1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17 Validation of the

18 Larval Amphibian Growth and Development Assay:

19 Integrated Summary Report

20

21

22

23

24

25 U.S. Environmental Protection Agency

26 Endocrine Disruptor Screening Program

27 Washington, D.C.

28

29 28 May 2013

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45
2

46 TABLE OF CONTENTS

47

48 EXECUTIVE SUMMARY.................................................................................................... 3

49 1 OBJECTIVE ........................................................................................................................... 5

50 2 INTRODUCTION .................................................................................................................. 6

51 2.1 Endocrine Disruptor Screening Program ......................................................................... 6

52 2.2 Validation......................................................................................................................... 8

53 3 OVERVIEW OF THE LAGDA ........................................................................................... 10

54 3.1 Summary of the LAGDA test protocol .......................................................................... 10

55 3.2 Purpose of the test and relevance of the LAGDA.......................................................... 11

56 3.3 Scientific basis for the test method ................................................................................ 11

57 4 PROTOCOL DEVELOPMENT........................................................................................... 12

58 4.1 Rationale for test species and exposure period .............................................................. 12

59 4.1.1 Species selection ..................................................................................................... 13

60 4.1.2 Test duration ........................................................................................................... 13

61 4.2 Test details...................................................................................................................... 14

62 4.3 Core Endpoints............................................................................................................... 17

63 4.4 Statistical Analyses ........................................................................................................ 18

64 5 INTER-LABORATORY EVALUATION OF THE LAGDA ............................................. 20

65 5.1 Overview of approach .................................................................................................... 20

66 5.2 Inter-laboratory data analysis......................................................................................... 20

67 5.3 Summary of results......................................................................................................... 21

68 5.3.1 Prochloraz ............................................................................................................... 21

69 5.3.2 4-tert-octylphenol ................................................................................................... 27

70 5.3.3 17β-trenbolone ........................................................................................................ 32

71 5.3.4 Benzophenone-2 ..................................................................................................... 35

72 6 DISCUSSION OF THE INTER-LABORATORY STUDIES ............................................. 40

73 6.1 Comparison of control performance .............................................................................. 40

74 6.2 Effectiveness of the LAGDA ......................................................................................... 42

75 6.3 Lessons learned from the inter-laboratory studies ......................................................... 46

76 6.4 Conclusions.................................................................................................................... 47

77 7 REFERENCES ..................................................................................................................... 47

78 8 APPENDICES ...................................................................................................................... 54

79 8.1 LAGDA Guideline ......................................................................................................... 54

80 8.2 Histological Development of the Gonad in Juvenile Xenopus laevis ............................ 87

81 8.3 Histopathology Guidance for Reading LAGDA Studies............................................... 91

82 8.4 Inter-laboratory validation study results ...................................................................... 100

83 8.4.1 Prochloraz results.................................................................................................. 100

84 8.4.2 4-tert-Octylphenol results ..................................................................................... 114

85 8.4.3 17-β Trenbolone results ........................................................................................ 121

86 8.4.4 Benzophenone-2 results........................................................................................ 125

87

88

89

90

91
3

92

93

94 EXECUTIVE SUMMARY

95

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

97 Amphibian Growth and Development Assay (LAGDA) aimed at characterizing potential 

98 disruption of the endocrine system by putative Endocrine Disrupting Chemicals. 

99

100 The LAGDA protocol describes a chronic toxicity test with an amphibian species that considers 

101 growth and development from fertilization through the early juvenile period. It also enables 

102 measurement of a suite of other endpoints that allows for diagnostic evaluation of endocrine 

103 disrupting chemicals or other types of developmental and reproductive toxicants. The LAGDA 

104 is a relatively long-term assay (normally 130 days or longer) that assesses early development, 

105 growth, and partial reproductive maturation. The test is designed to detect both endocrine and 

106 non-endocrine mechanisms by including diagnostic endpoints specific to key endocrine 

107 mechanisms. Endpoints evaluated during the course of the exposure include those indicative of 

108 generalized toxicity, mortality, abnormal behavior, and growth determinations (length and 

109 weight), as well as endpoints designed to characterize endocrine-specific modes of action 

110 targeting estrogen, androgen or thyroid-mediated physiological processes.

111

112 Individual and inter-laboratory evaluations of the LADGA were conducted to evaluate the 

113 practical transferability of the assay protocol and quantitative reproducibility of the results. The 

114 inter-laboratory validation evaluated the ability of four labs to conduct and evaluate the LAGDA 

115 assay. The following chemicals were evaluated across individual or multiple laboratories: 

116 prochloraz (aromatase inhibitor, AR agonist), 4-tert-octylphenol (ER agonist), 17-β trenbolone 

117 (AR agonist), and benzophenone-2 (ER agonist, TPO inhibitor). Prochloraz was tested in four

118 labs (A, B, C, and D), and 4-tert-octylphenol was tested in three labs (A, B, and C). Trenbolone 

119 and benzophenone-2 were tested in single labs (E and A, respectively), precluding their use in 

120 the inter-laboratory comparisons. However, these studies serve to demonstrate the 

121 responsiveness of the LAGDA to additional modes of action.

122

123 The results of the inter-laboratory validation were mixed. For larval growth and development, 

124 two laboratories, B and C, reported growth rates where confidence intervals were outside of one 

125 standard deviation from the overall mean weight for at least one chemical. Further investigation 

126 indicated that these laboratories did not closely follow the feeding regimens recommended in the 

127 LAGDA guidance, resulting in either excessive or inadequate growth of the developing larvae. 

128 Similarly, for juvenile growth and development, two studies had control juvenile weights more 

129 than one standard deviation form the overall mean in both juvenile males and females but in 

130 different directions: lab D’s prochloraz study had higher mean weights whereas the lab C’s 

131 prochloraz study had lower mean weights. Laboratory D used the recommended diet 

132 formulation but fed at a higher rate than what was recommended in the LAGDA guideline, and 

133 laboratory C used their own diet formulation but fed at a rate similar to that recommended in the

134 LAGDA guideline. This diet formulation was clearly inadequate for supporting growth rates 

135 consistent with the other studies and led to confounding effects on liver-somatic index (LSI) in 

136 their prochloraz study. The inconsistent findings related to growth and development observed 
4

137 can likely be corrected with more thorough guidelines regarding the dietary and feeding 

138 regimens.

139

140 The LAGDA was an effective test model. All four chemicals produced endocrine-related effects. 

141 Of the two chemicals available for inter-laboratory comparison, prochloraz resulted in thyroid

142 gland pathologies consistent with a hypothyroid condition in 3 of the 4 labs, and vitellogenin 

143 (VTG) induction and gonad/reproductive duct pathologies were noted in all 4 laboratories. The 

144 second chemical, 4-tert-octylphenol, produced thyroid gland pathologies consistent with a 

145 hypothyroid condition and delayed development in only 1 of the 3 laboratories. However, VTG 

146 production and mild gonad/reproductive duct pathologies were observed in all laboratory studies. 

147 17-β Trenbolone and benzopehone-2, although only tested in single laboratories, produced 

148 endocrine-related effects involving the thyroid gland, delayed metamorphosis, VTG production 

149 and reproductive tract pathologies.

150

151 There were several areas of inconsistency noted among the laboratories:

152  The growth and developmental differences related to feeding and diet have already been 

153 discussed. In addition, inconsistencies in pathology findings were observed for 

154 prochloraz. In the prochloraz studies, A and D gonad, oviduct and kidney samples were 

155 read by a different pathologist than the B and C samples. Interestingly, the A and D 

156 studies shared consistencies as did the B and C studies. It is not possible to definitively 

157 demonstrate this as a cause for the observed inconsistencies. However, it does point to 

158 the need for adherence to the pathology guidance document. Similar anomalies were 

159 observed with liver pathology for the prochloraz studies. The A, B and D studies were 

160 read by the same contracted pathologist whereas the C study was read by a different 

161 contracted pathologist. It is interesting to point out that similar treatment-related liver 

162 pathologies were reported for A, B and D whereas a different spectrum of treatment163 related pathologies were reported for C. It is worth noting that the animals in the C study

164 showed feeding-related reduced juvenile growth rates as compared to the other studies. It 

165 is possible that disease issues may have factored into the apparent inconsistencies in liver 

166 findings. Regardless, developing a pathology guidance document for both the liver and 

167 kidney is critical. 

168

169  Three laboratories used commercially available ELISA kits specific for human or canine 

170 T4 and were subsequently unsuccessful in quantifying T4. Although a methods for 

171 determining serum T4 in X. laevis using high pressure liquid chromatography and 

172 inductively-coupled plasma mass spectrometry (HPLC/ICP-MS) are available, US EPA 

173 Mid-Continent Ecology Division (MED ) has developed an extraction method which 

174 allowed for the use of these commercially available ELISA kits.

175

176  Absolute control VTG levels varied considerably between laboratories with coefficient of 

177 variation (CV) values close to, or exceeding 100%, and control means differed across 

178 labs by up to 74-fold. Although there were concentration-dependent increases in VTG 

179 production in response to prochloraz, 4-tert-octylphenol, and benzophenone-2, VTG data 

180 from all other studies are likely background levels and the VTG induction as a 

181 reproductive endpoint should be interpreted in the context of overall potential magnitude 

182 of response.
5

183

184 Overall, the authors conclude that the Tier 2 LAGDA is capable of sufficiently characterizing

185 potential disruption of the endocrine system by putative endocrine disrupting chemicals. 

186 However, careful attention to recommendations in the Test Guideline is necessary to ensure

187 consistent and reliable findings across multiple testing laboratories. 

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224 1 OBJECTIVE

225
6

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

227 validation process for the Larval Amphibian Growth and Development Assay (LAGDA) so that 

228 FIFRA Scientific Advisory Panel members may address the attached charge questions. 

229

230 2 INTRODUCTION 

231

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

233 U.S. Environmental Protection Agency (US EPA) to:

234

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

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

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

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

239

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

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

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

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

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

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

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

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

248 and the creation of similar programs in other countries, the Organization for Economic Co249 operation and Development (OECD) established the Endocrine Disrupters Testing and 

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

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

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

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

254 new and existing substances.

255

256 The LAGDA is one of the assays proposed as part of the EDSP Tier 2 and is the subject of this 

257 report. This report complements the attached supporting materials and is meant to provide the 

258 peer reviewers with the necessary information, and/or direction to necessary information, to 

259 address the peer review charge questions. It introduces the purpose of the LAGDA and how it 

260 fits into the EDSP, the scientific rationale for the assay, and an historical account of the 

261 development and optimization of the assay protocol. It also synthesizes the information gained 

262 during the validation process and addresses the advantages and limitations of this amphibian test 

263 method based on its strengths and weaknesses, practicality, reproducibility, reliability, and 

264 protocol transferability.

265

266 2.1 Endocrine Disruptor Screening Program 

267

268 To comply with its mandate, the US EPA chartered a Federal advisory committee, the Endocrine 

269 Disruptor Screening and Testing Advisory Committee (EDSTAC), to provide advice and 
7

270 guidance on the development of a screening and testing program. In 1998, it recommended to 

271 the EPA a conceptual two-tiered approach that involved screening and testing chemical 

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

273 (collectively referred to as EAT). The ultimate goal of these assays is to provide input into 

274 hazard identification to assess risk of adverse consequences to humans and wildlife (EDSTAC,

275 1998).

276

277 The US EPA submitted a proposal of the EDSP for public review and comment (FRN, 1998) as 

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

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

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

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

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

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

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

285 relationships for hazard identification using Tier 2 testing. 

286

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

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

289 • be relatively fast and efficient;

290 • be standardized and validated;

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

292 rate of false positives;

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

294 possible;

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

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

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

298

299 Together, the suite of Tier 1 assays would become a battery in which some endocrine axis 

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

301 estrogenic response). This redundancy will allow for a weight-of-evidence approach, as 

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

303 definitive, testing.

304

305 The following assays, recommended by EDSTAC, have undergone validation and have been 

306 adopted as the EDSP Tier 1 screening battery (Table 2.1-1). These assays are meant to detect 

307 chemicals that may affect the estrogen, androgen and thyroid hormone axes through any known 

308 modes of action.

309

310

311

312

313

314

315
8

316

317 Table 2.1-1. 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 anti-androgenic 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.

318

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

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

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

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

323

324  Two-generation Rat Reproduction Test

325  Medaka Multi-generation Test

326  Japanese Quail Two-generation Test

327  Mysid Two-generation Toxicity Test

328  Larval Amphibian Growth and Development Assay

329

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

331 remaining four Tier 2 tests are the subject of the FIFRA Scientific Review Panel for which the 

332 LAGDA is reviewed here.

333

334 2.2 Validation

335

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

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

338
9

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

340 laboratories. 

341 Relevance describes whether a test is meaningful and useful for a particular purpose (OECD 

342 996). For Tier I EDSP assays, relevance can be defined as the ability of an assay to detect 

343 chemicals with the potential to interact with the endocrine system.

344

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

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

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

348 LAGDA were based on the principles of validation developed by the U.S. Interagency 

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

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

351 follows:

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

353 of its proposed use, should be available.

354

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

356 effect and toxicity of interest must be addressed.

357

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

359 domain. It should be sufficiently detailed to enable the user to adhere to it and should 

360 include data analysis and decision criteria.

361

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

363 vary with time should have been evaluated.

364

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

366 reference chemicals preferably coded to exclude bias.

367

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

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

370

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

372

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

374

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

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

377 preferably, published in an independent, peer-reviewed publication.

378

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

380 attachment A (EDSP, 2007). Although attempts have been made to thoroughly comply with all 

381 validation criteria, the in vitro and in vivo screening assays in the Tier 1 battery are not 

382 replacement assays (Validation Criterion No. 6). Many of them are novel assays; consequently, 

383 large data bases do not exist as a reference to establish their predictive capacity (e.g., 

384 determination of false positive and false negative rates).
10

385

386 In general, the US EPA is following a five-part or stage validation process outlined by

387 ICCVAM (NIEHS, 1997). The US EPA believes that it is essential to recognize that this process 

388 was specifically developed for in vitro assays intended to replace in vivo assays. A rudimentary

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

390 ecological in vivo assays in Tiers 1 and 2 that have no suitable in vitro substitute. Nonetheless, 

391 the stages of the process outlined by the ICCVAM are as follows:

392

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

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

395 Paper (DRP) to explain the purpose of the test method, the context in which it will be 

396 used, and the scientific bases upon which the assay’s protocol, endpoints, and relevance 

397 rest (attachment B). The DRP reviewed the scientific literature for candidate protocols 

398 and evaluates them with respect to a number of considerations, such as whether candidate 

399 protocols meet the assay’s intended purpose, costs, and other practical considerations. 

400 The DRP also identified the developmental status and questions related to each protocol; 

401 the information needed to answer the questions; and, when possible, recommends an 

402 initial protocol for the initiation of the second stage of validation, standardization and 

403 optimization. 

404

405 During the second stage, standardization and optimization, studies were performed 

406 geared toward refining, optimizing, and standardizing the protocol, and initially assessing 

407 protocol transferability and performance. 

408

409 In the third stage, inter-laboratory validation, studies were conducted in several 

410 independent laboratories with the refined protocol. The results of these studies were used 

411 to determine inter-laboratory variability and to set or cross-check performance criteria. 

412

413 In the fourth stage, peer review, an independent scientific review by qualified experts.

414 EPA has developed extensive guidance on the conduct of peer reviews because the 

415 Agency believes that peer review is an important step in ensuring the quality of science 

416 that underlies its regulatory decisions (US EPA, 2006).

417

418 The final stage is regulatory acceptance, adoption for regulatory use by an agency after 

419 the other stages are completed. 

420 3 OVERVIEW OF THE LAGDA

421 3.1 Summary of the LAGDA test protocol

422

423 The LAGDA protocol describes a chronic toxicity test with an amphibian species that considers 

424 growth and development from fertilization through the early juvenile period. It also enables 

425 measurement of a suite of other endpoints that allows for diagnostic evaluation of endocrine 

426 disrupting chemicals (EDCs) or other types of developmental and reproductive toxicants. The 

427 LAGDA is a relatively long-term assay (normally 130 days or longer) that assesses early 

428 development, growth, and partial reproductive maturation.
11

429

430 The LAGDA is intended to serve as a higher tier test with an amphibian for collecting definitive 

431 concentration-response information on adverse effects suitable for use in ecological risk 

432 assessment. Specifically, the design enables the collection of amphibian hormone-regulated 

433 endpoint data (e.g., metamorphosis, gonadal development) and information concerning various 

434 aspects of reproductive biology and life-stage viability. 

435

436 The general experimental design entails exposing NF stage 8 (see Nieuwkoop and Faber, 1994 

437 for staging details) Xenopus laevis embryos to four different concentrations of a test chemical 

438 and a control until 10 weeks after the median time to NF stage 62 in the control, with one interim 

439 sub-sample at NF stage 62. There are four replicates in each test concentration with eight 

440 replicates for the control. Endpoints evaluated during the course of the exposure (at the interim 

441 sub-sample and final sample at the completion of the test) include those indicative of generalized 

442 toxicity, mortality, abnormal behavior, and growth determinations (length and weight), as well as 

443 endpoints designed to characterize endocrine-specific modes of action targeting estrogen, 

444 androgen or thyroid-mediated physiological processes. 

445

446 3.2 Purpose of the test and relevance of the LAGDA

447

448 The LAGDA is intended to identify and characterize the adverse consequences of aquatic 

449 exposure to substances which interfere with the normal development and growth of amphibians

450 through larval development and metamorphosis. It is an important assay for evaluating effects 

451 from exposure to contaminants during the sensitive larval stage, where effects on survival and 

452 development, including normal development of reproductive organs, may adversely affect 

453 populations. The test is designed to detect both endocrine and non-endocrine mechanisms by 

454 including diagnostic endpoints specific to key endocrine mechanisms. It should be noted that, to 

455 date, no validated assay exists that serves this function for amphibians. 

456

457 3.3 Scientific basis for the test method

458

459 Much of our current understanding of amphibian biology has been obtained using the laboratory 

460 model species X. laevis. This species can be routinely cultured in the laboratory; ovulation can 

461 be induced using human chorionic gonadotropin (hCG); and animal stocks are readily available 

462 from commercial breeders. 

463

464 Like all vertebrates, reproduction in amphibians is under the control of the hypothalamic 

465 pituitary gonadal (HPG) axis (Kloas and Lutz, 2006). Estrogens and androgens are mediators of 

466 this endocrine system, directing the development and physiology of sexually-dimorphic tissues. 

467 There are three distinct phases in the life cycle of amphibians when this axis is especially active: 

468 (1) gonadal differentiation during larval development, (2) development of secondary sex 

469 characteristics and gonadal maturation during the juvenile phase, and (3) functional reproduction 

470 of adults. Each of these three developmental windows are likely susceptible to endocrine 

471 perturbation by xenobiotics ultimately leading to a loss of reproductive fitness by the organisms. 

472
12

473 The gonads begin development at NF stage 43, when the bipotential genital ridge first develops 

474 (Nieuwkoop and Faber, 1994). Differentiation of the gonads begins at NF stage 52 when 

475 primordial germ cells either migrate to medullary tissue (males) or remain in the cortical region 

476 (females) of the developing gonads (Nieuwkoop and Faber, 1994). This process of sexual 

477 differentiation of the gonads was first reported to be susceptible to chemical alteration in 

478 Xenopus in the 1950s (Chang and Witschi, 1956; Gallien, 1953). Exposure of tadpoles to 

479 estradiol during this period of gonad differentiation results in sex reversal of males that when 

480 raised to adulthood are fully functional females (Villalpando and Merchant-Larios, 1990; Miyata 

481 et al., 1999). Functional sex reversal of females into males is also possible and has been reported 

482 following implantation of testis tissue in tadpoles (Mikamo and Witschi, 1963) or exposure to an 

483 aromatase inhibitor (Olmstead et al., 2009a). Historically, toxicant effects on gonadal 

484 differentiation have been assessed by histological examination of the gonads at metamorphosis,

485 and sex reversal could only be determined by analysis of sex ratios. Until recently, there had 

486 been no means by which to directly determine the genetic sex of Xenopus. However, recent 

487 establishment of sex-linked markers in X. laevis (Yoshimoto et al., 2008) and X. tropicalis

488 (Olmstead et al., 2010) makes it possible to determine genetic sex and allows for the direct 

489 identification of sex-reversed animals.

490

491 In males, juvenile development proceeds as testosterone blood levels increase corresponding 

492 with the development of secondary sex characteristics as well as testis development (Olmstead et 

493 al., 2009b). In females, estradiol is produced by the ovaries resulting in the appearance of 

494 vitellogenin in the plasma, vitellogenic oocytes in the ovary, and the development of the oviducts 

495 (Olmstead et al., 2009b). Oviducts are female secondary sex characteristics that function in 

496 oocyte maturation during reproduction (Wake and Dickie, 1998). Jelly coats are applied to the 

497 outside of oocytes as they pass through the oviduct and collect in the ovisac, ready for 

498 fertilization. Oviduct development appears to be regulated by estrogens as development 

499 correlates with blood estradiol levels in X. laevis (Tobias et al., 1998) and X. tropicalis

500 (Olmstead et al., 2009b). The development of oviducts in males following exposure to 

501 polychlorinated biphenyl compounds (PCBs) (Qin et al., 2007) and 4-tert-octylphenol (Porter et 

502 al., 2011) has been reported.

503

504 4 PROTOCOL DEVELOPMENT

505

506 4.1 Rationale for test species and exposure period

507

508 Short-term partial life cycle testing methods have been developed for amphibian species that 

509 evaluate chemical impacts on metamorphosis (OCSPP 890.1100: Amphibian Metamorphosis 

510 (Frog) Assay [US EPA, 2009]). However, there are no standardized tests for evaluating chronic 

511 effects on growth and development of amphibians. Ideally a test of this nature would be 

512 developed using a species that can be genetically sexed and would incorporate an exposure 

513 period encompassing embryonic development through reproduction. There are several factors 

514 that limit the current development of such a test, with the result being the proposed LAGDA as a 

515 compromise in terms of test species selection and test duration. 

516
13

517 4.1.1 Species selection

518

519 Ideally, the test species should be robust in the laboratory setting, be able to be genetically sexed,

520 and develop to maturity in a reasonable amount of time. Several reports demonstrate the 

521 potential for the well known laboratory model species X. laevis (Fort et al., 2004a; Pickford and 

522 Morris, 2003; Pickford et al., 2003) and X. tropicalis (Fort et al., 2004b; Porter et al., 2011;

523 Olmstead et al., 2009a) as species for evaluating developmental impacts of chronic chemical 

524 exposure. Although closely related, there are significant differences between these two species 

525 as they relate to use in a chronic testing method. 

526 1) Both species are widely cultured in the laboratory, although X. laevis has proven to be 

527 considerably more robust and more amenable to routine culture in most laboratory 

528 settings.

529 2) Both species can be genetically sexed due to the recent development of sex-linked 

530 markers. The marker for X. laevis is the DMW gene which is located on the chromosome 

531 which determines the female phenotype (Yoshimoto et al., 2008). The marker for X. 

532 tropicalis is a female-linked polymorphism derived from one female animal raised at the 

533 US EPA Mid-Continent Ecology Division (MED) (Olmstead et al., 2010). Unfortunately, 

534 because of the nature of this sex-linked polymorphism, a very limited population of 

535 animals currently exists, and this strain has proven to lack the robustness necessary for 

536 routine testing.

537 3) It has been reported that X. tropicalis reach sexual maturity within 6 months of complete 

538 metamorphosis (Hirsch et al., 2002). However, work conducted at MED has shown that 

539 a typical time to maturation is 8 months to 1 year under laboratory conditions (Olmstead 

540 et al., 2009b). X. laevis have a considerably longer maturation phase reaching sexual 

541 maturity within 1.5 to 2 years. 

542

543 Given that a robust strain capable of being genetically sexed does not currently exist for X.

544 tropicalis, X. laevis was chosen as the representative amphibian species for development of the 

545 LAGDA. 

546

547 4.1.2 Test duration

548

549 Initially the US EPA proposed the development of a testing protocol capable of addressing the 

550 impacts of chemical exposure on development, growth and reproduction in amphibians. Over 

551 the intervening years, the Agency has determined that, given the current state of the science, such 

552 a protocol could not be developed. Instead the Agency has developed a protocol that addresses 

553 many aspects of the original proposal, is scientifically sound, and is executable given the 

554 constraints of existing resources. 

555

556 Due to the lack of a standardized methodology, fecundity cannot be directly assessed in Xenopus

557 or any other amphibian species without the aid of exogenous hormones. Surrogate 

558 measurements that are likely to be related to fecundity (sperm count, oocyte count) were 

559 assessed throughout the maturation process in X. tropicalis, and it was determined that these

560 endpoints were highly variable (Olmstead et al., 2009b). Studies conducted with 4-tert561 octylphenol using a 40-week exposure protocol showed that conducting the exposure until sexual 
14

562 maturation did not offer additional information due to the high variability of these surrogate 

563 reproductive endpoints (Porter et al., 2011). Evidence indicates that X. laevis would not differ

564 greatly from X. tropicalis in this regard. Further, given the selection of X. laevis as the most 

565 viable test species, the exposure period necessary to encompass sexual maturation would need to 

566 be in excess of 78 weeks. The cost and effort required to conduct an assay of this length would 

567 be exceptionally high. Another important reality of aquatic testing is that longer assays run 

568 greater risks of catastrophic failures (e.g., disease, exposure system mechanical failures, etc.).

569

570 Since it is impractical to conduct the exposure up to sexual maturation, it was decided that the 

571 exposure would continue from NF stage 8 until significant hallmarks of gonad development are 

572 achieved. Toward this end, MED scientists have conducted studies to determine when the 

573 gonads show an appropriate level of maturation to allow identification of abnormalities in 

574 development of reproductive organs. From these studies it was determined that, by 8 weeks 

575 post-metamorphosis, nearly complete maturation of oocytes and spermatocytes had occurred,

576 males had developed seminiferous tubules and associated ducts, the female oviduct system was 

577 developing, and supporting cells and tissues such as granulosa cells, Sertoli and Leydig cells

578 were present (Appendix 8.2). Based on these findings, the US EPA designed a protocol for an 

579 exposure period which is initiated shortly after fertilization and continues through 8 weeks post580 metamorphosis. 

581

582 4.2 Test details

583

584 The general experimental design entails exposing de-jellied NF stage 8 X. laevis embryos to four

585 different concentrations of a test substance along with a control until 10 weeks after the median 

586 time to NF stage 62 (approximately two weeks before completion of metamorphosis in the 

587 control) with one interim sub-sample at NF stage 62. There are four replicates in each test 

588 concentration with eight replicates for the control. Endpoints evaluated during the course of the 

589 exposure include those indicative of generalized toxicity, mortality, abnormal behavior, and 

590 growth determinations (length and weight), as well as endpoints designed to characterize

591 endocrine-specific modes of action targeting estrogen, androgen or thyroid-mediated 

592 physiological processes. An assessment of the gonads two months post-metamorphosis is 

593 expected to yield histological changes indicative of alterations in differentiation or delayed 

594 development. The test details and performance criteria are summarized in Tables 4.2-1 and 4.2-2.

595

596

597

598

599

600

601

602
15

603 Table 4.2-1. Summary of method

Test animal Xenopus laevis

Initial larval stage Nieuwkoop and Faber (NF) stage 8

Exposure period Until 10 weeks after the median time to NF stage 62 in 

the control

Test concentrations Minimum of 4 different concentrations

Dilution water / laboratory control Any water that permits normal growth and development 

of X. laevis (e.g., spring water or charcoal-filtered tap 

water)

Replication 4 replicate test vessels / test concentration

8 replicate test vessels / control

Exposure regime Flow-through

Test system flow-rate Constant, in consideration of both the maintenance of 

biological conditions and chemical exposure (e.g., ≥10 

tank turnovers/day)

Test solution / test vessel 4 – 10 L (10 – 15 cm minimum water depth) / glass or 

stainless steel

Initial larval density 20 larvae / test vessel

Larval cull to adjust density after NF 

stage 66

10 juveniles / test vessel(determined randomly)

Feeding See LAGDA Appendix 2

Lighting Photoperiod 12 hr light : 12 hr dark

Intensity 600 to 2000 lux (lumens/m2) at the water surface

Water temperature 21o ± 1oC

pH 6.5 to 8.5

Dissolved oxygen (DO) concentration >3.5 mg/L (>40% of the air saturation)

Analytical chemistry schedule Initiation (day 0), and weekly thereafter for each 

replicate tank at each concentration 

604

605

606

607

608

609

610

611

612

613

614

615

616

617
16

618 Table 4.2-2. Performance criteria for the LAGDA.

Criterion Acceptable limits

Test concentrations Maintained at ≤ 20% CV(variability of measured 

test concentration) over the entire test 

Mortality in controls ≤ 20% mortality in any one replicate in the controls

Development in controls The mean time to NF stage 62 is ≤ 45 days, and the 

mean weight at NF stage 62 is 1.0 ± 0.2 g. The 

mean weight at test termination is 11.5 ± 3 g.

Dissolved Oxygen ≥ 40% air saturation 

pH pH should be maintained between 6.5-8.5. The interreplicate/inter-treatment differentials should not 

exceed 0.5. 

Water temperature Target temperature ± 1oC - the inter-replicate/intertreatment differentials should not exceed 1.0 oC 

Test concentrations without overt toxicity ≥ 2 

Replicate performance ≤ 2 replicates across the test can be compromised 

Special conditions for use of a solvent If a carrier solvent is used, both a solvent control 

and clean water control must be used.

Statistically significant differences between solvent 

control and clean water control groups are treated 

specially. See LAGDA guideline for more 

information.

619

620
17

621 4.3 Core Endpoints

622

623 The general experimental design entails exposing de-jellied NF stage 8 X. laevis embryos to four

624 different concentrations of a test substance and a control until 10 weeks after the median time to 

625 NF stage 62 in the control, with one interim sub-sample at NF stage 62. Table 4.3-1 summarizes 

626 the endpoints and timing of tissue/sample collection. 

627

628 Table 4.3-1 Summary of endpoint and collection stages

Apical Endpoints Daily Interim 

Sampling

Test 

Termination

Mortality x

Clinical signs of disease and/or toxicities x

Growth x x

Time to NF stage 62 x

Thyroid histology x

Serum T4 (thyroxine) x

Vitellogenin x

Genetic/histological sex comparison x

Gonad histology x

Liver histology x

Liver-somatic index x

Kidney histology x

629

630

631 Growth and overt toxicity endpoints 

632

633 Routine toxicity test endpoints of mortality, clinical signs of disease and/or toxicity, and growth 

634 are measured. In addition, liver and kidney histopathology and liver-somatic index (LSI) have

635 been included as measures of overt toxicity to the organism. Given the importance of the liver in 

636 metabolism and the kidney in clearance of xenobiotics, these tissues are often targets of overt 

637 toxicity associated with a chemical. Overt toxicity can confound and confuse the interpretation 

638 of endocrine studies if it is not clearly understood, so inclusion of these endpoints in the LAGDA

639 will ultimately assist in interpreting future data sets. 

640

641 Thyroid endpoints

642

643 Three endpoints have been included which are intended to identify impacts on the thyroid axis:

644 time to NF stage 62, thyroid histology, and serum thyroxine (T4) measures. In the AMA, the rate 

645 of development is recorded as an indication of impacts on the thyroid axis. In contrast to the 

646 AMA, which determines median stage achieved in a given amount of time (21 days), the 

647 LAGDA measures the time it takes for each individual to reach NF stage 62, a stage referred to 

648 as metamorphic climax. However, this measure should be interpreted with caution because non649 specific toxicity experienced prior to metamorphosis may impact the length of time it takes to 

650 reach NF stage 62. Thyroid histology, the hallmark for diagnosing thyroid disruption, is a focal 

651 endpoint in the AMA and has been validated extensively (Grim et al., 2009). Serum T4 has also 
18

652 been included as an endpoint for characterizing thyroid axis disruption in the LAGDA. The 

653 robustness of the diagnosis for thyroid impacts is enhanced with this three-pronged approach.

654

655 Reproductive endpoints

656

657 Vitellogenin

658

659 Vitellogenin (VTG) is a widely accepted biomarker of estrogenic chemicals. The measure is 

660 made using enzyme-linked immunosorbent assay (ELISA) methodology. Currently there are no 

661 commercially available antibodies. However, given the wealth of information for this protein

662 and the availability of cost-effective commercial antibody production services, it is reasonable 

663 that laboratories can easily develop an ELISA to make this measure (Olmlstead et al., 2009b). 

664 Details are provided in LAGDA Appendix 4. 

665

666 Gonad Histology

667

668 Gonad histology has been a standard measure for the EDSP Tier 1 Fish Short Term 

669 Reproduction Assay which exposes fathead minnows for 21 days. The value of histological 

670 analysis has been reviewed extensively, and a detailed internationally harmonized guidance 

671 document is available for fish (Johnson et al., 2009). This fish guidance has served as the basis 

672 for development of a LAGDA histopathology guidance document (Appendix 8.3).

673

674 Genetic Sex

675

676 DMW is a gene that has been shown to be linked to the female phenotype (Yoshimoto et al.,

677 2008). The presence of this gene (marker) indicates that the individual is genetically female. 

678 The presence or absence of the marker when compared back to the histological determination of 

679 sex can be used to identify if sex reversal has occurred (LAGDA Appendix 3). 

680

681 4.4 Statistical Analyses

682

683 The LAGDA generates two forms of data: 1) quantitative continuous data including growth and 

684 biochemical concentrations, and 2) ordinal data in the form of severity scores or developmental 

685 stages from histopathology evaluations. Data are typically collected in spreadsheets and then 

686 transferred to and analyzed with a statistical software package. Statistical analyses of the data 

687 should preferably follow procedures described in the document Current Approaches in the 

688 Statistical Analysis of Ecotoxicity Data: A Guidance to Application (OECD, 2006). Data for

689 continuous endpoints should be assessed for normality (preferably using the Shapiro-Wilk or 

690 Anderson-Darling test) and variance homogeneity (preferably using Levene’s test). Both tests 

691 are performed on the residuals from an ANOVA. Expert judgment can be used in lieu of these 

692 formal tests for normality and variance homogeneity, though formal tests are preferred. Where 

693 non-normality or variance heterogeneity is found, a normalizing, variance stabilizing 

694 transformation should be sought. If the data (perhaps after a transformation) are normally 

695 distributed with homogeneous variance, a significant treatment effect is determined from 

696 Dunnett’s test. If the data (perhaps after a transformation) are normally distributed with 
19

697 heterogeneous variance, a significant treatment effect is determined from the Tamhane-Dunnett 

698 or T3 test or from the Mann-Whitney-Wilcoxon U test. Where no normalizing transformation 

699 can be found, a significant treatment effect is determined from the Mann-Whitney-Wilcoxon U 

700 test using a Bonferroni-Holm adjustment to the p-values. The Dunnett test is applied 

701 independently of any ANOVA F-test, and the Mann-Whitney test is applied independently of 

702 any overall Kruskall-Wallis test. In addition, regardless of the results of the previous tests, a 

703 step-down Jonckheere-Terpstra (JT) trend test should also be applied to the data as long as a 

704 monotonic response was observed.

705

706 Mortality data should be analyzed for time periods encompassing larval development and 

707 juvenile development. Tadpoles that do not complete metamorphosis in the given time frame, 

708 those tadpoles that are in the larval sub-sample cohort, those juvenile frogs that are culled, and 

709 any animal that dies due to experimenter error should be treated as censured data. Mortality data 

710 expressed as the percentage that died should be arcsine square root transformed prior to analysis.

711

712 Time to metamorphosis data should be treated as time to event data, with any mortalities treated 

713 as censured data. Median time to completion of metamorphosis should be determined by 

714 Kaplan-Meier product-limit estimators and used in the statistical analysis.

715

716 Body weight and snout-vent lengths should be analyzed using the means of a given tank. Males 

717 and females are not sexually-dimorphic at the completion of metamorphosis, and data for the 

718 sexes are combined when analyzing larval sub-sampling data. Male and female juvenile body 

719 size data should be analyzed separately.

720

721 Liver weights should be expressed as a proportion of whole body weight (LSI), arcsine square 

722 root transformed, and analyzed separately for each sex.

723

724 Histopathology data is in the form of severity scores or developmental stages. A new test termed 

725 RSCABS (Rao-Scott Cochran-Armitage by Slices) uses a step-down Rao-Scott adjusted 

726 Cochran-Armitage trend test on each level of severity in a histopathology response. The Rao727 Scott adjustment incorporates the replicate vessel experimental design into the test. The “by 

728 slices” procedure incorporates the biological expectation that severity of effect tends to increase 

729 with increasing doses or concentrations, while retaining the individual subject scores and 

730 revealing the severity of any effect found. The RSCABS procedure not only determines which 

731 treatments are statistically different, that is, have more severe pathology than controls, but it also 

732 determines at which severity score the difference occurs providing much needed context to the 

733 analysis. Currently, this test can only be implemented by StatCHARRMS (explained below) 

734 which will soon be made freely available as an open-source R package. A detailed description of 

735 this test can also be found in the EDSP Tier 2 Medaka Multi-generation Test (MMT) Integrated 

736 Summary Report (ISR).

737

738

739

740
20

741 5 INTER-LABORATORY EVALUATION OF THE LAGDA

742

743 5.1 Overview of approach

744

745 Inter-laboratory evaluations of the LADGA were conducted to evaluate the practical 

746 transferability of the assay protocol and quantitative reproducibility of the results. The following 

747 laboratories participated in the inter-laboratory validation study: US EPA MED; U.S. Army 

748 Center for Environmental Health Research (ACEHR) in Fort Detrick, MD; IDEA Consultants, 

749 Inc., a Japanese environmental research facility that is contracted to the Japanese government;

750 ABC Laboratories, Inc. in Columbus, MO; and, Fort Environmental Laboratories (FEL) in 

751 Stillwater, OK. The latter two labs were sub-contractors to the US EPA contractor Battelle

752 Memorial Institute, a global research and development organization. Four known endocrine 

753 active chemicals were selected for testing: prochloraz, 4-tert-octylphenol, 17-β trenbolone and 

754 benzophenone-2. Table 5.1-1 summarizes which chemicals were tested in each participating 

755 laboratory. The participating laboratories were provided a draft protocol to follow. This 

756 protocol, in its revised form, can be found in Appendix 8.1. In some cases, minor changes were 

757 made, and any deviations that occurred are pointed out in the section below. 

758

759 Table 5.1-1 Outline of inter-laboratory participation

Prochloraz 4-tert-octylphenol 17-β trenbolone Benzophenone-2

Laboratory (aromatase inhibitor; 

AR antagonist)

(ER agonist) (AR agonist) (ER agonist; TPO inhibitor)

A X X X

B X X

C X X

D X

E X

760

761 5.2 Inter-laboratory data analysis

762

763 Following the studies, data were provided to MED for analysis using StatCHARRMS (Statistical 

764 analysis of Chemistry, Histopathology, And Reproduction endpoints including Repeated 

measures and Multi-generation Studies), a SAS®765 (SAS Institute, Cary, NC)-based program 

766 developed for MED by John Green (Dupont Applied Statistics Group, Newark, DE). For 

767 continuous data measured once per subject, normality (via Shapiro-Wilk test) and variance 

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

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

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

771 the data as long as a monotonic response was observed. For consistency across all LAGDA 

772 validation studies, the statistical test that indicated the lowest concentration significantly 

773 different than control was reported. Table 5.2-1 summarizes the statistical tests used by 

774 StatCHARRMS, while a thorough description of the StatCHARRMS package including 

775 statistical rationale can be found in the EDSP Tier 2 MMT ISR.
21

776

777

Table 5.2-1 StatCHARRMS decision logic for statistical tests on continuous data.

Shapiro-Wilk Levene’s Statistical test

Pass Pass Dunnett; Jonckheere-Terpstra

Fail Pass Modified Dunn’s; Jonckheere-Terpstra

Pass Fail Tamhane-Dunnett; Jonckheere-Terpstra

Fail Fail Jonckheere-Terpstra

778

779 Histopathology data were analyzed using the RSCABS test explained above implemented in 

780 StatCHARRMS. As with the continuous data, for consistency across all LAGDA validation 

781 studies, the lowest concentration exhibiting a pathology that was significantly different than 

782 control was reported. 

783 5.3 Summary of results

784 5.3.1 Prochloraz

785

786 Introduction

787

788 Prochloraz is an imidazole fungicide shown to have multiple mechanisms of action within the 

789 HPG axis. In vitro studies have shown that prochloraz has characteristics of both an antagonist 

790 of the androgen receptor and inhibitor of aromatase activity (Noriega et al., 2005; Villeneuve et 

791 al., 2007). In adult fish, in vivo exposure to prochloraz produces effects that are consistent with 

792 inhibition of aromatase (Ankley et al., 2005). Further, developmental exposure in rats produces 

793 a spectrum of male reproductive tract malformations which have been attributed to anti794 androgenic properties (Noriega et al., 2005; Blystone et al., 2007). Interestingly, females were 

795 not affected by developmental exposure in the aforementioned two studies. 

796

797 Results

798

799 Exposure levels and measured test concentrations

800

801 The high test concentration was set based on range finding experiments conducted by A. In 

802 those studies conducted with X. tropicalis, concentrations in excess of 200 μg/L resulted in 

803 mortality in early larval organisms. The high test concentration for the inter-laboratory 

804 prochloraz validation study was 180 μg/L with a 0.33 dilution factor resulting in nominal test 

805 concentrations of 180, 60, 20 and 6.7 μg/L. All four labs met the criteria of maintaining 

806 coefficients of variation (CVs) of ≤ 20% over the entire test for all concentrations with the 

807 exception of the 6.67 and 20 μg/L nominal concentrations at C which had CVs of 38 and 23%

808 and were on average 84 and 25% above nominal, respectively (Appendix 8.4.1, Table 8.4.1-1). 

809 Measured concentrations did not show any crossover among treatment levels in all labs except C 

810 which had several overlaps in treatment concentrations through week five of exposure. CV 

811 values between replicates could not be calculated on a weekly basis for B and C due to 

812 measurements of only one replicate tank per week. Treatment levels are referred to as nominal 

813 concentrations regardless of mean-measured concentrations reported by each of the labs.
22

814

815 Measure of General Toxicity 

816

817 The first prochloraz study was performed by lab A in the initial stages of protocol development.

818 The interim sampling to evaluate endpoints related to larval development and potential thyroid 

819 active agents consisted of sample collection at NF stage 66, or at the completion of 

820 metamorphosis. Following completion of the prochloraz study at lab A, it was decided that 

821 thyroid related endpoints were better assessed at NF stage 62 due to maximal constitutive levels 

822 of circulating thyroxine (T4) occurring at this stage. Therefore, interim assessments occurred at 

823 NF stage 66 for lab A while the other laboratories performed interim assessments at NF stage 62.

824

825 Larval and Juvenile Growth 

826

Summary of inter-laboratory prochloraz growth endpoints.

All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal 

regardless of mean-measured concentrations. Dash: Not significant.

Lab A D B C

Larval sample

Weight - 20 μg/L 180 μg/L -

Length - 180 μg/L 180 μg/L 60 μg/L*

Juvenile Males

Weight - 60 μg/L - -

Length - 60 μg/L - -

LSI 60 μg/L 6.7 μg/L 180 μg/L -

Juvenile Females

Weight - 180 μg/L - 60 μg/L*

Length - 180 μg/L 20 μg/L -

LSI 60 μg/L 20 μg/L 20 μg/L 180 μg/L

* Non-monotonic (i.e., the only treatment significantly different than control)

827

828

829 Several measures often associated with general toxicity have been incorporated into the test 

830 protocol. They include mortality, measures of growth (length and weight), LSI, and liver and 

831 kidney pathology. The responses of these endpoints were generally consistent across the four 

832 studies. When considered together these results demonstrate that prochloraz, at the higher end of 

833 the concentration range, was overtly toxic to the developing organism (Appendix 8.4.1, Table 

834 8.4.1-5).

835

836 Two laboratories (B, D) demonstrated impacts on growth in the larval and juvenile organisms in 

837 a concentration-dependent fashion; the other two did not. It is noteworthy that control length and 

838 weight were greatest in these two studies that showed growth effects (Appendix 8.4.1, Tables

839 8.4.1-3 and 8.4.1-5).

840

841 Three laboratories (A, D and B) showed significant decreases in both juvenile male and female 

842 LSI (Appendix 8.4.1, Table 8.4.1-5). This is consistent with the liver histopathology findings 

843 which indicated significant pathologies produced by high concentrations of prochloraz for these 

844 three laboratories (described below). In contrast, females in the C study showed an increase in

845 LSI at the highest test concentration. The increase in LSI at C may be due to an overall lack of 

846 juvenile growth across the entire study performed by C. Further, increased liver weight relative 

847 to body weight is reasonable given the unique spectrum of liver pathologies in this laboratory.
23

848

849 Liver and Kidney pathology 

850

Summary of inter-laboratory prochloraz liver and kidney pathology.

All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal 

regardless of mean-measured concentrations.

Lab A D B C

Juvenile Males

Liver pathology 60 μg/L 20 μg/L 20 μg/L 20 μg/L

Kidney pathology 180 μg/L 180 μg/L 6.7 μg/L 20 μg/L

Juvenile Females

Liver pathology 60 μg/L 20 μg/L 60 μg/L 20 μg/L

Kidney pathology 60 μg/L 60 μg/L 6.7 μg/L 20 μg/L

851

852 Consistencies in treatment-related findings were observed for hepatocellular degeneration and 

853 hepatocellular atrophy in three of the four labs (Appendix 8.4.1, Tables 8.4.1-7 and 8.4.1-8). A

854 and B showed significant hepatocellular degeneration in the 180 μg/L treatment in both male and 

855 female organisms. Hepatocellular atrophy was observed in the D and B studies at the 20 μg/L 

856 treatment level and above in both male and female organisms. 

857

858 The C study exhibited a different suite of treatment-related liver pathologies including basophilia, 

859 increased hepatocellular vacuolization, and granulomatous inflammation, all of which were 

860 significantly different than the control at 60 and 180 μg/L (Appendix 8.4.1, Tables 8.4.1-7 and 

861 8.4.1-8). Cellular hypertrophy was also observed in C liver samples and showed a significant 

862 concentration-dependent effect. Prevalence, severity, sensitivity and pathologies were similar 

863 between males and females with slight variation in severity and sensitivity. The stark difference 

864 between findings at C compared to the other three labs is difficult to explain. It cannot be ruled 

865 out that these organisms were impacted by an infectious disease. This would be consistent with 

866 the observation that control organisms failed to thrive in terms of growth. 

867

868 The most consistent treatment-related finding in the juvenile kidney was tubular amphophilic 

869 intracytoplasmic inclusions, which were significantly present in the 180 μg/L treatment at A, D

870 and C (Appendix 8.4.1, Tables 8.4.1-13 and 8.4.1-14). This response was consistent between 

871 males and females. Other pathologies were observed but lacked consistency between studies and 

872 often were not treatment related. 

873

874 Thyroid Endpoints

875

Summary of inter-laboratory prochloraz thyroid endpoints.

All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal 

regardless of mean-measured concentrations. Dash: Not significant.

Lab A D B C

Thyroid endpoints

Time to NF stage 62 - 20 μg/L* - -

Serum T4 60 μg/L - - 6.7 μg/L

Thyroid pathology 180 μg/L 20 μg/L - 6.7 μg/L

* Non-monotonic (i.e., the only treatment significantly different than control)

876

877

878 Time to NF stage 62
24

879

880 There were no treatment-related effects on time to NF stage 62 (NF stage 66 in the case of A) in 

881 all four participating laboratories (Appendix 8.4.1, Tables 8.4.1-2 and 8.4.1-3). 

882

883 Larval Thyroid Pathology 

884

885 A, D, and C studies showed mild but significant treatment-related effects in the thyroid gland 

886 consistent with compensation commonly observed following thyroid axis disruption including

887 follicular cell hypertrophy and hyperplasia (Appendix 8.4.1, Table 8.4.1-6). Although these 

888 pathologies were present in larvae at B, there were no clear concentration-dependent trends in 

889 their study. This was an unexpected finding for prochloraz as there are no published reports of 

890 thyroid axis disruption. 

891

892 Larval blood thyroxine

893

894 The D, B and C laboratories used commercially available T4 ELISA kits with specific antibodies 

895 for human or canine T4. These laboratories failed to optimize these kits for use with X. laevis

896 and consequently were unsuccessful in making these measurements; therefore, these data are not 

897 included in this analysis. Laboratory A measured T4 levels in serum using a high pressure liquid 

898 chromatography and inductively-coupled plasma mass spectrometry (HPLC/ICP-MS) method,

899 which has been previously optimized for use with X. laevis (Tietge et al., 2010; Tietge et al., 

900 2012). In the A study, a significant increase in T4 was observed in the 60 and 180 μg/L 

901 treatments (Appendix 8.4.1, Table 8.4.1-4). It is important to note that the few days prior to NF 

902 stage 66 are marked by a rapid and appreciable decrease in serum T4 levels. This, when 

903 considered with the liver pathology findings, may suggest that compromised liver function may 

904 have resulted in decreased metabolism and elimination of T4. 

905

906 Reproductive Endpoints

907

Summary of inter-laboratory prochloraz reproductive endpoints.

All endpoints with lowest concentration statistically different than control. Concentrations listed as nominal 

regardless of mean-measured concentrations. Dash: Not significant.

Lab A D B C

Juvenile Males

Plasma VTG 20 μg/L 180 μg/L - 6.7 μg/L

Gonad pathology 6.7 μg/L 6.7 μg/L 6.7 μg/L 20 μg/L

Duct pathology 20 μg/L 60 μg/L 20 μg/L 20 μg/L

Juvenile Females

Plasma VTG 6.7 μg/L 60 μg/L - 20 μg/L

Gonad pathology 180 μg/L 60 μg/L 20 μg/L 6.7 μg/L

Duct pathology 180 μg/L - 180 μg/L -

908

909

910 Juvenile plasma vitellogenin

911

912 A, D and C showed significant increases in both juvenile male and female plasma vitellogenin 

913 titers (Appendix 8.4.1, Table 8.4.1-5). B showed no significant difference between control and 

914 treated organisms but also reported titer values that were much lower on average than the other 

915 three laboratories. Although the VTG ELISA registered a measurable signal in control males 
25

916 and females in all labs, VTG production has been shown not to begin until later in development

917 (Olmstead, 2009b). Therefore, these control values likely reflect background noise within the 

918 assay. 

919

920 Gonad Histopathology 

921

922 As with the liver pathology, two different contract pathologists were responsible for reading the 

923 various studies. In terms of the testis and ovary, there appears to be some inconsistencies in the 

924 results across the laboratories. However, this may be more a result of the differences in sample 

925 processing and reading rather than a true difference in results among the four laboratories. Labs 

926 A and D studies were processed and read by Integrated Laboratory Systems, Research Triangle 

927 Park, NC whereas Labs B and C studies were processed and read by Experimental Pathology 

928 Laboratory, Sterling, VA. In both the testis and ovary, the A and D studies showed many 

929 consistencies in diagnosis whereas C and B share consistencies. In all cases, the genetic sex 

930 matched the phenotype determined by the pathologist and indicated that treatment did not result 

931 in sex reversal.

932

933 Testis

934

935 Of the ten observed pathologies showing significant concentration-dependent trends in at least 

936 one of the four studies, several important findings showed a consistent treatment-related

937 response across the studies. A treatment-related increase in germ cell degeneration was observed 

938 across all studies and showed significance in the A, B and C studies (Appendix 8.4.1, Table 

939 8.4.1-9). Germ cell degeneration in the testis was characterized by the presence of apoptotic 

940 germ cells (either as a single cell or small cluster of cells) or germ cell syncytia. Germinal 

941 epithelium thinning was observed in the A, C and D studies, and its prevalence and severity 

942 increased in a concentration-dependent fashion. Germinal epithelial thinning presented as 

943 scattered enlargement of seminiferous tubule lumina, with irregular attenuation of the germinal 

944 epithelium surrounding affected tubules (Figure 5.2-1). A significant increase in spermatogonia 

945 was also observed in these same studies, and the pathologist noted that, in those cases with 

946 moderate germinal epithelial thinning /loss, the increase in spermatogonia may reflect a relative 

947 increase due to the loss of spermatocytes/spermatids rather than an absolute increase in numbers. 

948 The degenerative testicular changes observed in these studies are consistent with the anti949 androgenic effects of prochloraz reported in species as divergent as fathead minnows (Ankley et 

950 al., 2005) and rats (Blystone et al., 2007). In particular, germinal epithelium thinning/loss with 

951 high doses of prochloraz has been described in rats following exposure during gonadal 

952 development (Noriega et al., 2005). 

953

954

955
26

956

957 Figure 5.2-1 A) Control group male from prochloraz study, provided for reference, 10X. B) 

958 Prochloraz 180 μg/L treatment group male, 10X. Gonadal degeneration thinning/ loss grade 2. 

959 The germinal epithelium lining of numerous spermatic tubules is thin to absent (black arrows) 

960 with occasional exposure of the underlying basement membrane (red arrow). C) 180 μg/L

961 treatment group male, 10X. Gonadal degeneration thinning/loss, Grade 4. Diffusely, the 

962 germinal epithelium is missing or thin with frequent exposure of the underlying basement 

963 membranes (black arrows).

964

965 Ovary 

966

967 Histopathological findings in the ovaries of prochloraz-exposed female frogs were inconsistent 

968 and minimal (Appendix 8.4.1, Table 8.4.1-10). This finding is consistent with those findings in 

969 rats in which only male gonads are affected following developmental exposure to prochloraz 

970 (Noriega et al., 2005). 

971

972 Reproductive Ducts

973

974 There was a very consistent response to prochloraz treatment across all four labs in oviduct 

975 development (Appendix 8.4.1, Tables 8.4.1-11 and 8.4.1-12). In males, significant 

976 concentration-dependent delays in oviduct involution were evident in all studies indicating 

977 interference with normal oviduct regression (Kelley, 1996), a finding similar to the feminization 

978 of the secondary sexual characteristics of male rats during perinatal prochloraz exposure 

979 (Noriega et al., 2005; Vingaard et al., 2005). Female oviduct development was not affected by 

980 treatment across all four studies. However, female Wolffian duct development was slightly 

A

B

C
27

981 advanced in the highest treatment at B, and minimal mononuclear inflammation of the Wolffian 

982 duct was significantly present in the highest treatment at A. 

983

984 5.3.2 4-tert-octylphenol

985

986 Introduction

987

988 The weak estrogen, 4-tert-octylphenol (OP), (Bonefeld-Jorgensen et al., 2007), is a degradation 

989 product of octylphenol polyethoxylate used as non-ionic surfactants in industrial and household 

990 settings (Ying et al., 2002). Vitellogenin has been shown to be elevated in male zebrafish (Ortiz991 Zarragoitia and Cajaraville, 2005), male eelpout (Rasmussen et al., 2005), male and female 

992 medaka (Seki et al., 2003), and in X. laevis primary cultured hepatocytes (Mitsui et al., 2007)

993 exposed to OP. Decreased sperm count and the presence of testis-ova have been observed in 

994 medaka exposed to OP (Seki et al. 2003); decreased gonadosomatic index and increased testis995 ova have been reported in eelpout exposed to OP (Rasmussen et al., 2005); and decreased 

996 spermatozoa and increased testis anomalies have been documented in sheepshead minnows

997 exposed to OP (Karels et al., 2003). Female minnows exposed to OP have been shown to 

998 produce a decreased percent of viable eggs (Karels et al., 2003). Kloas et al. (1999) reported 

999 that exposure of larval X. laevis to OP resulted in a significant shift in sex ratio towards females.

1000 These finding could not be reproduced in studies conducted with X. tropicalis (Porter et al., 

1001 2011). The only significant findings in the Porter et al. (2011) studies were induction of VTG 

1002 and oviduct development in adult males. Based on these previously published studies, OP was 

1003 used as a representative weak estrogen for testing the transferability of the LAGDA. 

1004

1005 Results

1006

1007 Exposure levels and measured test concentrations

1008

1009 Test concentrations were set based on the findings of Porter et al. (2011) and a short term 

1010 embryo/larval exposure conducted at MED. In these studies, concentrations above 60 μg/L 

1011 resulted in significant mortality in embryos and early stage larvae. For this inter-laboratory 

1012 study, the high test concentration was set at 50 μg/L with a 0.5 dilution factor resulting in 

1013 nominal test concentrations of 50, 25, 12.5 and 6.25 μg/L. All test concentrations at A were 

1014 maintained with CVs ≤ 20% (Appendix 8.4.2, Table 8.4.2-1). B maintained test concentrations 

1015 with CVs ≤ 20% except for the lowest concentration. C had CVs ≤ 20% in two of the four 

1016 concentrations and had measurable amounts of test chemical in control water on three different 

1017 occasions. Weekly CV values could not be calculated for B and C due to only one replicate tank 

1018 being measured per week. Lab C’s concentrations were on average higher than nominal with the 

1019 lowest concentration >140% of nominal. Lab B’s concentrations were on average lower than 

1020 nominal with all mean-measured concentrations being between 73 and 77% of nominal.

1021

1022 Measure of General Toxicity 

1023
28

1024 Mortality, measures of growth (length and weight), liver somatic index, and liver and kidney 

1025 pathology were generally consistent across the four studies. When considered together, results 

1026 demonstrate that OP had little impact on the general health of the organisms. 

1027

1028 Survival

1029

1030 All labs met the criteria of ≤ 20% mortality in any one control replicate that could not be 

1031 explained by technical error, and no treatment-related trends in survival were identified. Lab A

1032 experienced technical difficulties (drain obstruction and tank overflow) late in the study and lost 

1033 one 12.5 μg/L replicate and two 50 μg/L replicates following the post-NF stage 66 cull (data not 

1034 shown).

1035

1036 Larval and Juvenile Growth 

1037

Summary of inter-laboratory octylphenol growth endpoints. 

All endpoints with lowest concentration statistically different than control. 

Concentrations listed as nominal regardless of mean-measured concentrations. Dash: 

Not significant.

Lab A B C

Larval sample

Weight - - -

Length 6.25 μg/L* 50 μg/L -

Juvenile Males

Weight - - -

Length - - -

LSI - - 50 μg/L

Juvenile Females

Weight - - -

Length - - 12.5 μg/L*

LSI - - 25 μg/L

* Non-monotonic (i.e. the only treatment significantly different than control)

1038

1039 There were no treatment-related effects on larval or juvenile growth in any of the three studies. 

1040 Juvenile LSI was significantly decreased in octylphenol-exposed males and females from C 

1041 whereas no treatment-related effects on LSI were measured at A or B (Appendix 8.4.2 Tables

1042 8.4.2-2 and 8.4.2-4). However, it is again worth noting that lab C organisms were smaller on 

1043 average than the other labs and it is uncertain whether reduced growth factored into this finding.

1044

1045 Liver and Kidney pathology 

1046

Summary of inter-laboratory octylphenol liver and kidney pathology.

All endpoints with lowest concentration statistically different than control. 

Concentrations listed as nominal regardless of mean-measured concentrations. Dash: 

Not significant.

Lab A B C

Juvenile Males

Liver pathology - - -

Kidney pathology - 25 μg/L -

Juvenile Females

Liver pathology - - -

Kidney pathology 50 μg/L 50 μg/L 6.25 μg/L

1047
29

1048

1049 No treatment-related effects were present in any of the octylphenol-exposed livers from all three 

1050 studies. Granulomatous inflammation was observed in all three studies, but prevalence and 

1051 severity were comparable among treated and control samples. Cystic degeneration was observed 

1052 in one of the three studies (B), but prevalence and severity were also comparable among treated 

1053 and control samples (data not shown). These results are suggestive of response to an infectious 

1054 disease. 

1055

1056 Male kidney tubule dilation and mineralization were observed throughout all three studies

1057 (Appendix 8.4.2, Table 8.4.2-10). Prevalence and severity were comparable between treatment 

1058 and control samples in most cases. However, these pathologies were significantly attenuated 

1059 with octylphenol exposure at B. Minimal interstitial proteinaceous fluid was observed at B but 

1060 was essentially not remarkable. 

1061

1062 Female kidney tubule dilation and mineralization were observed throughout all three studies

1063 (Appendix 8.4.2, Table 8.4.2-11). Lab C male kidneys exhibited a similar pattern to B male 

1064 kidneys where these pathologies were attenuated with octylphenol treatment. Lab A showed a 

1065 significant, but slight increase in severity of tubule dilation in the 50 μg/L treatment which was

1066 inconsistent with the findings in the other studies. Lab B females did not show significant trends 

1067 in these pathologies but showed significant minimal to mild severity of edema, although 

1068 prevalence was very low.

1069

1070 Thyroid Endpoints

1071

Summary of inter-laboratory octylphenol thyroid endpoints.

All endpoints with lowest concentration statistically different than control. 

Concentrations listed as nominal regardless of mean-measured concentrations. Dash: 

Not significant.

Lab A B C

Thyroid endpoints

Time to NF stage 62 50 μg/L - -

Serum T4 25 μg/L - -

Thyroid pathology 12.5 μg/L - -

1072

1073

1074 Time to NF stage 62

1075

1076 There was a significant delay in time to NF stage 62 in the A study at the highest test 

1077 concentration but not in studies conducted by B and C (Appendix 8.4.2, Table 8.4.2-2).

1078 . 

1079 Larval thyroid pathology

1080

1081 In the B and C studies, there were no significant impacts on thyroid glands. The only 

1082 statistically significant findings were increases in follicular cell hypertrophy and hyperplasia in 

1083 the A study at test concentrations ≥ 12.5 and 50 μg/L, respectively (Appendix 8.4.2, Table 8.4.2-

1084 5). 

1085

1086 Larval blood thyroxine

1087
30

1088 As indicted above, failure to optimize the ELISA kits by B and C resulted in no reliable T4

1089 measurement data. Lab A elected to develop an extraction method which allowed for the use of

1090 these commercially available ELISA kits (LAGDA Guideline Appendix 7) with X. laevis serum.

1091 Lab A also measured T4 levels in serum using an HPLC/ICP-MS method (Appendix 8.4.2, Table 

1092 8.4.2-3). OP exposure resulted in a concentration-dependent decrease in T4 (Figure 5.2.2-1). 

1093 The two methods were in agreement in terms of control levels and effects of OP.

1094

1095

1096

1097 Figure 5.2.2-1. The top panel (A) shows the results for the ELISA method, and the bottom panel

1098 (B) shows results for the ICP-MS method. The detection limits were 2.5 ng/ml and 0.15 ng/ml for 

1099 the ELISA and ICP-MS methods, respectively. The measured T4 levels were consistent between 

1100 the two methods. * significant at p< 0.05

1101

1102

1103 Reproductive Endpoints

1104

Summary of inter-laboratory octylphenol reproductive endpoints.

All endpoints with lowest concentration statistically different than control. 

Concentrations listed as nominal regardless of mean-measured concentrations. Dash: 

Not significant.

Lab A B C

Juvenile Males

Plasma VTG 12.5 μg/L - 6.25 μg/L

Gonad pathology - 12.5 μg/L -

Duct pathology 6.25 μg/L 25 μg/L -

Juvenile Females

Method: ICP-MS

Nominal 4-tert-octylphenol concentration (ug/L)

con 6.25 12.5 25 50

Serum T4 concentration (ng/ml)

0

10

20

30

40

50

60

70

*

B

Method: ELISA

Control 6.25 12.5 25 50

Serum T4 concentration (ng/ml)

0

10

20

30

40

50

60

70

*

*

A
31

Plasma VTG - 50 μg/L 6.25 μg/L

Gonad pathology 12.5 μg/L - 12.5 μg/L

Duct pathology 50 μg/L 12.5 μg/L 6.25 μg/L

1105

1106

1107 Juvenile plasma vitellogenin

1108

1109 Vitellogenin titers showed a concentration-dependent increase in octylphenol-exposed males at 

1110 labs A and C (Appendix 8.4.2, Table 8.4.2-4). Female vitellogenin levels showed a 

1111 concentration- dependent increase at labs B and C.

1112

1113 Gonad histopathology

1114

1115 Genetic sex was determined using the DMW sex-linked marker. In the cases of laboratories B 

1116 and C, there were significant numbers of individuals in which genetic sex did not match 

1117 phenotypic sex as determined by histological analysis. There was no indication of a treatment1118 related effect given that the mismatches occurred randomly throughout the control and treatment 

1119 groups. Further, both phenotypic males and females were misidentified. Upon further 

1120 investigation, it was determined that some, but not all, of the observed mismatches from 

1121 laboratory C could be explained by a labeling error that was identified and corrected. In order to 

1122 determine the nature of the remaining discrepancies, tissue samples from both lab B and C 

1123 studies were sent to MED for re-analysis of genetic sex. In all cases, the analyses performed by 

1124 MED resolved the apparent mismatching. Thus, OP did not produce sex reversal in any of the 

1125 studies.

1126

1127 Testis

1128

1129 Effects of octylphenol exposure on male gonads were minimal. Mononuclear cellular infiltrate 

1130 was observed throughout all three studies but was not treatment-related, although statistically, 

1131 severity was significantly less than controls at B (Appendix 8.4.2, Table 8.4.2-6). There were 

1132 very few observations of minimally increased spermatogonia at B and C, but the 50 μg/L 

1133 treatment at B was significantly different than the control. Statistically, there was a slight 

1134 advancement of testicular development in the 50 μg/L treatment at B, and this advancement 

1135 showed consistency across all three labs, although it was not statistically significant at labs A or 

1136 C. 

1137

1138 Ovary

1139

1140 Unlike male gonads, female gonads experienced treatment-related increases in severity of 

1141 mononuclear cellular infiltrate which were significant (minimal to mild) in the 12.5, 25 and 50 

1142 μg/L treatments at labs A and C (Appendix 8.4.2, Table 8.4.2-7). Granulomatous inflammation 

1143 was minimal at labs B and C in the low treatments and control, but was absent in the 25 and 50 

1144 μg/L treatments.

1145

1146 Reproductive duct histopathology

1147
32

1148 There were significant delays in male oviduct involution at lab A (6.25, 12.5, 25, 50 μg/L) and 

1149 lab B (25, 50 μg/L) and evidence of inappropriate advancement in male oviduct maturation at the 

1150 50 μg/L treatment at B (Appendix 8.4.2, Table 8.4.2-8). Although there was a significant delay 

1151 in Wolffian duct development in the highest treatment at B, stage distributions were mostly 

1152 consistent across labs and similar between treatments and controls. Female oviduct development 

1153 was significantly accelerated with octylphenol exposure across all three labs (Appendix 8.4.2, 

1154 Table 8.4.2-9). However, sensitivity differed between labs: Lab A only showed a significant 

1155 effect in the 50 μg/L treatment whereas significant effects were exhibited down to 12.5 and 6.25 

1156 μg/L at B and C, respectively. Both effects on males and female ducts are consistent with an 

1157 estrogenic response, a commonly reported activity of OP. 

1158

1159 5.3.3 17β-trenbolone 

1160

1161 Introduction

1162

1163 Trenbolone is an androgen receptor agonist used in cattle production and has been measured in 

1164 aquatic systems near animal-feeding operations (e.g., stock yards). The effects of trenbolone 

1165 exposure on fish have been studied with respect to those physiological processes regulated via 

1166 the HPG axis mainly with respect to gonad differentiation and development in fish and 

1167 amphibian species (Orn et al., 2006; Holbech et al., 2006; Morthorst et al., 2010; Olmstead et al.,

1168 2012). 17β-trenbolone has been tested previously in the closely related amphibian species X. 

1169 tropicalis (Olmstead et al., 2012). In that study, the authors characterized the effects of aqueous 

1170 exposure to 17β-trenbolone during larval X. tropicalis development using an exposure protocol 

1171 similar to the LAGDA. Concentrations ≥ 100 ng/L resulted in a spectrum of androgenic 

1172 responses which included hypertrophy of the larynx resulting in late larval mortality, 

1173 development of nuptial pads, a mixed-sex phenotype in the ovaries of females, and hypertrophy 

1174 of the Wolffian duct.

1175

1176 Results

1177

1178 Exposure levels and measured test concentrations

1179

1180 Given the observed mortality in the studies of Olmstead et al. (2012) the highest test 

1181 concentration was set at 100 ng/L, with a 0.5 dilution factor resulting in nominal test 

1182 concentrations of 100, 50, 25 and 12.5 ng/L. Mean ± SD (n = 40) measured concentrations of 

1183 the 12.5, 25, 50 and 100 ng/L 17β-trenbolone treatments were 9.0 ± 2.8, 19.2 ± 5.8, 41.4 ± 9.7 

1184 and 79.9 ± 24.1 ng/L, respectively, over the duration of the experiment. CVs (averaged across 

1185 the duration of the exposure) were 31, 30, 23 and 30% for the 12.5, 25, 50 and 100 ng/L 

1186 treatments, respectively. No 17β-trenbolone was detected in control water.

1187

1188 Measure of General Toxicity 

1189

1190 Survival

1191
33

1192 All control tanks met the guideline performance criteria of ≤ 20% mortality and there were no 

1193 treatment-related effects on survival (data not shown).

1194

1195 Larval and Juvenile growth

1196

1197 There were no significant treatment-related effects on larval and juvenile growth as measured by 

1198 weight and length (Appendix 8.4.3, Tables 8.4.3-1 and 8.4.3-2). Further, there were no 

1199 significant treatment-related effects on LSI for either gender (Appendix 8.4.3, Table 8.4.3-2).

1200

1201 Liver and Kidney histopathology

1202

Summary of trenbolone liver and kidney pathology.

All endpoints with lowest concentration statistically different 

than control. Concentrations listed as nominal regardless of 

mean-measured concentrations. Dash: Not significant.

Lab A

Nominal conc. 100, 50, 25, 12.5 ng/L

Juvenile Males

Liver pathology 50 ng/L

Kidney pathology 50 ng/L

Juvenile Females

Liver pathology 100 ng/L

Kidney pathology -

1203

1204 Treatment-related effects were present in the liver. The prevalence of minimal cellular 

1205 hypertrophy of hepatocytes was significantly different than the control in the 50 and 100 ng/L 

1206 treatments for juvenile males and females, respectively (Appendix 8.4.3, Table 8.4.3-4). The 

1207 prevalence and severity of the only other finding in the liver, granulomatous inflammation, were

1208 comparable among treated and control samples.

1209

1210 Two common findings in male kidneys were renal tubule mineralization/casts (minimal to 

1211 moderate) and renal tubule dilation (minimal to mild). However, tubule dilation was 

1212 significantly attenuated in the 50 and 100 ng/L treatments whereas mineralization/casts were not 

1213 treatment-related (Appendix 8.4.3, Table 8.4.3-6). There was a low but statistically significant 

1214 occurrence of interstitial fibrosis (minimal to mild) in the 100 ng/L treatment when compared to 

1215 the control. There were no apparent effects of trenbolone exposure on the kidneys of juvenile 

1216 females (data not shown).

1217

1218 Thyroid Endpoints

1219

Summary of trenbolone thyroid endpoints.

All endpoints with lowest concentration statistically 

different than control. Concentrations listed as 

nominal regardless of mean-measured concentrations. 

Dash: Not significant.

Lab A

Nominal conc. 100, 50, 25, 12.5 ng/L

Larval sample

Time to NF stage 62 -

Serum T4 -

Thyroid pathology 25 ng/L

1220
34

1221

1222 Time to NF stage 62

1223

1224 Trenbolone exposure did not affect the time it took for larvae to reach NF stage 62 (Appendix 

1225 8.4.3, Table 8.4.3-1).

1226

1227 Larval Thyroid histopathology

1228

1229 The two categories of morphologic findings in thyroid gland tissue, both of which were common, 

1230 were follicular cell hypertrophy (mild to moderate) and follicular cell hyperplasia (mild to 

1231 moderate). The severity of follicular cell hypertrophy was slightly decreased in the 50 and 100 

1232 ng/L trenbolone treatment groups as compared to the control, but those differences were not 

1233 statistically significant (Appendix 8.4.3, Table 8.4.3-3). The prevalence and severity of follicular 

1234 cell hyperplasia in trenbolone-treated frogs were mild but significantly different than the control 

1235 in the 25, 50 and 100 ng/L treatments.

1236

1237 Larval blood thyroxine

1238

1239 There were no treatment-related effects on larval serum T4 levels as measured by HPLC-ICP-MS 

1240 analysis (Appendix 8.4.3, Table 8.4.3-1).

1241

1242 Reproductive Endpoints

1243

Summary of trenbolone reproductive endpoints.

All endpoints with lowest concentration statistically 

different than control. Concentrations listed as nominal 

regardless of mean-measured concentrations. Dash: Not 

significant.

Lab A

Nominal conc. 100, 50, 25, 12.5 ng/L

Juvenile Males

Plasma VTG 50 ng/L

Gonad pathology 100 ng/L

Duct pathology 100 ng/L

Juvenile Females

Plasma VTG -

Gonad pathology -

Duct pathology 50 ng/L

1244

1245

1246 Juvenile plasma vitellogenin

1247

1248 Trenbolone treatment did not affect vitellogenin titers in juvenile male or female frogs (data not 

1249 shown).

1250

1251 Gonad histopathology

1252

1253 In all cases, the genetic sex matched the phenotype determined by the pathologist and indicates 

1254 that treatment did not result in sex reversal. There were two significant treatment-related 

1255 findings in the testes of males: increased spermatogonia (minimal) and germ cell vacuolation 
35

1256 (minimal to mild) in the 100 ng/L treatment (Appendix 8.4.3, Table 8.4.3-5). Germ cell 

1257 vacuolation was characterized by clear cytoplasmic ballooning in small clusters of pachytene1258 phase (cell nuclei in prophase of meiosis) spermatocytes. There were no apparent effects of 

1259 trenbolone exposure on the ovaries of juvenile females (data not shown). 

1260

1261 Reproductive duct histopathology

1262

1263 There were treatment-related effects on oviduct development in both genders. Males exhibited

1264 accelerated regression of oviducts in the 100 ng/L treatment as compared to the control whereas 

1265 females experienced prodigious oviduct regression in the 50 and 100 ng/L treatments (Appendix 

1266 8.4.3, Table 8.4.3-7). There were no treatment-related effects on Wolffian duct development or 

1267 pathology in either gender. Given the importance of the oviduct as a female reproductive organ 

1268 necessary for application of the gel coat to the oocyte and release of the oocyte, these effects are 

1269 expected to have considerable consequence on reproductive capacity (Figure 5.2.3-1). 

1270

1271

1272

1273

1274

1275 Figure 5.2.3-1 A) Stage 2 oviduct (arrow) observed in control females. B) Stage 1 oviduct 

1276 (arrow) from a female exposed to 100 ng/L trenbolone. Stage 1 oviducts such as this were not 

1277 observed in any control females.

1278

1279 5.3.4 Benzophenone-2

1280

1281 Introduction

1282

1283 Benzophenone-2 (BP-2) is used as a uV filter in personal care products such as cosmetics and

1284 sunscreens and in numerous other products for uV protection. BP-2 has been found to be 

1285 estrogenic in vitro and in vivo and is capable of impacting reproduction in fish (Weisbord et al.,

1286 2007). In addition to this documented estrogenic activity, BP-2 has been shown to be a thyroid 

1287 peroxidase inhibitor, and exposure to BP-2 can result in disruption of the thyroid axis in aquatic 

1288 species (Thienpont et al., 2011). BP-2 provided a unique study in the LAGDA evaluation of a 

1289 mixed mode of action chemical which included thyroid disruption. Laboratory E was the only 

1290 laboratory to conduct a test with BP-2. Due to limitations in exposure capabilities at this 

1291 laboratory, there were several deviations from the guidance provided. Instead of running eight

A

B
36

1292 control replicates and four treatment replicates at four test concentrations and a control, this 

1293 study was conducted using three test concentrations and a control, each with four replicates. Also,

1294 the test was initiated with 30 embryos per tank rather than the recommended 25 embryos. 

1295 Although not completely consistent with the proposed test method, this study provides 

1296 significant value in terms of organism response and transferability of the approach. 

1297

1298 Results

1299

1300 Exposure levels and measured test concentrations

1301

1302 The high test concentration was 6 mg/L with a 0.5 dilution factor resulting in nominal test 

1303 concentrations of 6.0, 3.0 and 1.5 mg/L. Mean ± SD (n = 36) measured concentrations of the 1.5, 

1304 3.0 and 6.0 mg/L benzophenone-2 treatments were 1.6 ± 0.1, 3.5 ± 0.2 and 6.1 ± 0.1 mg/L, 

1305 respectively, over the duration of the experiment. CVs (averaged across the duration of the 

1306 exposure) were 4, 6 and 1% for the 1.5, 3.0 and 6.0 mg/L treatments, respectively. No 

1307 benzophenone-2 was detected in control water.

1308

1309 Measure of General Toxicity 

1310

1311 Survival

1312

1313 BP-2 treatment resulted in significant mortality by NF stage 62 at 6.0 mg/L (Jonckeere-Terpstra, 

1314 Table 5.2.6-1).

1315

Table 5.2.6-1

Mean percent mortality prior to completion of NF62 sample. 

Highlighted cell is significantly different than control.

Nominal BP2 conc. (mg/L) 0 1.5 3 6

n/tank 29 29 29 29

replicates 4 4 4 4

Mean % mortality 0 ± 0 1 ± 2 3 ± 7 38 ± 6

1316

1317 Following the NF stage 66 cull, 10 individuals were left in each tank to continue exposure until 

1318 test termination. Within this timeframe, significant mortality was experienced in the high test 

1319 concentration (Jonckeere-Terpstra, Table 5.2.6-2). 

1320

Table 5.2.6-2

Mean percent juvenile mortality post-cull. Highlighted cell is 

significantly different than control.

Nominal BP2 conc. (mg/L) 0 1.5 3 6

n/tank 10 10 10 10

replicates 4 4 4 4

Mean % mortality 0 ± 0 0 ± 0 5 ± 6 25 ± 13

1321

1322

1323

1324

1325
37

1326 Larval and Juvenile Growth 

1327

Summary of BP-2 growth endpoints.

All endpoints with lowest concentration statistically 

different than control. Concentrations listed as 

nominal regardless of mean-measured concentrations.

Lab E

Nominal conc. 6, 3, 1.5 mg/L

Larval sample

Weight 6 mg/L

Length 6 mg/L

Juvenile Males

Weight 6 mg/L

Length 6 mg/L

LSI 6 mg/L

Juvenile Females

Weight 6 mg/L

Length 6 mg/L

LSI 1.5 mg/L*

* Non-monotonic (i.e. the only treatment significantly different than control)

1328

1329

1330 There was a significant increase in larval growth in the 6 mg/L treatment as compared to the 

1331 control. There was also a significant delay in time to reach NF stage 62 in the 6 mg/L treatment

1332 (Appendix 8.4.4, Table 8.4.4-3). This increase in tadpole size is consistent with previous reports 

1333 following exposure to thyroid axis disruptors which produce a significant developmental delay 

1334 (Degitz et al., 2005; Tietge et al., 2005). Another potential contributor to increased growth in 

1335 the 6 mg/L treatment could be the significant mortality in that treatment allowing the survivors 

1336 more access to food. Growth in male and female juveniles was significantly reduced in the 6 

1337 mg/L treatment compared to the control, presumably due to overt toxicity (Appendix 8.4.4, 

1338 Table 8.4.4-4). LSI appeared to be affected by BP-2 exposure; however, statistical analyses were 

1339 inconsistent between genders due to non-monotonicity of the data in both cases. There was a 

1340 significant decrease in male LSI in the 6 mg/L treatment and a significant increase in female LSI 

1341 in the 1.5 mg/L treatment (Appendix 8.4.4, Table 8.4.4-4).

1342

1343 Liver and Kidney pathology 

1344

Summary of BP-2 liver and kidney pathology.

All endpoints with lowest concentration statistically 

different than control. Concentrations listed as 

nominal regardless of mean-measured concentrations.

Lab E

Nominal conc. 6, 3, 1.5 mg/L

Juvenile Males

Liver pathology 3 mg/L

Kidney pathology 3 mg/L

Juvenile Females

Liver pathology 3 mg/L

Kidney pathology 1.5 mg/L

1345

1346

1347 Treatment-related findings in the liver were limited to phenotypic females and genetic males 

1348 which showed mixed sex gonads (i.e., 3.0 and 6.0 mg/L treatments). These included 
38

1349 concentration-dependent increases in the prevalence and severity of basophilia, decreased 

1350 hepatocellular vacuolation, and intravascular proteinaceous fluid (Appendix 8.4.4, Table 8.4.4-6). 

1351 As compared to control frogs, frogs of the 3 and 6 mg/L treatment groups additionally had higher 

1352 prevalence of increased pigment, which also tended to be concentration-dependent. Furthermore, 

1353 the few findings of individual hepatocyte necrosis and foci of hepatocellular alterations were 

1354 restricted to frogs of the 3 and 6 mg/L treatment groups and 6 mg/L treatment group, 

1355 respectively. Overall, these liver changes are likely related to estrogenic activity of BP-2 and 

1356 significant up-regulation of vitellogenin synthesis (i.e., vitellogenesis).

1357

1358 BP-2 exposure caused profound effects in both genotypic male and female kidneys. These 

1359 effects were very similar between genders and widespread in the 3 and 6 mg/L treatments

1360 (Appendix 8.4.4, Table 8.4.4-8). However, these two treatments had 100% female phenotypes. 

1361 Interestingly, many of the pathologies had higher severity scores in the 3 mg/L treatment and 

1362 were slightly less severe in the 6 mg/L treatment.

1363

1364 Thyroid Endpoints

1365

Summary of BP-2 thyroid endpoints.

All endpoints with lowest concentration statistically 

different than control. Concentrations listed as 

nominal regardless of mean-measured concentrations.

Lab E

Nominal conc. 6, 3, 1.5 mg/L

Larval sample

Time to NF62 6 mg/L

Thyroid pathology 1.5 mg/L

1366

1367

1368 Time to NF stage 62

1369

1370 There was a significant delay in time to NF stage 62 in the 6 mg/L treatment (Appendix 8.4.4, 

1371 Table 8.4.4-3).

1372

1373 Larval thyroid pathology

1374

1375 There were significant treatment-related effects on thyroid tissue including increased prevalence 

1376 and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy (Appendix 

1377 8.4.4, Table 8.4.4-5). The 6 mg/L treatment exhibited glandular hypertrophy and extreme 

1378 follicular cell hypertrophy and hyperplasia in 100% of the individuals. As noted above, there 

1379 was also a significant delay in the rate of metamorphosis.

1380

1381 Larval blood thyroxine

1382

1383 In the BP-2 study, heparinized plasma samples as opposed to serum were analyzed for T4 levels

1384 using both the optimized ELISA described in LAGDA Guideline Appendix 7 and the 

1385 HPLC/ICP-MS method. There were no statistically significant effects on circulating T4 in either 

1386 sample set, and T4 levels from the two methods did not agree. The cause of the lack of 

1387 agreement between the two methods is unknown. Therefore, the data is not presented. The 

1388 effects of using plasma as opposed to serum for T4 analyses are currently under investigation.
39

1389

1390 Reproductive Endpoints

1391

Summary of BP-2 reproductive endpoints.

All endpoints with lowest concentration statistically 

different than control. Concentrations listed as 

nominal regardless of mean-measured concentrations.

Lab E

Nominal 

conc. 6, 3, 1.5 mg/L

Juvenile Males

Plasma VTG 1.5 mg/L

Gonad pathology 1.5 mg/L

Duct pathology 3 mg/L

Juvenile Females

Plasma VTG 1.5 mg/L

Gonad pathology 1.5 mg/L

Duct pathology 1.5 mg/L

1392

1393 Juvenile plasma vitellogenin

1394

1395 There was significant induction of vitellogenin in all treatments compared to the control and the 

1396 response was very consistent between genders (Appendix 8.4.4, Table 8.4.4-4). The overall 

1397 response was a > 5000-fold induction in the 6 mg/L treatment in both genetic males and females 

1398 compared to controls. This illustrates the estrogenic properties of BP-2 and emphasizes the 

1399 potential magnitude of response of this endpoint.

1400

1401 Gonad histopathology

1402

1403 Genotypic males experienced feminization of the gonad resulting in both intersex gonads (e.g., 

1404 presence of testicular oocytes and partial ovarian cavity) and sex-reversed gonads in the 1.5 

1405 mg/L treatment (Figure 5.2.4-1). In the 3 and 6 mg/L treatments, 100% of the genotypic males 

1406 had been completely sex reversed exhibiting only ovarian tissue (i.e., phenotypic females). In 

1407 genotypic females, there was a significant treatment-related delay in gonad development based 

1408 on differences in developmental staging of ovaries in control versus treated samples (Appendix 

1409 8.4.4, Table 8.4.4-7). Similarly, there was a treatment-related delay in ovary development in 

1410 sex-reversed genotypic males. Thinning of the germinal epithelium is specific to testis tissue,

1411 and significant increases in prevalence and severity of this pathology were observed in the 1.5 

1412 mg/L treatment. Mononuclear cellular infiltrate was observed in genotypic males and females 

1413 across all treatments and the control, but prevalence and severity were significantly attenuated in 

1414 the 3 and 6 mg/L treatments for genotypic females. Prevalence and severity of proteinaceous 

1415 fluid infiltrate were significantly increased in both genders in the 3 and 6 mg/L treatments.

1416
40

1417 

1418

1419 A B

1420

1421

1422

1423

1424

1425

1426

1427

1428 

1429

1430 Figure 5.2.4-1 A) Testis, ducts, and kidney from a control male, provided for reference. Scale 

1431 bar = 200μm. B) Testis of genetic male treated with 1.5 mg/L BP-2. The gonad consists 

1432 primarily of testicular tissue, but also contains several oocytes and a partial ovarian cavity

1433 (arrow). Scale bar = 50μm.

1434

1435

1436 Reproductive duct histopathology

1437

1438 Oviduct development was rather variable within and between genders. In genotypic females, 

1439 there was a significant delay in oviduct development in all treatments (Appendix 8.4.4, Table 

1440 8.4.4-9). However, in both genders, there were some individuals in the 1.5 and 3 mg/L 

1441 treatments that are two developmental stages more advanced than the others. Both genders 

1442 experienced advancement of Wolffian duct development in the 3 and 6 mg/L treatments. 

1443

1444 6 DISCUSSION OF THE INTER-LABORATORY STUDIES

1445

1446 6.1 Comparison of control performance

1447

1448 Larval growth and development

1449

1450 A summary of control larval weight, length, and time to NF stage 62 across all the inter1451 laboratory studies is shown in Table 6.1-1. The measurements are ranked by weight in 

1452 descending order from a maximum control larval NF stage 62 weight of 2.07 + 0.33 g to a 

1453 minimum of 0.46 + 0.14 g. The overall mean weight was 1.0 g with a standard deviation of 0.5 g. 

1454 Two studies, B-OP and C-OP, fell outside of one standard deviation from the overall mean 

1455 weight. Within studies conducted by B, mean larval weight doubled from the PZ study to the OP 

1456 study and resulted in notable incidences of scoliosis in the OP study. Unpublished feeding 

1457 studies performed at A have demonstrated that excess feeding through larval development leads 

1458 to higher incidences and increased severity of scoliosis, which is generally considered adverse 

1459 for toxicological studies. Upon follow-up investigation, it was reported that feeding rates were 

1460 increased in the B-OP study by 20-30% because they opted to start the test with 25% more 
41

1461 organisms per tank and subsequently increased feeding rates proportionately. Increasing the 

1462 number of organisms per tank for test initiation, in order to account for mortality, is acceptable 

1463 according to the LAGDA guideline. However, increasing feeding rates proportionately proved 

1464 to be excessive. The lowest rates of larval growth among the labs occurred in C-PZ and C-OP. 

1465 These studies also exhibited the highest level of variance in all three growth and development 

1466 metrics. This lab utilized its own established feeding rates which were lower than the 

1467 recommended feeding rates in the LAGDA guideline. These data and observations emphasize 

1468 the need for strict adherence to the guideline’s recommended feeding rates to achieve 

1469 comparable and optimal organism growth across studies. It is suggested that the criterion for 

1470 control larval growth be 1.0 ± 0.2 grams at NF stage 62, which should be reached in less than 45 

1471 days. 

1472

Table 6.1-1

Inter-laboratory control larvae growth and development performance comparison. 

Entries are listed in descending order according to weight (mean ± SD).

Study Weight (g) CV Length (mm) CV Time to NF62 (d) CV

A-PZ - - -

B-OP 2.07 ± 0.33 16 23.1 ± 1.3 6 42 ± 2 5

E-BP2 1.18 ± 0.18 15 22.2 ± 1.1 5 38 ± 5 13

B-PZ 1.08 ± 0.17 16 20.8 ± 1.0 5 41 ± 3 7

A-OP 0.96 ± 0.15 16 19.2 ± 1.0 5 42 ± 5 12

A-TB 0.82 ± 0.14 17 18.7 ± 1.1 6 40 ± 4 10

D-PZ 0.79 ± 0.13 17 18.8 ± 1.0 5 43 ± 2 5

C-PZ 0.62 ± 0.14 23 17.1 ± 1.4 8 44 ± 6 14

C-OP 0.46 ± 0.14 30 15.2 ± 1.6 11 41 ± 10 24

Overall means 1.0 ± 0.49 49 19.4 ± 2.6 13 41 ± 2 4

1473

1474

1475 Juvenile growth and development

1476

1477 As with the larval comparison, Table 6.1-2 contains juvenile male and female growth metrics 

1478 (length, weight, LSI) and VTG for each gender ranked by weight in descending order. Across all 

1479 studies, the male mean weight was 11.2 ± 3.9 g, and female mean weight was 11.7 ± 4.3 g. Two 

1480 studies had control juvenile weights more than one standard deviation from the overall mean in 

1481 both juvenile males and females but in different directions: D-PZ had higher mean weights, and 

1482 C-PZ had lower mean weights. Lab D used the recommended diet formulation but fed at a 

1483 higher rate than what was recommended in the LAGDA guideline. This was not necessarily 

1484 adverse; however, there was a significant treatment-related decrease in weight in this study 

1485 whereas studies from the other labs did not show this effect from prochloraz. Lab C used their 

1486 own diet formulation but fed at rates similar to that recommended in the LAGDA guideline. 

1487 This diet formulation was clearly inadequate for supporting growth rates consistent with the 

1488 other studies and led to confounding effects on LSI in their prochloraz study (explained in 

1489 Section 5.2.1). Body size does not become sexually dimorphic until later in development, so 

1490 significant differences in growth are not expected between genders at test termination. Therefore, 

1491 criteria for juvenile growth can be set the same for both sexes and is suggested to be 11.5 ± 3 g. 

1492

1493 It is worth noting the VTG levels in control juveniles. These values are an artifact of detection 

1494 limits within each ELISA and are 100 to 1000-fold less than levels in mature females (Olmstead 

1495 et al., 2009b) and 10 to 1000-fold less than levels induced by OP and BP-2 exposures. The 

1496 utility of this endpoint is discussed further in Section 6.3.

1497
42

1498 

Table 6.1-2

Inter-laboratory control juvenile growth performance and vitellogenin titer comparison. Entries are 

listed in descending order according to weight (mean ± SD).

Study Weight (g) CV Length (mm) CV LSI CV VTG (mg/mL) CV

Male

D-PZ 18.6 ± 4.1 22 53.0 ± 3.9 7 0.058 ± 0.005 9 0.01 ± 0.02 158

A-PZ 13.1 ± 2.0 15 47.2 ± 2.8 6 0.059 ± 0.010 17 0.03 ± 0.02 59

A-TB 12.3 ± 3.3 27 46.2 ± 4.1 9 0.060 ± 0.010 17 0.07 ± 0.07 108

B-PZ 11.5 ± 3.6 31 43.0 ± 4.5 11 0.057 ± 0.009 16 0.00 ± 0.00 176

E-BP2 11.3 ± 2.7 24 45.2 ± 3.3 7 0.046 ± 0.005 11 0.02 ± 0.03 150

A-OP 10.9 ± 2.7 25 43.1 ± 3.3 8 0.058 ± 0.008 14 0.00 ± 0.00 150

B-OP 10.1 ± 2.6 26 41.6 ± 3.5 8 0.058 ± 0.007 12 0.01 ± 0.01 107

C-OP 8.6 ± 2.6 30 41.1 ± 3.4 8 0.072 ± 0.008 11 0.01 ± 0.001 30

C-PZ 4.0 ± 0.8 20 32.8 ± 2.2 7 0.035 ± 0.007 20 0.01 ± 0.002 44

Overall ♂ means 11.2 ± 3.9 35 43.7 ± 5.5 12 0.056 ± 0.010 18 0.02 ± 0.02 122

Female

D-PZ 20.0 ± 4.6 23 55.0 ± 5.2 10 0.059 ± 0.006 10 0.02 ± 0.07 357

B-PZ 13.8 ± 5.0 36 46.4 ± 5.4 12 0.064 ± 0.014 22 0.01 ± 0.02 251

A-PZ 12.9 ± 2.6 20 47.2 ± 2.6 6 0.061 ± 0.007 12 0.03 ± 0.03 76

A-TB 12.9 ± 3.7 29 47.5 ± 5.1 11 0.059 ± 0.006 10 0.06 ± 0.07 116

A-OP 12.0 ± 2.7 23 44.7 ± 4.0 9 0.059 ± 0.012 20 0.00 ± 0.00 112

B-OP 10.9 ± 3.0 28 42.5 ± 4.0 9 0.059 ± 0.014 24 0.01 ± 0.01 105

E-BP2 10.8 ± 2.1 19 44.7 ± 3.2 7 0.045 ± 0.006 13 0.04 ± 0.11 323

C-OP 8.3 ± 1.7 21 40.8 ± 3.1 8 0.075 ± 0.007 9 0.04 ± 0.01 32

C-PZ 4.1 ± 0.8 20 32.9 ± 2.2 7 0.032 ± 0.006 19 0.03 ± 0.01 41

Overall ♀ means 11.7 ± 4.3 37 44.6 ± 5.9 13 0.057 ± 0.012 21 0.03 ± 0.02 76

1499

1500 6.2 Effectiveness of the LAGDA

1501

1502 In total, the results for four chemicals are presented in this report. Two of these chemicals, 4-

1503 tert-octylphenol and prochloraz, were tested in three and four laboratories respectively. The 

1504 other two test chemicals, benzophenone-2 and 17-β trenbolone, were tested in a single laboratory 

1505 which prevents their use in inter-laboratory comparisons. However, these studies serve to 

1506 demonstrate the responsiveness of the LAGDA to additional modes of action. Table 6.2-1 

1507 summarizes the important findings for all of the studies which were conducted. All four 

1508 chemicals tested produced endocrine-related effects in these studies. 

1509
43

1510 Table 6.2-1. 

1511 Summary of significant findings in LAGDA studies on 4 chemicals representing different modes of action. 

Chemical Growth effects and 

overt toxicity Thyroid effects Reproduction-related effects

Prochloraz

(4 labs)

Liver and kidney 

pathologies (A, C, D, 

B)

Thyroid gland pathologies 

consistent with hypothyroid 

condition (A, D, and C)

VTG induction 

Gonad/ reproductive duct 

pathologies (all labs)

4-tert-octylphenol

(3 labs)

Thyroid gland pathologies 

consistent with hypothyroid 

condition (A) 

Delayed development (A)

VTG induction 

Mild gonad/reproductive duct 

pathologies (all labs)

17-β Trenbolone

(A only)

Liver and kidney 

pathologies 

Thyroid gland pathologies Oviduct pathologies 

Benzophenone-2

(E only)

Mortality Thyroid gland pathologies 

consistent with hypothyroid 

condition

Delayed metamorphosis

VTG induction

Profound gonad/reproductive 

duct pathologies

1512

1513

1514 Prochloraz

1515

1516 At the highest test concentration, prochloraz produced liver and kidney pathologies consistent 

1517 with a toxic response across the four studies, although there was little evidence of an effect on 

1518 growth. LSI was also impacted by the highest prochloraz concentration across the studies. It 

1519 was also apparently decreased at two, three, or four concentrations in most studies, although it 

1520 appeared increased in C-PZ, but this was likely an artifact due to control juvenile growth in that 

1521 study being significantly less than in all other studies. 

1522

1523 Effects on the thyroid axis (thyroid gland change) were also observed across the studies although 

1524 the metamorphic rate was not altered. This suggests a successful compensatory response by the 

1525 thyroid axis. Prochloraz has not been previously shown to have impacts on the thyroid axis and 

1526 a review of the literature found no evidence of prior investigations. 

1527

1528 The degenerative testicular changes consistently observed across the four prochloraz studies is in 

1529 agreement with the anti-androgenic effects of prochloraz reported in species as divergent as 

1530 fathead minnows (Ankley et al., 2005) and rats (Blystone et al., 2007). In particular, germinal 

1531 epithelium thinning/loss with high doses of prochloraz has been described in rats (Noriega et al., 

1532 2005). In male frogs, sporadic development of stage 3 oviducts and the retention of stage 2 

1533 oviducts with increasing prochloraz exposure were observed across the four studies and indicate 

1534 interference with normal oviduct regression. Oviduct regression in males is known to be an 

1535 androgen regulated process in amphibians (Kelley, 1996), and this finding is similar to the 

1536 feminization of the secondary sexual characteristics of male rats during perinatal prochloraz 

1537 exposure (Vingaard et al., 2005). 

1538

1539 The LAGDA was successful in identifying prochloraz as anti-androgenic and demonstrated the 

1540 consequence of this mechanism of action on male gonad development. 

1541

1542

1543 4-tert-octylphenol
44

1544

1545 In general, OP produced few signs of overt toxicity. No consistent effects on growth, liver and 

1546 kidney pathology or LSI were observed. 

1547

1548 OP exposure resulted in VTG induction and female oviduct pathologies across all four studies. 

1549 These OP-induced changes are consistent with weak estrogenic activity. Vitellogenin has also

1550 been shown to be elevated in males and females of multiple fish species exposed to OP (Seki et

1551 al., 2002; Ortiz-Zarragoitia and Cajaraville, 2005; Rasmussen et al., 2005; Seki et al., 2002), in 

1552 X. laevis primary cultured hepatocytes exposed to OP (Mitsui et al., 2007), and following in vivo

1553 OP exposure in X. tropicalis (Porter et al., 2011).

1554

1555 In these inter-laboratory studies, no consistent findings were observed in the developing male 

1556 and female gonads. Previous studies that have employed exposure during gonad development

1557 have shown disruption in gonad development. These effects include decreased sperm count and 

1558 the presence of testis-ova in medaka (Seki et al., 2002), decreased gonadosomatic index and 

1559 increased testis-ova in eelpout (Rasmussen et al., 2005), and decreased spermatozoa and 

1560 increased testis anomalies in sheepshead minnows (Karels et al., 2003). In contrast, previous 

1561 studies with the X. tropicalis have shown no impact on male or female gonad development 

1562 (Porter et al., 2011). When taken together, it appears that these amphibian test species are less 

1563 sensitive to the endocrine disrupting effects of OP than are fish species. It must be pointed out 

1564 that it was not possible to test higher concentrations given the fact that concentrations above 60 

1565 μg/L produced significant levels of embryo mortality in X. tropicalis (unpublished data, Lab A). 

1566 It is difficult to make extensive comments regarding across-laboratory performance given that 

1567 minimal effects were observed, other than to note that OP produced a consistent weak estrogenic 

1568 response. This was indicated by VTG induction and advancement of oviduct development 

1569 across the four studies, a response that it is consistent with OP activity reported in the published 

1570 literature. 

1571

1572 17-β Trenbolone

1573

1574 There were no significant treatment-related effects on larval or juvenile growth, although a 

1575 concentration-dependent increase in hepatocyte hypertrophy was observed. This finding

1576 indicates that the test concentrations did not produce an appreciable level of overt toxicity. 

1577

1578 The effects produced by trenbolone in this study were rather mild. Female gonads were 

1579 unaffected by exposure whereas there was increased spermatogonia and germ cell vacuolation in 

1580 the testis at 100 ng/L. The most significant finding was that 17-β trenbolone induced the 

1581 regression of the female oviduct. The oviduct functions to transport mature oocytes from the 

1582 ovary to the external environment for fertilization by the male. Loss of or functional alterations 

1583 in the oviduct will have significant impacts on reproductive success and could lead to population 

1584 level impacts. There was also a mild increase in follicular cell hyperplasia at ≥ 25 ng/L although 

1585 no significant alterations in developmental rate were observed. 

1586

1587 In the absence of additional LAGDA studies with 17-β trenbolone, comparisons to the published

1588 literature served as a basis for evaluating the performance of the LAGDA in responding to 

1589 androgenic chemicals. Studies in zebrafish have demonstrated that aqueous exposure to 
45

1590 trenbolone results in complete sex reversal of females and production of all male cohorts (Orn et 

1591 al., 2006; Holbech et al., 2006; Morthorst et al., 2010). Ovo-testes characterized by ovaries 

1592 which develop tubules containing spermatocytes were induced in mosquitofish exposed to 

1593 trenbolone (Sone et al., 2005). Females exposed to trenbolone have been reported to acquire 

1594 male secondary sex characteristics such as tubercles in adult fathead minnows (Ankley et al.,

1595 2003) and papillary processes in Japanese medaka (Seki et al., 2006). Trenbolone exposure has 

1596 also been reported to reduce circulating estradiol and testosterone levels in fathead minnows 

1597 (Ankley et al., 2003). Decreased circulating vitellogenin in females has been demonstrated in a 

1598 number of different fish species exposed to this toxicant (Ankley et al., 2003; Sone et al., 2005;

1599 Seki et al., 2006). Test concentrations of 17-β trenbolone were set based on reported findings for 

1600 X. tropicalis (Olmstead et al., 2012). In that study, the authors reported a much more dramatic 

1601 impact of 17-β trenbolone on androgen responsive tissues. Concentrations ≥ 100 ng/L resulted in 

1602 a spectrum of androgenic responses which included hypertrophy of the larynx resulting in late 

1603 larval mortality, precocious development of nuptial pads, a mixed-sex phenotype in the ovaries 

1604 of females, and hypertrophy of the Wolffian duct. This comparison suggests that X. laevis may 

1605 be less sensitive than X. tropicalis to this synthetic androgen. Although the LAGDA showed a 

1606 lower sensitivity when compared to the literature, the LAGDA successfully identified the 

1607 endocrine disrupting potential and mode of action of trenbolone. Further, the LAGDA 

1608 demonstrated findings (female oviduct regression) which are useful in the context of risk 

1609 assessment. 

1610

1611 Benzophenone-2

1612

1613 BP-2 treatment resulted in significant increases in larval and juvenile mortality at the highest test 

1614 concentration. 

1615

1616 There were significant treatment-related effects on thyroid tissue including increased prevalence 

1617 and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy. The 6 mg/L 

1618 treatment exhibited remarkable pathologies in 100% of the subjects which were associated with a 

1619 significant delay in the rate of metamorphosis. BP-2 has been shown to be a thyroid peroxidase 

1620 inhibitor (Schmutzler et al., 2007), and exposure has been shown to result in disruption of the 

1621 thyroid axis in mammalian (Jarry et al., 2004) and aquatic species (Thienpont et al., 2011).

1622

1623 BP-2 has been found to be estrogenic in vitro (Molina-Molina et al., 2008) and in vivo (Kunz and 

1624 Fent, 2009) and is capable of impacting reproduction in fish (Weisbord et al., 2007). The results 

1625 of this study are consistent with these published reports. Plasma vitellogenin levels were 

1626 considerably elevated in all treatments (Appendix 8.4.4, Table 8.4.4-4). In addition to this 

1627 finding, liver and renal pathologies which were observed are consistent with estrogen exposure 

1628 (Pawlowski et al., 2004; Folmar et al., 2001) and are presumably a consequence of excessive 

1629 VTG accumulation in these organs. Genotypic males experienced feminization of the gonad in a

1630 concentration-dependent fashion; both intersex gonads and sex-reversed gonads were observed 

1631 in the 1.5 mg/L treatment whereas in the 3 and 6 mg/L treatment, 100% of the genotypic males 

1632 had been completely sex reversed exhibiting only ovary tissue. In genotypic females, there was 

1633 a significant treatment-related delay in gonad development. Similarly, there was a treatment1634 related delay in ovary development in sex-reversed genotypic males. 

1635
46

1636 6.3 Lessons learned from the inter-laboratory studies

1637

1638 Inconsistencies in Growth

1639

1640 Inconsistencies in organism growth were observed (see Section 6.1) which may have impacted 

1641 the results of the test chemicals. Diet formulation and feeding rates have been shown to be 

1642 correlated with organismal growth and developmental rates in the LAGDA. In one instance,

1643 excessive control growth was observed when all the studies were considered as a group. In this

1644 case, treatment-related effects on growth were observed. In two other studies (C-PZ and C-OP),

1645 control growth was reduced relative to the broader group, and this was a confounding factor in 

1646 interpreting chemical related effects on LSI. This laboratory chose to use its own feed 

1647 formulation in lieu of that recommended in the guidance. From this inter-laboratory comparison,

1648 it can be concluded that this formulation is inadequate to support appropriate growth and 

1649 development. The recommended diet and feeding rates in the LAGDA provide for optimal 

1650 growth, and the Agency strongly encourages adherence to the provided guidance. 

1651

1652 Inconsistencies in Pathology Findings 

1653

1654 Another area of inconsistency was in the pathology findings. Over the course of these studies,

1655 the contract laboratories hired to prepare and read slides changed resulting in different 

1656 pathologists conducting the assessments. In the prochloraz studies, Labs A and D gonad, oviduct 

1657 and kidney samples were read by a different pathologist than the Labs B and C studies. 

1658 Interestingly, the Lab A and D studies shared consistencies as did the Lab B and C studies. It is 

1659 not possible to definitively demonstrate this as a cause for the observed inconsistencies. 

1660 However, it does point to the need for adherence to the pathology guidance document. It is 

1661 necessary to point out that the gonad histopathology guidance document was not finalized when 

1662 the prochloraz studies were prepared and read. In the case of OP, the three studies were prepared 

1663 and read by one laboratory, and reported findings (although minimal) were much more consistent. 

1664

1665 Similar anomalies were observed with liver pathology for the prochloraz studies. Lab A, B and 

1666 D studies were read by the same contracted pathologist whereas Lab C study was read by a 

1667 different contracted pathologist. It is interesting to point out that similar treatment-related liver 

1668 pathologies were reported for Labs A, B and D whereas a different spectrum of treatment-related 

1669 pathologies were reported for Lab C. It is worth noting that the animals in the C study showed 

1670 feeding-related reduced juvenile growth rates as compared to the other studies. It is possible that 

1671 disease issues may have factored into the apparent inconsistencies in liver findings. Regardless,

1672 developing a pathology guidance document for both the liver and kidney is critical. The samples 

1673 from these studies will provide the necessary examples for development of such a guidance 

1674 document.

1675

1676 T4 Serum Measures 

1677

1678 The B, C and D laboratories used commercially available ELISA kits specific for human or 

1679 canine T4. These laboratories either did not attempt to optimize or failed in any such effort. As 

1680 such, the laboratories were unsuccessful in making these measurements and these data were not 

1681 included in this analysis. Lab A measured T4 levels in serum using an HPLC/ICP-MS method 
47

1682 which has been previously optimized for use with X. laevis (Tietge et al., 2010; Tietge et al.,

1683 2012). Additionally, Lab A has developed an extraction method which allows for the use of 

1684 these commercially available ELISA kits (See LAGDA Guideline Appendix 7) with X. laevis

1685 serum. In the Lab A OP study, both methods were used and compared. 

1686

1687 Unfortunately, it is not possible to make inter-laboratory comparisons of the effects of the test 

1688 chemicals on T4 since studies that used appropriate methods for measuring T4 were single 

1689 laboratory studies. However, the collection of control data indicates that both methods are 

1690 reproducible. Further, these methods were effective in accurately identifying alterations in T4. 

1691 In the future, laboratories conducting the assay must demonstrate a competence in making this 

1692 measurement. These studies should serve as a foundation for establishing performance criteria 

1693 for this endpoint. 

1694

1695 Vitellogenin (VTG)

1696

1697 Although the VTG ELISA registers a measurable signal in control males and females, this 

1698 should be interpreted with caution. These values are more likely background noise within the 

1699 assay. PZ, OP, and BP-2 exposures resulted in a concentration-dependent significant increase in 

1700 plasma VTG. However, the BP-2 data set provides the best representation of the magnitude of 

1701 induction that can occur with estrogenic chemical exposure in X. laevis. Further, with the level 

1702 of induction exhibited in the BP-2 study, CVs approached more acceptable values. Taken 

1703 together, this emphasizes that VTG data from all of the other studies are likely background levels,

1704 and VTG induction as a reproductive endpoint in this assay should be interpreted in the context 

1705 of overall potential magnitude of response.

1706

1707 6.4 Conclusions

1708

1709 The LAGDA protocol describes a chronic toxicity test with an amphibian species that considers 

1710 growth and development from fertilization through the early juvenile period. Individual and 

1711 inter-laboratory evaluations of the LADGA were conducted to evaluate the practical 

1712 transferability of the assay protocol and quantitative reproducibility of the results. 

1713

1714 Although the results of the inter-laboratory validation were mixed, the LAGDA was an effective 

1715 test model with endocrine-related effects characterized for all four chemicals evaluated. Overall, 

1716 the authors conclude that the Tier 2 LAGDA is capable of sufficiently characterizing potential 

1717 disruption of the endocrine system by putative endocrine disrupting chemicals. However, careful 

1718 attention to recommendations in the Test Guideline is necessary to ensure consistent and reliable 

1719 findings across multiple testing laboratories. 

1720 7 REFERENCES

1721

1722 Ankley GT, Jensen KM, Durhan EJ, Makynen EA, Butterworth BC, Kahl MD, Villeneuve DL, 

1723 Linnum A, Gray LE, Cardon M. 2005. Effects of two fungicides with multiple modes of action 

1724 on reproductive endocrine function in the fathead minnow (Pimephales promelas). Toxicological 

1725 Sciences 86(2):300-308.
48

1726

1727 Ankley GT, Jensen KM, Makynen EA, Kahl MD, Korte JJ, Hornung MW, Henry TR, Denny JS, 

1728 Leino RL, Wilson VS. 2003. Effects of the androgenic growth promoter 17‐β‐trenbolone on 

1729 fecundity and reproductive endocrinology of the fathead minnow. Environmental Toxicology 

1730 and Chemistry 22(6):1350-1360.

1731

1732 Blystone CR, Lambright CS, Howdeshell KL, Furr J, Sternberg RM, Butterworth BC, Durhan EJ, 

1733 Makynen EA, Ankley GT, Wilson VS. 2007. Sensitivity of fetal rat testicular steroidogenesis to 

1734 maternal prochloraz exposure and the underlying mechanism of inhibition. Toxicological 

1735 Sciences 97(2):512-519.

1736

1737 Bonefeld-Jørgensen EC, Long M, Hofmeister MV, Vinggaard AM. 2007. Endocrine-disrupting 

1738 potential of bisphenol A, bisphenol A dimethacrylate, 4-n-nonylphenol, and 4-n-octylphenol in 

1739 vitro: new data and a brief review. Environmental Health Perspectives 115(S-1):69.

1740

1741 Chang C, Witschi E. Genic control and hormonal reversal of sex differentiation in Xenopus; 

1742 1956. Royal Society of Medicine. p 140-144.

1743

1744 EDSP, 2007. Validation of Screening and Testing Assays Proposed for the EDSP. Draft version 

1745 5.4. U.S. Environmental Protection Agency Endocrine Disruptor Screening Program, available at 

1746 http://www.epa.gov/scipoly/oscpendo/pubs/edsp_validation_paper_v%205.4.pdf 21 pages.

1747

1748 EDSTAC, 1998. Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) 

1749 Final Report, http://www.epa.gov/endo/pubs/edspoverview/finalrpt.htm.

1750

1751 Folmar LC, Gardner GR, Schreibman MP, Magliulo-Cepriano L, Mills LJ, Zaroogian G, 

1752 Gutjahr-Gobell R, Haebler R, Horowitz DB, Denslow ND. 2001. Vitellogenin-induced 

1753 pathology in male summer flounder (Paralichthys dentatus). Aquatic Toxicology 51(4):431-441.

1754

1755 Fort DJ, Guiney PD, Weeks JA, Thomas JH, Rogers RL, Noll AM, Spaulding CD. 2004a. Effect 

1756 of methoxychlor on various life stages of Xenopus laevis. Toxicological Sciences 81(2):454-466.

1757

1758 Fort DJ, Thomas JH, Rogers RL, Noll A, Spaulding CD, Guiney PD, Weeks JA. 2004b. 

1759 Evaluation of the developmental and reproductive toxicity of methoxychlor using an anuran 

1760 (Xenopus tropicalis) chronic exposure model. Toxicological Sciences 81(2):443-453.

1761

1762 FRN (Federal Register Notice), 1998. Endocrine Disruptor Screening Program. Federal Register. 

1763 August 11, 1998. 63(154):42852-42855.

1764

1765 Gallien L. 1953. Total inversion of sex in Xenopus laevis Daud, following treatment with 

1766 estradiol benzoate administered during larval stage. Comptes rendus hebdomadaires des séances 

1767 de l'Académie des sciences 237(23):1565.

1768

1769 Grim KC, Wolfe M, Braunbeck T, Iguchi T, Ohta Y, Tooi O, Touart L, Wolf DC, Tietge J. 2009. 

1770 Thyroid histopathology assessments for the amphibian metamorphosis assay to detect thyroid1771 active substances. Toxicologic Pathology 37(4):415-424.
49

1772

1773 Hirsch N, Zimmerman LB, Grainger RM. 2002. Xenopus, the next generation: X. tropicalis

1774 genetics and genomics. Developmental Dynamics 225(4):422-433.

1775

1776 Holbech H, Kinnberg K, Petersen GI, Jackson P, Hylland K, Norrgren L, Bjerregaard P. 2006. 

1777 Detection of endocrine disrupters: evaluation of a fish sexual development test (FSDT). 

1778 Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 144(1):57-66.

1779

1780 Jarry H, Christoffel J, Rimoldi G, Koch L, Wuttke W. 2004. Multi-organic endocrine disrupting 

1781 activity of the UV screen benzophenone 2 (BP2) in ovariectomized adult rats after 5 days 

1782 treatment. Toxicology 205(1):87-93.

1783

1784 Johnson R, Wolf J, Braunbeck T. 2009. OECD Guidance document for the diagnosis of 

1785 endocrine-related histopathology of fish gonads. Organisation for Economic Co-operation and 

1786 Development, France.

1787

1788 Karels AA, Manning S, Brouwer TH, Brouwer M. 2003. Reproductive effects of estrogenic and 

1789 antiestrogenic chemicals on sheepshead minnows (Cyprinodon variegatus). Environmental 

1790 Toxicology and Chemistry 22(4):855-865.

1791

1792 Kelley DB. 9 Sexual differentiation in Xenopus laevis; 1996. London: The Society, 1960-1999.

1793

1794 Kloas W, Lutz I, Einspanier R. 1999. Amphibians as a model to study endocrine disruptors: II. 

1795 Estrogenic activity of environmental chemicals in vitro and in vivo. The Science of the Total 

1796 Environment 225(1-2):59.

1797

1798 Kloas W, Lutz I. 2006. Amphibians as model to study endocrine disrupters. Journal of 

1799 Chromatography A 1130(1):16-27.

1800

1801 Kunz PY, Fent K. 2006. Estrogenic activity of UV filter mixtures. Toxicology and Applied 

1802 Pharmacology 217(1):86-99.

1803

1804 Kunz PY, Fent K. 2009. Estrogenic activity of ternary UV filter mixtures in fish (Pimephales 

1805 promelas)—An analysis with nonlinear isobolograms. Toxicology and Applied Pharmacology 

1806 234(1):77-88.

1807

1808 Mikamo K, Witschi E. 1963. Functional sex-reversal in genetic females of Xenopus laevis, 

1809 induced by implanted testes. Genetics 48(10):1411.

1810

1811 Mitsui N, Tooi O, Kawahara A. 2007. Vitellogenin-inducing activities of natural, synthetic, and 

1812 environmental estrogens in primary cultured Xenopus laevis hepatocytes. Comparative 

1813 Biochemistry and Physiology Part C: Toxicology & Pharmacology 146(4):581-587.

1814

1815 Miyata S, Koike S, Kubo T. 1999. Hormonal reversal and the genetic control of sex 

1816 differentiation in Xenopus. Zoological Science 16(2):335-340.

1817
50

1818 Molina-Molina J-M, Escande A, Pillon A, Gomez E, Pakdel F, Cavaillès V, Olea N, Aït-Aïssa S, 

1819 Balaguer P. 2008. Profiling of benzophenone derivatives using fish and human estrogen 

1820 receptor-specific in vitro bioassays. Toxicology and Applied Pharmacology 232(3):384-395.

1821

1822 Morthorst JE, Holbech H, Bjerregaard P. 2010. Trenbolone causes irreversible masculinization 

1823 of zebrafish at environmentally relevant concentrations. Aquatic Toxicology 98(4):336-343.

1824

1825 NIEHS, 1997. Validation and Regulatory Acceptance of Toxicological Test Methods: A Report 

1826 of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods. 

1827 NIH Report 97-3981. Research Triangle Park, NC.

1828

1829 Nieuwkoop P, Faber J. 1994. Normal Table of Xenopus Laevis (Daudin)(Garland, New York). 

1830 ISBN 0-8153-1896-0.

1831

1832 Noriega NC, Ostby J, Lambright C, Wilson VS, Gray LE. 2005. Late gestational exposure to the 

1833 fungicide prochloraz delays the onset of parturition and causes reproductive malformations in 

1834 male but not female rat offspring. Biology of Reproduction 72(6):1324-1335.

1835

1836 OECD (Organization for Economic Cooperation and Development), 1996. Final Report of the 

1837 OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative 

1838 Toxicological Test Methods. Paris, France. 

1839

1840 OECD (Organization for Economic Cooperation and Development), 2005. Guidance Document 

1841 on the Validation of International Acceptance of New or Updated Test Methods for Hazard 

1842 Assessment. Series on Testing and Assessment Number 34.

1843

1844 OECD (2006). Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance 

1845 to Application Environmental Health and Safety Publications, Paris.

1846

1847 Olmstead AW, Kosian PA, Korte JJ, Holcombe GW, Woodis KK, Degitz SJ. 2009a. Sex 

1848 reversal of the amphibian, Xenopus tropicalis, following larval exposure to an aromatase 

1849 inhibitor. Aquatic Toxicology 91(2):143-150.

1850

1851 Olmstead AW, Korte JJ, Woodis KK, Bennett BA, Ostazeski S, Degitz SJ. 2009b. Reproductive 

1852 maturation of the tropical clawed frog: Xenopus tropicalis. General and Comparative 

1853 Endocrinology 160(2):117-123.

1854

1855 Olmstead AW, Lindberg-Livingston A, Degitz SJ. 2010. Genotyping sex in the amphibian, 

1856 Xenopus (Silurana) tropicalis, for endocrine disruptor bioassays. Aquatic Toxicology 98(1):60-

1857 66.

1858

1859 Olmstead AW, Kosian PA, Johnson R, Blackshear PE, Haselman J, Blanksma C, Korte JJ, 

1860 Holcombe GW, Burgess E, Lindberg‐Livingston A. 2012. Trenbolone causes mortality and 

1861 altered sexual differentiation in Xenopus tropicalis during larval development. Environmental 

1862 Toxicology and Chemistry 31(10):2391-2398.

1863
51

1864 Orn S, Yamani S, Norrgren L. 2006. Comparison of vitellogenin induction, sex ratio, and gonad 

1865 morphology between zebrafish and Japanese medaka after exposure to 17alpha-ethinylestradiol 

1866 and 17beta-trenbolone. Archives of Environmental Contamination and Toxicology 51(2):237.

1867

1868 Ortiz-Zarragoitia M, Cajaraville MP. 2005. Effects of selected xenoestrogens on liver 

1869 peroxisomes, vitellogenin levels and spermatogenic cell proliferation in male zebrafish. 

1870 Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 141(2):133-144.

1871

1872 Pawlowski S, Van Aerle R, Tyler C, Braunbeck T. 2004. Effects of 17a-ethinylestradiol in a 

1873 fathead minnow (Pimephales promelas) gonadal recrudescence assay. Ecotoxicology and 

1874 Environmental Safety 57(3):330-345.

1875

1876 Pickford DB, Hetheridge MJ, Caunter JE, Tilghman Hall A, Hutchinson TH. 2003. Assessing 

1877 chronic toxicity of bisphenol A to larvae of the African clawed frog (Xenopus laevis) in a flow1878 through exposure system. Chemosphere 53(3):223-235.

1879

1880 Pickford DB, Morris ID. 2003. Inhibition of gonadotropin-induced oviposition and ovarian 

1881 steroidogenesis in the African clawed frog (Xenopus laevis) by the pesticide methoxychlor. 

1882 Aquatic Toxicology 62(3):179-194.

1883

1884 Porter KL, Olmstead AW, Kumsher DM, Dennis WE, Sprando RL, Holcombe GW, Korte JJ, 

1885 Lindberg-Livingston A, Degitz SJ. 2011. Effects of 4-tert-octylphenol on Xenopus tropicalis in a 

1886 long term exposure. Aquatic Toxicology 103(3):159-169.

1887

1888 Qin Z-F, Qin X-F, Yang L, Li H-T, Zhao X-R, Xu X-B. 2007. Feminizing/demasculinizing 

1889 effects of polychlorinated biphenyls on the secondary sexual development of Xenopus laevis. 

1890 Aquatic Toxicology 84(3):321-327.

1891

1892 Rasmussen TH, Teh SJ, Bjerregaard P, Korsgaard B. 2005. Anti-estrogen prevents xenoestrogen1893 induced testicular pathology of eelpout (Zoarces viviparus). Aquatic Toxicology 72(3):177-194.

1894

1895 SAB-SAP, 1999. Science Advisory Board. EDSP. Review of the Endocrine Disruptor Screening 

1896 Program by a Joint Subcommittee of the Science Advisory Board and the FIFRA Scientific 

1897 Advisory Panel. U.S. Environmental Protection Agency. EPA-SAB-EC-99-013.

1898

1899 Schmutzler C, Bacinski A, Gotthardt I, Huhne K, Ambrugger P, Klammer H, Schlecht C, 

1900 Hoang-Vu C, Grüters A, Wuttke W. 2007. The ultraviolet filter benzophenone 2 interferes with 

1901 the thyroid hormone axis in rats and is a potent in vitro inhibitor of human recombinant thyroid 

1902 peroxidase. Endocrinology 148(6):2835-2844.

1903

1904 Seki M, Yokota H, Maeda M, Tadokoro H, Kobayashi K. 2003. Effects of 4‐nonylphenol and 

1905 4‐tert‐octylphenol on sex differentiation and vitellogenin induction in medaka (Oryzias latipes). 

1906 Environmental Toxicology and Chemistry 22(7):1507-1516.

1907
52

1908 Seki M, Fujishima S, Nozaka T, Maeda M, Kobayashi K. 2006. Comparison of response to 

1909 17β‐estradiol and 17β‐trenbolone among three small fish species. Environmental Toxicology and 

1910 Chemistry 25(10):2742-2752.

1911

1912 Sone K, Hinago M, Itamoto M, Katsu Y, Watanabe H, Urushitani H, Tooi O, Guillette Jr LJ, 

1913 Iguchi T. 2005. Effects of an androgenic growth promoter 17β-trenbolone on masculinization of 

1914 Mosquitofish (Gambusia affinis affinis). General and Comparative Endocrinology 143(2):151-

1915 160.

1916

1917 Thienpont B, Tingaud-Sequeira Al, Prats E, Barata C, Babin PJ, Raldúa D. 2011. Zebrafish 

1918 eleutheroembryos provide a suitable vertebrate model for screening chemicals that impair 

1919 thyroid hormone synthesis. Environmental Science & Technology 45(17):7525-7532.

1920

1921 Tietge JE, Butterworth BC, Haselman JT, Holcombe GW, Hornung MW, Korte JJ, Kosian PA, 

1922 Wolfe M, Degitz SJ. 2010. Early temporal effects of three thyroid hormone synthesis inhibitors 

1923 in Xenopus laevis. Aquatic Toxicology 98(1):44-50.

1924

1925 Tietge JE, Degitz SJ, Haselman JT, Butterworth BC, Korte JJ, Kosian PA, Lindberg-Livingston 

1926 AJ, Burgess EM, Blackshear PE, Hornung MW. 2012. Inhibition of the Thyroid Hormone 

1927 Pathway in Xenopus laevis by 2-mercaptobenzothiazole. Aquatic Toxicology 126(1):128-136.

1928

1929 Tobias ML, Tomasson J, Kelley DB. 1998. Attaining and maintaining strong vocal synapses in 

1930 female Xenopus laevis. Journal of Neurobiology 37(3):441-448.

1931

1932 USEPA, 2006. U.S. EPA Peer Review Handbook. EPA/100/B-06/002.

1933

1934 USEPA. 2009 Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1100: 

1935 Amphibian Metamorphosis (Frog). Office of Chemical Safety and Pollution Prevention [OCSPP] 

1936 formerly Prevention, Pesticides and Toxic Substances [OPPTS] (7101) EPA 740-C-09-002. 

1937 October 2009. http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0576-

1938 0002

1939

1940 Villalpando I, Merchant-Larios H. 1990. Determination of the sensitive stages for gonadal sex1941 reversal in Xenopus laevis tadpoles. Int J Dev Biol 34(2):281-285.

1942

1943 Villeneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, Newsted JL, Giesy 

1944 JP, Hecker M. 2007. Comparison of fathead minnow ovary explant and H295R cell-based 

1945 steroidogenesis assays for identifying endocrine-active chemicals. Ecotoxicology and 

1946 Environmental Safety 68(1):20-32.

1947

1948 Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H, 

1949 Dalgaard M, Nellemann C, Hass U. 2005. Perinatal exposure to the fungicide prochloraz 

1950 feminizes the male rat offspring. Toxicological Sciences 85(2):886-897.

1951

1952 Wake MH, Dickie R. 1998. Oviduct structure and function and reproductive modes in 

1953 amphibians. Journal of Experimental Zoology 282(4‐5):477-506.
53

1954

1955 Weisbrod CJ, Kunz PY, Zenker AK, Fent K. 2007. Effects of the UV filter benzophenone-2 on 

1956 reproduction in fish. Toxicology and Applied Pharmacology 225(3):255-266.

1957

1958 Ying G-G, Williams B, Kookana R. 2002. Environmental fate of alkylphenols and alkylphenol 

1959 ethoxylates—a review. Environment International 28(3):215-226.

1960

1961 Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida-Umehara C, Matsuda Y, 

1962 Takamatsu N, Shiba T, Ito M. 2008. A W-linked DM-domain gene, DM-W, participates in 

1963 primary ovary development in Xenopus laevis. Proceedings of the National Academy of Sciences

1964 105(7):2469-2474.

1965

1966

1967

1968

1969

1970

1971

1972

1973

1974

1975
54

1976 8 APPENDICES

1977

1978 8.1 LAGDA Guideline

1979

1980 GUIDELINE FOR THE TESTING OF CHEMICALS

1981

1982 The Larval Amphibian Growth and Development Assay

1983

1984 INTRODUCTION

1985

1986 The need to develop and validate an assay capable of identifying and characterizing the adverse 

1987 consequences of exposure to toxic substances, in amphibians, originates from concerns that 

1988 environmental levels of chemicals may cause adverse effects in both humans and wildlife. The 

1989 Food Quality Protection Act of 1996 requires EPA to develop and implement a program using 

1990 valid tests for determining the potential endocrine effects from pesticides. The EPA established 

1991 an advisory group, the Endocrine Disruptor Screening and Testing Advisory Committee 

1992 (EDSTAC), to obtain advice on developing this program. In accordance with EDSTAC’s 

1993 recommendations, EPA proposed a two-tiered approach to the program: Tier 1 would identify 

1994 the potential of a substance to interact with the endocrine system whereas Tier 2 would confirm 

1995 the interaction and characterize the effects. One of the Tier 2 tests recommended by EDSTAC is 

1996 an amphibian full life cycle test to evaluate the adverse consequences of putative endocrine 

1997 disrupting chemicals, especially those active within the thyroid and reproductive systems, on the 

1998 development, growth and reproduction of amphibians.

1999

2000 PRINCIPLE OF THE TEST 

2001

2002 The Larval Amphibian Growth and Development Assay (LAGDA) is intended to identify and 

2003 characterize the adverse consequences of exposure to substances which interfere with the normal 

2004 development and growth of amphibians through larval development and metamorphosis. It is an 

2005 important assay to address potential inciting contributors to amphibian population declines by 

2006 evaluating the effects from exposure to contaminants, both through endocrine and non-endocrine 

2007 mechanisms, during the larval stage that may adversely affect populations. It should be noted 

2008 that to date, no validated assay exists that serves this function for amphibians. 

2009

2010 The general experimental design entails exposing NF stage 8 Xenopus laevis embryos to four

2011 different concentrations of a test chemical and a control until 10 weeks after the median time to 

2012 NF stage 62 in the control with one interim sub-sample at NF stage 62 (See Nieuwkoop and 

2013 Faber 1994 for staging details). There are four replicates in each test concentration with eight 

2014 replicates for the control. Endpoints evaluated during the course of the exposure include those 

2015 indicative of generalized toxicity: mortality, abnormal behavior, and growth determinations

2016 (length and weight), as well as endpoints designed to characterize specific endocrine toxicity 

2017 modes of action targeting estrogen-, androgen-, or thyroid-mediated physiological processes. 

2018

2019
55

2020 DESCRIPTION OF THE METHODS

2021

2022 Environmental Conditions

2023

2024 The inflow of water is provided to tanks housing tadpoles and post-metamorphic juvenile frogs

2025 at a rate of ≥10 tank additions per day (4.0 – 10.0 L tanks). Water temperature is maintained at

2026 21±1°C with a photoperiod of 12 hours of light and 12 hours of dark. Dissolved oxygen (%), 

2027 tank temperature, and pH are measured twice weekly. Conductivity (μS), alkalinity (mg/L of 

2028 CaCO3), and hardness (mg/L of CaCO3) are measured once a month. For post-metamorphic 

2029 juvenile frogs, humidity levels should be held above 30%. The occurrences of lesions on the 

2030 head just posterior to the nostrils are indications of insufficient humidity levels. 

2031

2032 Exposure tanks should be siphoned on a daily basis to remove uneaten food and waste products. 

2033 Care should be used to minimize stress and trauma to the animals, especially during movement, 

2034 cleaning of aquaria, and manipulation. Stressful conditions/activities should be avoided such as 

2035 loud and/or incessant noise, tapping on aquaria, vibrations in the aquaria, excessive activity in 

2036 the laboratory, and rapid changes in environmental media (light availability, temperature, pH, 

2037 DO, water flow rates, etc.). 

2038

2039 Feeding 

2040

2041 Feed and feeding rates change with life stages of Xenopus, which is unique to this test species 

2042 and is a very important aspect of the LAGDA protocol. Excessive feeding during the larval 

2043 phase typically results in increased incidences and severity of scoliosis and should be avoided.

2044 Conversely, inadequate feeding during the larval phase results in highly variable developmental 

2045 rates among controls potentially compromising statistical power or confounding test results. 

2046 Therefore, adherence to the provided feeding regime is strongly advised (Appendix 2).

2047

2048 Larvae

2049

2050 The larval diet consists of Silver Cup Trout Starter (Nelson and Sons, Murray, UT, USA), 

2051 Spirulina algae discs (Wardley, Secaucus, NJ, USA) and TetraFin® flakes (Tetra Sales, 

2052 Blacksburg, VA, USA) blended together in culture water. This mixture is fed three times daily 

2053 on weekdays and once daily on weekends. Tadpoles are also fed 24-hour old live brine shrimp 

2054 (Bio-Marine® Brand, Bio-Marine, Hawthorne, CA, USA) twice daily on weekdays and once 

2055 daily on the weekends starting on day 8 post-fertilization. Sera Micron® (Sera GmbH, 

2056 Heinsberg, Germany) can be used in place of the diet previously described but feeding rates need 

2057 to be adjusted to meet control performance criteria. 

2058

2059 Juvenile 

2060

2061 Once metamorphosis is complete, the feeding regime consists of 3/32” premium sinking frog 

2062 food by Xenopus Express. For juveniles, the pellets are briefly run in a coffee grinder or blender 

2063 in order to reduce their size. Appendix 2 provides information regarding the type and amount of 

2064 feed used during the juvenile life stage. The animals should be fed once per day.

2065
56

2066 Control organisms must meet all of the following performance criteria in order to ensure 

2067 reproducibility and transferability of the LAGDA:

2068

2069 1) The mean time to NF stage 62 is ≤ 45 days.

2070 2) The mean weight at NF stage 62 is 1.0±0.2 grams.

2071 3) The mean juvenile weight at test termination is 11.5±3 grams.

2072

2073 Chemical Testability 

2074

2075 The LAGDA is based upon an aqueous exposure protocol whereby the test chemical is 

2076 introduced into the test chambers via a flow-through system. Flow-through methods however, 

2077 introduce constraints on the types of chemicals that can be tested, as determined by the 

2078 physicochemical properties of the compound. Therefore, prior to using this protocol, baseline 

2079 information about the chemical should be obtained that is relevant to determining its testability. 

2080 Both the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

2081 Mixtures and the USEPA Guidance Document on Special Considerations for Conducting 

2082 Aquatic Laboratory Studies should be consulted (OECD 2000; OCSPP 850.1000). 

2083 Characteristics which indicate that the chemical may be difficult to test in aquatic systems

2084 include: high octanol-water partitioning coefficients (log Kow), high volatility, susceptibility to 

2085 hydrolysis, and susceptibility to photolysis under ambient laboratory lighting conditions. Other 

2086 factors may also be relevant to determining testability and should be determined on a case by 

2087 case basis. If a successful test is not possible for the chemical using a flow-through test system, 

2088 then it is recommended that this chemical not be tested using this protocol. 

2089

2090 Exposure System 

2091

2092 A flow-through diluter system is required for this protocol. The system components should have 

water-contact components of glass, stainless steel, and/or Teflon®2093 . However, suitable plastics 

2094 can be utilized if they do not compromise the study. Exposure tanks should be glass or stainless 

2095 steel aquaria, equipped with standpipes that result in an approximate tank volume between 4.0 

2096 and 10.0 L and minimum water depth of 10 to 15 cm. The system should be capable of 

2097 supporting all exposure concentrations, a control, and a vehicle control, if necessary, with four 

2098 replicates per treatment (eight in the control). The flow rate to each tank should be constant in 

2099 consideration of both the maintenance of biological conditions and chemical exposure. It is 

2100 recommended that flow rates accommodate at least 10 tank turnovers per day to avoid chemical 

2101 concentration declines due to metabolism by both the test organisms and aquatic microorganisms. 

2102 The treatment tanks should be randomly assigned to a position in the exposure system to reduce 

2103 potential positional effects, including slight variations in temperature, light intensity, etc. 

2104 Fluorescent lighting should be used to provide a photoperiod of 12 hr light and 12 hr dark at an 

intensity that ranges from 600 to 2,000 lux (lumens/m22105 ) at the water surface. Water temperature 

2106 should be maintained at 21o ± 1°C, pH maintained between 6.5 to 8.5, and the dissolved oxygen 

2107 (DO) concentration maintained at >3.5 mg/L (>40% of the air saturation) in each test tank. At a 

2108 minimum, water temperature, pH and dissolved oxygen should be measured twice a week. 

2109 Temperature should be measured continuously in at least one test vessel. Humidity within the 

2110 system should be kept above 30%. Appendix 1 outlines the experimental conditions under 

2111 which the protocol should be executed. For further information on setting up flow-through 
57

2112 exposure systems, please refer to the ASTM Standard Guide for Conducting Acute Toxicity 

2113 Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (ASTM 2002) and 

2114 general aquatic toxicology texts.

2115

2116 Water Quality

2117

2118 Any water that is locally available (e.g. spring water or charcoal-filtered tap water) and permits 

2119 normal growth and development of X. laevis can be used. Because local water quality can differ 

2120 substantially from one area to another, analysis of water quality should be undertaken, 

2121 particularly, if historical data on the utility of the water for raising amphibian larvae is not 

2122 available. Special attention should be given that the water is free of copper, chlorine and 

2123 chloramines, all of which are toxic to amphibians. It is further recommended to analyze the 

2124 water to determine background levels of estrogenic and androgenic chemicals, fluoride, 

2125 perchlorate and chlorate (by-product of drinking water disinfection) as all of these may confound 

2126 the study outcome. Analyses should be performed before testing begins and the testing water 

2127 should normally be free from these chemicals.

2128

2129 Iodide Concentration in Test Water 

2130

2131 In order for the thyroid gland to synthesize thyroid hormones to support normal metamorphosis, 

2132 sufficient iodide needs to be available to the larvae through a combination of aqueous and dietary 

2133 sources. Currently, there are no empirically derived guidelines for minimum iodide 

2134 concentrations. However, iodide availability may affect the responsiveness of the thyroid system 

2135 to thyroid active agents and is known to modulate the basal activity of the thyroid gland, an 

2136 aspect that deserves attention when interpreting the results from thyroid histopathology. 

2137 Therefore, measured aqueous iodide concentrations from the test water should be reported. 

2138 Based on previous work, the protocol has been demonstrated to work well when test water iodide 

(I2139 ) concentrations ranged between 0.5 and 10 μg/L. Ideally, the minimum iodide concentration 

2140 in the test water should be 0.5 μg/L. If the test water is reconstituted from deionized water, 

2141 iodine must be added at a minimum concentration of 0.5 μg/L. Supplementation of the test water 

2142 with iodine or other salts must be noted in the report.

2143

2144 Analytical Chemistry 

2145

2146 Prior to conducting a study, the stability of the test compound should be evaluated using existing 

2147 information on its solubility, degradability, and volatility. Test solutions from each replicate 

2148 tank at each concentration should be sampled for analytical chemistry analyses at test initiation 

2149 (day 0), and weekly throughout the exposure period. It is also recommended that each test 

2150 concentration be analyzed during system preparation, prior to test initiation, to verify system 

2151 performance and that stock solutions be analyzed when they are changed, especially if the 

2152 volume of the stock solution does not provide adequate amounts of chemical to span the duration 

2153 of routine sampling periods. In the case of chemicals which cannot be detected at some or all of 

2154 the concentrations used in a test, stock solutions should be measured and system flow rates 

2155 recorded in order to calculate actual concentrations.

2156

2157 Chemical Delivery 
58

2158

2159 The method used to introduce the test chemical to the system can vary depending on its 

2160 physicochemical properties. Water soluble compounds can be dissolved in aliquots of test water 

2161 at a concentration which allows delivery at the target test concentration in a flow-through system. 

2162 Chemicals which are liquid at room temperature and moderately soluble in water can be 

2163 introduced using liquid:liquid saturator methods. Chemicals which are solid at room temperature 

2164 and are moderately soluble in water can be introduced using glass wool column saturators (Kahl

2165 et al. 1999). The preference is to use a carrier-free test system; however, different test chemicals 

2166 will possess varied physicochemical properties that will likely require different approaches for 

2167 preparation of chemical exposure water. All efforts should be made to avoid solvents or carriers 

2168 because: (1) certain solvents themselves may result in toxicity and/or undesirable or unexpected 

2169 responses, (2) testing chemicals above their water solubility (as can frequently occur through the 

2170 use of solvents) can result in inaccurate determinations of effective concentrations, and (3) the 

2171 use of solvents in longer-term tests can result in a significant degree of “biofilming” associated 

2172 with microbial activity. For difficult to test substances, a solvent may be employed as a last 

2173 resort, and the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances 

2174 and Mixtures should be consulted (OECD 2002) to determine the best method. The choice of 

2175 solvent will be determined by the chemical properties of the substance. Solvents which have 

2176 been found to be effective for aquatic toxicity testing include acetone, ethanol, methanol, 

2177 dimethyl formamide and triethylene glycol. In case a solvent carrier is used, solvent 

2178 concentrations should be below the chronic No Observed Effect Concentration (NOEC); the 

2179 OECD Guidance Document recommends a maximum of 100 μL/L and a recent review 

2180 recommends that solvent concentrations as low as 20 μL/L of dilution water be used (Hutchinson

2181 et al. 2006). If solvent carriers are used, appropriate solvent controls (clean water) must be 

2182 evaluated in addition to non-solvent controls. If it is not possible to administer a chemical via 

2183 the water, either because of physicochemical characteristics (low solubility) or limited chemical 

2184 availability, introducing it via the diet may be considered. Preliminary work has been conducted 

2185 on dietary exposures; however, this route of exposure is not commonly used. The choice of 

2186 method should be documented and analytically verified.

2187

2188 Establishing the High Test Concentration 

2189

2190 For the purposes of this test, results from Tier I amphibian studies should be used to what extent 

2191 possible in determining the high test concentration so as to avoid concentrations that are overtly 

2192 toxic. Prior to running the LAGDA a range finding experiment should be conducted. The 

2193 exposure should be initiated within 24 hours of fertilization and continue for 7-10 days. The test 

2194 concentrations should be set such that the intervals between test concentrations are no greater 

2195 than 1 log. The results of the range finding experiment should serve to set the highest test 

2196 concentration in the LAGDA. Once the highest test concentration is established a 50% dilution 

2197 series is to be used to establish the 3 lower test concentrations. 

2198

2199 Test Concentration Range 

2200

2201 There is a required minimum of four test concentrations and a clean water control (and vehicle 

2202 control if necessary). The minimum test concentration differential between the highest and 
59

2203 lowest should be about one order of magnitude. The maximum concentration separation should 

2204 be no greater than 0.5.

2205

2206 PROCEDURE 

2207

2208 Assay Overview

2209

2210 This assay is intended for use in determining the chronic effects of chemicals on amphibian 

2211 larval growth and development. The assay is initiated with newly spawned larvae and continues 

2212 into juvenile development. Animals are examined daily for mortality and any signs of abnormal 

2213 behavior. At NF stage 62, a sub-sample of animals is collected and various endpoints are 

2214 examined (see Table 1). After all animals have reached NF stage 66, a cull is carried out to 

2215 reduce the number of animals per tank, and the remaining animals continue exposure until 10 

2216 weeks after the median time to NF stage 62 in the control. At test termination additional 

2217 measurements are collected (see Table 1). 

2218

2219 Table 1. Timing of apical endpoint measurements over the course of the LAGDA.

Apical Endpoints Daily Interim 

Sampling

Test 

Termination

Mortality x

Clinical signs of disease and/or general toxicities x

Time to NF stage 62 x

Thyroid histology x

Serum thyroxine (T4) x

Morphometrics (growth) x x

Liver-somatic index (LSI) x

Vitellogenin x

Genetic/phenotypic sex ratios x

Gonad histology x

Reproductive duct histology x

Kidney histology x

Liver histology x

2220

2221 Test Initiation

2222

2223 It is preferred that animals for test initiation have previously been shown to produce offspring 

2224 that can be genetically sexed (see Appendix 3). After spawning of adults, embryos are collected, 

2225 cysteine treated to remove the jelly coat and screened for viability according to ASTM standards 

2226 (ASTM 2004). Cysteine treatment allows the embryos to be handled during screening without 

2227 sticking to surfaces. Screening takes place under a dissecting microscope using an appropriately 

2228 sized eye dropper to remove non-viable embryos. It is preferred that a spawn resulting in greater 

2229 than 70% viability be used for the test. Embryos from a single spawn are randomly distributed 

2230 into exposure treatment tanks containing an appropriate volume of dilution water until each tank 

2231 contains 20 embryos. Embryos should be carefully handled during this transfer in order to 

2232 minimize handling stress and to avoid any injury. At 96 hours post spawning, the tadpoles should 

2233 have moved up the water column and begun clinging to the sides of the tank. 
60

2234

2235 Mortality

2236

2237 All test tanks should be checked daily for dead animals and the numbers recorded for each tank. 

2238 The date, concentration and tank number for any observation of mortality is recorded. Dead 

2239 animals should be removed from the test tank as soon as observed. The developmental stage of 

2240 dead animals should be categorized as either pre-NF stage 58 (pre-forelimb emergence), NF 

2241 stage 58-NF stage 62, NF stage 63-NF stage 66 (between NF 62 and complete tail absorption), or 

2242 post-NF stage 66 (post-larval). Mortality rates exceeding 20% may indicate inappropriate test 

2243 conditions or overtly toxic effects of the test chemical. During the first few days of development 

2244 after the spawning event and during metamorphic climax, the animals tend to be most sensitive 

2245 to non-chemical induced mortality events.

2246

2247 Additional Observations 

2248

2249 The date, treatment, and tank number for any observation of abnormal behavior, grossly visible 

2250 malformations (e.g. scoliosis), or lesions should be recorded. Normal behavior for larval animals 

2251 is characterized by suspension in the water column with tail elevated above the head, regular 

2252 rhythmic tail fin beating, periodic surfacing, operculating, and being responsive to stimuli. 

2253 Abnormal behaviors would include, for example, floating on the surface, lying on the bottom of 

2254 the tank, inverted or irregular swimming, lack of surfacing activity, and being nonresponsive to 

2255 stimuli. For post-metamorphic animals, in addition to the above abnormal behaviors, gross 

2256 differences in food consumption between treatments should be recorded. Gross malformations 

2257 and lesions could include morphological abnormalities (e.g., limb deformities), hemorrhagic 

2258 lesions, abdominal edema, and bacterial or fungal infections, to name a few. These 

2259 determinations are qualitative and should be considered akin to clinical signs of disease/stress 

2260 and made in comparison to control animals. If the rate of occurrence is greater in exposed tanks 

2261 than in the controls, then these should be considered as evidence for overt toxicity.

2262

2263 Larval Sub-Sample

2264

2265 Those tadpoles that have reached NF stage 62 should be removed from the tanks and either 

2266 sampled or moved to the next part of the exposure in a new tank, or physically separated from 

2267 the remaining tadpoles in the same tank with a divider. Tadpoles are checked daily, and the time 

2268 at which an individual tadpole reaches NF stage 62 is recorded. The defining characteristic for 

2269 use in this assessment is the shape of the head. Once the head has become reduced in size such 

2270 that it is the same width as the trunk of the tadpole, then that individual would be counted as 

2271 having attained NF stage 62. One tadpole from every four removed from a given tank is 

2272 randomly selected for the larval sub-sampling. Those tadpoles not sampled are moved to new 

2273 tanks or physically separated from the remaining tadpoles for continued exposure. The random 

2274 procedure used must give each organism on test equal probability of being selected. This can be 

2275 achieved by using any randomizing method but requires that each tadpole be netted. 

2276

2277 For the larval sub-sampling, the following endpoints are obtained:

2278 1. Time to NF stage 62 (number of days between fertilization and NF stage 62)

2279 2. External abnormalities 
61

2280 3. Morphometrics (growth - weight and length)

2281 4. Thyroid histology

2282 5. Serum T4

2283

2284 Outline of Procedure for Larval Sampling:

2285

2286 1. Euthanize tadpole by immersion in Tricaine Methanesulfonate (MS-222) 

2287 appropriately buffered to achieve pH 7.0.

2288 2. Rinse and blot dry.

2289 3. Weigh the tadpole (mg) and measure snout to vent length (mm).

2290 4. Note any gross morphologic abnormalities and/or clinical signs of toxicity. 

2291 5. Collect blood from the heart using a non-heparinized capillary tube for serum 

2292 collection (~25 μL).

2293 6. Remove and discard the lower torso below the forelimbs. 

2294 7. Fix the upper torso for 48 hours in Davidson’s solution with agitation and then rinse 

2295 with tap water and store tissues in 10% NBF.

2296

2297 Euthanasia

2298

2299 NF stage 62 tadpoles should be euthanized by immersion in a 0.01% MS-222 solution (500 mL). 

2300 This solution should be buffered with sodium bicarbonate to a pH of approximately 7.0. 

2301 (Unbuffered MS-222 solution is acidic and irritating to frog skin resulting is poor absorption and 

2302 unnecessary additional stress to the organisms.) The characteristics of the water used to make 

2303 this solution may impact the pH and therefore the amount of sodium bicarbonate needed to 

2304 neutralize this solution. In general, however, a recipe of one part MS-222 to two parts of sodium 

2305 bicarbonate should be used as a starting point.

2306

2307 Using a mesh dip net, a tadpole is removed from the experimental chamber and transported to 

2308 the necropsy area in a transport container. The tadpole is placed into the euthanasia solution. It 

2309 is ready for necropsy when it is unresponsive to external stimuli such as pinching the hind limb 

2310 with a pair of forceps.

2311

2312 Growth: Weight and Length

2313

2314 Measurements of wet weight and snout to vent length for each tadpole (see Figure 1) should be 

2315 made immediately after it becomes non-responsive. These measurements are included in the test 

2316 protocol to assess possible effects of test substances on the growth rate of organisms and are 

2317 useful in detecting generalized toxicity. Tadpoles should be blotted dry before weighing to 

2318 remove excess adherent water. After measurements of body size are made, any gross 

2319 morphological abnormalities and/or clinical signs of toxicity should be noted, recorded, and any 

2320 digital documentation made. These include scoliosis, petechiae, hermorrhage, etc.

2321

2322 a. Larval sub-sampling (NF stage 62)

2323
62

2324

2325

2326

2327 b. NF stage 66 cull and juvenile sampling

2328

2329 Figure 1. Landmarks for measuring snout to vent length for the LAGDA.

2330

2331

2332 Blood Collection for T4 Analyses 

2333

2334 Blood is collected from the heart using a non-heparinized capillary tube for serum collection 

(~25 μL). The tube is sealed (e.g., with Crit-o-seal®2335 ) and placed on ice until clots form (1-4 hrs.), 

2336 at which time the tubes should be centrifuged to remove the clots from the serum. The capillary 

2337 tube is scored and broken at the serum-clot interface, the volume of serum collected for each 

2338 individual is recorded, and the serum is expelled into a microcentrifuge tube. Serum samples 

2339 from individuals are stored at -80°C for subsequent quantification of T4. Given the limited 

2340 volume of blood that can be collected from an individual NF stage 62 tadpole, analyses of 

2341 circulating T4 has to be performed on pooled samples consisting of serum collected from all 

2342 individuals in a tank (i.e., replicate). A pooled sample is formed by combining equal volumes of 

2343 serum from every individual in a tank. The volume of serum contributed by each individual to 

2344 the pool is equal to the least volume of serum collected among all individuals in a tank. 

2345 Professional judgment should be used to exclude samples with low yields of collected serum due 

2346 to technical difficulties. 

Snout-Vent Length

Snout vent length
63

2347

2348 Tissue Collection and Fixation for General Histology

2349

2350 For the sub-sampling, thyroid glands are assessed for histology. The lower torso below the 

2351 forelimbs is removed and discarded. The trimmed carcass is placed into individually labelled 

2352 histological cassettes and fixed in Davidson’s fixative. The volume of fixative in the container 

2353 should be at least 10 times the approximated volume of the tissues. Appropriate agitation or 

2354 circulation of the fixative should be achieved to adequately fix the tissues of interest. All tissues 

2355 remain in Davidson’s fixative for at least 48 hours, but no longer than 96 hours at which time 

2356 they are rinsed in deionized water and stored in 10% NBF. 

2357

2358 Davidson’s Fixative:

2359 a) Glacial acetic acid...............115 ml

2360 b) Formaldehyde (35-40%)........220 ml

2361 c) 95% Ethyl Alcohol...............330 ml

2362 d) Distilled water....................335 ml

2363

2364 10% Neutral Buffered Formalin:

2365 a) 100 ml formalin, full strength (37-40% formaldehyde)

2366 b) 6.5 g sodium phosphate dibasic (anhydrous)

2367 c) 4.0 g sodium phosphate monobasic

2368 d) 900 ml distilled water

2369

2370 Refer to Appendix 5 for procedural information on processing tissues for histological 

2371 examinations and Appendix 6 (Appendix 8.3 of ISR) for information regarding the interpretation 

2372 of diagnostic criteria.

2373

2374 End of Larval Exposure

2375

2376 Given the initial number of tadpoles, it is expected that there will likely be a small percentage of 

2377 individuals that do not develop normally and do not complete metamorphosis in a reasonable 

2378 amount of time. The larval portion of the exposure should not exceed 70 days. Any tadpoles 

2379 remaining at the end of this period should be euthanized by immersion in 0.02% buffered MS2380 222 (pH 7.0). They should be weighed, staged (Nieuwkoop and Faber, 1994), and any 

2381 developmental abnormalities noted.

2382

2383 Cull after NF stage 66

2384

2385 Ideally, only 10 individuals per tank should continue until termination of the exposure. 

2386 Therefore, after all animals have reached NF stage 66 or after 70 days (whichever occurs first), a 

2387 cull should be conducted. Animals that will not continue the exposure should be selected at 

2388 random. These animals are euthanized by immersion in a 0.02% MS-222 solution (500 mL)

2389 buffered with sodium bicarbonate to a pH of approximately 7.0. Measurements of wet weight 

2390 and snout to vent length (see Figure 1) for each froglet are made, and a gross necropsy is 

2391 conducted. The phenotypic sex (based on gonad morphology) is noted as female, male, or 

2392 indeterminate.
64

2393

2394 Juvenile Sampling 

2395

2396 At the end of the exposure period, the remaining animals are euthanized, and the following 

2397 endpoints are evaluated and recorded:

2398

2399 1. Morphometrics (growth - weight and length)

2400 2. Phenotypic/genotypic sex ratios

2401 3. Plasma vitellogenin

2402 4. Gonad histopathology

2403 5. Reproductive duct histopathology

2404 6. Liver histopathology

2405 7. Liver weight (Liver-somatic Index)

2406 8. Kidney histopathology

2407 Methods:

2408

2409 1. Euthanize frogs by injection of buffered 10% MS-222 in PBS (0.01 ml per g frog). 

2410 2. Measure whole body weight (g) and snout-vent length (mm).

2411 3. Record any developmental abnormalities and obvious signs of disease (e.g. scoliosis, 

2412 hemorrhage).

2413 4. Place the frog in dorsal recumbency.

2414 5. Expose the heart by removing the skin and associated cartilage.

2415 6. Carefully expose the heart by opening the pericardial sac without cutting any major 

2416 blood vessels.

2417 7. Snip a major blood vessel coming from the heart and collect blood using a heparinized 

2418 capillary tube for plasma collection (~50 μL).

2419 8. Open the abdominal cavity. Dissect out, weigh, and fix the liver. 

2420 9. Carefully remove the digestive organs (e.g. stomach, intestines) from the lower 

2421 abdomen.

2422 10. Assess gross morphological abnormalities in gonads.

2423 11. Remove a foot for a DNA sample and snap freeze in liquid nitrogen or dry ice. 

2424 12. Remove hind limbs and fix remaining carcass.

2425

2426 Euthanasia

2427

2428 Post-metamorphic frogs are euthanized by an intraperitoneal injection of 10% MS-222 in an 

2429 appropriately buffered solution (e.g., phosphate buffered solution). Dosage for frogs is 0.01 mL 

2430 per gram of frog. This can be approximated to 0.1 mL for a typical male-sized individual and 

2431 0.2 mL for a typical female-sized individual. Using this procedure, frogs may be sampled after 

2432 becoming unresponsive, usually around 2 min after injection. Samples should be collected from 

2433 frogs shortly after euthanasia. Therefore, unless multiple prosectors are available, numerous 

2434 frogs should not be euthanized simultaneously.

2435

2436 Growth: Weight and Length

2437

2438 Measurement of weight and length are identical to those outlined for the larval sub-sampling.
65

2439

2440

2441 Blood Collection for Vitellogenin Analyses

2442

2443 The euthanized juveniles are placed in dorsal recumbency. The heart is exposed by removing the 

2444 skin and cartilage that covers it. The pericardial sac is opened and the heart exposed. In order 

2445 for adequate blood collection to be made, it is imperative that care be taken not to cut or tear any 

2446 blood vessels during this process. The exposed heart should still be beating at this point. A 

2447 major blood vessel at the top of the heart is snipped, and blood is collected with a heparinized 

capillary tube. The tube is sealed (e.g., with Crit-o-seal®2448 ) and placed on ice until the samples can 

2449 be centrifuged. At the end of the sampling, tubes should be centrifuged to remove the blood 

2450 cells from the plasma. The capillary tube is scored and broken at the plasma-cell interface, and 

2451 the plasma is expelled into a microcentrifuge tube that has been coated with aprotinin. Plasma 

2452 samples are stored at -80°C for subsequent quantification of vitellogenin levels with an ELISA.

2453 Vitellogenin is an established estrogen responsive biomarker in a number of fish and frogs 

2454 species. If a valid commercially available ELISA kit exists for X. laevis vitellogenin it may be 

2455 used. Alternatively, an ELISA can be developed as described in Appendix 4.

2456

2457 Tissue Collection and Fixation for General Histology

2458

2459 Gonads and livers are collected for histological analysis during the final sampling. The 

2460 abdominal cavity is opened, and the liver is dissected out, weighed and fixed. Next, the digestive 

2461 organs (e.g. stomach, intestines) are carefully removed from the lower abdomen to reveal the 

2462 gonads. Any gross morphological abnormalities in the gonads should be noted. Finally, the hind 

2463 limbs should be removed, and the carcass should be fixed with the gonads left in situ. Collected 

2464 livers and carcasses are placed into labelled histological cassettes and fixed in Davidson’s 

2465 fixative. The volume of fixative in the container should be at least 10 times the approximated 

2466 volume of the tissues. All tissues remain in Davidson’s fixative for at least 48 hours, but no 

2467 longer than 96 hours at which time they are rinsed in de-ionized water and stored in 10% NBF. 

2468 The same fixatives used in the larval sub-sampling as described previously are used with adult 

2469 samples. Refer to Appendix 5 for details on histological procedures. 

2470

2471 Genetic Sexing

2472

2473 DNA samples from frogs can be obtained from any tissue. One hind limb removed during 

2474 dissection is easy to collect and store in a microcentrifuge tube. Tissue can be stored at -20°C 

2475 until DNA isolation. There are several commercially available kits for the isolation of DNA 

2476 from tissue that can be used. Testing for the presence or absence of a marker is done by PCR as 

2477 described in Appendix 3. Development of the markers used for genetic sexing is described in 

2478 Yoshimoto et al. 2008 (X. laevis). 

2479

2480

2481

2482

2483

2484
66

2485

2486 DATA COLLECTION AND REPORTING 

2487

2488 All data should be collected using electronic or manual systems which conform to good 

2489 laboratory practices (GLP). Study data should include: 

2490

2491 1. Test substance: Characterization of the test substance: physical-chemical properties; 

2492 information on stability and biodegradability. 

2493 2. Chemical observations and data: method and frequency of preparation of dilutions. 

2494 Test chemical information includes actual and nominal concentrations of the test 

2495 chemical, and in some cases, non-parent chemical, as appropriate. Test chemical 

2496 measurements may be required for stock solutions as well as for test solutions. 

2497 3. Solvent (if other than water): justification of the choice of solvent, and 

2498 characterization of solvent (nature, concentration used). 

2499 4. Test conditions: 

2500 a) Operational records: these consist of observations pertaining to the functioning 

2501 of the test system and the supporting environment and infrastructure. Typical 

2502 records include: ambient temperature, test temperature, photoperiod, status of 

2503 critical components of the exposure system (e.g. pumps, cycle counters, 

2504 pressures), flow rates, water levels, stock bottle changes, and feeding records. 

2505 General water quality parameters include: pH, dissolved oxygen, conductivity, 

2506 total iodine, alkalinity, and hardness. 

2507 b) Deviations from the test method: this information should include any 

2508 information or narrative descriptions of deviations from the test method. 

2509 5. Results: 

2510 a) Biological observations and data: these include daily observations of mortality, 

2511 food consumption, abnormal swimming behavior, lethargy, loss of equilibrium, 

2512 malformations, lesions, etc. Observations and data collected at predetermined 

2513 intervals include: growth (weight and length), clinical signs of disease or general 

2514 toxicities, time to metamorphosis, T4 and vitellogenin measurements, and genetic 

2515 sex.

2516 b) Histological data including narrative descriptions, as well as graded severity 

2517 and incidence scores of specific observations.

2518 6. Statistical analyses: Statistical analytical techniques and justification of techniques 

2519 used; results of the statistical analysis preferably in tabular form.

2520 7. Ad hoc observations: these observations should include narrative descriptions of the 

2521 study that do not fit into the previously described categories. 

2522

2523

2524

2525

2526

2527

2528

2529

2530
67

2531

2532 PERFORMANCE CRITERIA AND TEST ACCEPTABILITY/VALIDITY

2533

2534 Performance Criteria

2535

2536 Generally, gross deviations from the test method will result in unacceptable data for 

2537 interpretation or reporting. Therefore, the following criteria in Table 2 have been developed as 

2538 guidance for determining the quality of the test performed and the general performance of the 

2539 control organisms. 

2540

2541

2542 Table 2. Performance criteria for the LAGDA.

2543

Criterion Acceptable limits

Test concentrations Maintained at ≤ 20% CV(variability of 

measured test concentration) over the entire 

test 

Mortality in controls ≤ 20% mortality in any one replicate in the 

controls.

Development in controls The mean time to NF stage 62 is ≤ 45 days, 

and the mean weight at NF stage 62 is ≥ 1.0

± 0.2 g. The mean weight at test 

termination is 11.5 ± 3 g.

Dissolved Oxygen ≥ 40% air saturation 

pH pH should be maintained between 6.5-8.5. 

The inter-replicate/inter-treatment 

differentials should not exceed 0.5. 

Water temperature Target temperature ± 1oC - the interreplicate/inter-treatment differentials should 

not exceed 1.0 oC 

Test concentrations without overt toxicity ≥ 2 

Replicate performance ≤ 2 replicates across the test can be 

compromised 

Special conditions for use of a solvent If a carrier solvent is used, both a solvent 

control and clean water control must be 

used.

Statistically significant differences between 

solvent control and water control groups are 

treated specially. See below for more 

information.

2544

2545

2546

2547

2548

2549
68

2550

2551 Test Validity

2552

2553 The following requirements must be met to deem a test acceptable/valid: 

2554 1. For any given treatment (including controls), mortality cannot exceed 20%, otherwise 

2555 the replicate is considered compromised.

2556 2. At least two treatment levels, with all four uncompromised replicates, must be 

2557 available for analysis.

2558 3. At least two treatment levels without overt toxicity must be available for analysis. 

2559

2560 STATISTICAL ANALYSES

2561

2562 Statistical analyses of the data should preferably follow procedures described in the document 

2563 Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application 

2564 (OECD 2006). Data for continuous endpoints should be assessed for normality (preferably using 

2565 the Shapiro-Wilk or Anderson-Darling test) and variance homogeneity (preferably using the 

2566 Levene test). Both tests are performed on the residuals from an ANOVA. Expert judgment can 

2567 be used in lieu of these formal tests for normality and variance homogeneity, though formal tests 

2568 are preferred. Where non-normality or variance heterogeneity is found, a normalizing, variance 

2569 stabilizing transformation should be sought. If the data (perhaps after a transformation) are 

2570 normally distributed with homogeneous variance, a significant treatment effect is determined 

2571 from Dunnett’s test. If the data (perhaps after a transformation) are normally distributed with 

2572 heterogeneous variance, a significant treatment effect is determined from the Tamhane-Dunnett 

2573 or T3 test or from the Mann-Whitney-Wilcoxon U test. Where no normalizing transformation 

2574 can be found, a significant treatment effect is determined from the Mann-Whitney-Wilcoxon U 

2575 test using a Bonferroni-Holm adjustment to the p-values. The Dunnett test is applied 

2576 independently of any ANOVA F-test and the Mann-Whitney test is applied independently of any 

2577 overall Kruskall-Wallis test. In addition, regardless of the results of the previous tests, a step2578 down Jonckheere-Terpstra trend (JT) test should also be applied to the data as long as a 

2579 monotonic response was observed.

2580

2581 Mortality data should be analyzed for time periods encompassing larval development and the full 

2582 test. Tadpoles that do not complete metamorphosis in the given time frame, those tadpoles that 

2583 are in the larval sub-sample cohort, those juvenile frogs that are culled, and any animal that dies 

2584 due to experimenter error should be treated as censured data. Mortality data as expressed as the 

2585 percentage that died should be arcsine square root transformed prior to analysis.

2586

2587 Time to metamorphosis data should be treated as time to event data, with any mortalities treated 

2588 as censured data. Median time to completion of metamorphosis should be determined by

2589 Kaplan-Meier product-limit estimators and used in the statistical analysis.

2590

2591 Body weight and snout-vent lengths should be analyzed using the means of a given tank. Males 

2592 and females are not sexually-dimorphic at the completion of metamorphosis and data for the 

2593 sexes are combined when analyzing larval sub-sampling data. Male and female juvenile body 

2594 size data should be analyzed separately.

2595
69

2596 Vitellogenin plasma concentrations should be log transformed before statistical analysis and 

2597 separated by sex.

2598

2599 Liver weights should be normalized as proportions of whole body weights (LSI), arcsine square 

2600 root transformed and analyzed separately for each sex.

2601

2602 Use of compromised treatment levels 

2603

2604 Several factors are considered when determining whether a replicate or entire treatment 

2605 demonstrates overt toxicity and should be removed from analysis. Overt toxicity is defined as >5

2606 mortalities in any replicate that cannot be explained by technical error. Other signs of overt 

2607 toxicity include hemorrhage, abnormal behaviors, abnormal swimming patterns, anorexia, and 

2608 any other clinical signs of disease. For sub-lethal signs of toxicity, qualitative evaluations may 

2609 be necessary, and should always be made in reference to the clean water control group. 

2610

2611 Solvent controls 

2612

2613 The use of a solvent should only be considered as a last resort, when all other chemical delivery 

2614 options have been considered. If a solvent is used, then a clean water control must be run in 

2615 concert. At the termination of the test, an evaluation of the potential effects of the solvent must 

2616 be performed. This is done through a statistical comparison of the solvent control group and the 

2617 clean water control group. The most relevant endpoints for consideration in this analysis are 

2618 growth determinants (weight and length), as these can be affected through generalized toxicities. 

2619 If statistically significant differences are detected in these endpoints between the clean water 

2620 control and solvent control groups, determine the study endpoints for the response measures 

2621 using the clean water control. If there is no statistically significant difference between the clean 

2622 water control and solvent control for all measured response variables, determine the study 

2623 endpoints for the response measures using the pooled dilution-water and solvent controls.

2624

2625 REFERENCES

2626

2627 ASTM (2002). Standard Guide for Conducting Acute Toxicity Tests on Test Materials with 

2628 Fishes, Macroinvertebrates, and Amphibians. (A. S. f. T. a. Materials, ed., Philadelphia, PA.

2629

2630 ASTM (2004). Standard Guide for Conducting the Frog Embryo Teratogenesis Assay - Xenopus

2631 (FETAX).

2632

2633 Hutchinson, T. H., Shillabeer, N., Winter, M. J., and Pickford, D. B. (2006). Acute and chronic 

2634 effects of carrier solvents in aquatic organisms: a critical review. Aquat Toxicol 76, 69-92.

2635

2636 Kahl, M. D., Russom, C. L., DeFoe, D. L., and Hammermeister, D. E. (1999). Saturation units 

2637 for use in aquatic bioassays. Chemosphere 39, 539-551.

2638

2639 Nieuwkoop, P. D. and Faber, J., (1994). Normal Table of Xenopus laevis (Daudin). Garland

2640 Publishing, Inc, New York, NY.

2641
70

2642 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

2643 Mixtures.

2644

2645 OECD (2002). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

2646 Mixtures. In OECD Guidelines for the Testing of Chemicals, Volume 1, Number 5, Paris, France.

2647

2648 OECD (2006). Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance 

2649 to Application Environmental Health and Safety Publications, Paris.

2650

2651 USEPA. 1996 Endocrine Disruptor Screening Protram Test Guidelines OPPTS 850.1000: 

2652 Special Considerations for Conducting Aquatic Laboratory Studies. Office of Chemical Safety 

2653 and Pollution Prevention [OCSPP] formerly Prevention, Pesticides and Toxic Substances 

2654 [OPPTS] (7101) EPA 712-C-96-113. April 1996.

2655

2656 Yoshimoto, S., Okada, E., Umemoto, H., Tamura, K., Uno, Y., Nishida-Umehara, C., Matsuda, 

2657 Y., Takamatsu, N., Shiba, T., Ito, M. (2008). A W-linked DM-domain gene, DM-W, 

2658 participates in primary ovary development in Xenopus laevis. Proc Natl Acad Sci USA 

2659 105(7):2469-2474.

2660

2661

2662

2663

2664
71

2665 Appendix 1: Experimental Conditions for the LAGDA

2666

Test animal Xenopus laevis

Initial larval stage Nieuwkoop and Faber (NF) stage 8

Exposure period Until 10 weeks after the median time to NF 

stage 62 in the control

Test concentrations Minimum of 4 different concentrations

Dilution water / laboratory control Any water that permits normal growth and 

development of X. laevis (e.g., spring water or 

charcoal-filtered tap water)

Replication 4 replicate test vessels / test concentration

8 replicate test vessels / control

Exposure regime Flow-through

Test system flow-rate Constant, in consideration of both the 

maintenance of biological conditions and 

chemical exposure (e.g., flow rate sufficient to 

provide 10 tank volume additions/day) 

Test solution / test vessel 4 – 10 L (10 – 15 cm minimum water) / glass or 

stainless steel

Initial larval density 20 larvae / test vessel

Larval density after NF stage 66 cull 10 juveniles / test vessel (decided randomly)

Feeding See Appendix 2

Lighting Photoperiod 12 hr light : 12 hr dark

Intensity 600 to 2000 lux (lumens/m2) at the water 

surface

Water temperature 21o ± 1°C

pH 6.5 to 8.5

Dissolved oxygen (DO) concentration > 3.5 mg/L (> 40% of the air saturation)

Analytical chemistry schedule initiation (day 0), and weekly thereafter for each 

replicate tank at each concentration 

2667

2668

2669

2670
72

2671 Appendix 2: Diet Preparation 

2672

2673 Larval diet

2674

This diet, consisting of Trout Starter, algae/TetraFin®2675 , and brine shrimp, allows control 

2676 organisms to meet both of the following criteria:

2677 1) mean time to NF stage 62 is ≤ 45 days, and

2678 2) mean weight at NF stage 62 is ≥ 1.0±0.2 g

2679

2680 Food Preparation

2681

A. 1:1 (v/v) Trout Starter: algae/TetraFin®2682 :

2683 1. Trout Starter: blend 80 mL of Trout Starter and 300 mL of suitable filtered on high 20 

2684 seconds

2. Algae/TetraFin®2685 mixture: blend 12 g spirulina algae disks and 500 mL filtered LSW on 

high for 40 seconds, blend 12 g Tetrafin®2686 with 500 mL filtered LSW and then combine 

these to make up one L of 12 g/L spirulina algae and 12 g/L Tetrafin®2687

3. Combine equal volumes of the blended Trout Starter and the algae/TetraFin®2688 mixture

2689

2690 B. Brine shrimp: Fifteen mL brine shrimp eggs are hatched in 1 L of salt water (prepared by 

2691 adding 20 mL of NaCl to 1 L deionized water). After aerating 24 hours at room temperature 

2692 under constant light, the brine shrimp are harvested. Briefly, the brine shrimp are allowed to 

2693 settle for 30 min by stopping aeration. Cysts that float to the top of the canister are poured 

2694 off and discarded, and the shrimp are poured through the appropriate filters and brought up to 

2695 30 mL with LSW. 

2696

2697 Feeding Protocol

2698 Table 3 provides a reference regarding the type and amount of feed used during the larval stages

2699 of the exposure. The animals should be fed three times per day Monday through Friday and once 

2700 per day on the weekends*.

2701

2702 Table 3. Feeding regime for X. laevis larvae in flow-through conditions (Trout Starter, 

Algae/TetraFin®2703 Diets, and Brine Shrimp Quantities for 26 tadpoles).

Time 

Post-Fertilization 

(Day 0=Date of 

Injection)

Weekday

Feeding Volume

(3X per day)

Weekend

Feeding Volume

(1X per day)

Brine Shrimp**

2X/day

(1X per day 

weekend)

4 or 5 days***-14 

days 0.33 ml 1.2 ml

0.5 ml (d 8)**

(0.5 ml)*

2 weeks 0.67 ml 2.4 ml

1 ml (d 16)**

(1 ml)*

3 weeks 1.3 ml 4.0 ml 1 ml (1 ml)*
73

4 weeks 1.5 ml 4.0 ml 1 ml (1 ml)*

5 weeks 1.6 ml 4.4 ml 1 ml (1 ml)*

6 weeks 1.6 ml 4.6 ml 1 ml (1 ml)*

7 weeks 1.7 ml 4.6 ml 1 ml (1 ml)*

8-12 weeks 1.7 ml 4.6 ml 1 ml (1 ml)*

2704 * All numbers in parentheses are feeding rates for the weekend (only fed once/day). Brine shrimp are fed once per 

2705 day on the weekend, and the volume remains either 0.5 ml (8-15 days) or 1 ml (16 days).

2706 ** Brine Shrimp: 

2707 Add 0.5 ml of live brine shrimp to the diet twice per day (once per day on the weekend) after 8 days.

2708 Add 1.0 ml of live brine shrimp to the diet twice per day (once per day on the weekend) after 16 days.

2709 ***Start feeding when animals are free swimming.

2710

2711 Juvenile diet 

2712

2713 Once metamorphosis is complete, the feeding regime changes to 3/32” premium sinking frog 

2714 food by Xenopus Express. 

2715

2716 Food preparation

2717 Pellets are briefly run in a coffee grinder or blender in order to reduce the size of the pellets. 

2718

2719 Feeding protocol

2720 Table 4 provides a reference regarding the type and amount of feed used during juvenile and 

2721 adult life stages. The animals should be fed once per day. It should be noted that as animals 

2722 metamorph, they continue receiving a portion of the brine shrimp until >95% animals complete 

2723 metamorphosis. 

2724

2725 Table 4. Feeding regime for X. laevis post-metamorphic juveniles in flow-through 

2726 conditions. 

Weeks post-median metamorphosis 

date*

Crushed pellet 

volume (mL) per 

froglet

Whole pellet volume 

(mL) per froglet

As animals complete metamorphosis 0.05 0

0-1 0.05 0.05

2-3 0 0.2

4-5 0 0.3

6-9 0 0.4

2727 *The first day of week 0 is the median metamorphosis date.

2728
74

2729 Appendix 3: Genetic Sexing

2730

2731 X. laevis primers

2732

2733 DM-W marker

2734 Forward: 5’-CCACACCCAGCTCATGTAAAG-3’

2735 Reverse: 5’-GGGCAGAGTCACATATACTG-3’

2736

2737 Positive Control

2738 Forward: 5’-AACAGGAGCCCAATTCTGAG-3’

2739 Reverse: 5’-AACTGCTTGACCTCTAATGC-3’

2740

2741 DNA purification

2742

2743 Purify DNA from muscle or skin tissue using Qiagen DNeasy Blood and Tissue Kit (cat # 69506) 

2744 or similar product according to kit instructions. DNA can be eluted from the spin columns using 

2745 less buffer to yield more concentrated samples if deemed necessary for PCR. 

2746

2747 PCR

2748

Sample protocol using JumpStartTM 2749 Taq from Sigma is outlined below:

2750

Master Mix 1x (μL) [Final]

NFW 11 -

10X Buffer 2.0 -

MgCl2 (25mM) 2.0 2.5 mM

dNTP’s (10mM ea) 0.4 200 μM

Marker for primer (8 μM) 0.8 0.3 μM

Marker rev primer (8 μM) 0.8 0.3 μM

Control for primer (8 μM) 0.8 0.3 μM

Control rev primer (8 μM) 0.8 0.3 μM

JumpStartTM Taq 0.4 0.05 units/μl

DNA template 1.0 ~200 pg/μl

2751

2752 Note: When preparing Master Mixes, prepare extra to account for any loss that may occur while 

2753 pipetting (example: 25x should be used for only 24 reactions). 

2754

2755 Reaction: Master Mix 19.0 μL

2756 Template 1.0 μL

2757 Total 20.0 μL

2758

2759

2760

2761

2762

2763
75

Thermocycler Profile: 1. 94o2764 C 1 min

 2. 94o2765 C 30 sec

 3. 60o2766 C 30 sec

 4. 72o2767 C 1 min

2768 5. Goto step 2. 35 cycles

 6. 72o2769 C 1 min

 7. 4o2770 C

2771

2772 PCR products can be run immediately in a gel or stored at 4oC. 

2773

2774

2775 Agarose Gel Electrophoresis (3%)

2776

2777 50X TAE

2778 24.2 g Tris

2779 5.71 mL glacial acetic acid

2780 3.72 g Na2(EDTA)·2H2O

2781 Add water to 100 mL

2782

2783

2784 1X TAE

2785 392 mL H2O

2786 8 mL 50X TAE

2787

2788 3:1 Agarose

2789 3 parts NuSieve agarose GTG

2790 1 part Fisher agarose low EEO

2791

2792 Method

2793 1. Prepare a 3% gel by adding 1.2 g agarose mix to 43 mL 1X TAE. Swirl to disassociate 

2794 large clumps.

2795 2. Microwave agarose mixture until completely dissolved (avoid boiling over). Let cool 

2796 slightly.

2797 3. Add 1.0μL ethidium bromide (10 mg/mL). Swirl flask.

2798 4. Pour gel into mold with comb. Cool completely.

2799 5. Add gel to apparatus. Cover gel with 1X TAE.

2800 6. Add 1μL of 6x loading dye to each 10μl PCR product.

2801 7. Pipette samples into wells.

2802 8. Run at 160 constant volts for ~20 minutes.

2803

2804

2805

2806

2807

2808

2809

2810

2811
76

2812 LAGDA Appendix 4: Vitellogenin ELISA

2813

2814 Measurement of Vitellogenin (Vtg) in Xenopus laevis by ELISA

2815

2816 The procedure is based on an ELISA originally developed for fathead minnow Vtg (Parks et al., 

2817 1999, Comp. Biochem. Physiol C 123:113-125). See Olmstead et al. (2009) Gen. Comp. 

2818 Endocrinol. 160:117-123 for a description of the assay as modified for Vtg in X. tropicalis. The 

2819 method uses an antibody made against X. tropicalis Vtg, but we know it also works for X. laevis

2820 Vtg. 

2821

2822 Materials and Reagents:

2823 Preadsorbed 1o Antibody (Ab) serum: mix 1 part anti- X.tropicalis Vtg 1o Ab serum with 2 parts 

2824 control male plasma and leave at RT for ~ 75 minutes, put on ice for 30 min, centrifuge > 20K x 

2825 G for 1 h at 4 oC, remove supernatant, aliquot, store at -20 oC.

2826

2827 2o Antibody: Goat Anti-Rabbit IgG-HRP conjugate (e.g., Bio-Rad 172-1019)

2828

2829 Vtg Standard: purified X. laevis Vtg at 3.3 mg/ml.

2830

2831 TMB (3,3',5,5' Tetramethyl-benzidine) (e.g., KPL 50-76-00; or Sigma T0440)

2832

2833 Normal Goat Serum (NGS) (e.g., Chemicon S26-100ml)

2834

2835 96 well EIA polystyrene microtiter plates (e.g., ICN:76-381-04, Costar:53590, Fisher:07-200-35)

2836

2837 37 oC hybridization oven (or fast equilibrating air incubator) for plates, water bath for tubes

2838

2839 Other common laboratory equipment, chemicals, and supplies.

2840

2841 Recipes:

2842 Coating Buffer (50mM Carbonate Buffer, pH 9.6):

2843 1.26g NaHCO3

2844 0.68 g Na2CO3

2845 428 ml water

2846 10X PBS (0.1 M phosphate, 1.5 M NaCl):

2847 0.83g NaH2PO4H2O

2848 20.1 g Na2HPO47 H2O

2849 71 g NaCl

2850 810 ml water

2851 Wash Buffer (PBST):

2852 100 ml 10X PBS

2853 900 ml water

2854 Adjust pH to 7.3 with 1 M HCL

2855 Then add 0.5 ml Tween-20

2856 Assay Buffer:

2857 3.75 ml Normal Goat Serum (NGS)
77

2858 146.25 ml Wash Buffer

2859

2860 Sample collection:

2861 Blood is collected with heparinized microhematocrit tube and placed on ice. After centrifugation 

2862 for 3 min, the tube is scored, broken open, and the plasma expelled into 0.6 ml microcentrifuge 

2863 tubes which contain 0.13 units of lyophilized aprotinin. (These tubes are prepared in advance by 

2864 adding the appropriate amount of aprotinin, freezing, and lyophilizing in a speed-vac at low heat 

2865 until dry.) Place plasma at -80 C until analyzed.

2866

2867 Procedure for One Plate:

2868

2869 Coating the Plate:

2870 Mix 20 μl of purified Vtg with 22 ml of carbonate buffer (final 3 μg/ml). Add 200 μl to each 

2871 well of a 96-well plate using. Cover the plate with adhesive sealing film and allow to incubate 

2872 overnight at 4 oC (or 37 oC for 2 hours).

2873

2874 Blocking the Plate:

2875 Blocking solution is prepared by adding 2 ml of Normal Goat Serum (NGS) to 38 ml of

2876 carbonate buffer. Remove coating solution and shake dry. Add 350 μl of the blocking solution 

2877 to each well. Cover with adhesive sealing film and incubate at 37 oC for 2 hours (or at 4 C 

2878 overnight). 

2879

2880 Preparation of Standards:

2881 5.8 μl of purified Vtg standard is mixed with 1.5 ml of assay buffer in a 12 x 75 mm borosilicate 

2882 disposable glass test tube. This yields 12,760 ng/ml. Then a serial dilution is performed by 

2883 adding 750 μl of the previous dilution to 750 μl of assay buffer to yield final concentrations of 

2884 12,760, 6380, 3190, 1595, 798, 399, 199, 100, and 50 ng/ml. 

2885

2886 Preparation of Samples: 

2887 Start with a 1:300 (e.g., combine 1 μl plasma with 299 μl of assay buffer) or 1:30 dilution of 

2888 plasma into assay buffer. If a large amount of Vtg is expected, additional or greater dilutions 

2889 may be needed. Try to keep B/Bo within the range of standards. For samples without 

2890 appreciable Vtg, e.g., control males and immature females, use the 1:30 dilution. Samples 

2891 diluted less than this may show unwanted matrix effects. 

2892

2893 Additionally, it is recommended to run a positive control sample on each plate. This comes from 

2894 a pool of plasma containing highly induced levels of Vtg. The pool is initially diluted in NGS, 

2895 divided in aliquots and stored at -80 C. For each plate, an aliquot is thawed, diluted further in 

2896 assay buffer and run similar to a test sample.

2897

2898 Incubation with 1o Antibody:

2899 The 1o antibody is prepared by making a 1:2,000 dilution of preadsorbed 1o antibody serum in 

2900 assay buffer (e.g., 8 μl to 16 ml of assay buffer). Combine 300 μl of 1o antibody solution with 

2901 300 μl of sample/standard in a glass tube. The Bo tube is prepared similarly with 300 μl of assay 

2902 buffer and 300 μl of antibody. Also, a NSB tube should be prepared using 600 μl of assay buffer 
78

2903 only, i.e., no Ab. Cover the tubes with parafilm and vortex gently to mix. Incubate in a 37 oC 

2904 water bath for 1 hour. 

2905

2906 Washing the Plate: 

2907 Just before the 1o antibody incubation is complete, wash the plate. This is done by shaking out 

2908 the contents and patting dry on absorbent paper. Then fill wells with 350 μl of wash solution, 

2909 dump out, and pat dry. A multi-channel repeater pipette or plate washer is useful here. The wash 

2910 step is repeated two more times for a total of three washes.

2911

2912

2913 Loading the Plate: 

2914 After the plate has been washed, remove the tubes from the water bath and vortex lightly. Add 

2915 200 μl from each sample, standard, Bo, and NSB tube to duplicate wells of the plate. Cover plate 

2916 with adhesive sealing film and allow to incubate for 1 hour at 37 oC.

2917

2918

2919 Incubation with the 2o Antibody:

2920 At the end of the incubation from the previous step, the plate should be washed three times again, 

2921 like above. The diluted 2o antibody is prepared by mixing 2.5 μl of 2o antibody with 50 ml of 

2922 assay buffer. Add 200 μl of diluted 2o antibody to each well, seal like above, and incubate for 1 

2923 hour at 37 oC.

2924

2925 Addition of Substrate:

2926 After the incubation with the 2o antibody is complete, wash the plate three times as described 

2927 earlier. Then add 100 μl of TMB substrate to each well. Allow the reaction to proceed for 10 

2928 minutes, preferably out of bright light. Stop the reaction by adding 100 μl of 1 M phosphoric 

2929 acid. This will change the color from blue to an intense yellow. Measure the absorbance at 450 

2930 nm using a plate reader.

2931

2932 Calculate B/Bo:

2933 Subtract the average NSB value from all measurements. The B/Bo for each sample and standard 

2934 is calculated by dividing the absorbance value by the average absorbance of the Bo sample. 

2935

2936 Obtain the Standard Curve and Determine Unknown Amounts:

2937 Generate a standard curve with the aid of some computer graphing software (e.g., Slidewrite or 

Sigma Plot®2938 ) that will extrapolate quantity from B/Bo of sample based on B/Bo of standards. 

2939 Typically, the amount is plotted on a log scale and the curve has a sigmoid shape. However, it 

2940 may appear linear when using a narrow range of standards. Correct sample amounts for dilution 

2941 factor and report as mg Vtg/ml of plasma.

2942

2943 Determination of Minimum Detection Limits (MDL):

2944 Often, particularly in normal males, it will not be clear how to report results from low values. In

2945 these cases, the 95% "Confidence limits" should be used to determine if the value should be 

2946 reported as zero or as some other number. If the sample result is within the confidence interval 

2947 of the zero standard (Bo), the result should be reported as zero. The minimum detection level will 

2948 be the lowest standard which is consistently different from the zero standard; that is, the two 
79

2949 confidence intervals don't overlap. For any sample result which is within the confidence limit of 

2950 the minimum detection level, or above, the calculated value will be reported. If a sample falls 

2951 between the zero standard and the minimum detection level intervals, one half of the minimum 

2952 detection level should be reported for the value of that sample. 

2953

2954

2955

2956
80

2957 Appendix 5: Histology Protocol

2958

2959 Larval Subsampling (Stage 62)

2960

2961 Dissection

2962 - Froglets euthanized by submersion in MS-222

2963 - Wet weight is measured

2964 - Blood is collected directly from aorta

2965 - Legs are removed at knee joint

2966

2967 Fixation

2968 - Davidson’s 48 hours

2969 a) Glacial acetic acid...............115 ml

2970 b) Formaldehyde (35-40%)........220 ml

2971 c) 95% Ethyl Alcohol...............330 ml

2972 d) Distilled water....................335 ml

2973 - Rinse with distilled water

2974 - 10% neutral buffered formalin

2975

2976 Trimming

2977 Gonad:

2978 - Transverse cuts posterior to the arms and anterior to the pelvis

2979 Thyroid:

2980 - Transverse cut through middle of arms leaving some shoulder joint

2981 - Transverse cut anterior to eyes to remove snout

2982

2983 Processing

2984 Decalcification

2985 - By fixation in Davidson’s or Bouin’s

2986 Dehydration 

2987

2988 Thyroid and gonad (1 hour stations)

Station 1 2 3 4 5 6 7

Reagent 70% EtOH 80% EtOH 95% EtOH 95% EtOH

100% 

EtOH

100% 

EtOH

100% 

EtOH

Time 1 hour 1 hour 1 hour 1 hour 1 hour 1 hour 1 hour

retort 

temp -ambient- -ambient- -ambient- -ambient- -ambient- -ambient- -ambientp/v on on on on on on on

Station 8 9 10 11 12 13 14

Reagent Clear-rite Clear-rite Clear-rite Paraffin Paraffin Paraffin Paraffin

Time 1 hour 1 hour 1 hour 1 hour 1 hour 1 hour 1 hour

retort 

temp -ambient- -ambient- -ambient- 60 ̊ 60 ̊ 60 ̊ 60 ̊

p/v on on on on on on on

2989

2990 Embedment

2991 Gonad: 
81

2992 - Embed anterior side down.

2993 

2994

2995

2996 Thyroid: 

2997 - Embed posterior side down, with the nose pointing up. The nose can be trimmed off anterior 

2998 to the eyes if needed to fit into embedding mold.

2999

3000 Sectioning Protocol 

3001 Gonad: (4 μm sections)

3002 - Face block

3003 - Step section and collect one good section to be mounted on a glass slide at the following 

3004 levels:

3005

3006 

3007

3008 Thyroid: (4 μm sections)

3009 - Face block

3010 - Take 1 section, mount on slide, and inspect in microscope for both thyroid glands. 

3011 - No glands?

3012 - Take 150-200 micron step, examine a section for thyroid glands, and repeat until both glands 

3013 appear on sections.

3014 - Section has both glands?

3015 - Take 1 section, cut 50 microns, take 1 section, and mount together on slide.

3016 - Seal block 

3017

3018 Staining

3019 - H&E and coverslip with glass using a permanent mounting medium

3020

3021 Juvenile Sampling & Processing

3022

3023 Dissection

3024 - Frogs euthanized by MS-222 injection

3025 - Wet weight, length measured

3026 - Blood is collected from heart

3027 - Liver is removed, weighed and fixed 

3028 - One foot is removed for DNA analysis

3029 - Remaining carcass is placed in an appropriately sized tissue cassette and fixed
82

3030 -

3031

3032

3033

3034

3035

3036

3037

3038 Male Female

3039

3040 Fixation

3041 - Davidson’s 48 hours

3042 Glacial acetic acid...............115 ml

3043 Formaldehyde (35-40%)........220 ml

3044 95% Ethyl Alcohol...............330 ml

3045 Distilled water....................335 ml

3046 - Rinse with distilled water

3047 - 10% neutral buffered formalin

3048

3049 Trimming

3050 Testis/Kidney/ducts

3051 - Transverse cuts on anterior and posterior margins of testis.

3052

3053

3054

3055

3056

3057

3058

3059

3060 Ovary/Kidney/ducts

3061 - Transverse cuts on anterior and posterior margins of the ovary. 

3062

3063

3064

3065

3066

3067

3068

3069 Thyroid

3070 - Transverse cut through middle of arms leaving some shoulder joint

3071 - Transverse cut anterior to eyes to remove snout

3072

3073

3074

3075
83

3076

3077

3078

3079 Processing

3080 Decalcification

3081 Bone surrounding thyroid and kidney samples should be decalcified for 24-48 hours 

3082 depending on strength of decalcifier. Decalcification not necessary for ovaries.

3083 Dehydration

3084 **Ovaries (1⁄2 hour stations) Thyroid and kidney (1 hour stations)

Station 1 2 3 4 5 6 7

Reagent 70% EtOH 80% EtOH 95% EtOH 95% EtOH

100% 

EtOH

100% 

EtOH

100% 

EtOH

Time   **  **   ** 

retort 

temp -ambient- -ambient- -ambient- -ambient- -ambient- -ambient- -ambientp/v on on on on on on on

Station 8 9 10 11 12 13 14

Reagent Clear-rite Clear-rite Clear-rite Paraffin Paraffin Paraffin Paraffin

Time **   **  ** ** **

retort 

temp -ambient- -ambient- -ambient- 60 ̊ 60 ̊ 60 ̊ 60 ̊

p/v on on on on on on on

3085

3086 Embedment

3087 Testis/kidney & Oviduct/kidney: Embed anterior side down

3088 Ovaries: Embed normally; a large mold will be needed

3089 Thyroid: Embed posterior side down, nose up in large mold

3090

3091 Sectioning

3092 Testis/kidney & Oviduct/kidney: (4μm sections)

3093 - Face block

3094 - Step section and collect one good section to be mounted on a glass slide at the following 

3095 levels:

3096

3097 

3098

3099 Ovaries: (7μm sections)

3100 - Face block

3101 - On the side of the paraffin block, mark 1⁄4, 1⁄2, 3⁄4 way through entire ovary.
84

3102 - Take a ribbon of 2-3 sections at each mark, mount on slide.

3103

3104 Thyroid: (4μm sections)

3105 - Face block 

3106 - Take 1 section, mount on slide, and inspect in microscope for both thyroid glands. 

3107 - No glands?

3108 - Take 150-200 micron step, examine a section for thyroid glands, and repeat until both glands 

3109 appear on sections.

3110 - Section has both glands?

3111 - Take 1 section, cut 50 microns, take 1 section, and mount together on slide.

3112 - Seal block 

3113

3114 Staining

3115 - H&E and coverslip with glass using a permanent mounting medium

3116

3117

3118

3119

3120

3121

3122

3123

3124

3125

3126
85

3127 Appendix 6: Histopathology Guidance for Reading LAGDA Studies

3128

3129 See ISR Appendix 8.3

3130

3131

3132 Appendix 7: T4 ELISA

3133

3134 Adaptation of a Commercially Available ELISA Kit to Measure Total

3135 Thyroxine (T4) in the Serum/Plasma of Xenopus laevis.

3136

3137 Materials

3138

3139 T4 ELISA kit: (e.g.MP Biomedicals 07BC1007)

3140 Buffer: 0.01 M Tris-Cl, pH 7.3, 1% methanol

3141 95% ethanol 

3142 Vacuum centrifuge concentrator (e.g. Savant Speed Vac SVC100)

3143 Other common laboratory equipment

3144

3145 Background

3146

3147 The ELISA kits are based on a competition between T4 in a sample and a constant amount of 

3148 enzyme-labeled T4 to bind an immobilized antibody recognizing T4. There do not appear to be 

3149 any commercially available ELISA kits for the measurement of circulating T4 in Xenopus laevis. 

3150 There are kits available for human and several other species, but these kits appear to be highly 

3151 dependent on the use of standards made with homologous serum. The kits are formulated to be 

3152 compatible with the presence of proteins in the blood which bind T4. These proteins include 

3153 thyroxine binding globulin, transthyretin (prealbumin), and albumin. Initial attempts to measure 

3154 T4 in X. laevis using a commercially available kit produced results which were highly variable 

3155 and greatly different from the values obtained with non-immuno-metric methods (i.e. ICP-MS). 

3156 It was thought that this variability was to due to inappropriate interaction between the binding

3157 proteins present in the sample, the amount of T4 in the sample, and the labeled T4 included in the 

3158 kit. It was thought that these problems would be avoided if the T4 binding ability of the sample 

3159 proteins was removed. This also necessitated using a standard which was free of binding 

3160 proteins. Reviewing literature of early methods for T4 measurement indicated that a 

3161 deproteination step with acid or ethanol was often used. Consistent and sensible results were 

3162 obtained with the ELISA kits when this adaptation was used.

3163

3164

3165 Procedure

3166

3167 Follow the kit instructions, with these three exceptions:

3168

3169 1 Standards 

3170 A superstock solution of T4 at 154 μg/ml was made in 0.05 M Na2CO3. This was diluted 

3171 to 20 μg/dl (200 ng/ml) in buffer. From this, additional dilutions in buffer were made to 

3172 give standards of 0.25, 0.5, 1.0, 2.0, 4.0 and 10.0 μg/dl. 

3173
86

3174

3175 2 Extraction/deproteination

3176 Blanks (buffer), standards, and samples should be processed at room temperature as 

3177 follows. Add 60 μl of buffer, standards, or serum/plasma to a 0.6 ml micorcentrifuge 

3178 tube. If lesser amounts of sample are used, make up the volume to 60 μl with buffer. 

3179 Add 180 μl of ethanol and mix vigorously by vortexing. Repeat vortexing two more 

3180 times within a 10 minute period. Centrifuge for 10 minutes at 20,000 times g. Remove 

3181 supernatant to another tube and repeat extraction on pellet with another 180 μl of ethanol. 

3182 Centrifuge like above for 5 minutes. Combine supernatants and mix. Bring samples to 

3183 dryness in a vacuum centrifuge concentrator. Thoroughly resuspend residue in 60 μl of 

3184 buffer. Centrifuge for 10 minutes at 20,000 times g. Remove 55 μl to another 0.6 ml 

3185 tube. 

3186

3187 3 Combine samples/standards with T4-HRP conjugate before loading on the EIA

3188 Plate

3189

3190 Add 220 μl of diluted T4-conjugate to the 55 μl from above and mix. Add 125 μl to 

3191 duplicate wells of the EIA plate. Continue with kit instructions.

3192

3193

3194 Comments

3195

3196 Although this adaptation has only been tried with the kit from MP Biomedicals, it is expected 

3197 that kits from other suppliers that use a similar format should work as well.

3198

3199

3200

3201

3202

3203

3204

3205

3206

3207

3208

3209

3210

3211

3212

3213

3214

3215

3216

3217

3218

3219
87

3220 8.2 Histological Development of the Gonad in Juvenile Xenopus laevis

3221

3222 Chad A. Blanksma1, Allen W. Olmstead2, Sigmund J. Degitz2, Jonathan Haselman2, Rodney D. Johnson2 

3223 1ORISE Research Participation Program at U.S. Environmental Protection Agency, ORD, NHEERL, Mid-Continental 

3224 Ecology Division, Duluth, MN 

3225 2U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental 

3226 Effects Research Laboratory, Mid-Continental Ecology Division, Duluth, MN

3227

3228 Introduction

3229 As directed by the Food Quality Protection Act, the US Environmental Protection 

3230 Agency is developing a screening program for endocrine disrupting compounds. The Larval 

3231 Amphibian Growth and Development Assay (LAGDA) is a tier II test intended to identify and 

3232 characterize the adverse consequences of toxicants that interfere with normal growth and 

3233 development of larval and juvenile frogs with particular emphasis on disruption of estrogen, 

3234 androgen, and thyroid mediated processes. A particularly important aspect of this assay is the 

3235 detection of alterations in gonad differentiation and development. 

3236 Assessments of gonad malformations in anuran species have traditionally relied on gonad 

3237 histology of tadpoles at the moment of metamorphosis completion (NF stage 66). Preliminary 

3238 experiments using Xenopus exposed to three model endocrine disruptors possessing estrogenic, 

3239 androgenic, or anti-aromatase activities indicated that gonad histology at this developmental 

3240 stage was inadequate for the goals of the assay. Specifically the gonads were too immature, with 

3241 the detection of mixed sex phenotypes being subjective and not informative with respect to a 

3242 toxicant’s mechanism of action. In addition, exposure to endocrine disruptors such as 

3243 ethynylestradiol and fadrozole, which induce testicular oocytes in older animals, did not in frogs 

3244 sampled at metamorphosis.

3245 We propose that assessing the gonads at a later stage of development would allow for

3246 improved detection of aberrant gonad differentiation and development, yielding more 

3247 informative results for risk assessment purposes. In order to identify a more appropriate level of 

3248 gonad development for use in the LAGDA, we characterized normal gonad development in the 

3249 test species, X. laevis, at two week intervals from completion of metamorphosis until 12 weeks 

3250 post-metamorphosis (wpm).

3251

3252 Materials and Methods

X. laevis were cultured in-house at EPA-MED using filtered Lake Superior water at 21o3253 C 

3254 with 12:12 hour light:dark photoperiod. No fewer than 4 males and 4 females were subsampled 

3255 at completion of metamorphosis (NF stage 66) and at 2 week time periods up to 12 weeks post 

3256 metamorphosis (wpm). 

3257 Frogs were euthanized by injection of MS-222 in accordance with animal care and use 

3258 guidelines. Specimens were trimmed by removing the gastrointestinal tract and liver to expose 

3259 the gonad, kidney, and reproductive ducts, followed by transverse cuts to the anterior margin of 

3260 the gonad and the caudal margin of the kidneys. The trimmed trunks containing gonads were 

3261 processed for histological analysis. Samples were embedded with the anterior surface down for 

3262 transverse sectioning so gonads could be assessed for morphological changes along their 

3263 longitudinal axis. Sections were cut at a thickness of 5 microns and stained with H&E. 

3264 Incremental sectioning produced 4-6 slides per sample, arranged anterior to posterior. 

3265 A representative image of the anterior region of each sample’s gonad was taken using a 

3266 40x objective and each sample was assessed for developmental landmarks. 
88

3267

3268

3269

3270

3271

3272

3273

3274

3275

3276

3277

3278

3279

90

3287

3288 Gonadal Development Timeline

3289

3290

3291

3292

3293 Conclusions

3294 The evaluation of gonad development is an important endpoint for protocols that test 

3295 chemicals for possible endocrine disruption effects. At metamorphosis, NF stage 66, the gonads 

3296 are only partially developed and sex-specific germ cells cannot be identified histologically. 

3297 Extending the test protocols past metamorphosis allows more comprehensive histological 

3298 evaluation of possible effects on gonad development. This study provides information which can 

3299 be used to optimize amphibian EDC test protocols. Extending the test protocol to at least 6 

3300 weeks post-metamorphosis provides substantially more insight into possible EDC effects on 

3301 gonad development. When sampling at this time or later, the gonads express:

3302

3303  Nearly complete maturation of oocytes and spermatocytes 

3304  Developed seminiferous tubules and associated ducts in males

3305  Developing oviduct system in females

3306  Developed supporting cells and tissues such as granulosa cells, Sertoli and Leydig cells. 

3307

3308 References

3309 Nieuwkoop PD, J Faber. 1967 Normal table of Xenopus laevis (Daudin): A Systematical and 

3310 Chronoligical Survey of the Development from the Fertilized Egg Till the End of 

3311 Metamorphosis. 2nd ed. Amsterdam: North Holland

3312 Reproduction of Amphibians. Biological Systems in Vertebrates. 2009. Edited by Maria 

3313 Ogielska. Enfield (New Hampshire): Science Publishers
91

3314 Dumont, James N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in 

3315 laboratory maintained animals. Journal of Morphology Volume 136, Issue 2, pp. 153-179. 

3316 February 1972

3317

3318 8.3 Histopathology Guidance for Reading LAGDA Studies

3319

3320 Histopathology Guidance for Reading LAGDA Studies

3321 Chad A. Blanksma1, Rodney D. Johnson2 

3322 1ORISE Research Participation Program at U.S. Environmental Protection Agency, ORD, NHEERL, Mid-Continental Ecology 

3323 Division, Duluth, MN 

3324 2U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects 

3325 Research Laboratory, Mid-Continental Ecology Division, Duluth, MN

3326

3327 The slides shall be evaluated by a pathologist for treatment-induced effects. The pathologist 

3328 shall perform the evaluation according to the Best Practices Guideline: Toxicologic 

3329 Histopathology (Crissman et al. 2004). It is recommended that evaluation begin with examining

3330 representative slides from specimens selected from control and the two highest treatments to 

3331 determine likely treatment-related effects. Then, images of representative treatment-related 

3332 effects and associated severity scores shall be prepared to be used as references for scoring the 

3333 remainder of the specimens. Finally, after the initial evaluation, the study pathologist shall 

3334 perform a masked re-evaluation of all the specimens, but for only the previously observed 

3335 treatment-related lesions.

3336

3337 Each section shall be evaluated for gonad pathology, whereas kidney, reproductive duct, and 

3338 liver pathologies shall be based on the assessment of one representative section. The prevalence 

3339 and extent of the effects shall be assessed in the various treatments as compared to controls. 

3340 Effects data containing diagnoses and associated severity grades shall be reported in an Excel 

3341 spreadsheet similar to the example provided below. Diagnoses of treatment effects shall be 

3342 based on the columns in the example pathology spreadsheet. However, for pathologies not listed 

3343 in the spreadsheet, the pathologist shall provide new diagnostic criteria to the spreadsheet. 

3344 Pathology not related to treatment shall also be noted as a comment in the spreadsheet. Create 

3345 digital images of representative diagnostic features in an uncompressed file format. Images of 

3346 representative control tissues shall be included for comparison. Each image shall be at an 

3347 appropriate magnification to illustrate the features using the best judgment of the pathologist. An

3348 embedded calibrated scale bar shall be included in all pictures. The spreadsheet shall indicate 

3349 imaged sections.

3350

3351 A report shall be prepared which includes a short summary of the protocols used in slide 

3352 preparation and a description of treatment-related effects based on concentration responses and 

3353 comparisons with control frogs. The narrative report shall include tables, figures, figure legends, 

3354 and citations, as necessary to describe treatment effects. Citations, tables, figures, figure legends, 

3355 shall be placed at the end of the narrative report. Other supporting histopathology data, images, 

3356 and histology technical reports shall be provided as spreadsheets, images without legends or 

3357 annotations, and documents. The titles and file names of reports and documentation shall 

3358 include: laboratory; species name; developmental stage; test chemical; and tissue examined. The 
92

3359 file names of digital images shall identify the test chemical, treatment, and specimen ID. A 

3360 tabular cross-reference of image files and report figure numbers and titles shall be provided.

3361 General approach to reading studies

3362 From OECD guidance document for the Diagnosis of Endocrine-Related Histopathology of Fish 

3363 Gonads. (2009)

3364

3365 Studies are to be read by individuals experienced in reading toxicologic pathology studies, and 

3366 who are familiar with normal frog gonad histology, with gonadal physiology, and with general 

3367 responses of the gonads to toxicologic insult. Pathologists may be board certified (e.g., American 

3368 College of Veterinary Pathologists, The European Centre of Toxicologic Pathology, or other 

3369 certifying organizations); however, certification is not a requirement as long as the pathologist 

3370 has obtained sufficient experience with, and knowledge of, frog histology and toxicologic

3371 pathology. Technicians should not be used to conduct readings due to the subtle nature of some 

3372 changes and the need for subjective judgments based on past experience.

3373 It is recognized that there is a limited pool of pathologists with the necessary training and 

3374 experience that are available to read the gonadal histopathology for the Larval Amphibian 

3375 Growth and Development Assay. If an individual has toxicological pathology experience and is 

3376 familiar with gonadal histology in amphibian species, he/she may be trained to read the frog 

3377 assay. If pathologists with little experience are used to conduct the histopathological analysis, 

3378 informal peer review may be necessary.

3379 Pathologists are to read the studies non-blinded (i.e., with knowledge of the treatment group

3380 status of individual fish). This is because endocrine effects on histomorphology tend to be 

3381 incremental, and subtle differences between exposed and unexposed animals may not be 

3382 recognizable unless tissue sections from high dose animals can be knowingly compared to those 

3383 from controls. Thus, the aim of the initial evaluation is to ensure that diagnoses are not missed 

3384 (i.e., to avoid false-negative results). However, it is expected that any potential compound3385 related findings will be re-evaluated by the pathologist in a blinded manner prior to reporting 

3386 such findings, when appropriate. Certain diagnostic criteria, such as relative increases or 

3387 decreases in cell populations, cannot be read in a blinded manner due to the diagnostic 

3388 dependence on control gonads. As a rule, treatment groups should be evaluated in the following 

3389 order: control, high-dose, intermediate-dose, and low-dose.

3390 It is suggested that the pathologists be provided with all available information related to the 

3391 study prior to conducting their readings. Information regarding gross morphologic abnormalities, 

3392 mortality rates, and general test population performance and health are useful for pathologists to 

3393 provide comprehensive reports and to aid in the interpretation of findings. For a more 

3394 comprehensive discussion of standard reading approaches for toxicologic pathology studies, 

3395 please refer to the Society of Toxicologic Pathology Best Practices for reading toxicologic 

3396 histopathology studies (Crissman et al., 2004).

3397

3398 Severity Grading

3399 In toxicologic pathology, it is recognized that compounds may exert subtle effects on tissues that 

3400 are not adequately represented by simple binary (positive or negative) responses. Severity 

3401 grading involves a semi-quantitative estimation of the degree to which a particular 

3402 histomorphological change is present in a tissue section (Shackelford et al., 2002). The purpose 

3403 of severity grading is to provide an efficient, semi-objective mechanism for comparing changes 

3404 (including potential compound-related effects) among animals, treatment groups, and studies.
93

3405

3406 Severity grading will employ the following system:

3407 Not remarkable

3408 Grade 1 (minimal)

3409 Grade 2 (mild)

3410 Grade 3 (moderate)

3411 Grade 4 (severe)

3412 A grading system needs to be flexible enough to encompass a variety of different tissue changes. 

3413 In theory, there are three broad categories of changes based on the intuitive manner in which 

3414 people tend to quantify observations in tissue sections:

3415 (1) Discrete: these are changes that can be readily counted. Examples include atretic follicles,

3416 oocytes in the testis, and clusters of apoptotic cells.

3417 (2) Spatial: these are changes that can be quantified by area measurements. This includes lesions

3418 that are typically classified as focal, multifocal, coalescing, or diffuse. Specific examples include

3419 granulomatous inflammation and tissue necrosis.

3420 (3) Global: these are generalized changes that would usually require more sophisticated measurement 

3421 techniques for quantification. Examples include increased hepatocyte basophilia,

3422 Sertoli cell/interstitial cell hypertrophy, or quantitative alterations in cell populations.

3423 General severity grading scale

3424 Not Remarkable: This grade is used if there are no findings associated with a particular diagnostic 

3425 criterion.

3426 Grade 1: Minimal. Ranging from inconspicuous to barely noticeable but so minor, small, or

3427 infrequent as to warrant no more than the least assignable grade. For discrete changes, grade

3428 1 is used when there are fewer than 2 occurrences per microscopic field, or 1 - 2 occurrences

3429 per section. For multifocal or diffusely-distributed alterations, this grade is used for

3430 processes where ≤20 % of the tissue in the section is involved.

3431 Grade 2: Mild. A noticeable feature of the tissue. For discrete changes, grade 2 is used when

3432 there are 3 - 5 occurrences per microscopic field or per tissue section. For multifocal or diffusely3433 distributed alterations, this grade is used for processes where 20 - 50 % of the tissue in

3434 the section are involved.

3435 Grade 3: Moderate. A dominant feature of the tissue. For discrete changes, grade 3 is used

3436 when there are 6 - 8 occurrences per microscopic field or per tissue section. For multifocal or

3437 diffusely-distributed alterations, this grade is used for processes where 50 - 80 % of the tissue

3438 in the section are involved.

3439 Grade 4: Severe. An overwhelming feature of the tissue. For discrete changes, grade 4 is

3440 used when there are more than 9 occurrences per microscopic field or per tissue section. For

3441 multifocal or diffusely-distributed alterations, this grade is used for processes where > 80 %

3442 of the tissue in the section are involved.

3443 At least some of the histomorphological changes that have been associated with EDCs in fish are 

3444 considered to be exacerbations of “normal”, physiologic findings (e.g., oocyte atresia

3445 [Nagahama, 1983; Tyler and Sumpter, 1996]). At the discretion of the pathologist, the severity 

3446 of a given change should be scored according to one of the following two methods:

3447 (1) score compound-exposed animals relative to the severity of the same change in control 

3448 animals, or

3449 (2) score all animals relative to “normal” as determined by the pathologist’s experience.

3450 For each important (i.e., treatment-associated) finding, the method that was used should be stated 

3451 in the Materials and Methods section of the pathology narrative report. By convention, severity 

3452 grading should not be influenced by the estimated physiologic importance of the change, since 

3453 this would add a further layer of subjectivity to the findings that complicates inter-laboratory 
94

3454 results comparisons. For example, the presence of two oocytes in the testis should not be graded 

3455 as “severe”, even if the pathologist considers this finding to be highly significant in terms of 

3456 endocrine modulation.

3457 Data Recording.

3458 An Excel worksheet form has been created that includes worksheets for primary, secondary, and 

3459 additional diagnoses to facilitate histopathology data collection. In this worksheet, each data 

3460 entry cell represents an individual fish. Additional sheets are available for comments and 

3461 additional findings. For each fish, the pathologist records a severity score associated with the 

3462 diagnosis. Diagnostic criteria with non-remarkable findings shall be denoted using (-). If there is 

3463 no reasonably appropriate diagnostic term for a particular finding, the pathologist can create a 

3464 term that can be recorded in the “Additional diagnoses” worksheet. If insufficient tissue is 

3465 available for diagnosis, this should be recorded as IT (insufficient tissue). If a target tissue is 

3466 missing, this should be recorded as MT (missing tissue).

3467

3468 Histopathology Report Format.

3469 Each histopathology narrative report should contain the following five sections:

3470 Introduction

3471 Materials and Methods

3472 Results

3473 Discussion

3474 Summary/Conclusions

3475 (References)

3476 The Introduction section briefly outlines the experimental design. The Materials and Methods 

3477 section describes any items or procedures that are essentially different from Section 1: Post3478 mortem and Histotechnical Procedures. As applicable, specific severity grading criteria (see 

3479 Severity Grading) should also be listed in this section. The Results section should report findings 

3480 that are: (1) compound-related; (2) potentially compound-related; (3) novel or unusual. Detailed 

3481 histomorphological descriptions need only be included for findings that differ substantially from 

3482 diagnoses presented in Section 4 (Glossary and diagnostic criteria). It is intended that the Results 

3483 section should be as objective as possible (i.e., opinions and theories should be reserved for the 

3484 Discussion section). The Discussion section, which contains subjective information, should 

3485 address relevant findings that were reported in the Results section. Opinions and theories can be 

3486 included in this section, preferably backed by references from peer-reviewed sources, but 

3487 unsupported speculation should be avoided. The Summary/Conclusions section should 

3488 encapsulate the most important information from the Results and Discussion sections.

3489 Developmental Staging of Xenopus gonads and Reproductive ducts.

3490

3491 Xenopus Testis Staging Criteria (Figure 1.)

3492 Stage 1: Undifferentiated gonad. 

3493 Stage 2: Medullary region populated with individual primary spermatogonia and basophillic 

3494 undifferentiated somatic cells.

3495 Stage 3: Somatic and germ cells organize into early seminiferous tubules populated with primary 

3496 spermatogonia and cysts of secondary spermatogonia.

3497 Stage 4: Germ cell cysts contain developmental stages up to primary spermatocytes with 

3498 formation of rete testis; may have occasional spermatocysts that contain round or elongated 

3499 spermatids.
95

3500 Stage 5: All stages of spermatogenesis evident. Organization and development of seminiferous 

3501 tubules and rete testis apparent.

3502

3503 Figure 1. Example of staging system applied to Xenopus laevis testis. Bar = 50 μm.

3504 Xenopus Ovary Staging Criteria (Figure 2.)

3505 Stage 1: Undifferentiated gonad. Gonad consists of an undifferentiated structure, germ cells, and 

3506 somatic cells.

3507 Stage 2: Gonad identifiable as an ovary based on the presence of a discontinuously open lumen 

3508 lined with epithelial cells. The germ cells within the cortex (the region outside the epithelial 

3509 layer) consist of primary oogonia, cysts of primary mitotic oogonia, secondary oogonia, and very 

3510 early meiotic oocytes.

3511 Stage 3: First appearance of diplotene oocytes in cortex. Structure and shape of the ovary 

3512 similar to stage 1, but the appearance of diplotene oocytes is the first morphological difference 

3513 between male and female germ cells. The most prevalent germ cell types at this stage are cysts 

3514 of secondary oogonia and cysts of leptotene-pachytene primary meiocytes.

3515 Stage 4: Pre-vitellogenic (Dumont Stage I) diplotene oocytes are the most prevalent germ cell 

3516 type observed by area and absolute cell counts. The central lumen is proportionately smaller 

3517 while the whole ovary grows greatly in size and volume due to the growth of the oocytes. Cysts 

3518 in earlier stages of oogenesis become fewer in number and are located along the periphery of 

3519 ovary.

3520 Stage 5: Ovary consists almost entirely of vitellogenic oocytes (Dumont Stage IV). Pre3521 vitellogenic diplotene oocytes can be found along the periphery of the ovary and germ patches of 

3522 primary and secondary oogonia are sparse and difficult to locate along the germinal epithelium.
96

3523

3524 Figure 2. Staging system applied to Xenopus laevis ovaries. Bar = 50 μm (Stage 2 and 3); Bar = 100 

3525 μm (Stage 4); Bar = 1 mm (Stage 5).

3526 Xenopus Wolffian Duct Staging Criteria (Figure 3.)

3527 Stage 1: The Wolffian ducts are paired ducts each located along the lateral edges of the kidneys, 

3528 usually adjacent to the suspensory connective tissue of the organ. At this stage, the epithelium of 

3529 the Wolffian duct consists of a single layer of cuboidal cells.

3530 Stage 2: Pseudostratified epithelial cells line the duct asymmetrically. The height of the 

3531 pseudostratified epithelium is less than 3 times the height of the other epithelial cells.

3532 Stage 3: The asymmetric epithelial lining of the duct is more pronounced with the 

3533 pseudostratified cells greater than 3 times the height of the other epithelial cells.

3534 Stage 4: The duct is lined symmetrically with tall columnar epithelial cells.
97

3535

3536 Figure 3. Wolffian duct staging. W = Wolffian duct; K = kidney tubule; Bar = 20 μm.

3537 Oviduct Staging (Figure 4.)

3538

3539 Stage 1: A discrete, solid, fibrous tag with proliferative fibroblasts in the location of normal 

3540 oviduct formation.

3541

3542 Stage 2: Formation of a rudimentary oviduct with a central lumen surrounded by a single layer 

3543 of cuboidal to columnar epithelium. The ductular lumen is frequently filled with cell debris. 

3544

3545 Stage 3: Oviduct is comprised of an undulating, pseudostratified, ciliated columnar epithelium, a 

3546 discrete lumen, and patchy areas of glandular formation. Ductular lumen may contain cell debris. 

3547

3548 Stage 4: Size of oviduct enlarged with prominent gland formation.

3549
98

3550 9Figure 4. Oviduct Staging. W = Wolffian duct; G = glandular region; Bar = 100 μm.

3551 Diagnostic Criteria for Specific Histopathological findings in EDC tests.

3552

Diagnostic Criteria for Specific Histopathological Findings

Gonad

Testis Stage

1 Undifferentiated gonad

2 Individual primary spermatogonia and undifferentiated somatic cells populate the medullary region

3 Seminiferous tubules with primary spermatogonia and cysts of secondary spermatogonia

4

Primary spermatocytes with rete testis formation; may have occasional spermatocysts that contain 

round or elongated spermatids

5 All stages of spermatogenesis evident

Tubule Lumen 

Development 

Score

1 Less than 50% of seminiferous tubules have visible lumens

2 50% or greater seminiferous tubules have visible lumens

Ovary Stage

1 Undifferentiated gonad

2

Gonad identifiable as an ovary based on the presence of a discontinuously open lumen lined with 

epithelial cells; germ cells within the cortex consist of primary oogonia, cysts of primary mitotic 

oogonia, secondary oogonia, and very early meiotic oocytes

3

First appearance of diplotene oocytes in cortex; the most prevalent germ cell types at this stage are 

cysts of secondary oogonia and cysts of leptotene-pachytene primary meiocytes

4

Pre-vitellogenic (Dumont Stage I) diplotene oocytes are the most prevalent germ cell type observed 

by area and absolute cell counts; the central lumen is proportionately smaller while the whole ovary 

grows greatly in size and volume due to the growth of the oocytes; cysts in earlier stages of oogenesis 

become fewer in number and are located along the periphery of ovary

5

Ovary consists almost entirely of vitellogenic oocytes (Dumont Stage IV); pre-vitellogenic diplotene 

oocytes can be found along the periphery of the ovary and germ patches of primary and secondary 

oogonia are difficult to locate

Gonad 

Degeneration

1 A single degenerating germ cell to less than three clusters of degenerating germ cells per gonad

2 Three or more clusters of degenerating germ cells, but less than 25% of gonad affected

3 Gonad consists of greater than 25% but less than 50% of degenerating germ cells

Stage 1 Stage 2

Stage 3 Stage 4
99

4 Gonad consists of 50% or more of degenerating germ cells

Mononuclear 

Cell Infiltrates

1 Small, focal infiltrates in one gonad

2 Small focal infiltrates in both gonads, or large area of infiltrates in one gonad

3 Large areas of infiltrates in both gonads

4 Gonads contain greater than 50% of infiltrates by area

Kidney

Mineralization

1 Less than three small foci of mineralization per gonad

2 Three or more small foci of mineralization per gonad, or less than three large foci

3 Three or more large foci of mineralization per gonad

4 Mineralized deposits in 50% or more tubules

Tubule Dilation

1 Minimal to mild dilation of less than three tubule clusters

2 Mild to moderate dilation of three or more tubule clusters

3 Moderate dilation affecting 50% or more tubules

4 Massive dilation of one or more tubules

Oviduct Oviduct Stage

1 Duct is either a fibrous connective tissue tag, or is essentially non-existent

2 Duct has an epithelial cell lining, and is comparable in size to the Wolffian duct

3

Duct has an epithelial cell lining, and is substantially larger (1.5x or greater) than the size of the 

Wolffian duct

4

Duct is substantially larger (1.5x or greater) than the size of the Wolffian duct Epithelial cells have 

formed large, basophilic glandular structures

Wolffian 

Duct

Wolffian Duct 

Stage

1 Epithelial lining of Wolffian duct is completely or focally less than two cell layers thick

2

Epithelial lining of Wolffian duct is two cell layers thick or greater, but clear apical portion of cell (if 

present) is less than half the cell height

3

Epithelial lining of Wlffian duct is two cell layers thick or greater, and clear apical portion of the 

columnar cells is at least half the cell height but less than 2/3 of the cell height

4 The clear apical portion of the columnar cells represents 2/3 or greater of the total cell height

3553

3554 References:

3555

3556 Crissman, J.W., Goodman, D.G., Hildebrandt, P.K., Maronpot, R.R., Prater, D.A., Riley, J.H., Seaman,

3557 W.J., Thake, D.C. (2004) Best practices guideline: Toxicologic pathology. Toxicol. Pathol. 32,

3558 126-131.

3559 Johnson, R., Wolf, J., and Braunbeck T. (2009). “OECD Guidance Document for the Diagnosis 

3560 of Endocrine-Related Histopathology of Fish Gonads” Organization for Economic and 

3561 Cooperative Development, Paris, France. 

3562 Nieuwkoop PD, J Faber. 1967 Normal table of Xenopus laevis (Daudin): A Systematical and 

3563 Chronoligical Survey of the Development from the Fertilized Egg Till the End of Metamorphosis. 

3564 2nd ed. Amsterdam: North Holland

3565 Reproduction of Amphibians. Biological Systems in Vertebrates. 2009. Edited by Maria 

3566 Ogielska. Enfield (New Hampshire): Science Publishers

3567 Dumont, James N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in 

3568 laboratory maintained animals. Journal of Morphology Volume 136, Issue 2, pp. 153-179. 

3569 February 1972

3570
100

3571 8.4 Inter-laboratory validation study results

3572 8.4.1 Prochloraz results

3573

3574 Measured test concentrations

3575

3576 All four labs met the criteria of maintaining CVs of ≤20% over the entire test for all 

3577 concentrations with the exception of the 6.67 and 20μg/L nominal concentrations at C which had 

3578 CVs of 0.38 and 0.23 and were on average 84% and 25% above nominal respectively (Table 

3579 8.4.1-1). Measured concentrations did not show any crossover with other treatment levels in all 

3580 labs except C which had several overlaps in treatment concentrations through week five of 

3581 exposure. CV values between replicates could not be calculated on a weekly basis for B and C 

3582 due to measurements of only one replicate tank per week. Treatment levels will be referred to as 

3583 nominal concentrations regardless of mean-measured concentrations reported by each of the labs.

3584

Table 8.4.1-1

Interlaboratory comparison of measured prochloraz concentrations throughout the duration of the LAGDA (mean ± SD). NR: not 

reported. Highlighted cells have CVs >20%.

Nominal PZ concentration A D B C

0 μg/L <MQL NR <MQL <MQL

6.7 μg/L 6.1 ± 0.7 6.6 ± 0.4 6.3 ± 1.1 12.3 ± 4.7

20 μg/L 18.1 ± 2.2 20.2 ± 2.3 15.4 ± 2.4 24.9 ± 5.8

60 μg/L 52.8 ± 5.9 59.3 ± 4.7 50.1 ± 6.9 64.7 ± 12.2

180 μg/L 169.8 ± 14.0 174.6 ± 10.5 156 ± 22.0 163.9 ± 28.6

MQL 2.5 μg/L NR 3.24 μg/L 3.23 μg/L

3585

3586 Survival

3587

3588 All labs met the criteria of ≤20% mortality in any one control replicate that could not be 

3589 explained by technical error and no treatment-related trends in survival were identified (data not 

3590 shown).

3591

3592 Larval growth (NF66)

3593

3594 There were no significant effects on larval growth at A or time to completion of metamorphosis 

3595 (Table 8.4.1-2).

3596

Table 8.4.1-2

Larval growth and development (NF66) following prochloraz exposure at A (mean ± SD).

Nominal PZ concentration Weight (g) Length (mm) Time to NF66 (d)

0 μg/L 0.99 ± 0.16 21.0 ± 1.2 46 ± 2

6.7 μg/L 1.08 ± 0.22 21.7 ± 1.8 46 ± 3

20 μg/L 0.98 ± 0.11 21.0 ± 1.0 45 ± 2

60 μg/L 1.01 ± 0.21 20.8 ± 1.2 47 ± 3

180 μg/L 1.01 ± 0.14 20.8 ± 1.1 47 ± 5

3597

3598 Larval growth (NF62)

3599

3600 Prochloraz exposure at D caused a significant decrease in both snout-vent length and body 

3601 weight in the 180 μg/L treatment and caused a significant decrease in body weight in the 20 and 

3602 60 μg/L treatments (Jonckheere-Terpstra). There were no treatment-related effects on time to 

3603 completion of metamorphosis at D. Prochloraz exposure at B caused a significant decrease in 
101

3604 body weight and snout-vent length in the 180μg/L treatment (ANOVA with Dunnett). There 

3605 were no concentration-related effects on larval growth in the C study (Table 8.4.1-3).

3606

Table 8.4.1-3

Interlaboratory comparison of larval growth and development (NF62) following prochloraz exposure (mean 

± SD). Highlighted cells are significantly different than control (α = 0.05).

Lab D B C

Nominal PZ concentration Weight (g)

0 μg/L 0.791 ± 0.130 1.08 ± 0.17 0.616 ± 0.139

6.7 μg/L 0.742 ± 0.244 1.21 ± 0.22 0.627 ± 0.104

20 μg/L 0.722 ± 0.204 1.08 ± 0.22 0.640 ± 0.124

60 μg/L 0.723 ± 0.153 1.13 ± 0.14 0.707 ± 0.154

180 μg/L 0.708 ± 0.168 0.92 ± 0.18 0.658 ± 0.174

Length (mm)

0 μg/L 18.8 ± 1.0 20.8 ± 1.0 17.1 ± 1.4

6.7 μg/L 18.5 ± 1.8 21.3 ± 1.2 17.5 ± 1.0

20 μg/L 18.4 ± 1.6 20.7 ± 1.2 16.9 ± 1.3

60 μg/L 18.3 ± 1.0 21.0 ± 0.9 18.1 ± 1.3

180 μg/L 18.1 ± 1.2 19.6 ± 1.2 16.8 ± 1.3

Time to NF62 (d)

0 μg/L 43 ± 2 41 ± 3 44 ± 6

6.7 μg/L 42 ± 2 39 ± 2 44 ± 4

20 μg/L 41 ± 2 40 ± 3 43 ± 5

60 μg/L 43 ± 2 41 ± 3 45 ± 5

180 μg/L 42 ± 2 40 ± 2 45 ± 10

3607

3608

3609 Larval blood thyroxine

3610

3611 A measured T4 levels in serum using an HPLC/ICP-MS method whereas the other labs used an 

3612 ELISA method to measure T4 in plasma. The ELISA method was not optimized so data could 

3613 not be included. There was a significant increase in T4 concentrations in the 60 and 180μg/L 

3614 treatment at A (ANOVA with Dunnett, Table 8.4.1-4).

3615

Table 8.4.1-4

Interlaboratory comparison of larval blood thyroxine 

concentrations following prochloraz exposure (mean ± 

SD). Highlighted cells are significantly different than 

control (α = 0.05).

T4 (ng/mL)

Nominal PZ concentration Lab A (NF66)

0 μg/L 5.4 ± 1.7

6.7 μg/L 6.5 ± 1.9

20 μg/L 7.7 ± 1.9

60 μg/L 8.2 ± 2.7

180 μg/L 11.8 ± 3.3

3616

3617 Juvenile growth

3618

3619 Juvenile female weight and length was significantly impeded in the 180μg/L treatment at D

3620 whereas only length in the 20, 60 and 180 μg/L treatments showed a significant decrease at B

3621 (Jonckeere-Terpstra). Juvenile male weight and length showed a significant decrease in the 60 

3622 and 180 μg/L treatments also at D. The other labs showed no significant concentration-related 

3623 effect on juvenile growth (Table 8.4.1-5).

3624

3625 Juvenile liver-somatic index (LSI)

3626
102

3627 A, D and B all showed significant decreases in both male and female LSI (Table 8.4.1-5). C 

3628 showed a significant female LSI increase in the 180μg/L treatment. 

3629

3630 Juvenile plasma vitellogenin

3631

3632 A, D and C showed a significant increase in both juvenile male and female plasma vitellogenin 

3633 titers (Table 8.4.1-5). B showed no significant concentration-related trend but also reported titer 

3634 values that were much lower on average than the other three labs. 

3635

3636
103

Table 8.4.1-5

Interlaboratory comparison of juvenile growth and plasma vitellogenin titers following prochloraz exposure (mean ± SD). Highlighted cells are significantly different than control (α = 0.05).

Male Female

A D B C A D B C

Nominal PZ concentration Weight (g) Weight (g)

0 μg/L 13.1 ± 2.0 18.6 ± 4.1 11.5 ± 3.6 4.0 ± 0.8 12.9 ± 2.6 20.0 ± 4.6 13.8 ± 5.0 4.1 ± 0.8

6.7 μg/L 12.5 ± 2.6 19.4 ± 3.8 13.2 ± 5.4 3.7 ± 0.5 13.8 ± 3.4 18.2 ± 3.0 12.3 ± 5.0 3.7 ± 0.6

20 μg/L 11.8 ± 2.8 17.6 ± 3.8 12.9 ± 5.0 4.0 ± 1.1 14.1 ± 2.3 20.3 ± 4.9 12.2 ± 4.8 4.2 ± 1.1

60 μg/L 12.6 ± 2.3 16.1 ± 3.9 12.5 ± 3.3 3.7 ± 0.8 13.8 ± 2.6 18.1 ± 4.4 12.0 ± 5.4 3.4 ± 0.7

180 μg/L 12.5 ± 2.7 10.8 ± 5.2 11.4 ± 4.3 4.0 ± 1.2 13.3 ± 2.8 14.3 ± 7.1 11.4 ± 4.5 4.7 ± 1.6

Length (mm) Length (mm)

0 μg/L 47.2 ± 2.8 53.0 ± 3.9 43.0 ± 4.5 32.8 ± 2.2 47.2 ± 2.6 55.0 ± 5.2 46.4 ± 5.4 32.9 ± 2.2

6.7 μg/L 46.4 ± 3.0 53.9 ± 4.1 44.8 ± 6.7 32.5 ± 1.3 47.9 ± 4.0 51.4 ± 8.6 43.8 ± 6.8 32.2 ± 1.9

20 μg/L 45.1 ± 4.2 52.0 ± 3.8 44.7 ± 5.4 31.9 ± 2.5 47.8 ± 2.0 55.2 ± 4.7 43.3 ± 5.4 33.5 ± 2.9

60 μg/L 46.9 ± 2.5 50.7 ± 4.0 44.1 ± 3.8 32.5 ± 2.4 47.7 ± 2.9 52.9 ± 4.5 43.0 ± 6.9 32.1 ± 1.8

180 μg/L 46.4 ± 3.8 43.6 ± 8.5 42.7 ± 6.2 32.2 ± 3.0 48.1 ± 3.7 48.3 ± 9.8 42.2 ± 6.8 33.9 ± 3.9

LSI LSI

0 μg/L 0.059 ± 0.010 0.058 ± 0.005 0.057 ± 0.009 0.035 ± 0.007 0.061 ± 0.007 0.059 ± 0.006 0.064 ± 0.014 0.032 ± 0.006

6.7 μg/L 0.056 ± 0.012 0.053 ± 0.012 0.064 ± 0.015 0.031 ± 0.005 0.056 ± 0.013 0.057 ± 0.006 0.059 ± 0.014 0.032 ± 0.007

20 μg/L 0.055 ± 0.012 0.054 ± 0.007 0.053 ± 0.014 0.034 ± 0.006 0.060 ± 0.007 0.055 ± 0.004 0.055 ± 0.010 0.032 ± 0.006

60 μg/L 0.052 ± 0.007 0.049 ± 0.005 0.052 ± 0.007 0.032 ± 0.007 0.051 ± 0.006 0.051 ± 0.006 0.056 ± 0.010 0.031 ± 0.005

180 μg/L 0.050 ± 0.004 0.040 ± 0.009 0.048 ± 0.009 0.037 ± 0.005 0.052 ± 0.005 0.044 ± 0.007 0.048 ± 0.011 0.037 ± 0.006

VTG (mg/mL) VTG (mg/mL)

0 μg/L 0.03 ± 0.02 0.01 ± 0.02 0.00 ± 0.00 0.00 ± 0.00 0.03 ± 0.03 0.02 ± 0.07 0.01 ± 0.02 0.03 ± 0.01

6.7 μg/L 0.05 ± 0.06 0.02 ± 0.04 0.01 ± 0.02 0.01 ± 0.002 0.07 ± 0.10 0.01 ± 0.01 0.03 ± 0.05 0.03 ± 0.02

20 μg/L 0.05 ± 0.04 0.01 ± 0.02 0.00 ± 0.00 0.01 ± 0.001 0.07 ± 0.10 0.01 ± 0.01 0.00 ± 0.00 0.04 ± 0.01

60 μg/L 0.12 ± 0.13 0.02 ± 0.03 0.00 ± 0.00 0.01 ± 0.004 0.12 ± 0.13 0.04 ± 0.07 0.00 ± 0.01 0.09 ± 0.05

180 μg/L 0.10 ± 0.10 0.02 ± 0.02 0.00 ± 0.01 0.01 ± 0.002 0.25 ± 0.32 0.04 ± 0.07 0.01 ± 0.01 0.10 ± 0.04
104

Larval thyroid histopathology

Common treatment-related observations in larval thyroid glands across all four studies were follicular cell hypertrophy and hyperplasia. Although 

these pathologies were present in larvae at B, there were no clear concentration-dependent trends in their study. However, the other three labs 

showed significant increases in follicular cell hypertrophy and two of those labs also showed significant increases in follicular cell hyperplasia. 

Gland hypertrophy and increased or decreased follicular lumen area were observed minimally in the 180 μg/L treatments inconsistently throughout 

the labs, but each were found to be significantly different than control in the 180 μg/L treatment in at least one of the labs (Table 8.4.1-6).

Table 8.4.1-6

Prevalance (%) and severity of larval thyroid gland histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable, highlighted cells are significantly 

different than control based on RSCABS analysis; dash: not observed in any treatment). 

Lab A (NF66) D (NF62) B (NF62) C (NF62)

Prochloraz concentration (μg/L) 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 40 23 25 25 24 56 28 28 28 28 40 20 20 20 20 39 17 16 18 18

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Thyroid Gland Hypertrophy

NR - - - - - 100 100 100 100 93 - - - - - 100 100 100 100 94

Mild - - - - - 0 0 0 0 7 - - - - - 0 0 0 0 6

Moderate - - - - - 0 0 0 0 0 - - - - - 0 0 0 0 0

Severe - - - - - 0 0 0 0 0 - - - - - 0 0 0 0 0

Follicular Cell Hypertrophy

NR 38 65 28 28 8 93 86 89 71 46 85 70 95 75 70 31 18 6 11 11

Mild 58 35 68 60 75 7 14 11 29 54 13 30 5 20 30 56 35 56 28 39

Moderate 5 0 4 12 17 0 0 0 0 0 3 0 0 5 0 10 47 38 61 44

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 6

Follicular Cell Hyperplasia

NR 78 87 80 76 67 95 86 79 68 46 95 80 95 90 90 85 59 50 39 61

Mild 23 13 20 24 33 5 14 21 32 54 5 20 5 10 10 13 41 44 56 33

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 6 6 6

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Incresed Follicular Lumen Area

NR - - - - - 100 100 100 100 93 - - - - - - - - - -

Mild - - - - - 0 0 0 0 7 - - - - - - - - - -

Moderate - - - - - 0 0 0 0 0 - - - - - - - - - -

Severe - - - - - 0 0 0 0 0 - - - - - - - - - -

Decreased Follicular Lumen Area

NR 100 100 100 100 92 - - - - - - - - - - 100 94 100 100 100

Mild 0 0 0 0 8 - - - - - - - - - - 0 6 0 0 0

Moderate 0 0 0 0 0 - - - - - - - - - - 0 0 0 0 0

Severe 0 0 0 0 0 - - - - - - - - - - 0 0 0 0 0
105

Liver histopathology

Consistencies in treatment-related findings were hepatocellular degeneration and hepatocellular atrophy in two of the four labs. A and B showed 

significant hepatocellular degeneration in the 180 μg/L treatment whereas hepatocellular atrophy showed slightly higher sensitivity to prochloraz 

treatment at D and B with both labs showing significant effects in the 20, 60 and 180 μg/L treatments. C exhibited a different suite of treatmentrelated liver pathologies including basophilia, increased hepatocellular vacuolization and granulomatous inflammation, all of which were 

significantly different than control at 60 and 180 μg/L. Cellular hypertrophy was also observed in C samples and was significantly different than 

control in the 20, 60 and 180 μg/L treatments (Table 8.4.1-7). Prevalence, severity, sensitivity and pathologies were extremely similar between 

males and females with slight variation in severity and sensitivity (Tables 8.4.1-7 and 8.4.1-8). The stark difference between findings at C compared 

to the other three labs could be due to the fact that tissues were processed and evaluated by a different pathologist, or could be due to the extreme 

lack of growth of Cs organisms compared to the other three labs. 

Table 8.4.1-7

Prevalence (%) and severity of juvenile male liver histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly

different than control based on RSCABS analysis; dash: not observed in any treatment).

Lab A D B C

Prochloraz concentration (μg/L) 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 16 10 10 10 10 23 12 12 12 12 24 12 12 11 11 37 14 18 15 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Basophilia

NR - - - - - - - - - - - - - - - 100 100 100 67 0

Minimal - - - - - - - - - - - - - - - 0 0 0 27 35

Mild - - - - - - - - - - - - - - - 0 0 0 7 35

Moderate - - - - - - - - - - - - - - - 0 0 0 0 10

Severe - - - - - - - - - - - - - - - 0 0 0 0 20

Increased Hepatocellular Vacuolization

NR - - - - - - - - - - - - - - - 100 100 100 67 0

Minimal - - - - - - - - - - - - - - - 0 0 0 27 35

Mild - - - - - - - - - - - - - - - 0 0 0 7 35

Moderate - - - - - - - - - - - - - - - 0 0 0 0 10

Severe - - - - - - - - - - - - - - - 0 0 0 0 20

Granulomatous Inflammation

NR - - - - - - - - - - - - - - - 49 36 28 0 5

Minimal - - - - - - - - - - - - - - - 49 50 72 87 55

Mild - - - - - - - - - - - - - - - 3 14 0 13 35

Moderate - - - - - - - - - - - - - - - 0 0 0 0 5

Severe - - - - - - - - - - - - - - - 0 0 0 0 0

Cellular Hypertrophy

NR - - - - - - - - - - - - - - - 100 100 50 13 10

Minimal - - - - - - - - - - - - - - - 0 0 50 67 40

Mild - - - - - - - - - - - - - - - 0 0 0 20 50

Moderate - - - - - - - - - - - - - - - 0 0 0 0 0

Severe - - - - - - - - - - - - - - - 0 0 0 0 0

Hepatocellular Degeneration

NR 100 100 90 70 0 - - - - - 100 100 100 91 9 - - - - -

Minimal 0 0 10 30 10 - - - - - 0 0 0 9 73 - - - - -

Mild 0 0 0 0 70 - - - - - 0 0 0 0 18 - - - - -

Moderate 0 0 0 0 20 - - - - - 0 0 0 0 0 - - - - -

Severe 0 0 0 0 0 - - - - - 0 0 0 0 0 - - - - -
106

Hepatocellular Atrophy

NR - - - - - 100 100 75 25 0 100 100 83 27 18 - - - - -

Minimal - - - - - 0 0 8 17 0 0 0 8 27 18 - - - - -

Mild - - - - - 0 0 17 42 50 0 0 8 36 36 - - - - -

Moderate - - - - - 0 0 0 17 42 0 0 0 9 27 - - - - -

Severe - - - - - 0 0 0 0 8 0 0 0 9 27 - - - - -

Table 8.4.1-8

Prevalence (%) and severity of juvenile female liver histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different 

than control based on RSCABS analysis; dash: not observed in any treatment).

Lab A D B C

Prochloraz concentration (μg/L) 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 16 10 10 10 10 25 12 12 12 12 24 12 12 13 13 43 26 22 25 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Basophilia

NR - - - - - - - - - - - - - - - 100 100 100 48 0

Minimal - - - - - - - - - - - - - - - 0 0 0 28 25

Mild - - - - - - - - - - - - - - - 0 0 0 24 45

Moderate - - - - - - - - - - - - - - - 0 0 0 0 30

Severe - - - - - - - - - - - - - - - 0 0 0 0 0

Increased Hepatocellular Vacuolization

NR - - - - - - - - - - - - - - - 100 100 100 48 0

Minimal - - - - - - - - - - - - - - - 0 0 0 28 25

Mild - - - - - - - - - - - - - - - 0 0 0 24 50

Moderate - - - - - - - - - - - - - - - 0 0 0 0 25

Severe - - - - - - - - - - - - - - - 0 0 0 0 0

Granulomatous Inflammation

NR - - - - - - - - - - 96 100 100 100 100 44 42 27 12 5

Minimal - - - - - - - - - - 0 0 0 0 0 49 58 64 84 70

Mild - - - - - - - - - - 4 0 0 0 0 7 0 9 4 25

Moderate - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Severe - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Cellular Hypertrophy

NR - - - - - - - - - - - - - - - 100 100 77 12 15

Minimal - - - - - - - - - - - - - - - 0 0 23 88 35

Mild - - - - - - - - - - - - - - - 0 0 0 0 50

Moderate - - - - - - - - - - - - - - - 0 0 0 0 0

Severe - - - - - - - - - - - - - - - 0 0 0 0 0

Hepatocellular Degeneration

NR 100 100 90 60 0 - - - - - 100 100 92 100 31 - - - - -

Minimal 0 0 10 40 60 - - - - - 0 0 8 0 62 - - - - -

Mild 0 0 0 0 10 - - - - - 0 0 0 0 8 - - - - -

Moderate 0 0 0 0 30 - - - - - 0 0 0 0 0 - - - - -

Severe 0 0 0 0 0 - - - - - 0 0 0 0 0 - - - - -

Hepatocellular Atrophy

NR - - - - - 100 100 83 42 0 100 100 92 77 15 - - - - -

Minimal - - - - - 0 0 8 33 8 0 0 0 23 23 - - - - -

Mild - - - - - 0 0 0 17 33 0 0 8 0 46 - - - - -

Moderate - - - - - 0 0 8 0 33 0 0 0 0 8 - - - - -

Severe - - - - - 0 0 0 8 25 0 0 0 0 8 - - - - -
107

Male gonad histopathology

Histopathological findings in the testes of prochloraz-exposed male frogs were widespread and varied in consistency across laboratories. Of the ten 

observed pathologies showing significant concentration-dependent trends in at least one of the four labs, increased spermatogonia, germinal epithelial 

thinning, and gonadal degeneration (necrosis/apoptosis) were represented as significant findings in all labs except B. Interstitial cell 

hyperplasia/hypertrophy, asynchronous development within cysts, and intratubular eosinophilic droplets were represented as significant and

consistent between A and D but varied in sensitivity and severity. Interstitial fibrosis was also substantially observed at A and D but only showed a 

significant treatment-related effect in A samples at 180 μg/L. Mononuclear cellular infiltrate was observed (minimal to mild) across all four labs, but 

only showed significantly increased prevalence in A and B, although the labs differed in observed sensitivity. C showed significant, albeit slight, 

advancement of tubule lumen development in the 180 μg/L treatment whereas this observation was not noted in the A and D reports (different 

histopathology contract). Finally, B had a significant minimal increase in spermatids in the 180 μg/L treatment and was the only lab exhibiting this 

pathology (Table 8.4.1-9). 

Table 8.4.1-9

Prevalence (%) and severity of juvenile male gonad histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different 

than control based on RSCABS analysis; dash: not observed in any treatment). Note: “Severe” rating only shown if observed in at least one of the four studies.

Lab A D B C

Prochloraz concentration (μg/L) 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 36 25 24 24 25 39 25 22 21 18 42 22 23 13 15 37 14 18 15 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Testicular interstitial cell hyperplasia/hypertrophy

NR 83 68 96 50 16 85 68 77 71 44 100 100 96 100 100 - - - - -

Minimal 17 32 4 46 52 15 32 23 29 56 0 0 0 0 0 - - - - -

Mild 0 0 0 4 32 0 0 0 0 0 0 0 4 0 0 - - - - -

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - - - - -

Increased spermatids

NR - - - - - - - - - - 100 100 96 100 87 - - - - -

Minimal - - - - - - - - - - 0 0 4 0 13 - - - - -

Mild - - - - - - - - - - 0 0 0 0 0 - - - - -

Moderate - - - - - - - - - - 0 0 0 0 0 - - - - -

Increased spermatogonia

NR 94 96 92 88 76 90 80 82 86 39 - - - - - 100 93 94 87 70

Minimal 6 4 4 8 12 10 20 18 10 50 - - - - - 0 7 6 13 30

Mild 0 0 0 0 8 0 0 0 5 11 - - - - - 0 0 0 0 0

Moderate 0 0 4 4 4 0 0 0 0 0 - - - - - 0 0 0 0 0

Tubule lumen development

NR - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Minimal - - - - - - - - - - 95 100 96 85 93 54 64 44 33 15

Mild - - - - - - - - - - 5 0 4 15 7 46 36 56 67 85

Moderate - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Germinal epithelial thinning

NR 92 84 92 54 12 85 56 41 29 17 95 100 100 100 100 100 93 100 67 35

Minimal 0 8 4 38 40 8 36 45 43 11 2 0 0 0 0 0 7 0 27 25

Mild 6 4 4 8 16 5 4 9 14 22 2 0 0 0 0 0 0 0 7 30

Moderate 3 4 0 0 28 3 4 0 14 44 0 0 0 0 0 0 0 0 0 10

Severe 0 0 0 0 4 0 0 5 0 6 0 0 0 0 0 0 0 0 0 0

Gonadal degeneration (necrosis / apoptosis)

NR 92 64 96 83 32 77 56 73 71 50 50 9 4 0 13 32 14 0 0 5

Minimal 8 32 4 17 64 23 44 27 29 50 38 73 65 77 60 65 86 94 93 75

Mild 0 4 0 0 4 0 0 0 0 0 12 18 30 23 27 3 0 6 7 20
108

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Mononuclear cellular infiltrate

NR 92 92 79 88 56 69 44 73 33 72 81 27 52 62 53 86 79 89 93 90

Minimal 8 8 21 13 40 31 52 18 62 22 14 50 35 38 40 8 21 11 7 10

Mild 0 0 0 0 4 0 4 9 5 6 5 23 13 0 7 5 0 0 0 0

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Asynchronous development within cysts

NR 100 80 75 67 40 97 92 91 90 56 - - - - - - - - - -

Minimal 0 16 17 25 44 3 8 9 10 39 - - - - - - - - - -

Mild 0 4 8 8 12 0 0 0 0 6 - - - - - - - - - -

Moderate 0 0 0 0 4 0 0 0 0 0 - - - - - - - - - -

Interstitial fibrosis

NR 75 52 71 75 12 69 56 45 62 61 95 100 100 100 100 - - - - -

Minimal 19 40 29 21 36 28 40 50 29 39 5 0 0 0 0 - - - - -

Mild 6 8 0 0 52 3 4 5 10 0 0 0 0 0 0 - - - - -

Moderate 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 - - - - -

Intratubular eosinophilic droplets

NR 94 96 100 96 76 79 60 82 67 28 - - - - - - - - - -

Minimal 3 4 0 4 24 15 20 14 10 50 - - - - - - - - - -

Mild 3 0 0 0 0 5 16 5 24 17 - - - - - - - - - -

Moderate 0 0 0 0 0 0 4 0 0 6 - - - - - - - - - -

Female gonad histopathology

Histopathological findings in the ovaries of prochloraz-exposed female frogs were somewhat inconsistent and mostly minimal. Mononuclear cellular 

infiltrate was observed across all four laboratories (minimal to mild) but only showed significant concentration-related trends at A and C, though C 

exhibited higher sensitivity with all four treatments being significantly different than control. Germ cell degeneration showed significant 

concentration-related trends at B and C down to 20 and 6.7 μg/L respectively and was not observed in either A or D studies. Granulomatous 

inflammation, ovarian hypoplasia, and interstitial fibrosis were primarily observed at A and D (minimal) and only the 180 μg/L treatments were 

significantly different than control in one of the two labs for each diagnosis. Gonadal degeneration (necrosis/apoptosis) was only observed in the D

study (minimal) with the 60 and 180 μg/L treatments being significantly different than control. Finally, ovary stages were evaluated in the B and C 

studies and there appeared to be significant acceleration of ovary development, albeit slight, in Cs 60 and 180 μg/L treatments compared to control 

(Table 8.4.1-10).

Table 8.4.1-10

Prevalence (%) and severity of juvenile female gonad histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different 

than control based on RSCABS analysis; dash: not observed in any treatment). Note: There were no diagnoses rated “severe” so data not shown.

Lab A ACEHR B C

Prochloraz concentration (μg/L) 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 43 26 22 24 25 40 15 16 19 22 37 18 16 26 25 42 26 22 25 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Gonad Stage

1 - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

2 - - - - - - - - - - 0 0 0 4 0 2 0 0 0 5

3 - - - - - - - - - - 0 6 0 4 4 52 23 64 24 15

4 - - - - - - - - - - 100 94 100 92 96 45 77 36 76 80

5 - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Germ cell degeneration

NR - - - - - - - - - - 95 89 63 73 84 38 4 18 4 25

Minimal - - - - - - - - - - 5 11 38 27 16 62 81 50 88 50
109

Mild - - - - - - - - - - 0 0 0 0 0 0 15 32 8 25

Moderate - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Gonadal degeneration (necrosis / apoptosis)

NR - - - - - 80 67 88 47 45 - - - - - - - - - -

Minimal - - - - - 18 33 13 53 55 - - - - - - - - - -

Mild - - - - - 0 0 0 0 0 - - - - - - - - - -

Moderate - - - - - 3 0 0 0 0 - - - - - - - - - -

Mononuclear cellular infiltrate

NR 72 77 77 50 44 50 20 50 11 55 41 22 19 27 40 43 15 18 12 5

Minimal 26 15 18 50 48 40 53 44 79 41 46 67 81 62 44 57 50 59 64 90

Mild 2 8 5 0 8 10 27 6 11 5 14 11 0 12 16 0 35 23 24 5

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Granulomatous inflammation

NR 100 100 91 100 92 95 93 100 100 91 - - - - - - - - - -

Minimal 0 0 9 0 4 5 7 0 0 9 - - - - - - - - - -

Mild 0 0 0 0 0 0 0 0 0 0 - - - - - - - - - -

Moderate 0 0 0 0 4 0 0 0 0 0 - - - - - - - - - -

Ovarian hypoplasia

NR 95 92 95 96 72 95 100 100 95 95 - - - - - 100 96 100 100 100

Minimal 5 8 5 4 28 5 0 0 5 5 - - - - - 0 4 0 0 0

Mild 0 0 0 0 0 0 0 0 0 0 - - - - - 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 0 0 0 - - - - - 0 0 0 0 0

Interstitial fibrosis

NR 93 96 86 92 80 70 47 69 42 32 - - - - - - - - - -

Minimal 7 0 14 8 12 28 47 31 53 59 - - - - - - - - - -

Mild 0 4 0 0 8 0 7 0 5 9 - - - - - - - - - -

Moderate 0 0 0 0 0 3 0 0 0 0 - - - - - - - - - -

Male reproductive duct histopathology

Male oviducts exhibited very consistent responses to prochloraz treatment across all four labs. Significant concentration-dependent delays in 

involution were evident in the 60 and 180 μg/L treatments for all labs and down to 20 μg/L for A, B and C (Table 8.4.1-11). Male Wolffian duct 

stages were similar between prochloraz treatments and controls for the B and C studies (data not shown).

Table 8.4.1-11

Histological staging (%) of juvenile male oviducts from four different labs performing the LAGDA with prochloraz (highlighted cells are significantly different than control based on RSCABS analysis). 

Lab A D B C

Prochloraz concentration (μg/L) 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 43 25 20 23 24 39 25 22 21 18 42 22 23 13 15 37 14 18 15 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Oviduct stage

1 84 88 50 31 4 31 24 18 10 11 78 68 52 15 7 89 79 50 20 10

2 16 8 42 58 92 69 76 82 90 89 22 32 48 85 93 11 21 50 80 90

3 0 4 8 12 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
110

Female reproductive duct histopathology

There were no treatment-related effects on female oviduct development across all four studies. However, female Wolffian duct development was 

slightly advanced in the 180 μg/L treatment at B and minimal mononuclear inflammation of the Wolffian duct was significantly present in the 180 

μg/L treatment at A (Table 8.4.1-12).

Table 8.4.1-12

Histological staging and pathology (%) of juvenile female reproductive ducts from four different labs performing the LAGDA with prochloraz (highlighted cells are significantly different than control based on RSCABS 

analysis).

Lab A D B C

Prochloraz concentration (μg/L) 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 43 26 22 24 25 40 15 16 19 22 37 18 16 27 25 43 26 21 25 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Oviduct stage

1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 5 0 0

2 44 0 9 4 12 100 100 100 100 100 100 100 100 100 100 98 100 62 96 100

3 56 100 91 96 88 0 0 0 0 0 0 0 0 0 0 0 0 33 4 0

Wolffian duct stage

1 - - - - - - - - - - 0 6 6 4 12 74 92 95 84 65

2 - - - - - - - - - - 97 67 81 81 64 26 8 5 16 35

3 - - - - - - - - - - 3 28 13 15 24 0 0 0 0 0

Wolffian duct mononuclear inflammation

NR 98 96 95 96 80 - - - - - - - - - - - - - - -

Minimal 2 4 5 4 20 - - - - - - - - - - - - - - -

Mild 0 0 0 0 0 - - - - - - - - - - - - - - -

Moderate 0 0 0 0 0 - - - - - - - - - - - - - - -

Severe 0 0 0 0 0 - - - - - - - - - - - - - - -
111

Male kidney histopathology

The most consistent treatment-related finding in the juvenile male kidney was tubular amphophilic intracytoplasmic inclusions, which were 

significantly present in the 180 μg/L treatment at A, D and C. Tubule mineralization/casts were observed throughout the B and C studies but showed 

a significant increase in prevalence; however severity and sensitivity varied between the two labs. Presumably, tubule dilation is a response to 

mineralization and follows accordingly with significant increases in prevalence at B and C. The other diagnoses, tubule protein, interstitial 

proteinaceous fluid and edema were primarily mild in severity and not likely related to prochloraz treatment (Table 8.4.1-13).

Table 8.4.1-13

Prevalence (%) and severity of juvenile male kidney histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different 

than control based on RSCABS analysis; dash: not observed in any treatment).

A D B C

Prochloraz concentration (μg/L) 0 7 20 60 180 0 7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 37 25 24 26 25 39 25 22 21 18 43 22 23 13 15 37 14 18 15 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Tubules, protein

NR 92 88 96 96 92 67 60 64 81 67 93 100 100 100 100 - - - - -

Minimal 8 12 4 4 8 33 40 32 19 33 7 0 0 0 0 - - - - -

Mild 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 - - - - -

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - - - - -

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - - - - -

Interstitial fluid (proteinaceous)

NR 100 100 96 100 92 97 100 95 100 100 100 95 96 100 100 92 100 100 100 90

Minimal 0 0 4 0 8 3 0 5 0 0 0 5 4 0 0 8 0 0 0 10

Mild 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Amphophilic intracytoplasmic bodies in tubule 

epithelium

NR 100 100 100 100 44 100 100 100 95 33 - - - - - 100 100 100 100 40

Minimal 0 0 0 0 56 0 0 0 5 67 - - - - - 0 0 0 0 15

Mild 0 0 0 0 0 0 0 0 0 0 - - - - - 0 0 0 0 25

Moderate 0 0 0 0 0 0 0 0 0 0 - - - - - 0 0 0 0 20

Severe 0 0 0 0 0 0 0 0 0 0 - - - - - 0 0 0 0 0

Interstitial fluid (non-proteinaceous edema)

NR - - - - - - - - - - 47 64 65 62 73 97 86 100 100 100

Minimal - - - - - - - - - - 40 32 35 38 13 3 7 0 0 0

Mild - - - - - - - - - - 14 5 0 0 13 0 7 0 0 0

Moderate - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Severe - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Tubules, Dilation

NR - - - - - - - - - - 12 9 9 0 13 95 93 67 80 45

Minimal - - - - - - - - - - 53 45 35 15 27 5 7 28 20 45

Mild - - - - - - - - - - 35 45 57 85 60 0 0 6 0 10

Moderate - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Severe - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Tubules, mineralization / casts
112

NR - - - - - - - - - - 0 0 0 0 7 30 7 11 27 10

Minimal - - - - - - - - - - 5 0 0 0 7 65 57 78 67 35

Mild - - - - - - - - - - 49 23 22 0 7 5 36 11 7 55

Moderate - - - - - - - - - - 47 77 78 100 80 0 0 0 0 0

Severe - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Female kidney histopathology

Like juvenile male kidneys, tubular amphophilic intracytoplasmic inclusions were the most consistent finding across three of the four labs - severity 

and sensitivity were almost identical between genders. Tubule mineralization and dilation were again observed throughout the B and C studies, but 

only C exhibited significant increases in prevalence for both pathologies. Unlike male kidneys, renal interstitial fibrosis (minimal to mild) and 

glomerular protein (mild) showed significant increases in prevalence in female kidneys at A and D respectively. Interstitial fluid, both proteinaceous 

and non-proteinaceous edema, was primarily mild, inconsistent across laboratories and not likely related to prochloraz treatment (Table 8.4.1-14). 

Table 8.4.1-14

Prevalence (%) and severity of juvenile female kidney histopathological observations showing significant treatment-related effects in at least one of the four studies (NR: not remarkable; highlighted cells are significantly different 

than control based on RSCABS analysis; dash: not observed in any treatment).

Lab A D B C

Prochloraz concentration (μg/L) 0 7 20 60 180 0 7 20 60 180 0 6.7 20 60 180 0 6.7 20 60 180

n 43 26 22 24 25 41 15 16 19 22 37 18 16 27 25 43 26 21 25 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4 8 4 4 4 4

Glomerular protein

NR 100 100 95 92 84 93 93 100 95 91 - - - - - - - - - -

Minimal 0 0 5 8 16 5 7 0 5 9 - - - - - - - - - -

Mild 0 0 0 0 0 2 0 0 0 0 - - - - - - - - - -

Moderate 0 0 0 0 0 0 0 0 0 0 - - - - - - - - - -

Severe 0 0 0 0 0 0 0 0 0 0 - - - - - - - - - -

Interstitial fluid (proteinaceous)

NR 100 100 95 92 96 - - - - - 100 100 94 100 92 88 96 100 100 95

Minimal 0 0 5 4 4 - - - - - 0 0 6 0 8 12 4 0 0 5

Mild 0 0 0 4 0 - - - - - 0 0 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 - - - - - 0 0 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 - - - - - 0 0 0 0 0 0 0 0 0 0

Renal interstitial fibrosis

NR 88 85 77 96 88 68 40 56 47 45 100 94 100 96 92 - - - - -

Minimal 9 12 18 4 8 24 60 25 26 32 0 6 0 0 8 - - - - -

Mild 0 4 5 0 4 5 0 19 26 23 0 0 0 4 0 - - - - -

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - - - - -

Severe 2 0 0 0 0 2 0 0 0 0 0 0 0 0 0 - - - - -

Amphophilic intracytoplasmic bodies in tubule 

epithelium

NR 100 100 100 100 36 100 100 100 100 50 97 100 100 96 100 100 100 100 92 45

Minimal 0 0 0 0 64 0 0 0 0 50 0 0 0 4 0 0 0 0 4 15

Mild 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 4 20

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Interstitial fluid (non-proteinaceous edema)

NR - - - - - - - - - - 32 61 75 59 56 93 100 100 100 95

Minimal - - - - - - - - - - 38 39 19 30 40 7 0 0 0 5

Mild - - - - - - - - - - 27 0 6 11 4 0 0 0 0 0

Moderate - - - - - - - - - - 3 0 0 0 0 0 0 0 0 0

Severe - - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Tubules, Dilation
113

NR----------0 17 13 308 84 81 81 72 40

Minimal---------- 27 11 31 19 20 16 19 19 28 35

Mild---------- 73 72 56 48 720000 25

Moderate----------0004000000

Severe----------0000000000

Tubules, mineralization / casts

NR----------06070 37 15 248 10

Minimal----------06074 53 77 48 56 40

Mild---------- 19 22 31 26 1298 29 36 50

Moderate---------- 81 67 69 59 8400000

Severe----------0000000000
DRAFT

114

8.4.2 4-tert-Octylphenol results

Measured test concentrations

All test concentrations at A were maintained with CVs ≤ 20%. B maintained test concentrations 

with CVs ≤ 20% except for their lowest concentration. C had CVs ≤ 20% in two of the four 

concentrations and had measurable amounts of test chemical in control water on three different 

occasions (Table 8.4.2-1). Weekly CV values could not be calculated for B and C due to only 

one replicate tank being measured per week. C’s concentrations were on average higher than 

nominal with the lowest concentration > 140% of nominal. B’s concentrations were on average 

lower than nominal with all mean concentrations being between 73 and 77% of nominal.

Table 8.4.2-1

Interlaboratory comparison of measured octylphenol concentrations throughout the duration of the LAGDA 

(mean ± SD). Highlighted cells indicate CVs >20%.

Nominal OP concentration A B C

0 μg/L <MQL <MQL <MQLa

6.25 μg/L 6.3 ± 0.5 4.8 ± 1.3 8.9 ± 1.9

12.5 μg/L 12.6 ± 1.1 9.9 ± 1.5 15.8 ± 1.7

25 μg/L 25.0 ± 1.9 18.8 ± 3.2 29.3 ± 7.2

50 μg/L 53.9 ± 4.5 36.3 ± 6.9 53.0 ± 5.8

MQL 2.5 μg/L 3.08 μg/L 3.0 μg/L

a 3.15, 5.39 and 4.55 μg/L were reported on days 7, 49 and 56 respectively. 

Survival

All labs met the criteria of ≤ 20% mortality in any one control replicate that could not be 

explained by technical error and no treatment-related trends in survival were identified. A

experienced technical difficulties late in the study and lost one 12.5 μg/L replicate and two 50 

μg/L replicates following the post-NF66 cull (data not shown).

Larval growth (NF62)

There were no treatment-related effects on larval growth in any of the three studies (Table 8.4.2-

2). However, overall larval growth differed substantially between the three studies and excess 

larval growth at B caused 38% of the larval sample to have notable instances of scoliosis.

Table 8.4.2-2

Interlaboratory comparison of larval growth and development (NF62) following octylphenol 

exposure (mean ± SD). Highlighted cells are significantly different than control (α = 0.05).

Lab A B C

Nominal OP concentration Weight (g)

0 μg/L 0.96 ± 0.15 2.07 ± 0.33 0.461 ± 0.14

6.25 μg/L 1.05 ± 0.14 2.04 ± 0.31 0.475 ± 0.15

12.5 μg/L 0.90 ± 0.15 2.17 ± 0.49 0.540 ± 0.13

25 μg/L 1.00 ± 0.16 2.11 ± 0.33 0.494 ± 0.13

50 μg/L 0.93 ± 0.19 2.19 ± 0.32 0.466 ± 0.09

Length (mm)

0 μg/L 19.2 ± 1.0 23.1 ± 1.3 15.2 ± 1.6

6.25 μg/L 19.9 ± 0.9 23.6 ± 1.1 15.3 ± 1.7

12.5 μg/L 19.0 ± 1.0 23.4 ± 1.6 16.0 ± 1.5

25 μg/L 19.8 ± 1.1 23.1 ± 1.4 15.5 ± 1.3

50 μg/L 18.9 ± 1.4 24.1 ± 1.4 15.5 ± 1.4
DRAFT

115

Time to NF62 (d)

0 μg/L 42 ± 5 42 ± 2 41 ± 10

6.25 μg/L 41 ± 3 43 ± 2 43 ± 13

12.5 μg/L 44 ± 5 44 ± 3 39 ± 10

25 μg/L 43 ± 4 43 ± 2 39 ± 8

50 μg/L 44 ± 5 43 ± 2 39 ± 9

Larval blood thyroxine

A used an HPLC/ICP-MS method for measuring serum T4 whereas the other two labs used an 

ELISA method to measure T4 in plasma. The ELISA method was not optimized so data could 

not be included. Serum T4 levels were significantly decreased in the 25 and 50 μg/L treatments 

at A (Jonckeere-Terpstra, Table 8.4.2-3).

Table 8.4.2-3

Interlaboratory comparison of larval blood thyroxine 

concentrations following octylphenol exposure 

(mean ± SD). Highlighted cells are significantly 

different than control (α = 0.05).

T4 (ng/mL)

Nominal OP concentration A

0 μg/L 39.4 ± 14.7

6.25 μg/L 47.4 ± 12.8

12.5 μg/L 28.3 ± 8.5

25 μg/L 22.3 ± 13.0

50 μg/L 14.4 ± 8.7

Juvenile growth

There were no treatment-related effects on growth in either gender across all three labs. 

However, it is worth noting that again, C organisms were smaller on average than the other labs 

(Table 8.4.2-4).

Juvenile liver-somatic index (LSI)

Juvenile LSI was significantly decreased in octylphenol-exposed males and females from C 

whereas no treatment-related effects on LSI were measured at A or B (Table 8.4.2-4).

Juvenile plasma vitellogenin

Vitellogenin titers were significantly increased in octylphenol-exposed males from A (12.5, 25, 

50 μg/L) and C (6.25, 12.5, 25, 50 μg/L). Female vitellogenin levels were significantly 

increased at B (50 μg/L) and C (6.25, 12.5, 25, 50 μg/L). In most cases, CV values are close to, 

or exceed 100% and control means differ across labs by up to 74-fold (Table 8.4.2.4). 
DRAFT

116

Table 8.4.2-4

Interlaboratory comparison of juvenile growth and plasma vitellogenin titers following octylphenol exposure (mean ± SD). Highlighted cells are significantly different 

than control (α = 0.05).

Male Female

A B C A B C

Nominal OP 

concentration Weight (g) Weight (g)

0 μg/L 10.9 ± 2.7 10.1 ± 2.6 8.6 ± 2.6 12.0 ± 2.7 10.9 ± 3.0 8.3 ± 1.7

6.25 μg/L 11.1 ± 2.0 10.0 ± 2.5 8.8 ± 2.8 12.4 ± 3.1 11.5 ± 3.1 9.1 ± 2.2

12.5 μg/L 11.5 ± 3.0 10.4 ± 2.4 8.0 ± 1.6 11.7 ± 2.9 10.3 ± 2.6 9.6 ± 2.7

25 μg/L 11.3 ± 3.0 10.8 ± 2.6 8.4 ± 2.0 12.1 ± 2.4 9.2 ± 4.0 7.5 ± 1.9

50 μg/L 10.9 ± 1.5 10.1 ± 3.2 7.8 ± 2.5 11.6 ± 3.0 11.6 ± 4.0 7.9 ± 2.0

Length (mm) Length (mm)

0 μg/L 43.1 ± 3.3 41.6 ± 3.5 41.1 ± 3.4 44.7 ± 4.0 42.5 ± 4.0 40.8 ± 3.1

6.25 μg/L 44.1 ± 2.8 40.7 ± 4.1 41.4 ± 3.9 46.1 ± 3.7 43.7 ± 4.3 41.9 ± 3.5

12.5 μg/L 43.1 ± 4.1 42.7 ± 3.5 41.8 ± 2.3 44.3 ± 4.0 43.5 ± 4.8 44.2 ± 4.0

25 μg/L 43.5 ± 3.6 43.8 ± 3.7 42.2 ± 3.2 44.5 ± 3.2 41.4 ± 5.9 40.8 ± 3.6

50 μg/L 43.3 ± 2.2 41.6 ± 4.5 41.2 ± 3.6 43.9 ± 4.3 44.5 ± 5.1 41.0 ± 3.4

LSI LSI

0 μg/L 0.058 ± 0.008 0.058 ± 0.007 0.072 ± 0.008 0.059 ± 0.012 0.059 ± 0.014 0.075 ± 0.007

6.25 μg/L 0.060 ± 0.010 0.056 ± 0.006 0.066 ± 0.009 0.059 ± 0.008 0.059 ± 0.005 0.073 ± 0.009

12.5 μg/L 0.058 ± 0.007 0.058 ± 0.008 0.070 ± 0.008 0.058 ± 0.004 0.062 ± 0.007 0.073 ± 0.007

25 μg/L 0.062 ± 0.010 0.062 ± 0.006 0.066 ± 0.009 0.061 ± 0.008 0.057 ± 0.012 0.069 ± 0.009

50 μg/L 0.066 ± 0.011 0.058 ± 0.007 0.063 ± 0.010 0.062 ± 0.013 0.057 ± 0.004 0.067 ± 0.006

VTG (mg/mL) VTG (mg/mL)

0 μg/L 0.000 ± 0.001 0.013 ± 0.014 0.005 ± 0.001 0.000 ± 0.001 0.008 ± 0.008 0.036 ± 0.011

6.25 μg/L 0.000 ± 0.001 0.009 ± 0.009 0.007 ± 0.003 0.000 ± 0.001 0.006 ± 0.007 0.042 ± 0.013

12.5 μg/L 0.001 ± 0.002 0.015 ± 0.034 0.018 ± 0.021 0.001 ± 0.001 0.010 ± 0.014 0.055 ± 0.009

25 μg/L 0.002 ± 0.004 0.007 ± 0.006 0.021 ± 0.016 0.001 ± 0.000 0.015 ± 0.017 0.097 ± 0.026

50 μg/L 0.001 ± 0.002 0.014 ± 0.015 0.039 ± 0.019 0.001 ± 0.001 0.019 ± 0.028 0.143 ± 0.023

Larval thyroid histopathology

Follicular cell hypertrophy and hyperplasia were observed throughout all three studies but only 

showed significant treatment-related increases in severity at A. Mild decreases in colloid size 

were substantially observed only at A and showed significant attenuation in the 25 and 50 μg/L 

treatments (Table 8.4.2-5). 

Table 8.4.2-5

Prevalance (%) and severity of larval thyroid gland histopathological observations showing significant treatment-related effects in at least one of the three studies (NR: 

not remarkable, highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment). 

A B C

Nominal OP concentration (μg/L) 0 6.25 12.5 25 50 0 6.25 12.5 25 50 0 6.25 12.5 25 50

n 39 24 25 25 24 40 20 20 20 20 40 20 20 20 20

replicates 8 5 5 5 5 8 4 4 4 4 8 4 4 4 4

Follicular Cell Hypertrophy

NR 3 0 0 0 4 3 5 0 5 10 73 70 75 65 75

Mild 46 21 36 16 4 90 90 90 85 80 28 30 25 35 25

Moderate 51 75 56 68 71 8 5 10 10 10 0 0 0 0 0

Severe 0 4 8 16 21 0 0 0 0 0 0 0 0 0 0

Follicular Cell Hyperplasia

NR 74 58 56 76 58 83 70 75 90 85 85 80 80 65 80

Mild 26 42 44 24 33 18 30 25 10 15 15 20 20 35 20

Moderate 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Decreased Colloid

NR 36 46 28 72 71 98 100 100 100 100 - - - - -

Mild 62 50 72 28 29 3 0 0 0 0 - - - - -

Moderate 3 4 0 0 0 0 0 0 0 0 - - - - -

Severe 0 0 0 0 0 0 0 0 0 0 - - - - -
DRAFT

117

Liver histopathology

No treatment-related effects were present in any of the octylphenol-exposed livers from all three 

studies. Granulomatous inflammation was observed in all three studies, but prevalence and 

severity was comparable among treated and control samples. Cystic degeneration was observed 

in one of the three studies (B), but prevalence and severity was also comparable among treated 

and control samples (data not shown).

Male gonad histopathology

Effects of octylphenol exposure on male gonads were minimal. Mononuclear cellular infiltrate 

was observed throughout all three studies but was not treatment-related, although statistically, 

severity was significantly less than controls at B. There were very few observations of 

minimally increased spermatogonia at B and C, but the 180 μg/L treatment at B was significantly 

different than control. Statistically, there appears to be slight advancement of testicular 

development in the 180 μg/L treatment at B and shows consistency across all three labs, 

although not statistically significant at A or C (Table 8.4.2-6). 

Table 8.4.2-6

Prevalence (%) and severity of juvenile male gonad histopathological observations showing significant treatment-related effects in at least one of the three studies (NR: not 

remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment).

A B C

Nominal OP concentration (μg/L) 0 6.25 12.5 25 50 0 6.25 12.5 25 50 0 6.25 12.5 25 50

n 32 20 16 16 9 45 19 23 22 25 40 14 20 15 19

replicates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Gonad Stage

1 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0

2 0 0 0 0 0 4 0 0 0 12 0 0 0 0 0

3 0 0 0 0 0 4 5 4 0 0 0 0 0 0 0

4 34 30 31 44 11 69 68 70 55 52 13 0 30 7 5

5 66 70 69 56 89 20 26 26 41 36 88 100 70 93 95

Increased Spermatogonia

NR - - - - - 100 100 100 100 92 100 100 100 93 100

Minimal - - - - - 0 0 0 0 8 0 0 0 7 0

Mild - - - - - 0 0 0 0 0 0 0 0 0 0

Moderate - - - - - 0 0 0 0 0 0 0 0 0 0

Severe - - - - - 0 0 0 0 0 0 0 0 0 0

Mononuclear Cellular Infiltrate

NR 66 85 56 69 78 62 79 57 64 76 90 93 95 87 84

Minimal 31 15 38 31 22 29 21 43 32 24 10 7 5 13 16

Mild 3 0 6 0 0 9 0 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Female gonad histopathology

Unlike male gonads, female gonads experienced treatment-related increases in severity of 

mononuclear cellular infiltrate and was significant (minimal to mild) in the 12.5, 25 and 50 μg/L 

treatments at A and C. Granulomatous inflammation was minimal at B and C in the low 

treatments and control, but was absent in the 25 and 50 μg/L treatments (Table 8.4.2-7).

Table 8.4.2-7
DRAFT

118

Prevalence (%) and severity of juvenile female gonad histopathological observations showing significant treatment-related effects in at least one of the three studies (NR: 

not remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment).

A B C

Nominal OP concentration (μg/L) 0 6.25 12.5 25 50 0 6.25 12.5 25 50 0 6.25 12.5 25 50

n 30 20 16 21 12 33 20 17 18 15 40 24 20 25 20

replicates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Gonad Stage

1 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0

2 0 0 0 0 0 3 5 6 0 13 0 0 0 0 5

3 0 0 0 0 8 0 0 29 0 0 18 29 25 28 15

4 100 100 100 100 92 97 95 65 94 87 83 71 75 72 80

5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Mononuclear Cellular Infiltrate

NR 63 40 25 38 42 53 30 41 39 33 55 42 50 24 30

Minimal 33 60 56 57 50 44 55 47 61 53 38 42 25 44 50

Mild 3 0 19 5 8 3 15 12 0 13 8 17 25 32 20

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Granulomatous inflammation

NR - - - - - 97 95 94 100 100 93 96 95 100 100

Minimal - - - - - 3 5 6 0 0 5 4 5 0 0

Mild - - - - - 0 0 0 0 0 3 0 0 0 0

Moderate - - - - - 0 0 0 0 0 0 0 0 0 0

Severe - - - - - 0 0 0 0 0 0 0 0 0 0

Male reproductive duct histopathology

There were significant delays in male oviduct regression at A (6.25, 12.5, 25, 50 μg/L) and B (25, 

50 μg/L) and evidence of advanced male oviduct maturation in the 50 μg/L treatment at B. 

Although there was a significant delay in Wolffian duct development in the 50 μg/L treatment at 

B, stage distributions were somewhat consistent across labs and similar between treatments and 

controls (Table 8.4.2-8).

Table 8.4.2-8

Histological staging (%) of juvenile male reproductive ducts from three different labs performing the LAGDA with octylphenol (highlighted cells are significantly different than 

control based on RSCABS analysis). 

A B C

Nominal OPconcentration (μg/L) 0 6.25 12.5 25 50 0 6.25 12.5 25 50 0 6.25 12.5 25 50

n 32 20 16 16 9 45 19 23 22 25 40 14 20 15 19

replicates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Oviduct Stage

1 88 60 44 31 67 40 26 30 9 12 98 94 100 93 100

2 12 40 56 69 33 58 74 65 86 28 3 6 0 7 0

3 0 0 0 0 0 2 0 4 5 52 0 0 0 0 0

4 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0

Wolffian Stage

1 3 10 0 25 0 16 26 4 5 28 10 19 25 20 5

2 88 60 50 56 67 60 68 70 86 72 48 44 50 67 37

3 9 25 50 19 33 24 5 26 9 0 43 31 25 13 58

4 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0

Female reproductive duct histopathology

Female oviduct development was significantly accelerated with octylphenol exposure across all 

three labs. However, sensitivity differed between labs with A only showing a significant effect 

in the 50 μg/L treatment whereas significant effects were exhibited down to 12.5 and 6.25 μg/L 

at B and C respectively (Table 8.4.2-9).

 
DRAFT

119

Table 8.4.2-9

Histological staging (%) of juvenile female reproductive ducts from three different labs performing the LAGDA with octylphenol (highlighted cells are significantly different 

than control based on RSCABS analysis).

A B C

Nominal OP concentration (μg/L) 0 6.25 12.5 25 50 0 6.25 12.5 25 50 0 6.25 12.5 25 50

n 30 20 16 21 12 33 20 17 18 15 40 24 20 25 20

replicates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Oviduct Stage

1 0 0 0 0 0 0 0 0 0 7 0 0 0 0 5

2 87 80 75 67 25 85 70 65 56 0 98 79 50 64 57

3 13 20 25 33 75 15 30 35 44 93 3 21 50 36 38

Wolffian Stage

1 13 25 25 29 42 27 20 24 22 33 43 46 50 60 48

2 77 60 63 48 50 58 60 59 72 40 40 38 40 28 33

3 10 15 13 24 8 15 20 18 6 27 18 17 10 12 19

Male kidney histopathology

Male kidney tubule dilation and mineralization were observed throughout all three studies. 

Prevalence and severity was comparable between treatments and control in most cases. However, 

these pathologies were significantly attenuated with octylphenol exposure at B. Minimal 

interstitial proteinaceous fluid was observed at B but is essentially not remarkable (Table 8.4.2-

10). 

Table 8.4.2-10

Prevalence (%) and severity of juvenile male kidney histopathological observations showing significant treatment-related effects in at least one of the three studies (NR: not 

remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment).

A B C

Nominal OPconcentration (μg/L) 0 6.25 12.5 25 50 0 6.25 12.5 25 50 0 6.25 12.5 25 50

n 33 20 16 16 9 45 19 23 22 25 40 16 20 15 19

replicates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Interstitial Fluid (Proteinaceous)

NR - - - - - 100 100 100 100 96 - - - - -

Minimal - - - - - 0 0 0 0 4 - - - - -

Mild - - - - - 0 0 0 0 0 - - - - -

Moderate - - - - - 0 0 0 0 0 - - - - -

Severe - - - - - 0 0 0 0 0 - - - - -

Tubules, Dilation

NR 18 20 6 13 11 4 11 9 9 16 10 19 15 27 16

Minimal 58 55 63 56 22 36 32 52 45 48 33 44 40 20 53

Mild 24 25 31 25 67 60 58 39 45 36 58 38 45 53 32

Moderate 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Tubules, Mineralization / Casts

NR 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0

Minimal 21 20 19 19 0 0 5 0 0 0 0 6 0 0 0

Mild 73 70 69 63 100 42 47 61 64 60 33 88 50 80 58

Moderate 6 10 13 19 0 58 47 39 36 40 68 6 45 20 42

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Female kidney histopathology

Female kidney tubule dilation and mineralization were observed throughout all three studies. C 

exhibited a similar pattern to B male kidneys where these pathologies are attenuated with 

octylphenol treatment. A showed a significant, but slight increase in severity of tubule dilation 

in the 50 μg/L treatment which is inconsistent with the findings in the other studies. B females 

did not show significant trends in these pathologies, but showed significant minimal to mild 

severity of edema, although prevalence was very low making the finding essentially not 

remarkable (Table 8.4.2-11).
DRAFT

120

Table 8.4.2-11

Prevalence (%) and severity of juvenile female kidney histopathological observations showing significant treatment-related effects in at least one of the three studies (NR: 

not remarkable; highlighted cells are significantly different than control based on RSCABS analysis; dash: not observed in any treatment).

A B C

Nominal OP concentration (μg/L) 0 6.25 12.5 25 50 0 6.25 12.5 25 50 0 6.25 12.5 25 50

n 30 20 16 21 12 33 20 17 18 15 40 24 20 25 21

replicates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Interstitial Fluid 

(Non-Proteinaceous Edema)

NR - - - - - 91 100 100 94 93 - - - - -

Minimal - - - - - 9 0 0 6 0 - - - - -

Mild - - - - - 0 0 0 0 7 - - - - -

Moderate - - - - - 0 0 0 0 0 - - - - -

Severe - - - - - 0 0 0 0 0 - - - - -

Tubules, Dilation

NR 23 5 6 10 0 15 0 29 28 13 0 8 0 12 14

Minimal 43 55 69 48 42 33 35 41 17 20 15 42 35 56 62

Mild 33 40 25 43 58 52 65 29 56 67 85 50 65 32 24

Moderate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Tubules, Mineralization / Casts

NR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Minimal 23 15 31 10 8 3 0 0 6 0 0 4 0 4 0

Mild 57 75 50 67 75 45 35 47 33 60 25 63 45 60 38

Moderate 20 10 19 24 17 52 65 53 61 40 75 33 55 36 62

Severe 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
DRAFT

121

8.4.3 17-β Trenbolone results

Measured test concentrations

Mean ± SD (n = 40) measured concentrations of the 12.5, 25, 50 and 100 ng/L 17β-trenbolone 

treatments were 9.0 ± 2.8, 19.2 ± 5.8, 41.4 ± 9.7 and 79.9 ± 24.1 ng/L, respectively, over the 

duration of the experiment. Among-tank variability was ≤ 32% with the coefficient of variation 

(averaged across the duration of the exposure) of 31%, 30%, 23% and 30%, respectively for the 

12.5, 25, 50 and 100 ng/L treatments. No 17β-trenbolone was detected in the control water 

treatments.

Survival

All control tanks met the guideline performance criteria of ≤20% mortality and there were no 

treatment-related effects on survival (data not shown).

Larval growth

There were no significant treatment-related effects on larval growth and development (Table 

8.4.3-1).

Larval blood thyroxine

Serum samples were analyzed for T4 levels using the HPLC/ICP-MS method described 

previously. There were no treatment-related effects on larval serum T4 levels (Table 8.4.3-1).

Table 8.4.3-1

Larval growth and serum thyroxine concentrations following trenbolone exposure at A (mean ± SD).

NF62 larvae

Nominal TB concentration Weight (g) Length (mm) Time to NF62 (d) T4 (ng/mL)

0 μg/L 0.818 ± 0.140 18.7 ± 1.1 40 ± 4 23.4 ± 9.0

12.5 ng/L 0.835 ± 0.146 18.7 ± 1.0 41 ± 4 25.0 ± 10.5

25 ng/L 0.853 ± 0.154 18.8 ± 1.2 40 ± 5 25.1 ± 9.7

50 ng/L 0.913 ± 0.193 19.2 ± 1.3 41 ± 6 30.6 ± 9.1

100 ng/L 0.867 ± 0.166 18.8 ± 1.2 39 ± 4 24.2 ± 9.8

Juvenile growth

There were no significant treatment-related effects from 17β-trenbolone exposure on juvenile 

growth for either gender (Table 8.4.3-2).

Juvenile liver-somatic index (LSI)

There were no significant treatment-related effects on LSI for either gender (Table 8.4.3-2).

Juvenile plasma vitellogenin

Trenbolone treatment did not affect vitellogenin titers in juvenile males or females (data not 

shown).
DRAFT

122

Table 8.4.3-2

Juvenile growth and plasma vitellogenin titers following trenbolone exposure at A (mean ± SD). 

Highlighted cells are significantly different than control (α = 0.05).

Weight (g) Length (mm) LSI

Nominal TB concentration Female

0 μg/L 12.9 ± 3.7 47.5 ± 5.1 0.059 ± 0.006

12.5 ng/L 13.3 ± 2.6 47.3 ± 3.3 0.058 ± 0.010

25 ng/L 12.5 ± 3.2 46.4 ± 4.4 0.061 ± 0.006

50 ng/L 12.5 ± 3.1 46.7 ± 4.8 0.058 ± 0.008

100 ng/L 13.7 ± 3.6 49.0 ± 4.8 0.058 ± 0.009

Male

0 μg/L 12.3 ± 3.3 46.2 ± 4.1 0.06 ± 0.010

12.5 ng/L 11.4 ± 2.4 45.4 ± 3.2 0.058 ± 0.009

25 ng/L 11.9 ± 2.7 46.3 ± 4.0 0.057 ± 0.006

50 ng/L 13.0 ± 2.5 47.4 ± 4.0 0.057 ± 0.008

100 ng/L 11.7 ± 2.7 46.4 ± 3.6 0.058 ± 0.007

Thyroid histopathology

The two categories of morphologic findings in thyroid gland tissue, both of which were common, 

were follicular cell hypertrophy (mild to moderate) and follicular cell hyperplasia (mild to 

moderate). The severity of follicular cell hypertrophy was slightly decreased in the 50 and 100 

ng/L trenbolone treatment groups as compared to controls, but those differences are not 

statistically significant. The prevalence and severity of follicular cell hyperplasia in trenbolonetreated frogs were mild but significantly different than controls in the 25, 50 and 100 ng/L 

treatments (Table 8.4.3-3).

Table 8.4.3-3

Prevalance (%) and severity of larval thyroid gland histopathological 

observations in trenbolone-exposed larvae (NR: not remarkable, highlighted cells 

are significantly different than control based on RSCABS analysis).

Trenbolone concentration (ng/L) 0 12.5 25 50 100

n 40 25 24 25 25

replicates 8 5 5 5 5

Follicular Cell Hypertrophy

NR 3 12 13 16 4

Minimal 68 64 58 68 80

Mild 30 24 29 16 16

Moderate 0 0 0 0 0

Follicular Cell Hyperplasia

NR 43 52 25 44 48

Minimal 45 44 75 56 52

Mild 13 4 0 0 0

Moderate 0 0 0 0 0

Liver histopathology

Treatment-related effects were present in the liver. The prevalence of minimal cellular 

hypertrophy of hepatocytes was significantly different than controls in the 50 and 100 ng/L 

treatments. The prevalence and severity of the only other finding in the liver, granulomatous 

inflammation, was comparable among treated and control frogs (Table 8.4.3-4).
DRAFT

123

Table 8.4.3-4

Prevalence (%) and severity of juvenile liver histopathological observations following trenbolone exposure (NR: not 

remarkable; highlighted cells are significantly different than control based on RSCABS analysis).

Male Female

Trenbolone concentration (ng/L) 0 12.5 25 50 100 0 13 25 50 100

n 38 21 20 25 25 37 25 29 24 25

replicates 8 5 5 5 5 8 5 5 5 5

Granulomatous Inflammation

NR 53 67 55 52 52 46 40 69 67 48

Minimal 47 33 45 48 48 54 60 31 33 52

Mild 0 0 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 0 0 0

Cellular Hypertrophy

NR 100 100 100 88 80 100 100 100 100 76

Minimal 0 0 0 12 20 0 0 0 0 24

Mild 0 0 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 0 0 0

Male gonad histopathology

The only two significant treatment-related findings in the testes of males were increased 

spermatogonia (minimal) and germ cell vacuolation (minimal to mild) in frogs of the 100 ng/L

treatment (Table 8.4.3-5). Germ cell vacuolation was characterized by clear cytoplasmic 

ballooning in small clusters of pachytene phase spermatocytes. Other common findings in the 

testes included germ cell degeneration (minimal) and mononuclear cell infiltrates (minimal to 

mild). The prevalence and severity of these findings were essentially comparable between 

trenbolone-treated and control frogs. Tubule lumen development was also similar among the 

various treatment groups (data not shown).

Table 8.4.3-5

Prevalence (%) and severity of juvenile male gonad histopathological 

observations showing significant treatment-related effects following trenbolone 

exposure (NR: not remarkable; highlighted cells are significantly different than 

control based on RSCABS analysis;).

Trenbolone concentration (ng/L) 0 12.5 25 50 100

n 39 21 20 25 25

replicates 8 5 5 5 5

Increased spermatogonia

NR 97 100 100 100 76

Minimal 3 0 0 0 24

Mild 0 0 0 0 0

Moderate 0 0 0 0 0

Severe 0 0 0 0 0

Germ cell vacuolation

NR 100 95 95 96 76

Minimal 0 5 5 4 20

Mild 0 0 0 0 4

Moderate 0 0 0 0 0

Severe 0 0 0 0 0

Female gonad histopathology

There were no apparent effects of trenbolone exposure on the ovaries of juvenile females (data 

not shown). 

Male kidney histopathology
DRAFT

124

Two common findings in male kidneys were renal tubule mineralization/casts (minimal to 

moderate) and renal tubule dilation (minimal to mild). However, tubule dilation was 

significantly attenuated in the 50 and 100 ng/L treatments whereas mineralization/casts were not 

treatment-related. There was a low occurrence of interstitial fibrosis (minimal to mild) in the 

100 ng/L treatment, but was statistically different than control (Table 8.4.3-6).

Table 8.4.3-6

Prevalence (%) and severity of juvenile male kidney histopathological 

observations showing significant treatment-related effects following trenbolone 

exposure (NR: not remarkable; highlighted cells are significantly different than 

control based on RSCABS analysis).

Trenbolone concentration (ng/L) 0 12.5 25 50 100

n 39 21 20 25 25

replicates 8 5 5 5 5

Interstitial fibrosis 

NR 100 100 100 96 92

Minimal 0 0 0 4 4

Mild 0 0 0 0 4

Moderate 0 0 0 0 0

Severe 0 0 0 0 0

Tubules, Dilation

NR 18 24 10 0 8

Minimal 18 52 50 24 56

Mild 64 24 40 76 36

Moderate 0 0 0 0 0

Severe 0 0 0 0 0

Female kidney histopathology

There were no apparent effects of trenbolone exposure on the kidneys of juvenile females (data 

not shown).

Reproductive duct histopathology

There were treatment-related effects on oviduct development in both genders. Males 

experienced accelerated regression of oviducts in the 100 ng/L treatment as compared to controls 

whereas females experienced prodigious oviduct regression in the 50 and 100 ng/L treatments

(Table 8.4.3-7). There were no treatment-related effects on Wolffian duct development or 

pathology in either gender. 

Table 8.4.3-7

Histological staging of juvenile oviducts following trenbolone exposure (highlighted cells are significantly different than 

control based on RSCABS analysis). 

Male Female

Trenbolone concentration (ng/L) 0 12.5 25 50 100 0 12.5 25 50 100

n 39 21 20 25 25 38 25 29 24 25

replicates 8 5 5 5 5 8 5 5 5 5

Oviduct Stage

1 54 29 40 64 100 0 0 0 8 52

2 46 71 60 36 0 100 100 100 92 48

3 0 0 0 0 0 0 0 0 0 0
DRAFT

125

8.4.4 Benzophenone-2 results

Measured test concentrations

Mean ± SD (n = 36) measured concentrations of the 1.5, 3.0 and 6.0 mg/L benzophenone-2 

treatments were 1.6 ± 0.1, 3.5 ± 0.2 and 6.1 ± 0.1 mg/L, respectively over the duration of the 

experiment. CVs (averaged across the duration of the exposure) were 4%, 6% and 1%, 

respectively for the 1.5, 3.0 and 6.0 mg/L treatments. No benzophenone-2 was detected in the 

control water treatments.

Survival

The test was initiated with 30 embryos per tank. Following hatch on day 4, all tanks were 

balanced based on the lowest number left in any particular tank, which was 29 larvae. 

Subsequently, there was significant treatment-related mortality during the larval phase of the 

assay (Jonckeere-Terpstra, Table 8.4.4-1).

Table 8.4.4-1

Mean percent mortality prior to completion of NF62 sample. Highlighted cell is 

significantly different than control.

Nominal BP2 concentration (mg/L) 0 1.5 3 6

n/tank 29 29 29 29

replicates 4 4 4 4

Mean % mortality 0 ± 0 1 ± 2 3 ± 7 38 ± 6

Following the NF66 cull, 10 individuals were left in each tank to continue exposure until test 

termination. Within this timeframe, significant mortality was experienced in the high test 

concentration (Jonckeere-Terpstra, Table 8.4.4-2). 

Table 8.4.4-2

Mean percent juvenile mortality post-cull. Highlighted cell is significantly 

different than control.

Nominal BP2 concentration (mg/L) 0 1.5 3 6

n/tank 10 10 10 10

replicates 4 4 4 4

Mean % mortality 0 ± 0 0 ± 0 5 ± 6 25 ± 13

Larval growth and development

There was a significant increase in growth in the 6 mg/L treatment, but this could be a result of 

the significant mortality experienced by this treatment level allowing more food access for 

surviving larvae (Jonckeere-Terpstra, Table 8.4.4-3). There was also a significant delay in time 

to NF stage 62 in the 6 mg/L treatment as compared to control (Jonckeere-Terpstra, Table 8.4.4-

3). 

Larval blood thyroxine
DRAFT

126

In the case of the BP-2 study, heparinized plasma samples were analyzed for T4 levels as 

opposed to serum using both the optimized ELISA described in LAGDA guideline Appendix 7

and the HPLC/ICP-MS methods. There were no statistically significant effects on circulating T4

in either sample set and T4 levels from the two methods did not agree. It is unknown what 

caused the lack of agreement between the two methods in this case. Therefore, the data is not 

presented. The effects of using plasma as opposed to serum for T4 analyses are currently under 

investigation.

Table 8.4.4-3

Larval growth and serum thyroxine concentrations following BP2 exposure at E (mean 

± SD). Highlighted cells are significantly different than control (α = 0.05).

NF62 larvae

Nominal TB 

concentration Weight (g) Length (mm)

Time to NF62 

(d)

0 mg/L 1.18 ± 0.18 22.2 ± 1.1 38 ± 5

1.5 mg/L 1.05 ± 0.23 21.5 ± 1.3 37 ± 3

3 mg/L 1.18 ± 0.18 22.2 ± 1.3 38 ± 3

6 mg/L 1.92 ± 0.59 25.2 ± 2.1 49 ± 7

Juvenile growth

Growth was significantly reduced in the 6 mg/L treatment compared to controls for both males 

and females (Jonckeere-Terpstra, Table 8.4.4-4).

Juvenile liver-somatic index (LSI)

LSI appeared to be affected by BP-2 exposure; however statistical analyses were inconsistent 

between genders due to non-monotonicity of the data in both cases. There was a significant 

decrease in male LSI in the 6 mg/L treatment and a significant increase in female LSI in the 1.5 

mg/L treatment (modified Dunn’s, Table 8.4.4-4).

Juvenile plasma vitellogenin

There was significant induction of vitellogenin in all treatments compared to control and the 

response was very consistent between genders (Jonckeere-Terpstra, Table 8.4.4-4). The 

magnitude of induction is much higher than any of the other inter-laboratory studies and 

emphasizes the responsiveness of this endpoint to estrogenic compounds.

Table 8.4.4-4

Juvenile growth and plasma vitellogenin titers following BP2 exposure at E (mean ± SD). Highlighted 

cells are significantly different than control (α = 0.05).

Weight (g) Length (mm) LSI VTG (mg/mL)

Nominal BP2 

concentration Female

0 mg/L 10.8 ± 2.1 44.7 ± 3.2 0.045 ± 0.006 0.04 ± 0.11

1.5 mg/L 11.8 ± 3.1 47.4 ± 4.4 0.052 ± 0.006 60.2 ± 27.2

3 mg/L 9.5 ± 4.8 43.3 ± 7.3 0.057 ± 0.013 245.8 ± 61.1

6 mg/L 4.1 ± 1.2 33.4 ± 2.7 0.036 ± 0.009 250.9 ± 75.5

Male

0 mg/L 11.3 ± 2.7 45.2 ± 3.3 0.046 ± 0.005 0.02 ± 0.03

1.5 mg/L 11.1 ± 2.6 46.7 ± 4.1 0.052 ± 0.006 59.9 ± 23.5

3 mg/L 9.8 ± 5.5 43.0 ± 7.5 0.055 ± 0.018 232.9 ± 56.9

6 mg/L 4.4 ± 1.4 33.9 ± 3.5 0.035 ± 0.007 259.4 ± 35.6
DRAFT

127

Thyroid histopathology

There were significant treatment-related effects on thyroid tissue including increased prevalence 

and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy. The 6 mg/L 

treatment exhibited notable pathologies in 100% of the subjects (Table 8.4.4-5).

Table 8.4.4-5

Prevalance (%) and severity of larval thyroid gland histopathological 

observations in BP2-exposed larvae (NR: not remarkable, highlighted 

cells are significantly different than control based on RSCABS analysis).

NF62

Nominal BP2 concentration (mg/L) 0 1.5 3 6

n 20 20 20 18

replicates 4 4 4 4

Thyroid Gland, Hypertrophy

NR 100 95 25 0

Mild 0 5 75 0

Moderate 0 0 0 67

Severe 0 0 0 33

Follicular Cells, Hypertrophy

NR 50 30 5 0

Mild 50 70 50 0

Moderate 0 0 45 33

Severe 0 0 0 67

Follicular Cells, Hyperplasia

NR 80 50 15 0

Mild 20 50 65 0

Moderate 0 0 20 11

Severe 0 0 0 89

Liver histopathology

Treatment-related findings in the liver were limited to phenotypic females and genetic males

which showed mixed sex gonads (i.e. the 3.0 and 6.0 mg/L treatments). These included 

concentration-dependent increases in the prevalence and severity of basophilia, decreased 

hepatocellular vacuolation, and intravascular proteinaceous fluid. As compared to controls, BP2-treated frogs of the 3.0 and 6.0 mg/L treatment groups additionally had higher prevalences of 

increased pigment, which also tended to be concentration-dependent. Furthermore, the few 

findings of individual hepatocyte necrosis, and foci of hepatocellular alteration, were restricted to 

frogs of the 3.0 and 6.0 mg/L concentration groups, and 6.0 mg/L treatment group, respectively

(Table 8.4.4-6). Overall, these liver changes are likely related to estrogenic activity of BP-2 and 

significant up-regulation of vitellogenin production.

Table 8.4.4-6

Prevalence (%) and severity of juvenile liver histopathological observations following BP2 exposure (NR: not remarkable; highlighted cells are 

significantly different than control based on RSCABS analysis).

Male Female

Nominal BP2 concentration (mg/L) 0 1.5 3 6 0 1.5 3 6

n 20 20 18 14 20 20 20 16

replicates 4 4 4 4 4 4 4 4

Basophilia

NR 100 100 0 0 100 90 0 0

Minimal 0 0 28 0 0 10 25 0

Mild 0 0 33 0 0 0 45 25

Moderate 0 0 28 86 0 0 30 50
DRAFT

128

Severe 0 0 11 14 0 0 0 25

Decreased Hepatocellular Vacuolation

NR 100 100 0 0 100 90 0 0

Minimal 0 0 28 0 0 10 25 0

Mild 0 0 33 0 0 0 45 25

Moderate 0 0 28 86 0 0 30 50

Severe 0 0 11 14 0 0 0 25

Proteinaceous Fluid Intravascular

NR 100 100 6 0 100 100 5 6

Minimal 0 0 28 0 0 0 30 0

Mild 0 0 22 0 0 0 30 19

Moderate 0 0 28 79 0 0 10 69

Severe 0 0 17 21 0 0 25 6

Individual Hepatocyte Necrosis / Apoptosis

NR 100 100 94 93 100 100 100 88

Minimal 0 0 6 7 0 0 0 13

Mild 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0

Increased Pigment

NR 100 100 50 0 100 100 45 0

Minimal 0 0 50 100 0 0 55 100

Mild 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0

Focus of Hepatocellular Alteration

NR 100 100 100 79 100 100 100 88

Minimal 0 0 0 21 0 0 0 6

Mild 0 0 0 0 0 0 0 0

Moderate 0 0 0 0 0 0 0 6

Severe 0 0 0 0 0 0 0 0

Gonad histopathology

Genotypic males experienced feminization of the gonad resulting in a portion of individuals 

presenting with mixed sex gonads and others with complete sex reversal in the 1.5 mg/L 

treatment. 100% of genotypic males in the 3 and 6 mg/L treatments were functionally sex 

reversed presenting only ovary tissue. In genotypic females, there was a significant treatmentrelated delay in gonad development. Similarly, there was a treatment-related delay in ovary 

development in sex-reversed genotypic males. Thinning of the germinal epithelium is specific to 

testis tissue and significant increases in prevalence and severity were observed in the 1.5 mg/L 

treatment. Mononuclear cellular infiltrate was observed in genotypic males and females across 

all treatments and control, but prevalence and severity was significantly attenuated in the 3 and 6 

mg/L treatments for genotypic females. Prevalence and severity of proteinaceous fluid infiltrate 

significantly increased in both genders in the 3 and 6 mg/L treatments (Table 8.4.4-7). 

Table 8.4.4-7

Prevalence (%) and severity of juvenile male gonad histopathological observations showing significant treatment-related effects following 

BP2 exposure (NR: not remarkable; highlighted cells are significantly different than control based on RSCABS analysis ;).

Male Female

Nominal BP2 concentration (mg/L) 0 1.5 3 6 0 1.5 3 6

n 20 19 12 14 17 17 16 9

replicates 4 4 4 4 4 4 4 4

Gonad phenotype

Testis 1 100 37 0 0 0 0 0 0

2 0 32 0 0 0 0 0 0

3 0 0 0 0 0 0 0 0

4 0 0 0 0 0 0 0 0

Ovary 5 0 32 100 100 100 100 100 100

Gonad Stage

1 0 0 0 0 0 6 6 0
DRAFT

129

2 5 5 0 0 0 6 0 0

3 0 16 42 100 0 12 44 89

4 45 58 58 0 100 76 50 11

5 50 21 0 0 0 0 0 0

Testicular Oocytes

NR 100 53 0 0 - - - -

Minimal 0 11 0 0 - - - -

Mild 0 5 0 0 - - - -

Moderate 0 0 0 0 - - - -

Severe 0 0 0 0 - - - -

Germinal Epithelium,Thinning

NR 100 47 0 0 - - - -

Minimal 0 11 0 0 - - - -

Mild 0 5 0 0 - - - -

Moderate 0 5 0 0 - - - -

Severe 0 0 0 0 - - - -

Mononuclear cellular infiltrate

NR 90 79 83 86 47 35 88 100

Minimal 10 21 17 14 41 35 13 0

Mild 0 0 0 0 12 29 0 0

Moderate 0 0 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0

Proteinaceous Fluid Infiltrate

NR 100 100 8 0 100 94 0 0

Minimal 0 0 67 21 0 6 63 33

Mild 0 0 25 79 0 0 31 44

Moderate 0 0 0 0 0 0 6 22

Severe 0 0 0 0 0 0 0 0

Kidney histopathology

BP-2 exposure caused profound effects in both genotypic male and female kidneys. These 

effects were very similar between genders and widespread in the 3 and 6 mg/L treatments. 

However, these two treatments had 100% female phenotypes. Interestingly, many of the 

pathologies have higher severity scores in the 3 mg/L treatment and are slightly less severe in the 

6 mg/L treatment (Table 8.4.4-8).

Table 8.4.4-8

Prevalence (%) and severity of juvenile male kidney histopathological observations showing significant treatment-related effects following 

BP2 exposure (NR: not remarkable; dash: not observed; highlighted cells are significantly different than control based on RSCABS 

analysis).

Male Female

Nominal BP2 concentration (mg/L) 0 1.5 3 6 0 1.5 3 6

n 20 19 12 14 17 17 16 9

replicates 4 4 4 4 4 4 4 4

Glomerulomegaly

NR 100 100 0 0 100 88 6 22

Minimal 0 0 42 21 0 12 0 0

Mild 0 0 58 79 0 0 6 56

Moderate 0 0 0 0 0 0 38 22

Severe 0 0 0 0 0 0 50 0

Glomerular Protein

NR 100 100 17 0 100 82 0 0

Minimal 0 0 50 7 0 18 0 22

Mild 0 0 33 93 0 0 56 22

Moderate 0 0 0 0 0 0 31 56

Severe 0 0 0 0 0 0 13 0

Glomerulus, hypercellularity

NR 100 100 8 100 100 94 0 56

Minimal 0 0 67 0 0 6 13 33

Mild 0 0 25 0 0 0 25 11

Moderate 0 0 0 0 0 0 56 0

Severe 0 0 0 0 0 0 6 0

Tubules, Protein
DRAFT

130

NR 100 100 0 43 100 100 6 11

Minimal 0 0 8 0 0 0 6 22

Mild 0 0 75 36 0 0 75 44

Moderate 0 0 17 21 0 0 13 0

Severe 0 0 0 0 0 0 0 22

Interstitial Fluid (Proteinaceous)

NR 100 100 0 0 100 71 0 0

Minimal 0 0 8 14 0 29 13 0

Mild 0 0 92 86 0 0 56 44

Moderate 0 0 0 0 0 0 19 56

Severe 0 0 0 0 0 0 13 0

Interstitial Fibrosis 

NR 100 100 8 100 100 100 25 89

Minimal 0 0 33 0 0 0 19 11

Mild 0 0 50 0 0 0 50 0

Moderate 0 0 8 0 0 0 6 0

Severe 0 0 0 0 0 0 0 0

Regenerative blast cell hyperplasia

NR - - - - 100 100 25 44

Minimal - - - - 0 0 31 56

Mild - - - - 0 0 31 0

Moderate - - - - 0 0 13 0

Severe - - - - 0 0 0 0

Tubules, Dilation

NR 25 11 0 7 18 12 0 56

Minimal 55 79 0 36 71 47 13 22

Mild 20 11 92 50 12 41 50 0

Moderate 0 0 8 7 0 0 38 22

Severe 0 0 0 0 0 0 0 0

Tubules, Mineralization / Casts

NR 5 0 58 64 6 24 13 100

Minimal 30 42 8 29 35 24 25 0

Mild 65 53 33 7 59 53 63 0

Moderate 0 5 0 0 0 0 0 0

Severe 0 0 0 0 0 0 0 0

Reproductive duct histopathology

Oviduct development was rather variable within and between genders. In genotypic females, 

there was a significant delay in oviduct development in all treatments. However, in both 

genders, there are some individuals in the 1.5 and 3 mg/L treatments that are two stages more 

advanced than the others. Both genders experienced advancement of Wolffian duct development 

in the 3 and 6 mg/L treatments. Again, these were all phenotypic females (Table 8.4.4-9).

Table 8.4.4-9

Histological staging of juvenile reproductive ducts following BP2 exposure (NR: not remarkable; dash: not observed; highlighted cells are 

significantly different than control based on RSCABS analysis). 

Male Female

Nominal BP2 concentration (mg/L) 0 1.5 3 6 0 1.5 3 6

n 20 19 12 14 17 17 16 9

replicates 4 4 4 4 4 4 4 4

Oviduct Stage

Not present 5 0 0 0 0 0 0 0

1 95 89 92 93 6 35 81 89

2 0 0 0 7 88 0 0 0

3 0 0 0 0 6 0 0 0

4 0 11 8 0 0 65 19 0

Wolffian Stage

1 30 37 33 36 18 41 6 33

2 70 58 17 43 76 59 38 33

3 0 5 50 21 6 0 50 11

4 0 0 0 0 0 0 6 11