 Trenbolone Quail Study (87-XE-0DUZ-11) Performed by U.S. Army Public Health 

37 Command, 2011.