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MMT ISR 1 May 28, 2013

1 Validation of the Medaka Multigeneration Test:

2 Integrated Summary Report

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6 U.S. Environmental Protection Agency

7 Endocrine Disruptor Screening Program

8 Washington, D.C.

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21 Contents

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

23 OBJECTIVE ................................................................................................................................... 6

24 1 INTRODUCTION ...................................................................................................................... 6

25 1.1 Endocrine Disruptor Screening Program ......................................................................... 7

26 1.2 Validation......................................................................................................................... 8

27 2 HISTORICAL OVERVIEW OF FISH TESTS........................................................................ 11

28 2.1 Purpose of the Medaka Multigeneration Test in EDSP ................................................. 11

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

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31 2.2.1 Advantages of the medaka test model ..................................................................... 12

32 2.2.2 Conceptual basis for a multigeneration test paradigm........................................... 12

33 3 MEDAKA MULTIGENERATION TEST PROTOCOL DEVELOPMENT .......................... 13

34 3.1 Overview of the Medaka Multigeneration Test (MMT) Research Protocol.................. 14

35 3.2 MMT Endpoints............................................................................................................. 16

36 3.2.1 Genotypic Sex (FO, F1, F2)...................................................................................... 17

37 3.2.2 Survival (FO, F1, F2)................................................................................................... 17

38 3.2.3 Growth (FO, F1, F2).................................................................................................... 17

39 3.2.4 Reproduction (FO, F1, F2)........................................................................................... 17

40 3.2.5 Liver Vitellogenin mRNA (FO, F1, F2) ........................................................................ 18

41 3.2.6 Secondary Sex Characters (FO, F1, F2) ...................................................................... 18

42 3.2.7 Hatch Success (FO, F1, F2).......................................................................................... 18

43 3.2.8 Pathology and Gonad Phenotype (FO, F1, F2)............................................................ 18

44 3.3 Research MMT Timeline ............................................................................................... 19

45 3.3.1 F0; Study weeks 1-5................................................................................................. 20

46 3.3.2 F1 and F2; Weeks 1-3 .............................................................................................. 20

47 3.3.3 F1 and F2; Week 4................................................................................................... 21

48 3.3.4 F1 and F2; Weeks 4-6 .............................................................................................. 21

49 3.3.5 F1 and F2; Week 7................................................................................................... 21

50 3.3.6 F1 and F2; Weeks 7-10 ............................................................................................ 21

51 3.3.7 F1 and F2; Weeks 11-15 .......................................................................................... 21

52 3.3.8 F1 and F2; Week 16................................................................................................. 21

53 3.4 Statistical Analyses ........................................................................................................ 21

54 3.5 Data Expectations........................................................................................................... 23

55 4 MMT DATA SUMMARIES ................................................................................................ 24

56 4.1 Results with ER-agonists ............................................................................................... 26

57 4.1.1 17β-Estradiol (CERI/Japan)....................................................................................... 27

58 4.1.2 4-t-Octylphenol (US EPA/ORD/MED) ....................................................................... 27

59 4.1.3 4-t-Octylphenol (NIES/Japan) .................................................................................... 28

60 4.1.4 Comparison of 4-t-Octylphenol MMTs in Support of Validation ............................... 29

61 4.1.5 o,p′-DDT (CERI/ Japan)............................................................................................ 30

62 4.1.6 4-chloro-3-methylphenol (US EPA/ORD/MED)......................................................... 30

63 4.1.7 Tamoxifen (NIES/Japan)............................................................................................. 31

64 4.2 Results with AR-agonists............................................................................................... 32

65 4.2.1 17β-Trenbolone (US EPA/ORD/MED)....................................................................... 32

66 4.3 Results with Steriodogenesis Inhibitor........................................................................... 33
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67 4.3.1 Prochloraz (US EPA/ORD/MED)............................................................................... 33

68 4.4 Results with AR Antagonist........................................................................................... 34

69 4.4.1 Vinclozolin (MED)...................................................................................................... 34

70 4.5 Interlaboratory Evaluation of the MMT protocol - Vinclozolin .................................... 36

71 4.5.1 Background................................................................................................................. 36

72 4.5.2 Protocol........................................................................................................................ 37

73 4.5.3 Chemistry Data ............................................................................................................ 38

74 4.5.4 Control Data Supporting Validation ........................................................................... 39

75 4.5.6 Summary of Lab 1 Vinclozolin MMT........................................................................... 41

76 4.5.7 Summary of Lab 2 Vinclozolin MMT........................................................................... 42

77 4.5.8 Summary of Lab 3 Vinclozolin MMT........................................................................... 42

78 4.5.9 US EPA Perspective on the Interlaboratory Results ................................................... 42

79 5 OPTIMIZATION OF THE MMT AND THE PROPOSED MEDAKA REPRODUCTION 

80 TEST (MRT) PROTOCOL........................................................................................................... 43

81 5.1 Primary MMT Protocol Changes for development of MRT Protocol ........................... 43

82 5.1.1 Increased Replication for the Assessment of Treatment-Effects on Reproduction . 46

83 5.1.2 In-life Exposure Complete After F2 Hatch.............................................................. 52

84 5.1.3 Effect-endpoints Not Measured in F0...................................................................... 54

85 5.1.4 Measure Subadult Endpoints at 9 wpf .................................................................... 54

86 5.1.5 Test Temperature 25.5 ° C...................................................................................... 56

87 5.1.6 Pathology Analysis of Adult Fish Only.................................................................. 57

88 6 DISCUSSION/SUMMARY OF THE VALIDATION OF MMT/MRT TEST METHOD . 58

89 APPENDICES .............................................................................................................................. 59

90 REFERENCES ............................................................................................................................. 60

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94 EXECUTIVE SUMMARY

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

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

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

99 fish chronic toxicity tests has previously been summarized in an in-depth review (Appendix B). 

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

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

102 estrogen receptor agonists (17-estradiol, 4-t-octylphenol, o,p’-DDT,4-chloro-3-methylphenol, 

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

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

105

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

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

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

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

110 toxicants. The MMT is a relatively long-term assay (29 weeks of in-life exposure) that assesses 

111 hatch, growth, survival, gonadal development, and reproduction in 21⁄2generations (F0, F1, and 

112 part of F2). Vinclozolin was chosen for an interlaboratory demonstration of the protocol.

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

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

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

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

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

119 importance of an additional F2 generation; and d) develop a scientifically-defensible Tier 2 fish 

120 test protocol for characterizing concentration-response for ED endpoints. 

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

123 concentration-response for several known endocrine active substances. In the case of 4-t124 octylphenol, tests at different laboratories and at different times in one laboratory comported 

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

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

127 The median responses from F1 reproductive measurements, F1, F2 length and weight 

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

129 of the results suggest some of the significant inter-lab variability can be attributed to the 

130 relatively small size at termination of F1 and F2 fish in a single laboratory. The small size of this 

131 laboratory F1 females appears to have contributed to the high inter-lab variability in fecundity. 

132 Normalizing F1 fecundity for the body mass of the female eliminated the significant difference. 

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

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

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

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

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

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

139 MMTs, there was consistency in the observation of effects on the SSC. 
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140

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

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

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

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

145 should be followed without deviation. 

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

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

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

150 for evaluating effects on reproduction, and terminating the test after the embryos hatch in F2. 

151 Other proposed changes include minimizing the collection of endpoint data from F0, and 

152 evaluating pathology in only the F1 adults sampled after the assessment of reproduction.

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

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

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

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

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

159 routinely by testing laboratories.

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187 OBJECTIVE

188

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

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

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

192 the validation charge questions provided.

193

194 1 INTRODUCTION 

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

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

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

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

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

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

202

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

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

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

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

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

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

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

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

211 and the creation of similar programs in other countries, the Organization for Economic Co212 operation and Development (OECD) established the Endocrine Disrupters Testing and 

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

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

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

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

217 new and existing substances.

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

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

221 materials introduces the purpose of the MMT, the scientific rationale for the assay, the 

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

223 It also synthesizes the information gained during the validation process and addresses the 

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

225 reliability, protocol transferability and fit for purpose.

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231 1.1 Endocrine Disruptor Screening Program 

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

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

235 recommended to the EPA a conceptual two-tiered approach that involved screening and testing 

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

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

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

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

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

242 Advisory Board (SAB) and FIFRA Scientific Advisory Panel (SAB-SAP, 1999). A complete 

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

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

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

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

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

248 relationships for hazard identification using Tier 2 testing. 

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

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

252 • be relatively fast and efficient;

253 • be standardized and validated;

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

255 rate of false positives;

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

257 possible;

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

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

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

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

263 incorporated (e.g.. two different assays may cover some similar aspects of the estrogenic 

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

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

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267 The assays in Table 1-1, recommended by EDSTAC, have undergone validation and have been 

268 adopted as the EDSP Tier 1 screening battery (Table 1-1). These assays are meant to detect 

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

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

Assay Reason for Inclusion

Estrogen receptor binding or transcriptional 

activation assay

An in vitro test to detect chemicals that may affect the 

endocrine system by binding to the estrogen receptor.

Estrogen receptor transcriptional activation 

assay

An in vitro test to detect chemicals that may affect the 

endocrine system by binding to the estrogen receptor.

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

endocrine system by binding to the androgen receptor.

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

synthesis of the sex steroid hormones

Placental Aromatase Assay An assay to detect interference with aromatase.

Uterotrophic Assay An in vivo assay to detect estrogenic chemicals. 

Hershberger Assay An in vivo assay to detect androgenic and antiandrogenic 

chemicals. 

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

or through the HPG axis that controls the estrogen and 

androgen hormone systems. It is also enhanced to detect 

chemicals that interfere with the thyroid system. This 

assay could in part substitute for the female pubertal 

assay.

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

or through the HPG axis that controls the estrogen and 

androgen hormone systems. It is also enhanced to detect 

chemicals that interfere with the thyroid system.

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

with the thyroid hormone system. 

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

with the HPG axes.

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

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

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

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

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285  Two-generation Rat Reproduction Test

286  Medaka Multigeneration Test

287  Japanese Quail Two-generation Test

288  Mysid Two-generation Toxicity Test

289  Larval Amphibian Growth and Development Test

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

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

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294 1.2 Validation

295

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

297 method are evaluated for a particular use” (NIEHS, 1997; OECD, 1996).
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298 Reliability is defined as the reproducibility of results from an assay within and between 

299 laboratories. 

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

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

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

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

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

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

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

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

308 (OECD, 2005). These criteria as stated by ICCVAM (NIEHS, 1997) are as follows:

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

310 its proposed use, should be available.

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

312 effect and toxicity of interest must be addressed.

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

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

315 analysis and decision criteria.

316 4. Within-test, intra-laboratory and interlaboratory variability and how these parameters 

317 vary with time should have been evaluated.

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

319 chemicals preferably coded to exclude bias.

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

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

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

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

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

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

326 published in an independent, peer-reviewed publication.

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

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

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

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

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

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

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

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

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

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

337 ecological in vivo assays in Tiers 1 and 2 that are being considered as the standards and not as 
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338 alternatives to some other assay. Nonetheless, the stages of the process outlined by the 

339 ICCVAM are discussed below.

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

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

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

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

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

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

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

347 considerations. The DRP also identified the developmental status and questions related to each 

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

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

350 optimization”. During standardization and optimization, studies were performed geared toward 

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

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

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

354 studies were used to determine interlaboratory variability and to set or cross-check performance 

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

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

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

358 phase, “regulatory acceptance”, i.e., adoption for regulatory use by an agency. EPA has 

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

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

361 decisions (USEPA, 2006).

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

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

364 Appendix E: MRT Protocol) to be used as an in vivo assay for EDSP Tier-2 testing. The 

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

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

367 proposed MRT are presented.

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370 2 HISTORICAL OVERVIEW OF FISH TESTS

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

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

374 endocrine-specific endpoints in mind. Further, it is known that growth, reproduction, 

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

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

377 chemicals (EDCs) is relevant.

378

379 2.1 Purpose of the Medaka Multigeneration Test in EDSP

380

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

382 a test organism through endocrine-mediated pathways, and to quantitatively evaluate those 

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

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

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

386

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

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

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

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

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

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

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

394 fulfill this purpose, these tests are much longer-term studies designed to encompass critical life 

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

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

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

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

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

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

401 the Tier 2 testing should be conclusive, and a discernible cause-effect relationship should be 

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

403 elements should be part of the test:

404

405  establish exposure/concentrations/timing and effects relationships 

406  be sensitive and specific

407  assess relevant endpoints

408  include all life stages across generations

409  include a dose range for full characterization of effects

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

411 (GLP).

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413 2.2 Medaka multigeneration toxicity tests for the detection of possible endocrine 

414 disruption

415

416 2.2.1 Advantages of the medaka test model

417

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

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

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

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

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

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

424 are available in EPA/600/3-91/064 (Denny et al., 1991). Medaka are small (25 to 50 mm in total 

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

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

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

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

429 observation of an anesthetized fish under a low-power microscope is recommended for 

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

431

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

448

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451 2.2.2 Conceptual basis for a multigeneration test paradigm

452

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

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

455 maternal transfer of xenobiotic as well as endogenous factors to the embryo, in ovo (Adkins456 Regan et al., 2013; Almasi et al., 2012; Foran et al., 2000; Hayward and Wingfield, 2004; 

457 Henriksen et al., 2011; Lin et al., 2004; Metcalfe et al., 2000; Schwaiger et al., 2002; Schweitzer 
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458 et al., 2013). Further, organizational effects during development and maturation may not fully 

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

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

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

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

463 most efficiently maximize the adverse impact-potential of an endocrine active substance. The 

464 initial F0 generation is exposed during spawning to allow test compound and maternal factors to 

465 be transferred into the eggs of the F1 generation. This transfer establishes the initial chemical 

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

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

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

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

470 F0 generation is exposed after onset of reproduction, then some transfer of factors that present 

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

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

473 and matured prior to exposure. If that is the case, the F1 animals would be subject to a modified 

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

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

476 Their progeny (F2) would therefore be expected to receive the full spectrum of modified factors 

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

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

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

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

481 generally reviewed by Denslow and Sepulveda (Denslow and Sepúlveda, 2007). It is the sexual 

482 differentiation and reproductive competence of the F2 generation that would conceptually be 

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

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

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

486

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

488 sheepshead minnow (Cyprinodon variegatus) (Cripe et al., 2009; Cripe et al., 2010; Raimondo et 

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

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

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

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

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

494 investigated, the importance of inclusion of additional generations is somewhat equivocal 

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

496 epigenetic effects. Comparing sensitivities between activational and organizational effects is 

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

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

499 full F2 generation.

500

501 3 MEDAKA MULTIGENERATION TEST PROTOCOL DEVELOPMENT

502
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MMT ISR 14 May 28, 2013

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

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

505 protocol. This protocol incorporated redundant endpoint measurements at comparable 

506 developmental life-stages in all generations of the test, F0, F1, and F2. As such, it is considered a 

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

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

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

510

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

512

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

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

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

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

517 toxicants. The MMT is a relatively long-term assay (29 weeks of in-life exposure) that assesses 

518 hatch, growth, survival, gonadal development, and reproduction in 21⁄2 generations (F0, F1, and 

519 part of F2). The MMT is an extension of existing standard Fish Full Life-Cycle test protocols 

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

521

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

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

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

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

526 various EDC-induced adverse outcome pathways (AOPs); and, c) develop a scientifically527 defensible Tier 2 fish test protocol for characterizing concentration-response for ED endpoints. 

528 The project involved developing methods for evaluating population-relevant endpoints such as 

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

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

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

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

533 modes of action.

534

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

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

537 involving disruption of the hypothalamus-pituitary-gonadal (HPG) endocrine axis (i.e., 

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

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

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

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

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

543 outcomes through population models. The non-endocrine mediated endpoints typically measured 

544 in full life-cycle and early life-stage (ELS) chronic toxicity tests are also included in the MMT. 

545 These are used to evaluate the concentration-response of a chemical working through any 

546 endocrine or non-endocrine mediated AOP. The question of whether a chemical has endocrine547 mediated effects at lower concentrations than non-endocrine-mediated toxicities cannot be 

548 unambiguously addressed by the apical endpoints alone and will rely on other diagnostic 
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MMT ISR 15 May 28, 2013

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

550 SSC.

551

552 The test conditions for the research MMT are outlined in Table 3-1. Briefly, the MMT protocol 

553 consists of continuously exposing 21⁄2 generations of fish to the test chemical starting with 

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

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

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

557 endpoints include embryo hatch, growth, survival, fecundity and fertility, liver vitellogenin 

558 mRNA levels in both sexes, the number of anal fin papillae (a sexual phenotype marker), 

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

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

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

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

563 here in detail, precise dependable flow-through exposure and dilution systems, as well as high564 quality analytical chemistry, are an integral part of the protocol. The in-life duration of the MMT 

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

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

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

568 endpoint is evaluated.

569

570 Table 3-1. Test Conditions for the Medaka Multigeneration Test (MMT) Protocol

571

Recommended species Medaka (Oryzias latipes)

Test type Continuous flow-through

Water temperature 26 ± 1oC 

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

Photoperiod 16 h light, 8 h dark

Loading rate F0- 2 fish/replicate; F1 and F2- initiated with maximum of 40 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 12 volume replacements/day (15 ml/min flow).

Age of test organisms at initiation F0-11 weeks post-fertilization, F1 and F2 continuously from fertilization

No. of organisms per replicate F0- 2 adults; F1 and F2- 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 

F0- 12 for each test concentration and for control; F1 and F2- 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% saturation
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MMT ISR 16 May 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 O2/L. 

Exposure period Trans-generational test: F0from 11-16 weeks post-fertilization (wpf),), F1 and F2from 0-

16 wpf

Primary endpoints Survival: F0from 11-16 wpf, F1 and F2 from 1-3 wpf and from 3-16 wpf

Growth : length, weight, (F0, F1, F2 at 16 wpf termination; F1 and F2 7 wpf)

Hatch: F1and F2 only

Anal Fin Papillae: F0, F1, F2 assessed at 16 wpf termination; F1 and F2 assessed at7 wpf

Fecundity: assessed for each breeding pair T,W,Th,F of weeks 11-15 for F0, F1 and F2

Fertility: assessed for each breeding pair T,W,Th,F of weeks 11-15 for F0, F1 and F2

Vitellogenin: determined at 7 and 16 wpf for F0, F1 and F2

Optional endpoints Time to first spawn for F1 and F2 generations.

Test acceptability Dissolved oxygen >60% of saturation; mean temperature of 26 ± 1oC; 70% survival in 

control treatments of each generation; successful reproduction in at least 65% of control 

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

572

573

574 3.2 MMT Endpoints 

575

576 An overview of the endpoints incorporated into research MMT are listed in Table 3-2. Some of 

577 the endpoints are related to EDC effects while others are traditional ecotoxicology endpoints 

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

579

580 Table 3-2. Endpoint overview of the medaka multigeneration test (MMT)

Life-stage Endpoint Endocrinemediated

Non-EndocrineMediatedPopulation 

Relevance

ELS1(1 wpf2) 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

1 581 ELS: early life-stage

2 582 wpf: weeks post-fertilization

583

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

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

586 reproductive measures of fecundity and fertility. To help differentiate endocrine-mediated effects 

587 from systemic toxicity, additional endocrine-mediated effects such as liver vitellogenin mRNA 
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MMT ISR 17 May 28, 2013

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

589 histological evaluation of gonadal sex and intersex, and finally, histopathology evaluation of 

590 kidney, liver, gonad, and other tissues are also measured (see endpoint list in Table 3-2). All 

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

592 traditional ecotoxicology endpoints that may not be endocrine-mediated such as embryo hatch, 

593 growth, and survival are also measured. 

594

595 3.2.1 Genotypic Sex (FO, F1, F2)

596

597 The genetic sex of each fish, for each generation, is determined by non-destructively obtaining a 

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

599 presence or absence of the sex-determining gene (DMY) in each fish. Under normal conditions 

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

601

602 3.2.2 Survival (FO, F1, F2)

603

604 Survival is assessed daily for F0, F1, and F2 individuals throughout the MMT to quantify 

605 chemically-induced mortality. The survival rates of larval fish at 3 weeks post-fertilization (wpf) 

606 are evaluated in F1 and F2 based on the difference in the number of hatched embryos placed in 

607 each replicate and the number of survivors collected. Subadult survival (F1, F2; 8 wpf), and adult 

608 survival (F0, F1, F2; 16 wpf) are evaluated based on the number of fish present in each replicate at 

609 the sampling time for each life-stage (see Section 3.3 for additional information on sampling 

610 timeline).

611

612 3.2.3 Growth (FO, F1, F2)

613

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

615 incorporated into the MMT to allow the evaluation of possible non-endocrine mediated overt 

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

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

618 integrated evaluation of all the test data will allow discrimination of endocrine versus non619 endocrine mediated AOPs. In the MMT, growth is determined on days when fish are being 

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

621 the test protocol. Specifically, wet weight and length are measured in all F1 and F2 fish shortly 

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

623 Subadult growth is measured in the F1 and F2 fish remaining after reproductive pairs have been 

624 selected. Finally, adult growth is measured in F0, F1, and F2 after reproduction has been assessed 

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

626 nearest 1 mg and length to the nearest 0.1 mm.

627

628 3.2.4 Reproduction (FO, F1, F2)

629

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

631 each generation (F0, F1, and F2). On the morning of each day (Tuesday through Friday of each 
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MMT ISR 18 May 28, 2013

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

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

634

635 3.2.5 Liver Vitellogenin mRNA (FO, F1, F2)

636

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

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

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

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

641 is measured in adults at the end of each generation (F0, F1, and F2) and in subadults in F1 and F2. 

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

643 then compared to the gender-specific control data from each particular generation and life-stage. 

644

645 3.2.6 Secondary Sex Characters (FO, F1, F2)

646

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

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

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

650 estrogen receptor (ER) agonists (e.g., estradiol and 4-tert octylphenol) to lower the number of 

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

652 (e.g., 17β-trenbolone) can induce the development of papillae in females. In all three generations, 

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

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

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

656

657 3.2.7 Hatch Success (FO, F1, F2)

658

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

660 spawning pair. The developing eggs are continually agitated within the incubator by air bubbles. 

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

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

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

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

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

666 non-viable and discarded.

667

668 3.2.8 Pathology and Gonad Phenotype (FO, F1, F2)

669

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

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

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

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

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

675 control males will have testes with a few minimally developed oocytes. The prevalence of this 
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MMT ISR 19 May 28, 2013

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

677 be less than 2%. The assessment of gonad phenotype in subadults involves transverse oblique 

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

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

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

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

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

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

684 sections of multiple organs and tissues is used.

685

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

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

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

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

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

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

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

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

694 addition, an internet-based resource is being developed that would not only include 

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

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

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

698

699 3.3 Research MMT Timeline

700

701 The timeline for the MMT protocol is illustrated in Figure 3-1. The test begins with exposure of 

702 adult F0 fish, and continues the exposure through the full medaka life-cycle for two additional 

703 generations, F1 and F2. The test is complete after assessing reproductive endpoints in the F2

704 generation.

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

722
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MMT ISR 20 May 28, 2013

723 Figure 3-1. The time-line and endpoints in the research MMT. The MMT is 29 weeks long and spans 2 1⁄2 

724 generations; F0, F1, and F2. The rows to the right of each endpoint listed on the left indicate the study-week when the 

725 endpoint is measured in each generation. The life-stages of the test animals are color-coded.

MMT Exposure and endpoint timeline Gen

F0 1 2 3 4 5

F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

F2 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 F0 F1 F2

Fertility F0 F1 F2

Hatch F1 F2

Survival F0 F1 F1 F1 F2 F2 F2

Growth F0 F1 F1 F2 F2

Vitellogenin F0 F1 F1 F2 F2

Secondary sex F0 F1 F1 F2 F2

Histopathology F0 F1 F1 F2 F2

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

728 3.3.1 F0; Study weeks 1-5

729

730 Young adult breeding pairs (11 wpf; females >250 mg; males >200 mg) of the first generation 

731 (F0) are moved into the exposure system at the start of Study week 1 and continually exposed for 

732 the next five weeks. During this exposure period developing oocytes and sperm within the 

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

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

735 relative to each treatment levels. On Study Day 1, a minimum of six female-male breeding pairs 

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

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

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

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

740 F. The F0 breeding pairs are sampled at the start of Study week 6 for the endpoints listed in Table 

741 3-2 and Figure 3-3.

742

743 3.3.2 F1 and F2; Weeks 1-3

744

745 The first week for generations F1 and F2 (study weeks 3 and 15, respectively), eggs are collected 

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

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

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

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

750 20 eggs, from each breeding pair. Aeration is continued until 7 days post-fertilization (dpf), at 
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MMT ISR 21 May 28, 2013

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

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

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

754

755 3.3.3 F1 and F2; Week 4

756

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

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

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

760

761 3.3.4 F1 and F2; Weeks 4-6

762

763 The exposure continues as the larval fish develop into sub-adults.

764

765 3.3.5 F1 and F2; Week 7

766

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

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

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

770 outlined in Figure 3-3.

771

772 3.3.6 F1 and F2; Weeks 7-10

773

774 The exposure continues as the sub-adult breeding pairs develop into adults.

775

776 3.3.7 F1 and F2; Weeks 11-15

777

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

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

780

781 3.3.8 F1 and F2; Week 16

782

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

784 Figure 3-3.

785

786 3.4 Statistical Analyses

787

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

789 is measured once per subject (fish), e.g., vitellogenin and weight; 2) a continuous response that is 

790 measured multiple times per subject (breeding pair), e.g., fecundity which is measured on 21 

791 consecutive days; or, 3) ordinal data, such as histopathology findings that are expressed as a 
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MMT ISR 22 May 28, 2013

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

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

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

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

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

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

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

799 was developed.

800

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

802 Chemistry, Histopathology, And Reproduction endpoints including Repeated measures and 

Multigeneration Studies), a Statistical Analysis System (SAS®;SAS®803 Institute, Cary, NC)-based 

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

805 For continuous data measured once per subject, normality (via Shapiro-Wilks test) and variance 

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

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

808 the results of those two tests, a step-down Jonckheere-Terpstra trend (JT) test was also applied to 

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

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

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

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

813 analyses are presented. Table 3-3 summarizes the statistical tests used by StatCHARRMS, while 

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

815 in Appendix G: Statistics.

816

817

818 Table 3-3. Decision-tree determining the statistical test used by StatCHARRMS.

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

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

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

823 week (or day), and the treatment-by-week interaction are used for analysis. During the MMT 

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

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

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

827 weeks, against the controls, a Dunnett’s test is used to adjust for the family-wise error rate. For 

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

829 effect or the week-by-treatment interaction is significant at the 0.05 level, then the comparisons 

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

831 adjustment. Similarly, if the treatment-by-week interaction is significant at the 0.05 level, then 

832 treatments within the same week are compared at the 0.05 level unadjusted p-values. Finally, if 
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MMT ISR 23 May 28, 2013

833 the treatment-by-week interaction is not significant, then comparisons of weeks within a 

834 treatment or treatments within a week are compared using Bonferroni-Holm adjusted p-values.

835

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

837 termed RSCABS (Rao-Scott Cochran-Armitage by Slices). RSCABS uses a step-down Rao838 Scott adjusted Cochran-Armitage trend test on each level of severity in a histopathology 

839 response. The Rao-Scott adjustment incorporates the replicate vessel experimental design into 

840 the test. The by-slices procedure incorporates the biological expectation that severity of effect 

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

842 scores and revealing the severity of any effect found. The RSCABS procedure not only 

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

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

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

846 Statistics.

847

848 3.5 Data Expectations

849

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

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

852 and to non-specific, or at least non-EDC related, toxicity (e.g., growth parameters). It is 

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

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

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

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

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

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

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

860

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

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

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

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

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

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

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

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

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

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

871 completely resolve impairment of one gender versus the other.

872

873 Several ER agonists were tested during MMT development, ranging from strong (17β-estradiol) 

874 to very weak in vitro agonists (4-chloro-3-methylphenol). Within these MMTs, the data closely 

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

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

877 decreased SSC (in males), up-regulation of vitellogenin, and feminization of the gonad, all 
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MMT ISR 24 May 28, 2013

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

879 AOP.

880

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

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

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

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

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

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

887 agonist (17β-trenbolone) was used as a test agent. Near the concentrations that reduced 

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

889 agonism AOP including the presence of male-specific SSC in female fish, the reduction of 

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

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

892

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

894 which catalyzes the conversion of testosterone to 17β-estrogen. The literature suggests that 

895 aromatase inhibition reduces 17β-estrogen plasma concentrations, reduces vitellogenin uptake 

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

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

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

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

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

901 SSC and vitellogenin.

902

903 The responses of fish in various bioassay systems to AR antagonists have been somewhat 

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

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

906 17β-trenbolone have been suggested as a means to identify AR antagonists (Ankley et al., 

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

908 uncertain. 

909

910 Table 3-4. Contingency table for effects of EDCs with different MIEs on vitellogenin, secondary sexual 

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

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

MIE Male Female

Vitellogenin SSC Gonad Phenotype Vitellogenin SSC Gonad Phenotype

ER agonist ↑ ↓ feminized (↑) 

AR agonist (↑) ↓ ↑ masculinized 

AR antagonist (↓) ? ?

Steroidogenesis inhibitor ? ↓ ?

913

914 4 MMT DATA SUMMARIES

915

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

917 beginning with the MMT protocol presented in Section 3. Each successive iteration of the MMT, 
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MMT ISR 25 May 28, 2013

918 usually with a new/different test agent, was performed using a protocol slightly different than 

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

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

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

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

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

924 for reproduction endpoints, feeding protocols, egg incubation methods, histopathology methods, 

925 etc. Table 4-1 lists the MMTs that were performed in U.S. EPA or Japanese laboratories. 

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

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

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

929 increasing the absolute value of a critical endpoint.

930

931 Table 4-1. Chemicals run in the MMT protocol

Chemical Laboratory Protocol AOP’s MIE*

17β-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 

MMT1

ER binder/modulator; tissuedependent antagonist/partial 

agonist

17β-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 

MMT1 AR antagonist

Vinclozolin Interlab A Abbreviated 

MMT1 AR antagonist

Interlab B Abbreviated 

MMT1 AR antagonist

Interlab C Abbreviated 

MMT1 AR antagonist

4-t-Octylphenol US EPA/MED Full MMT2 ER agonist

1

932 Abbreviated MMT: same as Full MMT but without F2 reproduction

2

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

*

934 Molecular Initiating Event of an AOP

935

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

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

938 MMTs provided information regarding endpoints that might provide insight into intermediate 

939 markers (key events) that involve disruption of the HPG axis (endocrine-mediated chemical 

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

941 mediated pathways. Table 4-2 summarizes the LOECs determined in each MMT for both 
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MMT ISR 26 May 28, 2013

942 reproduction and the lowest LOEC for the non-reproduction endpoints, and representative 

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

944

945 Table 4-2. Lowest observable effect concentration (LOEC) in each MMT for reproduction and lowest LOEC for 

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

947 the literature for the same test agent.

948

Chemical Laboratory

MMT LOEC Published Results

Fecundity Biomarker Conc. Effect

17β-Estradiol CERI/Japan 28 ng/L F1 2.8 ng/L SSC F1

27.9 ng/La

Fecundity

4-t-Octylphenol EPA/MED 51 μg/L

F2

13 μg/L SSC F2

11.4 μg/Lb

Vitellogenin

13 μg/L Gonad F1

4-t-Octylphenol NIES/Japan 100 μg/L F1

6.25 μg/L SSC F1

11.4 μg/Lb

Vitellogenin

6.25 μg/L Vtg F1/ F2

o,p′-DDT CERI/Japan 1.9 μg/L F2 0.07 μg/L Vtg F1/ F2

0.1 μg/Lc

Vitellogenin

CMP EPA/MED

>345 

μg/L NA 88 μg/L Vtg F0

No data No data

Tamoxifen NIES/Japan 20 μg/L F1

1.3 μg/L Vtg F1/ F2

625 μg/Ld

Fecundity

1.3 μg/L SSC F2

17β-Trenbolone EPA/MED 32 ng/L F1

32 ng/L SSC F1

39.7 ng/Le

32 ng/L Vtg F1 Vitellogenin

32 ng/L Gonad F1

Prochloraz EPA/MED 25 μg/L F2

9 μg/L SSC F1

30 μg/Lf

Fecundity

9 μg/L Vtg F1/ F2

Vinclozolin EPA/MED

>136 

μg/L NA 17 μg/L SSC F1

2500 μg/Lg

Spematogenesis

4-t-Octylphenol EPA/MED 23 μg/L F1/ F2 In process 11.4 μg/LbVitellogenin

949 a: Seki et al., 2005

950 b: Seki et al., 2003a

951 c: Uchida et al., 2010

952 d: Sun et al., 2007a

953 e: Seki et al., 2006

954 f: Zhang et al., 2008

955 g: Kiparissis et al., 2003

956

957

958
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MMT ISR 27 May 28, 2013

959 4.1 Results with ER-agonists 

960

961 4.1.1 17β-Estradiol (CERI/Japan)

962

963 The primary reproductive endpoint (fecundity) was affected by exposure to 17β-estradiol, in both 

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

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

966 other hand, reproduction was not affected by 17β-estradiol exposure in F0, presumably 

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

968 reproductive impairment. 

969

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

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

972 17β-estradiol exposure reported in the literature (Cripe et al., 2009; Hirai et al., 2006; Raimondo 

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

974 was 2.8 ng/L, based on SSC of F1, XY subadult fish. This is the same LOEC reported from a 

975 fish full life-cycle test with medaka for F1 (Seki et al., 2005). In further support of the ER 

976 agonist AOP, vitellogenin production in male livers was induced by 17β-estradiol exposure 

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

978 well. Growth parameters generally were not altered by 17β-estradiol exposure suggesting that 

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

980 tables of the data can be found in the Appendix H: MMT Summary Reports on pp. 1-15.

981

982 4.1.2 4-t-Octylphenol (US EPA/MED)

983

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

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

986 with embryos) at a concentration of 11.4 μg/L (Seki et al., 2003a). The literature clearly 

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

988 2001; Laws et al., 2000). Therefore, it is not surprising that it decreased reproduction in F1 at 

989 102 μg/L and in F2 at 51 μg/L. At 102 μg/L, reproduction was essential shutdown while at 51 

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

991 reproduction was not affected by 4tOP exposure in F0, presumably highlighting a greater 

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

993

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

995 exposure. The overall LOEC for the MMT was 13 μg/L, based on SSC in F2 XY fish, which 

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

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

998 male SSC LOEC was lowered in XY fish in both F1 and F2 at both subadult and adult samplings. 

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

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

1001 XY fish increased at all samplings except F0 with LOECs ranging from 13 μg/L to 51 μg/L. 

1002
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MMT ISR 28 May 28, 2013

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

1004 EDC and non-EDC effects on multiple organs including gonad, liver, and kidney. Generally, the 

1005 observed pathologies in F0 were of low severity, occurred only after exposure to 102 μg/L, and 

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

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

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

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

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

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

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

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

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

1015 very few, if any, pathologies that would be linked to non-EDC toxicity. 

1016

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

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

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

1020 on pp. 16-38.

1021

1022

1023 4.1.3 4-t-Octylphenol (NIES/Japan)

1024

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

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

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

1028 MED versus 8 wpf at NIES), no data were collected in F0, and the suite of endpoints that were 

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

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

1031 could not be analyzed using StatCHARRMS.

1032

1033 Fecundity was reduced by about 80% in F1 after exposure to the highest concentration tested, 

1034 100 μg/L, which resulted in no fish in that treatment in F2. There were no changes in fecundity 

1035 observed in F2 (the highest concentration was 50 μg/L). These results are very similar to the 

1036 MED MMT where the LOEC for fecundity was 102 μg/L in F1 and 51 μg/L in F2.

1037

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

1039 SSCs in XY fish were reduced at a LOEC of 6.25 μg/L in F1 subadults and 25 μg/L in F2

1040 subadults, while vitellogenin production in XY fish was induced at a LOEC of 6.25 μg/L in F2

1041 and only 25 μg/L in F1. The mean hepatic vitellogenin concentration in the same treatment 

1042 across F1 and F2 generations was not very great, 4.8 ng/mg liver in F1 compared to 8 ng/mg liver 

1043 in F2, but F2 was high enough to be statistically different than controls. The endpoint that 

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

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

1046 reported in the NIES MMT so that this was a relatively insensitive endpoint. A comprehensive 
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MMT ISR 29 May 28, 2013

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

1048 Reports on pp. 39-50.

1049

1050

1051 4.1.4 Comparison of 4-t-Octylphenol MMTs in Support of Validation

1052

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

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

1055 NIES Japan provide just such an opportunity. In Table 4-3, the only two parameters in adult 

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

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

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

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

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

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

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

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

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

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

1066

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

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

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

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

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

1073 provided mutually corroborating information on the documented AOP of 4tOP (Table 4-4). 

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

1075 key component of the validation process.

1076

1077

1078

1079

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

Endpoint US EPA/MED LOEC NIES/Japan LOEC

Fecundity 51 μg/L 100 μg/L

SSC 13 μg/L 6.25 μg/L

Vitellogenin 25 μg/L 6.25 μg/L

Gonad Phenotype 25 μg/L 100 μg/L (limited data)

1081

1082
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MMT ISR 30 May 28, 2013

1083 4.1.5 o,p′-DDT (CERI/ Japan)

1084

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

1086 Research Institute) as a part of the US-Japan bilateral agreement to test o,p’-DDT with only the 

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

1088 agonist that would be expected to bioaccumulate (Metcalfe et al., 2000; Uchida et al., 2010). 

1089

1090 Reproduction was not effected by exposure in F0, even at the highest treatment 1.9 μg/L. 

1091 However, in F1, 1.9 μg/L o,p’-DDT did reduce fertility (but not fecundity) mostly resulting from 

1092 two pairs producing large numbers of non-viable eggs. The LOEC for fecundity and fertility in 

1093 F2 was 1.9 μg/L. In this treatment, fecundity was reduced from 20.3 eggs per pair each day to 14 

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

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

1096 reproductive output in the same 1.9 μg/L treatment of F1; however, the control mean in F2 was 

1097 higher than in F1, presumably providing greater statistical power in the analysis of the data.

1098

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

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

1101 modest and did not exhibit the classical dose-response: exacerbation of the response with 

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

1103 increased in adult XY fish in both F1 and F2, but only in the highest treatment, i.e., 1.9 μg/L. 

1104

1105 The secondary endpoints again corroborate the expectation of o,p’-DDT acting as an ER agonist. 

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

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

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

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

1110 μg/L o,p’-DDT across both F1 and F2 generation and at both developmental stages had testicular 

1111 oocytes while in adults in F2 and subadults in F1 had increased testicular oocytes as well. A 

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

1113 MMT Summary Reports on pp. 51-64.

1114

1115 4.1.6 4-chloro-3-methylphenol (US EPA/MED)

1116

1117 The full research MMT was performed with 4-chloro-3-methylphenol (CMP) as the test agent. 

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

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

1120 overt toxicity (Schmieder et al., 2004). 

1121

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

1123 for the possibility of vitellogenin gene expression in XX adult fish in F0 only in which a small 

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

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

1126 of close to 50 different measurements), none were consistently observed across generations or 
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MMT ISR 31 May 28, 2013

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

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

1129

1130 Post-MMT, MED performed a short-term test to establish acute lethality data for medaka to 

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

1132 toxicity/mortality or EDC-related impacts. Newly hatched fish were exposed for 35 days to 

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

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

1135 of EDC effects, was quantified at the end of the exposure. The 7-day and 14-day median lethal 

1136 concentrations to 50% of the fish tested (i.e., LC50 values) were calculated as 2840 μg/L and 

1137 1090 μg/L CMP, respectively. At the end of the 35-day exposure, only fish from control, 300 

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

1139 vitellogenin mRNA in livers of either XX or XY fish indicating CMP is overtly toxic, causing 

1140 mortality, before it would induce EDC-related effects. A comprehensive report including graphs 

1141 and tables of the data can be found in the Appendix H: MMT Summary Reports on pp. 65-80.

1142 4.1.7 Tamoxifen (NIES/Japan)

1143

1144 Tamoxifen is classified as a selective ER modulator (SERM) as it has multiple actions both as an 

1145 ER antagonist and in specific tissues/species as an ER agonist (Shang and Brown, 2002). After 

1146 binding to the ER, tamoxifen has been shown to act as both agonist and antagonist in various 

1147 species including the medaka, in which previous tests have shown increased plasma vitellogenin 

1148 in males but decreased plasma vitellogenin in females (Chikae et al., 2004; Sun et al., 2007a).

1149

1150 The MMT completed by NIES in Japan testing the effects of tamoxifen was an abbreviated 

1151 MMT protocol that went from F0 adults to F2 subadults. 

1152

1153 Fecundity was reduced by over 50% upon exposure to 20 μg/L tamoxifen, the highest 

1154 concentration tested. The AOP related endpoints were typically affected at a LOEC of 1.3 μg/L 

1155 in subadults. These included increased vitellogenin and decreased SSC in XY fish. Similar to 

1156 other studies (Chikae et al., 2004; Sun et al., 2007a), tamoxifen actually reduced vitellogenin in 

1157 female fish, specifically starting at 1.3 μg/L. The induction of vitellogenin in males was fairly 

1158 modest, a few times that of controls but not approaching the same vitellogenin concentration as 

1159 normal female fish. The maximum change in SSC was seen in F2 where the number of anal fin 

1160 papillae was reduced from about 80 per XY fish to approximately 25. These same endpoints had 

1161 slightly higher LOECs (from 1 to 2 treatment levels) in the adult life stage, due either to true 

1162 differences in the sensitivity of the life-stage, or to random variation causing statistical, but not 

1163 true biological differences in the response. Also note, the subadult sampling were not done at the 

1164 same point in development, F1 at 54 dpf and F2 at 65 dpf, which had considerable impact on 

1165 control values for these parameters.

1166

1167 In addition to the AOP (ER agonist; SERM) responsive endpoints, tamoxifen exposure reduced 

1168 growth at 54 dpf (F1) by about 30% in both XX and XY fish, but by less than 20% in XX fish 

1169 only at 65 dpf (F2). It is unclear from the data whether this difference is reflective of delayed 

1170 maturation, where the fish evidently grow to the same size regardless of exposure, or growth is 

1171 irreversibly affected by exposure to tamoxifen. Unfortunately, growth data, which would have 
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MMT ISR 32 May 28, 2013

1172 provided insight into this question was not recorded for adults. And, histopathology, which 

1173 would have provided information both about potential EDC impacts and non-EDC toxicity at the 

1174 organ/tissue level, was also not assessed. A comprehensive report including graphs and tables of 

1175 the data can be found in the Appendix H: MMT Summary Reports on pp. 81-94.

1176

1177 4.2 Results with AR-agonists

1178

1179 4.2.1 17β-Trenbolone (US EPA/MED)

1180

1181 17β-trenbolone acts as a potent agonist of androgen receptors in a variety of systems including 

1182 fish where it has been shown to reduce fecundity, vitellogenin, and generally induce 

1183 masculinization of both external SSCSSC and gonad phenotypes (Ankley et al., 2003; Orn et al., 

1184 2006; Sone et al., 2005; Wilson et al., 2002). In a 21-day exposure of medaka, the LOEC for 

1185 changes in SSC and decreased vitellogenin was 365 ng/L and 39.7 ng/L, respectively (Seki et 

1186 al., 2006). The MMT completed by EPA-MED testing the effects of trenbolone was done with 

1187 the full research MMT protocol.

1188

1189 The LOEC for reproduction, which occurred in F1, was 32 ng/L. At this concentration fecundity 

1190 was reduced to zero. In addition, 13 ng/L 17β-trenbolone seemed to reduce fecundity (control 

1191 fecundity equaled 25 eggs per pair-day versus ~11 eggs per pair-day in the treatment); however, 

1192 it was not statistically significant. No reduction in reproduction was observed in F2 (the highest 

1193 concentration present was 13 ng/L), and the mean fecundity in the 13 ng/L treatment was equal 

1194 to controls (~24 eggs per pair-day) and much higher than in the same treatment in F1 (~11 eggs 

1195 per pair-day).

1196

1197 The LOECs across the entire trenbolone MMT in the AOP-related endpoints ranged from 32 to 

1198 84 ng/L, predominately affecting XX fish. All the findings supported the expected response to 

1199 an AR agonist, i.e., the presence of anal fin papillae (the measure of SSC) in XX fish, decreased 

1200 vitellogenin gene expression in XX fish, and most dramatically, XX fish presenting complete 

1201 male phenotypes including functional testes. In the 84 ng/L treatment, essentially all XX fish 

1202 appeared male for all measurements of phenotype: gonad phenotype, copy number of the 

1203 vitellogenin gene, and SSC. In addition at 32 ng/L of 17β-trenbolone, the same effects were 

1204 observed at intermediate magnitudes except gonad phenotype in which, interestingly, intersex 

1205 gonads were never observed, only complete sex reversal. However, intermediate effects in 

1206 gonad phenotype were observed in a slightly different manner. For instance in F1 subadults, 16 

1207 of 16 XX fish exposed to 84 ng/L 17β-trenbolone had testes, while in the 32 ng/L treatment, only 

1208 7 of 17 XX fish had testes.

1209

1210 The observed pathologies further supported the AOP of 17β-trenbolone. First there were very 

1211 few pathologies found in XY fish. Second, many of the pathologies observed in XX fish could 

1212 be ascribed to an AR agonist exposure. For instance oocyte atresia and decreased yolk formation 

1213 were found in XX subadults and adults at mild to moderate severities. There were also 

1214 pathologies observed in the kidneys of XX fish at high prevalence and severity including tubule 

1215 epithelial hypertrophy and eosinophilia. Ironically, 17β-trenbolone starting at 32 ng/L so 

1216 completely caused sex reversal in the XX individuals that the pathologist observed no 
DRAFT

MMT ISR 33 May 28, 2013

1217 pathologies in the gonads of these animals, in spite of the fact that they were XX fish with testes,

1218 albeit, histologically normal testes. 

1219

1220 In general, the F0 generation was less sensitive than the F1. Because there were no viable fish 

1221 available in the 32 and 84 ng/L treatments of F2, no effects were observed in any of the 

1222 measurements. In addition, the LOECs obtained at the F1 subadult sampling were lower than 

1223 those of the F1 adult sampling. However, whether this was a result of a greater sensitivity at this 

1224 life stage or a result of the statistical impact of greater sample size per replicate at the subadult 

1225 sampling is difficult to determine from the data. 

1226

1227 The suite of biological impacts on the fish within the MMT could be linked across levels of 

1228 biological organization to the well documented mode of action of trenbolone as an AR agonist. 

1229 At the population/organismal level, fecundity was reduced starting at 32 ng/L. This presumably 

1230 reflects the effect trenbolone had on the phenotype of individual XX medaka. Starting at the 32 

1231 ng/L level, profound effects were observed on the phenotype at the individual/tissue level of 

1232 organization, were individual XX fish developed into phenotypic males. This was observed 

1233 across multiple levels of organization including molecular (vitellogenin), tissue (external SSCs 

1234 and pathology), and organ (gonad phenotype) all tethered back to the molecular initiating event, 

1235 i.e., trenbolone binding and activating the AR. A comprehensive report including graphs and 

1236 tables of the data can be found in the Appendix H: MMT Summary Reports on pp. 95-112.

1237

1238 4.3 Results with Steriodogenesis Inhibitor

1239

1240 4.3.1 Prochloraz (US EPA/ MED)

1241

1242 Prochloraz is a fungicide in which its efficacy is based on inhibition of sterol biosynthesis in 

1243 target organisms thereby limiting target organisms ability to synthesize cell walls. Because of 

1244 the conserved nature of the pathways involved, prochloraz also inhibits CYP19 aromatase in fish 

1245 which catalyzes the conversion of testosterone to 17β-estradiol both in the ovary and the brain 

1246 (Hinfray et al., 2006; Monod et al., 1993; Skolness et al., 2011). In addition, there is evidence 

1247 that prochloraz acts as an AR antagonist. In the 21-day fathead minnow reproductive assay, 

1248 prochloraz produces a suite of biological effects characteristic of an aromatase inhibitor and an 

1249 AR antagonist including alterations in various steroid levels, reduced vitellogenin in females, and 

1250 lower cumulative fecundity at 0.1 mg/L prochloraz (Ankley et al., 2005). The MMT completed 

1251 by EPA-MED testing the effects of prochloraz was done with the full research MMT protocol.

1252

1253 The LOEC for fecundity was 41 μg/L in F0 (the highest concentration tested); 25 μg/L in F2, and 

1254 no LOEC was determined at the highest remaining concentration (25 μg/L) in F1. Comparing the 

1255 mean fecundity in the 25 μg/L treatments of F1 versus F2, the means were very close: ~4 eggs per 

1256 pair-day in F1 and ~6 eggs per pair-day in F2. However, only the 25 μg/L treatment in F2 was 

1257 significant because the difference between the exposed group and the appropriate control was 

1258 different: ~10 eggs in F1 and ~20 in F2 as a result of lower than normal fecundity in the F1

1259 control breeding pairs. In fact, three of six breeding pairs in F1 did not spawn. It is anticipated 

1260 that the increased replicate structure of the medaka reproduction test (MRT), proposed as the 
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MMT ISR 34 May 28, 2013

1261 Tier 2 fish test (see section 5), would mitigate the reduction in statistical power caused by the 

1262 random distribution of non-spawning breeding pairs throughout test.

1263

1264 The LOECs across the entire prochloraz MMT of the AOP descriptive endpoints ranged from 5 

1265 to 41 μg/L, affecting both XY and XX fish. Prochloraz affected growth in relatively low 

1266 concentrations but the decreases observed in the subadult samples were 1) not very large in 

1267 absolute terms, and 2) were not present in the adults. In fact, adults in F0 were actually larger 

1268 than controls after exposure to prochloraz. In addition, there were decreased copies of the 

1269 vitellogenin gene in XX fish, which can be plausibly linked to the inhibition of aromatase, 

1270 resulting in a decrease in circulating 17β-estrogen that in the end lowered hepatic vitellogenin 

1271 expression. Lastly, there was a modest reduction of anal fin papillae in F1 subadult XY fish, but 

1272 this was not observed in F2 and even more interesting, it was not observed in adults of all three 

1273 generations.

1274

1275 Many of the pathologies observed in prochloraz MMT were not present consistently through 

1276 generations or between life stages. Therefore, interpretation of the histopathology data was not 

1277 as straightforward as some of the other MMTs. However, some generalities can be made. While 

1278 prochloraz at the concentrations tested did not seem to have much of an impact on the tissues and 

1279 organs of XY fish, it did seem to cause EDC-related pathologies in the ovaries of XX fish (i.e., 

1280 decreased yolk and follicular hyperplasia or hypertrophy). As more information is gathered 

1281 about pathologies associated with exposure to steriodogenesis inhibitors, a clearer picture of the 

1282 patterns of pathology may appear that would allow more comprehensive interpretation of 

1283 histopathology datasets like the one generated in the prochloraz MMT. 

1284

1285 Regardless, taken together, the MMT protocol was able to detect effects of prochloraz exposure 

1286 on the reproduction, the population level parameter. In addition, the MMT provided information 

1287 regarding the presumptive AOP that, while not as conclusive as say an ER agonist, does 

1288 contribute to a WOE approach that would describe prochloraz as a steriodogenesis inhibitor. A 

1289 comprehensive report including graphs and tables of the data can be found in the Appendix H: 

1290 MMT Summary Reports on pp. 113-133.

1291

1292 4.4 Results with AR Antagonist

1293

1294 4.4.1 Vinclozolin (US EPA/ MED)

1295

1296 Vinclozolin and its degradants (M1 and M2) are anti-androgens that, via primarily through 

1297 hydrolysis, also persist in the environment (the half-lives of M1 and M2 are over 180 days) 

1298 (Bayley et al., 2003; Makynen et al., 2000). Vinclozolin is a dicarboximide fungicide that is 

1299 commonly used on fruits, vegetables, ornamental plants, and turf grasses, and therefore is an 

1300 environmentally relevant EDC. The presumptive AOP initiated by vinclozolin is binding with 

1301 the androgen receptor, but not initiating the transcription of androgen-dependent genes 

1302 (Martinovic et al., 2008). 

1303

1304 Vinclozolin is readily hydrolyzed in the exposure water to M1 and M2 (Kelce et al., 1994; Wong 

1305 et al., 1995) which are more potent anti-androgens. Because of this, the characteristics of the 
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MMT ISR 35 May 28, 2013

1306 exposure system delivering toxicant to the aquaria (i.e., flow rates, residence time of toxicant, 

1307 etc.) can quantitatively change the exposure concentrations of the metabolites. Therefore, it 

1308 might be difficult to compare different aquatic bioassays with vinclozolin, especially if they have 

1309 different exposure parameters.

1310

1311 In F0, fecundity decreased in the 253 μg/L treatment starting in the second week of the 

1312 reproduction assessment which is the third week after the start of exposure to vinclozolin. The 

1313 mean for the entire three week reproduction assessment was just 7 eggs spawned per day in 253 

1314 μg/L, compared to 24 spawned eggs per day in controls. Statistically significant reductions in 

1315 the numbers of spawned eggs were also seen in all the other treatment groups (17, 33, 70, and 

1316 136 μg/L) in the last week of the assessment, but these reductions were not great in absolute 

1317 terms: controls at 24 eggs per day and all the treatments at approximately 15 spawned eggs per 

1318 day. For comparison, in a 21-day test measuring reproduction in fathead minnows, vinclozolin 

1319 reduced cumulative egg production by about a third at 60 μg/L.

1320

1321 In F1, there were no effects of vinclozolin exposure at the concentrations tested. The mean 

1322 fecundities observed were 25, 26, 20, 29 (only 1 pair), and 26 in the controls, 17, 33, 70, and 136 

1323 μg/L, respectively. In addition, there was almost no reproduction in the 253 μg/L treatment;; 

1324 however, there was only a single breeding pair in this treatment. 

1325 

1326 There was not an abundance of evidence linking vinclozolin to its AOP as an anti-androgen. The 

1327 only effect on vitellogenin gene expression was increased copy number in XY subadults exposed 

1328 to 136 μg/L (the highest concentration present) in F2 only. In this group, very high 

1329 measurements were made in a few individuals accounting for the statistical significance. 

1330 Possibly linked to the AOP, there was a consistent decrease in anal fin papillae in XY subadults 

1331 with a LOEC of 17 μg/L in F1 and 33 μg/L in F2. In these treatments, the SSC was reduced by a 

1332 25% and 40%, respectively. Presumably, vinclozolin exposure caused a developmental delay, as 

1333 the SSCs were similar to control values in adults in both F1 and F2.

1334

1335 While growth in subadults was not affected by vinclozolin, there was increased weight and 

1336 length in some of the adult sampling groups but not all. In F0, XY fish were heavier, 275 mg in 

1337 controls versus 350 mg at 33 μg/L;; however, this did not increase with higher concentrations of 

1338 vinclozolin. However, XY adult fish were not affected in F1. In addition, XX adult fish were 

1339 larger (weight) in both F0 and F1.

1340

1341 There were few pathologies, fewer that were consistent across generations, and those observed 

1342 were by-and-large of low severity. The most prominent pathology was tubular epithelial 

1343 eosinophilia of F1 subadult kidneys of XY fish which had a LOEC of 17 μg/L and a moderate (3) 

1344 severity at the same concentration. In F2, the same pathology at the same severity was present at 

1345 the LOEC of 70 μg/L. However, there was no evidence of vinclozolin-induced changes to the 

1346 gonad phenotype in the entire MMT. 

1347

1348 It needs to be noted that there were four F1, XY fish in the 33 μg/L treatment (the highest 

1349 treatment with XY fish available for histology) that had normal ovaries, no anal fin papillae, and 

1350 high vitellogenin levels (equal to normal XX levels). In addition, there were three F2 XY fish in 

1351 the 136 μg/L treatment (again the highest treatment with XY fish available for histology) also 
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MMT ISR 36 May 28, 2013

1352 had the same phenotypes, i.e., normal ovaries, no anal fin papillae, and high vitellogenin levels. 

1353 There are two explanations for these observations. First, DNA contamination into XX samples 

1354 from XY fish during the fin clip/DMY assessment procedure would result in this very pattern. 

1355 Second, the data could be appropriate and simply reflect a biological response to vinclozolin 

1356 exposure; however, this seems unlikely because certain aspects of the complete dataset do not 

1357 seem to follow the expected pattern from an EDC with a strong effect on the gonad. For 

1358 instance, there were no instances of XY fish with intersex gonads in the affected treatments or 

1359 the lower treatments. Additionally, there was not a suite of pathologies in the gonad or liver that 

1360 would be easily related to an EDC exposure. 

1361

1362 While our interpretation of the data points to contamination, it is impossible to make a definitive 

1363 statement without a re-assessment of DMY. This can be done on the fixed, paraffin-embedded 

1364 tissue remaining after histology, but we have been unable to successfully extract DNA of 

1365 sufficient quality to perform the DMY assessment on these samples. We suspect that the picric 

1366 acid in the Bouin’s fixative interfered with DNA extraction. Appendix H: MMT Summary 

1367 Reports on pp. 134-151.

1368

1369 4.5 Interlaboratory Evaluation of the MMT protocol - Vinclozolin

1370

1371 4.5.1 Background

1372

1373 An important aspect of the validation of a Tier 2 assay within the EDSCP is demonstrating the 

1374 reliability or reproducibility of the protocol both within and between individual laboratories. To 

1375 that end, the EPA Office of Science Coordination and Policy (OSCP), via contract, initiated an 

1376 interlaboratory evaluation of a proposed MMT protocol that involved three distinct laboratories 

1377 implementing the protocol with vinclozolin as the test agent. These studies allow the evaluation 

1378 of the comparability of test responses across multiple laboratories and are necessary to fully 

1379 evaluate the practical transferability of the protocol and quantitative reproducibility of the 

1380 method. Three laboratories performed the interlaboratory studies and are termed Lab 1, 2, or 3. 

1381

1382 Vinclozolin, an AR antagonist, has tested positive in the EDSP Tier 1 fish assay and would 

1383 therefore be a candidate for EDSP Tier 2 testing. It was also tested in a MMT performed by 

1384 EPA/MED (see 4.4.1) which provides a fourth independent assessment of the reproducibility of 

1385 the protocol. Vinclozolin does pose challenges as a test agent in a validation exercise. It readily 

1386 hydrolyzes in aqueous solution, producing several intermediates which are also potentially active 

1387 EDCs (especially M1 and M2, (Kelce et al., 1994; Wong et al., 1995)) therefore the total 

1388 endocrine effect may combine the effects of vinclozolin and its degradates. Adding to the 

1389 complexity, the relative proportion of vinclozolin and its degradates is time- and pH-dependent. 

1390 Thus, the residence time of the toxicant water in the dilution and delivery apparatus as well as 

1391 the exposure aquaria determines the relative proportion of the parent chemical and its degradates. 

1392 Another challenge posed by vinclozolin is the expected outcomes of an AR antagonist are not as 

1393 well defined as other AOPs like ER or AR agonism. 

1394

1395 A comprehensive report including graphs and tables of the data as well as a summary report 

1396 provided by the lead contractor can be found in the Appendix C: Vinclozolin Interlaboratory 
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MMT ISR 37 May 28, 2013

1397 Report. Excerpts of the interlaboratory summary report authored by the lead contractor are used 

1398 below and are indicated by the use of italics. Where appropriate an EPA perspective is also 

1399 included.

1400

1401

1402 4.5.2 Protocol 

1403

1404 A timeline for the vinclozolin interlaboratory study is illustrated in Figure 4-1. The test spans 

1405 three generations, starting with adults in F0 (minimally 350 mg for females and 250 mg for 

1406 males), going through F1 from fertilization to 14 wpf, and finally ending the study at 8 wpf of F2. 

1407 Fish are sampled as adults of F1 and as subadults (9 wpf) of F1 and F2. Each endpoint is 

1408 determined in the same manner as previously described for the MMT. The activity through the 

1409 course of the test is summarized below. 

1410

1411

1412 Figure 4-1. Timeline for the vinclozolin interlaboratory study. Three generations (F0, F1, and F2) are 

1413 represented by horizontal rows; within a row each small block represents the age of the fish in weeks post 

1414 fertilization (wpf). The total number of weeks of the test is indicated on the bottom of the figure.

1415

1416

1417

1418

1419

1420

1421

1422

1423

1424 Study Weeks 1-4: Unlike the initial research MMT protocol described above, fecundity and 

1425 fertility were not measured in F0 in this later version of the MMT. Consequently, the age of the 

1426 breeding adults for F0 was not as tightly specified as it as for F1 and F2. The specification 

1427 follows: The first generation (F0) adult breeding pairs (10 to 15 week post fertilization; females 

1428 > 300 mg; males > 250 mg) are exposed for four weeks during which time developing oocytes 

1429 and sperm accumulate the test chemical, associated metabolites, and possible parental factors as 

1430 defined by the relative treatment levels. The breeding pairs are placed in an exposure system 

1431 that has six treatment levels including controls with six replicate tanks per treatment level on 

1432 Study Day 1. Each replicate tank houses a single breeding fish pair for a test total of 36 breeding 

1433 pairs (6 treatments with 6 replicates per treatment). Study Week 4: On Study Day 22, eggs are

1434 removed from both the tanks and the females to guarantee an accurate collection the next day. 

1435 The following day (Study Day 23), eggs are: a) collected and pooled from each treatment, b) 

1436 systematically distributed to 6 incubators with a maximum of 20 eggs per incubator, and c) 

1437 incubators are placed in one of two “incubator tanks” set up for each treatment. On Study Day 

1438 24, the process is repeated using the second incubator tank per treatment. 

1439 Study Weeks 5-6: As eggs hatch on each day, hatchlings are pooled for each treatment and 

1440 systematically distributed to each of the six replicate tanks within the specific treatment. The 

1441 appropriate data are collected to calculate the hatching success of each incubator and each 

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 24
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MMT ISR 38 May 28, 2013

1442 collection day. On Study Day 29, the F0 breeding pairs are sacrificed. On Study Day 32 and 33, 

the aeration (agitation) of the eggs from the 1st and 2nd 1443 days of egg collection, respectively, is 

1444 turned off. 

1445 Study Week 7: On Study Day 46, the number of juvenile fish is culled to 10 per replicate tank 

1446 for a total of 60 per treatment. 

1447 Study Weeks 8-10: The exposure continues with the 10 fish per replicate as they develop into 

1448 sub-adults. Study Week 11: On Study Day 71, a small sample is taken from the caudal fin of 

1449 each fish to determine the genotypic sex of the individual. This information is used to establish 

1450 female-male breeding pairs. On Study Days 73 and 74 after the genotypic sex of each fish is 

1451 determined, a single breeding pair per replicate is randomly established while the 8 (nominal) 

1452 remaining fish per replicate are sampled for the various endpoints outlined in Table 1, sub-adults. 

1453 Study Weeks 12-13: The exposure continues as the sub-adult breeding pairs develop into 

1454 adults. 

1455 Study Weeks 14-17: If the test was started on a Monday then on Tuesday through Friday of 

1456 each week (Study Days 93-96, 100-103, 107-110, and 114-117), spawned eggs are counted and 

1457 assessed for fertility. On Monday of each week (Study Days 92, 99, 106, and 113), all eggs, 

1458 either held by the female or deposited in the tank are removed to allow for accurate counting 

1459 starting the next day. 

1460 Study Week 17 (repeat of Study Week 4): On Study Day 113, eggs are removed from both the 

1461 tanks and the females to guarantee an accurate collection the next day. The following day (Study 

1462 Day 114), eggs are; a) collected and pooled from each treatment, b) systematically distributed to 

1463 6 incubators with a maximum of 20 eggs per incubator, and c) incubators are placed in one of 

1464 two incubator aquaria set up for each treatment. On Study Day 115, the process is repeated 

1465 using the second incubator tank per treatment. Study Week 18: On Study Day 120, the F1

1466 breeding pairs are terminated and analyzed for the endpoints in Table 1, adults. 

1467 Study Weeks 18-19: As eggs hatch on each day, hatchlings are pooled for each treatment and 

1468 systematically distributed to each of the six replicate tanks within the specific treatment. The 

1469 appropriate data are collected to calculate the hatching success of each incubator and each 

1470 collection day. 

1471 Study Week 20: On Study Day 137, the number of juvenile fish is culled to 10 per replicate 

1472 tank for a total of 60 per treatment. 

1473 Study Weeks 21-23: The 10 fish per replicate are maintained and become sub-adults. 

1474 Study Week 24: On Study Days 164 and 165, all the fish are sampled for the various endpoints 

1475 in Table 1, sub-adults.

1476

1477 4.5.3 Chemistry Data

1478

1479 Below is summary of the measured vinclozolin concentrations for each of the laboratories. 

1480 Vinclozolin degradate concentrations were not measured. Two of the laboratories maintained 

1481 parent vinclozolin above the nominal concentrations and fairly close to each other; however, the 

1482 measured concentrations during the Lab 1 MMT were considerably lower than nominal, and 

1483 more importantly, the concentrations were much lower (~50%) than the other two MMTs. 

1484 Therefore, it is more appropriate to compare Lab 1 results to one treatment level lower in the Lab 

1485 2 and 3 MMTs. In addition, the measured concentrations of vinclozolin in all treatments during 

1486 the Lab 2 MMT more than doubled around day 100 of the test. 

1487
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MMT ISR 39 May 28, 2013

1488

1489

1490

1491

1492

1493

1494 Table 4-5. Dosing summary for the interlaboratory MMTs.

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 a4.85 12.6 30.9 81.6 197

Lab 2 μg/L <MQL b11.6 25.2 62.0 155 360

Lab 3 μg/L <MQL c8.9 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 111 114 109 111 111

a

1496 Minimum Quantifiable Limit (MQL) = 2.28 μg//L 

b

1497 Minimum Quantifiable Limit (MQL) = 1.14 μg//L 

c

1498 Minimum Quantifiable Limit (MQL) = 5.0 μg//L 

1499

1500

1501

1502 4.5.4 Control Data Supporting Validation

1503

1504 Comparison of control data from each of the laboratory studies provides a measure of the 

1505 natural variation for each of the variables and is summarized in Table 4-6. This analysis 

1506 indicated that for many endpoints, the control response was not consistent between laboratories. 

1507 The median responses from F1 reproductive measurements, F1, F2 length and weight 

1508 measurements were all significantly different (p < 0.01) between laboratories. Closer inspection 

1509 of the results suggest some of the significant inter-lab variability can be attributed to the 

1510 relatively small size at termination of the Lab 2 F1 and F2 fish. The small size of Lab 2 F1

1511 females appears to have contributed to the high inter-lab variability in fecundity. Normalizing 

1512 F1 fecundity for the body mass of the female (F1 total eggs / day / g day 84 female weight) 

1513 eliminated the significant difference (p=0.463). Two endpoints that showed greater consistency, 

1514 which were not statistically different between labs was F1 and F2 anal fin processes and hepatic 

1515 expression of VTG-1. However, it should be noted that successful measurement of anal fin 
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MMT ISR 40 May 28, 2013

1516 processes only occurred for two labs in both the F1 and F2 generation, limiting inter-lab 

1517 comparisons.

1518

1519

1520

1521 Table 4-6. Median Control Response by Chemical and the Kruskal-Wallis Test 

1522

Variable Lab 1 Lab 2 Lab 3 Kruskal-Wallis p-value

F1 Total Eggs/Day 27.1 10.02 34.71 0.005

F1 Total Eggs/Day/ day 84 female weight 170.4 121.5 169.4 0.463

F1 Viable Eggs/Day 21.73 9.68 33.67 0.005

F1 Proportion Viable 0.757 0.9614 0.97086 0.044

F1-Female Day 80/84 Weight (g) 0.161 0.089 0.207 0.002

F1 Female Day 80/84 Length (mm) -- 19.799 26.625 0.01

F1 Female Day 84 Total Processes 7.8 0 --

F1-Female Day 80/84 copies VTG/ng RNA 22289 1195 17470 0.076

F1 Male Day 80/84 Weight (g) 0.127 0.107 0.199 0.001

F1 Male Day 80/84 Length (mm) -- 21.78 26.01 0.004

F1 Male Day 84 Total Processes 26.02 14.73 -- 0.423

F1 Male Day 80/84 copies VTG/ng RNA 2.25 27 1850 0.075

F1 Female Day 119 Weight (g) -- 0.192 0.419 0.006

F1 Female Day 119 Length (mm) -- 27.08 32.4 0.006

F1 Feale Day 119 Total Processes 0 0 --

F1 Male Day 119 Weight (g) -- 0.227 0.389 0.004

F1 Male Day 119 Length (mm) -- 29.38 32.33 0.005

F1 Male Day 119 Total Processes 73.5 53.33 -- 0.025

F2 Female Termination Weight (g) 0.211 0.034 0.226 0.002

F2 Female Termination Length (mm) -- 15.4 27.3 0.004

F2 Female Termination Total Processes 10.1 -- 1.43 0.40

F2 Female Termination copies VTG/ng RNA 23650 0.5 43273 0.103

F2 Male Termination Weight (g) 0.193 0.028 0.222 0.002

F2 Male Termination Length (mm) -- 14.7 28.1 0.004

F2 Male Termination Total Processes 52.9 -- 59.1 1.0

F2 Male Termination copies VTG/ng RNA 1.22 2.50 1.75 0.912

1523

1524

1525 There are two issues that are plausible explanations for the control parameter variation between 

1526 laboratories. First, as mentioned above, is the small size of F1 and F2 fish in the Lab 2 MMT. 

1527 Certainly these fish were not developing like the fish in the other two labs or with what would be 

1528 expected to be a normal growth rate. Because of this, all control parameters of the Lab 2 MMT 

1529 fish would not be expected to meet minimum performance criteria. Also, most of the statistical 

1530 differences between MMT control fish are a result of Lab 2 fish being so much different than 

1531 Lab 1 and Lab 3 fish. Second, as mentioned in the interlaboratory summary report found in 

1532 Appendix C: Vinclozolin Interlaboratory Report, there was a high rate of unexplained genotype 

1533 to phenotype mismatch not only in control fish but across all treatments. Given the prevalence 
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MMT ISR 41 May 28, 2013

1534 of the mismatches, and that both XX-male, and XY-female mismatches were observed, points to 

1535 technical problem in the determination of the genetic sex of the fish. This may have been due to 

1536 problems with sample/label fidelity during tissue collection and processing. Errors in 

1537 determining the genetic sex of individual fish would likely skew many of the control parameters, 

1538 as all of them show some level of sexual dimorphism.

1539

1540 With respect to the issues identified above, EPA believes that the control data do indeed support 

1541 the reproducibility of the protocol as long as genotypic sex data (DMY) are obtained and 

1542 acceptable growth of control fish is achieved. At this time, these are presumed to be technical 

1543 issues involving the appropriate rearing of the animals and sample handling procedures. 

1544 However, the apparent flaws in the data from the interlaboratory also further highlight the need 

1545 for ongoing improvement in the detailed methods for accomplishing the protocol and specific 

1546 training leading to more effective transfer of technical skills required to perform the MMT 

1547 protocol successfully.

1548

1549 4.5.6 Summary of Lab 1 Vinclozolin MMT

1550

1551 In this MMT, vinclozolin did not lower the fecundity rate or the fertility (either the number of 

1552 fertile eggs or percent fertile eggs) of the breeding pairs in any of the treatments of either F0 or 

1553 F1. The F0 reproductive assessment was not part of the original study plan, but during the course 

1554 of the F0 loading phase, fecundity data were collected for six consecutive days and fertility data 

1555 were collected for only two consecutive days. In F1, both fecundity and fertility were assessed, 

1556 according to the protocol, for 21 consecutive days. 

1557

1558 The only affected endpoint that presumably would reflect an EDC exposure was SSC (anal fin 

1559 papillae). XY fish, across both F1 and F2 and in both subadults and adults, had fewer papillae 

1560 than controls. Because genetic sex of individual fish was unreliable, analysis of treatment effects 

1561 on SSC had to be by comparisons to other phenotypic sex markers (i.e., either the gonad 

1562 phenotype or the secondary sex phenotype), and to be completely accurate, these fish are referred 

1563 to as “males” or “females”, instead of the preferred assignment of “XX” or “XY”. Vinclozolin1564 treated subadult and adult male fish, as defined by the gonad or SSC phenotype, in both F1 and 

1565 F2, had fewer anal fin papillae than controls. It was not possible to observe changes in phenotypic 

1566 females, as increases in the number of anal fin papillae would define an individual as a 

1567 phenotypic male. However, in the phenotypic males, there was a decrease in the number of 

1568 papillae in response to vinclozolin exposure with a LOEC of 31 μg/L, and continuing in both the 

1569 82 and 197 μg/L treatments with similar effect magnitudes. The control males had a mean of 77 

1570 papillae while in these three treatments (31, 82, and 197 μg/L), the mean number of papillae 

1571 were 59, 53, and 52, respectively. In F1 subadult males, vinclozolin at a concentration of 197 

1572 μg/L reduced the mean number of anal fin papillae from 32 as seen in controls to a mean of 20 

1573 per fish. In F2, the LOEC was 5 μg/L and the reduction of papillae consistently decreased in a 

1574 dose-dependent manner to the highest concentration (197 μg/L). For unknown reasons, the mean 

1575 number of papillae in F1 control males was much lower than seen in the male controls of F2 (32 

1576 versus 73). The mean in males for each of the vinclozolin exposed groups of F1 was actually 

1577 similar (or often lower) to the corresponding treatment group of F2. Therefore, the LOEC and 

1578 the apparent increased sensitivity of F2 may have been a result of the further developed fish, as 
DRAFT

MMT ISR 42 May 28, 2013

1579 measured by SSC, in F2 as compared to the F1 fish. A comprehensive report including graphs 

1580 and tables of the data as well as a summary report can be found in the Appendix C: Vinclozolin 

1581 Interlaboratory Report.

1582 

1583 4.5.7 Summary of Lab 2 Vinclozolin MMT

1584

1585 In this MMT, vinclozolin did not lower the fecundity rate or the fertility (either the number of 

1586 fertile eggs or percent fertile eggs) of the breeding pairs in any of the treatments of F1, where 

1587 both fecundity and fertility were assessed, as per the protocol, for 21 consecutive days. The 

1588 mean fecundities observed were 10, 10, 8, 9, 7, and 7 in the controls, 12, 25, 62, 155, and 360

1589 μg/L, respectively. In addition, the fertility was high across all the treatments. 

1590

1591 In the other endpoints, vitellogenin gene expression was induced in XX fish of F1 only in the 

1592 highest treatment tested (360 μg/L) from a mean of 830 copies per ng of total RNA to 8910 

1593 copies. Growth (weight) was increased by vinclozolin exposure in XX fish of both generations 

1594 (F1 and F2) with a LOEC of 12 μg/L (the lowest treatment), and in XY fish in F1 only with a 

1595 LOEC of 62 μg/L.

1596

1597 4.5.8 Summary of Lab 3 Vinclozolin MMT

1598

1599 In the primary endpoint of the MMT, reproduction, no changes in fecundity or fertility were 

1600 observed upon exposure to vinclozolin. In F0, fecundity and fertility was based upon counts of 

1601 pooled eggs. Therefore the box plots presented represent inter-day variance of the entire 

1602 treatment and not inter-breeding pair variance. To avoid confusion, the individual data, daily 

1603 counts of whole treatments, were not plotted on the graph for F0. While there was not a 

1604 statistically significant reduction in fecundity or fertility, there was a trend of lower fertility with 

1605 vinclozolin exposure. In F1, there were no effects of vinclozolin exposure on either fecundity or 

1606 fertility at the concentrations tested. The mean fecundities observed were 35, 28, 34, 33, 28, and 

1607 37, in the control, 9 μg/L, 22 μg/L, 52 μg/L, 133 μg/L, and 333 μg/L treatments, respectively. 

1608 The fertility was high across all the treatments ranging from 92% to 98%. 

1609

1610 Of the other endpoints, anal fin papillae were reduced in XY fish in F2 subadults only. 

1611 Unfortunately, there was a mistake in sample handling with the F1 subadult anal fins so that no 

1612 counts of anal fin papillae were possible. Vinclozolin reduced the mean number of papillae in 

1613 the XY subadult fish of F2 at all concentrations tested (9 – 333 μg/L). The mean number of 

1614 papillae was 57 in the control and was reduced to a mean of 39, 42, 40, 33 and 19 papillae in the 

1615 9 μg/L, 22 μg/L, 52 μg/L, 133 μg/L, and 333 μg/L treatments, respectively.

1616

1617 4.5.9 EPA Perspective on the Interlaboratory Results 

1618

1619 Overall the interlaboratory was a success insomuch as all three labs did not see decreased 

1620 reproduction at the concentrations of vinclozolin tested. This is also unfortunate for the very 

1621 same reason. Other endpoints were not affected by vinclozolin with the exception of SSC in 

1622 which was, when available for assessment, negatively responsive to vinclozolin exposure, and in 
DRAFT

MMT ISR 43 May 28, 2013

1623 the Lab 2 MMT only, sporadic increases in weight and vitellogenin were observed. As a note, 

1624 SSC was also decreased in the vinclozolin MMT performed by EPA/MED. So while few 

1625 significant effects were observed in the three interlaboratory MMTs, there was consistency in the 

1626 observation of effects on the SSC. In addition, the control data suggest the protocol is 

1627 reproducible even though most of the measurements had statistical differences between the three 

1628 labs. Recall, one lab had unusually poor control growth that seemed to cause developmental 

1629 delay in control fish, which, in turn resulted in poor control reproduction. When those data are 

1630 removed, there was considerable consistency between the data of the other two laboratories.

1631

1632 Unfortunately, the previously discussed issues with the interlaboratory data cannot simply be 

1633 ignored, and they highlight that this is a relatively complicated procedure that even with 

1634 experience is not a trivial undertaking. Many of the directives within the protocol are derived 

1635 from extensive experience at EPA/MED lab, and without a priori knowledge, should be 

1636 followed without deviation. Otherwise, there is a relatively high risk of generating poor quality 

1637 data with high variances. The consequence is that it is difficult to discern treatment-related 

1638 effects with all the endpoints, but especially on reproduction. Low reproduction in controls 

1639 seriously compromises the statistical power of the MMT to detect decreases in fecundity or 

1640 fertility. In addition, knowing the genetic sex (DMY) of each fish is an incredibly powerful tool 

1641 for analysis of the data. To maximize the statistical power for each endpoint requires this 

1642 information. Proficiency, both in DNA preparation and in the determination of the presence or 

1643 absence of DMY, must be achieved prior to implementation of the MMT protocol. 

1644

1645 The suite of expertise required to perform a MMT is broad, and there are many nuances that need 

1646 to be recognized by performing labs. The limitations in the interlaboratory trial data are not 

1647 indicative of low protocol reproducibility, but rather to an inadequate transfer of the technical 

1648 details necessary to successfully execute the MMT. 

1649

1650 5 OPTIMIZATION OF THE MMT AND THE PROPOSED MEDAKA 

1651 REPRODUCTION TEST (MRT) PROTOCOL

1652

1653 5.1 Primary MMT Protocol Changes for development of MRT Protocol

1654

1655 The outcomes of the various MMT protocols with several different chemicals performed at the 

1656 EPA MED lab and by the NIES and CERI laboratories in Japan, have provided enough 

1657 information to provide guidance for the development of a medaka reproduction test (MRT) for 

1658 use as a fish test in Tier 2 of the EDSP. Two major changes are proposed, i.e., an increase in the 

1659 number of replicates per treatment for evaluating effects on reproduction, and terminating the 

1660 test after the embryos hatch in F2. Other proposed changes include minimizing the collection of 

1661 endpoint data from F0, and evaluating pathology in only the F1 adults sampled after the 

1662 assessment of reproduction. Figure 5-1 presents the proposed timeline and endpoint data for the 

1663 MRT. The test conditions for the proposed MRT protocol are presented in Table 5-1. Endpoint 

1664 measurements are essentially identical to those employed in the full MMT except that the sagittal 

1665 sectioning plane is recommended for the pathology assessment. Comparisons of various aspects 

1666 of the research MMT and the proposed MRT are presented Table 5-2. The rationale for the 

1667 recommended changes in the MMT for the proposed Tier 2 fish test of the EDSP, are presented 
DRAFT

MMT ISR 44 May 28, 2013

1668 in the following sections. Detailed protocols of the Proposed Medaka Reproduction Test are 

1669 presented in Appendix E: MRT Protocol.

1670

1671

1672 Figure 5-1. Proposed Medaka Reproduction Test (MRT) for Tier 2 The timeline and endpoints in the 

1673 MRT. The duration of the in-life exposure is 19 weeks and spans 1 full generation (F1). The F0 generation serves as 

1674 a loading phase for the F1 embryos. The test is terminated after the F2 embryos hatch.

1675

Exposure and Endpoint Timeline Gen

F0 1 2 3 4

F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

F2 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 F1

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 treatments

2 blocks for control treatments

Fertility F1

Hatch F1 F2

Survival F1 F1 F1

Growth F1 F1

Vitellogenin F1

Secondary sex F1 F1

Histopathology F1

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

1680 Table 5-1. Test Conditions for the Proposed MRT

1681

Recommended species Medaka (Oryzias latipes)

Test type Continuous flow-through

Water temperature 25.5 ± 1oC 

Illumination quality Fluorescent bulbs (wide spectrum and ~150 lumens)

Photoperiod 16 h light, 8 h dark

Loading rate F0- 2 fish/replicate; F1 and F2- 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 F0-11 weeks post-fertilization, F1 and F2 continuously from fertilization

No. of organisms per replicate F0- 2 adults; F1 and F2- 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 

F0- 24 for each test concentration and 48 for controls; F1 and F2- 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 

(postBrine Shrimp(mg dry 

Time 

(postBrine Shrimp(mg dry weight/fish/day)
DRAFT

MMT ISR 45 May 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: F0from 11-15 wpf, F1 and F2from 0-15 wpf

Primary endpoints Survival: F1 from 1-3 wpf and from 3-15 wpf

Growth : length, weight, (F1 at 9 wpf and 15 wpf 

Hatch: F1and F2 only

Anal Fin Papillae: F1 assessed at 9 wpf and 15 wpf 

Fecundity: F1 assessed daily for 21 days, 12-14 wpf

Fertility: F1 assessed daily for 21 days, 12-14 wpf

Vitellogenin: F1 only at 9wpf

Optional endpoints Time to first spawn for F1 generation.

Test acceptability Dissolved oxygen >60% of saturation; mean temperature of 25.5 ± 1oC; 70% survival 

in control treatments of each generation; successful reproduction in at least 65% of 

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

1682

1683

1684

1685

1686 Table 5-2 Comparison of the Research MMT to the Proposed Tier 2 Fish Reproduction Protocol

1687

Research MMT Proposed MRT Rationale

Replication 

structure 6 replicates/trtmnt 12 replicates/trtmnt24 control replicates

Statistical power for 

reproductive analysis

Generations 2 1⁄2 (F0, F1, partial F2) 1 1⁄2 (F0, F1, F2 hatch only)

Reduce test time, chemical 

amount, cost, risk of test 

failure

F0 for dose-level 

evaluation and to expose 

developing gametes with 

test chemical

F0 endpoints

Reproduction, Growth, 

Survival, Vtg, SSC, 

Pathology

Reproduction – qualitative 

only

Adequate dose-level 

evaluation

Survival Adequate dose-level 

evaluation

No other endpoints 

measured

F0 < sensitive than F1 and 

F2

F2 endpoints

Hatch, Growth, Survival, 

Vtg, SSC, Reproduction, 

Pathology

Hatch only

Ensure adequate time for 

deposition of test agent to 

egg.
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MMT ISR 46 May 28, 2013

No other endpoints 

measured

Substantial and consistent 

sensitivity differences 

between F1 and F2 were 

not observed for the 

chemicals tested

F1 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, Th, FF only). 

Better for statistics and test 

duration 

Temperature 26 25.5

Maximize development 

rate; minimize prevalence 

of XX males

Pathology F0 adult

F1, F2 subadult and adult 

F1 adult

Increased power for 

pathology analysis, 

pathology correlated with 

pair reproduction 

outcomes 

1688

1689

1690 5.1.1 Increased Replication for the Assessment of Treatment-Effects on Reproduction

1691

1692 The reproductive endpoints of fecundity and fertility are important endpoints for ecotoxicology 

1693 assessments, due to their implicit direct relationship to effects at the population-level. To assess 

1694 treatment effects on adult reproduction, the full MMT protocol employed 6 replicate aquaria for 

1695 each test treatment, including the controls. Fecundity data from the full MMTs had relatively 

1696 high variance. Thus, with only 6 breeding replicates per treatment, the statistical power for 

1697 detecting significant changes in fecundity was quite low. Some of the modifications of the 

1698 protocol were implemented to ostensibly increase the power of the protocol either by increasing 

1699 baseline fecundity or by reducing the variance in egg production. Such modifications included 

1700 increasing the test temperature, increasing growth with feeding rates or by increasing the 

1701 development time before assessing reproduction. These modifications were only marginally 

1702 successful. 

1703

1704 After running a number of MMTs and evaluating the reproduction data, it became clear that the 

1705 statistical power for detecting significant effects on fecundity in the full MMT was quite low. 

1706 The trends observed in the dose-responses for fecundity suggested that fecundity was being 

1707 affected at treatment levels lower than the statistical LOEC. In some cases, the fecundity had to 

1708 be reduced to nearly zero to be significantly different from the control fecundity values. It 
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MMT ISR 47 May 28, 2013

1709 appeared that the most reliable method to increase the statistical power of the test to detect 

1710 treatment effects on reproduction was through increasing the number of breeding pairs.

1711

1712 To determine the number of replicates required a power analysis was performed using the 

1713 reproductive performance of control fish from several of the MMTs run at the MED laboratory 

1714 (Appendix I: Power Analysis). MMT fecundity data have characteristics that complicate the 

1715 calculations of statistical power. For example, the data are not transformable to normally 

1716 distributed data; they are measured repeatedly over time with several replicates in each 

1717 treatment; and days without reproduction (i.e., where the number of spawned eggs is zero) are 

1718 randomly distributed in the data set. Because of these characteristics, a numerical solution for 

1719 power was calculated using Monte Carlo simulation methods.

1720

1721 In order to design the Monte Carlo simulation, various assumptions were made including that the 

1722 between aquarium within treatment mean fecundity is normally distributed, the within pair 

1723 fecundity follows a Poisson distribution (typical of count data), the control fecundity does not 

1724 change during the reproductive assessment, and finally the rate at which individual pairs produce 

1725 zero eggs is constant. This last assumption is important because the experience of the MED 

1726 laboratory has been that a certain percent of breeding pairs will not successfully spawn for 

1727 unknown reasons, and this can be daily, weekly, or for the entirety of the assessment.

1728

1729 Using these assumptions and the control data from previous MMTs, the following parameters 

1730 were calculated for the Monte Carlo simulation: the population mean = 21 eggs and the 

1731 population variance = 49. Also the rate at which the fecundity of a pair was zero can be 

1732 separated into three categories; one day, one week (greater than one day but less than 8 days), 

1733 and always (greater than 8 days). These were estimated to be 8/91 for one day, 5/91 for one 

1734 week, and 3/91 for all days.

1735

1736 The data were simulated in the R programming language incorporating the assumptions above to 

1737 generate a fecundity dataset of control plus 5 pre-assigned reductions in fecundity (i.e.,

1738 treatments) of 20%, 30% 40%, 50% and 70% reduction. This process was repeated 1000 times 

1739 for each pair and percent reduction combination. Next, all of the simulated data were analyzed 

1740 by the repeated measures ANOVA of the StatCHARRMS program. Two different replicate 

1741 structures were analyzed: 1) the number of replicates is equal in all treatments including the 

1742 controls (balanced design), and 2) based upon statistical recommendations from the OECD 

1743 (OECD, 2006), the number of control replicates was double the number of replicates in each 

1744 chemical treatment (2:1 design). The 2:1 design had substantially greater power and is the only 

1745 analysis presented here.

1746

1747 Graphic results are illustrated in Figure 5-2. Note that at 6 replicates (12 replicates in the 

1748 control), there is a small but noticeable probability of not detecting a reduction of 50%, about a 

1749 75% probability at detecting a reduction of 40%, less than 50% chance at detecting a reduction 

1750 of 30%, and very little chance at detecting a reduction of 20%. At 12 replicates (24 replicates in 

1751 the control) the probability of not detecting a reduction of 40% or greater is near zero, and there 

1752 is probability of greater than 80% of detecting a reduction as low 30%. 

1753

1754
DRAFT

MMT ISR 48 May 28, 2013

1755

1756

1757 Figure 5-2. Monte Carlo simulation of the statistical power to detect proportionate reductions in medaka fecundity 

1758 with different numbers of replicate breeding pairs. The experimental design represented is the 2:1 design where 

1759 control replicates are double of the number of replicates in the chemical treatments. The power is the probability of 

1760 detecting a treatment effects compared to the controls at an α-level of 0.05.

1761

1762 During the discussions on revising the replicate structure of the MMT, consideration was given 

1763 not only to the power analysis, but also to the possibility of mortality, especially in the control 

1764 replicates. It has been the experience of MED researchers that a very small percentage of the 

1765 adults, irrespective of treatment, may die, and in addition, even with skilled technicians, there is 

1766 a possibility of handling-induced mortalities as well. To be conservative, 12 breeding pairs 

1767 (replicates) in treatments and 24 control breeding pairs (replicates) was chosen to mitigate the 

1768 consequences to statistical power.

1769

1770 Reproduction Results from Two Octylphenol MMTs: 6 vs. 12 Replicate Breeding Pairs

1771 To evaluate the effect of increasing the replication structure of the MMT, two tests (designated 

1772 PTOP-02 and PTOP-03) were run at the EPA MED lab with the weak ER agonist 4-tert1773 octylphenol. PTOP-02, which employed 1 breeding pair in each of 6 replicate aquaria per 

1774 treatment, was started late in 2007. PTOP-03, which was started late in 2012, employed 12 

1775 replicate aquaria per treatment, except for the control treatment which had 24 replicates. The 

1776 chemical concentrations were nearly identical for the two tests (see Table 5-3). Reproductive 

1777 assessments were similar except that for PTOP-02 fecundity and fertility were measured 4 days 

1778 per week for 5 weeks and for PTOP-03 the measurements were taken for 21 consecutive days. 

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 2:1
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MMT ISR 49 May 28, 2013

1779 There were no treatment effects on F0 reproduction in PTOP-02 (Appendix H: MMT Summary 

1780 Reports on pp. 16-38) and reproduction endpoints were not measured in PTOP-03.

1781

1782 Table 5-3 Comparative Treatment Concentrations for two 4-t-Octylphenol MMTs

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

1784 *: S.D not calculated for this test yet

1785

1786

1787 Figure 5-3. Reproduction Results from 6- and 12- Replicate Test Protocols

1788

1789

1790

1791 The primary effect of octylphenol on reproduction is to reduce the fecundity and not the fertility 

1792 of treated fish. Note also that the control means for fecundity are similar in F1 and F2 for both 

F1 Reproduction

F1 Reproduction

F2 Reproduction

F2Reproduction

6 Reps

12 Reps

LOEC

LOEC

LOEC

LOEC
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MMT ISR 50 May 28, 2013

1793 tests and range from 17 to 21 eggs/pair/day. The LOEC for F1 fecundity in the 6 rep design was 

1794 102μg/L which reduced the fecundity to nearly 0. Because of this, there were insufficient 

1795 survivors to populate that treatment in F2. The LOEC for F2 fecundity in the same test was 

1796 51μg/L. There were no significant differences between the mean fecundity at any particular 

1797 treatment level across generations (i.e., F1 control fecundity of 17 vs. F2 control fecundity of 

1798 19.5; or F1 51μg/L fecundity of 14 vs. F2 51μg/L fecundity of 8).

1799

1800 In the 12 replicate test, the LOEC for fecundity was 23μg/L for both the F1 and F2 generations, 

1801 one treatment level lower than the LOEC for the 6 replicate test. Note also that the reduction in 

1802 fecundity observed at the LOEC was only about 7.5 eggs/pair/day in F1 and 5 eggs/pair/day in 

1803 F2. In summary, doubling the number of replicate breeding pairs increased the statistical power 

1804 of the test to detect significant treatment effects on fecundity.

1805

1806 The 12 replicates per treatment design also has increased power due to the fact that there are two 

1807 blocks of 12 replicates for the control treatment. There are a total of 84 replicate aquaria in the 

1808 reproductive phase of the test; ((5 chemical treatments) x 12 replicates + (2 control blocks x 12 

1809 replicates) = 84). While this may seem excessive, this number of replicates is necessary due to 

1810 the highly variable nature of fish reproduction. Furthermore, this design provides additional 

1811 insurance for the baseline probability of having non-spawning pairs within the test population 

1812 and for the inevitable loss of replicates due to technical accidents. The difference in the statistical 

1813 power for fecundity in the MRT with 12 breeding replicates for each of the chemical treatments 

1814 and 24 replicates in the controls is presented in Table 5-4. Note that in the MMT a 40% 

1815 reduction in fecundity will be detected with a probability of 66 %, and a 28% probability of 

1816 detecting a 30% reduction in fecundity. In contrast, with the MRT the probability of detecting 

1817 40% and 30% reductions in fecundity are 99% and 83% respectively.

1818

1819 Table 5-4 Statistical Power Comparison for Fecundity in the MMT and the proposed MRT

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

1822 When considering the number of fish employed in the two different test designs, it is important 

1823 to note that there are actually fewer fish used in the 12-replicate MRT design. First, the initial 

1824 phase of the protocol employs 6 replicates per treatment. And, the initial number of embryos 

1825 placed in incubators has been reduced from 30 to 20. Then, when starting the reproductive phase 

1826 of the test at 9 wpf, 4 fish per replicate are used to set up two breeding pairs rather than the 2 fish 

1827 for the single pair per replicate employed in the used in the 6-replicate design of the MMT. The 

1828 increase in statistical power afforded by the 12 replicate design is accomplished by reallocating 

1829 fish from the 9 wpf sampling into the analysis of treatment effects on reproduction. The 

1830 concomitant reduction in the statistical power of the non-reproductive endpoints (i.e., 6 fish per 
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MMT ISR 51 May 28, 2013

1831 replicate in the MRT compared with 8 per replicate in the MMT) is relatively small. Specifically, 

1832 Table 5-5 presents the statistical power of the MRT to detect changes in the reductions in the 

1833 biomarker endpoints (i.e., wet weight, length, vitellogenin and SSC) in fish sampled at 9 wpf. 

1834 Note that except for the female vitellogenin endpoint, the power to detect treatment effects in 

1835 these endpoints in the 9 wpf samples is similar or higher than the power to detect effects in 

1836 fecundity. Thus the statistical power of the endpoints appears to be reasonably balanced in 

1837 experimental design of the proposed MRT (Appendix I: Power Analysis).

1838

1839 Table 5-5. Statistical Power of Non-reproductive Endpoints in Proposed MRT

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 0.62 0.93 1.00 1.00 1.00

1840

1841

1842 Comparison of Animal Use between the MMT and MRT

1843

1844 An evaluation of the number of fish that are used in the research MMT protocol compared with 

1845 the proposed MRT indicates that even though the number of replicate breeding pairs is doubled 

1846 in the chemical treatments and is 4x high in the controls, the total number of fish sampled and 

1847 culled is less in the MRT; 2,520 fish in the MRT compared with 2,952 in the MMT. 

1848 Furthermore, if one discounts the portion of newly hatched, as suggested by the OECD 

1849 guidelines for fish embryo toxicity tests, eleutheroembryos which are not continued to the 

1850 juvenile and adult stages in this calculation, then there are far fewer fish employed in the MRT; 

1851 558 juvenile and/or adult fish in the MRT compared with the same 2,952 juvenile and adult fish 

1852 used in the research MMT. 

1853

1854 Estimates of person-hours required to perform the MMT and MRT

1855

1856 Two critical changes in the MRT compared with the MMT are the recommended increase in the 

1857 number of breeding replicates employed and, the completion of the in-life potion of the test after 

1858 embryo hatch in F2. The increase in replication requires an increase in the human resources 

1859 necessary to perform the MRT, especially during the assessment of fecundity and fertility. In 

1860 contrast, the decrease in the exposure time reduces the necessary human resources to perform the 
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MMT ISR 52 May 28, 2013

1861 test. A comparison of the time required to perform the MRT and the MMT is presented in Table 

1862 5-6.

1863

1864 Table 5-6. Human-Resource Estimates for In-life Portions of MMT and MRT (hours)

1865

Resource Category MMT MRT

Technician Hours 1197 939

Biologist Hours 424 297

Chemist Hours 590 382

Total Hours 2211 1618

1866

1867 Note that even with the replicate increase, the estimate of the total human resource time required 

1868 to run the in-life portion of the MRT is about 25% less than the MMT.

1869

1870 5.1.2 In-life Exposure Complete After F2 Hatch

1871

1872 The context of this discussion is whether the data from the F2 generation added significant value 

1873 to the understanding of treatment effects on the test population. A summary of the data from all 

1874 the MMTs performed by EPA or Japanese labs that included assessments of fecundity in both F1

1875 and F2 is presented in Table 5-7. With 17β-estradiol the LOEC for fecundity was lower by 1 

1876 treatment level in F1 compared to F2. In one of the three tests with 4-t-octylphenol, the LOEC in 

1877 F2 was 1 treatment level lower and in the other two the LOECs were the same in both 

1878 generations. Note that the LOEC in the 2012 octylphenol test, which had 12 breeding pairs per 

1879 treatment, was 1 treatment level lower than the previous test run by EPA, and that the LOECs 

1880 were the same in F1 and F2. The LOEC for the Japanese run o,p′-DDT test was not determinable 

1881 with a greater-than 1.9 μg/L value for F1 and 1.9 μg/L in F2. Lastly, the LOECs for fecundity in 

1882 F1 and F2 for the 17β-trenbolone test run by EPA were equal. In summary, for the six tests using 

1883 three different chemicals where effects on fecundity were measured in both the F1 and F2

1884 generations, the LOEC was lower in F1 in one test; lower in F2 in two tests, and the same in three 

1885 tests. In all cases, when the LOECs were different across generations, it was by one treatment 

1886 level. In the 2012 4-t-octylphenol test with 12 replicate breeding pairs, the LOECs were the same 

1887 in both generations and lower than the LOECs measured in the other two MMTs with 

1888 octylphenol. An MMT with more replication in the assessment of reproductive endpoints, and 

1889 without an assessment of reproduction in F2 (i.e., the proposed MRT) appears to detect effects on 

1890 fecundity as well or better than the information obtained in both generations of the research 

1891 MMT. Not only was the LOEC lower with 12 breeding pairs, but the LOEC in F1 and F2 were 

1892 the same. Additionally, the mean fecundity values in the control treatments and the LOEC 

1893 treatments were quite similar across generations. Finally, differences of the mean fecundity 

1894 levels in a particular treatment across generations were not statistically significant. 

1895

1896

1897

1898

1899

1900

1901

1902
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MMT ISR 53 May 28, 2013

1903 Table 5-7. LOECs for Fecundity in F1 compared with F2 in MMTs

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

17β-Trenbolone US EPA/MED 32 ng/L 32 ng/L

1905

1906 Comparisons of the lowest LOECs for the biomarker endpoints obtained from the subadult 

1907 samples in 6 MMTs is presented in Table 5-8. Note that the LOECs for the most sensitive 

1908 biomarker endpoints were the same in F1 and F2 except for one case, i.e., the US-run test with 

1909 octylphenol, were the LOEC for anal fin papillae was one treatment level lower in F2 than in F1.

1910

1911 Table 5-8. LOECs for Biomarker endpoints in F1 compared to F2 in MMTs

1912

Chemical Laboratory

Biomarker LOEC

Data through Endpoint

F1

Data through 

F2

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

17β-Trenbolone US EPA/MED 32 ng/L 32 ng/L Papillae and Liver 

vitellogenin

1913

1914 Occasionally, there is a LOEC for an endpoint in the F2 generation that is lower than the LOEC 

1915 for the same endpoint in F1. In all cases, the LOEC is different by only one treatment level. One 

1916 of the requirements for making valid comparisons of differences in the treatment effects among 

1917 generations is that the test conditions are the same in both generations. It is very difficult to 

1918 actually achieve this requirement, especially with respect to the chemical exposure 

1919 concentrations. Furthermore, in those cases where chemical concentrations are not the same 

1920 across generations, it is essentially not possible to discern whether the difference between the 

1921 LOECs are due to differences in test organism sensitivity or to differences in the chemical 

1922 exposure at a critical window in fishes life history.

1923

1924 When considering using multiple generations in a test protocol it is appropriate to consider 

1925 whether the additional data provided by each generation increases the quality of the risk 

1926 assessment, and whether the test protocol is likely to be completed successfully. In general, our 

1927 experience the longer a test continues, the greater the risk of incidents resulting in test failure. 

1928 The causes of failure include exposure and/or dilution pump malfunctions, malfunctions in 

1929 temperature control systems, disease, and, perhaps the most common problem -- maintaining 

1930 consistent treatment concentrations throughout the test. As the test progresses, heterotrophic 

1931 microbes which use the test chemical as an energy source, are selected for until they thrive and 
DRAFT

MMT ISR 54 May 28, 2013

1932 proliferate in the exposure system. These microbes metabolize the test chemical with subsequent 

1933 reductions in the treatment concentrations. To deal with this, the exposure system needs to be 

1934 periodically sterilized and, adjustments to the dilution system must be made frequently to 

1935 accommodate reductions in treatment concentrations. These adjustments are less arduous or 

1936 fewer in the shorter test protocol. 

1937

1938 5.1.3 Effect-endpoints Not Measured in F0

1939

1940 Results from full MMT tests indicate that the LOEC for any particular endpoint in F0 is never 

1941 lower than the LOEC from either F1 or F2 for that endpoint. In a few MMTs the F0 LOEC was 

1942 the same as the LOEC in F1 or F2, (see Table 5-9). Consequently, we recommend that it is not 

1943 necessary to measure endpoints in F0.

1944

1945 Table 5-9. Comparative LOEC values for measurement endpoints from several research MMTs across generations. 

1946 Green colored cells indicate the lowest LOEC among the three generations for that particular endpoint. Greater-than 

1947 values indicate the LOEC for that particular generation was higher than the highest concentration that still had fish. 

1948 Purple cells indicate that the LOEC is higher than the highest concentration tested.

1949

Endpoint F0 F1 F2

E2

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 μg/L 51 μg/L >51 μg/L

Weight-XX 102 μg/L 13 μg/L >51 μg/L

SSC >102 μg/L 102 μg/L 13 μg/L

Vitellogenin 51 μg/L 51 μg/L 51 μg/L

Gonad Phenotype 102 μg/L 13 μg/L 51 μg/L

Fecundity >102 μg/L 102 μg/L 51 μg/L

DDT

SSC >1.9 μg/L >1.9 μg/L >1.9 μg/L

Vitellogenin 1.9 μg/L 0.69 μg/L 0.22 μg/L

Gonad Phenotype >1.9 μg/L 1.9 μg/L 0.69 μg/L

Fecundity >1.9 μg/L >1.9 μg/L 1.9 μg/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 μg/L >25 μg/L >25 μg/L

Fecundity 41 μg/L >25 μg/L 25 μg/L

1950

1951

1952 5.1.4 Measure Subadult Endpoints at 9 wpf

1953

1954 At ORD/MED, the subadult sampling had been done, depending on tests, at times between 44 

1955 and 59 dpf. Within this time window, medaka are growing and developing rapidly enough that 
DRAFT

MMT ISR 55 May 28, 2013

1956 shifting the subadult sampling time a week earlier or later has dramatic impacts on the data 

1957 collected. For instance, the medaka growth curve is very steep at this time (Figure 5-4).

1958

1959 Figure 5-4. Medaka Growth-Curve in MMT and MRT protocols

1960

1961 Concurrent with the rapid growth observed, the development of SSC (i.e. anal fin papillae) 

1962 occurs around 40 dpf. Therefore by sampling subadults at 9 wpf, males will have high numbers 

1963 of anal fin papillae (Figure 5-5). In addition, development of the gonad is dramatic during this 

1964 phase of development as well, and there is a substantial increase in the production of 

1965 vitellogenin. In typical MMTs, the mean number of copies of vitellogenin per ng of total liver 

RNA was 2 x 105 at 6 wpf and 5 x 106 1966 at 7 wpf. Another case in point, the mean liver 

1967 vitellogenin concentration in subadult control females of the F1 generation in the tamoxifen 

1968 MMT performed by NIES/Japan was approximately 40 ng/mg liver while the mean of the 

1969 subadult females of F2 in the same MMT was approximately 1350 ng/mg liver. The difference 

1970 between the subadult samplings of F1 and F2 in the tamoxifen MMT was that in F1 the sampling 

1971 was done at 54 dpf while in F2 the subadult sampling was done at 65 dpf, a difference of 11 

1972 days.

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 WEIGHT
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MMT ISR 56 May 28, 2013

1974 Figure 5-5. Time-course of Anal-fin Papillae Development

1975

1976

1977 Based upon the information gathered, the recommendation is to have the subadult sampling take 

place during the 9th 1978 wpf so that the fish will have had a chance to development to produce 

1979 sufficiently high mean values with low variance in all the measured parameters.

1980

1981

1982 5.1.5 Test Temperature 25.5 ° C

1983

1984 Appropriate test temperature and the allowable variance both within an aquarium and between 

1985 aquaria have been discussed at bilateral meetings between US EPA and Japan Ministry of the 

1986 Environment. The recommended temperature and variance throughout the test is 25.5°C ± 

1987 0.5°C. Brief excursion from the mean by individual aquaria must not be more than 2°C. 

1988 Replicates within a treatment must not statistically different from each other, and treatments 

1989 within the test must not be statistically different from each other. This specification is based 

1990 upon 1) the historical range of temperatures used for toxicology testing with medaka, 2) the 

1991 temperature(s) for optimal reproduction, and 3) the need to minimize the occurrence of XX 

1992 males. 

1993

1994 Table 5-9 shows the fecundity of control fish in a MMT run at 24°C and one run with the 

1995 suggested specification. The higher control fecundity should result in greater statistical power 

1996 for this vital endpoint. 

1997

1998 Table 5-9. Control Fish Fecundity at 24 and 25.5 ° C.

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 Papillae
DRAFT

MMT ISR 57 May 28, 2013

2001 It has been documented in the literature (Nanda et al., 2003; Sato et al., 2005; Selim et al., 2009)

2002 and observed in our medaka culture that a small number of unexposed XX medaka will 

2003 spontaneously develop into normal phenotypic males. One factor that increases the likelihood of 

2004 this is increased temperature. Temperatures reported in the published literature typically report 

2005 effects at 32°C, a temperature not considered for the MRT. Thus, we initiated an ancillary study 

2006 to evaluate appropriate temperature to recommend for the MRT protocol. Figure 5-6 shows how 

2007 differences in temperature during development affect the prevalence of XX males in the 

2008 population. Note that higher temperatures cause an increase in the prevalence of XX males even 

2009 within a series of temperatures that seem to be appropriate for the MRT, i.e., 24 – 26°C. The 

2010 recommended temperature of 25.5° C for the MRT balances the competing objectives of 

2011 maximizing fecundity while minimizing the prevalence of XX males in controls. 

2012

2013

2014 Figure 5-6. Prevalence of XX Males with Increasing Temperature. The bars show the proportion of phenotypic 

2015 males that are either XX or XY out of 100%. The number boxes are the actual count of each genotype.

2016

2017

2018

2019

2020 5.1.6 Pathology Analysis of Adult Fish Only

2021 Another aspect of the 12 replicate test design is that the sample size for pathology analysis of 

2022 adult fish in the MRT is double compared to the 6 replicates in the MMT. This difference is 

2023 substantial. It provides pathology results for up to 12 fish per gender per treatment compared to 

2024 the MMTs 6. Also, because the pathology is done on adult fish sampled after the reproduction 

2025 evaluation, it provides information that can be correlated with the reproductive performance of 

2026 each specific breeding pair. This is in contrast with the subadult pathology analysis which does 

Prevalence of XX Males with Increasing Temperature

- 23 C 24 C 25 C 26 C 32 C -

0

20

40

60

80

100

149

1 4 6 7 60

170 138 109 149

 XX Males

 XY Males
DRAFT

MMT ISR 58 May 28, 2013

2027 not allow these correlations to be made. Evaluation of treatment-related effects on gonadal 

2028 development such as sex-reversal, and the prevalence testicular oocytes, is possible in either 

2029 adult or subadult specimens, however, only the adult samples provide data that can be used to 

2030 correlate pathology with reproduction. The increased sample sizes for adult pathology in the 12 

2031 replicate design increases both the statistical power and the relevance of the pathology endpoint 

2032 compared with the 6 replicate test design. With increased replication the sample size for 

2033 evaluating pathology is also increased which increases the statistical power of pathology 

2034 endpoints, especially with the development of the RSCABS statistical procedure. Furthermore, 

2035 the pathology analysis of the adult breeding pairs can be directly coordinated with their 

2036 fecundity/fertility outcomes.

2037

2038

2039 6 CONCLUSIONS

2040 The outcomes of the various MMT trials have provided enough information to recommend 

2041 a medaka reproduction test (MRT) for use as the fish test in Tier 2 of the EDSP. Two major 

2042 changes from the MMT are proposed, i.e., an increase in the number of replicates per 

2043 treatment for evaluating effects on reproduction, and terminating the test after the 

2044 embryos hatch in F2. Other proposed changes include minimizing the collection of endpoint 

2045 data from F0, and evaluating pathology in only the F1 adults sampled after the assessment 

2046 of reproduction.

2047

2048 Overall, the authors conclude that both the MMT and the MRT are transferable methods and are 

2049 capable of adequately characterizing potential disruption of the endocrine system by putative 

2050 endocrine disrupting chemicals. However, the MRT is recommended as the preferred EDSP 

2051 Tier 2 test method for fish because it is less resource intensive with improved statistical power, 

2052 appears to be as sensitive, and is better able to ensure consistent findings when performed 

2053 routinely by testing laboratories. 

2054

2055

2056

2057

2058

2059

2060

2061

2062

2063

2064

2065

2066

2067

2068

2069
DRAFT

MMT ISR 59 May 28, 2013

2070 APPENDICES 

2071

2072 Appendix A: Criteria for Validation of Assays in the EDSP (2007)

2073

2074 Appendix B: Detailed Review Paper

2075

2076 Appendix C: Vinclozolin Interlaboratory Summary Report

2077

2078 Appendix D: MMT Protocol

2079

2080 Appendix E: MRT Protocol

2081

2082 Appendix F: Pathology Guidance

2083

2084 Appendix G: Statistics

2085

2086 Appendix H: MMT Summary Reports

2087

2088 Appendix I: Power Analysis

2089

2090

2091

2092

2093

2094

2095

2096

2097

2098

2099

2100

2101

2102

2103

2104

2105

2106

2107

2108
DRAFT

MMT ISR 60 May 28, 2013

2109

2110

2111

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