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## Valida tion of the Japanese Quail Two

- G eneration 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 2013DRAFT

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- Generation ISR ii May 2 8

, 2013 

### Contents 1

Page 2

EXECUTIVE SUMMARY

................................ ................................ ................................ .......... 1 3

OBJECTIVE

................................ ................................ ................................ ................................ . 3 4

1 INTRODUCTION

................................ ................................ ................................ ............. 3 5

1.1 Endocrine Disr uptor Screening Program

................................ ................................ 4 6

1.2 Validation

................................ ................................ ................................ ................ 5 7

2 OVERVIEW OF THE JAPANESE QUAIL TWO

- GENERATION TEST

............... 8 8

2.1 Background on Endocrine Disruption in Avian Species

................................ ........ 8 9

2.2 Relevance of the Japanese Quail Two

- generation Test

................................ ........ 10 10

2.3 Purpose of the Japanese Quail Two

- generation Test

................................ ............ 11 11

2.4 Overview of Japanese Quail Biology & Reproduct ive Endocrinology

................ 11 12

2.4.1 Altricial versus precocial birds: Development Patterns and 13

Vulnerable Stages

................................ ................................ ..................... 11 14

2.4.2 Post Hatch Growth and Maturation

................................ .......................... 16 15

2.5 Rationale for the Assay

................................ ................................ ......................... 17 16

3 PROTOCOL DEVELOPMENT

................................ ................................ ................... 19 17

3.1 Summary of the Japanese Quail Two

- generation Test Protocol

........................... 19 18

3.2 Study Design, Test Chemicals and Dose Selection

................................ .............. 19 19

3.3 Preparation and Monitoring of the Test Die t

................................ ........................ 20 20

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

......... 21 21

3.5 Feeding

................................ ................................ ................................ .................. 22 22

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

................................ .................... 23 23

3.7 Experimental Design

................................ ................................ ............................. 23 24

3.8 Offspring

– Eggs

................................ ................................ ................................ ... 23 25

3.9 Egg Para meters

................................ ................................ ................................ ..... 25 26

3.10 Chicks

................................ ................................ ................................ ................... 26 27

3.11 Pathology

................................ ................................ ................................ .............. 26 28

3.12 Biochemical/endocrine Measurements and Observations

................................ .... 27 29

3.13 Behavioral Measurements

................................ ................................ ..................... 27 30

4 BACKGROUND AND RATIO NALE FOR SELECTION O F ENDPOINTS

.......... 29 31

4.1 Endpoints

................................ ................................ ................................ .............. 29 32

4.1.1 Survival

................................ ................................ ................................ ..... 29 33

4.1.2 Food Consumption

................................ ................................ .................... 30 34

4.1.3 Body Weight

................................ ................................ ............................. 30 35

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

production)

................................ ................................ ................................ 31 37

4.1.5 Sexual Maturation and Puberty

................................ ................................ . 31 38

4.1.6 Behavior (activity, lacrimation, rep roductive behaviors, etc.)

.................. 32 39

4.1.7 Fecundity (egg production)

................................ ................................ ....... 32 40

4.1.8 Fertilization Success

................................ ................................ ................. 33 41

4.1.9 Embryo viability

................................ ................................ ....................... 33 42

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

eggshell color)

................................ ................................ ........................... 33 44DRAFT

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4.1.11 Hatchability (ED15, hatch)

................................ ................................ ....... 35 1

4.1.12 Genotypic Sex Determination

................................ ................................ ... 35 2

4.1.13 Histopathology

................................ ................................ .......................... 35 3

4.1.14 Neuroendocrine Systems Regulating Reproduction, Metabolism, 4

and Stress

................................ ................................ ................................ .. 38 5

4.1.15 Key Neuroendocrine Measures for Endocrine Disruption

........................ 39 6

5 PROTOCOL OPTIMIZATIO N AND DEMONSTRATION

................................ ..... 42 7

5.1 Comparability with the Avian Reproduction Test Currently in Use

.................... 42 8

5.2 Egg Injection Studies

................................ ................................ ............................ 42 9

5.2.1 Atrazine (Experimental Pathology Laboratories 2006)

............................ 43 10

5.2.2 Trenbolone

................................ ................................ ................................ 43 11

5.2.3 Estradiol

................................ ................................ ................................ .... 45 12

5.2.4 Vinclozoli n (McGary et al. 2001)

................................ ............................. 46 13

5.2.5 Other Egg Injection Treatments

................................ ................................ 46 14

5.3 Reproduction and Two

- Generation Dietary Studies

................................ ............. 46 15

5.3.1 Perchlorate (Stafford 2011)

................................ ................................ ....... 46 16

5.3.2 Bisphenol A (Frey et al. 2011)

................................ ................................ .. 47 17

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

- Dietary 18

Study #1

................................ ................................ ................................ .... 48 19

5.3.4 Methoxychlor (dietary study with Japanese quail and bobwhite 20

quail comparisons)

................................ ................................ .................... 48 21

5.3.5 Two

- Generation Dietary 17

- Beta Estradiol (E2) Dosing Study

............... 49 22

5.3.6 Two

- Generation Endosulfan Dietary Test (Quinn et al. 2010)

................. 51 23

5.3.7 Two

- Generation Trenbolone Dietary Study (Quinn et al. 2011)

.............. 51 24

5.3.8 Two

- Generation 17β Trenbolone Dietary Study (Karouna

- Renier 25

and Henry, 2013)

................................ ................................ ...................... 52 26

5.4 Transferability of the Avian Reproduction Test to an Avian 27

Multi

- Generation Test

................................ ................................ ........................... 52 28

5.5 Lessons Learned from Optimization Studies

................................ ........................ 53 29

6 INTERLABORATORY EVAL UATION

................................ ................................ ..... 56 30

6.1 Vinclozolin

— Bayer Crop Science Study

................................ ............................. 56 31

6.1.1 Exposure Levels and Protocol

................................ ................................ .. 56 32

6.1.2 Sensitive Endpoints and Overall Conclusions

................................ .......... 56 33

6.2 Vinclozolin

– Wildlife International Study

................................ ........................... 58 34

6.2.1 Exposure Levels and Protocol

................................ ................................ .. 58 35

6.2.2 Sensitive Endpoints and Overall Conclusions

................................ .......... 58 36

6.3 Vinclozolin

– Smithers Viscient Carolina Research Center Study

....................... 59 37

6.3.1 Exposure Levels and Protocol

................................ ................................ .. 59 38

6.3.2 Sensitive Endpoints and Overall Conclusion

................................ ............ 59 39

6.3.3 General Advantages of Japanese Quail Two

- generation Test 40

Reproducibility of the assay

................................ ................................ ...... 60 41

6.3.4 Data Interpretation

................................ ................................ .................... 61 42

6.3.5 Other Considerations for Multi

- Generation Tests

................................ .... 64 43

7 CONCLUSIONS

................................ ................................ ................................ ............. 66 44

7.1 General Advantages of Japanese Quail Two

- generation Test

.............................. 66 45DRAFT

Avian Two

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, 2013 7.2 Limitations of the Japanese Quail Two

- generation Test

................................ ...... 66 1

7.3 Value

- added of Multi

- generation Exposure

................................ .......................... 66 2

7.4 Complementary Technologies to the Japanese Quail T wo

- generation Test

......... 67 3

8 REFERENCES

................................ ................................ ................................ ................ 68 4

9 LIST OF APPENDICES

................................ ................................ ................................ 89 5

6

APPENDIX A. VALIDATI ON OF SCREENING AND TESTING ASSAYS 7

PROPOSED FOR THE EDS P (JULY 2007)

................................ ................................ 89 8

APPENDIX B. DETAILED REVIEW PAPER FOR AVI AN TWO

- GENERATION 9

TOXICITY TEST (OECD NO. 74, AUGUST 2007)

................................ ................... 89 10

APPENDIX C. REPORTS ON THE INTER

- LABORATORY TRIALS FO R THE 11

JQTT

................................ ................................ ................................ ................................ 89 12

APPENDIX D. AVIAN TW O

- GENERATION TOXICITY TEST IN THE 13

JAPANESE QUAIL, OECD GUIDELINE FOR TESTIN G OF CHEMICALS, 14

DRAFT, SEPTEMBER 201 1

................................ ................................ ......................... 89 15

APPENDIX E. SUPPORTI NG TWO

- GENERATION DEMONSTRA TION 16

STUDIES

................................ ................................ ................................ ......................... 89 17

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### Tables

Page

Avian Two

- Generation ISR v May 2 8 , 2013 Table 1

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

.............. 5 1

Table 3

- 1. Housing Conditions.

................................ ................................ ............................. 22 2

Table 3

- 2. Recommended Nutritrional Values.

................................ ................................ ...... 22 3

Table 3

- 3. Conditions for Egg Storage, Incubation, and Hatching.

................................ ....... 24 4

Table 3

- 4. Typical Values for Reproductive Parameters in Japanese Quail.

......................... 26 5

Table 4

- 1. Summary of Significant Effects (p<0.05) of Trenbolone Acetate and p,p

6

DDE on Immune, Reproductive, and Behavioral Measures.

................................ 38 7

Table 5

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

Injection from the Studies Reviewed.

................................ ................................ ... 45 9

Table 6

- 1. Summary of Statistical Analyses Yielding Effects

– Bayer.

................................ 57 10

Table 6

- 2. Summary of Statistical Analyses Yielding Effects

– Wildlife International.

....... 58 11

Table 6

- 3. Sum mary of Statistical Analyses Yielding Effects

– Smithers Viscient.

............. 60 12

Table 6

- 4. Ranges of CV% Across Laboratories (by Category).

................................ ........... 62 13

Table 6

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

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

................ 63 15

16



### Figures 17

Figure 2

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

developmental events during embryogenesis with indications of endogenous 19

endocrine change and potential periods of vulnerability

................................ ...... 13 20

Figure 2

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

Quail

................................ ................................ ................................ ...................... 14 22

Figure 2

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

Quail

................................ ................................ ................................ ...................... 14 24

Figure 2

- 4. Maternal deposition of the phytoestrogen, genistein by th e quail hen into 25

eggs

................................ ................................ ................................ ....................... 15 26

Figure 2

- 5. Diagram of the reproductive axis in Japanese Quail

................................ ............ 16 27

28

29

30DRAFT

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### EXECUTIVE SUMMARY 1

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

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

- generation 3

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

Disrupting Chemicals (EDCs). Pertinent liter ature was previously summarized in an in

- depth 5

review completed by the Organisation for Economic Co

- Operation and Development (OECD) in 6

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

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

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

avian two

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

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

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

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

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

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

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

compounds. 17

Findings from the interlaboratory comparison were informative in evaluating the 18

performance criteria for the two

- generation t esting protocol. More importantly, the 19

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

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

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

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

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

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

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

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

multigenerational test for characterizing potential adverse endocrine

- mediated effects and to 28

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

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

endosulfan and trenbolone

. 31

T o summarize, t here are some key differences in avian species that provide strong 32

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

characteristics include : 34

 Metabolic systems 35

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

o Associated accelerated toxicokinetics 37

o Migratory associated energy drain and mobilization of lipid reserves 38

o Thyroid system function i n precocial and altricial birds

. 39

 Reproductive system 40

o Female has one functional ovary 41

o Altered gonadal differentiation results in ovotestes 42

 Hormones and behavior 43

o Males adversely impacted by xenoestrogens and androgenic compounds 44DRAFT

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o Female behavior (receptivity) less sensitive to xenoestrogens 1

 Sexual differentiation 2

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

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

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

 Growth and migration 6

o High metabolism requires sufficient nutrient utilization 7

o Rapid growth rate especially for migratory species 8

o Thyroid system critical for pre

- migratory fattening 9

 Lifespan 10

o Long

- lived birds produce few offspring annually o ver many years

. 11

A nalyzing c ore endpoints of survival, growth, and reproduction across generations will 12

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

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

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

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

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

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

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

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

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

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

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

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

adults. Some chemicals may exert epigenet ic e ffects that would have long

- term impacts on wild 25

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

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

transgenerational eff ects and isolate s embryonic effects of EDCs. In summary, each portion of 28

the multi

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

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

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

, thereby allowing 31

expression of potential adverse effects. 32

33DRAFT

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### OBJECTIVE 1

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

validation process for the Japanese Quail Two

- generation Test (JQTT) so that Federal Insecticide, 3

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

accompanying charge questions. 5



### 1 INTRODUCTION 6

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

requires the US EPA to: 8

9

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

s cientifically relevant information, to determine whether certain substances may 11

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

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

designate [21 U.S.C. 3 46a(p)]. 14

15

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

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

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

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

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

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

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

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

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

countries, the Organisation for Economic Co

- operation and Development (OECD) established 25

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

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

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

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

and on existing regulatory sy stems for new and existing substances. 30

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

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

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

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

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

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

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

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

weaknesses, practicality, reproducibility, reliability, and protocol transferability. 39DRAFT

Avian Two

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### 1.1 Endocrine Disruptor Screening Program 1

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

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

this committee recommended to the EPA a conceptual two

- tiered approach that involved 4

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

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

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

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

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

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

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

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

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

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

consequences resulting from possible endocrine disruption and establishment of dose

- response 15

relationships for hazard identification using Tier 2 testing. 16

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

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

 B e relatively fast and efficient 19

 B e standardized and validated 20

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

of false positives 22

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

possible 24

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

 Y ield data that can be interpreted as either negative or positiv e for determining the 26

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

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

redundancy is incorporated (e.g.

, two different assays may cover some similar aspects of the 29

estro genic response). This redundancy allow s for a weight

- of

- evidence (WOE) approach, as 30

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

definitive, testing. 32

The assays listed in Table 1

- 1, recommended by EDSTAC, have undergon e validation 33

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

chemicals that may affect the estrogen, androgen

, and thyroid (EAT) hormone axes through any 35

known modes of action. 36

37DRAFT

Avian Two

- Gene ration ISR 5 May 2 8 , 2013 Table 1

- 1

. Assays recommended for consideration for the Tier 1 screening battery. 1

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 rec eptor.

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 i n 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

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

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

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

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

 Two

- generation Rat Reproduction Test 7

 Medaka Multi

- generation Test 8

 Japanese Quail Two

- generation Test 9

 Mysid Two

- generation Toxicity Test 10

 Larval Amphibian Growth and Development Test. 11

The Two

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

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

for which the JQTT is reviewed in this report

. 14



### 1.2 Validation 15

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

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

. 17

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

laboratories

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

( OECD 1996 )

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

detect the effects of chemicals with the potential to interact with the endocrine system. 21DRAFT

Avian Two

- Gene ration ISR 6 May 2 8 , 2013 Validation is generall y recognized as necessary for the regulatory acceptance of new and 1

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

acceptance of these methods (OECD 2005a)

. The criteria used to guide the validation process for 3

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

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

and the OECD (OECD 2005a)

. These criteria

, as stated by ICCVAM ( NIEHS 1997 )

, are a s 6

follows: 7

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

its proposed use, should be available. 9

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

effect and toxicity of int erest must be addressed. 11

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

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

analysis and decision criteria. 14

4. Within

- test, intr a

- laboratory and inter

- laboratory variability and how these parameters 15

vary with time should have been evaluated. 16

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

chemicals preferably coded to exclude bias. 18

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

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

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

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

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

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

published in an indepen dent, peer

- reviewed publication. 25

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

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

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

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

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

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

In general, the EPA uses a five

- part or stage validation process outlined by ICCVAM 32

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

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

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

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

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

as follows: 38

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

, which 39

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

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

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

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

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

intended purpose, costs, and other practical considerations. The DRP also identified the 45DRAFT

Avian Two

- Gene ration ISR 7 May 2 8 , 2013 developmental status and questions related to each protocol; the information needed to answer 1

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

second stage of validation, standardization and optimization

. During standardization and 3

optimization, studies were performed that were geared toward refini ng, optimizing, and 4

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

inter

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

refined protocol. The results of these stu dies were used to determine inter

- laboratory variability 7

and to set or cross

- check performance criteria. The report s on the inter

- laboratory trials for the 8

JQTT are provided in Appendix C , and supporting two

- generation demonstration studies are 9

provided in Appendix E

. Inter

- laboratory validation is followed by peer review

, i.e., an 10

independent scientific review by qualified experts, and by regulatory acceptance

, i.e., adoption 11

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

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

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

. 14

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

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

the Tier

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

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

presented. 19

20DRAFT

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### 2 OVERVIEW OF THE JAPANESE QUAIL TWO

- GENERATION TEST 1



### 2.1 Background on Endocrine Disruption in Avian Species 2

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

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

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

EDCs

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

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

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

wel l as developmental characteristics resulting in differential sensitivities and changing lifetime 9

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

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

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

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

primarily by estradiol

- mediated mechanisms . Dev elopmentally, songbirds and other passerine 14

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

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

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

undergo sexual differentiation in ovo; similarly

, other physiological systems are also well 18

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

in a relatively immature state

. Finally, the potential for adverse effects of EDCs on long

- or 20

short

- lived birds is unknown especially in the context that long

- lived birds tend to have small 21

clutches and raise fewer chicks per year compared to short

- lived birds (Ottinger et al. 2003; 22

Stearns 1979)

. 23

Workshops, including the Wingspread Meetings and research publications

, have 24

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

avian species (Berg et al. 1999; Bowerman et al. 2000; Brunstrom and Halldin 1998; Cohen

26

Barnhouse et al. 2011; Corbitt et al. 2007; CM Custer et al. 2010a; TW Custer et al. 2010a; 27

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

2004)

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

of endocrine

- related effects on organ isms in areas in which dioxin, PCB

, or other chemicals 30

occurred

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

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

when attempt ing to separate other interacting factors in the animal’s environment

. Additional 33

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

simultaneous exposure to several chemicals

. It is therefore important to deve lop reliable and 35

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

the EDCs. 37

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

serve as indices of exposure

. Inherent in t he selection of endpoint s to be used as bioindicators is 39

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

populations as a whole

. Steroid hormones and EDCs disrupt embryonic development including 41

organ developme nt, sexual differentiation, reproduction and immune function, reproductive 42

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

al. 2005a; Fox and Grasman 1999; Franceschini et al. 2008; Ottinger et al. 2005; Quin n et al. 44DRAFT

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. 1

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

early embryonic development, later disease, and potential ep igenetic effects

. In addition to 3

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

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

impact across a range of species, includin g invertebrates

. 6

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

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

. 8

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

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

compounds in comparison to estradiol

. However, at any one time, wildlife species are actually 11

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

toxicants

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

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

. 14

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

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

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

Jr et al. 2007; Quinn et al. 2008; Quinn et al. 2007)

. This is in contrast to the commonly held 18

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

. 19

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

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

(Brunstrom and Halldin 1998; Cohen

- Barnhouse et al. 2011; Fry 1995; Grasman and Fox 2001; 22

Head et al. 2008; Ishihara et al. 2 003; Kavlock et al. 1996; Lavoie and Grasman 2007; McNabb 23

and Fox 2003)

. Therefore, an emerging approach for capturing the activity of a compound is 24

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

. This approach 25

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

fundamental actions and molecular targets underpinning these impacts

. 27

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

concentrations of toxicants originating from chemical applications and industrial spills

. Often 29

these occurrences are associated with high mortality

, thereby prompting federal and regional 30

responses aimed at protecting human and wildlife health

. However, less is known about non

31

lethal impacts of EDCs on wild birds and the potential for adverse effects translating even to the 32

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

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

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

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

2002; Darnerud 2003; Francesc hini et al. 2008)

. 37

Some extraordinary datasets also exist or are emerging from long

- term monitoring and 38

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

physiological and reproductive impacts of exposure to chemica ls found in those areas

. 40

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

EDCs as well as periods of sensitivity throughout the life cycle

. Information from many of these 42

studies will be discussed below in order t o put the proposed two

- generation test into the context 43

of testing in avian species

. Furthermore, it is important to consider unique characteristics of 44

avian physiology with emphasis on sexual differentiation, hormonal modulation of endocrine and 45DRAFT

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- Gene ration ISR 10 May 2 8 , 2013 behaviora l components of reproduction, and functional impacts of EDCs on neuroendocrine 1

regulatory systems, especially considering potential adverse outcomes. 2



### 2.2 Relevance of the Japanese Quail Two

- generation Test 3

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

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

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

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

(Adkins

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

. 8

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

respons ive to EDCs and have been investigated in detail in Japanese quail (Adkins

- Regan and 10

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

et al. 2003; Oshima et al. 2012; Ottinger et al. 2009a; Panzica et al. 2 007; Quinn Jr et al. 2007; 12

Quinn et al. 2009; Quinn et al. 2008; Thompson and Adkins

- Regan 1992; Wilhelms et al. 2006)

. 13

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

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

breeding

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

mechanism(s) of action and targets for a particular compound

. Although much of the emphasis 17

of EDC studies ha s bee n on the reproductive axis, it is clear that the thyroid, immune, and stress 18

axes are also potential targets of endocrine disruption

. Toxicological effects are also observed 19

with many EDCs; measures including activation of ethoxyresorufin

- O

- dealkylase (ERO D) are 20

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

. 21

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

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

(Cohen

- Barnhouse et al. 2011; Farmahin et al. 2012a; Head et al. 2008)

. Behavior appears to be 24

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

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

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

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

and Siegel 1973; Benoff and Siegel 1976; Cunningham et al. 1977; Cohen

- Parsons et al. 1973)

. 29

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

hormone and EDC manipulations on development and the reproductive axis (Adkins

- Regan et al. 31

1994; Balthazart et al. 2009; Cohen

- Barnhouse et al. 2011; Ottinger and Dean 2011; Schweitzer 32

et al. 2013)

. These studies have shown that the male Japanese quail is exquisitely sensitive to 33

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

reproductive behavior (Adkins

- Regan et al. 1982; Balthazar t et al. 2009; Halldin et al. 1999; 35

Schweitzer et al. 2012)

. As such, estradiol is very useful as a positive control and as a means of 36

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

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

Panzica et al. 2007; Watson et al. 1990; Yamashita et al. 2011)

. There is a range of effects of 39

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

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

development (Quinn Jr et al. 2007; Quinn et al. 2008; Quinn et al. 2007)

. It is crucial to have an 42

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

multigenerational test will provide some assurance for potential impacts to birds. 44DRAFT

Avian Two

- Gene ration ISR 11 May 2 8 , 2013 

### 2.3 Purpose of the Japanese Quail Two

- generation Test 1

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

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

unique to birds. The JQTT provides a multi

- generation testing protocol that contains each 4

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

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

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

offspring as the final step in the testing protocol. 8

The OECD Avian Two

- generation Toxicity Test Draft Guideline ( Appendix D

, 20 11 ) 9

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

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

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

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

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

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

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

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

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

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

thyroid hormone)

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

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

history. 22



### 2.4 Overview of Japanese Quail Biology & Reproductive Endocrinology 23

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

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

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

with elements that are sexually differentiated primarily post

- hatch and remains plastic throughout 27

the life cycle, showing seasonal regression and hormone induced seasonal neurogenesis ( Adkins

28

Regan et al. 1990; Adkins

- Regan and Ascenzi 1990; Adkins

- R egan et al. 1994; Smith et al. 1997; 29

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

contributes to a n apparent lifelong vulnerability yet potential resilience to EDC exposure 31

(Iwaniuk et al. 2006; Markman et al. 2008; Quaglino et al. 2002)

. S ome s tudies in passerines 32

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

on singing behavior (Adkins

- Regan and Ascenzi 1990; Adkins

- Regan et al. 1994; Corbitt et al. 34

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

of EDCs have also been observed in finches and the dark

- eyed junco (Hoogesteijn et al. 2008; 36

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

2004)

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

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

Avian species rely on genetic based sexual differentiation with w ell characterized 40

developmental events occurring during ontogeny for both precocial and altricial birds

. Moreover, 41

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

homozygotic sex (ZZ) and the female is the heterozygoti c (ZW)

. Current research provides more 43DRAFT

Avian Two

- Gene ration ISR 12 May 2 8 , 2013 insight into the actions of these gender

- determining genes and provides further indication that 1

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

. 2

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

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

expression between males and female (Smith et al. 2009)

. This would be translated into mak ing 5

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

impacts of EDCs may be more global across both males and females than in mammals

, at least 7

relative to gender specific effects

. A further consideration is the s exual differentiation of the song 8

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

effects

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

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

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

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

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

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

if exposed during embryonic development , with potential long

- term effects over the ir lifespan. 16

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

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

characterized in quail. 19

Precocial Birds

– Galliforms and in particular Japanese Quail 20

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

(Touart 2004)

. The domestic chicken is a know n sensitive model commensurate with the 22

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

dioxin like compounds (AhR; Cohen

- Barnhouse et al. 2011; Farmahin et al. 2012a; Farmahin et 24

al. 2012b; Head et al. 20 08)

. The EPA Avian Reproduction Test (OSCPP 850.2300; US EPA 25

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

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

ha s turned to evaluating physiological impacts and vulnerability of birds to suspected EDCs

. 28

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

sexual differentiation of the reproductive endocrine and functional comp etency of other 30

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

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

may be both short

- and long

- term impacts on the individu al (Ottinger et al

. 2002; Panzica et al

. 33

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

embryonically produced hormones direct sexual differentiation

. 35

During development, each level of the reproductive (hypothalamic

- pitutitary

- gonadal , or 36

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

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

development (accessory structures)

. The HPG axis initiates function midway in embryonic 39

development in both chickens and quail

. Observations by Woods and colleagues provided a 40

detailed profile of developmental events in the domestic chicken

. They found that plasma steroid 41

hormones, including es tradiol (E 2) and androgen (A), follow sexually dimorphic patterns in the 42

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

whereas estradiol levels slowly increase in females until hatching (Woods and Brazzill 1 981; 44

Woods et al. 1989; Woods et al. 1995; Woods and Thommes 1984)

. The relative concentrations 45DRAFT

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- Gene ration ISR 13 May 2 8 , 2013 of E 2 were higher in females, whereas males generally had higher androgen levels relative to 1

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

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

. 3

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

Japanese quail (Figures 2

- 1 to 2

- 3)

. Estradiol is a critical element in se xual differentiation in 5

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

estradiol/androgen

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

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

development. In female embryos, plasma E 2 rose until hatch and decreased, post

- hatch (Ottinger 9

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

14 to 17 (E D 14

- E D 17), during the 17

- day incubation period, and declined post hatch (Ottinger 11

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

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

differentiation of multiple organ and endocrine systems throughout incubation

. Once the 14

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

concentrations rise to make the gender

- related pattern in changes more evident, especially during 16

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

. 17

18

F igure 2

- 1

. Stages in Embryonic Development. Diagram shows timing of developmental 19

events during embryogenesis with indications of endogenous endocrine change 20

and potential periods of vulnerability 21

22DRAFT

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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

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

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

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

behavioral components of reproduction occur during this time

, organizing the HPG axis and 6

gender specific behaviors ( Adkins 1975; Adkins

- Regan and Watson, 1990; Ottinger et al 2005 )

. 7

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

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

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

hypothalamic hormone that regulates the HPG axis is gonadotro pin releasing hormone

- I (GnRH

11

I), which is produced by the cell bodies located in the pre

- optic septal region of the 12

hypothalamus

. Hypothalamic GnRH

- I levels rise between embryonic days 10 and 15

, a sharp 13

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

regulation of GnRH

- I (Li et al. 1991)

. B oth the endocrine and behavioral components of 15

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

details below)

. 17

In concert wit h the changing steroid hormones during embryonic development, the 18

adrenal axis and the thyroid axis develop and achieve function

. Circulating concentrations of 19

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

precocial birds, with a slight delay in this pattern for altricial birds

. Additionally, the steroid 21

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

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

2005a; Grasman 2010; Grasman et al. 2013)

. Clear effects of EDCs have been observed for the 24

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

hormones during embryonic developmen t. Additionally, the thyroid axis has been well 26DRAFT

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

development

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

(McNabb and Fox 2003). 4

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

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

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

Hayward and Wingfield 2004 )

. Mounting data point to both direct and epigenetic effects of 8

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

reproductive status, and stressors

. These steroids can affect the viability as well as developmental 10

characteristics of the chicks

, as discussed above

, and there is an extensive literature pertinent to 11

this topic (Lipar et al. 1999; Schwabl 1993, 1996a, b)

. Deposition of EDCs is layered on top of 12

these endogenously produced hormones from the hen

. Moreover, most maternally transferred 13

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

, wherea s, 14

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

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

al. 2010a; CM Custer et al. 2010b; TW Custer et al. 2010a, b)

. Si milarly, laboratory studies have 17

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

(Figure 2

- 4)

, all readily transfer from the hen into the egg and partition in the egg compartment 19

according to lipid solubility (Ad kins

- Regan et al. 2005; Lin et al. 2004; Thompson et al. 1967)

. 20

21

22

Figure 2

- 4

. Maternal deposition of the phytoestrogen, genistein by the quail hen into eggs 23DRAFT

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

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

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

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

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

by 8 to 10 weeks of age. This dimorphism is reflected in hypothalamic GnRH

- I levels, which 7

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

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

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

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

al. 1996; Watson and Adkins

- Regan 1989a, b) . Because this area in known to be important in 12

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

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

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

preoptic

- septal region (POA

- SL) contains many of the gonadot ropin releasing hormone (GnRH) 16

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

- SL 17

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

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

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

(Tsutsui et al

. 2013). All these characteristics of the HPG axis (Figure 2

- 5) and sexually 21

dimorphic neuro endocrine systems are vulnerable to EDC exposure, especially during 22

organization of the reproductive axis during embryonic sexual differentiation

. 23

24

Figure 2

- 5

. Diagram of the reproductive axis in Japanese Quail 25

26 Hypothalamus

GnRH

- I

Pituitary Gland

LH, FSH

Gonads

androgens, estrogens,

progestins The Hypothalamic

- Pituitary

-

Gonadal Axis



# +

+DRAFT

Avian Two

- Gene ration ISR 17 May 2 8 , 2013 Steroids or EDC treatment during embryonic development reveals a number of long

- term 1

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

metabolic e ndocrine systems

. Specifically, early steroid exposure or treatment with fadrozole or 3

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

accesssory structures (Elbrecht and Smith 1992; Perrin et al. 1995; Rissman et al. 1 984; Stoll et 5

al. 1993 )

. Some long

- term studies of embryos treated with steroids or EDCs have examined 6

mechanisms of the observed impacts (Schweitzer et al. 2012)

. These studies have shown that 7

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

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

. 9



### 2.5 Rationale for the Assay 10

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

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

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

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

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

challenges of environmental chemicals with thorough review of exis ting information and 16

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

et al. 2008 )

. 18

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

Japanese quail provide s an advantageous mod el compared to other avian models; this species is a 20

well

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

. 21

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

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

McNabb 2003) , including the following: 24

 Metabolic systems 25

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

o Associated accelerated toxicokinetics 27

o Migratory

- associated energy drain and mobilization of lipid reserves 28

o Thyroid system function in precocial and altricial birds 29

 Reproductive system 30

o Female has one functional ovary 31

o Altered gonadal differentiation results in ovotestes 32

 Hormones and behavior 33

o Males adversely impacte d by xenoestrogens and androgenic compounds 34

o Female behavior (receptivity) less sensitive to xenoestrogens 35

 Sexual differentiation 36

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

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

 Male s

― primary exposure to testosterone 39

 Females

― primary exposure to estradiol 40

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

 Growth and migration 42

o High metabolism requires sufficient nutrient utilization 43DRAFT

Avian Two

- Gene ration ISR 18 May 2 8 , 2013

o Rapid growth rate especially for migratory species 1

o Thyroid system critical for pre

- migratory fattening 2

 Lifespan 3

o Long

- lived birds produce few offspring annually over many years

. 4

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

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

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

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

hatchability, 14

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

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

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

, 11

thereby requiring an extended period of time for a multigeneration test

. 12

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

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

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

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

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

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

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

target vulnerabilities in b irds. An OECD draft Guideline for Testing of Chemicals

— Avian Two

20

generation Toxicity Test in the Japanese Quail was revised in September 2011 and continues to 21

be the latest working document for harmonizing a multi

- generational avian toxicity test. The 22

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

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

sensitivity of the test. 25

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

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

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

scope of a practical testing paradigm due to time constraints. In the f uture

, models of adverse 29

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

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

. 31

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

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

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

many species, and a broad range of reproductive strategies

. It is critical to determine if EDCs 35

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

. Japanese 36

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

literature on the biology and endocrinology of this species

. There are many strains; the outbred, 38

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

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

on stimulatory environmental conditions

. Further, this species does well in captivity and is easily 41

studied

. These characteristics are important to optimize detection of the adverse ef fects of 42

potential EDCs. 43DRAFT

Avian Two

- Gene ration ISR 19 May 2 8 , 2013 

### 3 PROTOCOL DEVELOPMENT 1

The sections below have been taken from the OECD Guideline for Testing of Chemicals

2

Avian Two

- generation Toxicity Test in the Japanese Quail (Draft 2011

, Appendix D )

. 3



### 3.1 Summary of the Japanese Quail Tw o

- generation Test Protocol 4

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

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

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

chemicals under field conditions. These data from a two

- generation test are principally intended 8

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

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

endocrine disruptor. 11

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

water if more appropriate for the chemical. This guideline descr ibes a two

- generation test. The 13

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

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

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

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

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

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

embryonic exposure. 20



### 3.2 Study Design, Test Chemicals and Dose Selection 21

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

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

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

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

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

concentration (LOEC) are determined for health, fitness

, and endocrine parameters. 27

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

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

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

, 30

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

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

during the acclimation period. 33

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

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

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

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

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

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

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

if injury is imminent. The number of pairs allocated to each group (for example 16) should be 41DRAFT

Avian Two

- Gene ration ISR 20 May 2 8 , 2013 sufficient to ensure that there are 12 breeding pairs in the F0 control group that have successfully 1

produced eggs at the end of the tr eatment period. Eggs are collected from F0 post

- treatment 2

week 5 (and possibly, week 8 and/or 9) to examine fertility. Broods from the post

- treatment 6 th 3

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

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

the 6 th and 7 th post

- treatment weeks, a treatment level does not produce at least 40 eggs, that 6

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

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

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

parental pens being paired with f emales from even numbered parental pens and even numbered 10

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

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

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

chemical exposure

. ) The F1 generation breeding birds are maintained until 6 to 7 weeks post

14

fertility. Eggs collected from F1 pairs during weeks 1 through 5 will be us ed to evaluate fertility. 15

Eggs collected from weeks 6 and 7 post

- pairing will be used to establish the F2 generation ; 16

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

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

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

. 19

Dietary concentrations 20

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

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

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

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

D ietary concentrations of the test substance should be chosen on the basis of 25

toxicological data from a range

- finding study or other preceding avian tests. Information may 26

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

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

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

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

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

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

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

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

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

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

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



### 3.3 Preparation and Monitoring of the Test Diet 38

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

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

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

. 41

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

used to ensure a uniform distribution. Acetone may be used to dissolve a test substance, provided 43DRAFT

Avian Two

- Gene ration ISR 21 May 2 8 , 2013 it is allowed to evaporate before feeding the diet to the birds . The amount of vehicle used should 1

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

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

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

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

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

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

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

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

frozen until use. 10

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

the start of the test or in parallel with the range

- finding study. Stability data from previous 12

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

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

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

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

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

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

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

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

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

of the test substance. 22

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

chicks are maintained on untreated diet. 24



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

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

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

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

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

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

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

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

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

behavior 34

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

and an egg

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

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

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

slanting floors and egg

- catchers or other measures to prevent breakage of eggs, are 39

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

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

should be of a size sufficient to prevent foot injury (trauma or pododermatitis) but large enough 42DRAFT

Avian Two

- Gene ration ISR 22 May 2 8 , 2013 to allow excreta to drop through. Measures should be taken to minimize spillage of diet ( e.g

., 1

covering the food troughs with a wire grid). 2

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

pairs (one male/one female). The parental cages should be distributed in such a way that cross

4

contaminati on and positional effects are avoided. Pen mate aggression can be a problem and 5

measures must be taken to minimize injuries, stress

, and mortality resulting from such aggression. 6

Pairs should be reintroduced daily to maintain fertility. 7

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

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

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

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

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

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

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

has been determined. 15

Guidance for housing conditions is provided in Table 3

- 1. 16

Table 3

- 1

. Housing Conditions. 17

Age

( week) Temperature

(°C) Relative Humidity

(%) Minimum Floor Area

(cm 2 /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



### 3.5 Feeding 19

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

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

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

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

specified concentrations. 24

Guidance for recommended nutritional values is provided in Table 3

- 2. 25

Table 3

- 2

. Recommended Nutritrional Values. 26

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

27DRAFT

Avian Two

- Gene ration ISR 23 May 2 8 , 2013 

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

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

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

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

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

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

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

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

should be from the sam e hatch. 9

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

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

Parameters for adult (F0 and F1), egg production (F0 and F1), and of fspring health (F1 and F2) 12

are evaluated by making statistical comparisons between treated groups and the control group. 13

The no observed effect concentration (NOEC) and the LOEC are determined for health, fitness

, 14

and endocrine parameters. 15



### 3.7 Experimental Des ign 16

The test species is the Japanese quail ( Coturnix japonica ). 17

During the treatment period, the parental birds are observed daily to detect any overt 18

signs of toxicity or other clinical signs. If there is an injury due to adult pairings, this should be 19

re corded and the breeding pair(s) should be re

- established as needed if possible. The following 20

observations are to be performed on adults during the study: 21

 Clinical signs of toxicity and health conditions should be evaluated at least once daily, 22

during the acclimation and treatment periods. Observations should include mortality 23

and clinical sign of toxicity such as lethargy, depression, wing droop, ruffled feathers, 24

lacrimation, runny feces, etc. Any injuries sustained and subsequent treatment should 25

also be recorded. Clearly moribund individuals or those otherwise in severe distress 26

should be immediately euthanized. 27

 Food consumption (per pair) should be recorded at least weekly as often as food is 28

replaced in the feeders. Any apparent food spillage should be noted. 29

 Body weights should be determined at least at the start of treatment and at the end of 30

the treatment period. There should be no significant difference in mean body weight 31

between test groups at the start of the treatment period. If there is a signi ficant 32

difference between test groups, birds should be again randomized. 33

Observations of secondary sex characteristics, e.g.

, male/rusty or female/mottled plumage, 34

production of the first egg or first foam), cloacal length, should be recorded beginning on the 4 th 35

week and every week after until distinctly dimorphic traits are expressed (F0 and F1 generations). 36

If plumage is not distinctly “male” or “female

, ” note the plumage as “ambiguous

. ” 37



### 3.8 Offspring

– Eggs 38

Eggs are collected at least once daily, numbered a ccording to pen of origin and stored; 39

broken eggs are numbered, recorded

, and then discarded. The total number of eggs laid per pen 40

should be accounted for at the end of the study. 41DRAFT

Avian Two

- Gene ration ISR 24 May 2 8 , 2013 The eggs are stored in a cold storage facility, for a maximum of 2 weeks, p rior to setting 1

in the incubator (see Table 3

- 3). Cool storage prevents embryo development and aids in 2

synchronizing development of embryos of eggs. Before placing the eggs in the incubator, they 3

are candled to check for abnormalities and fine cracks that can be identified only by candling. 4

After removing all cracked and abnormal eggs, the remaining eggs are equilibrated to room 5

temperature and set in the incubator. All cracked and abnormal eggs should be recorded. 6

Table 3

- 3

. Conditions for Egg Storage, Incubation, and Hatching. 7

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 5 th , 6 th , and 7 th post

- pairing weeks. 9

Eggs collected during the 5 th week will be candled to estimate fertility and embryo viability on 10

embryonic days (ED) 8 and 15, respectively. On ED 15, eggs will be opened and embryo weight 11

and sex will be recorded. Tissues wil l be collected from two ED 15 chicks per pen. In addition, 12

trunk blood and thyroid glands will be collected for biochemical analyses from two ED 15 chicks 13

per pen. If egg production from the 5 th week is limited (i.e.

, < 4 eggs with viable ED15 14

embryos/pen are produced), the priority is as follows: 1 st embryo for histology, 2 nd embryo for 15

biochemistry, 3 rd embryo for histology, 4 th embryo for biochemistry. If the eggs produced during 16

week 5 are not sufficient (e.g.

, < 3/pair) to estimate fertility, additiona l eggs can be collected 17

from breeding pairs during weeks 8 and 9. F1 birds are maintained on treated diet until test 18

termination. Eggs collected from weeks 6 and 7 will be set, artificially incubated, and allowed to 19

hatch to establish the F1 generation. Al l F2 offspring will be maintained on untreated diet and 20

survival through day 14 is recorded. 21

The onset of sexual maturity (egg laying or foam production) or absence thereof can be 22

recorded for F2 hatchlings. The timing of sexual maturity will be considered egg or foam 23

production in 90% of the control (i.e.

, birds maintained on an untreated diet) and appropriate 24

behavioral responses among paired birds. Observations will extend for an additional week 25

beyond sexual maturity of the control groups to estimate po tential pubertal delays among treated 26

birds. 27

During the study, one egg per pen is collected from odd number ed pens in odd numbered 28

weeks and from even numbered pens in even numbered weeks to evaluate eggshell strength and 29

thickness. If egg production is li mited during the 5 th (<5 eggs), 6 th , and 7 th weeks (<7 eggs total 30

for both weeks), forgo egg collection for shell measurements. Eggshell strength is measured in 31

Newtons using a strength tester. The egg is placed on its side on the test stand so the 32

compres sion head will contact the egg at the equator. To determine eggshell thickness, each egg 33

will be cut open around the equator and washed out; subsequently the shells are left to dry with 34

the membrane intact for at least 48 hours at room temperature. Shell t hickness is measured at a 35

minimum of four points around the girth using a calibrated micrometer. Eggshells are measured 36

to at least 0.01 mm and the mean value calculated per egg. Eggs other than those collected for 37

production of the next generation (post

- t reatment weeks 6 and 7), embryonic survival and 38

fertility (post

- treatment week 5, and if necessary post

- treatments weeks 8 and 9), and those 39

collected for eggshell thickness and strength (one egg/pen/2 weeks), can be discarded after 40DRAFT

Avian Two

- Gene ration ISR 25 May 2 8 , 2013 enumeration and examina tion for cracks or abnormalities. Egg yolks from eggs broken during 1

shell measurements can be used for biochemical analyses

. 2

Eggs collected from the F0 and F1 generation during the 5 th , 6 th , and 7 th post

- pairing 3

week s will be incubated to assess embryo via bility (5 th post

- pairing week) and establish the next 4

generation (6 th and 7 th post

- pairing weeks). Eggs collected from the F1 generation during post

5

pairing weeks 1 to 4 will also be incubated through embryonic day 8 to evaluate fertility. 6

Incubation is be st performed when eggs are set in an incubator with temperature and humidity 7

control and an automatic turning device. From the second day of incubation onwards, eggs 8

should be turned at least three times per day. If turned by hand, eggs should be turned an odd 9

number of times a day. 10

Fertility and embryo viability in all incubated eggs are determined by candling the eggs 11

after 8 days of incubation (ED 8) and 2 days before hatching at embryonic day 15 (ED 15). Eggs 12

will be transferred to a hatcher. During can dling, eggs should not be allowed to cool to room 13

temperature, since this may delay embryonic development. After hatching, chicks should be dry 14

before they are taken out of the hatcher. Those chicks that have not hatched within 15

approximately 24 hours of th e majority of chicks hatching should be considered unhatched. No 16

assistance should be given to chicks during hatching. 17



### 3.9 Egg Parameters 18

Prior to start of the treatment period, egg production of all F0 generation pairs of birds 19

available for the study (includ ing potential replacements) should be recorded. These data will be 20

used to exclude pairs that do not lay eggs from further study. Eggs will be discarded after 21

candling unless otherwise indicated. 22

During the treatment periods, the following information is r ecorded: 23

 Eggs laid 24

 Eggs cracked 25

 Eggs broken 26

 Description of all egg abnormalities 27

 Eggs set 28

 Eggshell strength 29

 Eggshell thickness 30

 Infertile eggs 31

 Early viability (ED8) 32

 Late viability (ED15) 33

 Eggs that hatch 34

 Eggs that do not hatch 35

 Fertile eggs. 36

If a pair is remo ved during the study due to death, injury, etc., data from all preceding 37

weeks are evaluated. If there is an injury due to adult pairings, this should be recorded and the 38

breeding pair(s) should be re

- established as needed if possible. If data from a pen i n which 39

mortality occurred or a bird was injured are used in study results, this should be justified. 40

Typical control values for reproductive parameters in Japanese quail are provided in 41

Table 3

- 4. If reproductive parameters of control birds do not meet th ese values, the test 42DRAFT

Avian Two

- Gene ration ISR 26 May 2 8 , 2013 procedure and husbandry conditions should be checked for potential problems and this 1

information should be recorded

. 2

Table 3

- 4

. Typical Values for Reproductive Parameters in Japanese Quail. 3

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

Source: OECD 4



### 3.10 Chicks 5

The following observations on F1 and F2 chicks are recorded during the treatment 6

periods: 7

 Day of hatch 8

 Hatching mortality 9

 Hatchling morphology and/or abnormalities 10

 Chick body weight at hatching 11

 Clinical signs of toxicity/disease 12

 Number of 14

- day

- old surviving chicks 13

 Chick body weight at 14 days after hatching 14

 Time to reproductive maturation (F2 optional) 15

 Phenotypic sex 16

 Genetic sex (F0, F1, F2) 17

 Mating behavior (F2 optional). 18



### 3.11 Pathology 19

The following observations on F1 and F2 chicks are recorded during the treatment 20

periods: 21

Adults

- All adults that survive to the end of the treatment period are euthanized wi th 22

gross pathology assessment. Adult birds that die, or are killed during the course of the treatment 23

period, will be subjected to the same procedures. Trunk blood will be collected for biochemical 24

analyses. The left thyroid gland is to be stored for gland ular T4 content. Organs including brain 25

(POM, pituitary, pineal gland), kidneys, liver, adrenal glands, reproductive organs and associated 26

structures, cloacal gland, bursa, and right thyroid gland should preserved in situ using 27

Davidson’s fixative for furt her histopathology. Any morphologic abnormality should be recorded 28

at the time of necropsy. Fixed organ (or group of organs) weight s will be collected when organs 29

are removed for histopathology. 30

Embryos

– For each mating pair, two late

- stage embryos (ED 15, 2 days prior to hatch) 31

that are not allowed to hatch (i.e.

, from 5 th week post

- treatment eggs) are to undergo necropsy 32

and tissues preserved for histopathology as described above for adults. Trunk blo od and thyroid 33

glands will be collected from two late

- stage embryos per pair for biochemical analyses. 34DRAFT

Avian Two

- Gene ration ISR 27 May 2 8 , 2013 Birds (adults or chicks) found in severe distress will be euthanized. If one member of a 1

pair dies or is euthanized during the treatment period, the othe r member of the pair is euthanized 2

as well. 3



### 3.12 Biochemical/endocrine Measurements and Observations 4

Thyroid hormone measurements

– Measurements of thyroid function include thyroid 5

gland weight, glandular thyroxine (T4) content, and thyroid histology/histopatho logy. Samples 6

will be collected from embryonic and adult life stages. Eggs collected from post

- treatment week 7

5 incubated to ED 15, can be used for embryonic measurements. After assessing late embryo 8

viability 2 days prior to hatch, the embryo is removed f rom the shell and necropsied. Thyroid 9

glands are dissected. Fixed thyroid glands for histology will be collected from two embryos and 10

unfixed thyroid glands for biochemistry will be collected from two embryos for each male

11

female pair. In addition, during necropsy of the adult Japanese quail, thyroid glands are dissected 12

and the left thyroid gland will be used for glandular T4 analysis and right gland used for 13

histological assessment as above.e following observations on F1 and F2 chicks are recorded 14

during the treatment periods: 15

Steroid hormone measurements

- Non

- invasive procedures are preferable for periodic 16

measurements of estradiol and testosterone in developing birds, adults, and eggs. One approach 17

for measuring steroid hormones in the developing birds and adults is through fecal/urate material. 18

Steroids are to be measured from fecal/urate material using radioimmunoassay (RIA) starting 19

Day 1 of adult pairing, and weekly thereafter. Adults are housed in pairs, and the fecal/urate 20

material shall be linked to the pen. Steroid hormones shall be analyzed in trunk blood collected 21

from two late

- stage embryos per male

- female pair, from blood samples collected from all 22

animals at necropsy, and from egg yolks resulting from eggshell thickness/strength measures. 23

Vit ellogenin measurements

– Vitellogenin is a protein that is generally controlled by 24

estrogen. It is present in females of oviparous species during egg development. It is not 25

expressed in normal males due to lack of estrogen ; however in males that have been exposed to 26

estrogenic contaminants, it is expected that vitellogenesis will be stimulated. Vitellogenin is to 27

be measured from trunk blood of the late embryos used for steroid and thyroid hormone analyses 28

and from adults at necropsy, when appropriate, to e valuate estrogenic potential of a test chemical. 29



### 3.13 Behavioral Measurements 30

Males

– Initiation of normal male mating behavior is important for successful 31

reproduction. Mating behavioral observations will be conducted, preferably beginning at the 32

onset of matu ration, on males of the F1 (F2, optional) generation. Trials shall be performed to 33

assess mating behaviors including time to mount attempts, number of mount attempts, and 34

number of successful cloacal contacts. Details on the behaviors and the background of the te sting 35

protocol can be found in Adkins ( 1972 ) and in Ottinger and Brinkley (1978). Behavioral 36

observations shall be conducted on naive birds raised singly or in same

- sex pairs. At sexual 37

maturity (i.e.

, egg laying in females and foam production in ma les), 7 naive male

- female pairs 38

will be introduced and behavior will be observed during three

- minute trials for three consecutive 39

days. Trials may be truncated after successful mating or if bird aggression becomes an issue. At 40

the conclusion of the trial, birds will be returned to their single or same

- sex housing. The same 41

individuals shall be re

- introduced for three consecutive days or until successful mating is 42DRAFT

Avian Two

- Gene ration ISR 28 May 2 8 , 2013 observed. If behavioral observations of the F1 generation suggest a treatment

- related effect, 1

o bservations may be extended to the F2 generation. 2

Handling

- related disturbance during introduction and separation of birds involved in 3

behavior observation trials may perturb egg production, and therefore, observational data should 4

be collected from distin ct pairs not used in collection of other data endpoints (e.g.

, egg 5

production, fertility, etc.). 6DRAFT

Avian Two

- Gene ration ISR 29 May 2 8 , 2013 

### 4 BACKGROUND AND RATIONALE FOR SELECTION OF ENDPOINTS 1



### 4.1 Endpoints 2

T he selection of endpoints depends on the dosing regimen (i.e.

, amount of compound 3

adm inistered and the timing of administration), projected dose

- response, the window of exposure 4

(i.e.

, life

- stage), projected meachnisms of action, potential endocrine activity or induction of 5

vitellogenin or other biochemical responses, and mode of exposure

, all of which are critical 6

variables in evaluating potential EDCs

. A s more information on EDC actions becomes available, 7

it will be possible to use the structure proposed in the framework offered by the Adverse 8

Outcomes Pathways to help elucidate both the targeted physiological systems vulnerable to a 9

chemical/chemical class and the potential for significant impacts to avian species . This approach 10

is not currently integrated into the testing paradigm, but may prove useful for providing s tructure 11

to interpreting the observations and findings. For example, the developing embryo is much more 12

sensitive to the effects of EDCs than the adult, and developmental defects (i.e.

, permanent central 13

and peripheral disruption) often persist into matura tion and adult life stages

. Conversely, adult 14

exposure do es not generally have as much impact

, or effects appear to be more transient, 15

especially in precocial species (Ottinger et al. 2008) . Advances in our understanding of 16

molecular mechanisms have reveal ed that EDCs often exert epigenetic effects

. This has changed 17

our understanding of both the activational and organizational effects of EDCs, allowing for the 18

possibility that exposure of the F0 (or the developing F1 ) generation can cause lasting effects in 19

multiple generations (Skinner et al. 2011) . Therefore, it is extremely important to understand the 20

physiology of the model (i.e.

, Japanese quail) and the species

- specific endocrine mechanisms 21

behind the selection of endpoints. 22

4.1.1 Survival 23

Survival is a basic endpoint for acute and chronic toxicity testing of compounds. 24

Determining the median lethal dose that will kill 50% of the population being tested (LD50) is 25

important for traditional toxicology studies. The OECD Avian Acute Toxicity Test (OECD; 26

Appendix F ) is intended to ascertain the relative toxicity of a compound over a dose

- response 27

curve, using a minimal number of animals that are exposed as adults. The LD 50 or LD 20 of a 28

compound provides information for risk assess ors to determine the potential hazar d from that 29

compound, which then can be utilized for regulatory purposes. In dietary studies, survival must 30

be monitored in order to assess health effects and other impacts that may occur over the time of 31

the study. If there are any deaths of adults or off spring during the experiment, it is important to 32

determine an apparent cause of death. If unexplained deaths occur during an experiment in the 33

treated groups, especially in a dose

- response fashion, it is possible that the test compound has 34

acute toxic effe cts at the doses tested. However, in the context of endocrine disruption studies, it 35

is unlikely that the doses administered will approach lethal concentrations. Indeed, the nature of 36

endocrine disruption studies is to determine the sublethal effects of co mpounds on the endocrine 37

systems of organisms (Clotfelter et al. 2004) . Because only minuscule concentrations of 38

endogenous hormones (pg

- ng/kg amounts; Vandenberg et al. 2012) are required for normal 39

endocrine function, even small perturbations in the syst em can cause sublethal effects that can 40

reduce reproductive function, alter behavior, and/or lead to adult morbidity (Ottinger et al. 2008; 41

Vandenberg et al. 2012) . For example, 50 ug/kg/day of BPA has been determined to be a safe 42

exposure by classic toxic ology testing (Vogel 2009) . However doses 100 to 1000 times lower 43DRAFT

Avian Two

- Gene ration ISR 30 May 2 8 , 2013 than that have been shown to induce feminization of the gonads in Japanese quail (with a single 1

injection into the egg), with no changes in survivability among groups (Oshima et al. 2012) . I t is 2

possible that changes in behavior due to ED C exposure as adults or during development could 3

lead to increased mortality in treated groups, for example, by increasing aggression (Laskey and 4

Edens 1985; Schlinger and Callard 1989) . Thus, it is important to examine causes of death 5

carefully. Moreover, it is critical to examine impacts on survivors, especially if a dose

- response 6

relationship is observed

, in order to reveal potential short

- and long

- term impacts. Although 7

survival may not be a sensitive end point in terms of EDCs

, it should be included in the JQTT as 8

a basic endpoint of toxic effects. The t oxic effects of compounds will vary between avian species 9

(Head et al. 2008; Meyers et al. 1992) and according to the mode of exposure (Hill and 10

Soares 198 7)

. 11

4.1.2 Food Consumption 12

Food consumption is a basic measure of the general health; changes in food intake can 13

indicate acute or chronic toxic effects of a compound. Additionally, if the route of chemical 14

exposure is through the feed, it is important to monitor the consumption carefully to determine 15

the exact exposure. Feeders can be weighed (or the amount of food measured) before and after 16

filling on a daily or weekly basis to determine the amount of food consumed (see above; OECD 17

Two

- Generation Toxicity Test). It is critical to have measurements on the stability of any 18

treatment compound; this stability may dictate the timing for feeding and changing the feed 19

being offered to the test subjects. Steps should be taken to minimize mass lost through spillage 20

(via feeder/cage design), and to estimate the amount spilled in order to detect reduced food 21

consumption. Reduced food consumption is often exhibited in animals in classic toxicological 22

studies and may be indicative of chemical toxicity. In terms of endoc rine disruption , appetite is 23

regulated by hormonal signaling in humans and animals; chemical signals from the adipose 24

tissue, gut, and other organs (i.e.

, leptin, gherilin, insulin) act in brain centers to regulate feeding 25

(Coll et al. 2007)

. Food intake r egulation in birds appears to be similar to that in mammals 26

(Denbow 1985)

. Estrogen has been shown to inhibit food uptake (Hirschberg 2012) and 27

progesterone promotes food intake (Wade 1975) . Further, a growing body of evidence points to 28

ED C s as potential o besogens, indicating that food consumption may be altered by exposure to 29

certain ED C s (Grun and Blumberg 2009) . Reduced food consumption has been associated with 30

EDC exposure in Japanese quail (Yamashita et al. 2011) and in rodents (Trentacoste et al. 2001 )

. 31

However, reduced feed intake may not occur until EDC doses are relatively high (Vandenberg et 32

al. 2012)

, so the effects on food consumption may have been due to general toxic effects, rather 33

than endocrine disruption. Because monitoring food intake is r elatively easy, and changes in 34

food consumption (especially in the lower dose groups) could indicate novel actions of EDCs as 35

disrupting the metabolic endocrine system, this endpoint should be included in the JQTT 36

protocol. Food intake may vary between str ains, diets, and laboratory environments, so these 37

aspects should be standardized as much as possible, with adequate controls. 38

4.1.3 Body Weight 39

Body weight is closely linked with food intake and overall health, and can be indicative 40

of toxicological effects . Th is endpoint is easily measured in Japanese quail, using a weigh box or 41

cage. Along with general toxic effects, changes in body weight over time can relate to metabolic 42

system disruption, thyroid axis disruption, and altered growth, all of which may be link ed to 43

endocrine actions (Boas et al. 2009; Park et al. 2006; Swedenborg et al. 2009) . For example, 44DRAFT

Avian Two

- Gene ration ISR 31 May 2 8 , 2013 perchlorate, a thyroid hormone system disruptor, significantly reduced the growth (measured in 1

body mass) of zebra finches dosed for the first 2 weeks of lif e (Rainwater et al. 2008) . These 2

birds also exhibited altered begging and fledging behaviors

, indicating that other endpoints may 3

be linked to altered body weight, depending on the mechanism of disruption (Rainwater et al. 4

2008) . Further, altered body weig ht during hatch/development could indicate eventual adult 5

morbidity (Siegel 1999) , which is an important measurement endpoint in EDC studies. Therefore, 6

body weight measurements are an important measure in the JQTT protocol, especially at the start 7

and end of the experiment for each generation tested. In particular, the growth of chicks to 8

adulthood should be closely monitored, preferably daily, to determine changes in growth rate; 9

this can be accomplished by measurement of body weight gain. Because the ave rage normal 10

increase in body weight has been recorded over a variety of strains of Japanese quail, body 11

weight becomes a reliable indicator of growth and health. 12

4.1.4 Secondary Sex Characteristics (plumage, cloacal gland area/fo a m production) 13

Males have several sex

- specific characteristics that occur in adults and reflect sufficient 14

circulating levels of testosterone. Japanese quail have a cloacal gland that is androgen dependent; 15

that is, sufficient circulating testosterone must be present to stimulate the glan d to grow larger 16

through both hyperplasia and hypertrophy, as well as to produce a glycoproteinaceous secretion, 17

termed cloacal gland foam (Ottinger and Brinkley 1978, 1979b; Seiwert and Adkins

- Regan 18

1998) . Measurements of the cloacal gland area have provi ded a reliable correlated measure for 19

the sexual maturation and reproductive status of an individual male (Ottinger and Brinkley 20

1979b)

. An estimate of the cloacal gland area is roughly approximated through the product of the 21

length times the width of the gland (Siopes and Wilson 1975) . The foam produced can be 22

estimated on a scale of 1 to 5 or a similar ordinal subjective measurement scale. The function of 23

the foam is for marking the male’s territory; the foam has been called the ” topping on the 24

dropping ” (Schleidt and Shalter 1972) . Since Japanese quail are ground dwellers and males 25

establish territories during breeding season, cloacal gland foam provides a marker delineating 26

their territory (Schleidt and Shalter 1972) . Further, the male Japanese quail doe s not have a 27

penile organ for transfer of the sperm to the female’s reproductive tract. Therefore, cloacal gland 28

foam serves as a vehicle to accomplish transfer between the male to the female’s cloacal vent 29

area. 30

4.1.5 Sexual Maturation and Puberty 31

Because Japan ese quail mature rapidly, this species is ideal for monitoring the effects of 32

potential endocrine disruptors on the initiation of sexual maturation and onset of full 33

reproductive function, including initiation of egg production. Starting at 5 weeks of age, birds 34

should be monitored for presence of cloacal gland foam in males as an initial indicator of 35

testicular activity and production of androgens. Exposure to EDCs may either accelerate or slow 36

sexual maturation. Further, depending on an EDC ’s action(s), t here may be differential effects in 37

males and females

. Briefly, males mature slightly earlier than females, showing the first evidence 38

of sexually dimorphic plumage as early as 3 weeks of age

. By 5 to 6 weeks of age, testicular 39

activity initiates

, begin nin g to produce increasing concentrations of testosterone leading to 40

increasing expression of reproductive endocrine function, courtship

, and mating behavior 41

(Ottinger and Brinkley 1978, 1979b)

. 42DRAFT

Avian Two

- Gene ration ISR 32 May 2 8 , 2013 4.1.6 Behavior (activity, lacrimation, reproductive behaviors, etc.) 1

A lthough the Japanese quail female exhibits a range of sex

- specific behaviors, these 2

behaviors are not readily quantifiable. Female specific behaviors include vocalizations at the 3

time of egg laying, separation from her mate, and contact calls to the chicks (Mills et al. 1997)

. 4

Males and females both exhibit aggressive behavior, which can be quantified (Ramenofsky 1985; 5

Schlinger et al. 1987) . Aggressive behavior can confound the behavioral testing paradigm if 6

either the male or female is excessively aggress ive, leading to damage of the other bird. If there 7

is aggression with damage to one of the pair, the pair must be separated. 8

Male courtship and mating behavior provides an excellent bioindicator of reproductive 9

competency

. Sexual behaviors are steroid horm on e dependent, meaning that there must be 10

sufficient circulating concentrations of androgens to enable the expression of male sexual 11

behavior. Moreover, male sexual behavior is an exquisitely sensitive endpoint for embryonic 12

exposure to estrogenic and high ly active androgen active compounds. As such, it is worthy of 13

further refinement as a useful responsive measurement. Birds should be housed in individual 14

cages at 5 weeks of age (prior to full sexual maturation). When most males (~90%) are 15

producing cloaca l gland foam, a 3

- minute test is conducted in which a receptive female ( control 16

diet female that is from the same population; non

- experimental) is introduced into the male’s 17

home cage. The timing and behavioral measures in this testing paradigm have been v alidated and 18

this method is in use by a number of laboratories (Ottinger and Brinkley 1978) . Latency to 19

mount, mount attempts, and cloacal contacts by the male are recorded. An individual may be 20

tested on 3 successive days if it is important to determine t he effects of experience on behavioral 21

responses. 22

Interpretation of the outcomes of behavioral testing relates to the potential mechanisms of 23

action of compounds under testing. More specifically, the female quail requires exposure to 24

estradiol during embry onic development and is masculinized by exposure to androgens between 25

embryonic days 12 and 18 . Conversely, males are demasculinized as evidenced by impaired 26

male sexual behavior as adults

, if they have been exposed to either estrogens or androgens during 27

this critical period (Adkins 1975, 1979) . As such, the male Japanese quail is exquisitely sensitive 28

to exposure to EDCs during sexual differentiation (Ottinger et al. 2008)

. 29

4.1.7 Fecundity (egg production) 30

Egg production and onset of laying are important measures of reproductive function in 31

birds, and are easily monitored by checking cages daily and marking eggs. A vian EDC studies 32

have shown reduced egg production (Scanes and McNabb 2003) as well as delay ed onset of egg 33

production (Rattner e t al. 1984) . Although some of these studies found reductions in fecundity 34

with adult exposure, indicating direct actions of EDCs on gonadal or HPG axis function, most 35

disruption occurs with either in ovo or maturation related exposures. Exposure during sen sitive 36

developmental periods, even to low doses of EDCs, can permanently disrupt the oviduct, as well 37

as central reproductive control (i.e.

, the reproductive brain), and eventual fecundity of the hen as 38

an adult. Therefore, it is important to monitor egg p roduction in both the parent generation and 39

the F1 generation, to assess both the activational and organizational effects of the EDCs being 40

tested. 41DRAFT

Avian Two

- Gene ration ISR 33 May 2 8 , 2013 4.1.8 Fertilization Success 1

EDCs may act directly on the gonads, gametes, or sperm

- host glands, potentially 2

result ing in decreased fertilization success

. This outcome would be observed in the number of 3

viable embryos at first candling

. Although it is possible to observe the early development of 4

blood vessels and see by candling the embryo as early as ED 3 to 5 in whit e

- shelled eggs, most 5

laboratories wait about a week (ED 7 to 8) to make a determination of fertility and embryonic 6

viability

. In the case of the lack of embryonic development, the egg is opened to determine if 7

there was a germinal disc, which is indicative of initial fertilization and early embryonic death

. 8

This is important to discern early lethality due to frank toxicity of some compounds or doses. As 9

such, decreased reproductive success in response to EDCs could be an indication of male or 10

female gamete disruption, or both, and assessing gender

- specific gamete disruption requires 11

specialized fertility tests (OECD 2007). Therefore, measurement of fertility by observing 12

viability of eggs produced by EDC exposed pairs and comparison to control animals provi des an 13

indication of potential sperm or ova disruption. 14

4.1.9 Embryo viability 15

Set eggs should be candled daily in order to monitor embryo viability. The timing of 16

EDC effects will depend on the targeted mechanism of action for that compound

. In the case of 17

toxi city, the embryo may not survive beyond the first few hours or days of development

. During 18

this time, the uptake of yolk (white and yellow yolk) is minimal and as such reflects primarily a 19

toxic effect of a compound or specific effects impairing the functi on of homeobox genes and 20

disruption of cellular lineages contributing to the formation of organ systems (Morrison et al. 21

2002 ; van de Lavoir et al. 2006, 2012; Carro et al. 2013a; Carro et al. 2013b)

. Later embryonic 22

effects and lethality are more likely t o relate to disruption of essential physiological systems, 23

such as impacting the thyroid endocrine axis and other systems essential for the development and 24

synchronization of endocrine and other physiological systems. 25

It has also been shown that a combinat ion of early and late embryo mortality is a stronger 26

indicator of toxicity that either alone (OECD 2007 ). Further, the embryo seems to be highly 27

dependent on diet (i.e.

, the ratio of n

- 6/n

- 3 fatty acids), so care should be taken to standardize the 28

diet (OE CD 2007)

. Therefore, it is important to monitor embryo viability throughout 29

development by regular candling of eggs

. Moreover, there are distinct strain differences in 30

Japanese quail

. A n extensive and excellent literature deals with a variety of physiologi cal 31

responses in Japanese quail that have been selected for multiple generations (Blohowaik et al

. 32

1984; 1985; Marks et al

. 1990; 1991; Hammad et al

. 1996; Yang et al

. 1999; Aggrey and Marks 33

2002) . In some cases, t hese highly selected strains mimic the Japanese quail strain found in 34

Europe, which has been selected for high body weight

. This heavier strain of Japanese quail 35

tends to have slower maturation and lower production characteristics via reduced fertility, 36

embryo viability, or egg production , simi lar to the heavier broiler breeder strain of domesticated 37

chickens

. Further, eggs produced by some strains have more coloration, thereby making it more 38

difficult to visualize the early embryo by candling

. 39

4.1.10 Eggshell parameters (abnormalities, eggshell streng th/thickness, eggshell color) 40

Eggshell thickness, strength, and other abnormalities can be both quantitatively and 41

quantitatively assessed, although some methods require more specialized equipment and training. 42DRAFT

Avian Two

- Gene ration ISR 34 May 2 8 , 2013 Gross abnormalities (such as apex abnormaliti es, calcium deposits, ridges) can be visually 1

assessed. Eggshell thickness is often measured using calipers or a micrometer. The egg contents 2

are removed

, the shell is rinsed and air

- dried , and then the shell thickness is measured, preferably 3

at the middle /widest portion of the eggshell. Scanning electron microscope (SEM) analysis has 4

been used to determine species

- specific differences in the eggshell matrix; these studies have 5

revealed species specific differences in the characteristics of the shell that a re adaptive for the 6

habitat or life history of that species or population (Igic et al. 2011; Morales et al. 2012)

. 7

However species

- specific morphological differences in the shell only become of importance if 8

birds inhabit areas that are sprayed with insect icides or other agents; in that case, the shell 9

permeability may alter embryonic exposure. 10

There is a long history of eggshell thinning as an endpoint for chemical exposure in birds. 11

As early as the 1960s, exposure to DDT and DDE was linked to eggshell thi nning and population 12

decline in many wild bird species (Rattner 2009) . This was thought to be an activational effect of 13

pesticide exposure, and the endocrine disruptive effects of DDT/DDE were not known. More 14

recently, it has been shown that exposure to ED Cs during development can result in eggshell 15

thinning in the eggs of adult birds (Berg et al. 2004; Holm et al. 2006) . Nestling female zebra 16

finches treated orally with estradiol benzoate had an increased incidence of cracked and broken 17

eggs as adults (Mil lam et al. 2001; Rochester et al. 2008) . In chickens, eggshell thinning has 18

been observed in females exposed in ovo to estrogenic ED C s (including DDT) (Berg et al. 2004; 19

Holm et al. 2006)

. The authors of these studies also proposed that, because of reduced frequency 20

of shell gland capillaries and reduced carbonic anhydrase activity in the uteri of these birds, 21

eggshell thinning may be due to organizational effects on the developing shell gland during 22

development (Berg et al. 2004; Holm et al. 2006) . Signifi cant eggshell thinning was also found, 23

however, in adult Japanese quail exposed to perchlorate, indicating that ED C s can also have 24

activational effects on this parameter. Although the eggshells were significantly thinner, the 25

authors state that they were n ot out of the ‘normal’ range for Japanese quail (Stafford 2011) . It is 26

unclear, however

, if birds from different laboratories have different eggshell parameter variances, 27

so any significant deviations from the control groups should be examined for adverse effects. 28

Indeed, it has been shown that egg quality can vary between birds, depending on the husbandry 29

methods of the laboratory (OECD 2007). Additionally, this study did not monitor the eggs of the 30

offspring (exposed during development) to see if there we re more substantial effects in the F1 31

generation (Stafford 2011) . Eggshell parameters are important endpoints for inclusion in the 32

JQTT. 33

H istorically, it is well documented that there are species differences in susceptibility to 34

eggshell thinning from ED C exposures (Lundholm 1997) , and there may be strain differences 35

within species (OECD 2007). It would be useful to determine the extent to which strain 36

differences occur , particularly for body weight and growth that often are negatively correlated 37

with repro duction

. If one species is selected (e. g. , Japanese quail) there should be as little 38

variation of strain and husbandry methods between laboratories as possible, in order to minimize 39

intra

- laboratory variation of eggshell parameters

. Again, there are insigh ts from studies in which 40

a number of generations in Japanese quail have been selected for growth, reproductive, 41

behavioral, and production characteristics (Blohowaik et al

. 1984; 1985; Marks et al

. 1990; 42

Hammad et al

. 1996; Yang et al

. 1999; Aggrey and Marks 2002). 43DRAFT

Avian Two

- Gene ration ISR 35 May 2 8 , 2013 4.1.11 Hatchability (ED15, hatch) 1

The Japanese quail chick begins to pip at ED15 in preparation for hatching at ED17

. This 2

process begins with the chick breaking through the egg membrane into the air cell. Once pipped, 3

the chick is able to vocal ize and often there is synchronizing in hatching of chicks in a nest or 4

incubator. Pipping is followed by the chick pecking a hole, similar to an escape hatch

, which 5

requires a great deal of energy. D uring this time

, thyroid hormones peak; corticosterone l evels 6

also increase (Tona et al. 2003) . The hatching success of chicks has been shown to be highly 7

sensitive to chemical perturbation, and thus hatchability is an important endpoint in EDC studies. 8

Again, husbandry plays an important role, as sub

- optimal h andling, nutrition, incubation 9

conditions, etc., can greatly affect hatchability (OECD 2007). 10

4.1.12 Genotypic Sex Determination 11

Genotypic sex is important when examining embryonic or early development (i.e.

, 12

feminization or masculinization of reproductive organs ), when the genetic sex of the bird may be 13

unclear, either because of an early developmental stage or masculinization/feminization of the 14

embryo (Berg et al. 1999) . Since the rusty

- red plumage of the male is not apparent until 3 weeks 15

of age, it may be nec essary to determine genetic sex prior (Tilmann and Capel 2002) . In 16

mammals, males are heterogametic (XY) and females are homogametic (XX). The testes

17

determining factor (the SRY gene) located on the Y chromosome initiates differentiation of the 18

testes and thus the steroidal milieu that promotes male development. In the absence of SRY, the 19

embryo will differentiate into a female (Sekido and Lovell

- Badge 2013) . In birds, the female is 20

the heterogemetic sex (ZW), and the male is homogametic (ZZ), with no SRY. Sexual 21

differentiation in birds, therefore, is not as straightforward and is less well characterized. Either 22

there is an equivalent ” ovary

- determining factor ” on the W chromosome for female development, 23

or the ” double dose ” of the Z chromosome initiates mal e development, or perhaps both are at play 24

(Smith et al. 2009) . It is thought that a double dose of DMRT1, located on the Z chromosome, 25

initiates male differentiation (Smith et al. 1997) . Several W

- specific genes are also implicated in 26

ovarian development, and thus may play a role in female sexual differentiation (Ayers et al. 27

2013) . Genotype can be determined by polymerase chain reaction (PCR) (Berg et al. 1999; 28

Morinha et al. 2012)

. 29

4.1.13 Histopathology 30

Many organ systems are highly sensitive to hormonal perturbations, particularly if 31

exposed during development. Further, organs are relatively easy to collect and prepare for 32

histological examination, and should be included in the analysis of EDC effects on the F0 and 33

F1/F2 generations. Reproductive organ (i .e.

, testes/ovaries/oviduct) weights and gross disruption 34

of organ morphology should be recorded, and the reproductive organs should be examined 35

histologically for microscopic disruption (i.e.

, disrupted follicular function or spermatogenesis) 36

(OECD 2007). In addition, thyroid glands, liver, and other organs should be examined, 37

depending on the projected target(s) for the EDC (see below for more discussion). Detailed 38

explanations of organ collection, preparation, and analysis for EDC endpoints are described in 39

the OECD 2011 DRP and Histopathology Guidance Document for the Avian 2

- Generation Test, 40

2012

. The organ systems that have been the focus of histological analyses have been those 41

considered likely targets for endocrine active and/or toxicological impact s of EDCs

. They 42

include the organs and organ systems listed below

. 43DRAFT

Avian Two

- Gene ration ISR 36 May 2 8 , 2013 Reproductive organs (gonads) are often subjected to histological analyses for evidence of 1

pathology

. For both males and females, initial wet weight provides some information about 2

potential abnormalities, specifically the retention of both male and female gonadal tissue as well 3

as other potential pathologies

. The sexual differentiation of the avian female involves regression 4

of one of the two primordial ovaries with its accompanying oviduct (Sturkie 1991)

. As such, a 5

critical observation for female birds is the presence/regression of the right ovary and oviduct; this 6

provides initial information about appr opriate sexual differentiation of the female reproductive 7

axis ( Van Tienhoven 1968; Muller et al. 1980; Sturkie 1991 )

. Because gonadal sexual 8

differentiation in birds relies on the differentiation of the female by genetic and then hormonal 9

drivers that pro mote ovarian development and sexual differentiation of the hypothalamus and 10

accessory sex structures, the female is vulnerable to impacts by EDCs

. Some of the impacts 11

noted have been for impaired sexual maturation with altered onset of egg production as we ll as 12

reduced egg production and potentially reduced fertility (Rissman et al. 1984)

. There are sperm

13

host glands in the oviduct of the avian female, in which sperm may reside for some weeks (Bakst 14

1998) , allowing continued fertility for ova produced as th e female sequentially lays the eggs 15

making up a complete clutch

. However, there are no data available for a potential impact of 16

EDCs on these glands ; altered function of these glands would contribute to impaired fertility of 17

the female in addition to any o ther direct effects on the gonads or on the HPG axis. 18

The ovary in the mature avian female has multiple follicles in varied stages of 19

recruitment and maturation, often likened to a cluster of grapes ( Van Tienhoven 1968 )

. The 20

stages in follicular developmen t and maturation mirror those observed in mammals; however, the 21

hormonal production by the follicle and hypothalamic endocrine support for ovulation differs 22

from mammals and other vertebrate classes

. Environmental signals stimulate the hypothalamic 23

GnRH sy stem that stimulates pituitary gland gonadotropin production (luteinizing hormone; LH 24

and follicle stimulating hormone; FSH), which in turn stimulate ovarian function

. Follicular 25

recruitment generates a sequential developmental pattern in follicular matura tion

. The small 26

white follicles produce primarily estradiol; as the follicle grows to secondary and then tertiary 27

follicles, steroidogenic cells in the thecal layer continue producing estradiol as well as secreting 28

increasing concentrations of progesterone (Bahr et al. 1983; Johnson and van Tienhoven 1980; 29

Johnson et al. 1987)

. Progesterone produced by the tertiary follicle provides the stimulations for 30

the pre

- ovulatory GnRH surge that promotes the LH peaks critical for ovulation

. Birds do not 31

have a lutea l phase in their ovulatory cycle; as such, there is no corpus luteum as in mammals

. 32

Histological analyses will provide information as to the status of ovarian function

. L inking 33

ovarian morphology to these components of ovarian function provides insight int o potential EDC 34

targets and actions

. 35

The avian male is also vulnerable to embryonic exposure to EDCs

. Early exposure to 36

EDCs also can impact gonadal development in a genetic male; however, there is less information 37

about these type s of long

- term impacts

. I n some studies, the gonadal histology attributed to 38

effects of steroid or EDC exposure appears to be ovotestes, which are found in the hatchling but 39

generally do not persist into the adult

. Gonadal histology for adult functional males includes 40

assessment o f mass, with staining of the interstitial Leydig cells that produce testosterone and 5

- 41

α dihydrotestosterone (5

- α DHT) and the seminiferous tubules including staining of viable 42

spermatocytes, numbers of cells in various stages of the spermatogenic cycle, an d overall 43

morphology of the gonad

. Conversely, there are some neural markers of EDC exposure 44

described further below

. 45DRAFT

Avian Two

- Gene ration ISR 37 May 2 8 , 2013 Histology of other organs has provided additional insights into the actions and impacts of 1

EDCs

. Liver histology can provide information on any abnormalities associated with toxic 2

effects

. In addition

, the activation of detoxification enzymes are excellent markers for initiation 3

of these enzyme systems; conduct of the ethoxyresorufin

- O

- deethylase (EROD) assay on liver 4

subsamples can provide supporting evidence for histopathology findings

. 5

Previous studies have shown a link between embryonic exposure to polychlorinated 6

biphenyls (PCBs) and cardiac malformations including decreased heart indices (heart weight as a 7

percent of body mass), thinn ing of the ventricular walls, and abnormal heart apexes (DeWitt et al. 8

2006; Walker and Catron 2000; Walker et al. 1997)

. Domestic chick embryos exposed to dioxin

9

like PCBs had increased ventricular

- septal defects, ventricular dilation, and higher overall heart 10

weight by embryonic day (ED) 10 (Carro et al. 2013a; Walker and Catron 2000)

. 11

Kidney histology can provide information about toxicological impacts of EDCs due to 12

the critical role of the kidneys in detoxification and elimination of water soluble tox icants

. Some 13

protocols have been used to determine kidney pathologies associated with endocrine disruption; 14

these observations support the use of kidney histopathology as a responsive endpoint for some 15

classes of EDCs (Scanes and McNabb 2003; Shibuya et al . 2005)

. 16

Pathophysiological measurements of the bursa provide a powerful tool to assess 17

endocrine disruption affecting the immune system

. There is evidence for cross

- talk between the 18

neuroendocrine and immune systems

. Further, the bursal size differs in m ales and females, with 19

smaller organ size in hatchling males

. The bursa of Fabricius is exquisitely sensitive to steroid 20

hormones, especially during development with testosterone having an immunosuppressive effect 21

(al

- Afaleq and Homeida 1998; Glick 1980; M arsh and Scanes 1994; Quinn Jr. and Ottinger 22

2006)

. Embryonic exposure to trenbolone acetate resulted in persistent effects on bursal 23

morphology with fewer, smaller follicles (Quinn et al. 2007; Table 4

- 1 )

. As may be seen in Table 24

5, consequences for the i mmune system from EDC exposure also relate to effects on the 25

reproductive axis

. Conversely, estradiol treatment resulted in larger bursal size; however, there 26

was evidence of histopathology in treated individuals (Quinn et al. 2009)

. Many of these studies 27

have also found a consistent relationship between bursal morphology and immune response

. As 28

such, bursal histopathology appears to be a reliable measure of impact to the immune system, 29

especially assessing impacts during embryonic treatment and the persist ence of these effects into 30

adulthood

. Moreover, immunosuppression in response to EDC exposure remains a critical 31

responsive endpoint that has been shown to be sensitive to endocrine disruption 32

(Grasman et al. 1996)

. 33

34DRAFT

Avian Two

- Gene ration ISR 38 May 2 8 , 2013 Table 4

- 1

. Summary of Significant Effects (p<0.05) of Trenbolone Acetate and p,p

- DDE on 1

Immune, Reproductive, and Behavioral Measures

. 2

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 μm g/g,

decreased at 50 μ g/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 μ g/g NA

Hatchling total leukocyte counts NA Increased in chicks

Hatchling di ff erential 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

“NA” denotes measures that were not affected by treatments. 3

Adapted from Quinn and Ottinger (2006). Reprinted with permission, The Journal of Poultry Science Vol 43: 1

- 11, 4

Japan Poultry Science Association, Tsukuba, Ibaraki, Japan

. 5

4.1.14 Neuroendocrine Systems Regulating Reproduction, Metabolism, and Stress 6

A great deal is known about sexual differentiation of male sexual behavior in Japanese 7

quail, which has proven to be sensitive to exogenous steroid exposure during embryonic 8

developmen t (Adkins 1979; Grasman et al. 1996; Halldin 2005; Halldin et al. 2005; Panzica et al. 9

2005; Panzica et al. 1996; Panzica et al. 2007; Schweitzer et al. 2013) . Embryonic exposures to 10

steroids or compounds that are estrogen or androgen active result in impa ired behavior in male 11

adults (Cornil et al. 2006; Ottinger et al. 2008; Panzica et al. 2007; Quinn et al. 2008)

. Further, it 12

has been shown that specific neural systems, including neurotransmitters (norepinephrine, 13

dopamine, serotonergic) and neuropeptides (vasotocinergic) are modified in response to 14

embryonic exposure to steroid hormones and to EDCs (Cornil et al. 2006; Ottinger et al. 2008; 15

Panzica et al. 2005) . Assessing the effects of embryonic exposure to EDCs on neurotransmitters 16

and neuropeptides can provide information about direct effects of these compounds on regulatory 17

systems that modulate reproductive, thyroid, and adrenal endocrine axes

. 18

In addition, metabolic systems have been investigated in detail in birds, especially for 19

domestic poultry

. T he thyroid system in birds has proven vulnerable to specific types of 20

endocrine disruption in which this endocrine system is a target

. Specifically, PCBs , flame 21

retardants, and other classes of EDCs have been implicated in having thyroid system actions tha t 22

impact birds, both in the laboratory and in field birds (Chen et al. 2009; Chen et al. 2008; Fernie 23

et al. 2005b; McNabb and Fox 2003; McNabb 2006; Wada et al. 2009; Webb and McNabb 2008)

. 24

The mechanisms of action exerted by these compounds in birds are not as clear; data from the 25DRAFT

Avian Two

- Gene ration ISR 39 May 2 8 , 2013 McNabb laboratory and others suggest that altered enzyme activity, specifically deiodinases

, 1

may accompany EDC exposure

, thereby reducing availability of bioactive thyroid hormones to 2

target tissues (Chen et al. 2009; Chen et al . 2008; Fernie et al. 2005b)

. It is not clear if 3

histological methods provide a sensitive method for revealing these EDC effects; however, 4

impaired production and release of thyroid hormones would be observed in abnormal thyroid 5

gland follicles size and di stribution

. Presumably, these abnormalities would also be reflected in 6

differences in circulating thyroxine (T4) and the more bioactive form, triiodothyronine (T3)

. 7

Moreover, there are changes in thyroid responsive genes with EDC exposure in cultured avian 8

neural cells (Vongphachan et al. 2011)

. Finally, the hypothalamic

- pituitary

- adrenal axis (HPA) is 9

another endocrine system potentially responsive to EDC challenges (Jönsson et al. 1994)

. In the 10

case of EDCs that also have toxicological effects, the HPA ax is is likely to be activated in 11

response to both EDC and toxic actions

, especially if there is sustained stress to the organism

. A 12

newer method of measuring corticosterone in feathers has recently been validated (Lattin et al. 13

2011)

. This may be an interes ting technique to determine if long

- term stress has occurred; a 14

method that may be especially useful to assess stress level in field birds over time

. 15

Seasonality and migration also complicate responses by birds to EDCs; both affect the 16

histology of EDC res ponsive organs and physiological systems

. Birds often exhibit seasonal 17

patterns in reproduction

, and species that migrate often show complete regression of the 18

reproductive axis with cessation of function followed by restimulation of the reproductive syste m, 19

or seasonal recrudescence for the next breeding cycle

. Initial stimulation of reproductive function 20

in birds relies on environmental triggers, termed zeitgebers

. I n the case of temperate zone species

, 21

this environmental cue is often photoperiod

. Birds a re generally long day breeders due to their 22

relatively short incubation and relatively quick maturation times for their offspring

. As such, 23

most photoperiodic birds become reproductive with day length longer than 12 hours

. Some 24

species are termed relativel y photoperiodic, meaning that they respond to changes in photoperiod 25

length to act as their cue

. There is a very large literature on this subject that deals with the neural 26

mechanisms, response via the eyes and pineal gland, and the hypothalamic response t o regulate 27

reproductive function; review of this literature is beyond the scope of this ISR

, but it is important 28

to recognize seasonal variations in energy demands due to reproduction, migration, and 29

environmental challenges especially as EDCs impact criti cal endocrine systems involved in these 30

seasonal and adaptive responses. 31

4.1.15 Key Neuroendocrine Measures for Endocrine Disruption 32

Hypothalamic Gonadotropin Releasing Hormone

- I (GnRH

- I) 33

Gonadotropin releasing hormone (GnRH ) is the primary hypothalamic hormone that 34

directs the reproductive or HPG axis. The form of GnRH that is a primary form in avian species 35

is termed GnRH

- I. This form of the peptide has been shown to regulate reproductive function in 36

birds ( Li and Ottinger 19 88 ; Li et al. 1991 ). In birds, there are approximately 1000 GnRH

- 1 37

neurons that are located in the pre

- optic/lateral septal region of the hypothalamus ( Kuenzel and 38

Masson 1988; McGary et al. 2001; Ottinger et al. 2002; Ottinger et al. 2008; Ottinger et a l. 39

2009b )

. These neurons send axonal projections to the median eminence, where the GnRH is 40

released to the portal vessels that carry hypothalamic hormones to gonadotropin producing cells 41

in the anterior pituitary gland

. Synthesis and secretion of GnRH is m odulated by 42

neurotransmitter and neuropeptide systems that respond to environmental signals and to internal 43

physiological modulators (see Figure 2

- 5 above for a diagram of the HPG axis)

. 44DRAFT

Avian Two

- Gene ration ISR 40 May 2 8 , 2013 Neurotransmitters, Neuropeptides, and Enzymes 1

Impact s to neurotransmi tter systems have been a subject of interest for many years

. In the 2

case of toxicity assessments, brain acetylcholine esterase (ACHase) has been used as a measure 3

of toxicant exposure (Rattner et al. 1986)

. However, it is not clear how responsive the 4

acety lcholine system is to endocrine disruption

. There is mounting evidence that 5

neurodegenerative diseases in humans may be linked to chemical exposures in mammals

, and it 6

would be expected that similar deleterious impacts would occur in avian neural systems

. 7

However, additional information is needed to understand if this type of long

- term impact 8

provides a bioindicator of adverse effects in birds

. Monoamines are key neurotransmitters that 9

regulate the reproductive and metabolic endocrine axes as well as modula te stress responses. 10

Analytical methods include High Pressure Liquid Chromatography

– Electrochemical Detection 11

or by ELISA. Specific monoamines of interest include: serotonin, 5

- hydroxyindolamine 12

(5HIAA), norepinephrine (NE), and dopamine (DA; Abdelnabi a nd Ottinger 2003) . Monoamines 13

are critical regulators of neuroendocrine function

. A number of studies in both mammals and 14

birds have shown that alterations in these neuroregulatory systems result in diminished 15

reproductive or metabolic endocrine function

. As mentioned earlier, steroids and EDCs impact 16

arginine vasotocin (AVT) and aromatase enzyme in brain regions that modulate reproduction and 17

sexual behavior (Panzica et al. 2005; Panzica et al. 2009; Panzica et al. 1996; 18

Panzica et al. 2007)

. 19

Behavioral M

odulation by Critical Brain Regions

– Effects of EDCs 20

As summarized earlier, behavior can serve as a sensitive measure of EDC exposure, 21

especially in precocial species

. The brain regions that modulate reproductive behavior in male 22

Japanese quail have been thoroughly characterized; these regions depend on androgen 23

stimulation (Adkins

- Regan et al. 1994; Watson et al. 1990; Watson and Adkins

- Regan 1989b ). 24

Songbirds and other altricial birds also show impacts of EDC exposure on avian brain 25

morphology (Iwaniuk e t al. 2006; Quaglino et al. 2002)

. Further, there is a large literature about 26

the impacts on steroid hormones on the sexual differentiation and function of the nuclei that 27

direct and modulate song control in songbirds (Quaglino et al. 2002; Wade and Arnold 2004)

. 28

These species show maturation and seasonal changes in reproductive behavior; these changes are 29

associated with recrudescence of neuroanatomical areas modulating singing behavior

. It is the 30

song control nuclei that exhibit with greater neuroplastici ty (Hoogesteijn et al. 2008)

, thereby 31

allowing songbirds greater adaptability to changing conditions and environmental challenges

. 32

Because of the neuroplasticity in songbirds, EDC effects may be more transient in songbirds; 33

however, there are insufficient data to ascertain if this is the case

. Nonetheless, EDC effects on 34

brain regions that modulate singing and other behaviors that are steroid dependent in both 35

pre cocial and altricial birds have been clearly documented

. 36

Other Biochemical Endpoints Potentially Reflective of Endocrine Disruption 37

Vitellogenin is a yolk protein that is produced in the liver in egg

- laying animals in 38

response to estrogen. In sexually matu re females, it is produced in response to endogenous 39

estrogen. However, in males and pre

- pubertal animals, vitellogenin production can indicate 40

exposure to exogenous estrogens (Heppell et al. 1995) . Tests of vitellogenin response have not 41

shown this to be a sensitive measure in birds, either relative to increased plasma vitellogenin in 42

males or in pre

- pubertal animals. 43DRAFT

Avian Two

- Gene ration ISR 41 May 2 8 , 2013 Oxidative damage has been linked to adverse effects of xenobiotics; however, few data 1

are available for birds to support selection as a glob al indicator of endocrine disruption in birds

. 2

Similarly, DNA damage could provide important information as an indicator of adverse effects 3

of toxicants (Dix et al. 2012)

. However, similar to oxidative damage, more information is needed 4

to ascertain the re liability of these measures as bioindicators of effects

. Finally, the receptor 5

based assays have provided additional information on potential mechanisms of action of EDCs 6

as well as important insights into the comparative sensitivity across avian species t o Ah receptor 7

activated compounds (Farmahin et al. 2013; Farmahin et al. 2012b; Herve et al. 2010; Inoue et al. 8

2009; Lattin et al. 2012; Lee et al. 2011a; Lee et al. 2011b; Manning et al. 2013; 9

Tazelaar et al. 2013; Yang et al. 2010)

. 10DRAFT

Avian Two

- Gene ration ISR 42 May 2 8 , 2013 

### 5 PROTOCOL OPTIMIZATION AND DEMONSTRATION 1



### 5.1 Comparability with the Avian Reproduction Test Currently in Use 2

The EPA A vian R eproduction T est ( OSCPP 850.2300; US EPA 2012 ) was designed 3

primarily as a reproductive toxicological test to determine if a test compound ha s ad verse effects 4

on classic reproductive parameters

. These include measures of reproductive function as well as 5

toxicological endpoint s

. Further, most are largely measures of effects on adults

. This testing 6

paradigm has the potential to detect more extreme adverse effects of EDCs, including some 7

measures of fitness such as number of offspring produced and thus may reveal endocrine 8

disruption. However, the Avian Reproduction T est may not detect endocrine disruption or low

9

dose effects on developing embryos. The studies reviewed below include egg injection studies as 10

well as one

- and two

- generation dietary tests. Statistically significant differences are discussed 11

below with the exception of trends, as noted in the text. 12



### 5.2 Egg Injection Studi es 13

A series of studies performed in a number of laboratories provide background 14

information about the effects, mechanisms of action, and relative impacts of various classes of 15

EDCs in birds

. Studies described below are for the most part those conducted und er controlled 16

laboratory conditions

. These experiments were designed to simulate maternal deposition of 17

EDCs into eggs prior to oviposition by the female

. There are complexities to consider with the 18

use of egg injection as a method of administering known c oncentrations of a compound

. First, 19

since the avian embryo develops outside constant maternal input as occurs in mammals, it is 20

assumed that the egg is a relatively closed system in which a known amount of compound is 21

inserted

. This is advantageous, especi ally with administration of the test compound prior to 22

incubation

, because there is opportunity for enzymes in the albumin and embryonic organs to 23

metabolize the compound into secondary substances similar to what would occur in wildlife 24

populations

. A seco nd consideration is the method of administration; specifically the vehicle, site 25

of administration, and movement of the compound through the egg compartments may vary with 26

the chemical characteristics of the test substance

. Third, it is critical to test fi eld relevant 27

concentrations as part of a dose response experiment to ascertain bioactivity as well as determine 28

potential mechanisms of action

. Finally, egg injection methods have improved to optimize 29

vehicles, injection volumes and sites, and timing of in jection to minimize treatment effects

. The 30

egg injection studies below provide information on the response of avian embryos to a dose 31

range of selected EDCs. 32

It is instructive to compare results with the avian embryo assay. The avian embryo assay 33

involves exposing the embryo via injection of the compound into the egg. While this method 34

may be less relevant to real world exposure than dietary exposure in the Avian Reproduction 35

Test (OCSPP 850.2300, EPA 2012) and maternal transfer in the Two

- Generation Test, it has an 36

advantage over the Avian Reproduction Test in that the embryo is exposed to the potential EDC; 37

thus developmental disruption can be examined. Because there is maternal transfer of 38

environmental chemicals to the yolk (Adkins

- Regan et al. 2005; Lin et al. 2004; Thompson et al. 39

1967), egg injection mimics this maternal transfer, although it is difficult to determine relevant 40

doses, without maternal transfer studies. Further, egg injection bypasses the metabolism of the 41

compound in the hen, and the po ssible actions of metabolites. The Avian Embryo Assay is a 42

bioassay that can involve dosing the egg at different times and comparing the results during 43DRAFT

Avian Two

- Gene ration ISR 43 May 2 8 , 2013 critical periods in development. However, there are concerns about how closely egg injections 1

mimic mate rnal deposition and with the selection of vehicles that do not interfere with discerning 2

impacts of test compound(s). There are a number of studies in the literature that address these 3

issues, and provide guidance on vehicles, injection sites and volumes, and relative uptake of 4

treatments (McKernan et al. 2009). 5

5.2.1 Atrazine (Experimental Pathology Laboratories 2006) 6

Exposure Levels and Protocol 7

On day 0 before incubation, eggs were injected at doses of 0, 0.05, 0.5, 5.0  g/egg, and 8

untreated. Endpoints included embryonic viability, hatching success, growth/weight gain, timing 9

of maturation, egg production, male reproductive behavior, genetic sexing of hatchlings and 10

adults , h ypothalamic dopamine/GnRH

- I, and brain aromatase (days 1 and 50) , f ecal steroids 11

(days 1 and 50) , p lasma steroids (day 50) , h istopathology of abnormal bursa (days 1 and 50)

, 12

aromatase activity or histopathology of gonads (days 1 and 50), thyroid weight, plasma and 13

gland thyroxine (T4) content (days 1 and 50)

, plasma T4 (days 1 and 50), cloacal gland area and 14

foam production (day 50) , i nitiation of egg production, hypothalmic/gonadal aromatase in half of 15

the controls, low, and high dose chicks (when available) and adults (half of the gonads sent to 16

EPL and half used for frozen tissue), and plasma vitellogenin in chicks and adults. 17

Sensitive Endpoints 18

Liver mass was reduced in hatchlings in all treatment groups; gonad weight was lower in 19

5 ug/egg group. Dopamine was decreased in the males at 0.5  g/egg an d 5

. 0  g/egg doses. 20

Circulating e stradiol was decreased in dosed females. 21

In adults, exposed males showed greater latency to mount with high dose. Low dose 22

males also had fewer mating attempts, while the medium dose had the most mount attempts, but 23

not nec essarily the greatest mating success. There was a trend of lower dopamine in the medium 24

and high dose groups. Aromatase activity was higher in the medium dose group. Females in the 25

low dose group had lower estradiol than the other groups. Males in the low dose group had 26

higher estradiol than the other groups. Androgen was reduced in all treatment groups. Some 27

sample sizes were too small for statistical analyses. 28

Overall Conclusions 29

Vinclozolin administered prior to incubation affected organ weights, with so me effects on 30

dopamine and circulating estradiol levels. Adult males had impaired sexual behavior, with some 31

effects noted for neuroendocrine systems and circulating steroids. 32

5.2.2 Trenbolone 33

Exposure Levels and Protocol 34

There were two trials for trenbolone. W i th Trial 1: 0.05, 0.5, 5, 50, and 125 μg/egg; and 35

Trial 2: 0.05, 0.5, 5, 50  g/egg injected at embryonic day 4 (i.e., ED 4 or ED 11, respectively

. 36

Adult tissues sampled included blood, brain (frozen), gonads and bursa ( weighed ) , testes (left 37

frozen and right fixed in Bouin’s). Hatchling tissues were blood, brain (frozen), bursa , and 38

gonads ( gross morphological deformities ). Other endpoints included chick behavior (motor 39

activity), onset of puberty (monitored starting at 5 wks for cloacal gland hypertroph y and foam 40DRAFT

Avian Two

- Gene ration ISR 44 May 2 8 , 2013 production in males and oviposition in females), egg production, embryo development and 1

fertility, male sexual behavior, steroid hormones (estradiol and androgens), aromatase enzyme 2

activity (brain), monoamines (brain), GnRH (brain), and tissue histology. 3

Sensitive Endpoints 4

There was increased embryo mortality at 50 and 125  g/egg (100% mortality in 125 5

 g/egg) ; surviving chicks showed delayed puberty, decreased bursa size at 5 and 50  g/egg (high 6

doses) with fewer bursal follicles , and a trend for reduced circulating androgens (see Table 5

- 1 7

for summary)

. Males in all treatment groups had significantly fewer successful copulations than 8

controls; treatment effects were observed in latency to mount and number of mating attempts. 9

There was a trend of an increase in aromatase activity. Males in the low dose group had higher 10

estradiol than the other groups. Androgen was reduced in all treatment groups. Females in the 11

low dose group had lower estradiol than the other groups. 12

Treatment at ED11 also resu lted in effects, many of which did not follow a dose

- response 13

relationship. There was increased mortality in the lowest dose, increased bursal index decreased 14

circulating steroid hormones, reduced ovarian weight and follicle number; with these and other 15

ef fects occurring in a non

- dose

- dependent pattern

. Altered neurotransmitter levels were observed 16

with specific doses in serotonin metabolite (5

- HIAA) as well as a dose related increase in 17

norepinephrine . Often, there have been non

- linear responses observed i n these endpoints; 18

however, this is sometime due to the number of individuals examined and could be assessed 19

through a power analysis to determine the vigor of that particular endpoint. Both are indicative of 20

long

- term effects on the neural systems that mo dulate both endocrine and behavioral components 21

of reproduction. Ovary weight was reduced in the 0.5 and in the 5.0  g/egg treatments. There 22

was a trend of reduction in follicles in the 0.5 and 5.0  g/egg groups and increased left testis 23

mass in 0.05  g/egg group. Statistics were conducted to compare groups with sufficient sample 24

size. A summary of findings is shown in Table 5

- 1. 25

Overall Conclusions 26

Trenbolone had both toxicological and endocrine disrupting effects that were not dose

27

related , suggesting that there may be a survivor effect . This would suggest that the more 28

sensitive birds responded and did not survive , thereby leaving less sensitive birds to be sampled 29

in the higher doses

. Trenbolone administered during development (egg injection) resulte d in 30

disrupted development of the immune system (smaller bursa; Quinn et al. 2007) as well as 31

disrupted reproductive parameters (delayed puberty, reduced cloacal glad development, reduced 32

reproductive behaviors) in males (Quinn et al. 2007)

. 33DRAFT

Avian Two

- Gene ration ISR 45 May 2 8 , 2013 Table 5

- 1

. Summary of Responses to Estradiol or Trenbolone with Day of Egg Injection from 1

the Studies Reviewed. 2

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.

5.2.3 Estradiol 3

E ffects of exogenous steroids on sexual differentiation in Japanese quail have been 4

examined in a number of studies . These studies have investigated the effects of exogenous 5

estradiol treatment on sexual differentiation in Japanese quail

. These studies have shown that 6

neural systems are sensitive to the effects of estradiol; however, few have examined 7

toxicological effects or studied the endocrine disrupting effects of exogenous estradiol in a dose 8

response manner

. Nonetheless, this literature is valuable because it does reveal some impacts of 9

excess estradiol beyond that maternally deposited or produced by the embryonic gonads and 10

adr enal glands. 11

Exposure Levels and Protocol 12

The dose response study included 0, 0.5, 50

, or 500  g per egg (20  l vehicle)

. Injections 13

were conducted at either ED4 or ED11, to determine early effects compared to impacts on the 14

HPG axis and other gender specific characteristics

. Studies in the older literature had higher 15

doses, often with more toxicity than endocrine disruption as a result

. 16

Sensitive Endpoints 17

Response to the treatment differed with the timing of exposure

. Early embryonic 18

treatment impa ired male sexual behavior and decreased bursa size

. Females had reduced egg 19

production

. Treatment at ED11 also reduced egg production in females, with altered circulating 20

steroid hormone levels

. Brain catecholamines were altered by treatments. A summary of findings 21

is shown in Table 5

- 1 above

. 22

Conclusions 23

Estradiol is particularly potent early in embryonic development

. Effects in females may 24

be due to direct effects on the ovary (Rissman et al. 1984)

. Male sexual behavior and associated 25

neural systems are exquisitely sensitive to exogenous estradiol treatment during embryonic 26

development

. This study supports findings from other studies demonstrating that estradiol can 27

act as a potent endocrine disruptor, acting on neural systems that regulate endocrine and 28

behavioral components of reproduction, as well as impact ing immune

- related endpoint s such as 29

the bursa

. Further, it is clear that early exposure has greater overall impacts

, which would be 30DRAFT

Avian Two

- Gene ration ISR 46 May 2 8 , 2013 similar to maternal deposition with embryonic exposure occurring th rough the entire 1

developmental process. 2

5.2.4 Vinclozolin (McGary et al. 2001) 3

Exposure Levels and Protocol 4

Eggs injected on Day 4 of incubation at doses of 0, 25, 50, or 100 ppm (calculated for a 10 g 5

egg), and untreated. Endpoints in hatchlings were steroid hormones and hypothalamic GnRH

- I. 6

Adult males were measured for cloacal gland, male sexual behavior, hypothalamic GnRH

- I, and 7

steroid concentrations (estradiol and androgens). 8

Sensitive Endpoints 9

Results showed tha t exposure to VIN significantly (p< 0.05) altered GnRH

- I in male hatchlings, 10

whereas GnRH

- I levels in females remained unaffected. Although steroid levels were unaltered 11

by any VIN treatment, maturation appeared affected, with male the onset of reproductive 12

behavior delayed, with lower numbe r of mounts and cloacal contacts being lower (p<0.05) in 13

VIN

- treated males at higher doses. 14

Overall Conclusions 15

Vinclozolin treatments impacted males at higher doses, indicating that the antiandrogenic actions 16

adversely affected male sexual differentiation. However, responses were significant primarily at 17

relatively high doses. 18

5.2.5 Other Egg Injection Treatments 19

Many other studies in birds have utilized egg injections as the method to administer 20

treatments

. Although these studies have not been detailed, there are numerous examples of both 21

short

- and long

- term impacts of endocrine active compounds that are environmentally available

. 22

Original papers and reviews may be found in the additional information provid ed in support of 23

this ISR

. 24



### 5.3 Reproduction and Two

- Generation Dietary Studies 25

The effects of EDCs have been studied in a number of dietary studies in quail, including 26

reproduction and multigenerational tests

. Selected studies are described below to demonstrat e 27

response to specific EDCs and to provide information on responsive endpoint s

. 28

5.3.1 Perchlorate (Stafford 2011) 29

Exposure Levels and Protocol 30

Adult Japanese quail were exposed to perchlorate in the diet for ~10 weeks at doses of 0, 31

13.8, 36.4, 64.7 and 144.1 mg a.i./kg body weight/day

. Birds were photostimulated for 19 days, 32

and eggs were collected for 10 weeks and incubated

. Adult and 14

- day juvenile tissues collected 33

included blood, heart, liver, spleen, head (with the occipital plate of the cranium removed), 34

cervical and thoracic cavity (including the glands extending along the neck to above the heart), 35

and caudodorsal cavity (including kidney, adrenals, and reproductive organs). Endpoints 36

included overall adult male/female body weight change, overall average adult feed consumption 37

expressed as grams per bird per day, total number of eggs laid, cracked eggs , non

- cracked eggs

, 38DRAFT

Avian Two

- Gene ration ISR 47 May 2 8 , 2013 eggs set into the incubator, eggs set of total eggs, viable embryos (and of total eggs set), live 1

embryos (and of total viable embryos), successful hatchlings (and of eggs laid set, of total eggs 2

laid, and of total live embryos), total number of 14

- day off spring survivors (and of total eggs set, 3

and of total successful hatchlings), mean hatchling body weight, mean 14

- day od offspring 4

survivor body weight, and mean eggshell thickness per hen. 5

Sensitive Endpoints 6

A trend of increased aggression was observed, male weight decreased with dose

, and 7

there was a dose

- related reduction in egg shell thickness at 250, 500, and 1000 ppm

. A reduction 8

in number of viable embryos, and viable embryos of eggs set was observed with significant 9

reduction at 100 ppm

. Offspring data were not rep orted. E ndpoints including those for thyroid 10

system impact were not measured in adults (i.e.

, T3 and T4 levels, and mRNA). 11

Conclusions 12

Perchlorate is expected to primarily affect the thyroid axis

, not necessarily the 13

reproductive axis . H owever, if the perchlorate is toxic in addition to having thyroid system 14

effects, then additional effects may be associated with toxicity

. Therefore, the response to 15

perchlorate relative to egg quality indicates a capacity for the compound to affect the 16

re productive system as well as the thyroid system

. More sensitive thyroid endpoints/behavioral 17

endpoints were not tested in offspring . P erchlorate has been shown to alter behavior in birds 18

exposed during development (Rainwater et al. 2008) , and change embryo nic thyroid levels (Chen 19

et al. 2008)

. 20

5.3.2 Bisphenol A (Frey et al. 2011) 21

Exposure Levels and Protocol 22

Adult Japanese quail were treated with BPA (0, 1, 30, 300

, and 1000 ppm) during a 4 23

week acclimation period, then photostimulated

. After 1 week, an 8

- week egg laying period was 24

initiated

. Body weight, feed consumption, egg quality and production, fertility, hatchability, and 25

14

- day survival were measured. Adults were euthanized after an 8

- week egg

- laying period, eggs 26

incubated and hatchlings reared to 14 day s. Adult and offspring tissues collected included blood, 27

head, heart, liver, spleen, kidneys, thyroid glands, bursa of Fabricius, adrenal glands, gonads and 28

associated structures (oviduct, shell gland, epididymis, ductus deferens)

, and cloacal gland area. 29

Sensitive Endpoints 30

No adverse effects were detected in this study. 31

Overall Conclusions 32

This was a classic reproduction study conducted in adult reproductive Japanese quail with 33

additional measurements to assess effects on immune, reproductive, thyroid, a nd stress axes, as 34

well as collection of additional measures of impacts to the reproductive system

. Organ weights 35

and gross morphology were used to check for possible effects on the liver, spleen thyroid, bursa, 36

and adrenal glands. These additional measure s would reveal significant impacts, but may not be 37

the most sensitive measures . O ther endpoints

, such as behavioral or neural measures , may reveal 38

endocrine disruption (Halldin et al. 2005; Panzica et al. 2005; Yigit and Daglioglu 2010)

. 39DRAFT

Avian Two

- Gene ration ISR 48 May 2 8 , 2013 5.3.3 Trenbolone Reprodu ction Dietary Study (Frey et al. 2012)

- Dietary Study #1 1

Exposure Levels and Protocol 2

Adult quail were exposed to trenbolone at doses of 0, 0.1, 0.4, 1.6

, and 6.4 ppm for 9 3

weeks

. Birds were acclimated for 4 wks, then photostimulated for 1 week followed by an 8

- 4

week egg

- laying/collection period

. Eggs were incubated and hatchlings were reared to 14 days. 5

Body weight, feed consumption, egg quality and production, fertility, hatchability, and 14

- day 6

viability were measured. Adult and offspring tissues collecte d included b lood, head, heart, liver, 7

spleen, kidneys, thyroid glands, bursa of Fabricius, adrenal glands, gonads and associated 8

structures (oviduct, shell gland, epididymis, ductus deferens)

, and cloacal gland area. 9

Sensitive Endpoints 10

There was no evidence of endocrine disruption using this one

- generation reproductive 11

test

. Organ weights we re reported and tissues appear to have been preserved, but there is no 12

mention of additional analyses. 13

Overall Conclusions 14

Test doses were determined in a range finding test using adults

. Based on the results, 15

doses were selected and administered to adult Japanese quail . The age and reproductive status of 16

the birds at the initiation of the study are unclear. In other protocols from this laboratory, the 17

photostimul ation just denotes lengthening the photoperiod from 15 to 18 hours of light/day

. As 18

such, the birds should be sexually mature and functional at the initiation of the treatment

. The 19

biochemistry for the chemical analysis is very thorough and verifies the ef ficacy of the test 20

compound

. Dietary trenbolone in adults did not result in endocrine disruption

. This may be due 21

to lower sensitivity of adults as well as a lack of impact of androgenic compound

. In adult males, 22

additional androgen would not impair the ma le system unless the concentrations and activity of 23

the compound was sufficient to shut down the reproductive axis via negative feedback to inhibit 24

hypothalamic GnRH

. Similarly, females would not be affected by trenbolone unless high levels 25

are sufficient to interfere with the preovulatory LH surge. 26

5.3.4 Methoxychlor (dietary study with Japanese quail and bobwhite quail 27

comparisons) 28

Exposure Levels and Protocol 29

Two studies were conducted to compare Japanese quail and northern bobwhite quail 30

exposed to dietary m ethoxychlor at 0, 5

, and 10 ppm according to the OECD two

- generation 31

protocol

. Japanese and northern bobwhite quail ( parental generation ; P) were raised under short 32

photoperiod until somatic maturity , then placed on diet containing low concentrations of 33

methoxychlor (MXC; 0, 5, and 10 ppm) and simultaneously photostimulated. A range of 34

measures were monitored including feed intake, egg production, egg quality, fertility, and 35

offspring viability

. In addition, other measures were selected for their potentia l to detect 36

endocrine disruption

. Chicks (F1) were raised on the same diet as their parents, and paired as 37

adults . S exual maturation, reproductive behavior

, and endocrine measures were examined, 38

similar to the parent generation. F2 chicks were raised on co ntrol feed and assessed for the same 39

endpoint s as the F1 birds. 40DRAFT

Avian Two

- Gene ration ISR 49 May 2 8 , 2013 Sensitive Endpoints 1

There were some inherent species differences, including slower maturation and lower 2

productivity in bobwhite quail

. These differences were observed consistently over all ge nerations 3

in both species

. No treatment effects were observed in either species for body weight, feed intake, 4

egg quality, or viability

. Effects of 5 ppm MXC were similar in both species, and no treatment 5

effects were observed for egg production, shell qua lity, fertility, and chick growth

. However, 6

both species showed delayed sexual maturation with treatment

. This was more pronounced in the 7

10 ppm MXC treatment, with significant (p<0.05) reduction in the number of birds achieving 8

optimal reproductive perfor mance. Sexual behavior was significantly (p<0.05) impaired by MXC 9

exposure in both F1 and F2 Japanese quail males; behavioral assessment was not conducted in 10

bobwhite males due to species differences in courtship and mating behavior

. A modified open 11

field test was developed and pilot data was collected to assess potential impairment of motor and 12

behavioral responses in young chicks

. This testing method has potential for Japanese quail, but 13

not for bobwhite quail, again due to species differences in behavior al responses

. 14

Although variable, plasma and fecal steroid hormones were significantly (p<0.05) 15

different with treatment. In the 5 ppm MXC treatment, there was an apparent bimodal response, 16

especially in maturing birds

. This is potentially an important mark er because gonadal steroids 17

typically are variable among individuals during sexual maturation whereas treated birds showed 18

reduced and uniformly lower plasma steroid hormones

. This variability in gonadal steroids may 19

provide an index of EDC adverse effects on maturation, which would be important for field 20

populations where timing of reproduction is likely to be a critical factor

. This maturation related 21

relationship between reproductive status and steroid hormones was weaker in bobwhite quail 22

even though MX C exposure was associated with delayed sexual maturation, a similar effect was 23

observed in Japanese quail

. Hypothalamic neurotransmitters and GnRH

- I reflected stage in 24

maturation and reproductive capability; however these measures were variable and warrant 25

further characterization to link individual responses. 26

Overall Conclusions 27

A number of effects were observed in this multigeneration study, especially for endpoints 28

that revealed reproductive impacts. These effects were more apparent in the Japanese quai l

, 29

which also included male reproductive behavior. One purpose of the study was to directly 30

compare Japanese quail and northern bobwhite quail in the same testing protocol with the same 31

dietary treatments . Measures not conducted in this study include d morp hological, histological 32

(gonad, bursa), immune, and hormonal (thyroid, vitellogenin, gonadotropins) measures

. Overall, 33

the two species had similar responses to the low levels of dietary MXC treatments

. Japanese 34

quail offer a fast maturing precocial avian m odel that is practical relative to time for completion 35

of a two

- generation dietary regimen

. 36

5.3.5 Two

- Generation Dietary 17

- Beta Estradiol (E2) Dosing Study 37

Exposure Levels and Protocol 38

Japanese quail in t he P1 generation were either given the treatment diet prior to sexual 39

maturation (P1A, 3 weeks old ; 10 weeks of egg laying), or after proven egg

- laying ability (P1, 5 40

weeks ; 10 weeks of egg laying)

. F or each group, the F1s were either exposed to the treated diet 41

from hatch to egg

- laying (F1a) or not exposed t o the dietary treatment (F1b). F2 birds did not 42

receive the treatment. Controls were used for P1A and P1B, and F1a controls were used as the 43DRAFT

Avian Two

- Gene ration ISR 50 May 2 8 , 2013 control group for the other F1 treated groups

. E2 doses were 0, 0.078, 0.31, 1.25, and 5.0 ppm in 1

the diet. 2

Sensiti ve Endpoints 3

T he P1 gene ration had elevated E2 levels in both P1A and P1B males and females. There 4

was a trend of decreased testosterone in males with elevated E2. P1B females had significantly 5

greater numbers of right oviducts than P1A females. T hyroid an d adrenal gland weight s were 6

greater in the P1B group with diffuse hypertrophy in the adrenal. P1A had fewer eggs

, thinner 7

eggshell thickness

, and higher yolk E2 concentration. P1A males had degenerative changes of 8

the testis and reduced sperm number . P1B m ales had testicular lesion s in the high dose group. 9

P1A males had earlier sexual maturation and P1A males had increased non

- male plumage with 10

E2 treatment. 11

In the F1 generation, the P1A F1 females had lower hatch and adult body weights

. F1a 12

females had inc reased thyroid gland weights and decrease d pancreas and ovary weights. P1A

13

F1a females had more active oocytes and increased oviduct weight. F1a females had later sexual 14

maturation. F1a birds had increased Day 15 viability of eggs and hatchling production. An 15

increase in the proportion of cracked shells was associated with P1B parental exposure. F1a hens 16

also had increased eggshell thickness

. F1 males (P1A

- F1a) had increased terminal body weight, 17

tibiotarsus weight, and pancreas, liver, and testis weights a nd weight indices. Cloacal gland 18

weight was reduced in the high E2 diet, and smaller in the P1A

- F1a males, although the area was 19

greater in these males. 20

F2 generation females (P1A

- F1a) from E2 treated birds had more surviving F2 offspring. 21

F1a birds expose d to E2

, P1A

- F1a F2 chicks had significantly or nearly significantly reduced 22

measures of growth (tibiotarsus diameter and tarsometatsus length) ovary

- to

- body weight ratios 23

and increased gross and body weight

- adjusted pancreas weights of the F1a 5 ppm treat ment 24

groups. F1A F2 chicks had greater thyroid gland and spleen weight. P1B

- F1a F2 female chicks 25

had increased adrenal gland mass. P1B

- F1b chicks had increased liver weights. F2 chicks with 26

P1A exposure had significantly lower ovary weights. F2 chicks with P1B exposures had 27

increased incidence of limb malformations

. The F1a

- F2 male chicks had a lower body weight, 28

and less bone growth than male offspring of F1b parents. These P1A

- F1a F2 males also had 29

increased thyroid, pancreas, liver and cloacal gland weig hts, and decreased testis weight. F1a F2 30

males had significantly decreased brain weight. 31

Overall Conclusions 32

Even though the P1A dosing regime did not have a large impact on the P1 generation, the 33

P1A offspring (F1 and F2 generations) had effects o n many i mportant endpoints, suggesting that 34

the P1A protocol (i.e.

, dosing the P1 generation from 3 to 4 weeks of age) would be the most 35

appropriate protocol for assessing reproductive and other impact of estrogen active compounds. 36

Further, exposure of the F1 generation is important to observe combined effects of embryonic 37

exposure followed by lifetime dietary exposure. The F2 birds provide insight into the impacts on 38

F1 females’ reproductive systems including maternal deposition into eggs produced b y these 39

females. Moreover, these potential sources of endocrine disruption are isolated from direct 40

dietary exposure in the F2 birds because they are fed only control diet

. 41DRAFT

Avian Two

- Gene ration ISR 51 May 2 8 , 2013 5.3.6 Two

- Generation Endosulfan Dietary Test ( Quinn et al. 2010

) 1

Exposure Levels and Prot ocol 2

Dietary endosulfan was administered at 0, 30, 60, 125, and 250 ppm

. The two

- generation 3

experimental protocol was followed, with measurement of the range of endpoint s described 4

above in the protocol

. 5

Sensitive Endpoints 6

There were differences in organ weights; however, many of the responses were non

7

linear relative to dose

. 8

Overall Conclusions 9

There was clear maternal transfer of endosulfan into the eggs, which resulted in 10

significant effects at specific doses, primarily in the F1 generation

. Observed effects included 11

body weight, brain weight, kidney weight, and thyroid weight

. There were some gender related 12

differences in response, with exposed females in the 60ppm treatment having smaller ovaries 13

(p=0.003) associated with fewer follicles (p=0.057)

. 14

5.3.7 T

wo

- Generation Trenbolone Dietary Study ( Quinn et al. 2011 ) 15

Exposure Levels and Protocol 16

Dietary exposure was 0, 0.5, 5, 10, and 20 ppm, beginning at 4 weeks of age in the 17

parental generation, followed by a F1 generation maintaining treatment groups and an untreated 18

F2 generation

. Measurement endpoint s were conducted according to the two

- generation protocol, 19

with some additional endpoint s for assessing reproduction

. The two highest doses are beyond 20

those used in the one

- generation dietary test described abo ve

. 21

Sensitive Endpoints 22

Females showed impacts from the two high doses, with reduced egg production in all 23

generations to the extent that there was no F2 generation in the 20ppm dose level

. Additionally, 24

females showed delayed sexual maturation in the F0 and F1 generations. 25

Males showed effects of the higher doses of trenbolone

. Although the timing of sexual 26

maturation was unaffected, courtship and copulatory behaviors were impacted

. There were minor 27

effects on sperm motion; otherwise no other sperm charac teristics were affected

. Similarly, 28

cloacal gland area differed only in the 0.5 ppm treatment, where there was reduced size

. Organ 29

weights were largely unaffected, with some bursal response noted. 30

Overall Conclusions 31

In males, sperm characteristics and qua lity were not the most sensitive measures of effect

. 32

Cloacal gland area reflected androgen stimulation; the low dose reduction may reflect inhibition 33

of the HPG axis with insufficient combined trenbolone (androgen active EDC) and endogenous 34

androgen stimul ation

. Male sexual behavior was affected; however, the results are somewhat 35

complex and this testing paradigm will need further refinement to develop a clear testing 36

protocol for multiple users

. The author compares these findings to injection of 37

dichlorodi phenyldicholoroethylene (p, p’

- DDE), which is an anti

- androgen

, conclud ing that both 38

inhibited male sexual behavior

. The bursa remains a potential bioindicator of immune function 39DRAFT

Avian Two

- Gene ration ISR 52 May 2 8 , 2013 impacts. Female reproductive function was a responsive endpoint , culminating in the lack of F2 1

offspring in the highest dose group

. In total, trenbolone exerted endocrine disrupting effects, 2

especially on the F1 generation. 3

5.3.8 Two

- Generation 17β Trenbolone Dietary Study (Karouna

- Renier and 4

Henry, 2013) 5

Exposure Levels and Protocol 6

Ef

fects of continuous exposure to 17β trenbolone (17βT) w ere assessed in three 7

successive generations of Japanese quail. For the F0 generation, 6 week

- old Japanese quail were 8

randomly assigned to one of six doses of 17ß

- trenbolone: 0, 1, 5, 10, 20

, and 40 mg /kg feed 9

(n=16 pairs per dose). Eggs were collected from F0 and F1over weeks 7 to 8 to generate pairs for 10

the next generation. F1 birds were assigned to the same dose as their parents and received dosed 11

feed from day 1 of hatch. The F2 birds were exposed s olely in ovo and were provided only 12

untreated feed. The 40 ppm dose was dropped after F0 since the pairs exposed to this dose were 13

unable to produce sufficient numbers of eggs to populate the F1 generation. F0, F1 and F2 14

parents were euthanized at 16 weeks of age to harvest tissues for genomic and biochemical 15

analysis. 16

Sensitive Endpoints 17

Significant changes were observed in liver vitellogenin (VTG) mRNA, which decreased 18

with dose in F0 females and increased in F1 females and F0 males

. Plasma testosterone l evels 19

were decreased in F0 females with dose and were higher in the F0 (10, 20 and 40 ppm) and F2 20

females (20 ppm)

. Plasma estradiol significantly increased in F0 and F1 males with dose; 21

females showed isolated differences in estradiol levels with generation and dose

. Fecal steroids 22

mirrored differences in plasma steroids with showed treatment related differences; whereas, 23

thyroid hormones did not differ with treatment. Bursa weights were lower across all treatments

. 24

Behavioral trials indicated diff erences in latency to mount and mounting success among the 25

males in the 40ppm group

. Behavior and some other endpoint s are undergoing further analysis

. 26



### 5.4 Transferability of the Avian Reproduction Test to an Avian 27



### Multi

- Generation Test 28

The previous Reproducti on Test accurately detects gross morphological and overall 29

reproductive impairment with dietary test chemical exposure

. These measurement endpoint s are 30

useful in detecting effects

, but do not discern endocrine disruption from frank toxicological 31

activity

. Additionally, the endpoints measured throughout the multi

- generational study and 32

especially in the F1 generation, (body weight, potentially pre

- pubertal organ histology) provide 33

assessment of both toxicological effects and endocrine disruption

. It is impor tant for more 34

sensitive endpoints, such as fertility, behavior, histolopathology of the gonads and bursa, 35

hormone concentrations, and neuroendocrine measures

, to be included to ascertain potential 36

short and long

- term endocrine disruption. In summary, the A vian Reproduction Test is not 37

appropriate to discern endocrine disruption from toxicological effects and the test is relatively 38

insensitive to more subtle EDC effects of environmental chemicals, especially at lower (i.e. 39

environmentally relevant) exposures

. A series of specific points addressing these items follows 40

in the next section. 41DRAFT

Avian Two

- Gene ration ISR 53 May 2 8 , 2013 

### 5.5 Lessons Learned from Optimization Studies 1

 Avian physiology and sexual differentiation must underpin measurement endpoint s 2

chosen for the testing paradigm 3

 Need consistency in test design (photoperiod, timing of initiation of both photoperiod 4

and dosing, dosing period, timing of collections, measures, and genetic strain ) 5

 Need to consider “low” and “environmentally relevant” doses 6

 Standard reproductive measures are relatively insensitive, especially in dietary studies 7

 The two

- generation test provides a means of dosing various life stages and focusing 8

on potential vulnerable periods 9

 Egg injection studies tend to have clearer effects because the dose received may be 10

more consiste nt or there is no chemical breakdown through the digestive tract ; it is 11

also possible that the primary route of exposure has not been delineated 12

 Behavioral assessments were variable so it was difficult to show clear dose related 13

differences; same for cloa cal gland measurements 14

 Male sexual behavior may be one of the most sensitive endpoint s to estrogenic 15

chemicals; however, the protocol must be standardized 16

 Species differences in behavioral measures 17

 18

Sampling Points and Schedule 19

 Egg injection studies showe d that the embryo is the most vulnerable stage for 20

precocial birds 21

 In a diet test, the F1 is expected to be the most affected because both embryonic and 22

dietary exposure occurs throughout the lifespan 23

 Sampling times: 24

o At hatch to ascertain embryonic/develop mental effects, 25

o During maturation to detect any acceleration or delay 26

o Monitor growth and achieving adult statu s 27

o Adult onset of reproduction 28

o Productivity during adult reproduction, once full reproductive function has 29

been attained (about 10 weeks of age) 30

o S ustained reproductive function and any evidence of health issues for at 31

least 6 weeks of production 32

Histopathological Analyses 33

 Histology is revealing if the suspected target has significant chronic effects 34

 May be more revealing for toxic actions of the com pounds 35

 Altered thyroid system function does not usually result in easily discernable 36

histological effects except if there is a goiter (collection of hormone precursors in the 37

glandular follicles) 38

Secondary Sex Characteristics 39

 Male cloacal gland area is testosterone dependent 40

 Plumage characteristic of male and female 41

 Body size and weight 42

 Relative body fat 43DRAFT

Avian Two

- Gene ration ISR 54 May 2 8 , 2013 Vitellogenin. 1

 Has not been readily detectable in hatchlings 2

 Not clearly a responsive endpoint 3

 More characterization is needed to delineat e when this would be a responsive 4

endpoint and in what situations; specifically does vitellogenin change in females and 5

how much do males respond? 6

Sex Steroid Measurements 7

 Plasma measurements may be more variable, but revealing with smaller volumes 8

from h atchlings 9

 Fecal samples reflect more consistent effects 10

 Responsive endpoint if the reproductive axis is affected 11

Thyroid hormone measurements 12

 Appropriate measurement endpoint for PCBs and PBDEs as well as other thyroid 13

active compounds 14

 Gland content reflec ts long term impacts; response can be increased (lack of secretion) 15

or decreased (impaired hormone synthesis) 16

 Plasma T3 and T4 reflect alterations in glandular function, axis function, as well as 17

altered enzymes (deiodinases) activity which are important i n the conversion of T4 to 18

T3 or rT3 (reverse T3)

. More information is needed to develop a predictive model for 19

chemical classes with predicted diagnostic responses 20

Exposure Analyses 21

 Analytical data are very useful in determining the amount of chemical that elicits 22

adverse effects 23

 Establish threshold of impact and relative sensitivity of species and/or life stage at the 24

time(s) of exposure 25

 Studies indicate that embryonic exposures are at much lower dose than previously 26

believed; meaning that the effective concentration of EDCs that result in adverse 27

effects are much lower when bioavailability and embryonic uptake are considered 28

o More data are needed to accurately predict effects

— suggest embryonic 29

bioassay as a range finder and initial assessment for potential mechanism 30

Statistical Analyses 31

 Need to consider and allow for non

- linear data analysis 32

 Analysis of many endpoint s may dilute detection of effects 33

o Primary responsive endpoint s must be identified for assessment 34

Parametric tests 35

 Preferred, but many of the data do not meet statistical assumptions 36

 Depending on the measurement endpoint , the statistical analyses must be pre

- planned 37

to accommodate a range of issues that are inherent in the types of data to be collected 38

 Assessment should include some acknowledgement of variability as an indicator of 39

effect; specifically there may be increased (due to individual differences in sensitivity) 40

or decreased variability (high mortality and survivor effect or delayed maturation) 41

associated with endocrine disruption 42DRAFT

Avian Two

- Gene ration ISR 55 May 2 8 , 2013 Non

- parametric tests 1

 Appropriate for behavioral tests and for ordinal data (cloacal gland area) 2

 Standardized methods should be prescribed so that there is consistency in analyses

. 3

4

5

6

7DRAFT

Avian Two

- Gene ration ISR 56 May 2 8 , 2013 

### 6 INTERLABORATORY EVALUATION 1

The following discussion summarizes the laboratory results and statistical analysis from 2

various studies intended to evaluate the protocol on both an intra

- laboratory (within a single 3

laboratory) and inter

- laboratory (across all laboratories) basis. The reports documenting these 4

studies and analyses are presented in Appendix C

. Supporting demonstration studies were also 5

sponsored by USEPA and performed by the U.S. Geological Survey and the U.S. Army, the 6

results of which are gi ven in Appendix E

. 7



### 6.1 Vinclozolin

— Bayer Crop Science Study 8

6.1.1 Exposure Levels and Protocol 9

Four treatment groups, each initially containing 18 pairs of Japanese quail, were fed diets 10

containing the test substance at 64, 160, 400 & 1000 ppm a.i. through two generati ons (F 0 and 11

F 1 ). A control group, also with 18 pairs initially, was maintained concurrently

. A third 12

generation (F 2 ) was reared without exposure to the test substance through their diet. The F 0 13

Japanese quail were genetically sexed after hatching and were 6 weeks old at test initiation

. The 14

F 0 generation was exposed to treated diets from week 6 of age through an egg laying cycle of 15

seven weeks

. Selected embryos (Hatch E, 5 th group of eggs set from F 0 hens) at embryo day15 16

(ED15) were sampled for possible an alysis of endocrine and histological impacts

. Hatched F 1 17

chicks (Hatch F & G, 6 th & 7 th group of eggs set from F 0 birds) were exposed to treated diets 18

from hatch through rearing to 14 days of age

. At one week of age surviving chicks were 19

genetically sexed

. Selected F 1 offspring (Hatch F & G) maintained on treated diets for grow

- out 20

to four weeks of age

. With exposure continuing, at three or four weeks of age the F 1 birds were 21

paired and monitored through a seven

- week reproduction cycle

. Selected F 1 embryos (Hatch E) 22

at embryo development day 15 (ED15) were sampled for possible analysis of endocrine and 23

histological impact

. Surviving F 2 chicks were genetically sexed at one week of age and reared to 24

14

- days of age on untreated diet

. Tissues of selected F 2 14

- d ay old survivors (Hatch F & G) 25

were sampled for possible analysis of histological impact

. Additional F 2 birds were selected and 26

reared to sexual maturation for mating behavior observations

. 27

For each phase, effects of exposure to the test substance on bird health, body weight and 28

feed con sumption were evaluated

. The effects on the number of eggs laid, viability of the 29

embryos, normal development of embryos, percent hatchability, offspring survival and body 30

weight, and eggshell thickness for both the F 0 and F 1 generations were evaluated

. Selected tissue 31

samples were collected from embryos, 14

- day old chicks and adults for evaluation of 32

biochemical endpoints such as serum steroid concentration

. In addition, observations were 33

conducted on subsets of F 1 and F 2 b irds to evaluate possible effects of exposure on mating 34

behavior

. 35

6.1.2 Sensitive Endpoints and Overall Conclusions 36

The summary of statistical analyses provided by Dr. Tim Springer (see Appendix C for 37

details) included 106 variables, some of which were subjected to multiple comparisons

. He 38

conducted two types of analyses for each variable

- by

- generation cell. First, response to each test 39

concentration was compared to the controls using Dunnett’s mul tiple comparison procedure. 40

These responses were subjected to a Jonkheere

- Terpstra trend test to provide an indication a 41

concentration

- related trend. When effects in one direction (i.e.

, increase or decrease) were clearly 42DRAFT

Avian Two

- Gene ration ISR 57 May 2 8 , 2013 deleterious, and not deleterious i n the other direction, a one

- sided Dunnett’s test was used. 1

Otherwise, a two

- sided test was performed

. The one

- sided or two

- sided test as well as the 2

direction of the test if one

- sided, is indicated in Table 6

- 1 . In either case, the cell

- wise type I 3

error rate was controlled at α = 0.05. The Jonkheere

- Terpstra trend test was performed 156 times 4

and showed that 7 of the significant trends were likely to reflect a false discovery rate. Similarly, 5

the Benjamini

- Hochberg approach was applied to the results of t he 624 p

- values from the 6

Dunnett’s test and revealed 13 false discoveries

. 7

Most variables did not reveal significant differences due to treatment (Table 6

- 1 ); body 8

weight and feed consumption showed some differences. The only other variables with p

- values 9

less than 0.05 for both Dunnett’s test and trend were Day14fE2 (day 14 female E2 concentration) 10

and AmT3 (adult male T3)

. As such, there were few significant e ffects of the vinclozolin at the 11

dietary doses in this study

. 12

Table 6

- 1

. Summary of Statistical Analyses Yielding Effects

– Bayer. 13

Obs Variable Name Generation N a of Dunnett p < 0.05 N b of JT p < 0.05 Both c

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

15DRAFT

Avian Two

- Gene ration ISR 58 May 2 8 , 2013 

### 6.2 Vinclozolin

– Wildlife International Study 1

6.2.1 Exposure Levels and Protocol 2

This trial was conducted according to the sam e protocol as described above. The primary 3

difference was that the Japanese quail used in this trial were from the Wildlife International quail 4

colony as compared to birds purchased from breeders by the other entities performing the inter

5

laboratory comparison two

- generation trials

. 6

6.2.2 Sensitive Endpoints and Overall Conclusions 7

Summary of statistical analyses ( provided by Dr. Tim Springer; see Appendix C for 8

detail s ) showed evidence of few effects due to dietary vinclozolin exposure for the 106 variables 9

analyzed (Table 6

- 2 )

. The analysis was identical to that performed for the Bayer Crop Science 10

multi

- generatio n study

. Seven variables gave Dunnett’s test p

- values of less than 0.05 for at least 11

one treatment group, and trend tests for nine variables suggested a concentration response trend 12

(i.e. p < 0.05). Variables with Dunnett p

- values < 0.05 were CloacaM4, Clo acaF6, CloacaM6, 13

and CloacaM7 (male or female c loacal area (mm 2 ) at 4, 6, and 7weeks) in the F1 generation. The 14

variables ThyF214DayM (Thyroid weight of day 14 birds in the F2 generation, AfE2 (adult 15

female E2) in the F0 generation and AfT (adult female te stosterone) in the F1 generation also 16

gave Dunnett p

- values < 0.05

. Table 6

- 2 shows variables with at least one p value less than 0.05 17

according to Dunnett’s test or p value for trend of less than 0.05 according to the Jonkheere

18

Terpstra trend test. The ni ne variables with Jonkheere

- Terpstra p values less 0.05 are shown. 19

Table 6

- 2

. Summary of Statistical Analyses Yielding Effects

– Wildlife International. 20

Obs Variable Name Generation N a of Dunnett p < 0.05 N b of JT p < 0.05 Both c

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.

21DRAFT

Avian Two

- Gene ration ISR 59 May 2 8 , 2013 When both the trend test and Dunnett’s tests have low p

- values, there is evidence for both 1

a relatively large effect and a concentration response trend

. Observing low probabilities for both 2

tests is stronger evidence for a meaningful response than detecting a large change without 3

concentration response, or slight concentration related trend where response is small. Only three 4

variables, CloacaM4 (male c loacal area (mm 2 ) at 4 weeks), CloacaM6 (male c loacal area (mm 2 ) 5

at 6 weeks) and AfT (adult female testosterone) for F1 generation birds, had at least one 6

Dunnett’s p

- value of less than 0.05, and also showed a concentration response trend with p < 7

0.05. 8



### 6.3 Vinclozolin

– Smithers Viscient Carolina Research Center Study 9

6.3.1 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 (F 0 and 12

F 1 )

. A control group, also with 18 pairs initially was maintained conc urrently. A third generation 13

(F 2 ) was reared without exposure to the test substance through their diet

. The approximate 14

duration of the study was eight to nine months

. Primary phases of the study included: production 15

of test birds (hatching, rearing and DN A sexing)

- 4 weeks, acclimation to reproduction test pens 16

- 2 weeks, F 0 egg production

- 7 weeks, F 0 post egg production (embryo sampling, adult 17

termination and sampling, and final incubation)

- 2 weeks, F 1 hatching and rearing to sexual 18

maturation (DNA s exing and selection of birds for second phase of reproduction and mating 19

behavior observations)

- 6 weeks, F 1 egg production

- 7 weeks, F 1 post egg production (embryo 20

sampling, adult termination and sampling, and final incubation)

- 2 weeks, F 2 hatching an d 21

rearing (DNA sexing and 14

- day old offspring sampling)

- 2 weeks, F 2 rearing to sexual 22

maturation (for mating behavior observations)

– 6 weeks

. Behavioral assessments were made for 23

males only in the control and high treatment groups for a subset of 7 mal es in each

. Blood 24

samples were drawn by cardiac puncture, including samples taken from ED 15 embryos

. Data 25

were collected for the following endpoint s: adult body weight, adult feed consumption, eggs laid 26

per pen per day (eggs laid/maximum laid)

- Days coun ted from test initiation for F0 and from the 27

first egg layed by any bird for F1 and potentially F2, eggs cracked of eggs laid, fertile eggs of eggs 28

incubated, viable embryos of eggs incubated

, live 2

- week embryos of viable embryos

, hatchability 29

of 2

- week e mbryos

, 14

- day old survivors of hatchlings , h atchlings of eggs incubated , hatchlings of 30

fertile eggs, hatchlings per pen per day (hatchlings/maximum set)

, 14

- day old survivors of eggs 31

incubated

, 14

- day old survivors per pen per day (14

- day olds/maximum set )

, hatchling body 32

weights

, 14

- day old survivor body weights

, egg shell thickness

, reproductive behavior , and 33

secondary sex characteristics/phenotype. 34

6.3.2 Sensitive Endpoints and Overall Conclusion 35

There were no treatment related effects noted

. No effects were observed in any 36

generation related to mating behavior or reproductive parameters

. As such, the NOEC was 37

determined to be 1000 a.i./kg feed, which was the highest concentration. 38

Statistical analyses of 106 variables ( provided by Dr. Tim Springer ; see Append ix C ) 39

revealed that 21 variables gave Dunnett’s test p

- values of less than 0.05 (Table 6

- 3), and the 40

trend tests for 9 variables suggested that a concentration response trend was present (i.e. p < 41

0.05)

. These data are shown below

. However, the Benjamini

- H ochberg procedure indicated that 42DRAFT

Avian Two

- Gene ration ISR 60 May 2 8 , 2013 a number of these differences might be false discoveries; similarly there is reduced confidence in 1

differences when The Jonkheere

- Terpstra Trend Test was applied. 2

Table 6

- 3

. Summary of Statistic al Analyses Yielding Effects

– Smithers Viscient. 3

Obs Variable Name Generation N a of Dunnett p

< 0.05 N b of JT p <

0.05 Both c

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 Atte m p ts2 F1 1 0 0

135 ChangeAt F1 1 0 0

Thyroid Weight

145 T h y F2 1 4DayM F2 1 0 0

146 T h y F2 1 4DayF F2 1 0 0

147 T h y AdultMa l e F0 1 0 0

148 T h y AdultMa l e F1 1 1 1

Bio Chem

155 Da y 14f E 2 F2 1 0 0

161 Da y 14 m T3 F2 1 0 0

168 AfT3 F1 1 0 0

169 AfT4 F0 0 0 0

170 AfT4 F1 0 1 0

171 A m E2 F0 1 0 0

174 A m T F1 1 1 1

176 A m T3 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. O therwise Both = N.

4

6.3.3 General Advantages of Japanese Quail Two

- generation Test Reproducibility of 5

the assay 6

Interlaboratory comparisons were conducted to determine if there were adverse 7

consequences of exposure to putative endocrine active substances in a two

- generation testing 8

paradigm and to ascertain reliability of the testing protocol. The interlaboratory t ests were 9

sponsored by EPA and conducted in three laboratories: Smithers Viscient LLC ( SV), Bayer 10

CropScience Inc

. ( later renamed SynTech , or ST), and Wildlife International (WLI)

. All these 11

laboratories carried out tests with vincolozlin at 64, 160, 400

, and 1 000 ppm concentration levels

. 12

The intra

- laboratory analyses were performed for each laboratory ’s data by WLI and have been 13

summarized above

. 14DRAFT

Avian Two

- Gene ration ISR 61 May 2 8 , 2013 The interlaboratory analysis compared first, control group results among the three 1

laboratories to assess the range of variation within laboratory and among laboratories and the 2

power analyses based on the control groups to detect alternatives fr o m the control groups within 3

a laboratory that can be detected with high power, based on average within laboratory variabilit y

. 4

Second, the treatment and control group results were compared across laboratories

. Third, 5

generations were compared across laboratories. 6

Comparison of the data from the three laboratories show s evidence for the following: 7

 There are clear differences in strains of Japanese quail in terms of body weight, and 8

related variables that contribute to somewhat large differences and ranges in 9

measurement endpoint s 10

 Strain differences may also be a factor in rate of maturation and reproductive 11

characteristics, 12

 Some variables also show intra

- assay variability; it is not clear if this is indicative of an 13

unreliable measurement endpoint or if the method for measurement requires refinement

. 14

o Behavioral measures may be improved with more precise testing methods, 15

specific ally consistency in the number and treatments subjected to testing and 16

potentially use of one testing day rather than three successive days 17

 The primary measures indicative of endocrine disruption require a multi

- generation test 18

protocol in order to discern vulnerability on specific life stages such as embryos as 19

opposed to adult stages of the life cycle

. 20

6.3.4 Data Interpretation 21

The inter

- laboratory comparison provides insights on sources of variation due to the 22

testing paradigm and other factors

. Vinclozolin di d not exert strong effects in any of the inter

23

laboratory trials

. Comparison of control birds across the three laboratories revealed that strain 24

differences are critical and in fact the Japanese quail strain that is closest to wild type tends to be 25

smaller , mature s more rapidly, and is generally more vigorous

. More highly selected strains may 26

also lose their photoresponsiveness and may show altered reproductive system function that 27

differs from wild birds

. In addition, comparison of control birds revealed variability in some of 28

the selected measurement endpoint s, which could be indicative of the level of power needed to 29

discern endocrine disruption

. Conversely, an EDC given in a dose response venue would be 30

expected to overcome individual variation in response. 31

V inclozolin, which was chosen for this inter

- laboratory comparison , did not exhibit a 32

substantial response in Japanese quail

. Based on the biological basis of sexual differentiation, 33

this is not a comp letely surprising outcome

. Although effects were observed with egg injection 34

experiments , the levels achievable by this type of direct treatment were effectively higher than 35

those associated with maternal deposition

. Further, the mechanism of action of vin clozolin is as 36

an anti

- androgen

. Sexual differentiation in avian species is genomic with females being the 37

heterogametic (ZW) sex and males being the homogametic (WW) sex

. The heterogametic female 38

relies o n estradiol exposure to differentiate; whereas, the male is often considered more similar 39

to the mammalian female as the homogametic sex

. It should be pointed out that similar to 40

mammals, both genders do produce steroid hormones necessary for development of accessory 41

sex structures

. However, an anti

- androg en would not be predicted to alter sexual differentiation 42

of the female hypothalamus and unlikely to impact the male except at high levels

. Conversely, 43DRAFT

Avian Two

- Gene ration ISR 62 May 2 8 , 2013 estrogen active and strong androgen active compounds did have adverse effects in both males 1

and females in the optimization studies

. Furthermore, thyroid active compounds also impact 2

avian species

. 3

Table 6

- 4 summarize s the coefficients of variability (CV) across laboratories by category

. 4

Table 6

- 5 summarizes and the ranges of percent deviations from control group means for 5

detections with 80% power

. For each parameter the CV across labs and the associated p

- value 6

for significance of the laboratory random variation (i.e.

, differences among laboratories) for each 7

generation were summarized

. The ranges of CVs we re compared across categories. The 8

following are some findings: 9

 The median of F1 CVs exceeds that of the F0 CVs for 27 of 57 (47%) comparsions

. This 10

is nearly 50%, so there is no systematic generation difference with respect to CVs. Some 11

of the highest CVs are in the F1 generation, particularly for mating behavior. 12

 The median of F2 CVs exceeds that of the F1 CVs in 15 of 24 (62.5%) comparisons

. This 13

possibly suggests some degradation in sensitivity in F2 compared to F1. 14

 The smaller CVs for body weight in the F2 generation may be due to thefact that adult 15

body weights were not measured for the F2 generation. 16

The ranges of percent deviations to attain high power were compared across categories. 17

F1 was compared to F0 for six of nine categor ies

. The F1 percent deviation exceeded the F0 18

percent deviation for 34 of 57 comparisons (60%). This possibly suggests some degradation in 19

sensitivity in F1 compared to F0

. The F2 percent deviation exceeded the F1 percent deviation for 20

14 of 24 comparisons (58%)

. This again possibly suggests some degradation in sensitivity in F2 21

compared to F1. 22

Table 6

- 4

. Ranges of CV% Across Laboratories (by Category). 23

F0 Generation F1 Generation F2 Generation # F1 >

F0 # F2 >

F1 Category CV Range Median CV Range Median CV Range Median

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

25DRAFT

Avian Two

- Generation ISR 63 May 2 8 , 2013 Table 6

- 5

. Ranges of Percent Deviations from Control Group Means for Detections with 80% Power and the Ratios Between 1

Generations (by Category)

. 2

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

4DRAFT

Avian Two

- Generation ISR 64 May 2 8 , 2013 6.3.5 Other Considerations for Multi

- Generation Tests 1

Low Doses, Non

-Linear Dose Responses, and Windows of Exposure 2

Determining endocrine disruptive effects of environmental chemicals requires a 3

drastically different approach than the approach to determining classic toxicological effects such 4

as lethality, weight changes, organ d isruption, and teratogenic mutagenic actions. In order to 5

understand the full extent of the endocrine disruptive effects of compounds, the design and 6

interpretation of the EDC studies need to be firmly based in the principles of endocrinology: 7

1) Windows of exposure

— the timing of exposure to EDCs is extremely important. Data 8

indicate that precocial birds are primarily vulnerable to EDC exposure during embryonic 9

development, likely due to sexual differentiation during embryonic and early post hatch 10

phases of development. Effects of embryonic exposure to EDCs are manifest during 11

sexual maturation and in adults. 12

13

2) Concentration Response

— endocrine responsive tissues and systems may be affected 14

differently than understood from classic toxicology studies. Even modest exposure 15

concentrations of EDCs can interact with endogenous hormones that are often at pg

16

ng/ml concentrations. Moreover, responses can be drastically different at different points 17

along a concentration response curve

. Results should be stud ied for trends and 18

appropriate statistical models will be needed. For example, there was a non

- linear 19

response to vinclozolin in Japanese quail in a study by McGary et al. (2001) . However, 20

the increase in GnRH peptide concentrations occurred at mid

- doses ( inverted “U” shaped 21

curve) and there was a “U” shaped response in copulatory behavior. Because these data 22

were not analyzed to determine non

- linear responses, there may have been less ability to 23

detect statistical significance. While it is important not to use statistical analysis to extract 24

significance when there is none, it is also important to take into account physiological 25

relevance (i.e.

, the non

- linear action of hormones) when deciding on the correct statistical 26

model. 27

28

3) Strain/Species Variability

— B ennett et al. (2001) state that “There are data that show 29

strains of Japanese quail may be quite variable in reproductive and growth characteristics. 30

For example, many of the strains common to Europe have been selected for rapid growth 31

and higher body weig ht. They may weigh 250 to 300 gm. In comparison, most the 32

random bred strains in the USA and Japan tend to have lower body weight (100 to 33

150 gm). Some comparisons between strains exist for endocrine characteristics, which 34

have shown that the heavier strai ns tend to be slower to mature, have lower productivity, 35

have shorter periods of peak production, and some strains have lower fertility (Van Krey 36

et al. 1983; Zelenka et al. 1984; Anthony et al. 1991; Lilja and Marks 1991; Marks and 37

Washburn 1991a, 1991b; Peebles and Marks 1991; Yang et al. 1998; Aggrey et al. 2003). 38

Conversely, some of the smaller birds, especially the Hawaiian feral colony may be more 39

flighty. The smaller strains are preferable for long

- term tests as the fertility and 40

productivity tend to stay at peak for a longer period of time than the larger strains. 41

However, even these birds show an age

- related decline in reproductive performance. It is 42

not clear which strains are more aggressive. Some of the larger strains may be more 43

sedentary, but c oincidentally may be less sexually aggressive as well. 44DRAFT

Avian Two

- Generation ISR 65 May 2 8 , 2013 1

4) Eggshells of Japanese quail tend to have mottled coloration. Some strains have primarily 2

light colored or white shells. This is desirable for easy candling of eggs, but is not a 3

critical factor to con sider. Current data suggest there are no differences in the shell 4

strength across strains. 5

6

5) Birds have altricial and precocial reproductive and developmental strategies. Altricial 7

species include song birds have post

- hatch differentiation of the song syste m and 8

copulatory behavior. Often, both males and female share incubation and taking care of 9

the young. Altricial birds appear to be less sensitive to effects of EDCs during embryonic 10

development, but may remain more vulnerable to EDC impacts as adults, wit h impaired 11

singing, mating, and parental behaviors. Altricial birds studied in the laboratory include 12

zebra finches, ring doves, and some other species; a number of studies have contributed 13

to a substantial literature. Precocial birds that have been studie d in the laboratory include 14

Japanese quail, mallard ducks, and domestic chickens. The advantage of precocial birds 15

as a model species for EDC studies is that these species are established models with a 16

great deal of information. They are easily managed in the laboratory and mature rapidly. 17

18

6) The process of sexual differentiation in birds is a unique process; thereby making it 19

critical to test an avian model for effects of EDCs. 20

21DRAFT

Avian Two

- Generation ISR 66 May 2 8 , 2013 

### 7 CONCLUSIONS 1



### 7.1 General Advantages of Japanese Quail Two

- generation Test 2

 Precocial avian model that matures rapidly 3

 Well characterized with a great deal of information available 4

 Multi

- generation test can be completed in 12 months for the in life portion of the 5

test 6

 Methods and assays have been validated for this species 7

 As more information becomes available, it is possible to develop straightforward 8

assays and tests that elucidate mechanisms 9

 Japanese quail appear moderately sensitive to the effects of EDCs

, thereby 10

providing an indication that is more broadly applicable across avian species 11

 Quail are precocial; chicks can be treated via dietary administration 12

 The wild type laboratory strain retains photoperiodicity and other characteristics 13

of wild populations 14

 The immune, adrenal, and thyroid systems have been well characterize d

. 15



### 7.2 Limitations of the Japanese Quail Two

- generation Test 16

 There are ontogenetic differences in the pattern and timing of hormone changes 17

during development for the reproduction and thyroid endocrine systems for 18

precocial and altricial birds 19

 Japanese quail may vary across strains, with birds selected for heavy body weight 20

maturing more slowly and having less robust reproductive function 21

 Japanese quail provide a model species that does not completely emulate all birds, 22

especially very long lived species

. 23



### 7.3 Valu e

- added of Multi

- generation Exposure 24

 Data indicate that adult birds are generally less vulnerable to adverse effects of 25

EDCs 26

 It is critical to have the F1 generation and then to observe the capability of the F1 27

to successfully reproduce viable offspring 28

 So me chemicals may exert epigenetic effects that would have long term impacts 29

on wild population over multiple generations, even without continued EDC 30

exposure

. The F2 generation provides a test for this type of adverse effect 31

 Each portion of the multi

- gener ation test protocol provides a unique treatment in 32

that the P0 has maturation and adult exposure, the F1 has embryonic and life long 33

exposure, and the F2 tests solely embryonic exposure

. Together, all the salient 34

and vulnerable life stages are tested. 35

 It is important to incorporate both endocrine and behavioral tests to discern 36

endocrine disruption at various targets, including neural and direct organ system 37

effects

. 38DRAFT

Avian Two

- Generation ISR 67 May 2 8 , 2013 

### 7.4 Complementary Technologies to the Japanese Quail Two

- generation 1



### Test 2

Other kinds of testing paradigms may be considered, but at this point, the two

- generation 3

Japanese qua i l test is recommended for detection of endocrine disruption. Moreover, current 4

technologies available for fish and mammals are now becoming available for avian species. 5

These technologies, including ELISAs and epigenetic endpoints, are expected to provide 6

sensitive and precise detection of more subtle alterations due to endocrine disruption. 7DRAFT

Avian Two

- Generation ISR 68 May 2 8 , 2013 

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### 9 List of Appendices 1

2



### APPENDIX A . VALIDATION OF SCRE ENING AND TESTING AS SAYS 3



### PROPOSED FOR THE EDS P ( JULY 2007) 4



### APPENDIX B . DETAILED REVIEW PA PER FOR AVIAN TWO

- GENERATION 5



### TOXICITY TEST ( OECD NO. 74

, AUGUST 200 7 ) 6



### APPENDIX C

. REPORTS ON THE INTER

- LABORATORY TRIALS FO R THE 7



### JQTT 8

C.1 Inter

- laboratory Statistical Analysis, 2013 9

10

C.2 Vinclozolin Study (TO35) by SynTech (Bayer CropScience), 2013 11

12

C.3 Vinclozolin Study (1 85

- 133) by Wildlife International, 2013 13

14

C.4 Vinclozolin Study (13888.4106) by Smithers Viscient, 2013 15

16

C.5 Statistical Analysis for Vinclozolin Study Performed at Bayer (185

- 133

- 1), prepared by 17

Wildlife International, 2013 18

19

C.6 Statistical Analysis for Vinclozolin Study Performed at Wildlife International (185

- 133

- 20

3), prepared by Wildlife International, 2013 21

22

C.7 Statistical Analysis for Vinclozolin Study Performed at Smithers Viscient (185

- 133

- 2), 23

prepared by Wildlife International, 2013 24

25



### APPENDIX D

. AV IAN TWO

- GENERATION TOXICITY TEST IN THE 26



### JAPANESE QUAIL, OECD GUIDELINE FOR TESTIN G OF CHEMICALS

, DRAFT, 27



### SEPTEMBER 2011 28



### APPENDIX E

. SUPPORTING TWO

- GENERATION DEMONSTRA TION 29



### STUDIES 30

E.1 DRAFT Trenbolone Quail Study Performed by U.S. Geological Survey, 2013 31

32

E .2 DRAFT Endosulfan Quail Study (87

- XE

- 08JG

- 10) Performed by U.S. Army Public 33

Health Command, 2010. 34

35

E.3 Trenbolone Quail Study (87

- XE

- 0DUZ

- 11) Performed by U.S. Army Public Health 36

Command, 2011. 37