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## Validation of the Medaka Multigeneration Test : 1



## Integrated Summary Report 2

3

4

5

U.S. Environmental Protection Agency 6

Endocrine Disruptor Screening Program 7

Washington, D.C. 8

9

10

11

12

13

14

15

16

17

28 May 2013 18

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### Contents 21

EXECUTIVE SUMMARY....................................................................................4 22

OBJECTIVE

................................ ................................ ................................ ................................ ... 6 23

1 INTRODUCTION

................................ ................................ ................................ ...................... 6 24

1.1 Endocrine Disr uptor Screening Program

................................ ................................ ......... 7 25

1.2 Validation

................................ ................................ ................................ ......................... 8 26

2 HISTORICAL OVERVIEW OF FISH TESTS

................................ ................................ ........ 11 27

2.1 Purpose of the Medaka Multigeneration Test in EDSP

................................ ................. 11 28

2.2 Medaka multigeneration toxicity tests for the detection of possible endocrine disruption 29

12 30

2.2.1 Advantages of the medaka test model

................................ ................................ ..... 12 31

2.2.2 Conceptual basis for a multigeneration test paradigm

................................ ........... 12 32

3 MEDAKA MULTIGENERATION TEST PROTOCOL DEVELOPMENT

.......................... 13 33

3.1 Overview of the Medaka Multigeneration Test (MMT) Research Protocol

.................. 14 34

3.2 MMT Endpoints

................................ ................................ ................................ ............. 16 35

3.2.1 Genotypic Sex (F O , F 1 , F 2 )

................................ ................................ ...................... 17 36

3.2.2 Survival (F O , F 1 , F 2 )

................................ ................................ ................................ ... 17 37

3.2.3 Growth (F O , F 1 , F 2 )

................................ ................................ ................................ .... 17 38

3.2. 4 Reproduction (F O , F 1 , F 2 )

................................ ................................ ........................... 17 39

3.2.5 Liver Vitellogenin mRNA (F O , F 1 , F 2 )

................................ ................................ ........ 18 40

3.2.6 Secondary Sex Characters (F O , F 1 , F 2 )

................................ ................................ ...... 18 41

3.2.7 Hatch Success (F O , F 1 , F 2 )

................................ ................................ .......................... 18 42

3.2.8 Pathology and Gonad Phenotype (F O , F 1 , F 2 )

................................ ............................ 18 43

3.3 Research MMT Timeline

................................ ................................ ............................... 19 44

3.3.1 F 0 ; Study weeks 1

- 5

................................ ................................ ................................ . 20 45

3.3.2 F 1 and F

2 ; Weeks 1

- 3

................................ ................................ .............................. 20 46

3.3.3 F 1 and F

2 ; Week 4

................................ ................................ ................................ ... 21 47

3.3.4 F 1 and F

2 ; Weeks 4

- 6

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

3.3.5 F 1 and F

2 ; Week 7

................................ ................................ ................................ ... 21 49

3.3.6 F 1 and F

2 ; Weeks 7

- 10

................................ ................................ ............................ 21 50

3.3.7 F 1 and F

2 ; Weeks 11

- 15

................................ ................................ .......................... 21 51

3.3.8 F 1 and F

2 ; Week 16

................................ ................................ ................................ . 21 52

3.4 Statistical Analyses

................................ ................................ ................................ ........ 21 53

3.5 Data Expectations

................................ ................................ ................................ ........... 23 54

4 MMT DATA SUMMARIES

................................ ................................ ................................ 24 55

4.1 Results with ER

- agonists

................................ ................................ ............................... 26 56

4.1.1 17β

- Estradiol (CERI/Japan)

................................ ................................ ....................... 27 57

4.1.2 4

- t

- Octylphenol (US EPA/ORD/MED)

................................ ................................ ....... 27 58

4.1.3 4

- t

- Octylphenol (NIES/Japan)

................................ ................................ .................... 28 59

4.1.4 Comparison of 4

- t

- Octylphenol MMTs in Support of Validation

............................... 29 60

4.1.5 o,p′

- DDT (CERI/ Japan)

................................ ................................ ............................ 30 61

4.1.6 4

- chloro

- 3

- methylphenol (US EPA/ORD/MED)

................................ ......................... 30 62

4.1.7 Tamoxifen (NIES/Japan)

................................ ................................ ............................. 31 63

4.2 Results with AR

- agonists

................................ ................................ ............................... 32 64

4.2.1 17β

- Trenbolone (US EPA/ORD/MED)

................................ ................................ ....... 32 65

4.3 Results with Steriodogenesis Inhibitor

................................ ................................ ........... 33 66DRAFT

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................................ ................................ ............... 33 67

4.4 Results with AR Antagonist

................................ ................................ ........................... 34 68

4.4.1 Vinclozolin (MED)

................................ ................................ ................................ ...... 34 69

4.5 Inte rlaboratory Evaluation of the MMT protocol

- Vinclozolin

................................ .... 36 70

4.5.1 Background

................................ ................................ ................................ ................. 36 71

4.5.2 Protocol

................................ ................................ ................................ ........................ 37 72

4.5.3 Chemistry Da ta

................................ ................................ ................................ ............ 38 73

4.5.4 Control Data Supporting Validation

................................ ................................ ........... 39 74

4.5.6 Summary of Lab 1 Vinclozolin MMT

................................ ................................ ........... 41 75

4.5.7 Summary of Lab 2 Vinclozolin MMT

................................ ................................ ........... 42 76

4.5.8 Summary of Lab 3 Vinclozolin MMT

................................ ................................ ........... 42 77

4.5.9 US EPA Perspective on the Interlaboratory Results

................................ ................... 42 78

5 OPTIMIZATION OF THE MMT AND THE PROPOSED MEDAKA REPRODUCTION 79

TEST (MRT) PROTOCOL

................................ ................................ ................................ ........... 43 80

5.1 Primary MMT Protocol Changes for development of MRT Protocol

........................... 43 81

5.1.1 Increased Replication for the Assessment of Treatment

- Effects on Reproduction

. 46 82

5.1.2 In

- life Exposure Complete After F 2 Hatch

................................ .............................. 52 83

5.1.3 Effect

- endpoints Not Measured in F 0

................................ ................................ ...... 54 84

5.1.4 Measure Subadult Endpoints at 9 wpf

................................ ................................ .... 54 85

5.1.5 Test Temperature 25.5 ° C

................................ ................................ ...................... 56 86

5.1.6 Pathology Analysis of Adult Fish Only

................................ ................................ .. 57 87

6 DISCUSSION/SUMMARY OF THE VALIDATION OF MMT/MRT TEST METHOD

. 58 88

APPENDICES

................................ ................................ ................................ .............................. 59 89

REFERENCES

................................ ................................ ................................ ............................. 60 90

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93DRAFT

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95

This Integrated Summary Report (ISR) provides the validation record of the EDSP Tier 2 96

Medaka Multigeneration Test (MMT) aimed at characterizing potential disruption of the 97

endocrine system by putative Endocrine Disrupting Chemicals (EDCs). Pertinent literature on 98

fish chronic toxicity tests has previously been summarized in an in

- depth review ( Appendix B). 99

R elevant studies completed as part of the validation process for the MMT are summarized that 100

help ed inform the ultimate design of the recommended protocol. Studies include tests with 101

estrogen receptor agonists (17 

- estradiol, 4

- t

- octylphenol, o,p ’

- DDT,4

- chloro

- 3

- methylphenol, 102

and tamoxifen), an androgen receptor agonist and antagonist (trenbolone and vinclozolin, 103

respectively), and a steroid synthesis modulator (prochloraz). 104

105

The research MMT protocol describes a 21⁄2 generation toxicity test with a small freshwater fish 106

that evaluates reproductive fitness as an integrated measure of toxicant effects a cross 21⁄2 107

consecutive generations using pairs of adult medaka . It also enables measurement of a suite of 108

other endpoints for diagnostic and quantitative evaluation of EDCs or other types of reproductive 109

toxicants. The M MT is a relatively long

- term assay ( 2 9 weeks of in

- life exposure ) that assesses 110

hatch, growth, survival, gonadal development, and reproduction in 21⁄2 generations (F 0

, F 1

, and 111

part of F 2 ). Vinclozolin was chosen for an interlaboratory demonstration of the protocol. 112

113

The research objectives were to : a) develop a multigeneration test protocol using the Japanese 114

medaka that could be used to evaluate the effects of endocrine disruption chemicals (EDCs) on 115

reproduction and reproductive development ; b) to include diagnostic or intermediate effects 116

en dpoints that could be evaluated for associations with the apical outcomes to help elucidate 117

various EDC

- induced adverse outcome pathways (AOPs); c) address the question of the relative 118

importance of an additional F 2 generation; and d ) develop a scientifi cally

- defensible Tier 2 fish 119

test protocol for characterizing concentration

- response for ED endpoints. 120

121

The MMT method was successfully performed in characterizing adverse effects and 122

concentration

- response for several known endocrine active substances. In the case of 4

- t

123

octylphenol, tests at different laboratories and at different times in one laboratory comported 124

very well. However, in the interlaboratory trial with vinclozolin, the comparative analysis 125

i ndicated that for many endpoints, the control response was not consistent between laboratories. 126

The median responses from F 1 reproductive measurements, F 1 , F 2 length and weight 127

measurements were all significantly different (p < 0.01) between laboratories. Closer inspection 128

of the results suggest som e of the significant inter

- lab variability can be attributed to the 129

relatively small size at termination of F 1 and F 2 fish in a single lab oratory . The small size of this 130

laboratory F 1 females appears to have contributed to the high inter

- lab variability in fecundity. 131

Normalizing F 1 fecundity for the body mass of the female eliminated the significant difference

. 132

Overall the i nterlaboratory can be considered comparable insomuch as all three labs did not see 133

decreased reproduction at the concentrations of vinclozolin tested. This is also unfortunate for 134

the very same reason. Other endpoints were also not significantly affected by vinclozolin with 135

the exception of SSC which was negative ly responsive to vinclozolin exposure in all labs that 136

recorded this endpoint . SSC was also decreased in the vinclozolin MMT performed by the US 137

EPA/ORD/MED. So while few significant effects were observed in the three interlaboratory 138

MMTs, there was cons istency in the observation of effects on the SSC. 139DRAFT

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Unfortunately, the previously discussed issues with the interlaboratory data cannot simply be 141

ignored, and they highlight that this is a relatively complicated procedure that even with 142

experience is not a trivial undertaking. Many of the directives within the protocol are derived 143

from extensive experience at the US EPA/ORD/MED lab, and without a priori knowledge, 144

should be followed without deviation. 145

146

The outcomes of the various MMT trials have provide d enough information to recommend a 147

medaka reproduction test (MRT) for use as the fish test in Tier 2 of the EDSP. Two major 148

changes from the MMT are proposed, i.e., an increase in the number of replicates per treatment 149

for evaluating effects on reproducti on, and terminating the test after the embryos hatch in F 2

. 150

Other proposed changes include minimizing the collection of endpoint data from F 0 , and 151

evaluating pathology in only the F 1 adults sampled after the assessment of reproduction. 152

153

Overall, the author s conclude that both the MMT and the MRT are transferable methods and are 154

capable of adequately characterizing potential disruption of the endocrine system by putative 155

endocrine disrupting c hemicals. However, the MRT is recommended as the preferred EDSP 156

Tier 2 test method for fish because it is less resource intensive with improved statistical power, 157

appears to be as sensitive, and is better able to ensure consistent findings when performed 158

routinely by testing laboratories. 159

160

161

162

163

164

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166

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168

169

170

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175

176

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OBJECTIVE 187

188

The objective of this integrated summary report (ISR) is to provide a detailed account of the 189

validation process for the Medaka Multigeneration Test (MMT) so that Federal Insecticide, 190

Fungicide and Rodenticide Act (FIFRA) Scientific Advisory Pan el (SAP) members may address 191

the validation charge questions provided

. 192

193

1 INTRODUCTION 194

Section 408(p) of the Federal Food Drug and Cosmetic Act (FFDCA) requires the 195

U.S. Environmental Protection Agency (EPA) to: 196

197

develop a screening program, using approp riate validated test systems and other 198

scientifically relevant information, to determine whether certain substances may have an 199

effect in humans that is similar to an effect produced by a naturally occurring estrogen, 200

or other such endocrine effect as the Administrator may designate [21 U.S.C. 346a(p)]. 201

202

Subsequent to passage of the Food Quality Protection Act in 1996, which amended FFDCA and 203

FIFRA, the EPA formed the Endocrine Disruptor Screening and Testing Advisory Committee 204

(EDSTAC), a committee of scie ntists and stakeholders that was charged by the EPA to provide 205

recommendations on how to implement its Endocrine Disruptor Screening Program (EDSP). The 206

EDSP is described in detail at the following website: http://www.epa.gov/scipoly/oscpendo/

. 207

Upon recomm endations from the EDSTAC (EDSTAC 1998), the EPA expanded the EDSP using 208

the Administrator’s discretionary authority to include the androgen and thyroid hormonal 209

systems as well as the endocrine systems of wildlife. Following broader international concern s 210

and the creation of similar programs in other countries, the Organization for Economic Co

211

operation and Development (OECD) established the Endocrine Disrupters Testing and 212

Assessment (EDTA) Task Force in 1998 within its Test Guidelines Program. EDTA is c harged 213

with developing an internationally harmonized testing strategy for the screening and testing of 214

endocrine disrupting chemicals, taking into account the consequences of such a testing strategy 215

on the development and validation of Test Guidelines, and on existing regulatory systems for 216

new and existing substances. 217

218

The MMT is one of the assays proposed as part of the EDSP Tier 2 and is the subject of this 219

report. This report, along with the detailed information provided in the attached supporting 220

mater ials introduces the purpose of the MMT, the scientific rationale for the assay, the 221

development and optimization of the assay protocol, as well as how it fits into the larger EDSP. 222

It also synthesizes the information gained during the validation process an d addresses the 223

advantages and limitations of this fish test method based on its, practicality, reproducibility, 224

reliability, protocol transferability and fit for purpose. 225

226

227

228

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1.1 Endocrine Disruptor Screening Program 231

232

To comply with its mandate, the EPA chartered a Federal advisory committee (EDSTAC) to 233

provide advice and guidance on the development of a screening and testing program. In 1998, it 234

recommended to the EPA a conceptual two

- tiered approach that involved scre ening and testing 235

chemical compounds for effects on the estrogen (E), androgen (A), and thyroid (T) hormone axes 236

collectively referred to as EAT. The ultimate goal of these assays is to provide input into hazard 237

identification to assess risk of adverse co nsequences to humans and wildlife (EDSTAC, 1998)

. 238

239

The EPA submitted a proposal of the EDSP for public review and comment in a Federal Register 240

Notice (FRN, 1998) as well as peer review by a joint subcommittee of the EPA Science 241

Advisory Board (SAB) and FI FRA S cientific A dvisory P anel ( SAB

- SAP , 1999)

. A complete 242

description of the program proposal can be found in the FRN. Briefly, the EDSP proposed by 243

EDSTAC allows for: 1) initial sorting and prioritization of chemical compounds, 2) 244

identification of chemic als for further testing using a Tier 1 screening battery that includes in 245

vitro and in vivo mammalian, amphibian and fish assays, and 3) characterization of adverse 246

consequences resulting from possible endocrine disruption and establishment of dose

- respons e 247

relationships for hazard identification using Tier 2 testing. 248

249

In comparison to the more refined, detailed, and definitive tests in Tier 2, the EDSTAC indicated 250

that the in vitro and in vivo screening assays in the Tier 1 battery should: 251

• be relatively f ast and efficient; 252

• be standardized and validated; 253

• be more sensitive than specific to minimize false negatives without an unreasonable 254

rate of false positives; 255

• be comprised of multiple endpoints that reflect as many modes of endocrine action as 256

possible; 257

• h ave a sufficient range of taxonomic groups among test organisms represented; and 258

• yield data that can be interpreted as either negative or positive for determining the 259

necessity and manner in which to conduct Tier 2 tests. 260

261

Together, the suite of Tier 1 a ssays form a battery in which some endocrine axis redundancy is 262

incorporated ( e.g

. . two different assays may cover some similar aspects of the estrogenic 263

response). This redundancy allows for a weight

- of

- evidence (WOE) approach, as recommended 264

by EDSTAC, to determine whether a chemical undergoes further, more definitive, testing. 265

266

The as says in Table 1

- 1, recommended by EDSTAC, have undergone validation and have been 267

adopted as the EDSP Tier 1 screening battery ( Table 1

- 1 ). These assays are meant to detect 268

chemicals that may affect the E, AE, A and TT hormone axes. 269

270

271

272

273

274

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277

Table 1

- 1

. Assays recommended for consideration for the Tier 1 screening battery. 278

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 HPG axis that controls the estrogen and

androgen hormone systems. It is also enhanced to detect

chemi cals 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

androge n 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.

279

A weight of the evidence (WOE) review of the Tier 1 screening battery and other scientifically 280

relevant information (OSRI), will be made to ascertain if a chemical is deemed sufficiently 281

en docrine active to prompt requirements for Tier 2 testing. The following tests, as generally 282

recommended by EDSTAC, are being considered as part of the EDSP Tier 2: 283

284

 Two

- generation Rat Reproduction Test 285

 Medaka Multigeneration Test 286

 Japanese Quail Two

- genera tion Test 287

 Mysid Two

- generation Toxicity Test 288

 Larval Amphibian Growth and Development Test 289

290

The Two

- generation Rat Reproduction Test (OCSPP 870.3800) has already been adopted. The 291

remaining four Tier 2 tests are the subject of this FIFRA Scientific Advisory Panel review. 292

293

1.2 Validation 294

295

Validation has been defined as “the process by which the reliability and relevance of a test 296

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

. 297DRAFT

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laboratories. 299

Relevance describes whether a test is meaningful and useful for a particular purpose 300

(OECD, 1996)

. For Tier I EDSP assays, relevance can be defined as the ability of an assay to 301

detect the effects of ch emicals with the potential to interact with the endocrine system. 302

Validation is generally recognized as necessary for the regulatory acceptance of new and revised 303

test methods, and is now an integral component of the international development and acceptance 304

of these methods (OECD, 2005) . The criteria used to guide the validation process for the MMT 305

were based on the principles of validation developed by the U.S. Interagency Coordinating 306

Committee for the Validation of Alternative Methods (ICCVAM) (NIEHS, 1997) and the OECD 307

(OECD, 2005)

. These criteria as stated by ICCVAM (NIEHS, 1997) are as follows: 308

1. The scientific and regulatory rationale for the test method, including a clear statement of 309

its proposed use, should be available. 310

2. The relation ship of the endpoints determined by the test method to the in vivo biologic 311

effect and toxicity of interest must be addressed. 312

3. A formal detailed protocol must be provided and must be available in the public domain. 313

It should be sufficiently detailed to enable the user to adhere to it and should include data 314

analysis and decision criteria. 315

4. Within

- test, intra

- laboratory and interlaboratory variability and how these parameters 316

vary with time should have been evaluated. 317

5. The test method’s performance mu st have been demonstrated using a series of reference 318

chemicals preferably coded to exclude bias. 319

6. Sufficient data should be provided to permit a comparison of the performance of a 320

proposed substitute test to that of the test it is designed to replace. 321

7 . The limitations of the test method must be described ( e.g ., metabolic capability). 322

8. The data should be obtained in accordance with Good Laboratory Practices (GLPs). 323

9. All data supporting the assessment of the validity of the test methods including the full 324

data set collected during the validation studies must be publicly available and, preferably, 325

published in an independent, peer

- reviewed publication. 326

The EPA has adopted these various validation criteria for the EDSP as described in Appendix A: 327

Crite ria for Validation (EDSP, 2007 ) which were considered met in the review of the Tier 1 328

assays. Validation of these assays proved a challenge to comply with Criterion No. 6 because 329

these are not replacement assays (Validation Criterion No. 6). Many of them are novel assays; 330

consequently, large data bases do not exist as a reference to establish their predictive capacity 331

( e.g ., determination of false positive and false negative rates). 332

In general, the EPA uses a five

- part or stage validation process outlined by ICCVAM (NIEHS, 333

1997) . The EPA also believes it is important to recognize that this process was specifically 334

developed for alternative assays intended to replace or refine existing assays. A rudimentary 335

p roblem confronting the EPA is how to adapt and work with this process for rodent and 336

ecological in vivo assays in Tiers 1 and 2 that are being considered as the standards and not as 337DRAFT

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ICCVAM are discussed below. 339

The first stage of the process as employed for the MMT was “ test development ” , an applied 340

research function which culminated in an initial protocol. As part of this phase, EPA drafted a 341

Detailed Review Paper (DR P) to explain the purpose of the test method , the context in which it 342

will be used, and the scientific bases upon which the assay’s protocol, endpoints, and relevance 343

rest (Appendix B: Detailed Review Paper). The DRP review ed the scientific literature for 344

candidate protocols and evaluate d them with respect to a number of considerations, such as 345

whether candidate protocols met the assay’s intended purpose, costs, and other practical 346

considerations. The DRP also identifie d the developmental status and quest ions related to each 347

protocol; the information needed to answer the questions; and, when possible, recommend ed an 348

initial protocol for the initiation of the second stage of validation, i.e ., “ standardization and 349

optimization ”

. During standardization and o ptimization, studies were performed geared toward 350

refining, optimizing, and standardizing the protocol, and initially assessing protocol 351

transferability and performance. In the third stage, i.e ., “ interlaboratory validation ” studies were 352

conducted in several independent laboratories with the refined protocol. The results of these 353

studies were used to determine interlaboratory variability and to set or cross

- check performance 354

criteria. The report on the interlaboratory trials for the MMT is provided in Appendix C: 355

Vinclozolin Interlaboratory Report . Interlaboratory validation is followed by the “ peer review ” 356

phase

, involving an independent scientific review by qualified experts, and by then by the final 357

phase, “ regulatory acc eptance ”

, i.e., adoption for regulatory use by an agency. EPA has 358

developed extensive guidance on the conduct of peer reviews because the Agency believes that 359

peer review is an important step in ensuring the quality of science that underlies its regulator y 360

decisions (USEPA, 2006)

. 361

The purpose of this ISR is to provide a summary of the development and validation of a n MMT 362

protocol (Appendix D: MMT Protocol) and a final proposed Medaka Reproduction Test (MRT ; 363

Appendix E: MRT Protocol) to be used as an in viv o assay for EDSP Tier

- 2 testing . The 364

reasoning and judgments leading to the various studies, and conclusions concerning the strengths 365

and limitations of the MMT research assay in its initial form, and advantages of the final 366

proposed MRT are presented. 367

368

369DRAFT

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371

Fish are an important vertebrate class in both freshwater and marine systems. Although many 372

fish toxicity test protocols are routinely used in regulatory testing, few have been designed with 373

endocrine

- specific endpoint s in mind. Further, it is known that growth, reproduction, 374

development, and other aspects of fish physiology and life cycle are under endocrine control

. 375

Thus, vertebrate test method that uses fish for evaluating potential effects of endocrine disrupting 376

ch emicals ( EDCs ) is relevant. 377

378

2.1 Purpose of the Medaka Multigeneration Test in EDSP 379

380

The purpose of definitive Tier 2 testing is to determine whether a test substance adversely affects 381

a test organism through endocrine

- mediated pathways, and to quantitativ ely evaluate those 382

effects with respect to the estrogen, androgen, and thyroid systems. In addition, the tests must 383

include exposure during the most sensitive life stages, provide the opportunity for identification 384

of dose

- response effects, and encompass a variety of taxa. 385

386

The recommended fish (Tier 2) test will characterize the nature, likelihood, and dose

- response 387

relationship of any adverse outcomes from potential endocrine disruption via E, AE, A, and/or 388

TT pathways. In combinat ion with other ecotoxicity tests in the EDSP Tier 2 and Part 158 389

studies, a comprehensive ecological risk assessment could be performed. The Tier 2 tests should 390

complement Tier 1; however, results from well run Tier 2 tests would supersede Tier 1 results. 391

Tier 2 is the final phase of the screening and testing program and, thus, provides more detailed 392

information regarding the endocrine disruption activity of a tested chemical or mixture. To 393

fulfill this purpose, these tests are much longer

- term studies d esigned to encompass critical life 394

states and processes, a broad range of doses, and administration by a relevant route of exposure. 395

In addition, the effects associated with an EDC may be latent and not manifested until later in 396

life or may not be apparen t until reproductive processes occur in an organism's life history. 397

Thus, tests for endocrine disruption will often encompass additional generations, including 398

effects on fertility and mating, embryonic development, sensitive neonatal growth and 399

developme nt, and transformation from the juvenile life state to sexual maturity. The results from 400

the Tier 2 testing should be conclusive, and a discernible cause

- effect relationship should be 401

manifest during the test if one exists from the chemical exposure. In summary, the following 402

elements should be part of the test: 403

404

 establish exposure/concentrations/timing and effects relationships 405

 be sensitive and specific 406

 assess relevant endpoints 407

 include all life stages across generations 408

 include a dose range for full c haracterization of effects 409

 capable of being routinely conducted in accordance with Good Laboratory Practices 410

(GLP). 411

412DRAFT

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disruption 414

415

2.2.1 Advantages of the medaka test model 416

417

The medaka ( Oryzias latipes ) is a freshwater fish belonging to the family of Asian rice fishes 418

(Adrianichthyidae) indigenous to areas of Japan, Taiwan, and southeastern Asia, where ambient 419

temperatures range from 5oC to 35oC (Kirchen and West, 1976) . The medaka has a long history 420

as an experimental animal and a complete presentation of their biology can be found in 421

Yamamoto (Yamamoto, 1975) , which lists over 1000 references dating from the early 1900s. 422

The culture and handling of the medaka has been exhau stively studied and detailed guidelines 423

are available in EPA/600/3

- 91/064 (Denny et al., 1991) . Medaka are small (25 to 50 mm in total 424

length) and are easy to rear and maintain in laboratory aquaria. Adults can be maintained in an 425

aquarium with little sp ace and can tolerate low dissolved oxygen and a wide range of 426

temperatures and salinity (Yamamoto, 1975) . Medaka are sexually dimorphic and although the 427

sex of fully grown fish can be determined by observing the outline of the anal and dorsal fins, the 428

ob servation of an anesthetized fish under a low

- power microscope is recommended for 429

confirmation and for sexing an immature fish (Yamamoto, 1975)

. 430

431

Medaka have a generation interval of 2 to 3 months, but can be induced to spawn throughout the 432

year by controlling the temperature and photoperiod. Spawning is highly predictable in time, 433

usually within 1 hr of first daylight (Hirshfield, 1980; Koger et al., 1999) . Measures of medaka 434

reproductive output, including gonadal morphology, fecundity, and fertility, demonstrate that 435

through laboratory control of temperature and photoperiod, reproductive capacity of breeding 436

groups can be calibrated before expo sure, thereby establishing a baseline from which to assess 437

effects (Koger et al., 1999) . Individual females can lay an average of 25 eggs/day for up to 4 438

months under proper conditions of photoperiod, temperature, and food supply (Hirshfield, 1980)

. 439

A cl uster typically contains 10 to 30 eggs that are attached to the female's vent by filaments for a 440

number of hours until they are brushed off onto a spawning substrate simulating aquatic plants 441

(Yamamoto, 1975) . Even when spawning substrates such as a spawn ing sponge are provided, 442

some females retain their eggs requiring manual stripping (Denny et al., 1991) . The eggs should 443

be collected as soon as possible after spawning to prevent their predation by adults. Fertilization 444

can be easily assessed with low m agnification because of the transparency of the egg chorion. 445

The egg incubation period is approximately 1 week when kept at 28oC (Yamamoto, 1975) , and 446

the embryos will tolerate a temperature range of 7oC to 38oC (Kirchen and West, 1976)

. 447

448

449

450

2.2.2 Concep tual basis for a multigeneration test paradigm 451

452

Endocrine active substances are known to disrupt endocrine regulated organizational as well as 453

activational processes in an organism. It is also known that in oviparous animals there is 454

maternal transfer of xenobiotic as well as endogenous factors to the embryo, in ovo (Adkins

455

Regan et al., 2013; Almasi et al., 2012; Foran et al., 2000; Hayward and Wingfield, 2004; 456

Henriksen et al., 2011; Lin et al., 2004; Metcalfe et al., 2000; Schwaiger et al., 2002; Schwei tzer 457DRAFT

MMT ISR 13 M a y 28

, 2013 et al., 2013)

. Further, organizational effects during development and maturation may not fully 458

manifest as adverse until a later life stage or in future progeny (reproduction or sex ratio) 459

(Adkins

- Regan et al., 1995; Edmunds et al., 2000; Quinn et al ., 2007; Quinn et al., 2006; 460

Yamamoto, 1958; Yamamoto, 1953) . Therefore, a multigeneration test is intended to evaluate 461

these possible trans

- generation effects. A Medaka Multigeneration Test design was conceived to 462

most efficiently maximize the adverse i mpact

- potential of an endocrine active substance. The 463

initial F 0 generation is exposed during spawning to allow test compound and maternal factors to 464

be transferred into the eggs of the F 1 generation. This transfer establishes the initial chemical 465

enviro nment of the embryo. Conceptually, beginning exposure at the egg stage alone would 466

limit chemical exposure due to the attenuation of chemical absorption by the egg chorion and it 467

will not present an altered state of maternal factors, e.g ., base estrogen levels to the embryo. 468

Any potential organizational perturbation to the embryo in ovo would therefore be missed. If an 469

F 0 generation is exposed after onset of reproduction, then some transfer of factors that present 470

potential organizational effects may b e considered. However, the maternal transfer at this stage 471

may be partially affected or otherwise modified since the parent would have normally developed 472

and matured prior to exposure. If that is the case, t he F 1 animals would be subject to a modified 473

receipt of maternal factor s as well as increased test compound accumulation from continuous 474

exposure post

- hatch and is expected to be fully affected in their development and maturation. 475

Their progeny (F 2 ) would therefore be expected to receive the full spectrum of modified factors 476

which may further express and manifest in developmental or maturational disturbances. The 477

distinction between activational effects to sexually differentiated fishes exposed to EDCs 478

compared to the organizational effe cts that happen where exposure in ovo of endogenous factors 479

and/or a bioaccumulated xenobiotic EDC that occurs during sexual differentiation has been 480

generally reviewed by Denslow and Sepulveda (Denslow and Sepúlveda, 2007)

. It is the sexual 481

differentiati on and reproductive competence of the F 2 generation that would conceptually be 482

most severely affected if there are organizational changes that result from a chemical’s endocrine 483

action to the developing embryo in ovo . If that is indeed the case, then a mu ltigeneration test 484

paradigm would be needed to fully characterize the potential endocrine disrupting impact

. 485

486

Multigeneration fish tests in some form have been performed on several fish species including 487

sheepshead minnow ( Cyprinodon vari e gatus ) (Cripe et al., 2009; Cripe et al., 2010; Raimondo et 488

al., 2009) , fathead minnow ( Pimephales promelas ) (Staples et al., 2011) , zebrafish ( Danio rerio ) 489

(Nakari and Erkomaa, 2003) , Chinese rare minnow ( Gobiocypris rarus ) (Zha et al., 2008)

, 490

Australian crimson

- spotted rainbowfish ( Melanotaenia fluviatilis ) (Holdway et al., 2008)

, 491

convict cichlid (

Amatitlania nigrofasciata ) (Newsome, 1980) , as well as in medaka (Patyna and 492

Cooper, 2000; Patyna et al., 1999) . From the few studies and few chemicals that have been 493

investi gated, the importance of inclusion of additional generations is somewhat equivocal 494

beyond the need to inform population models in probabilistic assessments or investigate putative 495

epigenetic effects. Comparing sensitivities between activational and organi zational effects is 496

important in assessing the additional value of multiple generation tests. A key objective of the 497

MMT method development is to address the question of the relative importance of an additional 498

full F 2 generation. 499

500

3 MEDAKA MULTIGENERATIO N TEST PROTOCOL DEVELOPMENT 501

502DRAFT

MMT ISR 14 M a y 28

, 2013 As discussed in Section 2 , the focus of the ISR is on multigeneration studies conducted using 503

medaka, a small fish test model . This section summarizes the initial full version of the MMT 504

protocol. This protocol incorporated redundant endpoint measurements at comparable 505

developmental life

- stages in all generations of the test, F 0 , F 1 , and F 2 . As such, it is considered a 506

research protocol to be used for evaluating the value added of various endpoints and additional 507

generations to a possible Tier 2 fish reproduction test protocol. A complete presentation of the 508

MMT research protocol is included as Appendix D: MMT Protocol. 509

510

3.1 Overview of the Medaka Multigeneration Test (MMT) Research Protocol 511

512

The research MMT protocol describ es a 21⁄2 generation toxicity test with a small freshwater fish 513

that evaluates reproductive fitness as an integrated measure of toxicant effects across 21⁄2 514

consecutive generations using pairs of adult medaka . It also enables measurement of a suite of 515

other en dpoints for diagnostic and quantitative evaluation of EDCs or other types of reproductive 516

toxicants. The M MT is a relatively long

- term assay ( 29 weeks of in

- life exposure ) that assesses 517

hatch, growth, survival, gonadal development, and reproduction in 21⁄2 generations (F 0

, F 1

, and 518

part of F 2 ). The M MT is an extension of existing standard Fish Full Life

- Cycle test protocols 519

developed by Japanese scientists (Seki et al., 2003b; Yokota et al., 2001)

. 520

521

The research objectives were to : a) develop a multigeneration test protocol using the Japanese 522

medaka that could be used to evaluate the effects of endocrine disruption chemicals (EDCs) on 523

reproduction and reproductive development ; b) to include diagnostic or intermediate effects 524

endpoints that coul d be evaluated for associations with the apical outcomes to help elucidate 525

various EDC

- induced adverse outcome pathways (AOPs); and

, c ) develop a scientifically

526

defensible Tier 2 fish test protocol for characterizing concentration

- response for ED endpoin ts. 527

The project involved developing methods for evaluating population

- relevant endpoints such as 528

fecundity, fertility, reproductive behavior, gonadal development, and the genotypic sex of 529

individual fish compared to phenotypic sex within each generation t ested. Also, a number of 530

molecular and histological measures that are diagnostic of initiating and subsequent key events 531

along AOPs were developed to allow endpoint comparisons across test designs and chemical 532

modes of action. 533

534

The MMT provides data that c an be used to simultaneously evaluate two general types of 535

adverse outcome pathways (AOPs) ending in reproductive impairment: a) those primarily 536

involving disruption of the hypothalamus

- pituitary

- gonadal (HPG) endocrine axis (i.e., 537

endocrine

- mediated EDCs) ; and, b) those that cause reductions in apical effects on survival, 538

growth, hatch, etc through other endocrine axes ( e.g., HPT) and non

- endocrine mediated toxicity 539

pathways. Some of the EDC endpoints, such as the presence of anal fin papillae in medaka 540

ma les, are biomarkers only minimally linked to adverse reproductive outcomes; whereas other 541

EDC endpoints such as fecundity and fertility can be directly linked to adverse population 542

outcomes through population models. The non

- endocrine mediated endpoints ty pically measured 543

in full life

- cycle and early life

- stage (ELS) chronic toxicity tests are also included in the MMT. 544

These are used to evaluate the concentration

- response of a chemical working through any 545

endocrine or non

- endocrine mediated AOP. The questio n of whether a chemical has endocrine

546

mediated effects at lower concentrations than non

- endocrine

- mediated toxicities cannot be 547

unambiguously addressed by the apical endpoints alone and will rely on other diagnostic 548DRAFT

MMT ISR 15 M a y 28

, 2013 biochemical ( e.g ., vtg induction), hist opathological ( e.g ., Leydig cell hyperplasia), or changes in 549

SSC. 550

551

The test conditions for the research MMT are outlined in Table 3

- 1. Briefly, the MMT protocol 552

consist s of continuously exposing 2 1⁄2 generations of fish to the test chemical starting with 553

mat uring adults in the first generation (F0) and exposing for several weeks to allow chemical 554

accumulation . There are 6 treatments (if a solvent is not used) ; five chemical treatments and a n 555

unexposed control treatment. There are six replicates aquaria in eac h treatment. The measured 556

endpoints includ e embryo hatch, growth, survival, fecundity and fertility

, liver vitellogenin 557

mRNA levels in both sexes

, the number of anal fin papillae (a sexual phenotyp e marker), 558

histological evaluation of the gonad phenotype, and histopathology evaluation of kidney, liver 559

and gonad

. All of these endpoints are evaluated in the context of the genetic sex of each fish

. 560

C hemical concentrations are held constant in each treatment throughout the bioassay to allow 561

endpoint sensitivity comparisons across and within generations. Thus, although not discussed 562

here in detail, precise dependable flow

- through exposure and dilution systems

, as well as high

563

quality analytical chemistry

, are an integral part of the protocol. The in

- life duration of the MMT 564

is nominally 29 weeks. Note that each generation overlaps with the subsequent generation by 565

three weeks. Details of the endpoints measurements are presented in Section 3.2, and a detailed 566

protocol and sampling timeline is in Section 3.3 indica ting when each MMT measurement 567

endpoint is evaluated. 568

569

Table 3

- 1

. Test Conditions for the Medaka Multigeneration Test (MMT) Protocol 570

571

Recommended species Medaka (Oryzias latipes)

Test type Continuous flow

- through

Water temperature 2 6 ± 1 o C

Illumination quality Fluorescent bulbs (wide spectrum and ~1100 lumens )

Photoperiod 16 h light, 8 h dark

Loa ding rate F 0 - 2 fish/replicate; F 1 and F 2 - initiated with maximum of 40 larvae/replicate eventually

culled to 2 fish/replic ate

Test replicate chamber size 18x9x15 cm

Test replicate solution volume ~1.8 liters

Volume exchanges of test solutions 12 volume replacements/day (15 ml/min flow).

Age of test organisms at initiation F 0 - 11 weeks post

- fertilization, F 1 and F 2 continuously from fertilization

No. of organisms per replicate F 0 - 2 adults; F 1 and F 2 - initiated with maximum of 40 larval fish eventually culled to 2

adults

No. of treatments 6 ( 5 toxicant treatment plus 1 control )

No. replicates per treatment 6

No. of organisms per test

concentration F 0

- 12 for each test concentration and for control; F 1 and F 2 - start with a maximum of

240 and eventually cull to 12 for fecundity assessment.

Feeding regime Fish were fed 24

- hour old brine shrimp ( Artemia spp.) nauplii at the following rates:

Time

(weeks post

- hatch) Brine Shrimp

(mg dry

weight/fish/day)

1 0.77

2 3.06

3 5.48

4 7.75

5 9.12

6

- 7 11.4

8

- 15 13.7

Aeration None unless DO reaches <60% saturationDRAFT

MMT ISR 16 M a y 28

, 2013 Dilution water 50 micron sand

- filtered Lake Superior Water, UV sterilized in the lab and further filtered

to 5 microns and degassed to 8.0 mg O 2 /L.

Exposure period Trans

- generational test: F 0 from 11

- 16 weeks post

- fertilization (wpf),), F 1 and F 2 from 0

- 16 wpf

Primary endpoints Survival: F 0 from 11

- 16 wpf, F 1 and F 2 from 1

- 3 wpf and from 3

- 16 wpf

Growth : length, weight, (F 0 , F 1, F 2 a t 16 wpf termination ; F 1 and F 2 7 wpf)

Hatch: F 1 and F 2 only

Anal Fin Papillae: F 0 , F 1, F 2 assessed a t 16 wpf termination ; F 1 and F 2 assessed at7 wpf

Fecundity: assessed for each breeding pair T,W,Th,F of weeks 11

- 15 for F 0 , F 1 and F 2 Fertility: assessed for each breeding pair T,W,Th,F of weeks 11

- 15 for F 0 , F 1 and F 2 Vitellogenin: determined at 7 and 16 wpf for F 0 , F 1 and F 2

Optional endpoints Time t o first spawn for F 1 and F 2 generations.

Test acceptability Dissolved oxygen > 60% of saturation; mean temperature of 2 6 ± 1 o C;  70% survival in

control treatments of each generation; successful reproduc tion in at least 6 5% of control

females with mean daily fecundity in the control treatment of at least 15 eggs.

572

573

3.2 MMT Endpoints 574

575

An overview of the endpoints incorporated into research MMT are listed in Table 3

- 2. Some of 576

the endpoints are related to EDC effects while others are traditional ecotoxic ology endpoints 577

generally considered to be a result of systemic toxicity, and not necessarily endocrine effects. 578

579

Table 3

- 2. Endpoint overview of the medaka multigeneration test (MMT) 580

Life

- stage Endpoint Endocrine

m ediated Non

- Endocrine

Mediated Population

Relevance

ELS 1 (1 wpf

2 ) Hatch Y Y

ELS ( 3 wpf) Survival Y Y

Subadult (8 wpf) Survival Y Y

Growth (weight) Y Y

Vitellogenin (Vtg) Y

Anal fin papillae Y

Intersex Y Maybe

Sex Reversal Y Y Y

Adult (12

- 16 wpf) Fecundity Y Y Y

Fertility Y Y Y

Adult (16 wpf) Survival Y Y

Growth (weight) Y

Vitellogenin (Vtg) Y

Anal fin papillae Y

Intersex Y Maybe

Sex Reversal Y Y Y

Histopathology Y Y Maybe

1 ELS: early life

- stage 581

2 wpf: weeks post

- fertilization 582

583

The p rimary emphasis in the MMT is on endpoints that can be associated with potential adverse 584

effects on population

- relevant parameters such as survival , gross development, growth and the 585

reproductive measures of fecundity and fertility . To help differentiate e ndocrine

- mediated effects 586

from systemic toxicity, additional endocrine

- mediated effects such as liver vitellogenin mRNA 587DRAFT

MMT ISR 17 M a y 28

, 2013 levels, the number of papillae on the anal fin (an external sexual phenotyp e marker), and the 588

histological evaluation of gonad al sex an d intersex, and finally, histopathology evaluation of 589

kidney, liver

, gon ad, and other tissues are also measured (see endpoint list in Table 3

- 2 ). All 590

endpoints are evaluated in the context of the genetic sex of each fish. As mentioned previously, 591

traditio nal ecotoxicology endpoints that may not be endocrine

- mediated such as embryo hatch, 592

growth, and survival are also measured. 593

594

3.2.1 Genotypic Sex (F O , F 1 , F

2 ) 595

596

The genetic sex of each fish, for each generation, is determined by non

- destructively obtaining a 597

small tissue (fin) sample and, using polymerase chain reaction (PCR) methods, identifying the 598

presence or absence of the sex

- determining gene (DMY) in each fish. Under normal conditions 599

XX individuals express the female phenotype and XY individ uals the male phenotype. 600

601

3.2.2 Survival (F O , F 1 , F

2 ) 602

603

Survival is assessed daily for F 0

, F 1

, and F 2 individuals throughout the M MT to quantify 604

chemically

- induced mortality. The s urvival rate s of larval fish at 3 weeks post

- fertilization (wpf) 605

are evalua ted in F 1 and F 2 based on the difference in the number of hatched embryos placed in 606

each replicate and the number of survivors collected. Subadult survival (F 1 , F 2 ; 8 wpf), and adult 607

survival (F 0 , F 1 , F 2 ; 16 wpf) are evaluated based on the number of fish p resent in each replicate at 608

the sampling time for each life

- stage (see Section 3.3 for additional information on sampling 609

timeline). 610

611

3.2.3 Growth (F O , F 1 , F

2 ) 612

613

A common toxic effect in response to chemical exposure is reduced growth , thus growth is 614

incorporated into the MMT to allow the evaluation of possible non

- endocrine mediated overt 615

toxicity as a possible explanation for treatment induced reductions in reproductive outcomes. 616

While it is true that endocrine processes can be linke d with growth outcomes, it is likely that an 617

integrated evaluation of all the test data will allow discrimination of endocrine versus non

618

endocrine mediated AOPs. In the M MT, growth is determined on days when fish are being 619

removed from aquaria to reduce t he number of fish per replicate when starting a new phase of 620

the test protocol. Specifically, wet weight and length are measured in all F 1 and F 2 fish shortly 621

after metamorphosis to juveniles as the number of fish per replicate aquarium are reduced to 10. 622

Subadult growth is measured in the F 1 and F 2 fish remaining after reproductive pairs have been 623

selected. Finally, adult growth is measured in F 0 , F 1 , and F 2 after reproduction has been assessed 624

and the fish in that generation are terminated. Growth is meas ured by measuring weight to the 625

nearest 1 m g and length to the nearest 0.1 mm

. 626

627

3.2.4 Reproduction (F O , F 1 , F

2 ) 628

629

The fecundity and fertility of reproductive pairs is assessed four days per week for 5 weeks in 630

each generation (F 0 , F 1 , and F 2 ). On the morning of each day (Tuesday through Friday of each 631DRAFT

MMT ISR 18 M a y 28

, 2013 week) egg clumps are removed from females and from the bottom of each aquarium and counted 632

for fecundity and evaluated under a dissecting microscope for fertility. 633

634

3.2.5 Liver Vitellogenin mRN A (F O , F 1 , F

2 ) 635

636

Vitellogenin (Vtg), a common biomarker of EDC exposure, is measured by determining the Vtg 637

mRNA copy number per nanogram (ng) of total mRNA in the liver by quantitative PCR 638

(QPCR). QPCR is highly sensitive and has a large dynamic range (~7 orders of magnitude), 639

making it an ideal tool for efficiently assessing concentrations of a target gene. Specifically, Vtg 640

is measured in adults at the end of each generation (F 0 , F 1 , and F 2 ) and in subadults in F 1 and F 2

. 641

The gene expression data in ea ch treatment are categorized by gender genotype (XX or XY) and 642

then compared to the gender

- specific control data from each particular generation and life

- stage. 643

644

3.2.6 Secondary Sex Characters (F O , F 1 , F

2 ) 645

646

Male medaka normally have anal fin papill ae that develop as a secondary sexual characteristic 647

(SSC) during the transition from subadult to adult. These bony structures are easily counted and 648

are responsive, negatively or positively, to various EDC exposures. In males, it is common for 649

estrogen receptor (ER) agonists ( e.g ., estradiol and 4

- tert octylphenol) to lower the number of 650

papillae or prevent their development altogether; conversely, androgen receptor (AR) agonists 651

( e.g ., 17

β

- trenbolone) can induce the development of papillae in females. I n all three generations, 652

during necropsy of adult and subadult samples, the portion of the fish posterior to the vent is 653

removed and fixed in Davidson’s solution. After fixation, the anal fin papillae are counted with 654

the aid of a dissecting microscope, an d recorded as the SSC measurement. 655

656

3.2.7 Hatch Success (F O , F 1 , F

2 ) 657

658

Eggs are placed in an incubator that is suspended in the same aquarium used to expose the 659

spawning pair. The developing eggs are continually agitated within the incubator by air bu bb les. 660

Incubators are checked daily for mortalities. Dead eggs are recorded and removed from the 661

incubators. T o promote hatching, the aeration of the incubators is stopped on the 8 th day and the 662

eggs settle to the bottom of the incubator. The majority of con trol eggs hatch over the next two 663

days. For each treatment, hatchlings are counted, and systematically distributed to each of the 664

replicate aquaria . Embryos that have not hatched by the 14 th day post fertilization are considered 665

non

- viable and discarded. 666

667

3.2.8 Pathology and Gonad Phenotype (F O , F 1 , F

2 ) 668

669

Particularly in the subadult samples, where the sample number is greater, but also in the adult 670

samples, the phenotype of the gonad is determined. At its simplest, the determination is either 671

ovary or t estis. However, an EDC exposure will often induce some level of intersex condition in 672

which a single gonad will contain both ovarian and testicular tissue of varying proportions, 673

presumably correlated to the potency of the EDC exposure. In addition a sma ll percentage of 674

control males will have testes with a few minimally developed oocytes . The prevalence of this 675DRAFT

MMT ISR 19 M a y 28

, 2013 appears to be somewhat strain

- dependent (Grim et al., 2007) , but in the EPA cultures, it seems to 676

be less than 2%. Th e assessment of gonad pheno type in subadults involves transverse oblique 677

sectioning of the gonad (sections of the kidney are often collected as well) to optimize the 678

sectioning plane. These sections are routinely hematoxylin and eosin (H&E) stained and viewed 679

by a trained pathologi st that categorizes them as one of the following: 1 = testis only; 2 = mostly 680

testicular tissue, some ovarian tissue; 3 = approximately equal prevalence of testicular and 681

ovarian tissues; 4 = mostly ovarian tissue, some testicular tissue; 5 = ovary only. The same 682

categories are used in the assessment of adults, but a sectioning protocol optimized to produce 683

sections of multiple organs and tissues is used. 684

685

While observations of histopathology are made on the subadult sectioned obliquely, the primary 686

histop athological analysis is done on the adult samples which can then be compared with the 687

reproductive output of the specific pair. Therefore, the adults are sectioned parallel to the 688

sagittal plane including one midline level and four symmetrical parasagitta l levels to produce 689

sections, primarily, of gonad, kidney, and liver while other secondary tissues can often be 690

observed as well. Diagnoses are scored for severity by the pathologist and these severity scores 691

are entered into StatCHARRMS for statistical a nalysis. Details of the histotechnical procedures 692

and pathology guidance documents have been provided ( Appendix F: Pathology Guidance). In 693

addition, an internet

- based resource is being developed that would not only include 694

histotechnical and pathology gu idance documents from various sources, but also would be an 695

interactive atlas to help harmonize diagnoses between independent pathologists. A description 696

of this resource is available in ( Appendix F: Pathology Guidance ). 697

698

3.3 Research MMT Timeline 699

700

The timeline for the MMT protocol is illustrated in Figure 3

- 1

. The test begins with exposure of 701

adult F 0 fish, and continues the exposure through the full medaka life

- cycle for two additional 702

generations, F 1 and F 2 . The test is complete after assessing reprod uctive endpoints in the F 2 703

generation. 704

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

722DRAFT

MMT ISR 20 M a y 28

, 2013 Figure 3

- 1 . T he time

- line and endpoints in the research MMT. The MMT is 29 weeks long and span s 2 1⁄2 723

generations; F 0 , F 1 , and F 2 . The rows to the right of each endpoint listed on the left indicate the study

- week when the 724

endpoint is measured in each generation. The life

- stages of t he test animals are color

- coded. 725

MMT Exposure and endpoint timeline

Gen F 0 1 2 3 4 5

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

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

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Lifestage Key Embryo Larvae Juvenile Subadult Adult

Endpoints

Fecundity F 0 F 1 F 2

Fertility F 0 F 1 F 2

Hatch F 1 F 2

Survival F 0 F 1 F 1 F 1 F 2 F 2 F 2

Growth F 0 F 1 F 1 F 2 F 2

Vitellogenin F 0 F 1 F 1 F 2 F 2

Secondary sex F 0 F 1 F 1 F 2 F 2

Histopathology F 0 F 1 F 1 F 2 F 2

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

726

727

3.3.1 F 0

; Study weeks 1

- 5 728

729

Young adult breeding pairs (11 w pf; females >250 mg; males >20 0 mg) of the first generation 730

(F 0 ) are moved into the exposure system at the start of Study week 1 and continually exposed for 731

the next five weeks . D uring this exposure period d eveloping oocytes and sperm within the 732

breeding adults accumulate the test chemical and/or its metabolites. Depending on toxicokinetic 733

factors, the concentration of the test chemical in the gametes may a steady state concentration 734

relative to each treatment levels. On Study Day 1, a minimum of six f emale

- male breeding pairs 735

are placed in an exposure system that has six tr eatment levels including controls with six 736

replicate aquaria per treatment concentration. Each replicate aquarium houses a single breeding 737

fish pair for a test total of 36 breeding pairs . The fecundity of each breeding pair is assessed for 738

the next 5 weeks by collecting all spawned eggs from each breeding pair on weekdays T, W, T, 739

F. The F 0 breeding pairs are sampled at the start of Study week 6 for the endpoints listed in Table 740

3

- 2 and Figure 3

- 3. 741

742

3.3.2 F 1 and F 2 ; Weeks 1

- 3 743

744

The first week for generations F 1 and F 2 (study weeks 3 and 15, respectively) , eggs are collected 745

from each breeding pair of the previous generation and a maximum of 20 eggs are loaded into a 746

glass incubator which is then placed in a separate incubator aquarium and sufficien t aeration is 747

supplied to the incubator to keep the eggs gently suspended in the water column of the incubator. 748

This process is repeated the next day, resulting in 2 incubators, each containing a maximum of 749

20 eggs, from each breeding pair. Aeration is con tinued until 7 days post

- fertilization (dpf), at 750DRAFT

MMT ISR 21 M a y 28

, 2013 which time the aeration is turned off and the eggs begin to hatch. Each day, the larval fish that 751

have hatched are collected, counted and placed in the incubator aquarium ( i.e ., same aquarium 752

and separate fo r each parental pair, but outside egg incubator enclosure). 753

754

3.3.3 F 1 and F 2 ; Week 4 755

756

At the start of week 4, all of the surviving larval fish from each treatment are counted, pooled 757

and redistributed to the same aquaria that contained the breeding pairs with a maximum of 10 758

fish per aquarium . The survival to 3 wpf is determined at this point. 759

760

3.3.4 F 1 and F 2 ; Weeks 4

- 6 761

762

Th e exposure continues as the larval fish develop into sub

- adults. 763

764

3.3.5 F 1 and F 2 ; Week 7 765

766

At the start of week 7, a small sample is taken from the caudal fin of each fish to determine the 767

genotypic sex of the individual. This information is used to randomly establish female

- male 768

breeding pairs. The fish not selected as breeding pairs are samp led for the various endpoints 769

outlined in Figure 3

- 3

. 770

771

3.3.6 F 1 and F 2 ; Weeks 7

- 10 772

773

The exposure continues as the sub

- adult breeding pairs develop into adults. 774

775

3.3.7 F 1 and F 2 ; Weeks 11

- 15 776

777

The fecundity of each breeding pair is assessed for the next 5 weeks by collecting all spawned 778

eggs from each breeding pair on T, W, Th, and FF. 779

780

3.3.8 F 1 and F 2 ; Week 16 781

782

The breeding pairs are sampled at the start of week 16 for the various endpoints outlined in 783

Figure 3

- 3. 784

785



### 3.4 Statistical Analyses 786

787

The biological data generated by the MMT are one of three types: 1) a continuous response that 788

is measured once per subject (fish)

, e.g., vitellogenin and weight ; 2) a continuous response that is 789

measured multiple times per subject (breeding pair)

, e.g

., fecundity which is measured on 21 790

consecutive days ; or, 3) ordinal data, such as histopathology findings that are expressed as a 791DRAFT

MMT ISR 22 M a y 28

, 2013 severity score

. These types of data are not unique to the MMT and, except for pathology data, 792

many other appropriate statisti cal methodologies have been developed to properly analyze them 793

depending on the characteristics of the data including normality, variance homogeneity, whether 794

the study design lends itself to hypothesis testing or regression analysis, etc. In order to ana lyze 795

the complete datasets from all the MMTs presented in this ISR, a somewhat statistically 796

conservative

, systematic approach was used as described below. For the prevalence/severity 797

score pathology data, a new statistical approach, described below and at tached as an appendix, 798

was developed. 799

800

All statistics were done with a program termed StatCHARRMS ( Stat istical analysis of 801

C hemistry, H istopathology, A nd R eproduction endpoints including R epeated measures and 802

M ultigeneration S tudies), a Statistical Analysi s System ( SAS ® ; SAS ® Institute, Cary, NC)

- based 803

program developed for this use by John Green (DuPont Applied Statistics Group, Newark, DE). 804

For continuous data measured once per subject, normality (via Shapiro

- Wilks test) and variance 805

homogeneity (via Leve ne’s test) were assessed and appropriate transforms applied by the 806

program to determine the appropriate statistic for hypothesis testing. In addition, regardless of 807

the results of those two tests, a step

- down Jonckheere

- Terpstra trend (JT) test was also a pplied to 808

the data as long as a monotonic response was observed. To be consistent across MMTs, the 809

statistical test that indicated the most sensitive lowest observed effect concentration ( LOEC ) was 810

reported. In addition, StatCHARRMS has the ability to an alyze continuous data across multiple 811

generations, but to simplify interpretation of the data within this ISR, only single generation 812

analyses are presented. T able 3

- 3 summarizes the statistical tests used by StatCHARRMS, while 813

a thorough description of t he StatCHARRMS package including statistical rationale can be found 814

in Appendix G: Statistics

. 815

816

817

Table 3

- 3

. Decision

- tree determining the statistical test used by StatCHARRMS. 818

Shapiro

- Wilks Levene’s Statistical test

Pass Pass Dunnett; JT

Fail Pass Modified Dunn’s; JT

Pass Fail Tamhane

- Dunnett; JT

Fail Fail JT

819

820

For fecundity and fertility, which are continuous responses that are measured multiple times, a 821

repeated measures analysis of variance (ANOVA) with two main effects, i.e ., treatment and 822

we ek (or day), and the treatment

- by

- week interaction are used for analysis. During the MMT 823

research program, transformations of the fecundity and fertility data never produced normally 824

distributions so the nonparametric analysis of a repeated measures ANOVA on the ranks of the 825

data is used. When comparing levels of the treatment effect, i.e ., treatments averaged across 826

weeks, against the controls, a Dunnett’s test is used to adjust for the family

- wise error rate. For 827

the week effect, i.e ., comparisons of we eks averaged across treatments, if either the week main 828

effect or the week

- by

- treatment interaction is significant at the 0.05 level, then the comparisons 829

of weeks averaged across treatments or within a treatment are done at the 0.05 level with no 830

adjustme nt. Similarly, if the treatment

- by

- week interaction is significant at the 0.05 level, then 831

treatments within the same week are compared at the 0.05 level unadjusted p

- values. Finally, if 832DRAFT

MMT ISR 23 M a y 28

, 2013 the treatment

- by

- week interaction is not significant, then compariso ns of weeks within a 833

treatment or treatments within a week are compared using Bonferroni

- Holm adjusted p

- values. 834

835

For histopathology data which are in the form of severity scores, StatCHARRMS uses a new test 836

termed RSCABS ( R ao

- S cott C ochran

- A rmitage b y S li ces). RSCABS uses a step

- down Rao

837

Scott adjusted Cochran

- Armitage trend test on each level of severity in a histopathology 838

response. The Rao

- Scott adjustment incorporates the replicate vessel experimental design into 839

the test. The by

- slices procedure in corporates the biological expectation that severity of effect 840

tends to increase with increasing doses or concentrations, while retaining the individual subject 841

scores and revealing the severity of any effect found

. The RSCABS procedure not only 842

determines which treatments are statistically different, i.e., have more severe pathology than 843

controls, but it also determines at which severity score the difference occurs providing much 844

needed context to the analysis. A detailed description of this test can be f ound in Appendix G: 845

Statistics. 846

847

3.5 Data Expectations 848

849

The types of data generated within the MMT are relevant to population dynamics ( e.g

., 850

reproduction), to the description of the affected AOP(s) ( e.g ., Vtg mRNA induction and SSC), 851

and to non

- specific, or at least non

- EDC related, toxicity ( e.g ., growth parameters). It is 852

expected that the complete suite of data from the MMT would not only define a LOEC for 853

reproduction but also provide valuable information regarding the affected AOP, perhaps to the 854

ex tent of providing a “fingerprint” to identify the test agent not only as an EDC but also as, for 855

instance, an ER agonist. Since the MMT measures apical endpoints and does not measure direct 856

consequences of the molecular initiating event (MIE) within an AO P, it does have limitations to 857

its ability to resolve the various known or postulated AOPs. However, information from the Tier 858

1 studies will provide relevant information regarding the AOP of the test chemical. 859

860

For ER agonists (or perhaps more appropriate ly ER activation), the pathway, after agonism of 861

the estrogen receptor, is expected to include, in male fish, production of vitellogenin (therefore 862

up

- regulated vitellogenin genes), feminized phenotypes of various somatic tissues normally 863

influenced by the HPG, developmental abnormalities and feminization of the testis including, at 864

the extreme, complete sex reversal, and finally impaired reproductive capacity (Ankley et al., 865

2010b) . While ER agonists, in particular strong ER agonists, presumably induce ad verse effects 866

in females, the affects appear to be less specific. They may impair ovary and/or brain 867

development which could reduce reproductive performance as well. The MMT does not assess 868

reproductive capacity of exposed males and females independently (for instance by forming a 869

breeding pair from an exposed male and an unexposed female) therefore, the MMT is unable to 870

completely resolve impairment of one gender versus the other. 871

872

Several ER agonists were tested during MMT development, ranging from stron g (17β

- e stradiol ) 873

to very weak in vitro agonists (4

- chloro

- 3

- methylphenol). Within these MMTs, the data closely 874

reflected effects to the measured parameters that would be expected. Typically, the end result of 875

exposure was reduced fecundity. Changes to biomarker endpoints were also observed including 876

decreased SSC (in males), up

- regulation of vitellogenin, and feminization of the gonad, all 877DRAFT

MMT ISR 24 M a y 28

, 2013 without adversely impacting growth. This suite of outcomes is consistent with an ER activation 878

AOP. 879

880

For AR agonis ts, the pathway after binding (the MIE) seems to include hormonal feedback that 881

results in the reduced synthesis of testosterone and estrogen which leads to reduced vitellogenin 882

production, impaired oocyte development, and finally impaired reproduction (An kley et al., 883

2010a) . In addition, AR agonists could be expected to produce masculinized somatic tissues 884

normally influenced by the HPG and developmental abnormalities and changes in the phenotype 885

of the ovary, including sex reversal. During the developme nt of the MMT, a single strong AR 886

agonist (17β

- trenbolone) was used as a test agent. Near the concentrations that reduced 887

fecundity, the MMT also provided data from biomarkers indicating the involvement of the AR 888

agonism AOP including the presence of male

- specific SSC in female fish, the reduction of 889

vitellogenin, and the development of testes in XX fish. Again, the suite of data seemed to 890

provide a hallmark pattern scientifically ascribable to the AR activation AOP. 891

892

Another MIE that leads to an AOP, alb eit not as well defined, is the inhibition of aromatase 893

which catalyzes the conversion of testosterone to 17 β

- estrogen. The literature suggests that 894

aromatase inhibition reduces 17 β

- estrogen plasma concentrations, reduces vitellogenin uptake 895

into oocytes, and reduces fecundity (Ankley et al., 2002; Sun et al., 2007b) . Indeed the MMT 896

with prochloraz, an aromatase inhibitor had the expected results: reduced fecundity and 897

decreased vitellogenin with no effect on the development of SSC in males. While far fr om 898

conclusive and needing further research, the MMT may serve as a means to differentiate between 899

AR agonists and aromatase inhibitor/steriodogenesis inhibitor based upon the responses of the 900

SSC and vitellogenin. 901

902

The responses of fish in various bioassay systems to AR antagonist s ha ve been somewhat 903

ambiguous (Bayley et al., 2003; Kiparissis et al., 2003; Makynen et al., 2000) . Enough so that 904

testing potential of AR antagonists to reduce or eliminate the effects of a model androgen like 905

17 β

- trenbolone hav e been suggested as a means to identify AR antagonists (Ankley et al., 906

2010a) . Therefore, expectations for any specific response to the AR antagonist, vinclozolin, are 907

uncertain. 908

909

Table 3

- 4. Contingency table for effects of EDCs with different MIEs on vitellogenin, secondary sexual 910

characteristics (SSC) and the histologically determined gonad phenotype. Arrows indicate direction of change and 911

parentheses indicate a change that can occur, albeit not consistently. Based on (Ankley et al., 2010b)

. 912

MIE Male Female

Vitellogenin SSC Gonad Phenotype Vitellogenin SSC Gonad Phenotype

ER agonist ↑ ↓ feminized (↑)

AR agonist (↑) ↓ ↑ masculinized

AR antagonist (↓) ? ?

Steroidogenesis inhibitor ? ↓ ?

913

4 MMT DATA SUMMARIES 914

915

This section provides a brief summary of the MMT tests performed in the U.S. and Japan 916

beginning with the MMT protocol presented in Section 3. Each successive iteration of the MMT, 917DRAFT

MMT ISR 25 M a y 28

, 2013 usually with a new/different test agent, was performed using a protocol sli ghtly different than 918

that used for the previous MMT. The differences were generally responses to one or more of the 919

following considerations; a) experience and data from previous tests, b) discussions with 920

Japanese scientists who were also performing MMT p rotocols, and c) results from ancillary 921

experiments designed to provide information to improve the MMT protocol. Examples of 922

modifications include: changes in the temperature of the test, duration and measurement times 923

for reproduction endpoints, feeding p rotocols, egg incubation methods, histopathology methods, 924

etc . Table 4

- 1 lists the MMTs that were performed in U.S. EPA or Japanese laboratories. 925

Generally, EDCs with different AOPs were tested in the successive MMTs. Modifications of the 926

MMT protocol were evaluated based on whether the change improved the efficiency of 927

performing the test, or the statistical power for detecting endpoint effects by reducing variance or 928

increasing the absolute value of a critical endpoint. 929

930

Table 4

- 1. Chemicals run in t he MMT protocol 931

Chemical Laboratory Protocol AOP’s MIE *

17 β

- Estradiol CERI/ Japan Full MMT ER agonist (strong)

4

- t

- Octylphenol US EPA/MED Full MMT ER agonist (weak)

4

- t

- Octylphenol NIES/Japan Full MMT ER agonist (weak)

o,p′

- DDT CERI/Japan Full MMT ER agonist (medium)

4

- chloro

- 3

- methylphenol US EPA/MED Full MMT ER agonist in vitro (weak)

Tamoxifen NIES/Japan Abbreviated

MMT 1 ER binder/modulator ; tissue

dependent antagonist/partial

agonist

17 β

- Trenbolone US EPA/MED Full MMT AR agonist

Prochloraz US EPA/MED Full MMT Aromatase inhibitor; AR

antagonist; steroid metabolism

modulator

Vinclozolin US EPA/MED Abbreviated

MMT 1 AR antagonist

Vinclozolin Interlab A Abbreviated

MMT 1 AR antagonist

Interlab B Abbreviated

MMT 1 AR antagonist

Interlab C Abbreviated

MMT 1 AR antagonist

4

- t

- Octylphenol US EPA/MED Full MMT 2 ER agonist

1 Abbreviated MMT: s ame as Full MMT but without F2 reproduction 932

2 Full MMT with additional replication for the assessment of reproduction in F1 and F2 933

* Molecular Initiating Event of an AOP 934

935

Taken en masse , the data indicate that the MMT is able to determine effect concentrations for 936

several chemicals with MIE for various AOPs that are relevant to reproduction. In addition, the 937

MMTs provided information regarding endpoints th at might provide insight into intermediate 938

markers (key events) that involve disruption of the HPG axis (endocrine

- mediated chemical 939

effects) and/or that cause reductions in survival, growth, hatch, etc . through non

- endocrine 940

mediated pathways. Table 4

- 2 summarizes the LOECs determined in each MMT for both 941DRAFT

MMT ISR 26 M a y 28

, 2013 reproduction and the lowest LOEC for the non

- reproduction endpoints, and representative 942

LOECs for similar measurements from the literature involving the same test agent. 943

944

Table 4

- 2. Lowest observable e ffect concentration ( LOEC ) in each MMT for reproduction and lowest LOEC for 945

any non

- reproductive endpoint (biomarker) and generation for which it was detected, compared to values reported in 946

the literature for the same test agent. 947

948

Chemical Laboratory MMT LOEC Published Results

Fecundity Biomarker Conc. Effect

17β

- Estradiol CERI/Japan 28 ng/L F 1 2.8 ng/L SSC F 1 27.9 ng/L a Fecundity

4

- t

- Octylphenol EPA/MED 51 μg/L

F 2 13 μg/L SSC F 2 11.4 μg/L b

Vitellogenin

13 μg/L Gonad F 1

4

- t

- Octylphenol NIES/Japan 100 μg/L F 1 6.25 μg/L SSC F 1 11.4 μg/L b Vitellogenin

6.25 μg/L Vtg F 1/ F 2

o,p′

- DDT CERI/Japan 1.9 μg/L F 2 0.07 μg/L Vtg F 1/ F 2 0.1 μg/L c Vitellogenin

CMP EPA/MED >345

μg/L NA 88 μg/L Vtg F 0 No data No data

Tamoxifen NIES/Japan 20 μg/L F 1 1.3 μg/L Vtg F 1/ F 2 625 μg/L d Fecundity

1.3 μg/L SSC F 2

17β

- Trenbolone EPA/MED 32 ng/L F 1 32 ng/L SSC F 1

39.7 ng/L e Vitellogenin 32 ng/L Vtg F 1

32 ng/L Gonad F 1

Prochloraz EPA/MED 25 μg/L F 2 9 μg/L SSC F 1 30 μg/L f Fecundity

9 μg/L Vtg F 1/ F 2

Vinclozolin EPA/MED >136

μg/L NA 17 μg/L SSC F 1 2500 μg/L g Spematogenesis

4

- t

- Octylphenol EPA/MED 23 μg/L F 1/ F 2 In process 11.4 μg/L b Vitellogenin

a: Seki et al., 2005 949

b: Seki et al., 2003a 950

c: Uchida et al., 2010 951

d: Sun et al., 2007a 952

e: Seki et al., 2006 953

f: Zhang et al., 2008 954

g: Kiparissis et al., 2003 955

956

957

958DRAFT

MMT ISR 27 M a y 28

, 2013 4.1 Results with ER

- agonists 959

960

4.1.1 17β

- Estradiol (CERI/Japan) 961

962

The primary reproductive endpoint (fecundity) was affected by exposure to 17β

- e stradiol , in both 963

F1 and F2 generations with a LOEC of 28 ng/L. In F1, fecundity was further reduced by 964

exposure to 84 ng/L (no breeding pairs were available for the 84 ng/L treatment in F2). On the 965

other hand, reproduction was not affected by 17β

- e stradiol exposure in F0, presumab ly 966

highlighting a greater sensitivity of organizational impacts over activational impacts on 967

reproductive impairment. 968

969

The secondary endpoints that provide information about the potential AOP (in this case 970

obviously identified as an ER agonist) responded in a manner and magnitude consistent with 971

17 β

- e stradiol exposure reported in the literature (Cripe et al., 2009; Hirai et al., 2006; Raimondo 972

et al., 2009; Seki et al., 2006; Seki et al., 2005) . The LOEC, considering all endpoints equally, 973

was 2.8 ng/L, b ased on SSC of F 1 , XY subadult fish. This is the same LOEC reported from a 974

fish full life

- cycle test with medaka for F 1 (Seki et al., 2005) . In further support of the ER 975

agonist AOP, vitellogenin production in male livers was induced by 17 β

- e stradiol exp osure 976

starting at 28 ng/L, and significant numbers of testicular oocytes started to appear at 28 ng/L as 977

well. Growth parameters generally were not altered by 17 β

- e stradiol exposure suggesting that 978

systemic toxicity was not observed in the MMT. A compreh ensive report including graphs and 979

tables of the data can be found in the Appendix H: MMT Summary Reports on pp. 1

- 15. 980

981

4.1.2 4

- t

- Octylphenol (US EPA/MED) 982

983

4

- t

- Octylphenol ( 4tOP) is a relatively strong estrogen that has been shown to induce vitellogenin 984

production and cause abnormal sex differentiation in medaka after a 60 day exposure (starting 985

with embryos) at a concentration of 11.4 μ g/L (Seki et al., 2003a) . The literatu re clearly 986

indicates that 4tOP is an ER agonist (Gray and Metcalfe, 1999; Gronen et al., 1999; Korner et al., 987

2001; Laws et al., 2000) . Therefore, it is not surprising that it decreased reproduction in F 1 at 988

102 μ g/L and in F 2 at 51 μ g/L. At 102 μ g/L, re production was essential shutdown while at 51 989

μ g/L, 4tOP reduced the reproductive output by approximately 50%. On the other hand, 990

reproduction was not affected by 4tOP exposure in F 0 , presumably highlighting a greater 991

sensitivity of developmental impacts over activational impacts on reproductive impairment. 992

993

Those endpoints that would be expected to be affected by an estrogen were responsive to 4tOP 994

exposure. The overall LOEC for the MMT was 13 μ g/L, based on SSC in F 2 XY fish, which 995

matches the publishe d LOECs from other protocols (Gray and Metcalfe, 1999; Knorr and 996

Braunbeck, 2002; Segner et al., 2003; Seki et al., 2003a) . In support of the ER agonist AOP, the 997

male SSC LOEC was lowered in XY fish in both F 1 and F 2 at both subadult and adult samplings. 998

Further, vitellogenin gene expression was induced in XY fish at both lifestages and in all 999

generations with LOECs of either 25 μ g/L or 51 μ g/L, and the prevalence of testicular oocytes in 1000

XY fish increased at all samplings except F 0 with LOECs ranging fro m 13 μ g/L to 51 μ g/L. 1001

1002DRAFT

MMT ISR 28 M a y 28

, 2013 Histological observations were also made to provide further information about both potential 1003

EDC and non

- EDC effects on multiple organs including gonad, liver, and kidney

. Generally, the 1004

observed pathologies in F 0 were of low seve rity , occurred only after exposure to 102 μ g/L, and 1005

were more prevalent in the kidney and liver than in the gonad. Of the pathologies reported, the 1006

majority were observed in XY individuals and could be linked to an ER agonist AOP: increased 1007

number and severity of testicular oocytes (LOEC = 13 μ g/L ), developmental perturbation within 1008

the gonad related to both cell types (immature spermatocysts; LOEC = 13 μ g/L) and structural 1009

malformations (efferent duct; LOEC = 13 μ g/L), testicular hypoplasia ( i.e., smaller than normal 1010

testes), and increased basophi lia in the liver, presumably related to the production of vitellogenin 1011

(LOEC = 51 μ g/L ). There were also pathologies observed in the kidneys of XY fish that are 1012

most likely a secondary pathology induced by high concentrations of vitellogenin in the 1013

bloods tream of XY fish that normally lack a clearance mechanism for vitellogenin. There were 1014

very few, if any, pathologies that would be linked to non

- EDC toxicity. 1015

1016

All of these findings demonstrate the MMTs potential to detect effects that are important b oth at 1017

the population level and at lower levels of biological organization. A comprehensive report 1018

including graphs and tables of the data can be found in the Appendix H: MMT Summary Reports 1019

on pp. 16

- 38. 1020

1021

1022

4.1.3 4

- t

- Octylphenol (NIES/Japan) 1023

1024

In conjunction with the 4tOP MMT completed by MED and as part of a bilateral agreement, 1025

NIES (National Institute for Environmental Studies) in Japan conducted a similar MMT, again 1026

with 4tOP as the test agent. The timing of the subadult samplings were a littl e different (7 wpf at 1027

MED versus 8 wpf at NIES), no data were collected in F 0 , and the suite of endpoints that were 1028

measured in the MED version of the MMT were reduced in the NIES MMT to just survival and 1029

SSC. Note, because the data provided by NIES were summary information and statistics, they 1030

could not be analyzed using StatCHARRMS. 1031

1032

Fecundity was reduced by about 80% in F 1 after exposure to the highest concentration tested, 1033

100 μ g/L, which resulted in no fish in that treatment in F 2 . There were no chan ges in fecundity 1034

observed in F 2 (the highest concentration was 50 μ g/L). These results are very similar to the 1035

MED MMT where the LOEC for fecundity was 102 μ g/L in F 1 and 51 μ g/L in F 2

. 1036

1037

The secondary endpoints that provide AOP information responded as wo uld be anticipated. 1038

SSCs in XY fish were reduced at a LOEC of 6.25 μ g/L in F 1 subadults and 25 μ g/L in F 2 1039

subadults, while vitellogenin production in XY fish was induced at a LOEC of 6.25 μ g/L in F 2 1040

and only 25 μ g/L in F 1 . The mean hepatic vitellogenin concentration in the same treatment 1041

across F 1 and F 2 generations was not very great, 4.8 ng/mg liver in F 1 compared to 8 ng/mg liver 1042

in F 2 , but F 2 was high enough to be statistically different than controls. The endpoint that 1043

seemed to respond differently between the MED and the NIES MMTs was gonad phenotype. 1044

While the MED MMT had many XY fish with 4tOP induced testicular oocytes, very few were 1045

reported in the NIES MMT so that this was a relatively insensitive endpoint. A comprehensive 1046DRAFT

MMT ISR 29 M a y 28

, 2013 report including graphs and tables of the data can be found in the Appendix H: MMT Summary 1047

Reports on pp. 39

- 50. 1048

1049

1050

4.1.4 Comparison of 4

- t

- Octylphenol MMTs in Support of Validation 1051

1052

Comparison of control parameters between MMTs can indicate whether the protocol was 1053

succes sfully transferred between laboratories. The 4tOP MMTs completed by EPA/MED and 1054

NIES Japan provide just such an opportunity. In Table 4

- 3 , the only two parameters in adult 1055

control fish that were measured the same in both MMTs are compared, showing simila r data 1056

across the two labs. Other data were collected in both, but are not directly comparable. For 1057

instance, vitellogenin was measured by QPCR to determine the copy number of vitellogenin 1058

mRNA in the MMT done at MED while NIES measured vitellogenin prot ein concentrations in 1059

the liver. Also, the subadult sampling done by MED was at 44 dpf while NIES sampled 1060

subadults at 56 dpf (or later as the data only indicate s 8 wpf). During this time, medaka are 1061

undergoing rapid development and growth, thus growth, SSC, and vitellogenin production all 1062

increase dramatically within this fairly small window. So while the mean SSC in NIES 1063

subadults was 69, the mean for MED subadults was only 27; however, as seen in the table by the 1064

adult life stage , they are very simila r regardless of lab. 1065

1066

Table 4

- 3. Comparison of adult control parameters in the two 4tOP MMTs. 1067

US EPA MED NIES Japan

F1 F2 F1 F2

Fecundity 17 ± 2 20 ± 5 29 ± 5 27 ± 3

SSC 83 ± 9 130 ± 5 116 ± 12 120 ± 19

1068

1069

In general, the two MMTs (MED and NIES) were successful demonstrations of the protocol’s 1070

reproducibility. Despite being run at different times, in different countries, and the language 1071

barrier between the two performing labs, they measured similar LOECs for reproduction and 1072

provided mutually co rroborating information on the documented AOP of 4tOP ( Table 4

- 4 ). 1073

These data indicate that the MMT is a reliable protocol which is reproducible between labs, a 1074

key component of the validation process. 1075

1076

1077

1078

1079

Table 4

- 4. LOECs from selected endpoints from the 4tOP MMTs of ORD and NIES. 1080

Endpoint US EPA/ MED LOEC NIES/Japan LOEC

Fecundity 51 μ g/L 100 μ g/L

SSC 13 μ g/L 6.25 μ g/L

Vitellogenin 25 μ g/L 6.25 μ g/L

Gonad Phenotype 25 μ g/L 100 μ g/L (limited data)

1081

1082DRAFT

MMT ISR 30 M a y 28

, 2013 4.1.5 o,p′

- DDT (CERI/ Japan) 1083

1084

Essentially, the full research MMT was implemented by CERI (Chemicals Evaluation and 1085

Research Institute) as a part of the US

- Japan bilateral agreement to test o,p’

- DDT with only the 1086

adult growth measurements being omitted. This te st agent was chosen because it is a known ER 1087

agonist that would be expected to bioaccumulate (Metcalfe et al., 2000; Uchida et al., 2010)

. 1088

1089

Reproduction was not effected by exposure in F 0 , even at the highest treatment 1.9 μ g/L. 1090

However, in F 1 , 1.9 μ g/L o,p’

- DDT did reduce fertility (but not fecundity) mostly resulting from 1091

two pairs producing large numbers of non

- viable eggs. The LOEC for fecundity and fertility in 1092

F 2 was 1.9 μ g/L. In this treatment, fecundity was reduced from 20.3 eggs per pair each day to 14 1093

while the mean number of fertile eggs in controls was 17.6 which was reduced to 6.9 fertile eggs 1094

per pair each day. Interestingly, these means for exposed breeding pairs were similar to the 1095

reproductive output in the same 1.9 μ g/L treatment of F 1 ; however, the control mean in F 2 was 1096

higher than in F 1 , presumably providing greater statistical power in the analysis of the data. 1097

1098

The overall LOEC for the MMT was 0.07 μ g/L;; however, this was based on an increase in the 1099

hepatic vitellogenin concentration in adult XX fish. The magnitude of this increase was very 1100

modest and did not exhibit the classical dose

- response: exacerbation of the response with 1101

increased concentration o f the test agent. The hepatic vitellogenin concentration was also 1102

increased in adult XY fish in both F 1 and F 2 , but only in the highest treatment, i.e ., 1.9 μ g/L. 1103

1104

The secondary endpoints again corroborate the expectation of o,p’

- DDT acting as an ER agon ist. 1105

The numbers of anal fin papillae (SSC) were lowered by more than 50% and the concentration of 1106

hepatic vitellogenin was increased in XY subadult fish in the 1.9 μ g/L treatment. The effect on 1107

SSC in subadults was lack of any observable papillae in the adult fish suggesting that this 1108

observation was due to a developmental delay. In addition, most of the XY fish exposed to 1.9 1109

μ g/L o,p’

- DDT across both F 1 and F 2 generation and at both developmental stages had testicular 1110

oocytes while in adults in F 2 an d subadults in F 1 had increased testicular oocytes as well. A 1111

comprehensive report including graphs and tables of the data can be found in the Appendix H: 1112

MMT Summary Reports on pp. 51

- 64. 1113

1114

4.1.6 4

- chloro

- 3

- methylphenol (US EPA/MED) 1115

1116

The full research MMT was performed with 4

- chloro

- 3

- methylphenol (CMP) as the test agent. 1117

In work done at MED, CMP binds to both recombinant human and rainbow trout ( Oncorhynchus 1118

mykiss ) ERs and induces rainbow trout vitellogenin mRNA at about the same co ncentrations as 1119

overt toxicity (Schmieder et al., 2004)

. 1120

1121

No effects were observed in the entire MMT that would be attributed to EDC exposure except 1122

for the possibility of vitellogenin gene expression in XX adult fish in F 0 only in which a small 1123

increase (~2X of controls) was observed starting at 88 μ g/L of CMP. Despite this, reproduction 1124

was not decreased in any of the generations, and of the three significant effects documented (out 1125

of close to 50 different measurements), none were consistently observe d across generations or 1126DRAFT

MMT ISR 31 M a y 28

, 2013 life stages. In addition, no indication of toxicity was observed via histopathology. Therefore, 1127

this MMT indicated that the LOEC for CMP was greater than 345 μ g/L. 1128

1129

Post

- MMT, MED performed a short

- term test to establish acute leth ality data for medaka to 1130

CMP to determine whether higher concentrations than 345 μ g/L of CMP cause overt 1131

toxicity/mortality or EDC

- related impacts. Newly hatched fish were exposed for 35 days to 1132

CMP at concentrations of 300, 600, 1200, 2400, and 4800 μ g/L . Survival as the measurement of 1133

toxicity was monitored throughout the exposure and vitellogenin gene expression, as the measure 1134

of EDC effects, was quantified at the end of the exposure. The 7

- day and 14

- day median lethal 1135

concentrations to 50% of the fi sh tested ( i.e ., LC 50 values) were calculated as 2840 μ g/L and 1136

1090 μ g/L CMP, respectively. At the end of the 35

- day exposure, only fish from control, 300 1137

μ g/L, 600 μ g/L, and 1200 μ g/L survived. There was no change in the copy number of 1138

vitellogenin mRNA in livers of either XX or XY fish indicating CMP is overtly toxic, causing 1139

mortality, before it would induce EDC

- related effects. A comprehensive report including graphs 1140

and tables of the data can be found in the Appendix H: MMT Summary Reports on pp. 65

- 80. 1141

4.1.7 Tamoxifen (NIES/Japan) 1142

1143

Tamoxifen is classified as a selective ER modulator (SERM) as it has multiple actions both as an 1144

ER antagonist and in specific tissues/species as an ER agonist (Shang and Brown, 2002) . After 1145

binding to the ER, tamoxifen has been shown to act as both agonist and antagonist in various 1146

species including the medaka, in which previous tests have shown increased plasma vitellogenin 1147

in males but decrease d plasma vitellogenin in females (Chikae et al., 2004; Sun et al., 2007a)

. 1148

1149

The MMT completed by NIES in Japan testing the effects of tamoxifen was an abbreviated 1150

MMT protocol that went from F 0 adults to F 2 subadults. 1151

1152

Fecundity was reduced by over 50% u pon exposure to 20 μ g/L tamoxifen, the highest 1153

concentration tested. The AOP related endpoints were typically affected at a LOEC of 1.3 μ g/L 1154

in subadults. These included increased vitellogenin and decreased SSC in XY fish. Similar to 1155

other studies (Chika e et al., 2004; Sun et al., 2007a) , tamoxifen actually reduced vitellogenin in 1156

female fish, specifically starting at 1.3 μ g/L. The induction of vitellogenin in males was fairly 1157

modest, a few times that of controls but not approaching the same vitellogenin concentration as 1158

normal female fish. The maximum change in SSC was seen in F 2 where the number of anal fin 1159

papillae was reduced from about 80 per XY fish to approximately 25. These same endpoints had 1160

slightly higher LOECs (from 1 to 2 treatment levels) in the adult life stage, due either to true 1161

differences in the sensitivity of the life

- stage, or to random variation causing statistical, but not 1162

true biological differences in the response. Also note, the subadult sampling were not done at the 1163

same point in development, F 1 at 54 dpf and F 2 at 65 dpf, which had considerable impact on 1164

control values for these parameters. 1165

1166

In addition to the AOP (ER agonist; SERM) responsive endpoints, tamoxifen exposure reduced 1167

growth at 54 dpf (F 1 ) by about 30% in both XX and XY fish, but by less than 20% in XX fish 1168

only at 65 dpf (F 2 ). It is unclear from the data whether this difference is reflective of delayed 1169

maturation, where the fish evidently grow to the same size regardless of exposure, or growth is 1170

irreversibly aff

ected by exposure to tamoxifen. Unfortunately, growth data, which would have 1171DRAFT

MMT ISR 32 M a y 28

, 2013 provided insight into this question was not recorded for adults. And, histopathology, which 1172

would have provided information both about potential EDC impacts and non

- EDC toxicity at the 1173

organ/tissue level, was also not assessed. A comprehensive report including graphs and tables of 1174

the data can be found in the Appendix H: MMT Summary Reports on pp. 81

- 94. 1175

1176

4.2 Results with AR

- agonists 1177

1178

4.2.1 17β

- Trenbolone (US EPA/MED) 1179

1180

17 β

- trenbolone acts as a potent agonist of androgen receptors in a variety of systems including 1181

fish where it has been shown to reduce fecundity, vitellogenin, and generally induce 1182

masculinization of both external SSCSSC and gonad phenotypes (Ankley et al., 2 003; Orn et al., 1183

2006; Sone et al., 2005; Wilson et al., 2002) . In a 21

- day exposure of medaka, the LOEC for 1184

changes in SSC and decreased vitellogenin was 365 ng/L and 39.7 ng/L, respectively (Seki et 1185

al., 2006)

. The MMT completed by EPA

- MED testing the effects of trenbolone was done with 1186

the full research MMT protocol

. 1187

1188

The LOEC for reproduction, which occurred in F 1

, was 32 ng/L. At this concentration fecundity 1189

was reduced to zero. In addition, 13 ng/L 17 β

- trenbolone seemed to reduce fecundity (contro l 1190

fecundity equaled 25 eggs per pair

- day versus ~11 eggs per pair

- day in the treatment); however, 1191

it was not statistically significant. No reduction in reproduction was observed in F 2 (the highest 1192

concentration present was 13 ng/L), and the mean fecundity in the 13 ng/L treatment was equal 1193

to controls (~24 eggs per pair

- day) and much higher than in the same treatment in F 1 (~11 eggs 1194

per pair

- day). 1195

1196

The LOEC s across the entire trenbolone MMT in the AOP

- related endpoints ranged from 32 to 1197

84 ng/L, predominately affecting XX fish. All the findings supported the expected response to 1198

an AR agonist, i.e ., the presence of anal fin papillae (the measure of SSC) in XX fish, decreased 1199

vitellogenin gene expression in XX fish, and most dramatically, XX fish presenting complete 1200

male phenotypes including functional testes. In the 84 ng/L treatment, essentially all XX fish 1201

appeared male for all measurements of phenotype: gonad phenotype, copy number of the 1202

vitellogenin gene, and SSC. In addition at 32 ng/L of 17 β

- trenbolone , the same effects were 1203

observed at intermediate magnitudes except gonad phenotype in which, interestingly, intersex 1204

gonads were never observed, only c omplete sex reversal. However, intermediate effects in 1205

gonad phenotype were observed in a slightly different manner. For instance in F 1 subadults, 16 1206

of 16 XX fish exposed to 84 ng/L 17 β

- trenbolone had testes, while in the 32 ng/L treatment, only 1207

7 of 17 XX fish had testes. 1208

1209

The observed pathologies further supported the AOP of 17 β

- trenbolone . First there were very 1210

few pathologies found in XY fish. Second, many of the pathologies observed in XX fish could 1211

be ascribed to an AR agonist exposure. For inst ance oocyte atresia and decreased yolk formation 1212

were found in XX subadults and adults at mild to moderate severities. There were also 1213

pathologies observed in the kidneys of XX fish at high prevalence and severity including tubule 1214

epithelial hypertrophy a nd eosinophilia. Ironically, 17 β

- trenbolone starting at 32 ng/L so 1215

completely caused sex reversal in the XX individuals that the pathologist observed no 1216DRAFT

MMT ISR 33 M a y 28

, 2013 pathologies in the gonads of these animals, in spite of the fact that they were XX fish with testes, 1217

albeit, histologically normal testes. 1218

1219

In general, the F 0 generation was less sensitive than the F 1 . Because there were no viable fish 1220

available in the 32 and 84 ng/L treatments of F 2 , no effects were observed in any of the 1221

measurements. In addi tion, the LOECs obtained at the F 1 subadult sampling were lower than 1222

those of the F 1 adult sampling. However, whether this was a result of a greater sensitivity at this 1223

life stage or a result of the statistical impact of greater sample size per replicate at the subadult 1224

sampling is difficult to determine from the data. 1225

1226

The suite of biological impacts on the fish within the MMT could be linked across levels of 1227

biological organization to the well documented mode of action of trenbolone as an AR agonist. 1228

At the population/organismal level, fecundity was reduced starting at 32 ng/L. This presumably 1229

reflects the effect trenbolone had on the phenotype of individual XX medaka. Starting at the 32 1230

ng/L level, profound effects were observed on the phenotype at t he individual/tissue level of 1231

organization, were individual XX fish developed into phenotypic males. This was observed 1232

across multiple levels of organization including molecular (vitellogenin), tissue (external SSCs 1233

and pathology), and organ (gonad phenot ype) all tethered back to the molecular initiating event, 1234

i.e ., trenbolone binding and activating the AR. A comprehensive report including graphs and 1235

tables of the data can be found in the Appendix H: MMT Summary Reports on pp. 95

- 112. 1236

1237

4.3 Results with Steriodogenesis Inhibitor 1238

1239

4.3.1 Prochloraz (US EPA/ MED) 1240

1241

Prochloraz is a fungicide in which its efficacy is based on inhibition of sterol biosynthesis in 1242

target organisms thereby limiting target organisms ability to synthesize cell walls. Because of 1243

th e conserved nature of the pathways involved, prochloraz also inhibits CYP19 aromatase in fish 1244

which catalyzes the conversion of testosterone to 17 β

- estradiol both in the ovary and the brain 1245

(Hinfray et al., 2006; Monod et al., 1993; Skolness et al., 2011)

. In addition, there is evidence 1246

that prochloraz acts as an AR antagonist. In the 21

- day fathead minnow reproductive assay, 1247

prochloraz produces a suite of biological effects characteristic of an aromatase inhibitor and an 1248

AR antagonist including alteratio ns in various steroid levels, reduced vitellogenin in females, and 1249

lower cumulative fecundity at 0.1 mg/L prochloraz (Ankley et al., 2005)

. The MMT completed 1250

by EPA

- MED testing the effects of prochloraz was done with the full research MMT protocol

. 1251

1252

The L OEC for fecundity was 41 μ g/L in F 0 (the highest concentration tested); 25 μ g/L in F 2 , and 1253

no LOEC was determined at the highest remaining concentration (25 μ g/L) in F 1 . Comparing the 1254

mean fecundity in the 25 μ g/L treatments of F 1 versus F 2 , the means wer e very close: ~4 eggs per 1255

pair

- day in F 1 and ~6 eggs per pair

- day in F 2 . However, only the 25 μ g/L treatment in F 2 was 1256

significant because the difference between the exposed group and the appropriate control was 1257

different: ~10 eggs in F 1 and ~20 in F 2 as a result of lower than normal fecundity in the F 1 1258

control breeding pairs. In fact, three of six b reeding pairs in F 1 did not spawn. It is anticipated 1259

that the increased replicate structure of the medaka reproduction test (MRT), proposed as the 1260DRAFT

MMT ISR 34 M a y 28

, 2013 Tier 2 fish test (see section 5) , would mitigate the reduction in statistical power caused by the 1261

random distribution of non

- spawning b reeding pairs throughout test. 1262

1263

The LOECs across the entire prochloraz MMT of the AOP descriptive endpoints ranged from 5 1264

to 41 μ g/L, affecting both XY and XX fish. Prochloraz affected growth in relatively low 1265

concentrations but the decreases observed in the subadult samples were 1) not very large in 1266

absolute terms, and 2) were not present in the adults. In fact, adults in F 0 we re actually larger 1267

than controls after exposure to prochloraz. In addition, there were decreased copies of the 1268

vitellogenin gene in XX fish, which can be plausibly linked to the inhibition of aromatase, 1269

resulting in a decrease in circulating 17 β

- estrogen that in the end lowered hepatic vitellogenin 1270

expression. Lastly, there was a modest reduction of anal fin papillae in F 1 subadult XY fish, but 1271

this was not observed in F 2 and even more interesting, it was not observed in adults of all three 1272

generations. 1273

1274

Many of the pathologies observed in prochloraz MMT were not present consistently through 1275

generations or between life stages. Therefore, interpretation of the histopathology data was not 1276

as straightforward as some of the other MMTs. However, some generali ties can be made. While 1277

prochloraz at the concentrations tested did not seem to have much of an impact on the tissues and 1278

organs of XY fish, it did seem to cause EDC

- related pathologies in the ovaries of XX fish ( i.e

., 1279

decreased yolk and follicular hyperp lasia or hypertrophy). As more information is gathered 1280

about pathologies associated with exposure to steriodogenesis inhibitors, a clearer picture of the 1281

patterns of pathology may appear that would allow more comprehensive interpretation of 1282

histopathology datasets like the one generated in the prochloraz MMT. 1283

1284

Regardless, taken together, the MMT protocol was able to detect effects of prochloraz exposure 1285

on the reproduction, the population level parameter. In addition, the MMT provided information 1286

regardi ng the presumptive AOP that, while not as conclusive as say an ER agonist, does 1287

contribute to a WOE approach that would describe prochloraz as a steriodogenesis i nhibitor . A 1288

comprehensive report including graphs and tables of the data can be found in the Appendix H: 1289

MMT Summary Reports on pp . 113

- 133. 1290

1291

4.4 Results with AR Antagonist 1292

1293

4.4.1 Vinclozolin ( US EPA/ MED ) 1294

1295

Vinclozolin and its degradants (M1 and M2) are anti

- androgens that, via primarily through 1296

hydrolysis, also persist in the environment (the half

- lives of M1 and M2 are over 180 days) 1297

(Bayley et al., 2003; Makynen et al., 2000) . Vinclozolin is a dicarboximi de fungicide that is 1298

commonly used on fruits, vegetables, ornamental plants, and turf grasses, and therefore is an 1299

environmentally relevant EDC. The presumptive AOP initiated by vinclozolin is binding with 1300

the androgen receptor, but not initiating the tra nscription of androgen

- dependent genes 1301

(Martinovic et al., 2008)

. 1302

1303

Vinclozolin is readily hydrolyzed in the exposure water to M1 and M2 (Kelce et al., 1994; Wong 1304

et al., 1995) which are more potent anti

- androgens. Because of this, the characteristics of the 1305DRAFT

MMT ISR 35 M a y 28

, 2013 exposure system delivering toxicant to the aquaria ( i.e., flow rates, residence time of toxicant, 1306

etc .) can quantitatively change the exposure concentrations of the metabol ites. Therefore, it 1307

might be difficult to compare different aquatic bioassays with vinclozolin, especially if they have 1308

different exposure parameters. 1309

1310

In F 0 , fecundity decreased in the 253 μg/L treatment starting in the second week of the 1311

reproduction as sessment which is the third week after the start of exposure to vinclozolin. The 1312

mean for the entire three week reproduction assessment was just 7 eggs spawned per day in 253 1313

μg/L, compared to 24 spawned eggs per day in controls. Statistically significan t reduc tions in 1314

the numbers of spawned eggs were also seen in all the other treatment groups (17, 33, 70, and 1315

136 μg/L) in the last week of the assessment, but these reduction s w ere not great in absolute 1316

terms: controls at 24 eggs per day and all the treat ments at approximately 15 spawned eggs per 1317

day. For comparison, in a 21

- day test measuring reproduction in fathead minnows, vinclozolin 1318

reduced cumulative egg production by about a third at 60 μg/L

. 1319

1320

In F 1 , there w ere no effects of vinclozolin exposure at the concentrations tested. The mean 1321

fecundities observed were 25, 26, 20, 29 (only 1 pair), and 26 in the controls, 17, 33, 70, and 136 1322

μg/L, respectively. In addition, there was almost no reproduction in the 253 μg/L treatment ;; 1323

however, there was only a single breeding pair in this treatment. 1324

1325

There was not an abundance of evidence linking vinclozolin to its AOP as an anti

- androgen. The 1326

only effect on vitellogenin gene expression was increased copy number in XY subadults exposed 1327

to 136 μg/L (th e highest concentration present) in F 2 only. In this group, very high 1328

measurements were made in a few individuals accounting for the statistical significance. 1329

Possibly linked to the AOP, there was a consistent decrease in anal fin papillae in XY subadult s 1330

with a LOEC of 17 μg/L in F 1 and 33 μg/L in F 2 . In these treatments, the SSC was reduced by a 1331

25% and 40%, respectively. Presumably, vinclozolin exposure caused a developmental delay, as 1332

the SSCs were similar to control values in adults in both F 1 and F 2

. 1333

1334

While growth in subadults was not affected by vinclozolin, there was increased weight and 1335

length in some of the adult sampling groups but not all. In F 0 , XY fish were heavier, 275 mg in 1336

controls versus 350 mg at 33 μg/L ;; however, this did not increa se with higher concentrations of 1337

vinclozolin. However, XY adult fish were not affected in F 1 . In addition, XX adult fish were 1338

larger (weight) in both F 0 and F 1

. 1339

1340

There were few pathologies, fewer that were consistent across generations, and those observe d 1341

were by

- and

- large of low severity. The most prominent pathology was tubular epithelial 1342

eosinophilia of F 1 subadult kidneys of XY fish which had a LOEC of 17 μg/L and a moderate (3) 1343

severity at the same concentration. In F 2 , the same pathology at the sa me severity was present at 1344

the LOEC of 70 μg/L . However, there was no evidence of vinclozolin

- induced changes to the 1345

gonad phenotype in the entire MMT. 1346

1347

It needs to be noted that there were four F 1 , XY fish in the 33 μg/L treatment (the highest 1348

treatmen t with XY fish available for histology) that had normal ovaries, no anal fin papillae, and 1349

high vitellogenin levels (equal to normal XX levels). In addition, there were three F 2 XY fish in 1350

the 136 μg/L treatment (again the highest treatment with XY fish av ailable for histology) also 1351DRAFT

MMT ISR 36 M a y 28

, 2013 had the same phenotypes

, i.e

., normal ovaries, no anal fin papillae, and high vitellogenin levels. 1352

There are two explanations for these observations. First, DNA contamination into XX samples 1353

from XY fish during the fin clip/DM Y assessment procedure would result in this very pattern. 1354

Second, the data could be appropriate and simply reflect a biological response to vinclozolin 1355

exposure ; h owever, this seems unlikely because certain aspects of the complete dataset do not 1356

seem to f

ollow the expect ed pattern from an EDC with a strong effect on the gonad. For 1357

instance, there were no instances of XY fish with intersex gonads in the a ffected treatments or 1358

the lower treatments. Additionally, there was not a suite of pathologies in the gonad or liver that 1359

would be easily related to an EDC exposure. 1360

1361

While our interpretation of the data points to contamination, it is impossible to make a definitive 1362

statement without a re

- assessment of DMY. This can be done on the fixed, paraffin

- embedded 1363

tissue remaining after histology, but we have been unable to successful ly extract DNA of 1364

sufficient quality to perform the DMY assessment on these samples. We suspect th at the picric 1365

acid in the Bouin’s fixative interfered with DNA extraction . A ppendix H: MMT Summary 1366

Reports on pp. 134

- 151. 1367

1368

4.5 Interlaboratory Evaluation of the MMT protocol

- Vinclozolin 1369

1370

4.5.1 Background 1371

1372

An important aspect of the validation of a Tier 2 assay within the EDSCP is demonstrating the 1373

reliability or reproducibilit y of the protocol both within and between individual laboratories. To 1374

that end, the EPA Office of Science Coordination and Policy (OSCP), via contract, initiated an 1375

interlaboratory evaluation of a proposed MMT protocol that involved three distinct laborat ories 1376

implementing the protocol with vinclozolin as the test agent. These studies allow the evaluation 1377

of the comparability of test responses across multiple laboratories and are necessary to fully 1378

evaluate the practical transferability of the protocol an d quantitative reproducibility of the 1379

method. Three laboratories perform ed the interlaboratory studies and are termed Lab 1, 2, or 3. 1380

1381

Vinclozolin, an AR antagonist, ha s tested positive in the EDSP Tier 1 fish assay and would 1382

therefore be a candidate fo r EDSP Tier 2 testing. It was also tested in a MMT performed by 1383

EPA/MED ( see 4.4.1) which provides a fourth independent assessment of the reproducibility of 1384

the protocol. Vinclozolin does pose challenges as a test agent in a validation exercise. It read ily 1385

hydrolyzes in aqueous solution, producing several intermediates which are also potentially active 1386

EDCs (especially M1 and M2, (Kelce et al., 1994; Wong et al., 1995) ) therefore the total 1387

endocrine effect may combine the effects of vinclozolin and its d egradates. Adding to the 1388

complexity, the relative proportion of vinclozolin and its degradates is time

- and pH

- dependent. 1389

Thus, the residence time of the toxicant water in the dilution and delivery apparatus as well as 1390

the exposure aquaria determines the relative proportion of the parent chemical and its degradates. 1391

Another challenge posed by vinclozolin is the expected outcomes of an AR antagonist are not as 1392

well defined as other AOPs like ER or AR agonism. 1393

1394

A comprehensive report including graphs and tables of the data as well as a summary report 1395

provided by the lead contractor can be found in the Appendix C: Vinclozolin Interlaboratory 1396DRAFT

MMT ISR 37 M a y 28

, 2013 Report. Excerpts of the interlaboratory summary report authored by the l ead contractor are used 1397

below and are indicated by the use of italics . Where appropriate an EPA perspective is also 1398

included. 1399

1400

1401

4.5.2 Protocol 1402

1403

A timeline for the vinclozolin interlaboratory study is illustrated in Figure 4

- 1

. The test spans 1404

three generations , starting with adults in F 0 (minimally 350 mg for females and 250 mg for 1405

males), going through F 1 from fertilization to 14 wpf, and finally ending the study at 8 wpf of F 2

. 1406

Fish are sampled as adults of F 1 and as subadults (9 wpf) of F 1 and F 2 . Each endpoint is 1407

determined in the same manner as previously described for the MMT. The activity through the 1408

cours e of the test is summarized below

. 1409

1410

1411

Figure 4

- 1. T imeline for the v inclozolin interlab oratory study

. Three generations (F0, F1, and F2) are 1412

represented by horizontal rows; within a row each small block represents the age of the fish in weeks post 1413

fertilization (wpf). The total number of weeks of the test is indicated on the bottom of the figure. 1414

1415

1416

1417

1418

1419

1420

1421

1422

1423

Study Weeks 1

- 4: Unlike the initial research MMT protocol described above, fecundity and 1424

fertility were not measured in F 0 in this later version of the MMT

. Consequently, the age of the 1425

breeding adults for F 0 was not as tightly specified as it as for F 1 and F 2

. The specification 1426

follows: The first generation (F 0 ) adult breeding pairs (10 to 15 week post fertilization; females 1427

> 300 mg; males > 250 mg) are exposed for four weeks during whi ch time developing oocytes 1428

and sperm accumulate the test chemical, associated metabolites, and possible parental factors as 1429

defined by the relative treatment levels. The breeding pairs are placed in an exposure system 1430

that has six treatment levels includi ng controls with six replicate tanks per treatment level on 1431

Study Day 1. Each replicate tank houses a single breeding fish pair for a test total of 36 breeding 1432

pairs (6 treatments with 6 replicates per treatment). Study Week 4: On Study Day 22, eggs are 1433

removed from both the tanks and the females to guarantee an accurate collection the next day. 1434

The following day (Study Day 23), eggs are : a) collected and pooled from each treatment, b) 1435

systematically distributed to 6 incubators with a maximum of 20 eggs per incubator, and c) 1436

incubators are placed in one of two “incubator tanks” set up for each treatment. On Study Day 1437

24, the process is repeated using the second incubator tank per treatment. 1438

Study Weeks 5

- 6: As eggs hatch on each day, hatchlings are p ooled for each treatment and 1439

systematically distributed to each of the six replicate tanks within the specific treatment. The 1440

appropriate data are collected to calculate the hatching success of each incubator and each 1441 Timeline Schematic

F0 1 2 3 4 Start with 10 to 15 wk old fish

Female wt = > 350 mg; male = >250 mg

F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14

F2 1 2 3 4 5 6 7 8

Study Wk 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24DRAFT

MMT ISR 38 M a y 28

, 2013 collection day. On Study Day 29, the F 0 breeding pairs are sacrificed. On Study Day 32 and 33, 1442

the aeration (agitation) of the eggs from the 1 st and 2 nd days of egg collection, respectively, is 1443

turned off. 1444

Study Week 7: On Study Day 46, the number of juvenile fish is culled to 10 per replicate tank 1445

for a total of 60 per treatment. 1446

Study Weeks 8

- 10: The exposure continues with the 10 fish per replicate as they develop into 1447

sub

- adults. Study Week 11: On Study Day 71, a small sample is taken from the caudal fin of 1448

each fish to determine the genotypic sex of the individual. This information is used to establish 1449

female

- male breeding pairs. On Study Days 73 and 74 after the genotypic sex of each fish is 1450

determined, a single breeding pair per replicate is randomly established while the 8 (nominal) 1451

remaining fish per replicate are sampled for the various endpoints outlined in Table 1, sub

- adults. 1452

Study Weeks 12

- 13: The exposure continues as the sub

- adult breeding pairs develop into 1453

adults. 1454

Study Weeks 14

- 17: If the test was started on a Monday then on Tuesday through Friday of 1455

each week (Study Days 93

- 96, 100

- 103, 107

- 110, and 114

- 117), spawned eggs are counted and 1456

assessed for fertility. On Monday of each week (Study Days 92, 99, 106, and 113), all eggs, 1457

either held by the female or deposited in the tank are removed to allow for accurate counting 1458

starting the next day. 1459

Study Week 17 (repeat of Study Week 4): On Study Day 113, eggs are removed from both the 1460

tanks and the females to guarantee an accurate collection the next day. The following day (Study 1461

Day 114), eggs are; a) collected and pooled from each treatment, b) systematically distributed to 1462

6 incubators with a maximum of 20 eggs per incubato r, and c) incubators are placed in one of 1463

two incubator aquaria set up for each treatment. On Study Day 115, the process is repeated 1464

using the second incubator tank per treatment. Study Week 18: On Study Day 120, the F 1 1465

breeding pairs are terminated and analyzed for the endpoints in Table 1, adults. 1466

Study Weeks 18

- 19: As eggs hatch on each day, hatchlings are pooled for each treatment and 1467

systematically distributed to each of the six replicate tanks within the specific treatment. The 1468

appropriate data are collected to calculate the hatching success of each incubator and each 1469

collection day. 1470

Study Week 20: On Study Day 137, the number of juvenile fish is culled to 10 per replicate 1471

tank for a total of 60 per treatment. 1472

Study Weeks 21

- 23: The 10 fish per replicate are maintained and become sub

- adults. 1473

Study Week 24: On Study Days 164 and 165, all the fish are sampled for the various endpoints 1474

in Table 1, sub

- adults. 1475

1476

4.5.3 Chemistry Data 1477

1478

Below is summary of the measured vinclozolin concentrations for each of the laboratories. 1479

Vinclozolin degradate concentrations were not measured. Two of the laboratories maintained 1480

parent vinclozolin above the nominal concentrations and fairly close to each other; however, the 1481

measured concentrations during the La b 1 MMT were considerably lower than nominal, and 1482

more importantly, the concentrations were much lower (~50%) than the other two MMTs. 1483

Therefore, it is more appropriate to compare Lab 1 results to one treatment level lower in the Lab 1484

2 and 3 MMTs. In add ition, the measured concentrations of vinclozolin in all treatments during 1485

the Lab 2 MMT more than doubled around day 100 of the test. 1486

1487DRAFT

MMT ISR 39 M a y 28

, 2013 1488

1489

1490

1491

1492

1493

Table 4

- 5. Dosing summary for the interlab oratory MMTs. 1494

1495

Nominal Units Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

Lab 1 μg/L 0 8.0 19 48 120 300

Lab 2 μg /L 0 8.0 19 48 120 300

Lab 3 μg /L 0 8.0 19 48 120 300

Mean Exposure Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

Lab 1 μg /L <MQL a 4.85 12.6 30.9 81.6 197

Lab 2 μg /L <MQL b 11.6 25.2 62.0 155 360

Lab 3 μg /L <MQL c 8.9 21.7 52.3 133 333

% CV Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

Lab 1 NA 0.88 1.70 6.21 10.1 29.2

Lab 2 NA 49.9 36.5 30.8 32.8 24.7

Lab 3 NA 14.6 10.1 10.9 10.4 8.5

% of Nominal Level 0 Level 1 Level 2 Level 3 Level 4 Level 5

Lab 1 NA 61 66 64 68 66

Lab 2 NA 145 133 129 129 120

Lab 3 NA 111 114 109 111 11 1

a Minimum Quantif iable Limit (MQL) = 2.28 μg //L 1496

b Minimum Quantifiable Limit (MQL) = 1.14 μg //L 1497

c Minimum Quantifiable Limit (MQL) = 5.0 μg //L 1498

1499

1500

1501

4.5.4 Control Data Supporting Validation 1502

1503

Comparison of control data from each of the laboratory studies provides a measure of the 1504

natural variation for each of the variables and is summarized in Table 4

- 6. This analysis 1505

indicated that for many endpoints, the control response was not consistent between laboratories. 1506

The median responses from F 1 reproductive measurements, F 1 , F 2 length and weight 1507

measurements were all significantly different (p < 0.01) between laboratories. Closer inspection 1508

of the results suggest some of the significant inter

- lab variability can be attributed to the 1509

relatively small size at termination of the Lab 2 F1 and F 2 fish. The small size of Lab 2 F 1 1510

females appears to h ave contributed to the high inter

- lab variability in fecundity. Normalizing 1511

F

1 fecundity for the body mass of the female ( F 1 total eggs / day / g day 84 female weight) 1512

eliminated the significant difference (p=0.463). Two endpoints that showed greater cons istency, 1513

which were not statistically different between labs was F 1 and F

2 anal fin processes and hepatic 1514

expression of VTG

- 1. However, it should be noted that successful measurement of anal fin 1515DRAFT

MMT ISR 40 M a y 28

, 2013 processes only occurred for two labs in both the F 1 and F

2 ge neration, limiting inter

- lab 1516

comparisons. 1517

1518

1519

1520

Table 4

- 6. Median Control Response by Chemical and the Kruskal

- Wallis Test 1521

1522

Variable Lab 1 Lab 2 Lab 3 Kruskal

- Wallis p

- value

F 1 Total Eggs/Day 27.1 10.02 34.71 0.005

F 1 Total Eggs/Day

/ day 84 female weight 170.4 121.5 169.4 0.463

F 1 Viable Eggs/Day 21.73 9.68 33.67 0.005

F 1 Proportion Viable 0.757 0.9614 0.97086 0.044

F 1

- Female Day 80/84 Weight (g) 0.161 0.089 0.207 0.002

F 1 Female Day 80/84 Length (mm)

-- 19.799 26.625 0.01

F 1 Fem ale Day 84 Total Processes 7.8 0

--

F 1

- Female Day 80/84 copies VTG/ng RNA 22289 1195 17470 0.076

F 1 Male Day 80/84 Weight (g) 0.127 0.107 0.199 0.001

F 1 Male Day 80/84 Length (mm)

-- 21.78 26.01 0.004

F 1 Male Day 84 Total Processes 26.02 14.73

-- 0.423

F 1 Male Day 80/84 copies VTG/ng RNA 2.25 27 1850 0.075

F 1 Female Day 119 Weight (g)

-- 0.192 0.419 0.006

F 1 Female Day 119 Length (mm)

-- 27.08 32.4 0.006

F 1 Fe ale Day 119 Total Processes 0 0

--

F 1 Male Day 119 Weight (g)

-- 0.227 0.389 0.004

F 1 Male Day 119 Length (mm)

-- 29.38 32.33 0.005

F 1 Male Day 119 Total Processes 73.5 53.33

-- 0.025

F 2 Female Termination Weight (g) 0.211 0.034 0.226 0.002

F 2 Female Termination Length (mm)

-- 15.4 27.3 0.004

F 2 Female Termination Total Processes 10.1

-- 1.43 0.40

F 2 Female Termination copies VTG/ng RNA 23650 0.5 43273 0.103

F 2 Male Termination Weight (g) 0.193 0.028 0.222 0.002

F 2 Male Termination Length (mm)

-- 14.7 28.1 0.004

F 2 Male Termination Total Processes 52.9

-- 59.1 1.0

F 2 Male Termination copies VTG/ng RNA 1.22 2.50 1.75 0.912

1523

1524

There are two issues that are plausible explanations for the control parameter variation between 1525

laboratories. First, as mentioned above, is the small size of F 1 and F 2 fish in the Lab 2 MMT. 1526

Certainly these fish were not developing like the fish in the other two labs or with what would be 1527

expected to be a normal growth rate. Because of this, all control parameters of the Lab 2 MMT 1528

fish would not be expected to meet mi nimum performance criteria. Also, most of the statistical 1529

differences between MMT control fish are a result of Lab 2 fish being so much different than 1530

Lab 1 and Lab 3 fish. Second, as mentioned in the interlab oratory summary report found in 1531

Appendix C: V inclozolin Interlab oratory Report, there was a high rate of unexplained genotype 1532

to phenotype mismatch not only in control fish but across all treatments. Given the prevalence 1533DRAFT

MMT ISR 41 M a y 28

, 2013 of the mismatches, and that both XX

- male , and XY

- female mismatches were observe d, points to 1534

technical problem in the determination of the genetic sex of the fish. This may have been due to 1535

problems with sample/label fidelity during tissue collection and processing. Errors in 1536

determining the genetic sex of individual fish would likely skew many of the control parameters, 1537

as all of them show some level of sexual dimorphism. 1538

1539

With respect to the issues identified above, EPA believes that the control data do indeed support 1540

the reproducibility of the protocol as long as genotypic sex data (DMY) are obtained and 1541

acceptable growth of control fish is achieved. At this time, these are presumed to be technical 1542

issues involving the appropriate rearing of the animals and sample handling procedures. 1543

However, the apparent flaws in the data from th e interlaboratory also further highlight the need 1544

for ongoing improvement in the detailed methods for accomplishing the protocol and specific 1545

training leading to more effective transfer of technical skills required to perform the MMT 1546

protocol successfully. 1547

1548

4.5.6 Summary of Lab 1 Vinclozolin MMT 1549

1550

In this MMT, vinclozolin did not lower the fecundity rate or the fertility (either the number of 1551

fertile eggs or percent fertile eggs) of the breeding pairs in any of the treatments of either F 0 or 1552

F 1 . The F 0 rep roductive assessment was not part of the original study plan, but during the course 1553

of the F 0 loading phase, fecundity data w ere collected for six consecutive days and fertility data 1554

w ere collected for only two consecutive days. In F 1 , both fecundity and fertility were assessed, 1555

according to the protocol, for 21 consecutive days. 1556

1557

The only affected endpoint that presumably would reflect an EDC exposure was SSC (anal fin 1558

papillae). XY fish, across both F 1 and F 2 and in both subadults and adults, had fewer papillae 1559

than controls. Because genetic sex of individual fish was unreliable, analysis of treatment effects 1560

on SSC had to be by comparisons to other phenotypic sex markers ( i.e., either the gonad 1561

phenotype or the secondary sex phenotype ), and to be completely accurate, these fish are referred 1562

to as “males” or “females”, instead of the preferred assignment of “XX” or “XY”. Vinclozolin

1563

treated subadult and adult male fish, as defined by the gonad or SSC phenotype , in both F 1 and 1564

F 2

, had fewer anal fin papillae than controls. It was not possible to observe changes in phenotypic 1565

females, as increases in the number of anal fin papillae would define an individual as a 1566

phenotypic male. However, in the phenotypic males , there was a decrease in the number of 1567

papillae in response to vinclozolin exposure with a LOEC of 31 μ g/L , and continuing in both the 1568

82 and 197 μ g/L treatments with similar effect magnitudes. The control males had a mean of 77 1569

papillae while in these three treatments (31, 82, and 197 μ g/L ), the mean number of papillae 1570

were 59, 53, and 52, respectively. In F 1 subadult males, vinclozolin at a concentr ation of 197 1571

μ g/L reduced the mean number of anal fin papillae from 32 as seen in controls to a mean of 20 1572

per fish. In F 2 , the LOEC was 5 μ g/L and the reduction of papillae consistently decreased in a 1573

dose

- dependent manner to the highest concentration (1 97 μ g/L ). For unknown reasons, the mean 1574

number of papillae in F 1 control males was much lower than seen in the male controls of F 2 (32 1575

versus 73). The mean in males for each of the vinclozolin exposed groups of F 1 was actually 1576

similar (or often lower) t o the corresponding treatment group of F 2 . Therefore, the LOEC and 1577

the apparent increased sensitivity of F 2 may have been a result of the further developed fish, as 1578DRAFT

MMT ISR 42 M a y 28

, 2013 measured by SSC, in F 2 as compared to the F 1 fish. A comprehensive report including graph s 1579

and tables of the data as well as a summary report can be found in the Appendix C: Vinclozolin 1580

Interlaboratory Report. 1581

1582

4.5.7 Summary of Lab 2 Vinclozolin MMT 1583

1584

In this MMT, vinclozolin did not lower the fecundity rate or the fertility (either the numb er of 1585

fertile eggs or percent fertile eggs) of the breeding pairs in any of the treatments of F 1 , where 1586

both fecundity and fertility were assessed, as per the protocol, for 21 consecutive days. The 1587

m ean fecundities observed were 10, 10, 8, 9, 7, and 7 in the controls, 12, 25, 62, 155, and 360 1588

μ g/L, respectively. In addition, the fertility was high across all the treatments. 1589

1590

In the other endpoints, vitellogenin gene expression was induced in XX fish of F1 only in the 1591

highest treatment tested (360 μ g/L) from a mean of 830 copies per ng of total RNA to 8910 1592

copies. Growth (weight) was increased by vinclozolin exposure in XX fish of both generations 1593

(F 1 and F 2 ) with a LOEC of 12 μ g/L (the lowest treatment), and in XY fish in F 1 only with a 1594

LOEC of 62 μ g/L. 1595

1596

4.5.8 Summary of Lab 3 Vinclozolin MMT 1597

1598

In the primary endpoint of the MMT, reproduction, no changes in fecundity or fertility were 1599

observed upon exposure to vinclozolin. In F 0 , fecundity and fertility was based upon counts of 1600

pooled eggs. Therefore th e box plots presented represent inter

- day variance of the entire 1601

treatment and not inter

- breeding pair variance. To avoid confusion, the individual data, daily 1602

counts of whole treatments, w ere n ot plotted on the graph for F 0 . While there was not a 1603

statis tically significant reduction in fecundity or fertility, there was a trend of lower fertility with 1604

vinclozolin exposure. In F 1 , there were no effects of vinclozolin exposure on either fecundity or 1605

fertility at the concentrations tested. The mean fecundit ies observed were 35, 28, 34, 33, 28, and 1606

37

, in the control, 9 μ g/L , 22 μ g/L , 52 μ g/L , 133 μ g/L , and 333 μ g/L treatments, respectively. 1607

The fertility was high across all the treatments ranging from 92% to 98%. 1608

1609

Of the other endpoints, anal fin papillae were reduced in XY fish in F 2 subadults only. 1610

Unfortunately, there was a mistake in sample handling with the F 1 subadult anal fins so that no 1611

counts of anal fin papillae were possible. Vinclozolin reduced the mean number of papillae in 1612

the XY subadult f ish of F 2 at all concentrations tested (9

– 333 μ g/L ). The mean number of 1613

papillae was 57 in the control and was reduced to a mean of 39, 42, 40, 33 and 19 papillae in the 1614

9 μ g/L , 22 μ g/L , 52 μ g/L , 133 μ g/L , and 333 μ g/L treatments, respectively. 1615

1616

4.5.9 EPA Perspective on the Interlaboratory Results 1617

1618

Overall the interlaboratory was a success insomuch as all three labs did not see decreased 1619

reproduction at the concentrations of vinclozolin tested. This is also unfortunate for the very 1620

same reason. Other endpoints were not affected by vinclozolin with the exception of SSC in 1621

which was, when available for assessment, negatively responsive to vinclozolin exposure, and in 1622DRAFT

MMT ISR 43 M a y 28

, 2013 the Lab 2 MMT only, sporadic increases in weight and vitellogenin were observed. As a note, 1623

SSC was also decreased in the vinclozolin MMT performed by EPA/MED. So while few 1624

significant effects were observed in the three interlaboratory MMTs, there was consistency in the 1625

observation of effects on the SSC. In addition, the control data sugg est the protocol is 1626

reproducible even though most of the measurements had statistical differences between the three 1627

labs. Recall, one lab had unusually poor control growth that seemed to cause developmental 1628

delay in control fish, which, in turn resulted i n poor control reproduction. When those data are 1629

removed, there was considerable consistency between the data of the other two laboratories. 1630

1631

Unfortunately, the previously discussed issues with the interlaboratory data cannot simply be 1632

ignored, and they h ighlight that this is a relatively complicated procedure that even with 1633

experience is not a trivial undertaking. Many of the directives within the protocol are derived 1634

from extensive experience at EPA/MED lab, and without a priori knowledge, should be 1635

fol lowed without deviation. Otherwise, there is a relatively high risk of generating poor quality 1636

data with high variances. The consequence is that it is difficult to discern treatment

- related 1637

effects with all the endpoints, but especially on reproduction. Low reproduction in controls 1638

seriously compromises the statistical power of the MMT to detect decreases in fecundity or 1639

fertility. In addition, knowing the genetic sex (DMY) of each fish is an incredibly powerful tool 1640

for analysis of the data. To maximize the statistical power for each endpoint requires this 1641

information. Proficiency, both in DNA preparation and in the determination of the presence or 1642

absence of DMY, must be achieved prior to implementation of the MMT protocol. 1643

1644

The suite of expertise req uired to perform a MMT is broad, and there are many nuances that need 1645

to be recognized by performing labs. The limitations in the interlaboratory trial data are not 1646

indicative of low protocol reproducibility, but rather to an inadequate transfer of the te chnical 1647

details necessary to successfully execute the MMT. 1648

1649

5 OPTIMIZATION OF THE MMT AND THE PROPOSED MEDAKA 1650

REPRODUCTION TEST (MRT) PROTOCOL 1651

1652

5.1 Primary MMT Protocol Changes for development of MRT Protocol 1653

1654

The outcomes of the various MMT protocols with several different chemicals performed at the 1655

EPA MED lab and by the NIES and CERI laboratories in Japan, have provided enough 1656

information to provide guidance for the development of a medaka reproduction test ( MRT) for 1657

use as a fish test in Tier 2 of the EDSP. Two major changes are proposed, i.e., an increase in the 1658

number of replicates per treatment for evaluating effects on reproduction, and terminating the 1659

test after the embryos hatch in F 2 . Other proposed ch anges include minimizing the collection of 1660

endpoint data from F 0 , and evaluating pathology in only the F 1 adults sampled after the 1661

assessment of reproduction. Figure 5

- 1 presents the proposed timeline and endpoint data for the 1662

MRT. The test conditions for the proposed MRT protocol are presented in Table 5

- 1. Endpoint 1663

measurements are essentially identical to those employed in the full MMT except that the sagittal 1664

sectioning plane is recommended for the pathology assessment. Comparisons of various aspects 1665

of the research MMT and the proposed MRT are presented Table 5

- 2 . The rationale for the 1666

recommended changes in the MMT for the proposed Tier 2 fish test of the EDSP, are presented 1667DRAFT

MMT ISR 44 M a y 28

, 2013 in the following sections . Detailed protocols of the Proposed Medaka Reproduct ion Test are 1668

presented in Appendix E: MRT Protocol. 1669

1670

1671

Figure 5

- 1. Proposed Medaka Reproduction Test (MRT) for Tier 2 The timeline and endpoints in the 1672

MRT . The duration of the in

- life exposure is 19 weeks and spans 1 full generation (F 1 ). The F 0 generatio n serves as 1673

a loading phase for the F 1 embryos. The test is terminated after the F 2 embryos hatch. 1674

1675

Exposure and Endpoint Timeline

Gen F 0 1 2 3 4

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

F 2 1 2

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Lifestage Key Embryo Larvae Juvenile Subadult Adult

Endpoints

Fecundity F 1 Experimental design: 7 blocks

where 1 block=12 replicate

aquaria for fecundity and fertility

measurements and 6 replicate

aquaria for the embryo thru

subadult endpoints

5 blocks for chemical treatment s

2 blocks for control treatment s Fertility F 1

Hatch F 1 F 2

Survival F 1 F 1 F 1

Growth F 1 F 1

Vitellogenin F 1

Secondary sex F 1 F 1

Histopathology F 1

Study Week 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

1676

1677

1678

1679

Table 5

- 1 . Test Conditions for the Proposed MRT 1680

1681

Recommended species Medaka ( Oryzias latipes )

Test type Continuous flow

- through

Water temperature 2 5.5 ± 1 o C

Illumination quality Fluorescent bulbs (wide spectrum and ~150 lumens )

Photoperiod 16 h light, 8 h dark

Loa ding rate F 0 - 2 fish/replicate; F 1 and F 2 - initiated with maximum of 20 larvae/replicate eventually

culled to 2 fish/replicate

Test replicate chamber size 18x9x15 cm

Test replicate solution volume ~1.8 liters

Volume exchanges of test solutions 16 volume replacements/day (20 ml/min flow).

Age of test organisms at initiation F 0 - 11 weeks post

- fertilization, F 1 and F 2 continuously from fertilization

No. of organisms per replicate F 0 - 2 adults; F 1 and F 2 - initiated with maximum of 20 larval fish eventually culled to 2

adults

No. of treatments 6 ( 5 toxicant treatments plus 1 control, and, a solvent control if needed )

No. replicates per treatment 6 per treatment for each toxicant treatment; 12 for control treatment; replication structure

doubled for fecundity assessment

No. of organisms per test

concentration F 0

- 24 for each test concentration and 48 for controls; F 1 and F 2 - start with a maximum

of 120 and eventually cull to 48 for fecundity assessment.

Feeding regime Fish were fed 24

- hour old brine shrimp ( Artemia spp.) nauplii at the following rates:

Time

(post

Brine Shrimp

(mg dry Time

(post

Brine Shrimp

(mg dry weight/fish/day)DRAFT

MMT ISR 45 M a y 28

, 2013 hatch) weight/fish/day) hatch)

Day 1 0.5 Day 12 4.2

Day 2 0.5 Day 13 4.5

Day 3 0.6 Day 14 4.8

Day 4 0.7 Day 15 5.2

Day 5 1.0 Day 16

- 19 5.6

Day 6 1.3 Week 4 7.7

Day 7 1.7 Week 5 9.0

Day 8 2.2 Week 6 11.0

Day 9 2.8 Week 7 13.5

Day 10 3.5 Week 8

- 15 22.5

Day 11 4.2

Aeration None unless DO reaches <60% saturation

Dilution water Filtered (5 micron)

, sterilized fresh surface water, or appropriately conditioned well

water.

Exposure period Trans

- generational test: F 0 from 11

- 15 wpf, F 1 and F 2 from 0

- 15 wpf

Primary endpoints Survival: F 1 from 1

- 3 wpf and from 3

- 15 wpf

Growth : length, weight, (F 1 a t 9 wpf and 15 wpf

Hatch: F 1 and F 2 only

Anal Fin Papillae: F 1 assessed a t 9 wpf and 15 wpf

Fecundity: F 1 assessed daily for 21 days, 12

- 14 wpf

Fertility: F 1 assessed daily for 21 days, 12

- 14 wpf

Vitellogenin: F 1 only at 9wpf

Optional endpoints Time to first spawn for F 1 generation.

Test acceptability Dissolved oxygen > 60% of saturation; mean temperature of 25.5 ± 1 o C ;  70% survival

in control treatments of each generation; successful reproduc tion in at least 6 5% of

control females with mean daily fecundity in the control treatment of at least 15 eggs.

1682

1683

1684

1685

Table 5

- 2 Comparison of the Research MMT to the Proposed Tier 2 Fish Reproduction Protocol 1686

1687

Research MMT Proposed MRT Rationale

Replication

structure 6 replicates/trtmnt 12 replicates/trtmnt

24 control replicates Statistical power for

reproductive analysis

Generations 2 1⁄2 (F 0 , F 1 , partial F 2 ) 1 1⁄2 (F 0 , F 1 , F 2 hatch only) Reduce test time, chemical

amount, cost, risk of test

failure

F 0 for dose

- level

evaluation and to expose

developing gametes with

test chemical

F 0 endpoints Reproduction, Growth,

Survival, Vtg, SSC,

Pathology Reproduction

– qualitative

only Adequate dose

- level

evaluation

Survival Adequate dose

- level

evaluation

No other endpoints

measured F 0 < sensitive than F 1 and

F 2

F 2 endpoints Hatch, Growth, Survival,

Vtg, SSC, Reproduction,

Pathology Hatch only Ensure adequate time for

deposition of test agent to

egg.DRAFT

MMT ISR 46 M a y 28

, 2013 No other endpoints

measured Substantial and consistent

sensitivity differences

between F 1 and F 2 were

not observed for the

chemicals tested

F 1 endpoints Hatch, Growth, Survival

Subadult: Vtg, SSC,

pathology Hatch, Growth, Survival

Subadult: Vtg, SSC Increased replication

allows evaluation of

biomarker endpoints with

good statistical power

Adult: Reproduction,

Pathology Adult: Reproduction,

Pathology Increased replication

increases statistical power

for reproduction and

pathology evaluation

Adult: Reproduction Three weeks continuous

(21 consecutive days) vs

. 5

weeks (T

, W

, T h, FF only).

Better for statistics and test

duration

Temperature 26 25.5 Maximize development

rate; minimize prevalence

of XX males

Pathology F 0 adult

F 1 , F 2 subadult and adult F 1 adult Increased power for

pathology analysis,

pathology correlated with

pair reproduction

outcomes

1688

1689

5.1.1 Increased Replication for the Assessment of Treatment

- Effects on Reproduction 1690

1691

The reproductive endpoints of fecundity and fertility are important endpoi nts for ecotoxicology 1692

assessments, due to their implicit direct relationship to effects at the population

- level. To assess 1693

treatment effects on adult reproduction, the full MMT protocol employed 6 replicate aquaria for 1694

each test treatment, including the co ntrols. Fecundity data from the full MMTs had relatively 1695

high variance. Thus, with only 6 breeding replicates per treatment, the statistical power for 1696

detecting significant changes in fecundity was quite low. Some of the modifications of the 1697

protocol were implemented to ostensibly increase the power of the protocol either by increasing 1698

baseline fecundity or by reducing the variance in egg production. Such modifications included 1699

increasing the test temperature, increasing growth with feeding rates or by incr easing the 1700

development time before assessing reproduction. These modifications were only marginally 1701

successful. 1702

1703

After running a number of MMTs and evaluating the reproduction data, it became clear that the 1704

statistical power for detecting significant effe cts on fecundity in the full MMT was quite low. 1705

The trends observed in the dose

- responses for fecundity suggested that fecundity was being 1706

affected at treatment levels lower than the statistical LOEC. In some cases, the fecundity had to 1707

be reduced to nearl y zero to be significantly different from the control fecundity values. It 1708DRAFT

MMT ISR 47 M a y 28

, 2013 appeared that the most reliable method to increase the statistical power of the test to detect 1709

treatment effects on reproduction was through increasing the number of breeding pairs. 1710

1711

To determine the number of replicates required a power analysis was performed using the 1712

reproductive performance of control fish from several of the MMTs run at the MED laboratory 1713

(Appendix I: Power Analysis). MMT fecundity data have characteristics tha t complicate the 1714

calculations of statistical power. For example, the data are not transformabl e to normally 1715

distributed data; they are measured repeatedly over t ime with several replicates in each 1716

treatment ; and days without reproduction ( i.e., where the n umber of spawned eggs is zero) are 1717

randomly distributed in the data set. Because of these characteristics, a numerical solution for 1718

power was calculated using Monte Carlo simulation methods. 1719

1720

In order to design the Monte Carlo simulation, various assumpti ons were made including that the 1721

between aquarium within treatment mean fecundity is normally distributed, the within pair 1722

fecundity follows a Poisson distribution (typical of count data), the control fecundity does not 1723

change during the reproductive asses sment, and finally the rate at which individual pairs produce 1724

zero eggs is constant. This last assumption is important because the experience of the MED 1725

laboratory has been that a certain percent of breeding pairs will not successfully spawn for 1726

unknown r easons, and this can be daily, weekly, or for the entirety of the assessment. 1727

1728

Using these assumptions and the control data from previous MMTs, the following parameters 1729

were calculated for the Monte Carlo simulation: the population mean = 21 eggs and the 1730

p opulation variance = 49. Also the rate at which the fecundity of a pair was zero can be 1731

separated into three categories; one day, one week (greater than one day but less than 8 days), 1732

and always (greater than 8 days). These were estimated to be 8/91 for one day, 5/91 for one 1733

week, and 3/91 for all days. 1734

1735

The data were simulated in the R programming language incorporating the assumptions above to 1736

generate a fecundity dataset of control plus 5 pre

- assigned reductions in fecundity ( i.e., 1737

treatments) of 20%, 30% 40%, 50% and 70% reduction. This process was repeated 1000 times 1738

for each pair and percent reduction combination. Next, all of the simulated data were analyzed 1739

by the repeated measures ANOVA of the StatCHARRMS program. Two diffe rent replicate 1740

structures were analyzed: 1) the number of replicates is equal in all treatments including the 1741

controls (balanced design), and 2) based upon statistical recommendations from the OECD 1742

(OECD, 2006) , the number of control replicates was double the number of replicates in each 1743

chemical treatment (2:1 design). The 2:1 design had substantially greater power and is the only 1744

analysis presented here. 1745

1746

Graphic results are illustrated in F igure 5

- 2. Note that a t 6 replicates (12 replicates in the 1747

contro l), there is a small but noticeable probability of not detecting a reduction of 50%, about a 1748

75% probability at detecting a reduction of 40%, less than 50% chance at detecting a reduction 1749

of 30%, and very little chance at detecting a reduction of 20%. At 12 replicates (24 replicates in 1750

the control) the probability of not detecting a reduction of 40% or greater is near zero, and there 1751

is probability of greater than 80% of detecting a reduction as low 30%. 1752

1753

1754DRAFT

MMT ISR 48 M a y 28

, 2013 1755

1756

Figure 5

- 2

. Monte Carlo simulation of the statistical power to detect proportionate reductions in medaka fecundity 1757

with different numbers of replicate breeding pairs. The experimental design represented is the 2:1 design where 1758

control replicates are double of the num ber of replicates in the chemical treatments. The power is the probability of 1759

detecting a treatment effects compared to the controls at an α

- level of 0.05. 1760

1761

During the discussions on revising the replicate structure of the MMT, consideration was given 1762

not only to the power analysis, but also to the possibility of mortality, especially in the control 1763

replicates. It has been the experience of MED researchers that a very small percentage of the 1764

adults, irrespective of treatment, may die, and in addition

, even with skilled technicians, there is 1765

a possibility of handling

- induced mortalities as well. To be conservative, 12 breeding pairs 1766

(replicates) in treatments and 24 control breeding pairs (replicates) was chosen to mitigate the 1767

consequences to statistical p ower. 1768

1769

Reproduction Results from Two Octylphenol MMTs: 6 vs. 12 R eplicate B reeding P airs 1770

To evaluate the effect of increasing the replication structure of the MMT, two tests (designated 1771

PTOP

- 02 and PTOP

- 03) were run at the EPA MED lab with the weak ER agon ist 4

- tert

1772

octylphenol. PTOP

- 02, which employed 1 breeding pair in each of 6 replicate aquaria per 1773

treatment, was started late in 2007. PTOP

- 03, which was started late in 2012, employed 12 1774

replicate aquaria per treatment, except for the control treatment w hich had 24 replicates. The 1775

chemical concentrations were nearly identical for the two tests (see Table 5

- 3). Reproductive 1776

assessments were similar except that for PTOP

- 02 fecundity and fertility were measured 4 days 1777

per week for 5 weeks and for PTOP

- 03 the measurements were taken for 21 consecutive days. 1778 0

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

1pr 2pr 3pr 4pr 5pr 6pr 7pr 8pr 9pr 10pr 11pr 12pr Power 

## Medaka Fecundity

2:1 Experimental Design

20% Reduction 2:1

30% Reduction 2:1

40% Reduction 2:1

50% Reduction 2:1

70% Reduction 2:1DRAFT

MMT ISR 49 M a y 28

, 2013 There were no treatment effects on F 0 reproduction in PTOP

- 02 (Appendix H: MMT Summary 1779

Reports on pp. 16

- 38) and reproduction endpoints were not measured in PTOP

- 03. 1780

1781

Table 5

- 3 Comparative Treatment Concen trations for two 4

- t

- Octylphenol MMTs 1782

1783

Treatment PTOP

- 02

Conc (μg/L)

and (S.D.) PTOP

- 03

Conc (μg/L)*

1 0 (0) 0

2 6 (0.6) 6

3 13 (1.5) 11

4 25 (2.6 23

5 51 (5.6) 49

6 102 (8.5) 98

*: S.D not calculated for this test yet 1784

1785

1786

Figure 5

- 3. Reproduction Results from 6

- and 12

- Replicate Test Protocols 1787

1788

1789

1790

The primary effect of octylphenol on reproduction is to reduce the fecundity and not the fertility 1791

of treated fish. Note also that the control means for fecundity are similar in F 1 and F 2 for both 1792 

## F1 Reproduction

F1 Reproduction F2 Reproduction

F2Reproduction 6 Reps

12 Reps LOEC LOEC

LOEC LOECDRAFT

MMT ISR 50 M a y 28

, 2013 tests and range from 1 7 to 21 eggs/pair/day. The LOEC for F 1 fecundity in the 6 rep design was 1793

102μg/L which reduced the fecundity to nearly 0. Because of this, there were insufficient 1794

survivors to populate that treatment in F 2 . The LOEC for F 2 fecundity in the same test was 1795

51 μg/L. There were no significant differences between the mean fecundity at any particular 1796

treatment level across generations ( i.e., F 1 control fecundity of 17 vs. F 2 control fecundity of 1797

19.5; or F 1 51μg/L fecundity of 14 vs. F 2 51μg/L fecundity of 8). 1798

1799

In the 12 replicate test, the LOEC for fecundity was 23μg/L for both the F 1 and F 2 generations, 1800

one treatment level lower than the LOEC for the 6 replicate test. Note also that the reduction in 1801

fecundity observed at the LOEC was only about 7.5 eggs/pair/day i n F 1 and 5 eggs/pair/day in 1802

F 2 . In summary, doubling the number of replicate breeding pairs increased the statistical power 1803

of the test to detect significant treatment effects on fecundity. 1804

1805

The 12 replicates per treatment design also has increased power d ue to the fact that there are two 1806

blocks of 12 replicates for the control treatment. There are a total of 84 replicate aquaria in the 1807

reproductive phase of the test; ((5 chemical treatments) x 12 replicates + (2 control blocks x 12 1808

replicates) = 84 ) . While this may seem excessive, this number of replicates is necessary due to 1809

the highly variable nature of fish reproduction. Furthermore, this design provides additional 1810

insurance for the baseline probability of having non

- spawning pairs within the test popula tion 1811

and for the inevitable loss of replicates due to technical accidents. The difference in the statistical 1812

power for fecundity in the MRT with 12 breeding replicates for each of the chemical treatments 1813

and 24 replicates in the controls is presented in Ta ble 5

- 4. Note that in the MMT a 40% 1814

reduction in fecundity will be detected with a probability of 66 %, and a 28% probability of 1815

detecting a 30% reduction in fecundity. In contrast, with the MRT the probability of detecting 1816

40% and 30% reductions in fecund ity are 99% and 83% respectively. 1817

1818

Table 5

- 4 Statistical Power Comparison for Fecundity in the MMT and the proposed MRT 1819

1820

Fecundity

Reduction

(%) MMT MRT

Balanced

design

6 replicates 2:1

design

12 replicates

20 0.17 0.40

30 0.28 0.83

40 0.66 0.99

50 0.83 1

70 0.91 1

1821

When considering the number of fish employed in the two different test designs, it is important 1822

to note that there are actually fewer fish used in the 12

- replicate MRT design. First, the initial 1823

phase of the protocol employs 6 replicate s per treatment. And, the initial number of embryos 1824

placed in incubators has been reduced from 30 to 20. Then, when starting the reproductive phase 1825

of the test at 9 wpf, 4 fish per replicate are used to set up two breeding pairs rather than the 2 fish 1826

for the single pair per replicate employed in the used in the 6

- replicate design of the MMT. The 1827

increase in statistical power afforded by the 12 replicate design is accomplished by reallocating 1828

fish from the 9 wpf sampling into the analysis of treatment effec ts on reproduction. The 1829

concomitant reduction in the statistical power of the non

- reproductive endpoints ( i.e ., 6 fish per 1830DRAFT

MMT ISR 51 M a y 28

, 2013 replicate in the MRT compared with 8 per replicate in the MMT) is relatively small. Specifically, 1831

Table 5

- 5 presents the statistical power of the MRT to detect changes in the reductions in the 1832

biomarker endpoints ( i.e ., wet weight, length, vitellogenin and SSC) in fish sampled at 9 wpf. 1833

Note that except for the female vitellogenin endpoint, the power to detect treatment effects in 1834

these endpoints in the 9 wpf samples is similar or higher than the power to detect effects in 1835

fecundity. Thus the statistical power of the endpoints appears to be reasonably balanced in 1836

experimental design of the proposed MRT (Appendix I: Power Analysis)

. 1837

1838

Tabl e 5

- 5. Statistical Power of Non

- reproductive Endpoints in Proposed MRT 1839

Statistical power of MRT to detect proportionate differences in non

- reproductive endpoints

compared with control values ( α =0.05)

10% 20% 30% 40% 50% Trend

Wet Weight Reduction

Female 0.14 0.67 1.00 1.00 1.00 1.00

Male 0.10 0.53 0.95 0.99 1.00 1.00

Length Reduction

Female 0.99 1.00 1.00 1.00 1.00 1.00

Male 0.99 1.00 1.00 1.00 1.00 1.00

Secondary Sex (reductions in anal fin papillary processes)

Male 0.01 0.25 0.87 0.99 1.00 1.00

Vitellogenin Reduction

Female 0.01 0.09 0.59 0.99 1.00 1.00

Vitellogenin Increase

2X 5X 10X 20X 50X Trend

Male 0. 10 0.6 2 0.9 3 1.00 1.00 1.00

1840

1841

Comparison of Animal Use between the MMT and MRT 1842

1843

An evaluation of the number of fish that are used in the research MMT protocol compared with 1844

the proposed MRT indicates that even though the number of replicate breeding pairs is doubled 1845

in the chemical treatments and is 4x high in the controls, the total number of fish sampled and 1846

culled is less in the MRT; 2,520 fish in the MRT compared with 2,952 in the MMT. 1847

Furthermore, if one discounts the portion of newly hatched , as suggested by the OECD 1848

guidelines for fish embryo toxicity tests, eleutheroembryos which are not continued to the 1849

juvenile and adu lt stages in this calculation, then there are far fewer fish employed in the MRT; 1850

558 juvenile and/or adult fish in the MRT compared with the same 2,952 juvenile and adult fish 1851

used in the research MMT. 1852

1853

Estimates of person

- hours required to perform the M MT and MRT 1854

1855

Two critical changes in the MRT compared with the MMT are the recommended increase in the 1856

number of breeding replicates employed and, the completion of the in

- life potion of the test after 1857

embryo hatch in F 2 . The increase in replication requires an increase in the human resources 1858

necessary to perform the MRT, especially during the assessment of fecundity and fertility. In 1859

contrast, the decrease in the exposure time reduces the necessary human resources to perform the 1860DRAFT

MMT ISR 52 M a y 28

, 2013 test. A comparison of the time required to perform the MRT and the MMT is presented in Table 1861

5

- 6. 1862

1863

Table 5

- 6

. Human

- R esource Estimates for In

- life Portions of MMT and MRT (hours) 1864

1865

Resource Category MMT MRT

Technician Hours 1197 939

Biologist Hours 424 297

Chemist Hours 590 382

Total Hours 2211 1618

1866

Note that even with the replicate increase, the estimate of the total human resource time required 1867

to run the in

- life portion of the MRT is about 25% less than the MMT. 1868

1869

5.1.2 In

- life Exposure Complete After F 2 Hatch 1870

1871

The context of this discussion is whether the data from the F 2 generation added significant value 1872

to the understanding of treatment effects on the test population. A summary of the data from all 1873

the MMTs performed by EPA or Japanese labs that included assessme nts of fecundity in both F 1 1874

and F 2 is presented in Table 5

- 7. With 17 β

- e stradiol the LOEC for fecundity was lower by 1 1875

treatment level in F 1 compared to F 2 . In one of the three tests with 4

- t

- o ctylphenol , the LOEC in 1876

F 2 was 1 treatment level lower and in the other two the LOECs were the same in both 1877

generations. Note that the LOEC in the 2012 octylphenol test, which had 12 breeding pairs per 1878

treatment, was 1 treatment level lower than the previous test run by EPA, and th at the LOECs 1879

were the same in F 1 and F 2 . The LOEC for the Japanese run o,p′

- DDT test was not determinable 1880

with a greater

- than 1.9 μg/L value for F 1 and 1.9 μg/L in F 2 . Lastly, the LOECs for fecundity in 1881

F 1 and F 2 for the 17 β

- t renbolone test run by EPA were equal. In summary, for the six tests using 1882

three different chemicals where effects on fecundity were measured in both the F 1 and F 2 1883

generations, the LOEC was lower in F 1 in one test; lower in F 2 in two tests, and the same in three 1884

tests. In all cases, whe n the LOECs were different across generations, it was by one treatment 1885

level. In the 2012 4

- t

- o ctylphenol test with 12 replicate breeding pairs, the LOECs were the same 1886

in both generations and lower than the LOECs measured in the other two MMTs with 1887

octylp henol. An MMT with more replication in the assessment of reproductive endpoints, and 1888

without an assessment of reproduction in F 2 ( i.e ., the proposed MRT) appears to detect effects on 1889

fecundity as well or better than the information obtained in both generat ions of the research 1890

MMT. Not only was the LOEC lower with 12 breeding pairs, but the LOEC in F 1 and F 2 were 1891

the same. Additionally, the mean fecundity values in the control treatments and the LOEC 1892

treatments were quite similar across generations. Finally, differences of the mean fecundity 1893

levels in a particular treatment across generations were not statistically significant. 1894

1895

1896

1897

1898

1899

1900

1901

1902DRAFT

MMT ISR 53 M a y 28

, 2013 Table 5

- 7. LOECs for Fecundity in F1 compared with F2 in MMTs 1903

1904

Chemical Laboratory Fecundity LOEC

Data through F1 Data through F2

17β

- Estradiol CERI/Japan 28 ng/L >28 ng/L

4

- t

- Octylphenol US EPA/MED

- 2007 102 μg/L 51 μg/L

4

- t

- Octylphenol NIES/Japan 100 μg/L 100 μg/L (>50)

4

- t

- Octylphenol US EPA/MED

- 2012 (12 reps) 23 μg/L 23 μg/L

o,p ′

- DDT CERI/Japan >1.9 μg/L 1.9 μg/L

17 β

- Trenbolone US EPA/MED 32 ng/L 32 ng/L

1905

Comparisons of the lowest LOECs for the biomarker endpoints obtained from the subadult 1906

samples in 6 MMTs is presented in Table 5

- 8. Note that the LOECs for the most sensitive 1907

biomarker endpoints were the same in F 1 and F 2 except for one case, i.e., the US

- run test with 1908

octylphenol, were the LOEC for anal fin papillae was one treatment level lower in F 2 than in F 1

. 1909

1910

Table 5

- 8 . LOECs for Biomarker endpoints in F1 compared to F2 in MMTs 1911

1912

Chemical Laboratory Biomarker LOEC

Endpoint Data through

F 1 Data through

F 2 17β

- Estradiol CERI/Japan 2.8 ng/L 2.8 ng/L Papillae

4

- t

- Octylphenol US EPA/MED 25 μg/L 13 μg/L Papillae

4

- t

- Octylphenol NIES/Japan 6.25 μg/L 6.25 μg/L Liver vitellogenin

o,p ′

- DDT CERI/Japan 0.07 μg/L 0.07 μg/L Liver vitellogenin

Tamoxifen NIES/Japan 1.3 μg/L 1.3 μg/L Liver vitellogenin

17 β

- Trenbolone US EPA/MED 32 ng/L 32 ng/L Papillae and Liver

vitellogenin

1913

Occasionally, there is a LOEC for an endpoint in the F 2 generation that is lower than the LOEC 1914

for the same endpoint in F 1 . In all cases, the LOEC is different by only one treatment level. One 1915

of the requirements for making valid comparisons of differences in the treatment effects among 1916

generations is that the test conditions are the same in both generations. It is very difficult to 1917

actually achieve this requirement, especially with respect to the chemical exposure 1918

concentrations. Furthermore, in those cases where chemical concentrations are not the same 1919

across generations, it is essentially not possible to discern whether the difference between the 1920

LOECs are due to differences in test organism sensitivity or to differences in the chemical 1921

exposure at a critical window in fishes life history. 1922

1923

When considering u sing multiple generations in a test protocol it is appropriate to consider 1924

whether the additional data provided by each generation increases the quality of the risk 1925

assessment, and whether the test protocol is likely to be completed successfully. In genera l, our 1926

experience the longer a test continues , the greater the risk of incidents resulting in test failure. 1927

The causes of failure include exposure and/or dilution pump malfunctions, malfunctions in 1928

temperature control systems, disease, and, perhaps the most common problem

-- maintaining 1929

consistent treatment concentrations throughout the test. As t he test progresses, heterotrophic 1930

microbes which use the test chemical as an energy source, are selected for until they thrive and 1931DRAFT

MMT ISR 54 M a y 28

, 2013 proliferate in the exposure system. These microbes metabolize the test chemical with subsequent 1932

reductions in the treatment c oncentrations. To deal with this, the exposure system needs to be 1933

periodically sterilized and , adjustments to the dilution system must be made frequently to 1934

accommodate reductions in treatment concentrations. These adjustments are less arduous or 1935

fewer in the shorter test protocol. 1936

1937

5.1.3 Effect

- endpoints Not Measured in F 0 1938

1939

Results from full MMT tests indicate that the LOEC for any particular endpoint in F 0 is never 1940

lower than the LOEC from either F 1 or F 2 for that endpoint

. In a few MMTs the F 0 LOEC was 1941

the same as the LOEC in F 1 or F 2 , ( see T able 5

- 9 )

. C onsequently

, we recommend that it is not 1942

necessary to measure e ndpoints in F 0

. 1943

1944

Table 5

- 9. Comparative LOEC values for measurement endpoints from several research MMTs across generations. 1945

Green colored cells indicate the lowest LOEC among the three generations for that particular endpoint. Greater

- than 1946

values indicate the LOEC for that particular generation was higher than the highest concentration that still had fish. 1947

P urple cells indicate that the LOEC is higher than the highest concentration tested. 1948

1949

Endpoint F 0 F 1 F 2

E 2 SSC >84 ng/L 28 ng/L >28 ng/L

Vitellogenin 28 ng/L 28 ng/L 28 ng/L

Fecundity >84 ng/L 28 ng/L >28 ng/L

4tOP MED Weight

- XY >102 μ g/L 51 μ g/L >51 μ g/L

Weight

- XX 102 μ g/L 13 μ g/L >51 μ g/L

SSC >102 μ g/L 102 μ g/L 13 μ g/L

Vitellogenin 51 μ g/L 51 μ g/L 51 μ g/L

Gonad Phenotype 102 μ g/L 13 μ g/L 51 μ g/L

Fecundity >102 μ g/L 102 μ g/L 51 μ g/L

DDT SSC >1.9 μ g/L >1.9 μ g/L >1.9 μ g/L

Vitellogenin 1.9 μ g/L 0.69 μ g/L 0.22 μ g/L

Gonad Phenotype >1.9 μ g/L 1.9 μ g/L 0.69 μ g/L

Fecundity >1.9 μ g/L >1.9 μ g/L 1.9 μ g/L

trenbolone SSC 84 ng/L 84 ng/L >13 ng/L

Vitellogenin >84 ng/L 32 ng/L >13 ng/L

Gonad Phenotype >84 ng/L 84 ng/L >13 ng/L

Fecundity 84 ng/L 32 ng/L >13 ng/L

prochloraz Weight 41 μ g/L >25 μ g/L >25 μ g/L

Fecundity 41 μ g/L >25 μ g/L 25 μ g/L

1950

1951

5.1.4 Measure Subadult Endpoints at 9 wpf 1952

1953

At ORD/MED, the subadult sampling had been done, depending on tests, at times between 44 1954

and 59 dpf. Within this time window, medaka are growing and developing rapidly enough that 1955DRAFT

MMT ISR 55 M a y 28

, 2013 shifting the subadult sampling time a week earlier or later has dramatic im pacts on the data 1956

collected. For instance, the medaka growth curve is very steep at this time (Figure 5

- 4)

. 1957

1958

Figure 5

- 4. Medaka Growth

- Curve in MMT and MRT protocols 1959

1960 Concurrent with the rapid growth observed, the development of SSC (i.e. anal fin papillae) 1961

occurs around 40 dpf. Therefore by sampling subadults at 9 wpf, males will have high numbers 1962

of anal fin papillae (Figure 5

- 5). In addition, development of the gon ad is dramatic during this 1963

phase of development as well, and there is a substantial increase in the production of 1964

vitellogenin. In typical MMTs, the mean number of copies of vitellogenin per ng of total liver 1965

RNA was 2 x 10 5 at 6 wpf and 5 x 10 6 at 7 wpf. Another case in point, the mean liver 1966

vitellogenin concentration in subadult control females of the F1 generation in the tamoxifen 1967

MMT performed by NIES/Japan was approximately 40 ng/mg liver while the mean of the 1968

subadult females of F2 in the same MMT w as approximately 1350 ng/mg liver. The difference 1969

between the subadult samplings of F1 and F2 in the tamoxifen MMT was that in F1 the sampling 1970

was done at 54 dpf while in F2 the subadult sampling was done at 65 dpf, a difference of 11 1971

days. 1972

1973 0.000 0.050 0.100 0.150 0.200 0.250 0.300 0.350 0.400 0.450

16 37 58 79 Weight (g)

Age as days post fertilization

MEDAKA WEIGHTDRAFT

MMT ISR 56 M a y 28

, 2013 Figure 5

- 5. Time

- course of Anal

- fin Papillae Development 1974

1975

1976

Based upon the information gathered, the recommendation is to have the subadult sampling take 1977

place during the 9 th wpf so that the fish will have had a chance to development to produce 1978

sufficiently high mean values with low variance in all the measured parameters. 1979

1980

1981

5.1.5 Test Temperature 25.5 ° C 1982

1983

Appropriate test temperature and the allowable varianc e both within an aquarium and between 1984

aquaria have been discussed at bilateral meetings between US EPA and Japan Ministry of the 1985

Environment . The recommended temperature and variance throughout the test is 25.5 ° C ± 1986

0.5 ° C. Brief excursion from the mean by individual aquaria must not be more than 2 ° C

. 1987

Replicates within a treatment must not statistically different from each other, and treatments 1988

within the test must not be statistically different from each other. T his specification is based 1989

upon 1) the historical range of temperatures used for toxicol ogy testing with medaka, 2) the 1990

temperature(s) for optimal reproduction, and 3) the need to minimize the occurrence of XX 1991

males. 1992

1993

Table 5

- 9 shows the fecundity of control fish in a MMT run at 24 ° C and one run with the 1994

suggested specification. The higher control fecundity should result in greater statistical power 1995

for this vital endpoint. 1996

1997

Table 5

- 9

. Control Fish Fecundity at 24 and 25.5 ° C. 1998

MMT at 24 ° C MMT at 25.5 ° C

F0 F1 F2 F0 F1 F2

Fecundity (mean/pair

- day) 26.3 13.9 26.4 37.8 32.5 39.1

Standard Deviation 1.4 6.2 1.6 2.9 2.6 2.8

1999

2000 0

20 40 60 80 100 120

23 30 37 44 51 58 65 72 Mean Papillae per Fish

Age (dpf) Anal Fin PapillaeDRAFT

MMT ISR 57 M a y 28

, 2013 It has been documented in the literature (Nanda et al., 2003; Sato et al., 2005; Selim et al., 2009) 2001

and observed in our medaka culture that a small number of unexposed XX medaka will 2002

spontaneously develop into normal phenotypic males. One factor that increases the likelihood of 2003

this is increased temperature. Temperatures reported in the published lite rature typically report 2004

effects at 32 ° C, a temperature not considered for the MRT. Thus, we initiated an ancillary study 2005

to evaluate appropriate temperature to recommend for the MRT protocol. Figure 5

- 6 shows how 2006

differences in temperature during developm ent affect the prevalence of XX males in the 2007

population. Note that higher temperatures cause an increase in the prevalence of XX males even 2008

within a series of temperatures that seem to be appropriate for the MRT, i.e., 24

– 26 ° C. The 2009

recommended temperat ure of 25.5° C for the MRT balances the competing objectives of 2010

maximizing fecundity while minimizing the prevalence of XX males in controls. 2011

2012

2013

Figure 5

- 6. Prevalence of XX Males with Increasing Temperature. The bars show the proportion of phenotypic 2014

m ales that are either XX or XY out of 100%. The number boxes are the actual count of each genotype. 2015

2016

2017

2018

2019

5.1.6 Pathology Analysis of Adult Fish Only 2020

Another aspect of the 12 replicate test design is that the sample size for pathology analysis of 2021

adult fish in the MRT is double compared to the 6 replicates in the MMT. This difference is 2022

substantial. It provides pathology results for up to 12 fish per ge nder per treatment compared to 2023

the MMTs 6. Also, because the pathology is done on adult fish sampled after the reproduction 2024

evaluation, it provides information that can be correlated with the reproductive performance of 2025

each specific breeding pair. This is in contrast with the subadult pathology analysis which does 2026DRAFT

MMT ISR 58 M a y 28

, 2013 not allow these correlations to be made. Evaluation of treatment

- related effects on gonadal 2027

development such as sex

- reversal, and the prevalence testicular oocytes, is possible in either 2028

adult or subadult specimens, however, only the adult samples provide data that can be used to 2029

correlate pathology with reproduction. The increased sample sizes for adult pathology in the 12 2030

replicate design increases both the statistical power and the relevance of the pathology endpoint 2031

compared with the 6 replicate test design. With increased replication the sample size for 2032

evaluating pathology is also increased which increases the statistical power of pathology 2033

endpoints, especially with the development of the RS CABS statistical procedure. Furt hermore, 2034

the pathology analysis of the adult breeding pairs can be directly coordinated with their 2035

fecundity/fertility outcomes

. 2036

2037

2038

6 CONCLUSIONS 2039

The outcomes of the various MMT trials have provided enough information to recommend 2040

a medaka reproduction test ( MRT ) for use as the fish test in Tier 2 of the EDSP. Two major 2041

changes from the MMT are proposed, i.e., an increase in the number of replicates per 2042

treatment for evaluating e ffects on reproduction, and terminating the test after the 2043

embryos hatch in F 2 . Other proposed changes include minimizing the collection of endpoint 2044

data from F 0 , and evaluating pathology in only the F 1 adults sampled after the assessment 2045

of reproduction. 2046

2047

Overall, the authors conclude that both the MMT and the MRT are transferable methods and are 2048

capable of adequately characterizing potential disruption of the endocrine system by putative 2049

endocrine disrupting chemicals. However, the MRT is recommended as the preferred EDSP 2050

Tier 2 test method for fish because it is less resource intensive with improved statistical power, 2051

appears to be as sensitive, and is better able to ensure consistent findings when performed 2052

routinely by testing laboratories. 2053

2054

2055

2056

2057

2058

2059

2060

2061

2062

2063

2064

2065

2066

2067

2068

2069DRAFT

MMT ISR 59 M a y 28

, 2013 APPENDICES 2070

2071

Appendix A: Criteria for Valida tion of Assays in the EDSP (2007 ) 2072

2073

Appendix B: Detailed Review Paper 2074

2075

Appendix C: Vinclozolin Interlaboratory Summary Report 2076

2077

Appendix D: MMT Protocol 2078

2079

Appendix E: MRT Protocol 2080

2081

Appendix F: Pathology G uidance 2082

2083

Appendix G: Statistics 2084

2085

Appendix H: MMT Summary Reports 2086

2087

Appendix I: Power Analysis 2088

2089

2090

2091

2092

2093

2094

2095

2096

2097

2098

2099

2100

2101

2102

2103

2104

2105

2106

2107

2108DRAFT

MMT ISR 60 M a y 28

, 2013 2109

2110

2111

REFERENCES 2112

2113

Adkins

- Regan, E., Banerjee, S.B., Correa, S.M., Schweitzer, C., 2013. Maternal effects in quail 2114

and zebra finches: Behavior and hormones. Gen Comp Endocrinol. 2115

2116

Adkins

- Regan, E., Ottinger, M.A., Park, J., 1995. Maternal transfer of estradiol to egg yolks 2117

alters sexual differentiation of avian offspring. Journal of Experimental Zoology 271, 466

- 470. 2118

2119

Almasi, B., Rettenbacher, S., Muller, C., Brill, S., Wagner, H., Jenni, L., 2012. Maternal 2120

corticosterone is transferred into the egg yolk. Gen Comp Endocrinol 178, 139

- 144. 2121

2122

Ankley, G., Jensen, K.M., Kahl, M.D., Durhan, E.J., Makynen, E.A., Cavallin, J.E., Martinovic, 2123

D., Wehmas, L.C. , Mueller, N.D., Villeneuve, D.L., 2010a. Use of chemical mixtures to 2124

differentiate mechanisms of endocrine action in a small fish model. Aquatic Toxicology. 2125

2126

Ankley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D., Mount, 2127

D.R ., Nichols, J.W., Russom, C.L., Schmieder, P.K., Serrano, J.A., Tietge, J.E., Villeneuve, 2128

D.L., 2010b. Adverse outcome pathways: a conceptual framework to support exotoxicology 2129

research and risk assessment. Environmental Toxicology and Chemistry 29, 730

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2131

Ankley, G.T., Jensen, K.M., Durhan, E.J., Makynen, E.A., Butterworth, B.C., Kahl, M.D., 2132

Villeneuve, D.L., Linnum, A., Gray, L.E., Cardon, M., Wilson, V.S., 2005. Effects of two 2133

fungicides with multiple modes of action on reproductive endocrine function in the fathead 2134

minnow (Pimephales promelas). Toxicol Sci 86, 300

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