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# Validation of the 17



# Larval Amphibian Growth and Development Assay : 18



# Integrated Summary Report 19

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U.S. Environmental Protection Agency 25

Endocrine Disruptor Screening Program 26

Washington, D.C. 27

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28 May 201 3 29

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T ABLE OF CONTENTS 46

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EXEC UTIVE SUMMAR Y.................................................................................................... 3 48

1 OBJECTIVE

................................ ................................ ................................ ........................... 5 49

2 INTRODUCTION

................................ ................................ ................................ .................. 6 50

2.1 Endocrine Disruptor Screening Program

................................ ................................ ......... 6 51

2.2 Validation

................................ ................................ ................................ ......................... 8 52

3 OVERVIEW OF THE LAGDA

................................ ................................ ........................... 10 53

3.1 Summary of the LAGDA test protocol

................................ ................................ .......... 10 54

3.2 Purpose of the test and relevance of the LAGDA

................................ .......................... 11 55

3.3 Scientific basis for the test method

................................ ................................ ................ 11 56

4 PROTOCOL DEVELOPMENT

................................ ................................ ........................... 12 57

4.1 Rationale for test species and exposure period

................................ .............................. 12 58

4.1.1 Species selection

................................ ................................ ................................ ..... 13 59

4.1.2 Test duration

................................ ................................ ................................ ........... 13 60

4.2 Test det ails

................................ ................................ ................................ ...................... 14 61

4.3 Core Endpoints

................................ ................................ ................................ ............... 17 62

4.4 Statistical Analyses

................................ ................................ ................................ ........ 18 63

5 INTER

- LABORATORY EVALUATION OF THE LAGDA

................................ ............. 20 64

5.1 Overview of approach

................................ ................................ ................................ .... 20 65

5.2 Inter

- laboratory data analysis

................................ ................................ ......................... 20 66

5.3 Summary of results

................................ ................................ ................................ ......... 21 67

5.3.1 Prochloraz

................................ ................................ ................................ ............... 21 68

5.3.2 4

- tert

- oct ylphenol

................................ ................................ ................................ ... 27 69

5.3.3 17β

- trenbolone

................................ ................................ ................................ ........ 32 70

5.3.4 Benzophenone

- 2

................................ ................................ ................................ ..... 35 71

6 DISCUSSION OF THE INTER

- LABORATORY STUDIES

................................ ............. 40 72

6.1 Comparison of control performance

................................ ................................ .............. 40 73

6.2 Effectiveness of the LAGDA

................................ ................................ ......................... 42 74

6.3 Lessons learned from the inter

- laboratory studies

................................ ......................... 46 75

6.4 Conclusions

................................ ................................ ................................ .................... 47 76

7 REFERENCES

................................ ................................ ................................ ..................... 47 77

8 APPENDICES

................................ ................................ ................................ ...................... 54 78

8.1 LAGDA Guideline

................................ ................................ ................................ ......... 54 79

8.2 Histolog ical Development of the Gonad in Juvenile Xenopus laevis

............................ 87 80

8.3 Histopathology Guidance for Reading LAGDA Studies

................................ ............... 91 81

8.4 Inter

- laboratory validation study results

................................ ................................ ...... 100 82

8.4.1 Prochloraz results

................................ ................................ ................................ .. 100 83

8.4.2 4

- tert

- Octylphenol results

................................ ................................ ..................... 114 84

8.4.3 17

- β Trenbolone results

................................ ................................ ........................ 121 85

8.4.4 Benzophenone

- 2 results

................................ ................................ ........................ 125 86

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# EXECUTIVE SUMMARY 94

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

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

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

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

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

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

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

is a relatively long

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

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

non

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

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

generalized toxicity

, mo rtality, abnormal behavior, and growth determination s (length and 108

weight) , as well as endpoints designed to characterize endocrine

- specific modes of action 109

targeting estrogen, androgen or thyroid

- mediated physiological processes. 110

111

Individual and i nter

- laboratory evaluations of the LADGA were conducted to evaluate the 112

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

inter

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

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

prochloraz (aromatase inhibitor , AR agonist ) , 4

- tert

- octylphenol (ER agonist), 17

- β t renbolone 116

(AR agonist)

, and benzophenone

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

lab s (A, B, C, and D), and 4

- tert

- octylphenol was tested in three lab s (A, B, and C)

. Trenbolone 118

and b enzophenone

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

the inter

- laboratory comparisons. H owev er, these studies serve to demonstrate the 120

responsiveness of the LAGDA to additional modes of action. 121

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The results of the inter

- laboratory validation were mixed. For larval growth and development

, 123

two laboratories

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

sta n dard deviation from the overall mean weight for at least one chemical. Further investigation 125

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

LAGDA guidance, resulting in either excessive or inadequate growth of the developing larva e

. 127

Similarly, for j uvenile growth and development , t wo studies had control juvenile weights more 128

than one standard deviation form the o verall mean in both juvenile ma les and females but in 129

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

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

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

laboratory C use d their own diet formulation but fed at a rate similar to th at recommended in the 133

LAGDA guideline. This diet formulation was cl ea rly inadequate for supporting growth rates 134

consistent with t he other studies and led to confounding effects on liver

- somatic index ( LSI ) in 135

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

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

regimens

. 138

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The LAGDA was an effective test model. All four chemicals produced endocrine

- related effects. 140

Of the two chemicals available for inter

- laboratory comparison, p rochloraz resulted in thyroid 141

gland pathologies consistent with a hypothyroid condition in 3 of the 4 labs

, and vitellogenin 142

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

second chemical

, 4

- tert

- octylphenol

, produced thyroid gland pathologies consistent with a 144

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

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

17

- β Trenbolone and benzopehone

- 2, although only tested in single laboratories, produce d 147

endocrine

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

and reproductive tract pathologies. 149

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

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

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

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

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

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

demonstrate this as a cause for the observed inconsistencies

. H owever

, it does point to 157

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

observed with liver pathology for the prochloraz studies. The A

, B and D studies were 159

read by the same contracted pathologist whereas the C stud y w as read by a different 160

contracted pathologist. It is interesting to point out that similar treatment

- related liver 161

pathologies were reported for A

, B and D whereas a different spectrum of treatment

162

related pathologies were reported for C. It is worth noting that the animals in the C stud y 163

showed feeding

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

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

findings. Regardless

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

kidney is criti cal

. 167

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

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

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

i nductively

- coupled plasma ma ss spectrometry (HPLC/ICP

- MS) are available, US EPA 172

Mid

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

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

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

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

labs by up to 74

- fold. Although there were concentration

- dependent increases in VT G 178

production in response to prochloraz, 4

- tert

- octylphenol , and benzophenone

- 2 , VTG data 179

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

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

of response. 182

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

potential disruption of the endocrine system by putative e ndocrine d isrupting c hemicals. 185

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

consistent and reliable findings across multiple testing laboratories. 187

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# 1 O BJECTIVE 224

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

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

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

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# 2 INTRODUCTION 230

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

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

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

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

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

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

239

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

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

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

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

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

. 244

Upon recommendations from the EDSTAC (EDSTAC

, 1998), the EPA expanded the EDSP 245

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

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

and the creation of similar programs in other countries, the Organi z ation for Economic Co

248

operation and Dev elopment (OECD) established the Endocrine Disrupters Testing and 249

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

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

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

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

new and existing substances. 254

255

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

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

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

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

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

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

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

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

protocol transferability. 264

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# 2.1 Endocrine Disruptor Screening Program 266

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

, the Endocrine 268

Disruptor Screening and Testing Advisory Committee (EDSTAC)

, to provide advice and 269

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

the EPA a conceptual two

- tiered approach that involved screening and testing chemical 271

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

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

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

, 274

1998). 275

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The US EPA submitted a proposal of the EDSP for public review and comment (FRN

, 1998) as 277

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

Scientific Advisory Panel (SAB

- SAP

, 1999). A complete description of the program proposal 279

can be found in the Federal Register Notice (FRN

, 1998). Briefly, the EDSP proposed by 280

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

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

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

consequences resulting from possible endocrine disruption and establishment of dose

- response 284

relationships for hazard identification using Tier 2 testing. 285

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

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

• be relatively fast and efficient; 289

• be standardized and validated; 290

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

rate of false positives; 292

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

possible; 294

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

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

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

298

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

redundancy is incorporated ( e.g

. , two different assays may cover some similar aspects of the 300

estrogenic response). This redundancy will allow for a weight

- of

- evidence approach, as 301

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

definitive, testing. 303

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

adopted as the EDSP Tier 1 screening battery ( Table 2.1

- 1 ). These assays are meant to detect 306

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

modes of action. 308

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Table 2.1

- 1

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

# 317

Assay Reason for Inclusion

Estrogen receptor binding or transcriptional

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

system by binding to the estrogen receptor.

Estrogen receptor transcriptional activation

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

system by binding to the estrogen rec eptor.

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

system by binding to the androgen receptor.

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

the sex steroid hormones

Placental Aromatase Assay An assay to detect interference with aromatase.

Uterotrophic Assay An in vivo assay to detect estrogenic chemicals.

Hershberger Assay An 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 f emale

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

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

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

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

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

323

 Two

- generation Rat Reproduction Test 324

 Medaka Multi

- generation Test 325

 Japanese Quail Two

- generation Test 326

 Mysid Two

- generation Toxicity Test 327

 Larval Amphibian Growth and Development Assay 328

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The Two

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

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

LAGDA is reviewed here. 332

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# 2.2 Validation 334

335

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

method are evaluated for a particular use” ( NIEHS

, 1997; OECD

, 1996 )

. 337

338

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

laboratories. 340

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

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

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

344

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

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

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

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

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

and the OECD (OECD 2005 )

. These criteria as stated by ICCVAM ( NIEHS 1997 ) are as 350

follows: 351

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

of its proposed use, should be available. 353

354

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

effect and toxicity of interest must be addressed. 356

357

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

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

include data analysis and decision criteria. 360

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4. Within

- test, intra

- laboratory and inter

- laboratory variability and how these parameters 362

vary with time should have been evaluated. 363

364

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

reference chemicals preferably coded to exclude bias. 366

367

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

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

370

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

372

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

374

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

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

p referably, published in an independent, peer

- reviewed publication. 377

378

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

attachment A (EDSP

, 2007 )

. Although attempts have been made to thoroughly comply with all 380

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

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

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

., 383

determination of false positive and false negative rates). 384

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In ge neral, the US EPA is following a five

- part or stage validation process outlined by 386

ICCVAM (NIEHS

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

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

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

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

the stages of the process ou tlined by the ICCVAM are as follows: 391

392

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

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

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

used, and the scientific bases upon which t he assay’s protocol, endpoints, and relevance 396

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

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

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

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

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

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

optimization

. 403

404

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

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

pro tocol transferability and performance. 407

408

In the third stage, inter

- laboratory validation

, studies were conducted in several 409

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

to determine inter

- laboratory variabili ty and to set or cross

- check performance criteria. 411

412

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

. 413

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

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

that underlies its regulatory decisions (US EPA

, 2006)

. 416

417

The final stage is regulatory acceptance

, adoption for regulatory use by an agency af ter 418

the other stages are completed

. 419



# 3 OVERVIEW OF THE LAGDA 420



# 3.1 Summary of the LAGDA t est p rotocol 421

422

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

growth and development fr o m fertilization through the early juvenile period . It also enables 424

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

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

LAGDA is a relatively long

- term assa y (normally 130 days or longer) that assesses early 427

development, growth, and partial reproducti ve maturation. 428

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429

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

concentration

- response information on adverse ef fects suitable for use in ecological risk 431

assessment. Specifically, the design enables the collection of amphibian hormone

- regulated 432

endpoint data ( e.g.

, metamorphosis, g onadal development ) and information concerning various 433

aspects of reproductive biolog y and life

- stage viability. 434

435

T he general experimental design entails exposing NF stage 8 ( s ee Nieuwkoop and Faber

, 1994 436

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

and a control until 10 weeks after the median time to NF stage 62 in the control

, with one interim 438

sub

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

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

sub

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

toxicity

, mortality, abnormal behavior, and growth determination s (length and weight) , as well as 442

endpoints designed to characterize endocrine

- specific modes of action targeting estrogen, 443

androgen or thyroid

- mediated physiological processes. 444

445



# 3.2 Purpose of the test and r elevance of the LAGDA 446

447

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

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

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

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

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

populations

. The test is designed to detect both endocrine and non

- endocrine mechanisms by 453

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

, to 454

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

456



# 3.3 Scientific basis for the test method 457

458

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

model specie s X. laevis

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

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

from commercial breeders. 462

463

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

pituitary gonadal ( HPG ) axis (Kloas and Lutz

, 2006). Estrogens and androgens are mediators of 465

this endocrine system, directing the development and physiology of sexually

- dimorphic tissues. 466

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

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

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

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

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

472

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

(Nieuwkoop and Faber

, 1994). Diff erentiation of the gonads begins at NF stage 52 when 474

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

(females) of the developing gonads (Nieuwkoop and Faber

, 1994). This process of sexual 476

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

Xenopus in the 1950s (Chang and Witschi

, 1956; Gallien

, 1953). Exposure of tadpoles to 478

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

raised to adulthood are fully functional females (Villalpando and Merchant

- Larios , 1990; Miyata 480

et al

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

following implantation of testis tissue in tadpoles (Mikamo and Wits chi

, 1963) or exposure to an 482

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

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

, 484

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

been no means by which to directly determine the genetic sex of Xenopus

. However, recent 486

establishment of sex

- linked markers in X. la e vis ( Yoshimoto et al

. , 2008 ) and X. tropicalis 487

( O lmstead et al

. , 2010 ) mak es it possible to determine genetic sex and allows for the direct 488

identification of sex

- reversed animals

. 489

490

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

with the development of secondary sex characte ristics as well as testis d evelopment (Olmstead et 492

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

vitellogenin in the plasma, vitellogenic oocytes in the ovary

, and the development of the oviducts 494

(Olmstead e t al

. , 2009b ). Oviducts are female secondary sex characteristics that function in 495

oocyte maturation during reproduction (Wake and Dickie

, 1998). Jelly coats are applied to the 496

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

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

correlates with blood estradiol levels in X. laevis (Tobias et al

. , 1998) and X. tropicalis 499

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

polychlorinated biphenyl compounds ( PCBs ) (Qin et al

. , 2007) and 4

- tert

- octylphenol (Porter et 501

al

. , 2011 ) has been reported. 502

503



# 4 PRO TOCOL DEVELOPMENT 504

505



# 4.1 Rationale for test species and exposure period 506

507

Short

- term p artial life cycle testing methods have been developed for amphibian species that 508

evaluate chemical impacts on metamorphosis ( O CS PP 890.1100 : Amphibian Metamorphosis 509

(Frog) Assay [ US EPA

, 2009 ]) . However, there are no standardized tests for evaluating chron ic 510

effects on growth and development of amphibian s

. Ideally a test of this nature would be 511

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

period encompass ing embryonic development through reproduction

. There are several fact or s 513

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

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

516

# 13

4.1.1 Species selection 517

518

Ideally

, the test species should be robust in the laboratory setting, be able to be genetic ally sex ed

, 519

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

potential for the well known laboratory model species X. laevis (Fort et al

. , 2004a; Pickford and 521

Morris , 2003; Pickford et al

. , 2003) and X. tropicalis ( Fort et al

. , 2004b; Porter et al

. , 2011; 522

O lmstead et al

., 2009 a ) as species for evaluating developmental impacts of c h ronic chemical 523

exposure. Although closely related

, ther e are significant differences between these two species 524

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

1) Both species are widely cultured in the laboratory

, although X. laevis has proven to be 526

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

settings. 528

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

- linked 529

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

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

. 531

tropicalis is a female

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

US EPA Mid

- C ontinent Ecology Division (MED) (O l mstead et al ., 20 10). Unfortunately, 533

because of the nature of this sex

- linked polymorphism, a very limited population of 534

animals currently exists

, and this strain has proven to lack the r obustness necessary for 535

routine testing. 536

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

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

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

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

maturity within 1.5 to 2 years. 541

542

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

. 543

tropicalis

, X. l a e vis was chosen as the representative amphibian species for development of the 544

LAGDA

. 545

546

4.1.2 Test duration 547

548

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

impacts of chemical exposure on development, growth and reproduction in a m phibians. Over 550

the intervening years

, the A gency has determined that

, given the current state of the science

, such 551

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

many aspects of the original proposal, is scientifically sound

, and is executable given t he 553

constraints of existing resources. 554

555

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

or any other amphibian species without the aid o f exogenous hormones

. Surrogate 557

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

assessed throughout the maturation process in X. tropicalis

, and it was determined that these 559

endpoints were highly variable ( O lmstead et al

. , 2009 b ). S tudies conducted with 4

- tert

560

o ctylphenol using a 40

- week exposure protocol showed that conducting the exposure until sexual 561

# 14

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

reproductive endpoints ( P orter et al

. , 2011 ). Evidence indicates that X. laevis would not differ 563

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

viable test species

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

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

be exceptionally high. Another important reality of aquatic testing is that l onger assay s run 567

greater risks of catastrophic failures ( e.g

. , disease, exposure system mechanical failures, etc .). 568

569

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

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

achieved. Toward this end, MED scientist s have conducted studies to determ ine when the 572

gonad s show an appropriate level of matur ation to allow identif ication of abnormalities in 573

development of reproductive organs

. From these studies it was determine d that

, by 8 weeks 574

post

- metamorphosis

, n early complete maturation of oocytes and spermatocytes had occurred

, 575

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

developing

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

were present ( A ppendix 8.2 ). Base d on these findings , the US EPA designed a protocol for an 578

exposure period which is initiated shortly after fertilization and continue s through 8 weeks post

579

metamorphosis

. 580

581



# 4.2 Test details 582

583

T he general experimental design entails exposing de

- jellied NF stage 8 X

. laevis embryos to four 584

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

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

control ) with one interim sub

- sample at NF stage 62

. There are four replicates in each test 587

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

exposure include those indicative of generalized toxicity

, mortality, abnormal behavior, and 589

growth determ ination s (length and weight) , as well as endpoints designed to characterize 590

endocrine

- specific modes of action targeting estrogen, androgen or thyroid

- mediated 591

physiological processes. An assessment of the gonads two months post

- metamorphosis is 592

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

development. The test details and performance criteria are summarized in T able s 4.2

- 1 and 4.2

- 2

. 594

595

596

597

598

599

600

601

602

# 15

Table 4.2

- 1 . Summary of method 

# 603

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 c ull 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/m 2 

# ) at the water surface

Water temperature 2 1 o ± 1 o C

pH 6.5 to 8.5

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

Analytical chemistry schedule I nitiation (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

Table 4.2

- 2

. Performance criteria for the LAGDA. 

# 618

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 inter

- replicate/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 clean water control groups are treated

specially. See LAGDA guideline for more

information

. 619

620

# 17



# 4.3 Core Endpoints 621

622

T he general experimental design entails exposing de

- jellied NF stage 8 X

. laevis embryos to four 623

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

NF stage 62 in the control

, with one interim sub

- sample at NF stage 62

. Table 4.3

- 1 summarizes 625

the endpoint s and timing of tissue/sample collection. 626

627

Table 4.3

- 1 Summary of endpoint and collection stages 

# 628

Apical Endpoints Daily Interim

Sampling Test

Termination

Mortality x

Clinical signs of disease and/or toxicities x

G rowth x x

Time to NF stage 62 x

Thyroid histology x

Serum T 4 

# (thyroxine) x

Vitellogenin x

Genetic/ histological sex comparison x

Gonad histology x

Liver histology x

Liver

- somatic index x

Kidney histology x

629

630

Growth and overt toxicity endpoints 631

632

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

are measured. In addition, l iver and kidney histopatholo gy and liver

- somatic index (LSI) have 634

been included as measure s of overt toxicity to the organism

. Given the importance of the liver in 635

metabolism and the kidney in clearance of xenobiotic s

, these tiss u e s are o ften targets of overt 636

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

of endocrine studies if it is not clearly under stood , so i nclusion of these endpoints in the LA GD A 638

will ultimately assist in interpreting future data set s

. 639

640

Thyroid endpoints 641

642

Three endpoints have been included which are intend ed to identify impact s on the thyroid axis : 643

t ime to NF stage 62

, t hyroid histology

, and serum thyroxine ( T 4 

# ) measures. In t he AMA

, the rate 644

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

AMA

, which determine s median stage achieved in a given amount of time ( 21 days )

, the 646

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

as metamorphic climax. However, this measure should be interpreted with caution b ecause non

648

specific toxicity experienced prior to metamorphosis m a y impact the length of time it take s to 649

reach NF stage 62. Thyroid histology

, the hallmark for diagnosing thyroid disruption

, is a focal 650

endpoint in the AMA and has been validated extensively ( Grim et al ., 2009 )

. Serum T 4 

# has also 651

# 18

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

robustness of the diagnosis for thyroid impa cts is enhanced with this three

- pronged approach

. 653

654

Reproductive endpoints 655

656

Vitellogenin 657

658

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

made using enzyme

- linked immuno so r bent assay ( ELISA ) methodology. Currently there are no 660

commercially available antibod ies . However, give n the wealth of information for this protein 661

and the availability of cost

- effective commercial antibody product ion services

, it is reasonable 662

that laboratories can easily develop an ELISA to make this measure (Olmlstead et al

. , 2009b)

. 663

D etail s are provided in LAGDA A ppendix 4

. 664

665

Gonad Histology 666

667

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

Reproduction Assay which exposes fathead minnow s for 21 day s

. The value of histological 669

analysis has been review ed extensive ly

, and a detailed international ly harmonized guida nce 670

document is available for fish ( Johnson et al

. , 2009). This fish guidance has served as the basis 671

for development of a LAGDA h istopathology guidance document ( A ppendix 8.3)

. 672

673

Genetic Sex 674

675

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

. , 676

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

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

sex c an be used to identify if sex r eversal has occurred (LAGDA Appendix 3)

. 679

680



# 4.4 Statistical Analyses 681

682

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

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

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

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

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

Statistical Analysis of Ecotoxicity Data: A Guidance to Application (OECD

, 2006)

. Data for 688

continuous endpoints should be assessed for normality (prefera bly using the Shapiro

- Wilk or 689

Anderson

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

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

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

non

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

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

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

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

# 19

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

- Dunnett 697

or T 3 test or from the Mann

- Whitney

- Wilcoxon U test. Where no normalizing transformation 698

can be found, a significant treatment effect is determined from the Mann

- Whitney

- Wilcoxon U 699

test using a Bonferroni

- Holm adjustment to the p

- values. The Dunnett test is applied 700

independently of any ANOVA F

- test

, and the Mann

- Whitney test is applied independently of 701

any overall Kruskall

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

step

- down Jonckheere

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

monotonic response was observed

. 704

705

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

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

those tad poles that are in the larval sub

- sample cohort, those juvenile frogs that are culled, and 708

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

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

711

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

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

Kaplan

- Meier product

- limit estimators an d used in the statistical analysis. 714

715

Body weight and snout

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

and females are not sexually

- dimorphic at the completion of metamorphosis

, and data for the 717

sexes are combined when analyzing larval sub

- sampling data. Male and female juvenile body 718

size data should be analyzed separately. 719

720

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

root transformed

, and analyzed separately for each sex. 722

723

H istop athology data is in the form of severity scores or developmental stages

. A new test termed 724

RSCABS ( R ao

- S cott C ochran

- A rmitage b y S lices) uses a step

- down Rao

- Scott adjusted 725

Cochran

- Armitage trend test on each level of severity in a histopathology response. The Rao

726

Scott adjustment incorporates the replicate vessel experimental design into the test. The “by 727

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

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

revealing the severity of any effect fou nd

. The RSCABS procedure not only determines which 730

treatments are statistically different, that is

, have more severe pathology than controls, but it also 731

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

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

which will soon be made freely available as an open

- source R package. A detailed des cription of 734

this test can also be found in the EDSP Tier 2 Medaka Multi

- generation Test (MMT) Integrated 735

Summary Report (ISR). 736

737

738

739

740

# 20



# 5 INTER

- LABORATORY EVALUATION OF THE LAGDA 741

742



# 5.1 O verview of approach 743

744

Inter

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

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

laboratories participated in the inter

- laboratory validation study: US EPA MED ; U.S. Army 747

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

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

ABC Laboratories, Inc. in Columbus, MO ; and

, Fort Environmental Laboratories (FEL) in 750

Stillwat er, OK. T he latter two labs were sub

- contractors to the US EPA contractor Battelle 751

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

active chemicals were selected for testing: p rochloraz, 4

- tert

- octylphenol, 1 7

- β t renbolone and 753

b enzophenone

- 2

. Table 5 .1

- 1 summarizes which chemicals were tested in each participating 754

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

protocol, in its revised form, can be found in Appendix 8.1. In some cases

, minor changes were 756

made

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

758

Table 5.1

- 1 Outline of i nter

- laboratory p articipation 

# 759

Prochloraz 4

- tert

- o ctylphenol 17

- β t renbolone 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



# 5.2 Inter

- laboratory data analysis 761

762

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

analysis of C hemistry, H istopathology, A nd R eproduction endpoints including R epeated 764

measures and M ulti

- generation S tudies), a SAS ® 

# (SAS Institute, Cary, NC)

- based program 765

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

continuous data measured once per subject, normality (via Shapiro

- Wilk test) and variance 767

homogeneity (via Levene’s test) were assessed

, and appropriate transforms were applied by the 768

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

the results of those two tests, a step

- down Jonckheere

- Terpstra (JT) trend test was also applied to 770

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

validation studies, the statis tical test that indicated the lowest concentration significantly 772

different than control was reported. T able 5.2

- 1 summarizes the statistical tests used by 773

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

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

. 775

# 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

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

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

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

control was reported. 782



# 5.3 Summary of results 783

5.3.1 Prochloraz 784

785

Introduction 786

787

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

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

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

al

., 2007 ). In adult fish

, in vivo exposure to prochloraz produces effects that are consistent with 791

inhibition of aromatase (Ankley et al

., 2005). Further, developmental exposure in rats produces 792

a spectrum of male reproduc tive tract malformation s which have been attributed to anti

793

androgenic properties (Noriega et al

. , 2005; Blystone et al ., 2007). Interestingly

, females were 794

not affected by developmental exposure in the aforementioned two studies

. 795

796

Results 797

798

Exposure levels and m easured test concentrations 799

800

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

those studies conducted with X . t ropicalis

, concentrations in excess of 200 μg/L resulted in 802

morta lity in early larval organisms. The high test concentration for the inter

- laboratory 803

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

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

coeff

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

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

and were on average 84 and 25% above nominal

, respectively ( Appendix 8.4.1, T able 8.4.1

- 1 ). 808

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

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

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

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

concentrations regardless of mean

- measured concentrations reported by each of the labs. 813

# 22

814

Measure of General Toxicity 815

816

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

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

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

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

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

of circulating thyroxine (T 4 

# ) occurring at this stage

. Therefore, interim assessments occurred at 822

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

824

Larval and Juvenile Growth 825

826

Summary of inter

- laboratory prochloraz growth endpoints.

A ll endpoints with lowest co ncentration 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

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

protocol. They include mortality, measure s of growth (length and weight), LSI

, and liver and 830

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

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

the concentration range, was overtly toxic to the dev eloping organism (Appendix 8.4.1, T able 833

8.4.1

- 5)

. 834

835

Two laboratories ( B

, D ) demonstrated impacts on growth in the larval and juvenile organisms in 836

a concentration

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

weight wer e greatest in these two studies that showed growth effects ( Appendix 8.4.1 , T able s 838

8.4.1

- 3 and 8.4.1

- 5 )

. 839

840

Three laboratories ( A

, D and B ) showed significant decreases in both juvenile male and female 841

LSI (Appendix 8.4.1, T able 8. 4.1

- 5) . This is consistent with the liver histopathology findings 842

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

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

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

juvenile growth across the entire study performed by C

. Further

, increased liver weight relative 846

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

# 23

848

L iver and Kidney pathology 849

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

Consistencies in treatment

- related findings were observed for hepatocellular degeneration and 852

hepatocellular atrophy in three of the four labs ( Appendix 8.4.1 , T able s 8.4 .1

- 7 and 8.4.1

- 8)

. A 853

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

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

treatment level and above in both male and female organism s

. 856

857

The C study exhibited a different suite of treatment

- related liver pathologies including basophilia, 858

increased hepatocellular vacuolization

, and granulomatous inflammation, all of which were 859

significantly different than the control at 60 and 180 μg/L (Appendix 8 .4.1, T able s 8.4 .1

- 7 and 860

8.4.1

- 8)

. Cellular hypertrophy was also observed in C liver s amples and showed a significant 861

concentration

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

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

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

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

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

867

The most consistent treatment

- related finding in the juvenile kidney was tubular amphophilic 868

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

, D 869

and C ( Appendix 8.4.1 , T able s 8.4 .1

- 13 and 8.4.1

- 14 )

. This response was consistent between 870

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

often were not treatment related. 872

873

Thyroid Endpoints 874

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 T 4 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

Time to NF s tage 62 878

# 24

879

T here were no treatment

- related e ffects on time to NF stage 62 ( NF stage 66 in the case of A ) in 880

all four participating laboratories ( Appendix 8.4.1 , T able s 8.4.1

- 2 and 8.4.1

- 3 )

. 881

882

Larval Thyroid Pathology 883

884

A

, D , and C studies showed mild but significant treatment

- related effects in the thyroid gland 885

consistent with compensation commonly observed fo llowing thyroid axis disruption including 886

follicular cell hypertrophy and hyperplasia ( Appendix 8.4.1, T able 8.4.1

- 6 )

. Although these 887

pathologies were present in larvae at B , there were no clear concentration

- dependent trends in 888

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

thyroid axis disruption. 890

891

Larval blood thyroxine 892

893

The D

, B and C laboratories used commercially a vailable T 4 

# ELISA kit s with specific antibodies 894

for human or canine T 4



# . T h ese laboratories failed to optimize these kits for use with X. laevis 895

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

included in this analysis. Laboratory A measured T 4 

# levels in serum using a high pressure liquid 897

chromatography and inductively

- coupled plasma mass spectrometry ( HPLC/ICP

- MS ) method

, 898

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

., 899

2012 ). In the A study, a significant increase in T 4 

# was observed in the 60 and 180 μg/L 900

treatment s ( Appendix 8.4.1, T able 8.4.1

- 4 )

. It is important to note that the few days prior to NF 901

stage 66 are marked by a rapid and appreciable decrease in serum T 4 

# levels. This, when 902

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

have resulted in decreased metabolism and elimination of T 4



# . 904

905

Reproductive Endpoints 906

907

Summary of int er

- 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

Juvenile plasma vitellogenin 910

911

A

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

titers ( Appendix 8.4.1 , T able 8.4.1

- 5)

. B showed no significant difference between control and 913

treated organism s but also reported titer values that were much lower on a verage than the other 914

three laboratories

. Although the VTG ELISA register ed a measurable signal in control males 915

# 25

and females in all labs

, VTG prod uction has been shown not to begin until later in development 916

(Olmstead, 2009b )

. Therefore, these control values likely reflect background noise within the 917

assay. 918

919

Gonad Histopathology 920

921

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

various studies. In terms of the testis and ovary

, there appears to be some inconsistencies in the 923

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

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

A and D studies were processed and read by I ntegrated L aboratory S ystems, Research Triangle 926

Park, NC wh ereas Labs B and C studies were processed and read by E xperimental Pathology 927

Laboratory, Sterling, VA

. In both the testis and ovary

, the A and D studies showed many 928

consistencies in diagnosis wh ereas C and B share consistencies. In all cases

, the genetic sex 929

matched the phenotype determined by the pathologis t and indicate d that treatment did not result 930

in sex reversal. 931

932

Testis 933

934

Of the ten observed pathologies showing significant c oncentration

- dependent trends in at least 935

one of the four studies

, several important finding s showed a consistent treatment

- related 936

response a cross the studies. A treatment

- related increase in germ cell degeneration was observed 937

across all studies and showed significance in the A

, B and C studies ( Appendix 8.4.1 , T able 938

8.4.1

- 9 )

. Germ cell degeneration in the testis was characterized by the presence of apoptotic 939

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

epithelium thinning was observed in the A

, C and D studies

, and its prevalen ce and severity 941

increased in a concentration

- dependent fashion. G erminal epithelial thinning presented as 942

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

epithelium surrounding affected tubules ( Figure 5.2

- 1 ) . A significant increase in spermatogonia 944

was also observed in these same studies

, and the pathologist noted that

, i n those cases with 945

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

increase due to the l oss of s permatocytes/spermatids rather than an absolute increase in numbers. 947

The degenerative testicular changes observed in these studies are consistent with the anti

948

androgenic effects of prochloraz reported in species as divergent as fathead minnows (A nkley et 949

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

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

development (Noriega et al ., 2005). 952

953

954

955

# 26

956 Figure 5 .2

- 1 A) C ontrol group male f ro m p rochloraz study, provided for reference , 10X . B) 957

Prochloraz 18 0 μg/L treatment group male , 10X . Gonadal degeneration thinning/ loss grade 2. 958

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

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

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

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

membranes (black arrows)

. 963

964

Ovary 965

966

Histopathological findings in the ovaries of prochloraz

- exposed female frogs were inconsistent 967

and minimal (Appendix 8.4.1

, T able 8.4.1

- 10 ). This finding is consistent with those finding s in 968

rats in which only male gonads ar e a ffected following developmental exposure to prochloraz 969

(Noriega et al ., 2005). 970

971

Reproductive Ducts 972

973

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

development ( Appendix 8.4.1, T ables 8.4 .1

- 11 and 8.4.1

- 12 )

. In males, significant 975

concentration

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

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

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

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

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

# A

B

C

# 27

advanced in the highest treatment at B

, and minimal mononuclear inflammation of the W o lffian 981

duct was significantly present in the highest treatment at A

. 982

983

5.3.2 4

- tert

- o ctylphenol 984

985

Introduction 986

987

The weak estrogen, 4

- tert

- octylphenol (OP), ( Bonefeld

- Jorgensen et al ., 2007 ), is a degradation 988

product of octylphenol polyethoxylate us ed as non

- ionic surfactants in industrial and household 989

settings ( Ying et al ., 2002 ). Vitellogenin has been shown to be elevated in male zebrafish (Ortiz

990

Zarragoitia and Cajaraville

, 2005) , male eelpout (Rasmussen et al

. , 2005) , male and female 991

medaka (Seki et al

. , 2003 ) , and in X. laevis primary cultured hepatocytes (Mitsui et al

. , 2007) 992

exposed to OP

. Decreased sperm count and the presence of testis

- ova h ave been observed in 993

medaka exposed to OP (Seki et al . 2003 ) ; decreased gonadosomatic index and increased testis

994

ova have been reported in eelpout exposed to OP (Rasmussen et al

. , 2005) ; and decreased 995

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

ex posed to OP (Karels et al

. , 2003)

. Female minnows exposed to OP have been shown to 997

produce a decreased percent of viable eggs (Kar els et al

. , 2003)

. Kloas et al . (1999) reported 998

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

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

. , 1000

2011 ). The only signif icant find ing s in the Porter et al

. ( 2011 ) studies were induction of VTG 1001

and ov i duct development in adult males. Based on these previously published studies

, OP was 1002

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

1004

Results 1005

1006

Exposure levels and measure d test concentrations 1007

1008

Test concentrations were set based on the findings of Porter e t al

. ( 2011 ) and a short term 1009

embryo/larval exposure conducted at MED. In these studies

, concentrations above 60 μg/L 1010

resulted in significant mortality i n embryos and early stage larvae . For this inter

- laboratory 1011

study

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

nominal test concentrations of 5 0, 25

, 12.5 and 6. 25 μg/L. All test concentrations at A were 1013

maintained with CVs ≤ 20% ( Appendix 8.4.2 , Table 8.4.2

- 1)

. B maintained test concentrations 1014

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

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

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

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

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

nominal with all mean

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

1021

Measure of General Toxicity 1022

1023

# 28

M ortality, measure s of growth (length and weight), liver somatic index, and liver and kidney 1024

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

demonstrate t hat OP had little impact on the general health of the organism s

. 1026

1027

Survival 1028

1029

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

explained by technical error

, and no treatment

- related trends in survival were identifi ed. Lab A 1031

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

one 12.5 μg/L replicate and two 50 μg/L replicates following the post

- NF stage 66 cull (data not 1033

shown)

. 1034

1035

Larval and Juvenile G rowth 1036

1037

Summary of int er

- 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

There were no treatment

- related effects on larval or juvenile growth in any of the three studies. 1039

Juvenile LSI was significantly decreased in octylphenol

- exposed males and females from C 1040

whereas no treatment

- related effects on LSI were measured at A or B ( Appendix 8.4.2 T able s 1041

8.4.2

- 2 and 8.4.2

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

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

1044

L iver and Kidney pathology 1045

1046

Summary of int er

- 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

No treatment

- related effects were present in any of the octylphenol

- exposed livers from all three 1049

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

severity w ere comparable among treated and control samples. Cystic de generation was observed 1051

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

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

disease. 1054

1055

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

( Appendix 8.4.2

, T able 8 .4.2

- 10) . Prevalence and severity w ere comparable between treatment 1057

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

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

was essentially not remarkable. 1060

1061

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

( Appendix 8.4.2

, T able 8.4.2

- 11)

. Lab C male kidneys exhibited a similar pattern to B male 1063

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

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

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

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

prevalence was very low

. 1068

1069

Thyroid Endpoints 1070

1071

Summary of int er

- 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 T 4 25 μg/L

- - Thyroid pathology 12.5 μg/L

- - 

# 1072

1073

Time to NF s tage 62 1074

1075

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

concentration but not in studies conducted by B and C (Appendix 8.4.2, T able 8.4.2

- 2)

. 1077

. 1078

Larval t hyroid pathology 1079

1080

In the B and C studies

, there were no significant impacts on thyroid gland s . The only 1081

statistically significant find ing s were increases in follicular cell hypertrophy and hyperplasia in 1082

the A s tudy at test concentrations ≥ 12.5 and 50 μ g/L

, respectively (Appendix 8.4.2, T able 8.4.2

- 1083

5)

. 1084

1085

Larval blood thyroxine 1086

1087

# 30

As indicted above

, failure to optimize the ELISA kits by B and C resulted in no reliable T 4 

# 1088

measur ement data

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

these commercially available EL I S A kits ( LAGDA Guideline A ppendix 7 ) with X. laevis serum

. 1090

Lab A also measured T 4 

# levels in serum using an HPLC/ICP

- MS method (Appendix 8.4.2, T able 1091

8.4.2

- 3)

. OP exposure resulted in a concentration

- dependent decrease in T 4 

# (F igure 5.2 .2

- 1 )

. 1092

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

. 1093

1094

1095

1096

Figure 5.2

. 2

- 1

. The top panel (A) shows the results for the EL I S A me thod

, and the bottom panel 1097

(B) shows results for the ICP

- MS method. The detection limits were 2.5 ng/ml and 0 .15 ng/ml for 1098

the ELISA and ICP

- MS method s

, respectively. The measured T 4 

# level s wer e consistent between 1099

the two methods

. * significant at p < 0 .05 1100

1101

1102

Reproductive Endpoints 1103

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

# 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

Juvenile plasma vitellogenin 1107

1108

Vitellogeni n titers showed a concentration

- dependent increase in octylphenol

- exposed males at 1109

labs A and C (Appendix 8.4.2, T able 8.4.2

- 4) . Female vitellogenin levels showed a 1110

concentration

- dependent increase at labs B and C

. 1111

1112

G onad histopathology 1113

1114

Genetic sex was de termined using the DMW sex

- linked marker. In the cases of laboratories B 1115

and C

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

phenotypic sex as determined by histological analysis. There was no indication of a treatment

1117

related effect given that the mismatches occurred randomly throughout the control and treatment 1118

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

investigation

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

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

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

studies were sent to MED for re

- analysis of geneti c sex. In all cases, the analyses performed by 1123

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

studies. 1125

1126

Testis 1127

1128

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

was observed throughout all three studies but was not treatment

- related, although statistically, 1130

severity was significantly less than controls at B (Appendix 8.4.2, T able 8.4.2

- 6) . There were 1131

very few observations of minimally increased spermatogonia at B an d C , but the 5 0 μg/L 1132

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

advancement of testicular development in the 5 0 μg/L treatment at B

, and this advancement 1134

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

C

. 1136

1137

Ovary 1138

1139

Unlike male gonads, female gonads experienced treatment

- related increases in severity of 1140

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

μg/L treatments at labs A and C (Appendix 8.4.2, T able 8.4.2

- 7) . Granulomatous inflammation 1142

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

μg/L treatment s

. 1144

1145

Reproductive duct histopathology 1146

1147

# 32

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

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

50 μg/L treatment at B ( Ap pendix 8.4.2, T able 8 .4.2

- 8) . Although there was a significant delay 1150

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

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

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

Table 8.4.2

- 9) . However, sensitivity differed between labs : Lab A only show ed a significant 1154

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

μg/L at B and C

, respectively. Both effects on males and female ducts are consistent with an 1156

estrogenic response, a commonly reported activity of OP. 1157

1158

5.3.3 17β

- t renbolone 1159

1160

Introduction 1161

1162

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

aquatic systems near animal

- feeding operations ( e.g ., stock yards)

. The effects of trenbolone 1164

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

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

amphibian species (O rn et al ., 2006; Holbech et al ., 2006; Morthorst et al

., 2010 ; Olmstead et al

., 1167

2012). 1 7

β

- trenbolone has been tested previously in the closely related amphibian species X. 1168

tropicalis (Olmstead et al

., 2012). In that study

, the authors characterized the effects of aqueous 1169

exposure to 17

β

- trenbolone during larval X. tropicalis development using an exposur e protocol 1170

similar to the LAGDA

. C oncentrations ≥ 100 ng/L resulted in a spectrum of androgenic 1171

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

development of nuptial pads, a mixed

- sex phenotype in the ovaries of f em ales, and hypertrophy 1173

of the W olffian duct. 1174

1175

Results 1176

1177

Exposure levels and measured test concentrations 1178

1179

Given the observed mortality in the studies of Olmstead et al

. ( 2012 ) the high est test 1180

concentration was set at 1 00 n g/L

, with a 0. 5 dilution factor resulting in nominal test 1181

concentrations of 100

, 5 0, 2 5 and 12.5 n g/L. Mean ± SD (n = 40) measured concentrations of 1182

the 12.5, 25, 50 and 100 ng/L 17 β

- trenbolone treatments were 9

. 0 ± 2.8, 19.2 ± 5. 8 , 41.4 ± 9.7 1183

and 79. 9 ± 24.1 ng/L, resp ectively, over the duration of the experiment. CVs (averaged across 1184

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

treatments , respectively . No 17 β

- trenbolone was detected in control water. 1186

1187

Measure of General Tox icity 1188

1189

Survival 1190

1191

# 33

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

treatment

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

1194

Larval and Juvenile growth 1195

1196

There were no significant treatment

- related effects on larval and juvenile growth as measured by 1197

weight and length ( Appendix 8.4.3, T able s 8 .4.3

- 1 and 8.4.3

- 2 ). Further

, there were no 1198

significant treatment

- related effects on LSI for either gender ( Appendi x 8.4.3, T able 8.4.3

- 2 ). 1199

1200

Liver and Kidney histopathology 1201

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

Treatment

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

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

treatments for juvenile males and females, respectively (Appendix 8.4.3, Table 8.4.3

- 4)

. The 1206

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

comparable among treated and control samples

. 1208

1209

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

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

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

treatment

- related (Appendix 8.4.3, T able 8.4.3

- 6) . There was a low but st atistically significant 1213

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

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

females (data not shown). 1216

1217

Thyroid Endpo ints 1218

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 T 4 - Thyroid pathology 25 ng/L



# 1220

# 34

1221

Time to NF s tage 62 1222

1223

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

8.4.3, T able 8.4.3

- 1)

. 1225

1226

Larval Thyroid histopathology 1227

1228

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

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

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

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

statistically significant ( Appendix 8.4.3, T able 8.4.3

- 3)

. The prevalence and severity of follicular 1233

cell hype rplasia in trenbolone

- treated frogs were mild but significantly different than the control 1234

in the 25, 50 and 100 ng/L treatments

. 1235

1236

Larval blood thyroxine 1237

1238

There were no treatment

- related effects on larval serum T 4 

# levels as measured by HPLC

- ICP

- MS 1239

analysis ( Appendix 8.4.3, T able 8.4.3

- 1 ). 1240

1241

Reproductive Endpoints 1242

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

Juvenile plasma vitellogenin 1246

1247

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

shown). 1249

1250

G onad histopathology 1251

1252

In all case s

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

that treatment did not result in sex reversal. There were t wo significant treatment

- related 1254

fin dings in the testes of male s : increased spermatogonia (minimal) and germ cell vacuolation 1255

# 35

(minimal to mild) in the 100 ng/L treatment ( Appendix 8.4.3, T able 8 .4.3

- 5 ). Germ cell 1256

vacuolation was characterized by clear cytoplasmic ballooning in small clusters of pachytene

1257

phase (cell nuc lei in prophase of meiosis) spermatocytes . There were no apparent effects of 1258

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

1260

Reproductive duct histopathology 1261

1262

There were treatment

- related effects on oviduct development in both genders. Males ex hibited 1263

accelerated regression of oviducts in th e 100 n g/L treatment as compared to the control whereas 1264

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

8.4.3, T able 8.4.3

- 7 )

. There were no tr eatment

- related effects on Wolffian duct development or 1266

pathology in either gender. Given the importance of the oviduct as a female reproductive organ 1267

necessary for application of the gel coat to the oocyte and release of the oocyte

, these effects are 1268

exp ected to have considerable consequence on reproductive capacity ( F igure 5 .2.3

- 1 )

. 1269

1270

1271

1272

1273

1274

Figure 5 .2.3

- 1 A) Stage 2 oviduct (arrow) observed in control females. B) Stage 1 oviduct 1275

(arrow) from a female exposed to 100 ng/L trenbolone. Stage 1 oviducts such as this were not 1276

observed in any control females. 1277

1278

5.3.4 Benzophenone

- 2 1279

1280

Introduction 1281

1282

B enzophenone

- 2 (BP

- 2) is used as a uV filter in personal care products such as cosmetics and 1283

sunscreens and in numerous other products for u V protection. BP

- 2 has been found to be 1284

estrogenic in vitro and in vivo and is capable of impacting reproduction in fish (Weisbord et al

., 1285

2007). In addition to this documented estrogenic activity

, BP

- 2 has been shown to be a thyroid 1286

peroxidase inhibitor

, and exposure to BP

- 2 can result in disruption of the thyroid axis in a quatic 1287

species (Thienpont et al

., 2011). BP

- 2 provided a unique study in the LAGDA evaluation of a 1288

mixed mode of action chemical which included thyroid disruption. Laboratory E was the only 1289

laboratory to conduct a test with BP

- 2. Due to limitations in e xposure capabiliti es at this 1290

laboratory

, there were several deviations from the guidance provided. Instead of running eight 1291 

# A B

# 36

control replicates and four treatment replicates at four test concentrations and a control, this 1292

study was conducted using three te st concentrations and a control, each with four replicates. Also

, 1293

the test was initiated with 30 embryos per tank rather than the recommend ed 25 embryos. 1294

Although not completely consistent with the proposed test method, t his study provides 1295

significant va lue in term s of organism response and transferability of the approach. 1296

1297

Results 1298

1299

Exposure levels and measured test concentrations 1300

1301

The high test concentration was 6 m g/L with a 0. 5 dilution factor resulting in nominal test 1302

concentrations of 6.0

, 3.0 and 1.5 m g/L. Mean ± SD (n = 36 ) measured concentrations of the 1.5, 1303

3.0 and 6.0 m g/L benzophenone

- 2 treatments were 1.6 ± 0.1

, 3.5 ± 0.2 and 6.1 ± 0.1 mg/L, 1304

respectively

, over the duration of the experiment . C Vs (averaged across the d uration of the 1305

exposure ) were 4, 6 and 1 % for the 1.5

, 3.0 and 6.0 m g/L treatments , respectively . No 1306

benzophenone

- 2 was detected in control water. 1307

1308

Measure of General Toxicity 1309

1310

Survival 1311

1312

BP

- 2 treatment resulted in significant mortality by NF stage 62 at 6.0 mg/L (Jonckeere

- Terpstra, 1313

T able 5.2.6

- 1 ). 1314

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

Following the NF stage 66 cull, 10 individuals were left in each tank to continue exposure until 1317

test termination. Within this timeframe, significant mortality was experienced in the high test 1318

concentrat ion (Jonckeere

- Terpstra, T able 5.2.6

- 2 ). 1319

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

Larval and Juvenile G rowth 1326

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

There was a significant increase in larval growth in the 6 mg/L treatment as compared to the 1330

control

. There was also a significant delay in time to reach NF stage 62 in the 6 mg/L treatment 1331

( Appendix 8.4.4, Table 8.4.4

- 3)

. This increase in tadpole size is consistent with previous reports 1332

following exposure to thyroid axis disruptors which produce a significant d evelopmental delay 1333

(Degitz et al ., 2005; Tietge et al

., 2005). Another potential contributor to increased growth in 1334

the 6 mg/L treatment could be the significant mortality in that treatment allowing the survivors 1335

more access to food. Growth in male and female juveniles was significantly reduced in the 6 1336

mg/L treatm ent compared to the control, presumably due to overt toxicity ( Appendix 8.4.4

, 1337

T able 8.4.4

- 4 ). LSI appeared to be affected by BP

- 2 exposure; however, statistical analyses were 1338

inconsistent between genders due to non

- monotonicity of the data in both cases. There was a 1339

significant decrease in male LSI in the 6 mg/L treatment and a significant increase in female LSI 1340

in the 1.5 mg/L treatment ( Appendix 8.4.4 , T able 8 .4.4

- 4). 1341

1342

L iver and Kidney pathology 1343

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

Treatment

- related findings in the liver were limited to phenotypic female s and genetic males 1347

which showed mixed sex gonads ( i.e

. , 3.0 and 6.0 mg/L treatments)

. T hese included 1348

# 38

concentration

- dependent increases in the prevalence and severity of basophilia, decreased 1349

hepatocellular vacuolation, and intravascular proteinaceous fluid ( Appendix 8.4.4, Table 8.4.4

- 6)

. 1350

As compared to contro l frogs , frogs of the 3 and 6 m g/L treatment groups additionally had higher 1351

prevalence of increased pigment, which also tended to be concentration

- dependent. Furthermore, 1352

the few findings of individual hepatocyte necrosis and foci of hepatocellular alteration s were 1353

restri cted to frogs of the 3 and 6 mg/L treatment groups and 6 mg/L treatment group, 1354

respectively. Overall, these live r changes are likely related to estrogenic activity of BP

- 2 and 1355

significant up

- regulation of vitellogenin synthesis ( i.e ., vitellogenesis)

. 1356

1357

BP

- 2 exposure ca used profound effects in both genotypic male and female kidneys. These 1358

effects were very similar between genders and widespread in the 3 and 6 mg/L treatments 1359

( Appendix 8.4.4, T able 8.4.4

- 8) . However, these two treatments had 100% female phenotypes. 1360

Int erestingly, many of the pathologies ha d higher severity scores in the 3 mg/L treatment and 1361

were slightly less severe in the 6 mg/L treatment. 1362

1363

Thyroid Endpoints 1364

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

Time to NF stage 62 1368

1369

There was a significant delay in time to NF stage 62 in the 6 mg/L treatment (Appendix 8.4.4, 1370

Table 8.4.4

- 3)

. 1371

1372

Larval t hyroid pathology 1373

1374

There were significant treatment

- related effects on thyroid tissue including increased prevalence 1375

and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy ( Appendix 1376

8.4.4, Table 8.4.4

- 5) . The 6 mg/L treatment exhibited glandular hypertrophy and extreme 1377

follicular cell hypertrophy and hyperplasia in 100% of the indi viduals

. As noted above

, there 1378

was also a significant delay in the rate of metamorphosis

. 1379

1380

Larval blood thyroxine 1381

1382

In the BP

- 2 study, heparinized p lasma samples as opposed to serum were analyzed for T 4 

# levels 1383

using both the optimized ELISA described in L AGDA Guideline Appendix 7 and the 1384

HPLC/ICP

- MS method . There were no statistically significant effects on circulating T 4 

# in either 1385

sample set

, and T 4 

# levels from the two methods did not agree

. The cause of the lack of 1386

agreement between the two methods is unknown . Therefore, the data is not presented. The 1387

effects of using plasma as opposed to serum for T 4 

# analyses are currently under investigation. 1388

# 39

1389

Reproductive Endpoints 1390

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

Juvenile plasma vitellogenin 1393

1394

There was significant induction of vitellogenin in all treatments compared to the control and the 1395

response was very consistent between genders ( Appendix 8.4.4 , Table 8. 4.4

- 4 ). The overall 1396

response was a > 5000

- fold induction in the 6 mg/L treatment in both genetic males and females 1397

compared to c ontrols. This illustrates the estrogenic properties of BP

- 2 and emphasizes the 1398

potential magnitude of response of this endpoint

. 1399

1400

G onad histopathology 1401

1402

Genotypic males experienced feminization of the gonad resulting in both i ntersex gonads ( e.g

., 1403

presence of testicular oocytes and partial ovarian cavity) and sex

- reversed gonads in the 1.5 1404

mg/L treatment (F igure 5.2.4

- 1)

. In the 3 and 6 mg/L treatment s , 100% of the genotypic males 1405

had been completely sex reversed exhibiting only ovar ian tissue ( i.e ., phenotypic females) . In 1406

genoty pic females, there was a significant treatment

- related delay in gonad development based 1407

on differences in developmental staging of ovaries in control versus treated samples ( Appendix 1408

8.4.4, T able 8.4.4

- 7)

. Similarly, there was a treatment

- related delay in ovary development in 1409

sex

- reversed genotypic males. Thinning of the germinal epithelium is specific to testis tissue

, 1410

and significant increases in prevalence and severity of this pathology were observed i n the 1.5 1411

mg/L treatment. Mononuclear cellular infiltrate was observed in genotypic males and females 1412

across all treatments and the control, but prevalence and severity w ere significantly attenuated in 1413

the 3 and 6 mg/L treatments for genotypic females. P revalence and severity of proteinaceous 1414

fluid infiltrate w ere significantly increased in both genders in the 3 and 6 mg/L treatments. 1415

1416

# 40

1417

1418

A B 1419

1420

1421

1422

1423

1424

1425

1426

1427

1428

1429

Figure 5.2.4

- 1 A) Testis, ducts, and kidney from a control male, provided f or reference

. Scale 1430

bar = 200μm . B) Testis of genetic male treated with 1.5 mg/L BP

- 2. The gonad consists 1431

p rimarily of testicular tissue , but also contains several oocytes and a partial ovarian cavity 1432

(arrow ). Scale bar = 50μm

. 1433

1434

1435

Reproductive duct histopathology 1436

1437

Oviduct development was rather variable within and between genders. In genotypic females, 1438

there was a significant delay in oviduct development in all treatments (Appendix 8.4.4, Table 1439

8.4.4

- 9 ) . However, in both genders, there were some individuals in the 1.5 and 3 mg/L 1440

treatments that are two developmental stages more advanced than the others. Both genders 1441

experienced advancement of Wolffian duct development in the 3 and 6 mg/L treatments. 1442

1443



# 6 DISCUSSION OF THE INTER

- LAB ORATORY STUDIES 1444

1445



# 6.1 Comparison of control performance 1446

1447

Larval growth and development 1448

1449

A summary of control larval weight, length, and time to NF stage 62 across all the inter

1450

laboratory studies is shown in T able 6. 1

- 1

. The measurements are ranked by weight in 1451

descending order from a maximum control larval NF stage 62 weight of 2.07 + 0.33 g to a 1452

minimum of 0.46 + 0.14 g. T he overall mean weight was 1.0 g with a standard deviation of 0.5 g. 1453

Two studies , B

- OP and C

- OP, fell outside of one standard deviation from the overall mean 1454

weight. Within studies conducted by B , mean larval weight doubled from the PZ study to the OP 1455

study and resulted in notable incidences of scoliosis in the OP study. Unpublished feeding 1456

studies perfo rmed at A have demonstrated that excess feeding through larval development leads 1457

to high er incidence s and increased severity of scoliosis, which is generally considered adverse 1458

for toxicological studies. Upon follow

- up investigation, it was reported that feeding rates were 1459

increased in the B

- OP study by 20

- 30% because they opted to start the test with 25% more 1460

# 41

organisms per tank and subsequently increased feeding rates proportionately. Increasing the 1461

number of organisms per tank for test initiation, in or der to account for mortality, is acceptable 1462

according to the LAGDA guideline. However, increasing feeding rates proportionately proved 1463

to be excessive. The lowest rate s of larval growth among the labs occurred in C

- PZ and C

- OP. 1464

These studies also exhibi ted the highest level of variance in all three growth and development 1465

metrics. This lab utilized its own established feeding rates which were lower than the 1466

recommended feeding rates in the LAGDA guideline. T hese data and observations emphasize 1467

the need for strict adherence to the guideline ’s recommended feeding rates to achieve 1468

comparable and optimal organism growth across studies . It is suggested that the criterion for 1469

control larval growth be 1.0 ± 0.2 grams at NF stage 62, which should be reached in less than 45 1470

days. 1471

1472

Table 6.1

- 1

Inter

- laboratory control larvae growth and development performance comparison.

Entries are listed in de scending 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 1 6 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 1 6 20.8 ± 1.0 5 41 ± 3 7

A

- OP 0.96 ± 0.15 1 6 19.2 ± 1.0 5 42 ± 5 1 2

A

- TB 0.82 ± 0.14 17 18.7 ± 1.1 6 40 ± 4 10

D

- PZ 0.79 ± 0.13 1 7 18.8 ± 1.0 5 43 ± 2 5

C

- PZ 0.62 ± 0.14 2 3 17.1 ± 1.4 8 44 ± 6 1 4

C

- OP 0.46 ± 0.14 30 15.2 ± 1.6 1 1 41 ± 10 24

Overall means 1.0 ± 0.49 49 19.4 ± 2.6 13 41 ± 2 4



# 1473

1474

Juvenile growth and development 1475

1476

As with the larval comparison

, T able 6.1

- 2 contains juvenile male and female growth metrics 1477

(length, weig h t , LSI) and VTG for each gender ranked by weight in de scending order. Across all 1478

studies, t he male mean weight was 11.2 ± 3.9 g

, and female mean weight was 11.7 ± 4.3 g

. Two 1479

studies had control juvenile weights more than one standard deviation from the overall mean in 1480

both juvenile males and females but in different directions : D

- PZ had higher mean weights

, and 1481

C

- PZ had lower mean weights

. Lab D used the recommended diet formulation but fed at a 1482

higher rate than what was recommended in the LAGDA guideline. This was not necessarily 1483

adverse; however

, there was a significant treatment

- related dec rease in weight in this study 1484

whereas studies from the other labs did not show this effect from prochloraz

. Lab C used their 1485

own diet formulation but fed at rates similar to th at recommended in the LAGDA guideline. 1486

This diet formulation was clearly inadequate for supporting growth rates consistent with the 1487

other studies and led to confounding effects on LSI in their prochloraz study ( explained in 1488

S ection 5.2.1 ). Body size does not become sexually dimorphic until later in development, so 1489

significant differences in growth are not expected between genders at test termination. Therefore, 1490

criteria for juvenile growth can be set the same for both sexes and is suggested to be 11.5 ± 3 g. 1491

1492

It is worth noting the VTG levels in control juveniles . These values are an artifact of detection 1493

limits within each ELISA and are 100 to 1000

- fold less than levels in mature females (Olmstead 1494

et al

. , 2009 b ) and 10 to 1000

- fold less than levels induced by OP and BP

- 2 exposures

. T he 1495

utility of this endpoint is discussed further in Section 6.3

. 1496

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 (m g/mL) CV

Male

D

- PZ 18.6 ± 4.1 22 53.0 ± 3.9 7 0.058 ± 0.005 9 0.01 ± 0.0 2 158

A

- PZ 13.1 ± 2.0 15 47.2 ± 2.8 6 0.059 ± 0.010 17 0.03 ± 0.0 2 59

A

- TB 12.3 ± 3.3 2 7 46.2 ± 4.1 9 0.060 ± 0.010 1 7 0.07 ± 0.0 7 108

B

- PZ 11.5 ± 3.6 31 43.0 ± 4.5 1 1 0.057 ± 0.009 16 0.00 ± 0.00 176

E

- BP2 11.3 ± 2.7 2 4 45.2 ± 3.3 7 0.046 ± 0.005 11 0.02 ± 0.0 3 150

A

- OP 10.9 ± 2.7 2 5 43.1 ± 3.3 8 0.058 ± 0.008 14 0.00 ± 0.00 150

B

- OP 10.1 ± 2.6 2 6 41.6 ± 3.5 8 0.058 ± 0.007 12 0.01 ± 0.0 1 107

C

- OP 8.6 ± 2.6 30 41.1 ± 3.4 8 0.072 ± 0.008 11 0.0 1 ± 0.00 1 30

C

- PZ 4.0 ± 0.8 20 32.8 ± 2.2 7 0.035 ± 0.007 20 0.01 ± 0.00 2 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 35 7

B

- PZ 13.8 ± 5.0 36 46.4 ± 5.4 12 0.064 ± 0.014 22 0. 01 ± 0.02 25 1

A

- PZ 12.9 ± 2.6 20 47.2 ± 2.6 6 0.061 ± 0.007 12 0.0 3 ± 0.03 7 6

A

- TB 12.9 ± 3.7 29 47.5 ± 5.1 11 0.059 ± 0.006 10 0.0 6 ± 0.0 7 11 6

A

- OP 12.0 ± 2.7 23 44.7 ± 4.0 9 0.059 ± 0.012 2 0 0. 00 ± 0.00 112

B

- O P 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 32 3

C

- OP 8.3 ± 1.7 21 40.8 ± 3.1 8 0.075 ± 0.007 9 0.04 ± 0.0 1 3 2

C

- PZ 4.1 ± 0.8 20 32.9 ± 2.2 7 0.032 ± 0.006 19 0.03 ± 0.0 1 4 1

Overall ♀ means 11.7 ± 4.3 37 44.6 ± 5.9 13 0.057 ± 0.012 21 0.03 ± 0.02 76



# 1499



# 6.2 Effectiveness of the LAGDA 1500

1501

In total

, the results for four chemicals are presented in this report. Two of these chemicals, 4

- 1502

tert

- octylphenol and prochloraz

, were tested in three and four laboratories respectively. The 1503

other two test chemicals, b enzophenone

- 2 and 17

- β t renbolone, were tested in a single laboratory 1504

which prevents their use in inter

- laboratory comparison s . However, these studies serve to 1505

demonstrate the responsiveness of the LAGD A to additional modes of action. T able 6.2

- 1 1506

summar izes the important find ing s for all of the studies which were conducted

. All four 1507

chem icals tested produced endocrine

- related effects in these studies. 1508

1509

# 43

Table 6.2

- 1

. 

# 1510

Summary of significant findings in LAGDA studies on 4 chemicals representing different modes of action. 

# 1511

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 me t amorphosis VTG induction

Profound gonad/reproductive

duct pathologies



# 1512

1513

Prochloraz 1514

1515

At the highest test concentration

, prochloraz produced liver and kidney pathologies consistent 1516

with a toxic response across the four studies, although there was little evidence of an effect on 1517

growth. LSI was also impacted by the highest prochloraz concentration across the studies

. It 1518

was also apparently decreased at two, three, or four concentration s in most studies, although it 1519

ap peared increased in C

- PZ

, but this was likely an artifact due to control juvenile growth in that 1520

study being significantly less than in all other studies

. 1521

1522

Effects on the thyroid axis ( thyroid gland change) were also observed across the studies although 1523

the metamorphic rate was not altered . This suggest s a successful compensatory response by the 1524

thyroid axis. Prochloraz has not been previously shown to have impacts on the thyroid axis and 1525

a review of the literature found no evidence of prior investigati ons. 1526

1527

The degenerative testicular changes consistently observed across the four prochloraz studies is in 1528

agreement with the anti

- androgenic effects of prochloraz reported in species as divergent as 1529

fathead minnows (Ankley et al ., 2005) and rats (Blystone et al ., 2007). In particular, germinal 1530

epithelium thinning/loss with high doses of prochloraz ha s been described in rats (Noriega et al

., 1531

2005). In male frogs, sporadic development of stage 3 oviducts and the retention of stage 2 1532

oviducts with increasing prochloraz exposure w ere observed across the four studies and indicate 1533

interference with normal oviduct regression

. Oviduct regression in males is known to be an 1534

androgen regulated process in amphibians (Kelley, 1996)

, and this finding is similar to the 1535

feminization of the secondary sexual characteristics of male rats during perinatal prochloraz 1536

exposure (Vingaard et al ., 2005). 1537

1538

The LAGDA was successful in identifying proch loraz as anti

- androgen ic and demonstrated the 1539

consequence of this mechanism of action on male gonad development. 1540

1541

1542

4

- tert

- octylphenol 1543

# 44

1544

In general

, OP produced few signs of overt toxicity. No consistent effects on growth, liver and 1545

kidney pathology or LSI were observed. 1546

1547

OP exposure resulted in VTG induction and female oviduct pat hologies across all four studies. 1548

These OP

- induced changes are consistent with weak estrogenic activity. V itellogenin has also 1549

be en shown to be elevated in males and females of multiple fish species e xposed to OP (Seki et 1550

al

. , 2002 ; Ortiz

- Zarragoitia and Cajaraville

, 2005 ; Rasmussen et al

. , 2005 ; Seki et al.

, 2002 ) , in 1551

X. laevis primary cultured hepatocytes e xposed to OP (Mitsui et al

. , 2007)

, and following in vivo 1552

OP exposure in X. tropicalis (Porter et al

., 2011)

. 1553

1554

In these inter

- laboratory studies

, no consistent findings were observed in the developing male 1555

and female gonads. P revious studies that have employed exposure during gonad development 1556

have shown disrupt ion in gonad development. These effects include d ecreased sperm count and 1557

the presence of testis

- ova in med aka (Seki et al

. , 2002) , decreased gonadosomatic index and 1558

increased test is

- ova in eelpout (Rasmussen et al

. , 2005) , and decreased spermatozoa and 1559

increased testis anomalies in sheepshead minnows (Karels et al

. , 2003)

. In contrast, p revio us 1560

studies with the X. tropicalis have shown no impact on male or female gonad development 1561

(Porter et al

., 2011 ). When taken together

, it appears that these amphibian test species are less 1562

sensitive to the endocrine disrupting effects of OP than are fish species. It must be pointed out 1563

that it was not possible to test high er concentration s given the fact that concentrations above 60 1564

μg/L produced significant levels of embryo mortality in X. tropicalis (unpublished data

, Lab A )

. 1565

It is difficult to make extensive comments regarding across

- laboratory performance g iven that 1566

minimal effects were observed

, other than to note that OP produced a consistent weak estrogenic 1567

response

. This was indic a ted by VTG induction and advancement of oviduct development 1568

across the four studies , a response that it is consistent with OP activity reported in the published 1569

literature

. 1570

1571

17

-

β Trenbolone 1572

1573

There were no significant treatment

- r elated effects on larval or juvenile growth

, although a 1574

concentration

- dependent increase in hepatocyte hypertrophy was observed. This find ing 1575

indicate s that the test concentrations did not produce an appreciable level of overt toxicity. 1576

1577

The effects prod uced by t renbolone in this study were rather mild. Female gonads were 1578

unaffected by exposure wh ereas there was increased spermatogonia and germ cel l vacuolation in 1579

the testis at 100 ng/L

. The most sig nificant finding was that 17

- β t renbolone induced the 1580

regression of the female oviduct. The oviduct functions to transport mature oocytes from the 1581

ovary to the external environment for fertilization by the male. Loss of or functional alterations 1582

in the oviduct will have significant imp acts on reproductive success and c ould lead to population 1583

level impacts. There was also a mild increase in follicular cell hyperplasia at ≥ 25 ng/L although 1584

no significant alterations in developmental rate were observed

. 1585

1586

In the ab sence of additional LA GDA studies with 17

- β trenbolone

, comparison s to the published 1587

literature served as a basis for evaluating the performance of the LAGDA in responding to 1588

androgenic chemicals. Studies in zebrafish have demonstrated that aqueous exposure to 1589

# 45

trenbolone resul ts in complete sex reversal of females and production of all male cohorts ( O rn et 1590

al ., 2006; Holbech et al ., 2006; Morthorst et al

., 2010). Ovo

- testes characterized by ovaries 1591

which develop tubules containing spermatocytes were indu ced in mosquitofish exposed to 1592

t renbolone (Sone et al

., 2005). Females exposed to trenbolone have been reported to acquire 1593

mal e secondary sex characteristics such as tubercles in adult fathead minnows (Ankley et al

. , 1594

2003) and papillary processes in Japanese medaka (Seki et al

., 2006). Trenbolone exposure has 1595

also been reported to reduce circulating estradiol and testosterone levels in fathead minnows 1596

(Ankley et al

. , 2003). Decreased circulating vitellogenin in females has been demonstrated in a 1597

number of different fish spe cies exposed to this toxicant (Ankley et al

. , 2003; Sone et al ., 2005; 1598

Seki et al

., 2006)

. Test concentrations of 1 7

- β t renbolone were set based on reported finding s for 1599

X. tropicalis (Olmstead et al

., 2012)

. In that study

, the authors reported a much mo re dramatic 1600

impact of 17

-

β t renbolone on androgen responsive tissues. C oncentrations ≥ 100 ng/L resulted in 1601

a spectrum of androgenic responses which included hypertrophy of the larynx resulting in late 1602

larval mortality, precocious development of nuptial pads, a mixed

- sex phenotype in the ovaries 1603

of females, and hypertrophy of the W olffian duct. This comparison suggests that X

. laevis may 1604

be less sensitive than X. tropicalis to this synthetic androgen . Although the LAGDA show ed a 1605

l ower sensitivity when compared to the literature

, the LAGDA successfully identified the 1606

endocrine disrupting potential and mode of action of t renbolone

. Further, the LAGDA 1607

demonstrated findings (female oviduct regression) which are useful in the context o f risk 1608

assessment. 1609

1610

Benzophenone

- 2 1611

1612

BP

- 2 treatment resulted in significant increase s in larval and juvenile mortality at the highest test 1613

concentration

. 1614

1615

There were significant treatment

- related effects on thyroid tissue including increased prevalence 1616

and severity of follicular cell hypertrophy and hyperplasia and gland hypertrophy. The 6 mg/L 1617

treatment exhibited remarkable pathologies in 100% of the subjects which w ere associated with a 1618

significant delay in the rate of metamorphosis

. BP

- 2 has been shown to be a thyroid peroxidase 1619

inhibitor ( Schmutzler et al

., 2007)

, and exposure has been shown to result in disruption of the 1620

thyroid axis in mammalian ( Jarry et al

., 2004) and aquatic species (Thienpont et al

., 2011). 1621

1622

BP

- 2 has been found to be estrogenic in vitro ( Molina

- Molina et al

., 2008) and in vivo (Kunz and 1623

Fent

, 2009) and is capable of impacting reproduction in fish (Weisbord et al

., 2007). The results 1624

of this study are consistent with these published reports. P lasma v itellogenin lev els were 1625

considerably elevated in all treatments ( Appendix 8.4.4, Table 8 .4.4

- 4 ). In addition to this 1626

finding

, liver and renal pathologies which were observed are consistent with estrogen exposure 1627

( Pawlowski et al ., 2004; Folmar et al

., 2001 ) and are presumably a consequence of excessive 1628

VTG accumulation in these organs

. Genotypic males experienced feminizat ion of the gonad in a 1629

concentration

- dependent fashion ; both intersex gonads and sex

- reversed gonads were observed 1630

in the 1.5 mg/L treatme nt whereas in the 3 and 6 mg/L treatment, 100% of the genotypic males 1631

had been completely sex reversed exhibiting only ovary tissue. In genotypic females, there was 1632

a significant treatment

- related delay in gonad development. Similarly, there was a treatm ent

1633

related delay in ovary development in sex

- reversed genotypic males. 1634

1635

# 46



# 6.3 Les s ons l earned fr o m the in ter

- laboratory s tudies 1636

1637

Inconsistencies in Growth 1638

1639

Inconsistencies in organism growth were observed ( see S ection 6.1 ) which may have impacted 1640

the results of the test chemicals. Diet formulation and feeding rates have been shown to be 1641

correlated with organismal growth and developmental rates in the LAGDA. In on e instance

, 1642

excessive control growth was observed when all the studies were considered as a group. In this 1643

case

, treatment

- related effects on growth were observed. In two other studies ( C

- PZ and C

- OP)

, 1644

control growth was reduced relative to the broader group

, and this was a confounding factor in 1645

interpreting ch emical related effects on LSI. This laboratory chose to use its own feed 1646

formulation in lieu of th at recommended in the guidance. F r o m this inter

- laboratory comparison

, 1647

it can be concluded that this formulation is inadequate to support appropriate growth and 1648

development. The recommended diet and feeding rates in the LAGDA provide for optimal 1649

growth

, and the Agency strongly encourage s adherence to the provided guidance. 1650

1651

Inconsistencies in Pathology Findings 1652

1653

Another area of inconsistency was in the pat hology findings. Over the course of these studies

, 1654

the contract laboratories hired to prepare and read slides changed resulting in different 1655

pathologist s conducting the assessment s . In the prochloraz studies

, Labs A and D gonad, oviduct 1656

and kidney sample s were rea d by a different pathologist tha n the Labs B and C studies. 1657

Interestingly, the Lab A and D studies shared consistencies as did the Lab B and C studies. It is 1658

not possible to definitively demonstrate this as a cause for the observed inconsistencies

. 1659

H owever

, it does point to the need for adherence to the pathology guidance document. It is 1660

necessary to point out that the gonad histopathology guidance document was not finalized when 1661

the prochloraz studies were prepared and read. In the case of OP

, the three studies were prepared 1662

and read by one laboratory

, and reported findings ( although minimal ) were much more consistent. 1663

1664

Similar anomalies were observed with liver pathology for the prochloraz studies. Lab A

, B and 1665

D studies were read by the sa me contracted pathologist where as Lab C study was read by a 1666

different contracted pathologist. It is interesting to point out that similar treatment

- relate d liver 1667

pathologies were re ported for Labs A

, B and D wh ereas a different spectrum of treatment

- related 1668

pathologies were reported for Lab C . It is worth noting that the animals in the C stud y showed 1669

feeding

- related reduced juvenile growth rates as compared to the other studies. It is possible that 1670

disease issues may have factored into the apparent inconsistencies in liver finding s

. Regardless

, 1671

developing a pathology guidance document for both the liver and kidney is critical . The samples 1672

from these studies will provide the necessary ex ample s for development of such a guidance 1673

document. 1674

1675

T

4 

# Serum Measures 1676

1677

The B

, C and D laboratories used commercially available ELISA kit s specific for human or 1678

canine T 4



# . These laboratories either did not attempt to optimize or failed in any such effort. As 1679

such

, the laboratories were unsuccessful in mak ing these measurements and th e s e data were not 1680

included in this analysis. Lab A measured T 4 

# levels in serum using an HPLC/ICP

- MS method 1681

# 47

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

., 1682

2012 ). Additionally, Lab A has develop ed an extraction method which allow s for the use of 1683

these commercially available ELISA kits ( See LAGDA Guideline A ppendix 7 ) with X. laevis 1684

seru m. In the Lab A OP study

, both methods were used and compared. 1685

1686

Unfortunately, it is not pos sible to make inter

- laboratory comparison s of the effects of the test 1687

chemicals on T 4 

# since studies that used appropriate methods for measuring T 4 

# were single 1688

laboratory studies. However, the collection of control data indicate s that both methods are 1689

rep roducible . Further

, these methods were effective in accurately identifying alteration s in T 4



# . 1690

In the future, labo ratories conducting the assay must demonstrate a competence in making this 1691

measurement. These studies should serve as a foundation for estab lishing performance criteria 1692

for this endpoint. 1693

1694

V

itellogenin ( VTG ) 1695

1696

Although the VTG ELISA registers a measurable signal in control males and females

, this 1697

should be interpreted with caution. These values are more likely background noise within the 1698

assay. P Z

, OP , and BP

- 2 exposure s resulted in a concentration

- depende nt significant increase in 1699

plasma VTG. However, the BP

- 2 data set provides the best re presentation of the magnitude of 1700

induction that can occur with estrogenic chemical exposure in X. lae vis . Further, with the level 1701

of induction exhibited in the BP

- 2 study , CVs approach ed more acceptable values. Taken 1702

together, t his emphasizes that VTG da ta from all of the other studies are likely background le vels

, 1703

and VTG induction as a reproductive endpoint in this assay should be interpreted in the context 1704

of overall potential magnitude of response. 1705

1706



# 6.4 Conclusions 1707

1708

The LAGDA protocol describes a chron ic toxicity test with an amphibian species that considers 1709

growth and development from fertilization through the early juvenile period

. Individual and 1710

i nter

- laboratory evaluations of the LADGA were conducted to evaluate the practical 1711

transferability of the assay protocol a nd quantitative reproducibility of the results. 1712

1713

Although the results of the inter

- laboratory validation were mixed, the LAGDA was an effective 1714

test model with endocrine

- related effects characterized for all four chemicals evaluated

. Ove rall, 1715

the authors conclude that the Tier 2 LAGDA is capable of sufficiently characterizing potential 1716

disruption of the endocrine system by putative e ndocrine d isrupting c hemicals. However, careful 1717

attention to recommendations in the Test Guideline is nece ssary to ensure consistent and reliable 1718

findings across multiple testing laboratories. 1719



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1939

Villalpando I, Merchant

- Larios H. 1990. Determination of the sensitive stages for gonadal sex

1940

reversal in Xenopus laevis tadpoles. Int J Dev Biol 34(2):281

- 285. 1941

1942

Villeneuve DL, Ankley GT , Makynen EA, Blake LS, Greene KJ, Higley EB, Newsted JL, Giesy 1943

JP, Hecker M. 2007. Comparison of fathead minnow ovary explant and H295R cell

- based 1944

steroidogenesis assays for identifying endocrine

- active chemicals. Ecotoxicology and 1945

Environmental Safety 68 (1):20

- 32. 1946

1947

Vinggaard AM, Christiansen S, Laier P, Poulsen ME, Breinholt V, Jarfelt K, Jacobsen H, 1948

Dalgaard M, Nellemann C, Hass U. 2005. Perinatal exposure to the fungicide prochloraz 1949

feminizes the male rat offspring. Toxicological Sciences 85(2):886

- 897. 1950

1951

Wake MH, Dickie R. 1998. Oviduct structure and function and reproductive modes in 1952

amphibians. Journal of Experimental Zoology 282(4

‐ 5):477

- 506. 1953

# 53

1954

Weisbrod CJ, Kunz PY, Zenker AK, Fent K. 2007. Effects of the UV filter benzophenone

- 2 on 1955

reprod uction in fish. Toxicology and A pplied P harmacology 225(3):255

- 266. 1956

1957

Ying G

- G, Williams B, Kookana R. 2002. Environmental fate of alkylphenols and alkylphenol 1958

ethoxylates

— a review. Environment International 28(3):215

- 226. 1959

1960

Yoshimoto S, Okada E, Umemoto H, Tamura K, Uno Y, Nishida

- Umehara C, Matsuda Y, 1961

Takamatsu N, Shiba T, Ito M. 2008. A W

- linked DM

- domain gene, DM

- W, participates in 1962

primary ovary development in Xenopus laevis . Proceedings of the National Academy of Sciences 1963

105(7):2469

- 2474. 1964

1965

1966

1967

1968

1969

1970

1971

1972

1973

1974

1975

# 54



# 8 APPENDICES 1976

1977



# 8.1 LAGDA Guideline 1978

1979



# GUIDELINE FOR THE TESTING OF CHEMICALS 1980

1981

The Larval Amphibian Growth and Development Assay 1982

1983

INTRODUCTION 1984

1985

The need to develop and validate an assay capable of identifying and characterizing the adverse 1986

consequences of exposure to toxic substances, in amphibians, originates from concerns that 1987

environmental levels of chemicals may cause adverse effects in both humans and wildlife. The 1988

Food Quality Protection Act of 1996 requires EPA to develop and implement a program using 1989

valid tests for determining the potential endocrine effects from pesticides. The EPA established 1990

an advisory group, the Endocrine Disruptor Screening and Testing Advisory Committee 1991

(EDSTAC), to obtain advice on developing th is p rogram. In accordance with EDSTAC’s 1992

recommendations, EPA proposed a two

- tiered approach to the program: Tier 1 would identify 1993

the potential of a substance to interact with the endocrine system whereas Tier 2 would confirm 1994

the interaction and characterize the effects. One of the Tier 2 tests recommended by EDSTAC is 1995

an amphibian full life cycle test to evaluate the adverse consequences of putative endocrine 1996

disrupting chemicals, especially those active within the thyroid and reproductive systems, on the 1997

de velopment, growth and reproduction of amphibians. 1998

1999

PRINCIPLE OF THE TEST 2000

2001

The Larval Amphibian Growth and Development Assay (LAGDA) is intended to identify and 2002

characterize the adverse consequences of exposure to substances which interfere with the norma l 2003

development and growth of amphibians through larval development and metamorphosis . It is an 2004

important assay to address potential inciting contributors to amphibian population declines by 2005

evaluating the effects from exposure to contaminants, both through endocrine and non

- endocrine 2006

mechanisms, during the larval stage that may adversely affect populations . It should be noted 2007

that to date, no validated assay exists that serves this function for amphibians. 2008

2009

The general experimental design entails exposin g NF stage 8 Xenopus la e vis embryos to four 2010

different concentrations of a test chemical and a control until 10 weeks after the median time to 2011

NF stage 62 in the control with one interim sub

- sample at NF stage 62 (See Nieuwkoop and 2012

Faber 1994 for staging de tails) . There are four replicates in each test concentration with eight 2013

replicates for the control . Endpoints evaluated during the course of the exposure include those 2014

indicative of generalized toxicity: mortality, abnormal behavior, and growth determina tion s 2015

(length and weight) , as well as endpoints designed to characterize specific endocrine toxicity 2016

modes of action targeting estrogen

- , androgen

- , or thyroid

- mediated physiological processes. 2017

2018

2019

# 55

DESCRIPTION OF THE METHOD S 2020

2021

Environmental Conditions 2022

2023

The inflow of water is provided to tanks housing tadpoles and post

- metamorphic juvenile frogs 2024

at a rate of ≥10 tank additions per day (4.0

– 10.0 L tanks) . Water temperature is maintained at 2025

21 ±1 ° C with a photoperiod of 12 hours of light and 12 hours of dark. Dissolved oxygen (%), 2026

tank temperature, and pH are measured twice weekly. C onductivity (μS), alkalinity (mg/L of 2027

CaCO 3 

# ), and hardness (mg/L of CaCO 3 

# ) are measured once a month

. For post

- metamorphic 2028

juvenile frogs, humidity levels should be held above 3 0%. The occurrences of lesions on the 2029

head just posterior to the nostrils are indications of insufficient humidity levels. 2030

2031

Exposure tanks should be siphoned on a daily basis to remove uneaten food and waste products. 2032

Care should be used to minimize str ess and trauma to the animals , especially during movement, 2033

cleaning of aquaria, and manipulation. Stressful conditions/activities should be avoided such as 2034

loud and/or incessant noise, tapping on aquaria, vibrations in the aquaria, excessive activity in 2035

t he laboratory, and rapid changes in environmental media (light availability, temperature, pH, 2036

DO, water flow rates, etc .). 2037

2038

Feeding 2039

2040

Feed and feeding rates change with life stages of Xenopus , which is unique to this test species 2041

and is a very important aspect of the LAGDA protocol. Excessive feeding during the larval 2042

phase typically results in increased incidences and severity of scoliosis and should be avoided. 2043

Conversely, inadequate feeding during the larval phase results in highly variable developmental 2044

rates among controls potentially compromising statistical power or confounding test results. 2045

Therefore, adherence to the provided feeding regime is strongly a dvised ( Appendix 2 ). 2046

2047

Larvae 2048

2049

The larval diet consists of Silver Cup Trout Starter (Nelson and Sons, Murray, UT, USA), 2050

Spirulina algae discs (Wardley, Secaucus, NJ, USA) and TetraFin® flakes (Tetra Sales, 2051

Blacksburg, VA, USA) blended together in culture water. This mixture is fed three times daily 2052

on weekdays and once daily on weekends. Tadpoles are also fed 24

- hour old live brin e shrimp 2053

(Bio

- Marine® Brand, Bio

- Marine, Hawthorne, CA, USA) twice daily on weekdays and once 2054

daily on the weekends starting on day 8 post

- fertilization. Sera Micron® (Sera GmbH, 2055

Heinsberg, Germany) can be used in place of the diet previously described bu t feeding rates need 2056

to be adjusted to meet control performance criteria. 2057

2058

Juvenile 2059

2060

Once metamorphosis is complete, the feeding regime consists of 3/32” premium sinking frog 2061

food by Xenopus Express. For juveniles, the pellets are briefly run in a c offee grinder or blender 2062

in order to reduce the ir size. Appendix 2 provides information regarding the type and amount of 2063

feed used during the juvenile life stage. The animals should be fed once per day. 2064

2065

# 56

Control organisms must meet all of the following pe rformance criteria in order to ensure 2066

reproducibility and transferability of the LAGDA: 2067

2068

1) The mean time to NF stage 62 is ≤ 45 days. 2069

2) The mean weight at NF stage 62 is 1.0±0.2 grams. 2070

3) The mean juvenile weight at test termination is 11.5 ±3 grams. 2071

2072

Che mical Testability 2073

2074

The LAGDA is based upon an aqueous exposure protocol whereby the test chemical is 2075

introduced into the test chambers via a flow

- through system. Flow

- through methods however, 2076

introduce constraints on the types of chemicals that can be tested, as determined by the 2077

physicochemical properties of the compound. Therefore, prior to using this protocol, baseline 2078

information about the che mical should be obtained that is relevant to determining its testability. 2079

Both the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 2080

Mixtures and the USEPA Guidance Document on Special Considerations for Conducting 2081

Aquatic Lab oratory Studies should be consulted (OECD 2000 ; OCSPP 850.1000 )

. 2082

Characteristics which indicate that the chemical may be difficult to test in aquatic systems 2083

include: high octanol

- water partitioning coefficients (log K

ow 

# ), high volatility, susceptibility to 2084

hydrolysis, and susceptibility to photolysis under ambient laboratory lighting conditions. Other 2085

factors may also be relevant to determining testability a nd should be determined on a case by 2086

case basis. If a successful test is not possible for the chemical using a flow

- through test system, 2087

then it is recommended that this chemical not be tested using this protocol. 2088

2089

Exposure System 2090

2091

A flow

- through dilute r system is required for this protocol . The system components should have 2092

water

- contact components of glass, stainless steel, and/or Teflon ® 

# . However, suitable plastics 2093

can be utilized if they do not compromise the study. Exposure tanks should be glass or stainless 2094

steel aquaria, equipped with standpipes that result in an approximate tank volume between 4.0 2095

and 10.0 L and minimum water depth of 10 to 15 cm. The system should be capable of 2096

supporting all exposure concentrations, a control, and a vehicle control, if necessary, with four 2097

replicates per treatment (eight in the control) . The flow rate to each tank should be constant in 2098

consideration of both the maintenance of biological conditions and chemical exposure. It is 2099

recommended that flow rates acc ommodate at least 10 tank turnovers per day to avoid chemical 2100

concentration declines due to metabolism by both the test organisms and aquatic microorganisms. 2101

The treatment tanks should be randomly assigned to a position in the exposure system to reduce 2102

po tential positional effects, including slight variations in temperature, light intensity, etc

. 2103

Fluorescent lighting should be used to provide a photoperiod of 12 hr light and 12 hr dark at an 2104

intensity that ranges from 600 to 2,000 lux (lumens/m 2 

# ) at the w ater surface. Water temperature 2105

should be maintained at 2 1 o ± 1°C, pH maintained between 6.5 to 8.5, and the dissolved oxygen 2106

(DO) concentration maintained at >3.5 mg/L (> 4 0% of the air saturation) in each test tank. At a 2107

minimum, water temperature, pH a nd dissolved oxygen should be measured twice a week

. 2108

Temperature should be measured continuously in at least one test vessel. Humidity within the 2109

system should be kept above 30%. Appendix 1 outlines the experimental conditions under 2110

which the protocol should be executed. For further information on setting up flow

- through 2111

# 57

exposure systems, please refer to the ASTM Standard Guide for Conducting Acute Toxicity 2112

Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians (ASTM 2002) and 2113

general aquatic toxicology texts. 2114

2115

Water Quality 2116

2117

Any water that is locally available ( e.g . spring water or charcoal

- filtered tap water) and permits 2118

normal growth and development of X. laevis can be used. Because local water quality can differ 2119

substantially from one area to another, analysis of water qua lity should be undertaken, 2120

particularly, if historical data on the utility of the water for raising amphibian larvae is not 2121

available. Special attention should be given that the water is free of copper, chlorine and 2122

chloramines, all of which are toxic to amphibians . It is further recommended to analyze the 2123

water to determine background levels of estrogenic and androgenic chemicals, fluoride, 2124

perchlorate and chlorate (by

- product of drinking water disinfection) as all of these may confound 2125

the study outcome . Analys e s should be performed before testing begins and the testing water 2126

should normally be free from these chemicals. 2127

2128

Iodide Concentration in Test Water 2129

2130

In order for the thyroid gland to synthesize thyroid hormones to support normal metamorphosis, 2131

sufficient iodide needs to be available to the larvae through a combination of aqueous and dietary 2132

sources. Currently, there are no empirically derived guidelines for minim um iodide 2133

concentrations. However, iodide availability may affect the responsivene ss of the thyroid system 2134

to thyroid active agents and is known to modulate the basal activity of the thyroid gland, an 2135

aspect that deserves attention when interpreting the results from thyroid histopathology. 2136

Therefore, measured aqueous iodide concentrati ons from the test water should be reported. 2137

Based on previous work, the protocol has been demonstrated to work well when test water iodide 2138

(I



# ) concentrations ranged between 0.5 and 10 μg/L. Ideally, the minimum iodide concentration 2139

in the test water sho uld be 0.5 μg/L. If the test water is reconstituted from deionized water, 2140

iodine must be added at a minimum concentration of 0.5 μg/L. Supplementation of the test water 2141

with iodine or other salts must be noted in the report. 2142

2143

Analytical Chemistry 2144

2145

Prior to conducting a study, the stability of the test compound should be evaluated using existing 2146

information on its solubility, degradability, and volatility. Test solutions from each replicate 2147

tank at each concentration should be sampled for analytical chem istry analyses at test initiation 2148

(day 0), and weekly throughout the exposure period. It is also recommended that each test 2149

concentration be analyzed during system preparation, prior to test initiation, to verify system 2150

performance and that stock solution s be analyzed when they are changed, especially if the 2151

volume of the stock solution does not provide adequate amounts of chemical to span the duration 2152

of routine sampling periods. In the case of chemicals which cannot be detected at some or all of 2153

the con centrations used in a test, stock solutions should be measured and system flow rates 2154

recorded in order to calculate actual concentrations. 2155

2156

Chemical Delivery 2157

# 58

2158

The method used to introduce the test chemical to the system can vary depending on its 2159

physicoch emical properties. Water soluble compounds can be dissolved in aliquots of test water 2160

at a concentration which allows delivery at the target test concentration in a flow

- through system. 2161

Chemicals which are liquid at room temperature and moderately solubl e in water can be 2162

introduced using liquid:liquid saturator methods. Chemicals which are solid at room temperature 2163

and are moderately soluble in water can be introduced using glass wool column saturators (Kahl 2164

et al. 1999) . The preference is to use a carrier

- free test system; however

, different test chemicals 2165

will possess varied physicochemical properties that will likely require different approaches for 2166

preparation of chemical exposure water. All efforts should be made to avoid solvents or carriers 2167

because: (1) certain solvents themselves may result in toxicity and/or undesirable or unexpected 2168

responses, (2) testing chemicals above their water solubility (as can frequently occur through the 2169

use of solvents) can result in inaccurate determinations of effective concentrations, and (3) the 2170

use of solvents in longer

- term tests can result in a significant degree of “biofilming” associated 2171

with microbial activity. For difficult to test substances, a solvent may be employed as a last 2172

resort, and the OECD Gu idance Document on Aquatic Toxicity Testing of Difficult Substances 2173

and Mixtures should be consulted (OECD 2002) to determine the best method. The choice of 2174

solvent will be determined by the chemical properties of the substance. Solvents which have 2175

bee n found to be effective for aquatic toxicity testing include acetone, ethanol, methanol, 2176

dimethyl formamide and triethylene glycol. In case a solvent carrier is used, solvent 2177

concentrations should be below the chronic No Observed Effect Concentration (NOE C); the 2178

OECD Guidance Document recommends a maximum of 100 μL/L and a recent review 2179

recommends that solvent concentrations as low as 20 μL/L of dilution water be used (Hutchinson 2180

et al. 2006) . If solvent carriers are used, appropriate solvent controls (clean water) must be 2181

evaluated in addition to non

- solvent controls. I f it is not possible to administer a chemical via 2182

the water, either because of physicochemical characteristics (low solubility) or limited chemical 2183

availability, introducing it via the diet may be considered. Preliminary work has been conducted 2184

on dietary exposures; however, this route of exposure is not commonly used. The choice of 2185

method should be documented and analytically verified. 2186

2187

Establishing the High Test Concentration 2188

2189

For the purposes of this test, results from Tier I amphibian studies should be used to what extent 2190

possible in determining the high test concentration so as to avoid concentrations that are overtly 2191

toxic. Prior to running the LAGDA a range finding experiment should be conducted. The 2192

exposure should be initiated within 24 hours o f fertilization and continue for 7

- 10 days. The test 2193

concentrations should be set such that the intervals between test concentrations are no greater 2194

than 1 log. The results of the range finding experiment should serve to set the highest test 2195

concentratio n in the LAGDA. Once the highest test concentration is established a 50% dilution 2196

series is to be used to establish the 3 lower test concentrations. 2197

2198

Test Concentration Range 2199

2200

There is a required minimum of four test concentrations and a clean water co ntrol (and vehicle 2201

control if necessary). The minimum test concentration differential between the highest and 2202

# 59

lowest should be about one order of magnitude. The maximum concentration separation should 2203

be no greater than 0.5. 2204

2205

PROCEDURE 2206

2207

Assay Overview 2208

2209

This assay is intended for use in determining the chronic effects of chemicals on amphibian 2210

larval growth and development. The assay is initiated with newly spawned larvae and continues 2211

into juvenile development. Animals are examined daily for mortality and any signs of abnormal 2212

behavior. At NF stage 62, a sub

- sample of animals is collected and various endpoints are 2213

examined (see Table 1). After all animals have reached NF stage 66, a cull is carried out to 2214

reduce the number of animals per tank, and the remaining animals continue exposure until 10 2215

weeks after the median time to NF stage 62 in the control. At test termination additional 2216

measurements are collected (see Table 1). 2217

2218

Table 1 . Timing of apical endpoint measurements ov er the course of the LAGDA. 2219

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 ( T 4 

# ) 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

Test Initiation 2221

2222

It is preferred that animals for test initiation have previously been shown to produce offspring 2223

that can be genetically sexed (see Appendix 3). After spawning of adults, embryos are collected, 2224

cysteine treated to remove the jelly coat and screened for viability according to ASTM standards 2225

(A STM 2004). Cysteine treatment allows the embryos to be handled during screening without 2226

sticking to surfaces. Screening takes place under a dissecting microscope using an appropriately 2227

sized eye dropper to remove non

- viable embryos. It is preferred that a spawn resulting in greater 2228

than 70% viability be used for the test. Embryos from a single spawn are randomly distributed 2229

into exposure treatment tanks containing an appropriate volume of dilution water until each tank 2230

contains 20 embryos

. Embryos shou ld be carefully handled during this transfer in order to 2231

minimize handling stress and to avoid any injury. At 96 hours post spawning, the tadpoles should 2232

have moved up the water column and begun clinging to the sides of the tank. 2233

# 60

2234

Mortality 2235

2236

All test ta nks should be checked daily for dead animals and the numbers recorded for each tank. 2237

The date, concentration and tank number for any observation of mortality is recorded. Dead 2238

animals should be removed from the test tank as soon as observed. The developm ental stage of 2239

dead animals should be categorized as either pre

- NF stage 58 (pre

- forelimb emergence), NF 2240

stage 58

- NF stage 62, NF stage 63

- NF stage 66 (between NF 62 and complete tail absorption), or 2241

post

- NF stage 66 (post

- larval ). Mortality rates exceedi ng 20% may indicate inappropriate test 2242

conditions or overtly toxic effects of the test chemical. During the first few days of development 2243

after the spawning event and during metamorphic climax, the animals tend to be most sensitive 2244

to non

- chemical induced mortality events. 2245

2246

Additional Observations 2247

2248

The date, treatment, and tank number for any observation of abnormal behavior, grossly visible 2249

malformations ( e.g. scoliosis) , or lesions should be recorded. Normal behavior for larval animals 2250

is characterized by suspension in the water column with tail elevated above the head, regular 2251

rhythmic tail fin beating, periodic surfacing, operculating, and being responsive to stimul i

. 2252

Abnormal behaviors would include, for example, floating on the surface, lying on th e bottom of 2253

the tank, inverted or irregular swimming, lack of surfacing activity, and being nonresponsive to 2254

stimul i . For post

- metamorphic animals , in addition to the above abnormal behaviors , gross 2255

differences in food consumption between treatments shoul d be recorded. Gross malformations 2256

and lesions could include morphological abnormalities ( e.g ., limb deformities), hemorrhagic 2257

lesions, abdominal edema, and bacterial or fungal infections, to name a few. These 2258

determinations are qualitative and should be considered akin to clinical signs of disease/stress 2259

and made in comparison to control animals. If the rate of occurrence is greater in exposed tanks 2260

than in the controls, then these should be considered as evidence for overt toxicity. 2261

2262

Larval Sub

- Sample 2263

2264

Those tadpoles that have reached NF stage 62 should be removed from the tanks and either 2265

sampled or moved to the next part of the exposure in a new tank, or physically separated from 2266

the remaining tadpoles in the same tank with a divider. Tadpoles are che cked daily, and the time 2267

at which an individual tadpole reaches NF stage 62 is recorded. The defining characteristic for 2268

use in this assessment is the shape of the head. Once the head has become reduced in size such 2269

that it is the same width as the trunk of the tadpole, then that individual would be counted as 2270

having attained NF stage 62. One tadpole from every four removed from a given tank is 2271

randomly selected for the larval sub

- sampling. Those tadpoles not sampled are moved to new 2272

tanks or physically separated from the remaining tadpoles for continued exposure. The random 2273

procedure used must give each organism on test equal probability of being selected. This can be 2274

achieved by using any randomizing method but requires that each tadpole be netted. 2275

2276

For the larval sub

- sampling, the following endpoints are obtained: 2277

1. Time to NF stage 62 (number of days between fertilization and NF stage 6 2 ) 2278

2. External abnormalities 2279

# 61

3 . Morphometrics (growth

- weight and length) 2280

4 . Thyroid h istology 2281

5. Ser um T 4 

# 2282

2283

Outline of Procedure for Larval Sampling: 2284

2285

1. Euthanize tadpole by immersion in T ricaine Methanesulfonate ( MS

- 222) 2286

appropriately buffered to achieve pH 7.0

. 2287

2 . Rinse and blot dry. 2288

3 . Weigh the tadpole (mg) and me asure snout to vent length (mm). 2289

4

. Note any gross morphologic abnormalities and /or clinical signs of toxicity

. 2290

5. C ollect blood from the heart using a non

- heparinized capillary tube for serum 2291

collection (~25 μL )

. 2292

6. Remove and discard the lower torso below the forelimbs. 2293

7. Fix the upper torso for 48 hours in Davidson’s solution with agitation and then rinse 2294

with tap water and store tissues in 10% NBF

. 2295

2296

Euthanasia 2297

2298

NF stage 62 tadpoles should be euthanized by immersion in a 0.01% MS

- 222 solution (500 mL). 2299

This solution should be buffered with sodium bicarbonate to a pH of approximately 7.0. 2300

(Unbuffered MS

- 222 solution is acidic and irritating to frog skin resulting is poor absorption and 2301

unnecessary additional stress to the organisms.) The characteristi cs of the water used to make 2302

this solution may impact the pH and therefore the amount of sodium bicarbonate needed to 2303

neutralize this solution. In general, however, a recipe of one part MS

- 222 to two parts of sodium 2304

bicarbonate should be used as a startin g point. 2305

2306

Using a mesh dip net, a tadpole is removed from the experimental chamber and transported to 2307

the necropsy area in a transport container. The tadpole is placed into the euthanasia solution. It 2308

is ready for necropsy when it is unresponsive to exte rnal stimuli such as pinching the hind limb 2309

with a pair of forceps. 2310

2311

Growth: Weight and Length 2312

2313

Measurements of wet weight and snout to vent length for each tadpole (see Figure 1) should be 2314

made immediately after it becomes non

- responsive. These measurem ents are included in the test 2315

protocol to assess possible effects of test substances on the growth rate of organisms and are 2316

useful in detecting generalized toxicity. Tadpoles should be blotted dry before weighing to 2317

remove excess adherent water. After m easurements of body size are made, any gross 2318

morphological abnormalities and/or clinical signs of toxicity should be noted, recorded, and any 2319

digital documentation made. These include scoliosis, petechiae, hermorrhage, etc

. 2320

2321

a. Larval sub

- sampling (NF sta ge 62) 2322

2323

# 62

2324

2325

2326

b. NF stage 66 cull and juvenile sampling 2327

2328

Figure 1. Landmarks for measuring snout to vent length for the LAGDA. 2329

2330

2331

Blood Collection for T 4 

# Analyses 2332

2333

Blood is collected from the heart using a non

- heparinized capillary tube for serum collection 2334

(~25 μL ) . The tube is sealed ( e.g ., with Crit

- o

- seal ® 

# ) and placed on ice until clots form (1

- 4 hrs.), 2335

at which time the tubes should be centrifuged to remove the clots from the serum. The capillary 2336

tube is scored and broken at the serum

- clot interface, the volume of se rum collected for each 2337

individual is recorded, and the serum is expelled into a microcentrifuge tube. Serum samples 2338

from individuals are stored at

- 80°C for subsequent quantification of T 4 

# . Given the limited 2339

volume of blood that can be collected from an individual NF stage 62 tadpole, analyses of 2340

circulating T 4 

# has to be performed on pooled samples consisting of serum collected from all 2341

individuals in a tank ( i.e ., replicate). A pooled sample is formed by combining equal volumes of 2342

serum from every indiv idual in a tank. The volume of serum contributed by each individual to 2343

the pool is equal to the least volume of serum collected among all individuals in a tank. 2344

Professional judgment should be used to exclude samples with low yields of collected serum du e 2345

to technical difficulties. 2346 Snout

- Vent Length 

# Snout vent length

# 63

2347

Tissue Collection and Fixation for General Histology 2348

2349

For the sub

- sampling, t hyroid glands are assessed for histology

. The lower torso below the 2350

forelimbs is removed and discarded. The trimmed carcass is placed into individually labelled 2351

histological cassettes and fix ed in Davidson’s fixative. The volume of fixative in the container 2352

should be at least 10 times the approximated volume of the tissues. Appropriate agitation or 2353

circulation of the fixative should be achi eved to adequately fix the tissues of interest. All tissues 2354

remain in Davidson’s fixative for at least 48 hours, but no longer than 96 hours at which time 2355

they are rinsed in deionized water and stored in 10% NBF. 2356

2357

Davidson’s Fixative: 2358

a) Glacial acetic acid...............115 ml 2359

b) Formaldehyde (35

- 40%)........220 ml 2360

c) 95% Ethyl Alcohol...............330 ml 2361

d) Distilled water....................335 ml 2362

2363

10% Neutral Buffered Formalin: 2364

a) 100 ml formalin, full strength (37

- 40% formaldehyde) 2365

b) 6.5 g sodium phosphate dibasic (anhy drous) 2366

c) 4.0 g sodium phosphate monobasic 2367

d) 900 ml distilled water 2368

2369

Refer to Appendix 5 for procedural information on processing tissues for histological 2370

examinations and Appendix 6 (Appendix 8.3 of ISR) for information regarding the interpretation 2371

o f diagnostic criteria. 2372

2373

End of Larval Exposure 2374

2375

Given the initial number of tadpoles, it is expected that there will likely be a small percentage of 2376

individuals that do not develop normally and do not complete metamorphosis in a reasonable 2377

amount of time

. The larval portion of the exposure should not exceed 70 days. Any tadpoles 2378

remaining at the end of this period should be euthanized by immersion in 0.0 2% buffered MS

2379

222 (pH 7.0) . They should be weighed, staged (Nieuwkoop and Faber, 1994) , and any 2380

devel opmental abnormalities noted. 2381

2382

Cull after NF stage 66 2383

2384

Ideally, only 10 individuals per tank should continue until termination of the exposure. 2385

Therefore, after all animals have reached NF stage 66 or after 70 days (whichever occurs first), a 2386

cull should be conducted. Animals that will not continue the expos ure should be selected at 2387

random. These animals are euthanized by immersion in a 0.0 2 % MS

- 222 solution (500 mL) 2388

buffered with sodium bicarbonate to a pH of approximately 7.0. Measurements of wet weight 2389

and snout to vent length (see Figure 1) for each fro glet are made, and a gross necropsy is 2390

conducted. The phenotypic sex (based on gonad morphology) is noted as female, male, or 2391

indeterminate. 2392

# 64

2393

Juvenile Sampling 2394

2395

At the end of the exposure period, the remaining animals are euthanized

, and the following 2396

en dpoints are evaluated and recorded: 2397

2398

1. Morphometrics (growth

- weight and length) 2399

2 . Phenotypic/genotypic sex ratios 2400

3. Plasma vitellogenin 2401

4

. Gonad histopathology 2402

5. Reproductive duct histopathology 2403

6. Liver histopathology 2404

7

. Liver weight (Liver

- somatic Index) 2405

8. Kidney histopathology 2406

Methods: 2407

2408

1. Euthaniz e frogs by injection of buffered 10% MS

- 222 in PBS (0.01 ml per g frog). 

# 2409

2. Measure whole body weight (g) and snout

- vent length (mm). 2410

3. Record any developmental abnormalities and obvious signs of disease ( e.g . scoliosis, 2411

hemorrhage). 2412

4

. Place the frog in dorsal recumbency

. 2413

5. Expose the heart by removing the skin and associated cartilage. 2414

6 . Carefully expose the heart by opening the pericardial sac without cutting any major 2415

blo od vessels. 2416

7

. Snip a major blood vessel coming from the heart and collect blood using a heparinized 2417

capillary tube for plasma collection (~50 μL )

. 2418

8 . Open the abdominal cavity. Dissect out, weigh, and fix the liver. 2419

9 . Carefully remove the digestive organs ( e.g . stomach, intestines) from the lower 2420

abdomen. 2421

10

. Assess gross morphological abnormalities in gonads. 2422

11. Remove a foot for a DNA sample and snap freeze in liquid nitrogen or dry ice

. 2423

12. Remove hind limbs and fix remaining carcass. 2424

2425

Euthanasia 2426

2427

Post

- metamorphic frogs are euthanized by an intraperitoneal injection of 10% MS

- 222 in an 2428

appropriately buffered solution ( e.g ., phosphate buffered solution). Dosage for frogs is 0.01 mL 2429

per gram of frog. This can be approximated to 0.1 mL for a typical male

- sized individual and 2430

0.2 mL for a typical female

- sized individual. Using this procedure, frogs may be sampled after 2431

becoming unresponsive, usually around 2 min after injection. Samples should be collected from 2432

frogs shortly after euthanasia . Therefore, unless multiple prosectors are available, numerous 2433

frogs should not be euthanized simultaneously. 2434

2435

Growth: Weight and Length 2436

2437

Measurement of weight and length are identical to th ose outlined for the larval sub

- sampling. 2438

# 65

2439

2440

Blood Collection for Vitellogenin Analyses 2441

2442

The euthanized juveniles are placed in dorsal recumbency. The heart is exposed by removing the 2443

skin and cartilage that covers it. The pericardial sac is opened and th e heart exposed. In order 2444

for adequate blood collection to be made, it is imperative that care be taken not to cut or tear any 2445

blood vessels during this process. The exposed heart should still be beating at this point. A 2446

major blood vessel at the top of the heart is snipped, and blood is collected with a heparinized 2447

capillary tube. The tube is sealed ( e.g ., with Crit

- o

- seal ® 

# ) and placed on ice until the samples can 2448

be centrifuged. At the end of the sampling, tubes should be centrifuged to remove the bl ood 2449

cells from the plasma. The capillary tube is scored and broken at the plasma

- cell interface, and 2450

the plasma is expelled into a microcentrifuge tube that has been coated with aprotinin. Plasma 2451

samples are stored at

- 80°C for subsequent quantification of vitellogenin levels with an ELISA. 2452

Vitellogenin is an established estrogen responsive biomarker in a number of fish and frogs 2453

species. If a valid commercially available ELISA kit exists for X. laevis vitellogenin it may be 2454

used. Alternatively, an ELIS A can be developed as described in Appendix 4. 2455

2456

Tissue Collection and Fixation for General Histology 2457

2458

Gonads and livers are collected for histological analysis during the final sampling . The 2459

abdominal cavity is opened, and the liver is dissected out, wei ghed and fixed. Next, the digestive 2460

organs ( e.g . stomach, intestines) are carefully removed from the lower abdomen to reveal the 2461

gonads. Any gross morphological abnormalities in the gonads should be noted. Finally, the hind 2462

limbs should be removed, and the carcass should be fixed with the gonads left in situ

. Collected 2463

livers and carcasses are placed into labelled histological cassettes and fixed in Davidson’s 2464

fixative. The volume of fixative in the container should be at least 10 times the approximate d 2465

volume of the tissues. All tissues remain in Davidson’s fixative for at least 48 hours, but no 2466

longer than 96 hours at which time they are rinsed in de

- ionized water and stored in 10% NBF. 2467

The same fixatives used in the larval sub

- sampling as described previously are used with adult 2468

samples. Refer to Appendix 5 for details on histological procedures

. 2469

2470

Genetic Sexing 2471

2472

DNA samples from frogs can be obtained from any tissue. One hind limb removed during 2473

dissection is easy to collect and store in a microcentrifuge tube. Tissue can be stored at

- 20°C 2474

until DNA isolation. There are several commercially available kits for the isolation of DNA 2475

from tissue that can be used. Testing for the presence or absence of a marker is done by PCR as 2476

described in Appendix 3. Development of the markers used for genetic sexing is described in 2477

Yoshimoto et al . 2008 ( X. laevis ). 2478

2479

2480

2481

2482

2483

2484

# 66

2485

DATA COLLECTION AND REPORTING 2486

2487

All data should be collected using electronic or manual systems which conform to good 2488

laboratory practices (GLP). Study data should include: 2489

2490

1. Test substance: Characterization of the test substance: physical

- chemical p roperties; 2491

information on stability and biodegradability. 2492

2. Chemical observations and data : method and frequency of preparation of dilutions. 2493

Test chemical information includes actual and nominal concentrations of the test 2494

chemical, and in some cases, non

- parent chemical, as appropriate. Test chemical 2495

measurements may be required for stock solutions as well as for test solutions. 2496

3. Solvent (if other than water) : justification of the choice of solvent, and 2497

characterization of solvent (nature, conce ntration used). 2498

4. Test conditions: 2499

a) Operational records: these consist of observations pertaining to the functioning 2500

of the test system and the supporting environment and infrastructure. Typical 2501

records include: ambient temperature, test temperat ure, photoperiod, status of 2502

critical components of the exposure system ( e.g . pumps, cycle counters, 2503

pressures), flow rates, water levels, stock bottle changes, and feeding records. 2504

General water quality parameters include: pH, dissolved oxygen , conductivity, 2505

total iodine, alkalinity, and hardness. 2506

b) Deviations from the test method: this information should include any 2507

information or narrative descriptions of deviations from the test method. 2508

5. Results: 2509

a) Biological observations and da ta: these include daily observations of mortality, 2510

food consumption, abnormal swimming behavior, lethargy, loss of equilibrium, 2511

malformations, lesions, etc . Observations and data collected at predetermined 2512

intervals include: growth (weight and length), cl inical signs of disease or general 2513

toxicities, time to metamorphosis, T 4 

# and vitellogenin measurements, and genetic 2514

sex

. 2515

b) Histological data including narrative descriptions, as well as graded severity 2516

and incidence scores of specific observations. 2517

6. Statistical analyses: Statistical analytical techniques and justification of techniques 2518

used; results of the statistical analysis preferably in tabular form. 2519

7. Ad hoc observations : these observations should include narrative descriptions of the 2520

study that do not fit into the previously described categories. 2521

2522

2523

2524

2525

2526

2527

2528

2529

2530

# 67

2531

PERFORMANCE CRITERIA AND TEST ACCEPTABILITY/VALIDITY 2532

2533

Performance Criteria 2534

2535

Generally, gross deviations from the test method will result in unacceptable data for 2536

interpretation or reporting. Therefore, the following criteria in Table 2 have been developed as 2537

guidance for determining the quality of the test performed and the gener al performance of the 2538

control organisms. 2539

2540

2541

Table 2 . Performance criteria for the LAGDA. 2542

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 ≤ 4 5 days,

and the mean weight at NF stage 6 2 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 T arget temperature ± 1oC

- the inter

replicate/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

Test Validity 2551

2552

The following requirements must be met to deem a test acceptable/valid: 2553

1. For any given treatment (including controls), mortality cannot exceed 20%, otherwise 2554

the replicate is considered compromised. 2555

2. At least two treatment levels, with all four uncompromised replicates, must be 2556

available for analysis. 2557

3. At least two treatment levels without overt toxicity must be available for analysis. 2558

2559

STATISTICAL ANALYSES 2560

2561

Statistical analyses of the data should preferably follow procedures described in the document 2562

Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application 2563

(OECD 2006)

. Data for continuous endpoin ts should be assessed for normality (preferably using 2564

the Shapiro

- Wilk or Anderson

- Darling test) and variance homogeneity (preferably using the 2565

Levene test). Both tests are performed on the residuals from an ANOVA. Expert judgment can 2566

be used in lieu of these formal tests for normality and variance homogeneity, though formal tests 2567

are preferred. Where non

- normality or variance heterogeneity is found, a normalizing, variance 2568

stabilizing transformation should be sought. If the data (perhaps after a transf ormation) are 2569

normally distributed with homogeneous variance, a significant treatment effect is determined 2570

from Dunnett’s test. If the data (perhaps after a transformation) are normally distributed with 2571

heterogeneous variance, a significant treatment effe ct is determined from the Tamhane

- Dunnett 2572

or T3 test or from the Mann

- Whitney

- Wilcoxon U test. Where no normalizing transformation 2573

can be found, a significant treatment effect is determined from the Mann

- Whitney

- Wilcoxon U 2574

test using a Bonferroni

- Holm adj ustment to the p

- values. The Dunnett test is applied 2575

independently of any ANOVA F

- test and the Mann

- Whitney test is applied independently of any 2576

overall Kruskall

- Wallis test. In addition, regardless of the results of the previous tests, a step

2577

down Jonck heere

- Terpstra trend (JT) test should also be applied to the data as long as a 2578

monotonic response was observed

. 2579

2580

Mortality data should be analyzed for time periods encompassing larval development and the full 2581

test. Tadpoles that do not complete metamorpho sis in the given time frame, those tadpoles that 2582

are in the larval sub

- sample cohort, those juvenile frogs that are culled, and any animal that dies 2583

due to experimenter error should be treated as censured data. Mortality data as expressed as the 2584

percentag e that died should be arcsine square root transformed prior to analysis. 2585

2586

Time to metamorphosis data should be treated as time to event data, with any mortalities treated 2587

as censured data. Median time to completion of metamorphosis should be determined by 2588

Kaplan

- Meier product

- limit estimators and used in the statistical analysis. 2589

2590

Body weight and snout

- vent lengths should be analyzed using the means of a given tank. Males 2591

and females are not sexually

- dimorphic at the completion of metamorphosis and data f or the 2592

sexes are combined when analyzing larval sub

- sampling data. Male and female juvenile body 2593

size data should be analyzed separately. 2594

2595

# 69

Vitellogenin plasma concentrations should be log transformed before statistical analysis and 2596

separated by sex. 2597

2598

Live r weights should be normalized as proportions of whole body weights (LSI), arcsine square 2599

root transformed and analyzed separately for each sex. 2600

2601

Use of compromised treatment levels 2602

2603

Several factors are considered when determining whether a replicate or entire treatment 2604

demonstrates overt toxicity and should be removed from analysis. Overt toxicity is defined as > 5 2605

mortalities in any replicate that can not be explained by technical error. Other signs of overt 2606

toxicity include hemorrhage, abnormal behavio rs, abnormal swimming patterns, anorexia, and 2607

any other clinical signs of disease. For sub

- lethal signs of toxicity, qualitative evaluations may 2608

be necessary, and should always be made in reference to the clean water control group. 2609

2610

Solvent controls 2611

2612

Th e use of a solvent should only be considered as a last resort, when all other chemical delivery 2613

options have been considered. If a solvent is used, then a clean water control must be run in 2614

concert. At the termination of the test, an evaluation of the po tential effects of the solvent must 2615

be performed. This is done through a statistical comparison of the solvent control group and the 2616

clean water control group. The most relevant endpoints for consideration in this analysis are 2617

growth determinants (weight and length), as these can be affected through generalized toxicities. 2618

If statistically significant differences are detected in these endpoints between the clean water 2619

control and solvent control groups, determine the study endpoints for the response meas ures 2620

using the clean water control. If there is no statistically significant difference between the clean 2621

water control and solvent control for all measured response variables, determine the study 2622

endpoints for the response measures using the pooled dilut ion

- water and solvent controls. 2623

2624

REFERENCES 2625

2626

ASTM (2002). Standard Guide for Conducting Acute Toxicity Tests on Test Materials with 2627

Fishes, Macroinvertebrates, and Amphibians. (A. S. f. T. a. Materials, ed., Philadelphia, PA. 2628

2629

ASTM (2004) . Standard Guide for Conducting the Frog Embryo Teratogenesis Assay

- Xenopus 2630

(FETAX). 2631

2632

Hutchinson, T. H., Shillabeer, N., Winter, M. J., and Pickford, D. B. (2006). Acute and chronic 2633

effects of carrier solvents in aquatic organisms: a critical review. Aqu at Toxicol 76 , 69

- 92. 2634

2635

Kahl, M. D., Russom, C. L., DeFoe, D. L., and Hammermeister, D. E. (1999). Saturation units 2636

for use in aquatic bioassays. Chemosphere 39 , 539

- 551. 2637

2638

Nieuwkoop, P. D. and Faber, J., ( 1994 ) . Normal Table of Xenopus laevis ( Daudin ). Garland 2639

Publishing, Inc, New York, NY. 2640

2641

# 70

OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 2642

Mixtures. 2643

2644

OECD (2002). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 2645

Mixtures. In OECD Guidelines fo r the Testing of Chemicals, Volume 1, Number 5, Paris, France. 2646

2647

OECD (2006). Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance 2648

to Application Environmental Health and Safety Publications, Paris. 2649

2650

USEPA. 1996 Endocrine Disruptor Screening Protram Test Guidelines OPPTS 850.1000: 2651

Special Considerations for Conducting Aquatic Laboratory Studies. Office of Chemical Safety 2652

and Pollution Prevention [OCSPP] formerly Prevention, Pesticides and Toxic Substances 2653

[OPPTS] (7101) E PA 712

- C

- 96

- 113. April 1996. 2654

2655

Yoshimoto, S., Okada, E., Umemoto, H., Tamura, K., Uno, Y., Nishida

- Umehara, C., Matsuda, 2656

Y., Takamatsu, N., Shiba, T., Ito, M. (2008). A W

- linked DM

- domain gene, DM

- W, 2657

participates in primary ovary development in Xenopus la evis. Proc Natl Acad Sci USA 2658

105(7):2469

- 2474. 2659

2660

2661

2662

2663

2664

# 71

Appendix 1: Experimental Conditions for the LAGDA 2665

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

Tes t system flow

- rate Constant

, in consideration of both the

maintenance of biological conditions and

chemical exposure ( e.g

., flow rate sufficient to

pro vide 10 tank volume additions/day)

T est 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 2 000 lux (lumens/m 2 

# ) at the water

surface

Water temperature 2 1 o ± 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

Appendix 2 : Diet P reparation 2671

2672

L arval diet 2673

2674

This diet

, consisting of Trout Starter, algae/TetraFin ® 

# , and brine shrimp , allows control 2675

organisms to meet both of the following criteria: 2676

1) mean time to NF stage 62 is ≤ 45 days, and 2677

2) mean weight at NF stage 62 is ≥ 1.0±0.2 g 2678

2679

Food Preparation 2680

2681

A. 1:1 (v/v) Trout Starter: algae/TetraFin ® 

# : 2682

1. Trout Starter: blend 80 mL of Trout Starter and 300 mL of suitable filtered on high 20 2683

seconds 2684

2. Algae/TetraFin ® 

# mixture: blend 12 g spirulina algae disks and 500 mL filtered LSW on 2685

high for 40 seconds, blend 12 g Tetrafin ® 

# with 500 mL filtered LSW and then combine 2686

these to mak e 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 ® 

# mixture 2688

2689

B. Brine shrimp: Fifteen mL brine shrimp eggs are hatched in 1 L of salt water (prepared by 2690

adding 20 mL of NaCl t o 1 L deionized water). After aerating 24 hours at room temperature 2691

under constant light, the brine shrimp are harvested. Briefly, the brine shrimp are allowed to 2692

settle for 30 min by stopping aeration. Cysts that float to the top of the canister are po ured 2693

off and discarded, and the shrimp are poured through the appropriate filters and brought up to 2694

30 mL with LSW. 2695

2696

Feeding Protocol 2697

Table 3 provides a reference regarding the type and amo unt of feed used during the larval stages 2698

of the exposure . The animals should be fed three times per day Monday through Friday and once 2699

per day on the weekends*. 2700

2701

Table 3. Feeding regime for X. laevis larvae in flow

- through conditions (Trout Starter, 2702

Algae/TetraFin ® 

# Diets, and Brine Shrimp Quantities for 26 tad poles). 2703

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)*

* All numbers in parentheses are feeding rates for the weekend (only fed once/day). Brine shrimp are fed once per 

# 2704

day on the weekend, and the volume remains either 0.5 ml (8

- 15 days) or 1 ml (16 days). 

# 2705

** Brine Shrimp: 

# 2706

Add 0.5 ml of live brine shrimp to the diet twice per day (once per day on the weekend) after 8 days. 

# 2707

Add 1.0 ml of live brine shrimp to the diet twice per day (once per day on the weekend) after 16 days. 

# 2708

***Start feeding when animals are free swimming. 

# 2709

2710

Juvenile diet 2711

2712

Once metamorphosis is co mplete, the feeding regime changes to 3/32” premium sinking frog 2713

food by Xenopus Express. 2714

2715

Food preparation 2716

Pe llets are briefly run in a coffee grinder or blender in order to reduce the size of the pellets. 2717

2718

Feeding protocol 2719

Table 4 provides a reference regarding the type and amount of feed used during juvenile and 2720

adult life stages. The animals should be fed once per day. It should be noted that a s animals 2721

metamorph, they continue receiving a portion of the brine shrimp until >95% animals c omplete 2722

metamorphosis. 2723

2724

Table 4. Feeding regime for X. laevis post

- metamorphic juveniles in flow

- through 2725

conditions. 2726

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

*The first day of week 0 is the median metamorphosis date. 2727

2728

# 74

Appendix 3 : Genetic Sexing 2729

2730

X. laevis primers 2731

2732

DM

- W marker 2733

Forward: 5’

- CCACACCCAGCTCATGTAAAG

- 3’ 2734

Reverse: 5’

- GGGCAGAGTCACATATACTG

- 3’ 2735

2736

Positive Control 2737

Forward: 5’

- AACAGGAGCCCAATTCTGAG

- 3’ 2738

Reverse: 5’

- AACTGCTTGACCTCTAATGC

- 3’ 2739

2740

DNA purification 2741

2742

Purify DNA from muscle or skin tissue using Qiagen DNeasy Blood and Tissue Kit (cat # 69506) 2743

or similar product accordin g to kit instructions. DNA can be eluted from the spin columns using 2744

less buffer to yield more concentrated samples if deemed necessary for PCR. 2745

2746

PCR 2747

2748

Sample protocol using JumpStart TM 

# Taq from Sigma is outlined below: 2749

2750

Master Mix 1x (μL) [Final]

NFW 11

-

10X Buffer 2.0

- MgCl 2 

# (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 p rimer (8 μM) 0.8 0.3 μM

Control rev primer (8 μM) 0.8 0.3 μM

JumpStart TM 

# T aq 0.4 0.05 units/μl

DNA template 1.0 ~200 pg/μl

2751

Note: When preparing Master Mixes, prepare extra to account for any loss that may occ ur while 2752

pipetting (example: 25x should be used for only 24 reactions). 2753

2754

Reaction: Master Mix 1 9.0 μL 2755

Template 1.0 μL 2756

Total 20.0 μL 2757

2758

2759

2760

2761

2762

2763

# 75

Thermocycler Profile: 1. 94 o 

# C 1 min 2764

2. 94 o 

# C 30 sec 2765

3. 60 o 

# C 30 sec 2766

4. 72 o 

# C 1 min 2767

5. Goto step 2. 35 cycles 2768

6. 72 o 

# C 1 min 2769

7. 4 o 

# C 2770

2771

PCR products can be run immediately in a gel or stored at 4oC. 2772

2773

2774

Agarose Gel Electrophoresis (3%) 2775

2776

50X TAE 2777

24.2 g Tris 2778

5.71 mL glacial acetic acid 2779

3.72 g Na 2 

# (EDTA)·2H 2 

# O 2780

Add water to 100 mL 2781

2782

2783

1X TAE 2784

392 mL H 2 

# O 2785

8 mL 50X TAE 2786

2787

3:1 Agarose 2788

3 parts NuSieve agarose GTG 2789

1 part Fisher agarose low EEO 2790

2791

Method 2792

1. Prepare a 3% gel by a dd ing 1.2 g agarose mix to 43 mL 1X TAE. Swirl to disassociate 2793

large clumps. 2794

2. Microwave agarose mixture until completely dissolved (avoid boiling over). Let cool 2795

slightly. 2796

3. Add 1.0μL ethidium bromide (10 mg/mL). Swirl flask. 2797

4. Pour gel into mold with c omb. Cool completely. 2798

5. Add gel to apparatus. Cover gel with 1X TAE. 2799

6. Add 1μL of 6x loading dye to each 10μl PCR product. 2800

7. Pipette samples into wells. 2801

8. Run at 160 constant volts for ~20 minutes. 2802

2803

2804

2805

2806

2807

2808

2809

2810

2811

# 76

LAGDA Appendix 4: Vitellogenin ELISA 

# 2812

2813



# Measurement of Vi tellogenin (Vtg) in Xenopus laevis by ELISA 2814

2815

The procedure is based on an ELISA originally developed for fathead minnow Vtg (Parks et al., 2816

1999, Comp. Biochem. Physiol C 123:113

- 125). See Olmstead et al . (2009) Gen. Comp. 2817

Endocrinol. 160:117

- 123 for a de scription of the assay as modified for Vtg in X

. tropicalis . The 2818

method uses an antibody made against X. tropicalis Vtg, but we know it also works for X. laevis 2819

Vtg

. 2820

2821

Materials and Reagents: 2822

Preadsorbed 1o Antibody (Ab) serum: mix 1 part anti

- X.tropicalis Vtg 1 o Ab serum with 2 parts 2823

control male plasma and leave at RT for ~ 75 minutes, put on ice for 30 min, centrifuge > 20K x 2824

G for 1 h at 4 oC, remove supernatant, aliquot, store at

- 20 oC. 2825

2826

2o Antibody: Goat Anti

- Rabbit IgG

- HRP conjugate ( e.g

. , Bio

- Rad 172

- 1019 ) 2827

2828

Vtg Standard: purified X

. laevis Vtg at 3.3 mg/ml. 2829

2830

TMB (3,3',5,5' Tetramethyl

- benzidine) ( e.g

. , KPL 50

- 76

- 00; or Sigma T0440) 2831

2832

Normal Goat Serum (NGS) ( e.g

. , Chemicon S26

- 100ml) 2833

2834

96 well EIA polystyrene microtiter plates ( e.g

. , ICN:7 6

- 381

- 04, Costar:53590, Fisher:07

- 200

- 35) 2835

2836

37 oC hybridization oven (or fast equilibrating air incubator) for plates, water bath for tubes 2837

2838

Other common laboratory equipment, chemicals, and supplies. 2839

2840

Recipes: 2841

Coating Buffer (50 mM Carbonate Buffer, pH 9.6): 2842

1.26g NaHCO 3 

# 2843

0.68 g Na 2 

# CO 3 

# 2844

428 ml water 2845

10X PBS (0.1 M phosphate, 1.5 M NaCl): 2846

0.83g NaH 2 

# PO 4 

#  H 2 

# O 2847

20.1 g Na 2 

# HPO 4 

#  7 H 2 

# O 2848

71 g NaCl 2849

810 ml water 2850

Wash Buffer (PBST): 2851

100 ml 10X PBS 2852

900 ml water 2853

Adjust pH to 7.3 with 1 M HCL 2854

Then add 0.5 ml Tween

- 20 2855

Assay Buffer: 2856

3.75 ml Normal Goat Serum (NGS) 2857

# 77

146.25 ml Wash Buffer 2858

2859

Sample collection: 2860

Blood is collected with heparinized microhematocrit tube and placed on ice. After centrifugation 2861

for 3 min, the tube is scored, broken open, and the pla sma expelled into 0.6 ml microcentrifuge 2862

tubes which contain 0.13 units of lyophilized aprotinin. (These tubes are prepared in advance by 2863

adding the appropriate amount of aprotinin, freezing, and lyophilizing in a speed

- vac at low heat 2864

until dry.) Place plasma at

- 80  C until analyzed. 2865

2866



# Procedure for One Plate: 2867

2868

Coating the Plate: 2869

Mix 20 μl of purified Vtg with 22 ml of carbonate buffer (final 3 μg/ml). Add 200 μl to each 2870

well of a 96

- well plate using. Cover the plate with adhesive sealing film and allo w to incubate 2871

overnight at 4 oC (or 37 oC for 2 hours). 2872

2873

Blocking the Plate: 2874

Blocking solution is prepared by adding 2 ml of Normal Goat Serum (NGS) to 38 ml of 2875

carbonate buffer. Remove coating solution and shake dry. Add 350 μl of the blocking solution 2876

to each well. Cover with adhesive sealing film and incubate at 37 oC for 2 hours (or at 4  C 2877

overnight). 2878

2879

Preparation of Standards: 2880

5.8 μl of purified Vtg standard is mixed with 1.5 ml of assay buffer in a 12 x 75 mm borosilicate 2881

disposable glass test tube. This yields 12,760 ng/ml. Then a serial dilution is performed by 2882

adding 750 μl of the previous dilution to 750 μl of assay buffer to yield final concentrations of 2883

12,760, 6380, 3190, 1595, 798, 399, 199 , 100, and 50 ng/ml. 2884

2885

Preparation of Samples : 2886

Start with a 1:300 ( e.g ., combine 1 μl plasma with 299 μl of assay buffer) or 1:30 dilution of 2887

plasma into assay buffer. If a large amount of Vtg is expected, additional or greater dilutions 2888

may be needed. Try to keep B/B

o 

# within the range of stand ards. For samples without 2889

appreciable Vtg, e.g ., control males and immature females, use the 1:30 dilution. Samples 2890

diluted less than this may show unwanted matrix effects. 2891

2892

Additionally, it is recommended to run a positive control sample on each plate. This comes from 2893

a pool of plasma containing highly induced levels of Vtg. The pool is initially diluted in NGS, 2894

divided in aliquots and stored at

- 80 C. For each plate, an aliquot is thawed, diluted further in 2895

assay buffer and run similar to a test sa mple. 2896

2897

Incubation with 1o Antibody: 2898

The 1o antibody is prepared by making a 1:2,000 dilution of preadsorbed 1o antibody serum in 2899

assay buffer ( e.g ., 8 μl to 16 ml of assay buffer). Combine 300 μl of 1o antibody solution with 2900

300 μl of sample/standard in a glass tube. The B

o 

# tube is prepared similarly with 300 μl of assay 2901

buffer and 300 μl of antibody. Also, a NSB tube should be prepared using 600 μl of assay buffer 2902

# 78

only, i.e ., no Ab. Cover the tubes with parafilm and vortex gently to mix. Incubate in a 37 oC 2903

water bath for 1 hour. 2904

2905

Washing the Plate: 2906

Just before the 1o antibody incubation is complete, wash the plate. This is done by shaking out 2907

the contents and patting dry on absorbent paper. Then fill wells with 350 μl of wash solution, 2908

dump out, and pat dry. A multi

- channel repeater pipette or plate washer is useful here. The wash 2909

step is repeated two more times for a total of three washes. 2910

2911

2912

Lo ading the Plate: 2913

A fter the plate has been washed, remove the tubes from the water bath and vortex lightly. Add 2914

200 μl from each sample, standard, B

o 

# , and NSB tube to duplicate wells of the plate. Cover plate 2915

with adhesive sealing film and allow to incub ate for 1 hour at 37 oC. 2916

2917

2918

Incubation with the 2o Antibody : 2919

At the end of the incubation from the previous step, the plate should be washed three times again, 2920

like above. The diluted 2o antibody is prepared by mixing 2.5 μl of 2o antibody with 50 ml of 2921

as say buffer. Add 200 μl of diluted 2o antibody to each well, seal like above, and incubate for 1 2922

hour at 37 oC. 2923

2924

Addition of Substrate: 2925

After the incubation with the 2o antibody is complete, wash the plate three times as described 2926

earlier. Then add 100 μl of TMB substrate to each well. Allow the reaction to proceed for 10 2927

minutes, preferably out of bright light. Stop the reaction by adding 100 μl of 1 M phosphoric 2928

acid. This will change the color from blue to an intense yellow. Measure the absorbance a t 450 2929

nm using a plate reader. 2930

2931

Calculate B/B

o 

# : 2932

Subtract the average NSB value from all measurements. The B/B

o 

# for each sample and standard 2933

is calculated by dividing the absorbance value by the average absorbance of the B

o 

# sample. 2934

2935

Obtain the Standard Cu rve and Determine Unknown Amounts: 2936

Generate a standard curve with the aid of some computer graphing software ( e.g ., Slidewrite or 2937

Sigma Plot ® 

# ) that will extrapolate quantity from B/B

o 

# of sample based on B/B

o 

# of standards. 2938

Typically, the amount is plotted on a log scale and the curve has a sigmoid shape. However, it 2939

may appear linear when using a narrow range of standards. Correct sample amounts for dilution 2940

factor and report as mg Vtg/ml of plasma. 2941

2942

Determination of Minimum Detection Limits (MDL): 2943

Often, particularly in normal males, it will not be clear how to report results from low values. In 2944

these cases, the 95% "Confidence limits" should be used to determine if the value should be 2945

reported as zero or as some other number. If the sample result is wit hin the confidence interval 2946

of the zero standard (B

o 

# ), the result should be reported as zero. The minimum detection level will 2947

be the lowest standard which is consistently different from the zero standard; that is, the two 2948

# 79

confidence intervals don't overla p. For any sample result which is within the confidence limit of 2949

the minimum detection level, or above, the calculated value will be reported. If a sample falls 2950

between the zero standard and the minimum detection level intervals, one half of the minimum 2951

de tection level should be reported for the value of that sample. 2952

2953

2954

2955

2956

# 80

Appendix 5 : Histology Protocol 2957

2958

Larval Subsampling (Stage 62) 2959

2960

Dissection 2961

- Froglets euthanized by submersion in MS

- 222 2962

- Wet weight is measured 2963

- Blood is collected directly from aorta 2964

- Legs are removed at knee joint 2965

2966

Fixation 2967

- Davidson’s 48 hours 2968

a) Glacial acetic acid...............115 ml 2969

b) Formaldehyde (35

- 40%)........220 ml 2970

c) 95% Ethyl Alcohol...............330 ml 2971

d) Distilled water....................335 ml 2972

- Rinse with distilled water 2973

- 10% neutral buffered formalin 2974

2975

Trimming 2976

Gonad: 2977

- Transverse cuts posterior to the arms and anterior to the pelvis 2978

Thyroid: 2979

- Transverse cut through middle of arms leaving some shoulder joint 2980

- Transverse cut anterior to eyes to remove snout 2981

2982

Processing 2983

Decalcification 2984

- By fixation in Davidson’s or Bouin’s 2985

Dehydration 2986

2987

Thyroid and gonad (1 hour stations) 2988

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

- - ambient

p/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

Embedment 2990

Gonad: 2991

# 81

- Embed anterior side down. 2992

2993

2994

2995

Thyroid: 2996

- Embed posterior side down, with the nose pointing up. The nose can be trimmed off anterior 2997

to the eyes if needed to fit into embedding mold. 2998

2999

Sectioning Protocol 3000

Gonad: (4 μm sections) 3001

- Face block 3002

- Step section and collect one good section to be mounted on a glass slide at the following 3003

levels: 3004

3005

3006

3007

Thyroid: (4 μm sections) 3008

- Face block 3009

- Take 1 section, mount on slide, and inspect in microscope for both thyroid glands. 3010

- No glands? 3011

- Take 150

- 200 micron step, examine a section for thyroid glands, and repeat until both glands 3012

appear on sections. 3013

- Section has both glands? 3014

- Take 1 sect ion, cut 50 microns, take 1 section, and mount together on slide. 3015

- Seal block 3016

3017

Staining 3018

- H&E and coverslip with glass using a permanent mounting medium 3019

3020

Juvenile Sampling & Processing 3021

3022

Dissection 3023

- Frogs euthanized by MS

- 222 injection 3024

- Wet weight, length measured 3025

- Blood is collected from heart 3026

- Liver is removed, weighed and fixed 3027

- One foot is removed for DNA analysis 3028

- Remaining carcass is placed in an appropriately sized tissue cassette and fixed 3029

# 82

- 3030

3031

3032

3033

3034

3035

3036

3037

Male Female 3038

3039

Fixation 3040

- Davidson’s 48 hours 3041

Glacial acetic acid...............115 ml 3042

Formaldehyde (35

- 40%)........220 ml 3043

95% Ethyl Alcohol...............330 ml 3044

Distilled water....................335 ml 3045

- Rinse with distilled water 3046

- 10% neutral buffered formalin 3047

3048

Trimming 3049

Testis/Kidney/ducts 3050

- Transverse cuts on anterior and posterior margins of testis. 3051

3052

3053

3054

3055

3056

3057

3058

3059

Ovary/Kidney/ducts 3060

- Transverse cuts on anterior and posterior margins of the ovary. 3061

3062

3063

3064

3065

3066

3067

3068

Thyroid 3069

- Transverse cut through middle of arms leaving some shoulder joint 3070

- Transverse cut anterior to eyes to remove snout 3071

3072

3073

3074

3075

# 83

3076

3077

3078

Processing 3079

Decalcification 3080

Bone surrounding thyroid and kidney samples should be decalcified for 24

- 48 hours 3081

depending on strength of decalcifier. Decalcification not necessary for ovaries. 3082

Dehydration 3083

**Ovaries (1⁄2 hour stations) Thyroid and kidney (1 hour stations) 3084

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

- - ambient

p/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

Embedment 3086

Testis/kidney & Oviduct/kidney: Embed anterior side down 3087

Ovaries: Embed normally; a large mold will be needed 3088

Thyroid: Embed posterior side down, nose up in large mold 3089

3090

Sectioning 3091

Testis/kidney & Oviduct/kidney: (4μm sections) 3092

- Face block 3093

- Step section and collect one good section to be mounted on a glass slide at the following 3094

levels: 3095

3096

3097

3098

Ovaries: (7μm sections) 3099

- Face block 3100

- On the side of the paraffin block, mark 1⁄4, 1⁄2, 3⁄4 way through entire ovary. 3101

# 84

- Take a ribbon of 2

- 3 sectio ns at each mark, mount on slide. 3102

3103

Thyroid: (4μm sections) 3104

- Face block 3105

- Take 1 section, mount on slide, and inspect in microscope for both thyroid glands. 3106

- No glands? 3107

- Take 150

- 200 micron step, examine a section for thyroid glands, and repeat until both gl ands 3108

appear on sections. 3109

- Section has both glands? 3110

- Take 1 section, cut 50 microns, take 1 section, and mount together on slide. 3111

- Seal block 3112

3113

Staining 3114

- H&E and coverslip with glass using a permanent mounting medium 3115

3116

3117

3118

3119

3120

3121

3122

3123

3124

3125

3126

# 85

Appendix 6: Histopathology Guidance for Reading LAGDA Studies 

# 3127

3128

See ISR Appendix 8.3 

# 3129

3130

3131

Appendix 7: T 4 

# E L I S A 

# 3132

3133



# Adaptation of a Commercially Available E L I S A Kit to Measure Total 3134



# Thyroxine (T 4 

# ) in the Serum/Plasma of Xenopus laevis

. 3135

3136

Materials 3137

3138

T 4 

# E L I S A kit: ( e.g .MP Biomedicals 07BC1007) 3139

Buffer: 0.01 M Tris

- Cl, pH 7.3, 1% methanol 3140

95% ethanol 3141

Vacuum centrifuge c oncentrator ( e.g . Savant Speed Vac SVC100) 3142

Other common laboratory equipment 3143

3144

Background 3145

3146

The E L I S A kits are based on a competition between T 4 

# in a sample and a constant amount of 3147

enzyme

- labeled T 4 

# to bind an immobilized antibody recognizing T 4 

# . There do not appear to be 3148

any commercially available E L I S A kits for the measurement of circulating T 4 

# in Xenopus laevis

. 3149

There are kits available for human and several other spec ies, but these kits appear to be highly 3150

dependent on the use of standards made with homologous serum. The kits are formulated to be 3151

compatible with the presence of proteins in the blood which bind T 4 

# . These proteins include 3152

thyroxine binding globulin, tr ansthyret in (prealbumin), and albumin. Initial attempts to measure 3153

T 4 

# in X

. laevis using a commercially available kit produced results which were highly variable 3154

and greatly different from the values obtained with non

- immuno

- metric methods ( i.e . ICP

- MS). 3155

It was thought that this variability was to due to inappropriate interaction between the binding 3156

proteins present in the sample, the amount of T 4 

# in the sample, and the labeled T 4 

# included in the 3157

kit. It was thought that these problems would be avoid ed if the T 4 

# binding ability of the sample 3158

proteins was removed. This also necessitated using a standard which was free of bin ding 3159

proteins. Reviewing literature of early methods for T 4 

# measurement indicated that a 3160

deproteination step with acid or ethano l was often used. C onsistent and sensible results were 3161

obtained with the E L I S A kits when this adaptation was used. 3162

3163

3164

Procedure 3165

3166

Follow the kit instructions, with these three exceptions: 3167

3168

1 Standards 3169

A superstock solution of T 4 

# at 154 μg/ml was made in 0.05 M Na 2 

# CO 3 

# . This was diluted 3170

to 20 μg/dl (200 ng/ml) in buffer. From this, additional dilutions in buffer were made to 3171

give standards of 0.25, 0.5, 1.0, 2.0, 4.0 and 10.0 μg/dl. 3172

3173

# 86

3174

2 Extraction/deproteination 3175

Blanks (buffer), standards, and samples should be processed at room temperature as 3176

follows. Add 60 μl of buffer, standards, or serum/plasma to a 0.6 ml micorcentrifuge 3177

tube. If lesser amounts of sample are used, make up the volume to 60 μl with buffer. 3178

Add 180 μl of ethanol and mix vigorously by vortexing. Repeat vortexing two more 3179

times within a 10 minute period. Centrifuge for 10 minutes at 20,000 times g. Remove 3180

supernatant to another tube and repeat extraction on pellet with another 180 μl of etha nol. 3181

Centrifuge like above for 5 minutes. Combine supernatants and mix. Bring samples to 3182

dryness in a vacuum centrifuge c oncentrator. Thoroughly resuspend residue in 60 μl of 3183

buffer. Centrifuge for 10 minutes at 20,000 times g. Remove 55 μl to anothe r 0.6 ml 3184

tube. 3185

3186

3 Combine samples/standards with T 4 

# - HRP conjugate before loading on the EIA 3187

Plate 3188

3189

Add 220 μl of diluted T 4



# - conjugate to the 55 μl from above and mix. Add 125 μl to 3190

duplicate wells of the EIA plate. Continue with kit instructions. 3191

3192

3193

Comments 3194

3195

Although this adaptation has only been tried with the kit from MP Biomedicals, it is expect ed 3196

that kits from other suppliers that use a similar format should work as well. 3197

3198

3199

3200

3201

3202

3203

3204

3205

3206

3207

3208

3209

3210

3211

3212

3213

3214

3215

3216

3217

3218

3219

# 87



# 8.2 Histological Development of the Gonad in Juvenile Xenopus laevis 3220

3221

Chad A. Blanksma1, Allen W. Olmstead2, Sigmund J. Degitz2, Jonathan Haselman2, Rodney D. Johnson2 

# 3222

1ORISE Research Participation Program at U.S. Environmental Protection Agency, ORD, NHEERL, Mid

- Continental 

# 3223

Ecology Division, Duluth, MN 

# 3224

2U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental 

# 3225

Effects R esearch Laboratory, Mid

- Continental Ecology Division, Duluth, MN 

# 3226

3227

I ntroduction 3228

As directed by the Food Quality Protection Act, the US Environmental Protection 3229

Agency is developing a screening program for endocrine disrupting compounds. The Larval 3230

Amphibia n Growth and Development Assay (LAGDA) is a tier II test intended to identify and 3231

characterize the adverse consequences of toxicants that interfere with normal growth and 3232

development of larval and juvenile frogs with particular emphasis on disruption of es trogen, 3233

androgen, and thyroid mediated processes. A particularly important aspect of this assay is the 3234

detection of alterations in gonad differentiation and development. 3235

Assessments of gonad malformations in anuran species have traditionally relied on go nad 3236

histology of tadpoles at the moment of metamorphosis completion (NF stage 66). Preliminary 3237

experiments using Xenopus exposed to three model endocrine disruptors possessing estrogenic, 3238

androgenic, or anti

- aromatase activities indicated that gonad histol ogy at this developmental 3239

stage was inadequate for the goals of the assay. Specifically the gonads were too immature, with 3240

the detection of mixed sex phenotypes being subjective and not informative with respect to a 3241

toxicant’s mechanism of action. In addit ion, exposure to endocrine disruptors such as 3242

ethynylestradiol and fadrozole, which induce testicular oocytes in older animals, did not in frogs 3243

sampled at metamorphosis. 3244

We propose that assessing the gonads at a later stage of development would allow for 3245

improved detection of aberrant gonad differentiation and development, yielding more 3246

informative results for risk assessment purposes. In order to identify a more appropriate level of 3247

gonad development for use in the LAGDA, we characterized normal gonad de velopment in the 3248

test species, X

. laevis , at two week intervals from completion of metamorphosis until 12 weeks 3249

post

- metamorphosis (wpm). 3250

3251

Materials and Methods 3252

X

. laevis were cultured in

- house at EPA

- MED using filtered Lake Superior water at 21 o 

# C 3253

with 12: 12 hour light:dark photoperiod. No fewer than 4 males and 4 females were subsampled 3254

at completion of metamorphosis (NF stage 66) and at 2 week time periods up to 12 weeks post 3255

metamorphosis (wpm). 3256

Frogs were euthanized by injection of MS

- 222 in accord ance with animal care and use 3257

guidelines. Specimens were trimmed by removing the gastrointestinal tract and liver to expose 3258

the gonad, kidney, and reproductive ducts, followed by transverse cuts to the anterior margin of 3259

the gonad and the caudal margin of the kidneys. The trimmed trunks containing gonads were 3260

processed for histological analysis. Samples were embedded with the anterior surface down for 3261

transverse sectioning so gonads could be assessed for morphological changes along their 3262

longitudinal ax is. Sections were cut at a thickness of 5 microns and stained with H&E. 3263

Incremental sectioning produced 4

- 6 slides per sample, arranged anterior to posterior. 3264

A representative image of the anterior region of each sample’s gonad was taken using a 3265

40x ob

jective and each sample was assessed for developmental landmarks. 3266

# 88

3267

3268

3269

3270

3271

3272

3273

3274

3275

3276

3277

3278

3279

# 90

3287

Gonadal Development Timeline 3288

3289

3290

3291

3292

Conclusions 3293

The evaluation of gonad development is an important endpoint for protocols that test 3294

chemicals for possible endocrine disruption effects. At metamorphosis, NF stage 66, the gonads 3295

are only partially developed and sex

- specific germ cells cannot be identified histologically. 3296

Extending the test protocols past metamorphosis allows more comprehensive histological 3297

evaluation of possible effects on gonad development. This study provides information which can 3298

be used to optimize amphibian EDC test protocols. Extending the test protocol to at least 6 3299

weeks post

- metamorphosis provides substantially more insight into possible EDC effects on 3300

gonad development. When sampling at this time or later, the gonads express: 3301

3302

 Near ly complete maturation of oocytes and spermatocytes 3303

 Developed seminiferous tubules and associated ducts in males 3304

 Developing oviduct system in females 3305

 Developed supporting cells and tissues such as granulosa cells, Sertoli and Leydig cells. 3306

3307

References 3308

Ni euwkoop PD, J Faber. 1967 Normal table of Xenopus laevis (Daudin): A Systematical and 3309

Chronoligical Survey of the Development from the Fertilized Egg Till the End of 3310

Metamorphosis. 2nd ed. Amsterdam: North Holland 3311

Reproduction of Amphibians. Biological Sys tems in Vertebrates. 2009. Edited by Maria 3312

Ogielska. Enfield (New Hampshire): Science Publishers 3313

# 91

Dumont, James N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in 3314

laboratory maintained animals. Journal of Morphology Volume 136, Issu e 2, pp. 153

- 179. 3315

February 1972 3316

3317



# 8.3 Histopathology Guidance for Reading LAGDA Studies 3318

3319

Histopathology Guidance for Reading LAGDA Studies 3320

Chad A. Blanksma1, Rodney D. Johnson2 

# 3321

1ORISE Research Participation Program at U.S. Environmental Protection Agency, ORD, NHEERL, Mid

- Continental Ecology 

# 3322

Division, Duluth, MN 

# 3323

2U.S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects 

# 3324

Research Laboratory, Mid

- Continental Ecology Division, Duluth, MN 

# 3325

3326

The slides shall be evaluated by a pathologist f or treatment

- induced effects

. The pathologist 3327

shall perform the evaluation according to the Best Practices Guideline: Toxicologic 3328

Histopathology (Crissman et al. 2004). It is recommended that evaluation begin with examining 3329

representative slides from specimens selected from control and the two highest treatments to 3330

determine likely treatment

- related effects. Then, images of representative treatment

- related 3331

effects and associated severity scores shall be prepared to be used a s references for scoring the 3332

remainder of the specimens. Finally, after the initial evaluation, the study pathologist shall 3333

perform a masked re

- evaluation of all the specimens, but for only the previously observed 3334

treatment

- related lesions. 3335

3336

Each sectio n shall be evaluated for gonad pathology, whereas kidney , reproductive duct, and 3337

liver pathologies shall be based on the assessment of one representative section. The prevalence 3338

and extent of the effects shall be assessed in the various treatments as compare d to controls. 3339

Effects data containing diagnoses and associated severity grades shall be reported in an Excel 3340

spreadsheet similar to the example provided below. Diagnoses of treatment effects shall be 3341

based on t he columns in the example pathology spreads heet. However, for pathologies not listed 3342

in the spreadsheet, the pathologist shall provide new diagnostic criteria to the spreadsheet. 3343

Pathology not related to treatment shall also be noted as a comment in the spreadsheet. Cre ate 3344

digital images of repre sentative diagnostic features in an uncompressed file format . Images of 3345

representative control tissues shall be included for comparison. Each image shall be at an 3346

appropriate magnification to illustrate the features using the best judgment of the pathologi st. An 3347

embedded calibrated scale bar shall be included in all pictures . The spreadsheet shall indicate 3348

imaged sections. 3349

3350

A report shall be prepared which includes a short summary of the protocols used in slide 3351

preparation and a description of treatment

- rel ated effects based on concentration responses and 3352

comparisons with control frogs. The narrative report shall include tables, figures , figure legends, 3353

and citations, as necessary to describe treatment effects. Citations, tables, figures, figure legends, 3354

sh all be placed at the end of the narrative report. Other supporting histopathology data, images, 3355

and histology technical reports shall be provided as spreadsheet s, images without legends or 3356

anno tations, and documents . The titles and file names of reports a nd documentation shall 3357

include : laboratory; species name; developmental stage; test chemical; and tissue examined . The 3358

# 92

file names of digital images shall identify the test chemical, treatment, and specimen ID. A 3359

tabular cross

- reference of image files and r eport figure numbers and titles shall be provided

. 3360

General approach to reading studies 3361

From OECD guidance document for the Diagnosis of Endocrine

- Related Histopathology of Fish 3362

Gonads. (2009) 3363

3364

Studies are to be read by individuals experienced in reading to xicologic pathology studies, and 3365

who are familiar with normal frog gonad histology, with gonadal physiology, and with general 3366

responses of the gonads to toxicologic insult. Pathologists may be board certified ( e.g

. , American 3367

College of Veterinary Pathologists, The European Centre of Toxicologic Pathology, or other 3368

certifying organizations); however, certification is not a requirement as long as the pathologist 3369

has obtained sufficient experie nce with, and knowledge of, frog histology and toxicologic 3370

pathology. Technicians should not be used to conduct readings due to the subtle nature of some 3371

changes and the need for subjective judgments based on past experience. 3372

It is recognized that there is a limited pool of pathologists with the necessary trainin g and 3373

experience that are available to read the gonadal histopathology for the Larval Amphibian 3374

Growth and Development Assay . If an individual has toxicological pathology experience and is 3375

familiar with gonadal histology in amphibian species, he/she may be t rained to read the frog 3376

assay . If pathologists with little experience are used to conduct the histopathological analysis, 3377

informal peer review may be necessary. 3378

Pathologists are to read the studies non

- blinded ( i.e

. , with knowledge of the treatment group 3379

status of individual fish). This is because endocrine effects on histomorphology tend to be 3380

incremental, and subtle differences between exposed and unexposed animals may not be 3381

recognizable unless tissue sections from high dose animals can be knowingly co mpared to those 3382

from controls. Thus, the aim of the initial evaluation is to ensure that diagnoses are not missed 3383

( i.e ., to avoid false

- negative results). However, it is expected that any potential compound

3384

related findings will be re

- evaluated by the path ologist in a blinded manner prior to reporting 3385

such f

indings, when appropriate. Certain diagnostic criteria, such as relative increases or 3386

decreases in cell populations, cannot be read in a blinded manner due to the diagnostic 3387

dependence on control gonads. As a rule, treatment groups should be evaluated in the following 3388

order: control, high

- dose, intermediate

- dose, and low

- dose. 3389

It is suggested that the pathologists be provided with all available information related to the 3390

study prior to conducting their re adings. Information regarding gross morphologic abnormalities, 3391

mortality rates, and general test population performance and health are useful for pathologists to 3392

provide comprehensive reports and to aid in the interpretation of findings. For a more 3393

compreh ensive discussion of standard reading approaches for toxicologic pathology studies, 3394

please refer to the Society of Toxicologic Pathology Best Practices for reading toxicologic 3395

histopathology studies (Crissman et al ., 2004). 3396

3397

Severity Grading 3398

In toxicologic pathology, it is recognized that compounds may exert subtle effects on tissues that 3399

are not adequately represented by simple binary (positive or negative) responses. Severity 3400

grading involves a semi

- quantitative estimation of the degree to which a particu lar 3401

histomorphological change is present in a tissue section (Shackelford et al ., 2002). The purpose 3402

of severity grading is to provide an efficient, semi

- objective mechanism for comparing changes 3403

(including potential compound

- related effects) among animals , treatment groups, and studies. 3404

# 93

3405

Severity grading will employ the following system: 3406

Not remarkable 

# 3407

Grade 1 (minimal) 

# 3408

Grade 2 (mild) 

# 3409

Grade 3 (moderate) 

# 3410

Grade 4 (severe) 

# 3411

A grading system needs to be flexible enough to encompass a variety of different tissue changes. 3412

In theory, there are three broad categories of changes based on the intuitive manner in which 3413

people tend to quantify observations in tissue sections: 3414

(1) Discrete : these are changes that can be readily counted. Examples include atretic follicles, 

# 3415

oocytes in the testis, and clusters of apoptotic cells. 

# 3416

(2) Spatial : these are changes that can be quantified by area measurements. This includes lesions 

# 3417

that are typically classified as focal, multifocal, coalescing, or diffuse. Specific exampl es include 

# 3418

granulomatous inflammation and tissue necrosis. 

# 3419

(3) Global : these are generalized changes that would usually require more sophisticated measurement 

# 3420

techniques for quantification. Examples include increased hepatocyte basophilia, 

# 3421

Sertoli cell/interstitial cell hypertrophy, or quantitative alterations in cell populations. 

# 3422

General severity grading scale 3423

 Not Remarkable : This grade is used if there are no findings associated with a particular diagnostic 

# 3424

criterion. 

# 3425

 Grade 1: Minimal . Ranging from inconspicuous to barely noticeable but so minor, small, or 

# 3426

infrequent as to warrant no more than the least assignable grade. For discrete changes, grade 

# 3427

1 is used when there are fewer than 2 occurrences per microscopic field, or 1

- 2 occurrences 

# 3428

per section. For multifocal or diffusely

- distributed alterations, this grade is used for 

# 3429

processes where ≤20 % of the tissue in the section is involved. 

# 3430

 Grade 2: Mild . A noticeable feature of the tissue. For discrete changes, grade 2 is used when 

# 3431

there are 3

- 5 occurrences per microscopic field or per tissue section. For multifocal or diffusely



# 3432

distributed alterations, this grade is used for processes where 20

- 50 % of the tissue in 

# 3433

the section are involved. 

# 3434

 Grade 3: Moderate . A dominant feature of the tissue. For discrete changes, grade 3 is used 

# 3435

when there are 6

- 8 occurrences per microscopic field or per tissue section. For multifocal or 

# 3436

diffusely

- distributed alterations, this grade is used for processes where 50

- 80 % of the tissue 

# 3437

in the section a re involved. 

# 3438

 Grade 4: Severe. An overwhelming feature of the tissue. For discrete changes, grade 4 is 

# 3439

used when there are more than 9 occurrences per microscopic field or per tissue section. For 

# 3440

multifocal or diffusely

- distributed alterations, this grade is used for processes where > 80 % 

# 3441

of the tissue in the section are involved. 

# 3442

At least some of the histomorphological changes that have been associated with EDCs in fish are 3443

considered to be exacerbations of “normal”, physiologic findings ( e.g ., oocyte atresia 3444

[Nagahama, 1983; Tyler and Sumpter, 1996]). At the discretion of the pathologist, the severity 3445

of a given change should be scored according to one of the following two methods : 3446

(1) score compound

- exposed animals relative to the severity of the same change in control 3447

animals, or 3448

(2) score all animals relative to “normal” as determined by the pathologist’s experience. 3449

For each important ( i.e ., treatment

- associated) finding, the method that was used should be stated 3450

in the Materials and Methods section of the pathology narrative report. By convention, severity 3451

grading should not be influenced by the estimated physiologic importance of the change, since 3452

this would add a further layer of subjectivity to the findings that complicates inter

- laboratory 3453

# 94

resul ts comparisons. For example, the presence of two oocytes in the testis should not be graded 3454

as “severe”, even if the pathologist considers this finding to be highly significant in terms of 3455

endocrine modulation. 3456

Data Recording

. 3457

An Excel worksheet form has b een created that includes worksheets for primary, secondary, and 3458

additional diagnoses to facilitate histopathology data collection. In this worksheet, each data 3459

entry cell represents an individual fish. Additional sheets are available for comments and 3460

addi tional findings. For each fish, the pathologist records a severity score associated with the 3461

diagnosis. Diagnostic criteria with non

- remarkable findings shall be denoted using (

- ). If there is 3462

no reasonably appropriate diagnostic term for a particular find ing, the pathologist can create a 3463

term that can be recorded in the “Additional diagnoses” worksheet. If insufficient tissue is 3464

available for diagnosis, this should be recorded as IT (insufficient tissue). If a target tissue is 3465

missing, this should be recor ded as MT (missing tissue). 3466

3467

Histopathology Report Format. 3468

Each histopathology narrative report should contain the following five sections: 3469

Introduction 3470

Materials and Methods 3471

Results 3472

Discussion 3473

Summary/Conclusions 3474

(References) 3475

The Introduction section briefly outlines the experimental design. The Materials and Methods 3476

section describes any items or procedures that are essentially different from Section 1: Post

3477

mortem and Histotechnical Procedures. As applicable, specific severity grading criteria (see 3478

Severity Grading) should also be listed in this section. The Results section should report findings 3479

that are: (1) compound

- related; (2) potentially compound

- related; (3) novel or unusual. Detailed 3480

histomorphological descriptions need only be included for f indings that differ substantially from 3481

diagnoses presented in Section 4 (Glossary and diagnostic criteria). It is intended that the Results 3482

section should be as objective as possible ( i.e ., opinions and theories should be reserved for the 3483

Discussion sectio n). The Discussion section, which contains subjective information, should 3484

address relevant findings that were reported in the Results section. Opinions and theories can be 3485

included in this section, preferably backed by references from peer

- reviewed sources , but 3486

unsupported speculation should be avoided. The Summary/Conclusions section should 3487

encapsulate the most important information from the Results and Discussion sections. 3488

D evelopmental S taging of Xenopus gonads and R eproductive ducts. 3489

3490

Xenopus Testis Sta ging Criteria (Figure 1.) 

# 3491

Stage 1: Undifferentiated gonad. 

# 3492

Stage 2: Medullary region populated with individual primary spermatogonia and basophillic 3493

undifferentiated somatic cells

. 3494

Stage 3: Somatic and germ cells organize into early semin i ferous tubules populated with primary 3495

spermatogonia and cysts of secondary spermatogonia

. 3496

Stage 4: Germ cell cysts contain developmental stages up to primary spermatocytes with 3497

formation of rete testis; may have occasional spermatocysts that contain round or elongated 3498

sp ermatids. 3499

# 95

Stage 5: All stages of spermatogenesis evident. Organization and development of seminiferous 3500

tubules and rete testis apparent

. 3501

3502

Figure 1. Example of staging system applied to Xenopus laevis testis. Bar = 50 μm

. 

# 3503

Xenopus Ovary Staging Criteria ( Figure 2. ) 3504

Stage 1: Undifferentiated gonad. Gonad consists of an undifferentiated structure, germ cells, and 3505

somatic cells. 3506

Stage 2: Gonad identifiable as an ovary based on the presence of a discontinuously open lumen 3507

lined with epithelial cells. The germ cells within the cortex (the region outside the epithelial 3508

layer) consist of primary oogonia, cysts of primary mitotic oogonia, secondary oogonia, and very 3509

early meiotic oocytes. 3510

Stage 3: First appearance of diplotene oocytes in cortex. Structure and sha pe of the ovary 3511

similar to stage 1, but the appearance of diplotene oocytes is the first morphological difference 3512

between male and female germ cells. The most prevalent germ cell types at this stage are cysts 3513

of secondary oogonia and cysts of leptotene

- pa chytene primary meiocytes. 3514

Stage 4: Pre

- vitellogenic (Dumont Stage I) diplotene oocytes are the most prevalent germ cell 3515

type observed by area and absolute cell counts. The central lumen is proportionately smaller 3516

while the whole ovary grows greatly in siz e and volume due to the growth of the oocytes. Cysts 3517

in earlier stages of oogenesis become fewer in number and are located along the periphery of 3518

ovary. 3519

Stage 5: Ovary consists almost entirely of vitellogenic oocytes (Dumont Stage IV). Pre

3520

vitellogenic d iplotene oocytes can be found along the periphery of the ovary and germ patches of 3521

primary and secondary oogonia are sparse and difficult to locate along the germinal epithelium. 3522

# 96

3523

Figure 2. Staging system applied to Xenopus laevis ovaries. Bar = 50 μm (St age 2 and 3); Bar = 100 

# 3524

μm (Stage 4); Bar = 1 mm (Stage 5). 

# 3525

Xenopus W o lffian Duct Staging Criteria ( Figure 3. ) 3526

Stage 1: The W o lffian ducts are paired ducts each located along the lateral edges of the kidneys, 3527

usually adjacent to the suspensory connective tissue of the organ. At this stage, the epithelium of 3528

the W o lffian duct consists of a single layer of cuboidal cells

. 3529

Stage 2: Pseudostratified epithelial cells line the duct asymmetrically. The height of the 3530

pseudostratified epithelium is less than 3 tim es the height of the other epithelial cells

. 3531

Stage 3: The asymmetric epithelial lining of the duct is more pronounced with the 3532

pseudostratified cells greater than 3 times the height of the other epithelial cells. 3533

Stage 4: The duct is lined symmetrically wi th tall columnar epithelial cells. 3534

# 97

3535

Figure 3. Wolffian duct staging. W = Wolffian duct; K = kidney tubule; Bar = 20 μm. 

# 3536

Oviduct Staging ( Figure 4. ) 3537

3538

Stage 1: A discrete, solid, fibrous tag with proliferative fibroblasts in the location of normal 3539

oviduct formation. 3540

3541

Stage 2: Formation of a rudimentary oviduct with a central lumen surrounded by a single layer 3542

of cuboidal to columnar epithelium. The ductular lum en is frequently filled with cell debris. 3543

3544

Stage 3: Oviduct is comprised of an undulating, pseudostratified, ciliated columnar epithelium, a 3545

discrete lumen, and patchy areas of glandular formation. Ductular lumen may contain cell debris. 3546

3547

Stage 4: Siz e of oviduct enlarged with prominent gland formation. 3548

3549

# 98

9 Figure 4. Oviduct Staging. W = Wolffian duct; G = glandular region; Bar = 100 μm. 

# 3550

Diagn ostic Criteria for Specific Histopathological findings in EDC tests. 3551

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 o vary

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 W o lffian duct

3 Duct has an epithelial cell lining, and is substantially larger (1.5x or greater) than the size of the

W o lffian duct

4 Duct is substantially larger (1.5x or greater) than the size of the W o lffian duct Epithelial cells have

formed large, basophilic glandular structures

W o lffian

Duct W o lffian Duct

Stage 1 Epithelial lining of W o lffian duct is completely or focally less than two cell layers thick

2 Epithelial lining of W o lffian 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

References: 3554

3555

Crissman, J.W., Goodman, D.G., Hildebrandt, P.K., Maronpot, R.R., Prater, D.A., Riley, J.H., Seaman, 

# 3556

W.J., Thake, D.C. (2004) Best practices guideline: Toxicologic pathology. Toxicol. Pathol. 32, 

# 3557

126

- 131. 

# 3558

Johnson, R., Wolf, J., and Braunbeck T. (2009). “OECD Guidance Document for the Diagnosis 3559

of Endocrine

- Related Histopathology of Fish Gon ads” Organization for Economic and 3560

Cooperative Development, Paris, France. 3561

Nieuwkoop PD, J Faber. 1967 Normal table of Xenopus laevis (Daudin): A Systematical and 3562

Chronoligical Survey of the Development from the Fertilized Egg Till the End of Metamorphos is. 3563

2nd ed. Amsterdam: North Holland 3564

Reproduction of Amphibians. Biological Systems in Vertebrates. 2009. Edited by Maria 3565

Ogielska. Enfield (New Hampshire): Science Publishers 3566

Dumont, James N. Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in 3567

laboratory maintained animals. Journal of Morphology Volume 136, Issue 2, pp. 153

- 179. 3568

February 1972 3569

3570

# 100



# 8.4 Inter

- laboratory validation study results 3571

8.4.1 Prochloraz results 3572

3573

Measured test concentrations 3574

3575

All four labs met the criteria of maintaining CVs of ≤20% over the entire test for all 3576

concentrations with the exception of the 6.67 and 20μg/L nominal concentrations at C which had 3577

CVs of 0.38 and 0.23 and were on average 84% and 25% above nominal respect ively (T able 3578

8.4.1

- 1 ). Measured concentrations did not show any crossover with other treatment levels in all 3579

labs except C which had several overlaps in treatment concentrations through week five of 3580

exposure. CV values between replicates could not be cal culated on a weekly basis for B and C 3581

due to measurements of only one replicate tank per week. Treatment levels will be referred to as 3582

nominal concentrations regardless of mean

- measured concentrations reported by each of the labs. 3583

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

Survival 3586

3587

All labs met the criteria of ≤20% mortality in any one control replicate that could not be 3588

explained by technical error and no treatment

- related trends in survival were identified (data not 3589

shown). 3590

3591

Larval growth (NF66) 3592

3593

There were no significant effects on larval grow th at A or time t o completion of metamorphosis 3594

(T able 8.4.1

- 2 ). 3595

3596

Table 8.4.1

- 2

L arval 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

Larval growth (NF62) 3598

3599

Prochloraz exposure at D caused a significant decrease in both snout

- vent length and body 3600

weight in the 180 μg/L treatment and caused a significant decrease in body weight in the 20 and 3601

60 μg/L treatments (Jonckheere

- Terpstra). There were no treatment

- related effects on time to 3602

completion of metamorphosis at D . Prochloraz exposure at B caused a significant decrease in 3603

# 101

body weight and snout

- vent length in the 180μg/L treatment (ANOVA with Dunnett). There 3604

were no concentration

- related effects on larval growth in the C study (Tabl e 8.4.1

- 3 ). 3605

3606

Table 8.4.1

- 3

Interlaboratory comparison of l arval 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

Larval blood thyroxine 3609

3610

A measured T 4 

# levels in serum using an HPLC/ICP

- MS method whereas the other labs used an 3611

ELISA method to measure T 4 

# in plasma. The ELISA method was not optimized so data could 3612

not be included. T here was a significant increase in T 4 

# concentrations in the 60 and 180μg/ L 3613

treatment at A (ANOVA with Dunnett

, Table 8.4.1

- 4 ). 3614

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).

T 4 (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

Juvenile growth 3617

3618

Juvenile female weight and length was significantly impeded in the 180μg/L treatment at D 3619

whereas only length in the 20, 60 and 180 μg/L treatments showed a significant decrease at B 3620

(Jonckeere

- Terpstra). Juvenile male weight and length showed a significa nt decrease in the 60 3621

and 180 μg/L treatments also at D . The other labs showed no significant concentration

- rela ted 3622

effect on juvenile growth (Table 8.4.1

- 5 ). 3623

3624

Juvenile liver

- somatic index (LSI) 3625

3626

# 102

A

, D and B all showed significant decrease s in both male and female LSI (Table 8.4.1

- 5 ). C 3627

showed a significant female LSI increase in the 180μg/L treatment. 3628

3629

Juvenile plasma vitellogenin 3630

3631

A

, D and C showed a significant increase in both juvenile male and fema le plasma vitellogenin 3632

titers (T able 8.4.1

- 5 ). B s howed no significant concentration

- related trend but also reported titer 3633

values that were much lower on average than the other three labs. 3634

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 (m g/mL) VTG (m g/mL)

0 μg/L 0. 0 3 ± 0.02 0.0 1 ± 0.02 0.00 ± 0.00 0.00 ± 0.00 0. 0 3 ± 0. 0 3 0. 0 2 ± 0. 0 7 0.01 ± 0. 0 2 0. 0 3 ± 0. 0 1

6.7 μg/L 0.05 ± 0.0 6 0.0 2 ± 0.04 0.0 1 ± 0.0 2 0.01 ± 0.002 0. 0 7 ± 0. 10 0. 0 1 ± 0. 0 1 0. 0 3 ± 0. 0 5 0. 0 3 ± 0. 0 2

20 μg/L 0.05 ± 0.04 0.0 1 ± 0.02 0.00 ± 0.00 0.01 ± 0.00 1 0. 0 7 ± 0.10 0.01 ± 0.01 0. 00 ± 0. 00 0. 0 4 ± 0.01

60 μg/L 0. 1 2 ± 0. 13 0.02 ± 0.0 3 0.00 ± 0.00 0.01 ± 0.00 4 0. 12 ± 0. 13 0. 0 4 ± 0. 0 7 0. 00 ± 0.01 0. 0 9 ± 0. 0 5

180 μg/L 0. 10 ± 0. 10 0.0 2 ± 0.02 0.00 ± 0.01 0. 01 ± 0. 00 2 0.25 ± 0.32 0. 0 4 ± 0. 0 7 0.01 ± 0.01 0.1 0 ± 0. 0 4

# 104

Larval t hyroid histopathology

Common treatment

- related observations in larval thyroid glands across all four studies were follicular cell hypertrophy and hyperplasia. Alth ough

these pathologies were present in larvae at B , there were no clear concentration

- dependent trends in their st udy. However, the other three labs

showed significant increases in follicular cell hypertrophy and two of those labs also showed significant increases in follic ular 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 treatmen t 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, hig hlighte d 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

r eplicates 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

M ild

- - - - - 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

L iver 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 sensi tivity 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 treatment

related 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 hypertr ophy 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 variatio n 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 cou ld be due to the e xtreme

lack of growth of C s 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; hig h lighted 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

r eplicates 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 significant ly differen t

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

r eplicates 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 g onad 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 conce ntration

- 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/h ypertrophy, 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 diffe red 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 signif icant minimal increase in spermatids in the 180 μg/L treatment and was the only lab ex hibiting 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; high lighted 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

r eplicates 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 eos i n o philic 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 g onad histopathology

Histopathological findings in the ovaries of prochloraz

- exposed female frogs were somewhat inconsistent and mostly minimal. Mononuclear cellular

infilt rate 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 sho wed 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 e valuated in the B and C

studies and there appeared to be significant a cceleration of ovary development, albeit slight, in C s 60 and 180 μg/L treatme nts 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; hi ghlighted 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

r eplicates 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 i n

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).

Tabl e 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 base d 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 develop ment 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 si gnificantly different tha n 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 s tage

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 k idney 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 t he B and C studies but showed

a significant increase in prevalence; however severity and sensitivity varied between the two labs. Presumably, tubule dilat ion 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; hig hlighted 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 k idney 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 obse rved throughout the B and C studies, but

only C exhibited significant increases in prevalence for both pathologies. Unlike male kidneys, renal interstitial fibrosis (minima l 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; h ighlighted 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 30 8 84 81 81 72 40

Minimal

- - - - - - - - - - 27 11 31 19 20 16 19 19 28 35

Mild

- - - - - - - - - - 73 72 56 48 72 0 0 0 0 25

Moderate

- - - - - - - - - - 0 0 0 4 0 0 0 0 0 0

Severe

- - - - - - - - - - 0 0 0 0 0 0 0 0 0 0

Tubules, mineralization / casts

NR

- - - - - - - - - - 0 6 0 7 0 37 15 24 8 10

Minimal

- - - - - - - - - - 0 6 0 7 4 53 77 48 56 40

Mild

- - - - - - - - - - 19 22 31 26 12 9 8 29 36 50

Moderate

- - - - - - - - - - 81 67 69 59 84 0 0 0 0 0

Severe

- - - - - - - - - - 0 0 0 0 0 0 0 0 0 0DRAFT 

# 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 v alues 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 nomina l 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 <MQL a

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 s ubstantially between the three studies and excess

la r val 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 ± 1.4DRAFT 

# 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 T 4 

# whereas the other two labs used an

ELISA method to measure T 4 

# in plasma

. The ELISA method was not optimized so data could

not be included. Serum T 4 

# 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).

T 4 (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 wa s 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 case s, CV values are close to,

or exceed 100% and control means differ acros s 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 (m g/mL) VTG (m g/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. 0 14 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. 01 9 0.001 ± 0.001 0.019 ± 0.028 0.143 ± 0. 023



# Larval t hyroid 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 at tenuation in the 25 and 50 μg/L

treatments (Table 8.4.2

- 5)

.

Table 8.4.2

- 5

Pre valance (%) 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 RSCA BS 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

rep licates 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

Mod erate 51 75 56 68 71 8 5 10 10 10 0 0 0 0 0

Sev ere 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

Mil d 26 42 44 24 33 18 30 25 10 15 15 20 20 35 20

Mod erate 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

- - - - -

Mil d 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

L iver 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 an d 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 g onad histopathology

Effects of octylphenol exposure on male g onads 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 increas ed 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 la bs,

although not statistically significant at A or C (Table 8.4.2

- 6 ).

Table 8.4.2

- 6

Pre valence (%) 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 RSCAB S 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

rep licates 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 3 4 30 31 44 11 69 68 70 55 52 13 0 30 7 5

5 6 6 70 69 56 89 20 26 26 41 36 88 100 70 93 95

Inc reased Spermatogonia

NR

- - - - - 100 100 100 100 92 100 100 100 93 100

Min imal

- - - - - 0 0 0 0 8 0 0 0 7 0

Mild

- - - - - 0 0 0 0 0 0 0 0 0 0

Mod erate

- - - - - 0 0 0 0 0 0 0 0 0 0

Sev ere

- - - - - 0 0 0 0 0 0 0 0 0 0

Mon onuclear Cellular Infiltrate

NR 66 85 56 69 78 62 79 57 64 76 90 93 95 87 84

Min imal 31 15 38 31 22 29 21 43 32 24 10 7 5 13 16

Mil d 3 0 6 0 0 9 0 0 0 0 0 0 0 0 0

Mod erate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Sev ere 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0



# Female g onad 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

- 7DRAFT 

# 118

Pre valence (%) 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 RSC ABS 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

rep licates 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 1 00 100 100 100 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

Mon onuclear Cellular Infiltrate

NR 63 40 25 38 42 53 30 41 39 33 55 42 50 24 30

Min imal 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

Sev ere 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Gra nulomatous inflammation

NR

- - - - - 97 95 94 100 100 93 96 95 100 100

Min imal

- - - - - 3 5 6 0 0 5 4 5 0 0

Mil d

- - - - - 0 0 0 0 0 3 0 0 0 0

Mod erate

- - - - - 0 0 0 0 0 0 0 0 0 0

Sev ere

- - - - - 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 be tween treatments and

controls (Table 8.4.2

- 8 ).

Table 8.4.2

- 8

His tological staging (%) of juvenile male reproductive ducts from three different labs performing the LAGDA with octylphenol (hi ghlighted 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

rep licates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Ovi duct Stage

1 88 60 44 31 67 40 26 30 9 12 98 94 100 93 100

2 1 2 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

Wol ffian Stage

1 3 10 0 25 0 16 26 4 5 28 10 19 25 20 5

2 8 8 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 diffe rent

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

rep licates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Ovi duct Stage

1 0 0 0 0 0 0 0 0 0 7 0 0 0 0 5

2 8 7 80 75 67 25 85 70 65 56 0 98 79 50 64 57

3 1 3 20 25 33 75 15 30 35 44 93 3 21 50 36 38

Wol ffian Stage

1 1 3 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 k idney 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

Pre valence (%) 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 RSCA BS 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

rep licates 8 5 4 5 3 8 4 4 4 4 8 4 4 4 4

Interstitial Fluid (Proteinaceous)

NR

- - - - - 100 100 100 100 96

- - - - -

Min imal

- - - - - 0 0 0 0 4

- - - - -

Mil d

- - - - - 0 0 0 0 0

- - - - -

Mod erate

- - - - - 0 0 0 0 0

- - - - -

Sev ere

- - - - - 0 0 0 0 0

- - - - -

Tub ules, Dilation

NR 18 20 6 13 11 4 11 9 9 16 10 19 15 27 16

Min imal 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

Sev ere 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Tub ules, Mineralization / Casts

NR 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0

Min imal 21 20 19 19 0 0 5 0 0 0 0 6 0 0 0

Mil d 73 70 69 63 100 42 47 61 64 60 33 88 50 80 58

Mod erate 6 10 13 19 0 58 47 39 36 40 68 6 45 20 42

Sev ere 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0



# Female k idney 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

d id not show significant trends in these pathologies, but showed significant minimal to mild

severity of edema, although prevalence was very low making the findi ng essentially not

remarkable (Table 8.4.2

- 11 ).DRAFT 

# 120

Table 8.4.2

- 11

Prevalence (%) an d 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

rep licates 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

- - - - -

Min imal

- - - - - 9 0 0 6 0

- - - - -

Mil d

- - - - - 0 0 0 0 7

- - - - -

Mod erate

- - - - - 0 0 0 0 0

- - - - -

Severe

- - - - - 0 0 0 0 0

- - - - -

Tub ules, Dilation

NR 23 5 6 10 0 15 0 29 28 13 0 8 0 12 14

Min imal 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

Mod erate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Sev ere 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Tub ules, Mineralization / Casts

NR 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Min imal 23 15 31 10 8 3 0 0 6 0 0 4 0 4 0

Mil d 57 75 50 67 75 45 35 47 33 60 25 63 45 60 38

Mod erate 20 10 19 24 17 52 65 53 61 40 75 33 55 36 62

Sev ere 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0DRAFT 

# 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 d uration of the exposure) of 31%, 30 %, 23% and 30%, respectively for the

12.5, 25, 50 and 100 ng/L tre atments. 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 lar val 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 o n larval serum T 4 

# 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) T 4 (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 17 β

- trenbolone exposure on juvenile

growth for either gender ( Table 8.4.3

- 2 ).

Juvenile liver

- somatic index (LSI)

There were no significant treatment

- related eff ects 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 a nd 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 trenbolone

treated frogs were mild but significantly different than cont rols in the 2 5, 50 and 100 ng/L

treatments (T able 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

r eplicates 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

Tab le 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 g onad histopathology

The only t wo significant treatment

- related fin dings in the testes of male s 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 findin gs 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 an alysis;).

Trenbolone concentration (ng/L) 0 12.5 25 50 100

n 39 21 20 25 25

r eplicates 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 g onad histopathology

There were no apparent effects of trenbolone exposure on the ovaries of juvenile females (data

not shown).

Male k idney histopathologyDRAFT 

# 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 m ineralization/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

r eplicates 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 k idney 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 th e 100 n g/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 0DRAFT 

# 125

8.4.4 B enzophenone

- 2 results

Measured test concentrations

Mean ± SD (n = 36 ) measured concentrations of the 1.5, 3.0 and 6.0 m g/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 . C Vs (averaged across the d uration of the exposure) were 4%, 6% and 1 %,

respectively for the 1.5

, 3.0 and 6.0 m g/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 nu mber 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 a lso a significant delay in time

to NF stage 62 in the 6 mg/L treatment as compared t o control (Jonckeere

- Terpstra, Table 8.4.4

-

3 ).

Larval blood thyroxineDRAFT 

# 126

In the case of the BP

- 2 study, heparinized plasma samples were analyzed for T 4 

# 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 T 4 

# in either sample set and T 4 

# levels from the two methods did n ot 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 T 4 

# 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 fema les (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 gend ers (Jonckeere

- Terpstra, Table 8.4.4

- 4 ). The

magnitude of induction is much higher than any of the other inter

- laboratory studies and

emphasiz es 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 ± 35.6DRAFT 

# 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 female s and genetic males

which showed mixed sex gonads ( i.e . the 3.0 and 6.0 mg/L treatments)

. T hese included

concentration

- dependent increases in the prevalence and severity of basophilia, decreased

hepatocellular vacuolation, and intravascular proteinaceous fluid. As compared to controls, BP

2

- 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 live r 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; high lighted 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 50DRAFT 

# 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



# G onad histopathology

Genotypic males experienced feminization of the gonad resulting in a portion of indi viduals

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

reve rsed presenting only ovary tissue. In genotypic females, there was a significant treatment

related 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 preval ence 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 0DRAFT 

# 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



# K idney 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, ProteinDRAFT 

# 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 oth ers. 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; highlight ed 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