UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the 

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

Environmental Fat~~fec~~~n ~ Office of Pesticide Program 

THROUGH: Jess Rowland ~ S ~~~ Deputy Division Director 

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

EPA has completed its Weight of Evidence (WoE) assessment evaluating results of the 

Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. 

The WoE documents for the 52 chemicals are listed in Attachment A along with the chemical 

and report identifiers. 

Page I of2 

Page 1 of 59
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 59
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: CAPTAN 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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ii 

TABLE OF CONTENTS 

Abbreviations...................................................................................................iii 

Executive Summary...........................................................................................1 

I. Introduction.......................................................................................3 

II. Sources of Scientific Data and Technical Information......................................4 

A. EDSP Tier 1 Screening Assays............................................................4 

B. Other Scientifically Relevant Information (OSRI)......................................4 

III. Weight of Evidence (WoE) Evaluation.......................................................4 

A. EDSP Tier 1 Screening Assays.............................................................7 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................8 

1. Effects on Estrogen Pathway.....................................................8 

2. Effects on Androgen Pathway..................................................13 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17 

IV. Committee’s Assessment of Weight of Evidence..........................................19 

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................19 

1. Tier 1 in vivo Assays.............................................................20 

2. OSRI...............................................................................21 

B. Estrogen Pathway...........................................................................23 

C. Androgen Pathway.........................................................................23 

D. Thyroid Pathway...........................................................................23 

E. Conclusions.................................................................................23 

V. EDSP Tier 2 Testing Recommendations....................................................24 

VI. References.......................................................................................25 

LIST OF TABLES: 

 Table 1. Tier 1 Assay for Captan......................................................................7 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for Captan........................................10 

 Table 3. Androgenic/Anti-Androgenic Pathway for Captan......................................14 

 Table 4. Thyroid Pathway for Captan...............................................................18 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................28 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................40 

APPENDIX 3. References Not Utilized in the Captan WoE Analysis.................................50 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

HPLC/MS/MS High Pressure Liquid Chromatography/Mass Spectroscopy 

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

Methods 

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

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v 

Abbreviation Terminology 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

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vi 

Abbreviation Terminology 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 51 

Executive Summary 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

perturbations of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid 

(HPT) axes. In addition to the available Tier 1 data, other scientifically relevant information 

(OSRI), including general toxicity data and open literature studies of sufficient quality were 

considered in this weight of evidence (WoE) assessment. 

In determining whether captan interacts with E, A or T hormone pathways, the number and type 

of effects induced, the magnitude and pattern of responses observed across studies, taxa and 

sexes were considered. Additionally, the conditions under which effects occur were considered, 

in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in 

general systemic toxicity or overt toxicity. 

On December 3, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of captan with the E, A or T signaling pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For the estrogen pathway, captan was negative in the ER, ERTA and steriodogenesis assays, and 

was equivocal in the aromatase assay. There were no effects observed in females in the FSTRA. 

No estrogen-related effects were seen in any of the mammalian of wildlife Part 158 studies. In 

the female pubertal assay, while delayed development (increase in age at VO) and decreased 

ovarian weights were observed at the low dose, it is noted that a 9% decrease in body weight and 

clinical signs of toxicity were observed suggesting that these effects were due to a dose that was 

on the cusp of being overtly toxic. Additionally, there were no other corroborating evidence in 

the Tier 1 assays or OSRI to suggest that captan potentially interacts with the estrogen pathway. 

There was no convincing evidence of potential interaction of captan with the androgen pathway. 

Captan was negative in the AR binding and steriodogenesis assays. No androgen-related effects 

were observed in the antagonist portion of the assay, the agonist portion of the assay could not be 

evaluated due to overt toxicity and/or excess mortality. There were no treatment-related effects 

in males in the FSTRA, except for an increase in body weight and length at the high 

concentration. In the male pubertal assay, there was an isolated finding observed (decreased 

ventral prostate weight); however, there were no complementary responses in this assay at a dose 

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Page 2 of 51 

that was at the cusp of overt toxicity. In addition, there were no androgen-related effects seen in 

any of the mammalian and wildlife Part 158 studies. 

In the AMA, there were no thyroid-related effects observed. In the male pubertal assay and Part 

158 mammalian studies, there were no thyroid-related effects observed in the absence of overt 

toxicity. In the female pubertal assay, while a decrease in T4 was observed, there were no other 

effects observed at this dose (which was on the cusp of overt toxicity). Collectively, there is no 

convincing evidence of a potential thyroid interaction in mammals or wildlife. 

Based on the weight of evidence evaluation, mammalian and wildlife EDSP Tier 2 studies are 

not recommended for captan since there was no convincing evidence of potential interaction with 

the estrogen, androgen or thyroid pathways. 

 

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Page 3 of 51 

I. Introduction 

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is designed to provide 

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

(E), androgen (A) or thyroid (T) signaling pathways. This interaction includes agonism and 

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

perturbations of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid 

(HPT) axes. In addition to the available Tier 1 data, other scientifically relevant information 

(OSRI), including general toxicity data and open literature studies of sufficient quality were 

considered in this weight of evidence (WoE) assessment. 

In determining whether captan interacts with E, A or T hormone pathways, the number and type 

of effects induced, the magnitude of responses, and the pattern of responses observed across 

studies, taxa, and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On December 3, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of captan with the E, A or T signaling pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Captan, a fungicide, has a water solubility of 5.1 mg/L at 25°C, with a log Kow of 2.8 and a 

bioconcentration factor of 126. The vapor pressure of captan is 9×10−8 mm Hg at 25°C, and 

vapor-phase captan will be degraded rapidly in the atmosphere (t1⁄2 = 1.5 hours). Captan is not 

expected to be susceptible to direct photolysis. Captan is expected to have high to moderate 

mobility in soil but volatilization from moist or dry soil surfaces, or from water surfaces, is not 

expected to be an important fate process. Captan undergoes rapid abiotic hydrolysis at 

environmental pH values of 5-9, with half-lives of 18.8 hours, 4.9 hours and 8.3 minutes 

measured in water at pH values of 5, 7 and 9, respectively. 

The available information considered in determining the potential interaction of captan with the 

E, A, T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant 

information (OSRI) such as general toxicity studies and/or other published articles. These data 

are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency, 

using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The 

EDSP Tier 2 studies recommendations are presented in Section V. 

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the 

agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix 

1. 

B. Other Scientifically Relevant Information (OSRI) 

In response to the Agency’s Test Orders, data believed to be relevant to one or more of the Tier 1 

assays were submitted as OSRI by the Test Order recipients and/or the public. This included 

studies published in the open literature and/or data submitted to support pesticide registration 

(e.g., Part 158 Guideline studies). The Agency’s review of the initial OSRI is provided in the 

Report of the Endocrine Disruptor Review Team on captan (USEPA, 2010). Since then, the 

Agency has also conducted a more recent search of available scientific literature for any 

additional relevant information. Summaries of the available OSRI are presented in Appendix 2. 

Additionally, literature/studies considered but not utilized for the WoE analysis are listed in 

Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

The principles, criteria and approach used in the WoE determination on the potential of a 

substance to interact with endocrine-related processes (i.e., E, A or T hormone pathways) were 

as described in the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA 

Scientific Advisory Panel (SAP (USEPA, 2013). The weight of evidence process identifies how 

the individual lines of evidence are assembled and integrated along two concepts (i.e., 

complementarity and redundancy) within the conceptual framework of an adverse outcome 

pathway). Broadly, there are four main steps outlined in the guidance which provide the 

foundation for WoE evaluations. The first step is to evaluate the individual studies for their 

scientific quality and relevance in evaluating potential endocrine interaction(s). The second step 

is to integrate the data along different levels of biological organization while examining the 

extent of complementarity (i.e., the concordance of endpoints within an assay that measures 

multiple endpoints) and redundancy (i.e., the concordance of endpoints/responses across assays) 

in the observed responses across these different levels of biological organization. The third step 

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the 

EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other 

scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 

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consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays were 

designed specifically to evaluate a number of key biological events including potential effects on 

receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other 

effects on the HPG and HPT axes. OSRI may include published literature studies as well as 

studies conducted under USEPA (often referred to as Part 158 data) or OECD guidelines 

submitted in support of pesticide registrations. Each study is evaluated for scientific quality and 

relevance for informing interactions with the E, A or T pathway. Additionally, the consistency 

of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple 

endpoints are measured in each assay. 

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the 

relative degree of diagnostic utility of a specific endpoint for discerning whether or not the 

chemical has interacted with the endocrine system, are considered. The collective response of the 

individual endpoints, as well as the conditions under which they were expressed, are considered 

when evaluating an overall indication of potential interaction as measured by the study. 

The second step in this WoE process is to formulate hypotheses and integrate the available data 

along different levels of biological organization. Two keys elements in the integration of data, as 

well as characterizing the extent to which the available data support a hypothesis that a chemical 

has the potential to interact E, A or T pathways, are the concepts of complementarity and 

redundancy. These two concepts provide a basis for considering the plausibility, coherence, 

strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are 

meant to evaluate whether or not a chemical can interact with E, A and T consisting of different 

levels of biological organization from a molecular initiating event, such as receptor binding, 

through potential adverse effects in apical endpoints such as sexual development and fecundity at 

the whole organism level. The extent of expression of responses at higher levels of biological 

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

After the data have been assembled and integrated, the third step is to characterize the main lines 

of evidence along with the conclusions; this characterization involves three components. The 

first component is whether the data provide relevant, robust and consistent evidence in terms of 

complementarity and redundancy as well as biological plausibility. Second, is at what level of 

biological organization were the responses observed and whether organisms exhibit 

compensatory responses at higher levels of biological organization? Finally, under what 

conditions did the responses occur including consideration of whether the responses were 

observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic 

toxicity introduces uncertainty in the ability to distinguish effects specifically related to an 

endocrine-related effect from a non-endocrine toxic response. 

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This uncertainty in distinguishing whether the responses were endocrine-related was discussed at 

the FIFRA SAP meeting in July 2013 that evaluated scientific issues associated with the WoE 

evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “In 

summary, the Panel agreed that little, if any, weight should be placed on signs of endocrine 

disruption in the presence of overt toxicity. All effects in endocrine sensitive tissues should be 

evaluated in terms of primary interactions with the endocrine system vs. secondary effects 

related to toxicity in non-endocrine organs or overall disruptions in homeostasis” (USEPA, 

2013). 

For these WoE analyses, overt toxicity was generally defined in accordance with EPA’s current 

approach as used by OPP in reviewing 40 CFR Part 158 studies for both human and ecological 

risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential 

evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as 

tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight 

decreases of ≥10% in mammals. Additionally, other effects including morphological (e.g., organ 

weights/histopathology), biochemical (e.g., blood chemistry), and other clinical signs (e.g., 

lethargy) were also considered when evaluating overt toxicity, especially if the effects were 

extreme. In some instances, one parameter (i.e., death or >10% decrease in mammalian body 

weight) was sufficient to consider a dose/concentration to be overtly toxic. However, in other 

instances, more than one parameter was needed to determine overt toxicity. For example, in the 

FSTRA, generally, body weight decreases were considered along with other responses when 

assessing potential overt toxicity. Additionally, effects which were considered to be signs of 

systemic toxicity were also captured and these effects were generally considered as less severe 

forms of toxicity (e.g., organ weights, blood chemistry). The circumstances for which a 

dose/concentration was considered overtly toxic for a particular study are described in Section 

IV.A. 

In summary, EPA considers multiple lines of evidence in including the observed responses in 

the Tier 1 assays and OSRI in the context of a chemical’s physical/chemical properties and its 

known modes of action in its overall characterization of a chemical’s potential to interact with 

the E, A or T pathway. Adequately addressing the three main components described above is 

fundamental to the WoE process and in determining whether additional data are needed. In 

addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their 

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account.

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 

assays and OSRI which are depicted in Tables 2 - 4. These tables contain data that are 

considered scientifically and biologically relevant with regard to a treatment-related effect 

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which supports a conclusion of whether or not a substance has the potential to interact with the 

E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the 

text for each respective pathway (E, A or T) but are not reported in the table for E, A or T. 

A. EDSP Tier 1 Screening Assays 

The Tier 1 assays submitted in response to the agency’s test order for captan are shown below in 

Table 1. 

Table 1. Tier 1 Screening Assays for Captan 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied 

(MRID 48615103) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 890.1300; 

OECD 455 

Requirement Satisfied 

(MRID 48615104) 

AR Binding Assay (Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied 

(MRID 48615101) 

Steroidogenesis Assay (Human cell line H295R) OSCPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID 48615106) 

Aromatase Assay (human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied 

(MRID 48615102) 

Uterotrophic Assay (Rat) OSCPP 890.1600; 

OECD 440 

Requirement Satisfied 

(MRID 48615107) 

Hershberger Assay (Rat) OSCPP 890.1400; 

OECD 441 

Requirement Not 

Satisfied1 

(MRID 48615105) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID 48669502) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID 48669503) 

Fish Short-term Reproduction Assay OSCPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID 48669501) 

Amphibian Metamorphosis Assay (Frog) OSCPP 890.1100; 

OECD 231 

Requirement Satisfied 

(MRID 49136701) 

1

This assay is not satisfied due to lack of surviving animals in the agonist phase of the assay. A repeat study is not 

required since no convincing evidence of androgen-related effects were seen in the other Tier 1 assays and will not 

impact the risk assessment. 

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B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis 

1. Effects on Estrogen Pathway 

The potential for captan to interact with the E hormone pathway is summarized in Table 2. The 

various targets of the estrogen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This table 

also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the 

presence of overt toxicity are discussed in the text but are not reported in the table and not 

considered further in the WOE assessment. 

Captan was negative (not interactive) in the Tier 1 in vitro ER binding and ERTA assays. In the 

Tier 1 steroidogenesis assay, a 9 and 25% decrease in testosterone concentration was observed in 

one of two runs at 10 and 100 μM, respectively. Whereas in an additional third run, precipitation 

was observed at these concentrations. In the in vitro Tier 1 aromatase assay, captan was 

classified as equivocal based on a decrease of 40% aromatase activity at the highest tested 

concentration (10−5 M). In addition, captan was demonstrated to be negative in several published 

ER transactivation studies [Kojima et al., 2004, 2005; and Okubo et al., 2004, although antiestrogenic activity was observed in the latter study at high concentrations (10 and 100 μM]. 

Captan was negative in the Tier 1 uterotrophic assay. 

In the female pubertal assay, overt toxicity was observed at the high dose (600 mg/kg/day) 

characterized as decreases in body weight (10%), clinical signs of toxicity (abnormal breathing 

and decreased movement), and death (2 animals). Also, at the low dose (300 mg/kg/day), 

clinical signs of toxicity were observed that were at the cusp of overt toxicity (9% decreased 

body weight, abnormal breathing and decreased movement). In this study, the mean age at 

vaginal opening (VO) was delayed by 4.0 and 5.3 days at the low and high doses (300 and 600 

mg/kg/day, respectively), and the mean age at first vaginal estrus was delayed by 2.5 days at the 

low dose and by 5.2 days at the high dose. Effects observed in female reproductive organ weight 

included: decreased ovarian weights in the low and high dose (18 and 23%, respectively) and 

uterus weights (wet, 35% and blotted, 34%) at the high dose group. Additionally, decreased 

pituitary weights were observed at the low (18%) and high (19%) dose groups. No treatmentrelated, histopathological findings were observed in the uterus. In the ovaries, the number of 

ovarian follicles (small, medium, antral and atretic) and follicular cysts were not different 

between control and treated females, however, the number of corpora lutea were decreased by 

30% and 29% in the low- and high-dose groups, respectively. 

No estrogen-related effects were seen in the mammalian Part 158 toxicity studies. 

In the FSTRA, there were no effects on females. In the Part 158 studies, no estrogen-related 

effects were observed in the avian reproduction toxicity studies in bobwhite quail and mallard 

ducks. 

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Page 9 of 51 

In an OSRI life cycle study in fathead minnows by Hermanutz et al. (1973), treatment-related 

reproductive effects included effects on fecundity at the medium and medium-high 

concentrations (16.8 and 39.5 μg/L, respectively). Additionally, effects on survival were 

observed at the high concentration, and effects on growth (body length) in parental fish and 30-

day old hatchlings were also observed. However, there were several limitations in this study 

including: use of a surfactant in addition to the acetone co-solvent; test concentrations were 

increased several times during the course of the study; and, low statistical power to detect 

differences between test groups. 

Page 17 of 59
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Page 10 of 51 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Captan Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Captan1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen levels 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

EDSP Tier 1 Assay 

ER Binding (MRID 48615103) N ERTA (MRID 48615104) NAromatase (MRID 48615102) E Steroidogenesis (MRID 48615106) N4 Uterotrophic (MRID 48615107) N Female Pubertal Rat (MRID 48669502) N ↓18% (L) N ↓18 % (L) ↑2.5 days (L) Age: ↑4.0 days (L) FSTRA (MRID 48669501) NE N N N N N 

OSRI 

Receptor Transactivation (Kojima et al., 2004) 

N 

Receptor Transactivation (Kojima et al., 2005) 

N 

Receptor Transactivation (Okubo et al., 2004) 

N 
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Page 11 of 51 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Captan Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Captan1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen levels 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Vitellogenin 

One-generation reproduction (Rat; MRID 00120315) 

NE NE NE NE N 

Three-generation reproduction (Rat; MRID 00125293) 

NE NE NE NE N 

Developmental toxicity (Rat; MRID 48126603) 

NE N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00120316) 

NE N N N 

Chronic toxicity/ carcinogenicity (Rat; MRID 00161230) 

NE N N N 

Chronic toxicity (Dog; MRID 40893604) 

NE N N NE 

Avian reproduction (Quail; MRID 00098295/48126601) 

N 

Avian reproduction (Duck; MRID 00098296/48126602) 

N 

1. Key to responses: Low treatment (L), Medium-low treatment (ML), Medium treatment (M), Medium-high treatment (MH), High 

or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable i

weight are absolute unless otherwise indicated. KW= Kidney weight 
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Page 12 of 51 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OS

this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an X 

details see Section IV. A 

4. A 9 and 25% decrease in testosterone concentration was observed in one of two runs at 10 and 100 μM, respectively. Whereas in a

precipitation was observed at these concentrations. 

5. Decreased bodyweight of 11 and 15%, respectively at the low and high dose. 

6. Based on clinical signs and body weight decreases ≥10% in the high-dose animals 

N Negative findings (in vitro)/No effect (in vivo) 

E Equivocal assay response NE Not examined 
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2. Effects on Androgen Pathway 

The potential for captan to interact with the androgen pathway is summarized in Table 3. The 

various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for androgenic, anti-androgenic or HPG axis effects. This 

table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in 

the presence of overt toxicity are discussed in the text but are not reported in the table and not 

considered further in the WOE assessment. 

Captan was classified as negative in the in vitro Tier 1 androgen receptor (AR) binding assay. In 

the Tier 1 steroidogenesis assay, a 9% decrease in testosterone concentration was observed in 

one of two runs at 10 μM, whereas in an additional third run, precipitation was observed at this 

concentration. In addition, no other significant changes in testosterone production was observed 

in the remaining test concentrations up to 100 μM. 

In the Tier 1 Hershberger assay, in the agonist portion of the assay both doses were overtly toxic 

(exhibiting severe clinical signs of toxicity, death or were sacrificed due to clinical signs). In the 

antagonist phase of the assay, no effects on AST weights were seen at the low dose (100 

mg/kg/day) in the absence of overt toxicity. However at the mid and high doses (200 and 400 

mg/kg/day), either all animals died or were sacrificed due to severe clinical signs. 

In the male pubertal rat study, overt toxicity was observed at the high dose (200 mg/kg/day) 

characterized as decreases in body weight (13%), clinical signs of toxicity (rales, abnormal 

breathing and ungroomed) and death (2 animals). Also, at the low dose (100 mg/kg/day), 

clinical signs of toxicity were observed that were at the cusp of overt toxicity (9% decreased 

body weight, rales and alopecia). In this study, serum testosterone concentrations were 

decreased at the low (↓23) and high (42%) dose, although the change in the low-dose group was 

not statistically significant. Age at attainment of preputial separation (PPS) was not different 

from control, but weight at PPS was decreased in a manner similar to final body weight changes 

(↓8 and 13%, at low and high dose, respectively). Effects observed in male reproductive organ 

weights were decreased ventral prostate (↓15 and 20%, respectively) and LABC (↓15%; high 

dose) weights. 

In the FSTRA, no androgen-related effects were observed. In this study, although increases in 

vitellogenin (VTG) concentrations at the mid and high treatment were observed in male fish, the 

results were highly variable and not attributable to treatment. No androgen-related effects were 

determined in the mammalian or wildlife Part 158 toxicity studies. 

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Page 14 of 51 

Table 3: Androgenic/Anti-Androgenic Pathway for Captan Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Captan1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48615101) N Steroidogenesis (MRID 48615106) N4 Hershberger (MRID 48615105) N5 N5 Male Pubertal Rat (MRID 48669503) N N N N N N 

VP: 

↓15% 

(L) 

N 

FSTRA (MRID 48669501) NE N N N N N 

OSRI 

Receptor Transactivation (Kojima et al., 

2004)

N 

One-generation reproduction (Rat; MRID 00120315) 

NE NE NE NE NE NE N 

Three-generation reproduction (Rat; MRID 00125293) 

NE NE NE NE NE NE N 
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Page 15 of 51 

Table 3: Androgenic/Anti-Androgenic Pathway for Captan Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Captan1Study Type/ Literature Citation 

AR Binding 

AR Activation 

Steroidogenesis 

Sex Steroid 

hormones 

Testes Weight/GSI 

Gonad Staging and 

Histopathology 

Epididymides 

Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex 

Organ Weights/ 

2° Sex 

Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at 

PPS 

Vitellogenin 

Chronic toxicity/ carcinogenicity (Rat; MRID 00120316) 

N N NE N N NE 

Chronic toxicity/ carcinogenicity (Rat; MRID 00161230) 

N N NE N N NE 

Chronic toxicity/ carcinogenicity (Dog; MRID 40893604) 

N N NE N NE N 

Avian reproduction (Quail; MRID 00098295/48126601) 

 N 

Avian reproduction (Duck; MRID 00098296/48126602) 

 N 

1. Key to responses: Low treatment (L), Medium-Low treatment (ML), Medium treatment (M), Medium high treatment (MH), High tr

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in thi

are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), D

Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). CS= clinical signs 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSR

this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI is indicated by an X i

see Section IV. A 
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Page 16 of 51 

4. A 9% decrease in testosterone concentration was observed in one of two runs at 10 μM, whereas in an additional third run, precipitati

concentration. In addition, no other significant changes in testosterone production was observed in the remaining test concentrations 

5. In the agonist phase, both doses were overtly toxic (exhibiting severe clinical signs of toxicity, death or were sacrificed due to clinica

phase of the assay, no effects on AST weights were seen at the low dose (100 mg/kg/day) in the absence of overt toxicity. Animals at 400 mg/kg/day) died or were sacrificed due to severe clinical signs and body weight loss. 

6. Based on clinical signs (rales and/or abnormal breathing) and decreased body weight (≥10%) in the high-dose animals. In addition, tw

euthanized after being found moribund. 

N Negative findings (in vitro)/No effect (in vivo)

NE Not examined 
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C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis 

The current EDSP Tier 1 battery does not have a specific in vitro assay to detect chemicals with 

the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it 

does include three in vivo assays that provide data to detect changes in the HPT axis, i.e., the 

pubertal female and male (rat) assays and the AMA (frog). 

The potential for captan to interact with thyroid regulation is summarized in Table 4. The 

various targets of the thyroid pathway across the relevant Tier 1 assays are delineated so as to 

facilitate determination of potential for thyroid or HPT axis effects. This table also includes 

HPT-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of 

overt toxicity are discussed in the text but are not reported in the table and not considered 

further in the WOE assessment. 

There were no treatment-related effects on the thyroid hormonal pathway in the Tier 1 male 

pubertal rat assay in the absence of overt toxicity. It is noted that at the high dose, there was an 

18% decrease in serum T4 in the presence of overt toxicity and without any other corresponding 

thyroid related effects. In the Tier 1 female pubertal assay, multiple effects were observed in the 

presence of or on the cusp of overt toxicity (moribund sacrifices, clinical signs and significant 

decreases in body weight). Serum T4 concentrations were decreased in a dose-dependent manner 

by 27 and 42% in the low- and high-dose groups (300 and 600 mg/kg/day, respectively), and 

thyroid stimulating hormone (TSH) concentrations were decreased in a non-dose-dependent 

manner. In the thyroid, follicular cell height was increased in the high-dose animals, and there 

was a corresponding decrease in the colloid area of thyroid follicles. A similar, though reduced, 

trend in increased follicular cell height at the low dose was noted. Finally, absolute and adjusted 

pituitary weights were decreased by 18% at the low dose, and absolute, adjusted and relative 

pituitary weights were decreased by 19-28% in the high-dose group. 

There were no treatment-related effects of captan in the AMA. No thyroid-related effects were 

observed in the mammalian Part 158 studies. 

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Page 18 of 51 

Table 4. Thyroid Pathway for Captan.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Captan1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (± or asynchronous, HLL) Growth (BW, SVL) Systemic Toxicity Observed2 

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Female Pubertal 

Rat (MRID 

48669502) 

N N ↓27% 

(L) N ↓18% (L) 

BW: ↓9% 

 CS 

 ( L, H) 

BW4 

(H) 

Male Pubertal Rat 

(MRID 48669503) N N N N N CS 

 (L, H) 

BW5

(H) 

AMA 

(MRID 49136701) N N N N 

OSRI

Chronic toxicity/ 

carcinogenicity 

(Rat; MRID 

00120316) 

N N N X 

(M, H)

X 

(M, H) 

Chronic toxicity/ 

carcinogenicity 

(Rat; MRID 

00161230) 

N N N X 

(H) 

X 

 (H)

Chronic toxicity/ 

carcinogenicity 

(Dog; MRID 

40893604) 

N N NE X 

 (H) N 

1. Key to responses: Low treatment (L), Medium treatment (M), High treatment (H). Arrows (↓ or 

↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely 

evaluated or is not applicable in this assay. CS= clinical signs, BW= body weight 

2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic 

toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A 

3. The overt toxicity in the Tier 1 assays are presented in this column (e.g. ↓BW). The overt toxicity for the OSRI 

is indicated by an X in this column. For details see Section IV. A 

4. Based on clinical signs and body weight decreases ≥10% in the high-dose animals 

5. Based on clinical signs (rales and/or abnormal breathing) and decreased body weight (≥10%) in the high-dose 

animals. In addition, two high-dose animals were euthanized after being found moribund. 

N Negative findings 

NE Not examined 

 

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IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight of evidence (WoE) determination on the 

potential of captan to interact with endocrine related processes (i.e., E, A, or T signaling 

pathways) as well as recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

are considered, along with other scientifically relevant information (e.g., 40 CFR Part 158 test 

guidelines and published or publicly available peer-reviewed studies). WoE analysis in the 

context of the EDSP follows the Agency’s guidance (USEPA, 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and 

OSRI), then performing an integrated analysis of those lines of evidence. 

The WoE evaluation includes considerations of biological plausibility of the findings from the 

different lines of evidence by examining the consistency, coherence, and interrelationships 

among the measured endpoints within and across studies. The available findings from standard 

toxicology studies on the substance may contribute to the WoE evaluation in helping elucidate if 

effects seen in the Tier 1 assay are related to perturbations of the endocrine system per se or 

alternatively a sequelae of systemic effects. Endocrine modes of action may elicit a number of 

phenotypic consequences other than those evaluated in the Tier 1 assays. 

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to 

whether or not the responses are related through an endocrine pathway, therefore non-endocrine 

toxic responses (including but not limited to mortality or body weight changes) are also 

considered in this WoE evaluation. The FIFRA Scientific Advisory Panel (SAP) that evaluated 

scientific issues associated with weight of evidence evaluation of the results of the Tier 1 assays 

stated that “In summary, the Panel agreed that little, if any, weight should be placed on signs of 

endocrine disruption in the presence of overt toxicity. All effects in endocrine sensitive tissues 

should be evaluated in terms of primary interactions with the endocrine system vs. secondary 

effects related to toxicity in non-endocrine organs or overall disruptions in homeostasis” 

(USEPA, 2013).

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI 

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 and OSRI 

studies are described below. Generally, one parameter (i.e., death or >10% decrease in 

mammalian body weight) was sufficient for a dose/concentration to be considered overtly toxic. 

However, in other instances, more than one parameter was needed to determine overt toxicity. 

Effects which were considered to be signs of systemic toxicity were generally less severe forms 

of toxicity (e.g., changes in organ weights or blood chemistry). 

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1. Tier 1 in vivo Assays 

In the 21-day AMA, Day 7 mortality was not reported for all groups, but no mortalities were 

reported in the negative control and low-concentration groups. No mortalities were reported in 

these groups at Day 21; mortalities in the remaining groups were no more than 1.7 or 3.3%. 

There were no treatment-related clinical signs. 

In the 21-day FSTRA, the survival rates in the negative and solvent control groups were 88 and 

96%, respectively; overall survival was 100% in all captan treatment groups. An increase in male 

body weight at the high-treatment concentration, and increases in male body length at the 

medium- and high-treatment levels were observed relative to the negative control group. There 

were no treatment-related effects on secondary sex characteristics or clinical signs. 

In the Hershberger assay, two animals in the low-dose group (200 mg/kg/day) died before study 

termination in the androgen agonist portion: one animal was found dead (suspected dosing error) 

and one animal was euthanized because of breathing difficulties. All animals in the high-dose 

group (400 mg/kg/day) were euthanized prior to study termination: four animals were euthanized 

due to abnormal breathing and body weight loss and the remainder were humanely euthanized 

prior to the potential onset of breathing difficulties and initiation of or continued weight loss. In 

the anti-androgen portion, one animal in the 100 mg/kg/day group was found dead on Day 1 with 

no obvious sign of dosing error. All animals in the medium- and high-dose groups (200 and 400 

mg/kg/day, respectively) were humanely euthanized or found dead prior to study termination: 

euthanasia was conducted due to body weight loss, or prior to the potential onset of breathing 

difficulties and weight loss. It was noted that moribund animals all exhibited abnormal breathing 

that was severe enough to result in rapid weight loss and, at necropsy, the stomach and/or 

intestines were severely distended with air. 

In the androgen agonist assay, clinical signs of abnormal breathing were observed in two rats in 

the low-dose group. Body weights were decreased by 9-10% on Days 4 and 7 and decreased by 

12% on Day 11. In the anti-androgen assay, one animal in the low-dose (100 mg/kg/day) group 

exhibited clinical signs of toxicity including lethargy and decreased movement, abnormal 

breathing and thin appearance. One additional animal in this group was found to have rales and 

another exhibited lethargy. Final body weights were comparable to the negative control; 

however, body weight gains were decreased by 49%. 

In the female pubertal assay, two animals in the high-dose group (600 mg/kg/day) were found 

moribund and euthanized on Study Days 5 and 17; all other rats survived until scheduled 

sacrifice. Cage-side and clinical observations included decreased movement (single occasion at 

the low dose) and abnormal breathing in several low- and high-dose animals (including the 

euthanized animals). In addition, final body weights were decreased by 9 and 10% in the lowand high-dose groups, respectively, yielding decreased overall body weight gains of 14% and 

15%, respectively. 

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Page 21 of 51 

In the male pubertal assay, two high-dose animals (200 mg/kg/day) were found moribund with 

abnormal breathing and were euthanized on Study Days 18 and 27. Cage-side and clinical 

observations included one animal with alopecia (100 mg/kg/day), one un-groomed animal (high 

dose), and two animals with rales (one each in the low- and high-dose groups). Mean unadjusted 

and adjusted final body weights were decreased by 9 and 13%, respectively, resulting in 

decreased unadjusted and adjusted overall body weight gains of 11 and 16%, respectively. 

In the uterotrophic assay, all animals survived until scheduled termination, with the exception of 

one animal in the 1000 mg/kg/day group that was euthanized moribund on Day 3. There were no 

treatment-related clinical findings in the surviving rats. Final body weights were decreased by 

15% on Day 3 in the high-dose (1000 mg/kg/day) group, resulting in an overall (Days 1-4) body 

weight loss of 216%. Final body weights in the 313 mg/kg/day group were decreased (not 

significant; NS) by 11%, with an overall body weight loss (p<0.05) of 10.2 g. The decreased 

body weights indicate that the dose levels tested were adequate. Uterine weights in the captan 

treatment groups were comparable to the vehicle controls. 

2. OSRI 

No systemic or overt toxicity was observed in the one-generation reproduction toxicity study in 

rats up to the highest dose (25 mg/kg/day). 

There were no reported treatment-related effects on mortality in the P, F1 or F2 parental animals 

in the three-generation reproduction toxicity study in rats. Differences in mortality were 

observed in the F2a litter at LDs 1 and 4, and in the F3a litter at LD 4, for the medium-high dose 

pups (250 mg/kg/day); and in the F2a and F3a litters at LDs 1 and 4, and in the F2b litter at LD 7, 

for the high-dose pups (500 mg/kg/day), but the actual decreases were not reported. Decreased 

parental body weights were observed in all groups, except the low-dose (25 mg/kg/day) animals. 

Decreases in the medium-low dose group (100 mg/kg/day) were occasional, and ranged from 15-

18% in males and 5-9% in females. Body weight decreases in the medium-high and high-dose 

parental groups ranged from 8-19% and 12-37%, respectively. Decreases in mean litter weights 

were observed in all treatment groups, with decreases up to 44% in the high-dose pups. Litter 

weight decreases in the low-dose group were <10%. Food consumption was decreased in all 

treatment groups except for the low-dose F1 males and the low- and medium-low dose F2

females. 

In the prenatal developmental toxicity study in rats, no mortalities occurred during the study, and 

clinical signs were limited to increased hair loss and lack of grooming in some high-dose dams. 

Maternal body weights in the high-dose dams were decreased throughout treatment (GD 7-15) 

and on GD 17 by 5-10%. Body weight gains were decreased during GD 0-20 and 6-20 by 8 and 

10%, respectively. During GD 7-9, food consumption was decreased by 47%, with decreases of 

Page 29 of 59
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Page 22 of 51 

17% during GD 10-13 and 14-16. Food consumption recovered with an increase of 17% during 

GD 17-20. 

In the combined chronic toxicity/carcinogenicity study in rats, survival was generally similar 

between the control and treatment groups until Week 70 for the high-dose animals (250 

mg/kg/day); further decreases in survival were noted in the medium-dose males and females 

(100 mg/kg/day) at Weeks 86 and 94, respectively, and at Weeks 94 and 82, respectively, in the 

low-dose males and females (25 mg/kg/day). No treatment-related clinical signs were observed 

during the study. Dose-related decreases in mean body weights for both sexes were observed at 

the medium- (↓6-20%) and high-doses (↓8-19%) throughout the study. Feed consumption was 

only slightly decreased in the medium- and high-dose animals. 

In another combined chronic toxicity/carcinogenicity study in rats, no treatment-related effects 

were observed for mortality or clinical signs. In the high-dose animals, mean body weight 

decreases in the high-dose group (98 mg/kg/day) ranged from 12-14% in males and 6-8% in 

females through Week 16, with similar decreases throughout the study; however, the final body 

weight decreases at 24 months were 8 and 10% in males and females, respectively. Food 

consumption was reported to be slightly lower in the high-dose animals. 

In the chronic toxicity study in dogs, no treatment-related effects were observed for mortality, 

body weights or food consumption. All dogs survived but increased incidences of emesis and 

soft/mucoid stools were observed in the high-dose animals (300 mg/kg/day). 

In the avian reproduction toxicity study in bobwhite quail, no treatment-related mortalities were 

observed; one mortality each occurred in the medium- and high-treatment groups due to physical 

injury. Evidence of stress including lacerated scalps, head lesions and signs of head and toepicking were observed in the control and all treatment groups. No treatment-related effects on 

body weight were observed. Feed consumption was reported to be decreased in the 300 mg/kg 

and 1000 mg/kg treatment groups, although the decreases were not apparent at each 

measurement interval. 

In the avian reproduction toxicity study in mallard ducks, no treatment-related effects on 

mortality, body weight or feed consumption were observed. One hen in the low-treatment group 

died during the study; the death was attributed to the stress of egg production. 

In a published full life cycle study in Fathead minnows by Hermanutz et al. (1973), survival of 

parental fish was decreased in the mean-measured 63.5 μg/L (high concentration) group (↓98%). 

The parental fish length and the F1 fish survival and length were decreased in the mean-measured 

39.5 μg/L (medium-high concentration) group. Percent hatchability of embryos from unexposed 

parents incubated at 63.5 μg/L captan was similar to that of control embryos, but all larvae died 

5-8 days after hatching. There also was 100% mortality of 1-day-old larvae from unexposed 

Page 30 of 59
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Page 23 of 51 

parents incubated at 63.5 μg/L captan within 24 hours. Survival of 3-day-old hatchlings in the 

medium-high concentration group was decreased by approximately 50%. 

B. Estrogen Pathway 

Captan was negative in the ER, ERTA and steriodogenesis assays, and was equivocal in the 

aromatase assay. There were no effects observed in females in the FSTRA. No estrogen-related 

effects were seen in any of the mammalian of wildlife Part 158 studies. In the female pubertal 

assay, while delayed development (increase in age at VO) and decreased ovarian weights were 

observed at the low dose, it is noted that a 9% decrease in body weight and clinical signs of 

toxicity were observed suggesting that these effects were due to a dose that was on the cusp of 

being overtly toxic. Additionally, there were no other corroborating evidence in the Tier 1 

assays or OSRI to suggest that captan potentially interacts with the estrogen pathway. 

C. Androgen Pathway 

There was no convincing evidence of potential interaction of captan with the androgen pathway. 

Captan was negative in the AR binding and steriodogenesis assays. No androgen-related effects 

were observed in the antagonist portion of the assay, the agonist portion of the assay could not be 

evaluated due to overt toxicity and/or excess mortality. There were no treatment-related effects 

in males in the FSTRA, except for an increase in body weight and length at the high 

concentration. In the male pubertal assay, there was an isolated finding observed (decreased 

ventral prostate weight); however, there were no complementary responses in this assay at a dose 

that was at the cusp of overt toxicity. In addition, there were no androgen-related effects seen in 

any of the mammalian and wildlife Part 158 studies. 

D. Thyroid Pathway 

In the AMA, there were no thyroid-related effects observed. In the male pubertal assay and Part 

158 mammalian studies, there were no thyroid-related effects observed in the absence of overt 

toxicity. In the female pubertal assay, while a decrease in T4 was observed, there were no other 

effects observed at this dose (which was on the cusp of overt toxicity). Collectively, there is no 

convincing evidence of a potential thyroid interaction in mammals or wildlife. 

E. Conclusions 

Captan demonstrates no convincing evidence of potential interaction with the estrogen, androgen 

or thyroid pathways in mammals or wildlife. 

Page 31 of 59
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Page 24 of 51 

V. EDSP Tier 2 Testing Recommendations 

Based on the weight of evidence evaluation, mammalian and wildlife EDSP Tier 2 studies are 

not recommended for captan since there was no convincing evidence of potential interaction with 

the estrogen, androgen or thyroid pathways. 

Page 32 of 59
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Page 25 of 51 

VI. References 

Beavers, J.B. (1980a) One-generation reproduction study-- Bobwhite quail: Captan technical 

(SX-1086). Report No. S-1717. Unpublished study conducted by Wildlife International Ltd. for 

Chevron Chemical Company. (MRID 00098295/48126601) 

Beavers, J.B. (1980b) One-generation reproduction study-- Mallard duck: Captan technical 

(SX-1086). Report No. S-1716. Unpublished study conducted by Wildlife International Ltd. for 

Chevron Chemical Company. (MRID 00098296/48126602) 

Blair, M. (1988) One-year oral toxicity study in dogs with captan technical. Unpublished study 

conducted by International Research and Development Corporation for ICI Americas Inc., 

Project No. IRDC 153-198. (MRID 40893604) 

Davis, J. P. (2011) The Uterotrophic Assay (OPPTS 890.1600); Captan. Unpublished study 

conducted by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study No.: 

C198-100, December 20, 2011. (MRID 48615107) 

Davis, J. P. (2012) The Hershberger Bioassay (OPPTS 890.1400); Captan. Unpublished study 

conducted by Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study No.: 

C198-200, January 5, 2012. (MRID 48615105) 

Davis, J.P. (2012) Pubertal development and thyroid function in intact juvenile/peripubertal 

female rats (OPPTS 80.1450); Captan. Unpublished study conducted by Integrated Laboratory 

Systems, Inc., Durham, NC. Laboratory Study No.: C198-300, June 20, 2012. (MRID 48669502) 

Davis, J.P. (2012) Pubertal development and thyroid function in intact juvenile/peripubertal male 

rats (OPPTS 890.1500); Captan. Unpublished study conducted by Integrated Laboratory 

Systems, Inc., Durham, NC. Laboratory Study No.: C198-301, June 21, 2012. (MRID 48669503) 

Goldenthal, E.I. (1982) Two-year oral toxicity/carcinogenicity study of captan in rats. 

Unpublished study conducted by International Research and Development Corporation for 

Stauffer Chemical Company. (MRID 00120316) 

Gordon, E. (2013). Captan: A weight of evidence evaluation of the U.S. EPA’s endocrine 

screening program Tier 1 screening battery. Elliot Gordon Consulting, LLC. Project No. 

83913/1. Captan Task Force, Raleigh, NC 

Hermanutz, R.O., Mueller, L.H., and Kempfert, K.D. (1973) Captan toxicity to Fathead minnows 

(Pimephales promelas), Bluegills (Lepomis macrochirus) and Brook trout (Salvelinus 

fontinalis). J. Fish. Res. Board Can. 30: 1811-1817. (MRID 00057846). 

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Kojima, H., Iida, M., Katsura, E., Kanetoshi, A., Hori, Y., and Kobayashi, K. (2003) Effects of a 

diphenyl ether-type herbicide, chlornitrofen, and its amino derivative on androgen and estrogen 

receptor activities. Environ. Health Perspect. 111:497–502. 

Kojima, H., Katsura, E., Takeuchi, S., Niiyami, K., and Kobayashi, K. (2004) Screening for 

estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using 

Chinese hamster ovary cells. Environ. Health Perspect. 112 (5): 524-531. 

Kojima, M., K Fukunaga, M. Sasaki, M. Nakamura, M. Tsuji, T. Nishiyama. (2005) Evaluation 

of estrogenic activities of pesticides using an in vitro reporter gene assay. Int. J. Environ. Health 

Res. 15:271-280. 

Lee, M.R. (2013) Captan- Amphibian Metamorphosis Assay with African Clawed Frog 

(Xenopus laevis) Following OPPTS Test Guideline 890.1100 and OECD Test Guideline No. 

231. Unpublished study conducted by Smithers Viscient, Wareham, Massachusetts. Laboratory 

report number 14017.6109. May 22, 2013. (MRID 49136701) 

Okubo, T., Yokoyama, Y., Kano, K., Soya, Y., and Kano, I. (2004) Estimation of estrogenic and 

antiestrogenic activities of selected pesticides by MCF-7 cell proliferation assay. Arch. Environ. 

Contam. Toxicol. 46:445-453. 

Rubin, Y. (1987) Captan: teratology study in the rat. Unpublished study conducted by Life 

Science Research Israel Ltd., Ness Ziona, Israel. Laboratory Project ID.: MAK/097/CAP, 

January 8, 1987. (MRID 48126603) 

Schardein, J.L. (1982a) Three-generation reproduction study in rats. Unpublished study 

conducted by International Research and Development Corporation for Stauffer Chemical 

Company. (MRID 00125293) 

Schardein, J.L. (1982b) One-generation reproduction study in rats with captan. Unpublished 

study conducted by International Research and Development Corporation for Stauffer Chemical 

Company. (MRID 00120315) 

Til, H.P., Kuper, C.F., and Folke, H.E. (1983) Life-span oral carcinogenicity study of Merpan in 

rats. Unpublished study conducted by CNO Institute TNO for Makhteshim Chemical Works Ltd. 

(MRID 00161230) 

USEPA (2010) CAPTAN - Report of the Endocrine Disruptor Review Team- Test Order#: 

EDSP-081301-163 and 164. December 9, 2010. Office of Chemical Safety and Pollution 

Prevention. U.S. Environmental Protection Agency, Washington, D.C. 

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USEPA (2013) Transmittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting 

held July 30 - August 1, 2013 on Weight-of-Evidence: Evaluating Results of EDSP Tier 1 

Screening. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection 

Agency, Washington, DC. 

Wagner, H. (2012) Captan: Steroidogenesis (Human Cell Line – H295R). Unpublished study 

conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No. 9141V-100374STER, 

January 4, 2012. (MRID 48615106) 

Wilga, P.C. (2012) Captan: Human Recombinant Aromatase Assay. Unpublished study 

conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9141V-100374AROM, 

January 4, 2012. (MRID 48615102) 

Willoughby, J.A. (2012) Captan: Androgen Receptor Binding Assay Using Rat Prostate Cytosol. 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9141V100374ARB, January 5, 2012. (MRID 48615101) 

Willoughby, J.A. (2012) Captan: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol. 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. Laboratory Study No: 9141V100374ERB, January 5, 2012. (MRID 48615103) 

Willoughby, J.A. (2012) Supplemental Information - Laboratory Proficiency Data for ERTA 

assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. 

Unpublished study conducted by CeeTox, Inc., Kalamazoo, MI. July19, 2012. (MRID 

48843501) 

Willoughby, J.A. (2012) Captan: Estrogen Receptor Transcriptional Activation (Human Cell 

Line HeLa-9903)). Unpublished study conducted by CeeTox, Kalamazoo, MI. Laboratory 

Report No. 9141V-100374ERTA, January 5, 2012. (MRID 48615104) 

York, D.O. (2012) Captan- Short-Term Reproduction Assay with Fathead Minnow (Pimephales 

promelas) Following OPPTS 890.1350 and OECD 229 Guidelines. Unpublished study 

conducted by Smithers Viscient, Wareham, Massachusetts. Laboratory report number 

14017.6110. June 25, 2012. (MRID 48669501) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 49136701) of captan on amphibian metamorphosis of African clawed 

frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian larvae at 

Nieuwkoop-Faber (NF) stage 51 (80/control and treatment group; 20/replicate) were exposed to 

nominal concentrations of captan (93% purity) at 0 (negative and solvent [0.0030 mL/L 

dimethylformamide (DMF)] controls), 0.00012, 0.0012, and 0.012 mg a.i./L. Mean-measured 

concentrations were <0.0000091 (<LOQ; controls), 0.000064, 0.00077, and 0.0073 mg a.i./L; 

reviewer-calculated time-weighted average (TWA) measured concentrations were <0.0000091 

(<LOQ; controls), 0.000060, 0.00074, and 0.0072 mg a.i./L. The test system was maintained at 21 

to 23o

C and a pH of 6.9 to 7.8. 

There were no significant differences (p>0.05) between the negative control and solvent control 

for any of the endpoints. Unless otherwise indicated, all effects are reported based on comparison 

to the negative control. 

Throughout the 21-day exposure period, mortality did not exceed 3% in any treatment group. 

Spinal deformities were observed in the controls and all treatment groups with incidence rates of 

11 to 32%; this was not attributed to exposure to captan. There were no significant effects of 

treatment on body weight or snout-vent length. 

There were no significant effects (p>0.05) on normalized (for snout-vent length) hind limb length 

(HLL) on Day 7 or 21 at any of the treatment levels. There was no significant (p>0.05) acceleration 

or delay of median NF developmental stage at 7 or 21 days at any treatment level; no asynchronous 

development was observed. Late-stage tadpoles were found in both control groups and all 

treatment groups with no apparent pattern in their occurrence; there were 3, 4, 4, 2, and 1 

individuals at NF 61 in the negative control, solvent control, and low, mid, and high treatment 

groups, respectively. Thyroid gland histopathology findings were observed in both controls and 

all treatment groups. Histopathological effects included mild to moderate follicular cell 

hypertrophy and mild follicular cell hyperplasia in the controls and all treatment groups. There 

were no apparent treatment-related patterns in the histopathological findings. 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48615101), ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, 

which was conducted to measure the binding of a single concentration of [3

H]-R1881 (1 nM) in 

the presence of increasing concentrations (logarithmic increase from 10−11 to 10−3 M) of captan 

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(93% purity, Lot #91138103). DMSO was used as a vehicle at a final assay concentration of 

approximately 3.2%. A total of three runs were performed, and each run included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard. 

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

cytosol was present in reasonable numbers and was functioning with appropriate affinity for the 

radio-labeled reference androgen (R1881) prior to routinely conducting AR competitive binding 

experiments. Saturation binding data were not originally provided in the study report; however, 

summarized saturation binding data (MRID 48843501) from the performing laboratory were 

submitted following a request by the Agency. The dissociation constant (Kd) for [3

H]-R1881 

was 0.613±0.041 nM and the estimated Bmax was 0.817±0.049 fmol/100 μg protein for the single 

batch of prostate cytosol that was prepared. The mean and individual Kd and Bmax values were 

below the expected ranges reported in the EPA validation program (0.685 to 1.57 nM and 7 to 16 

fmol/100 μg protein, respectively). However, confidence in these numbers is high based on the 

goodness of fit (R2

 = 0.957-0.984) and the small variation among runs. 

In the competitive binding experiment, the estimated mean log IC50s for R1881 (−9.0 M) and the 

weak positive control, dexamethasone (−4.6 M), were within the expected ranges. The mean 

relative binding affinity (RBA) for the weak positive control was 0.004%. Confidence in these 

numbers is high due to the small variation. Drift in the study assay could not be assessed 

because all solvent control tubes occurred at the beginning of the run. All performance criteria 

were generally met. 

Precipitation of captan was visually observed at a concentration of 10−3 M in Run 1, therefore the 

second and third runs were performed at a maximum test concentration of 10−4 M. Captan had 

no effect on specific binding of the [3H]-ligand over the concentration range of 10−11 to 10−4 M; 

therefore, a log IC50 and RBA could not be calculated for captan as it did not result in 50% 

reduction in binding at any concentration. 

Based on the results from the three runs, captan is classified as a Non-Binder in the Androgen 

Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48615102), captan (93% a.i. Batch #91138103 ) 

was incubated with human recombinant aromatase and tritiated androstenedione ([1β3

H(N)]-

androst-4-ene-3,17-dione; [3

H]ASDN) in dimethyl sulfoxide (DMSO) at log concentrations of 

10-10 to 10-3 M or 10-11 to 10-5 M for 15 minutes to assess the potential of captan to inhibit 

aromatase activity. 

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Aromatase activity was determined by measuring the amount of tritiated water produced at the 

end of a 15-minute incubation for each concentration of chemical. Tritiated water was quantified 

using liquid scintillation counting (LSC). Three runs were conducted, and each run included a 

full activity control, a background activity control, a positive control series (10−10 to 10−5

 M) 

with a known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN), and the test chemical series 

(10−11 to 10−5

 M) with three repetitions per concentration. In Run 1, precipitation occurred 

during the 15-minute incubation period with the two highest concentrations (10−3

 and 10−4 M) of 

captan tested; these results were not used for data interpretation. Subsequently, for Runs 2 and 3, 

the highest test concentration that did not result in precipitation, 10-5 M, was used as the upper 

limit. 

Aromatase activity in the full activity controls averaged 0.611 ± 0.026 nmol·mg-protein−1·min−1. 

The response of each full activity control within a run was between 90 to 110% of the average 

full activity. Activity in the background controls ranged 0.25 to 0.37% and averaged 0.30% of 

the full activity controls. 

For the positive control substance (4-OH ASDN), aromatase activity results were within the 

recommended ranges for the top of the curve, bottom curve, Hill slope, log IC50, and coefficient 

of variation for replicates of each concentration within runs. The estimated log IC50 for 4-OH 

ASDN averaged -7.27 M and the Hill slope was -0.94. 

For captan, aromatase activity averaged 0.613 ± 0.005 nmol·mg-protein−1

·min−1 at 10−11 M and 

0.368 ± 0.013 nmol·mg-protein−1·min−1 at 10−5 M. The average lowest portion of the response 

curve across the runs for captan was between 50 and 75% activity (60.3% at 10-5M), indicating 

an equivocal response. 

Based on the data from the average response curve, captan is classified as Equivocal for 

aromatase inhibition in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 48615103) for captan (93% purity, Batch 

#91138103), uterine cytosol from Sprague Dawley rats was used as the source of ER for a 

competitive binding experiment, which was conducted to measure the binding of a single 

concentration of [3

H]-17β-estradiol (1 nM) in the presence of increasing concentrations (10−11 to 

10−4

 M) of captan. DMSO was used as the solvent vehicle at a final assay concentration of 4%. 

A total of four runs were conducted, and each run included 19-norethindrone as a weak positive 

control, octyltriethoxysilane as a negative control, and 17β-estradiol as the natural ligand 

reference material. One run was not valid and was excluded from the analysis because the 

positive control data were outside the recommended criteria ranges. 

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A saturation binding experiment was conducted to demonstrate that the ER isolated from rat 

uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity 

for the radiolabeled reference estrogen prior to conducting ER competitive binding experiments. 

Saturation binding data were not originally provided in the study report; however, summarized 

saturation binding data (MRID 48843501) from the performing laboratory were submitted 

following a request by the Agency. The protein concentrations used in the saturation binding 

runs varied between each run, and were approximately 3- to 6-fold greater than recommended 

(160 to 320 μg versus 50±10 μg). The mean dissociation constant (Kd) for [3

H]-17β-estradiol 

was 0.331±0.061 nM and the estimated Bmax was 74.55±3.03 fmol/100 μg protein for the 

prepared rat uterine cytosol. The Kd for each run was within the expected guideline range of 

0.03 to 1.5 nM. 

In the competitive binding experiment, the estimated mean log IC50 was −9.0 M for 17β-estradiol 

and −5.5 M for the weak positive control. The mean RBA was 0.0295% for the weak positive 

control. All performance criteria were generally met; however, octyltriethoxysilane displaced 

more than 25% of [3

H]-17β-estradiol from the ER at the highest concentration tested (10-3 M) in 

two of the three assay runs. Total [3H]-17β-estradiol binding was >75% at all soluble 

concentrations (≤ 10-4 M) of captan tested in all three valid competitive binding runs. A log IC50

and RBA could not be determined for captan since displacement did not exceed 50% at any test 

concentration. 

Based on the results from the three runs, captan is classified as Not Interactive in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48615104), hERα-HeLa-9903 

cells cultured in vitro were exposed to captan (93% a.i., Lot # 91138103) at logarithmically 

increasing concentrations from 10−11 to 10−4 M and 10−12 to 10−5 in DMSO (0.1%) for 24 hours in 

two independent runs, respectively. The experiments were performed using 96-well plates and 

each captan concentration was tested in 6 wells/plate in each run. Cells were exposed to the test 

agent for 24±2 hours at 37±2°C to induce reporter (luciferase) gene products. Luciferase 

expression in response to activation of the estrogen receptor by captan was measured using a 

proprietary luciferase assay. 

Captan was tested up to the limit of cytotoxicity, which was observed at concentrations ≥10-5M 

in both runs. At concentrations up the 10-6 M, the RTA for captan was ≤4.9%, indicating that it 

is negative for estrogenic activity under the conditions of this assay. 

In the main assays, the responsiveness of the cells to the very weak positive control 

17α-methyltestosterone was lower than the expected values, indicating a decreased sensitivity of 

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the assay to very weak agonists. Furthermore, 17α-methyltestosterone responses reached only 

6.3-7.9% PC and were similar to those of the negative control corticosterone, which averaged 

2.6-3.7% PC. Therefore, under the conditions of this study, the reviewer cannot determine if 

captan is a very weak estrogen. Nevertheless, the responses of captan (4.3-4.9% PC were not 

comparable to the response of the weak estrogen control, 17α-estradiol (117-124% PC) and the 

RPCMax < PC10 in both assay runs. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48669501) of captan with fathead minnow 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (20 spawning 

groups; 2 males and 4 females/replicate; 4 replicates/treatment; ~21 weeks old) were exposed to 

captan (94.9% purity) at nominal concentrations of 0 (negative and solvent [12 μL/L 

dimethylformamide, DMF] controls), 0.00023, 0.0023, and 0.023 mg a.i./L; mean-measured 

concentrations were <0.000014 (<LOQ; controls), 0.00011, 0.00099, and 0.010 mg a.i./L. The 

test system was maintained at 24 to 26°C and a pH of 7.0 to 7.7. 

Unless otherwise indicated, all effects are reported based on comparison to the negative (clean 

water) control. 

Overall survival in the negative and solvent controls was 88 and 96%, respectively; overall survival 

was 100% in all captan treatment groups and was not significantly different (p>0.05) from the 

negative control. Significant increases (p<0.05) in male body weight at the high treatment level 

and in male body length at the mid and high treatment levels were observed relative to the negative 

control. There were no treatment-related effects on secondary sex characteristics or clinical signs 

(i.e., behavioral and other sublethal effects including (discoloration, changes in dorsal nape pad, 

lethargy, loss of equilibrium). 

In the negative and solvent controls, spawning occurred at least every 4 days in three out of four 

replicates in the negative control and two out of four replicates in the solvent control. Mean 

fecundity was 12 and 9.2 eggs/female/day/replicate (negative control range: 10-13; solvent control 

range: 3.1-14), respectively, and fertilization success was 96 and 97%, respectively. Fecundity and 

fertilization success was not significantly different (p>0.05) in any treatment group relative to the 

negative control. 

Plasma vitellogenin (VTG) was significantly increased (p<0.05; Mann-Whitney U test) in males 

at the mid and high treatment levels relative to the negative control; however, the increase was not 

concentration-responsive. There was no significant effect (p>0.05) on female plasma VTG at any 

treatment level. 

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Treatment-related effects on gonadal histopathology were not observed in males or females. 

Furthermore, there were no significant changes (p>0.05) in gonado-somatic index (GSI) for either 

sex. Nuptial tubercle score was comparable between the negative control and treatment groups for 

males; no nuptial tubercles were observed in females. Sex steroid levels were not measured. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48615105) screening for androgenic activity, captan (93% a.i., 

Batch/lot# 91138103) in 1% carboxymethylcellulose (CMC) was administered daily via oral 

gavage to groups of eight 59- or 60-day old, castrated male Sprague Dawley rats at dose levels of 

0 (vehicle), 200, or 400 mg/kg/day. An androgenic positive control group consisted of eight 

castrated rats exposed to the vehicle by oral gavage plus 0.4 mg/kg/day of testosterone 

propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, captan in 1% CMC was administered daily via 

oral gavage to groups of eight 59- or 60-day old, castrated male Sprague Dawley rats at dose 

levels of 0 (vehicle), 100, 200, or 400 mg/kg/day in conjunction with a daily dose of reference 

androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive control group 

consisted of eight castrated rats exposed to 3 mg/kg/day flutamide (FT, via oral gavage) and 0.4 

mg/kg/day TP (s.c. injection). The positive control group for the androgen agonist assay served 

as the negative control group for the anti-androgen assay. 

For both components of the assay, body weights were determined daily. The animals were dosed 

for 10 consecutive days and terminated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues were collected and weighed. 

In the androgen agonist assay, two animals in the 200 mg/kg/day group died before study 

termination: one animal was found dead (suspected dosing error), and one animal was euthanized 

because of breathing difficulties. All animals in the 400 mg/kg/day group were euthanized prior 

to study termination: four animals were euthanized due to abnormal breathing and body weight 

loss and the remainder were humanely euthanized prior to the potential onset of breathing 

difficulties and initiation of or continued weight loss. All animals in the vehicle control group 

survived to scheduled termination. 

In the anti-androgen assay, one animal in the 100 mg/kg/day group was found dead on Day 1 

with no obvious sign of dosing error. All animals in the 200 mg/kg/day group were humanely 

euthanized prior to study termination: two animals were euthanized due to abnormal breathing 

and body weight loss, and the remaining six animals were humanely euthanized due to body 

weight loss or prior to the potential onset of breathing difficulties and weight loss. All animals in 

the 400 mg/kg/day group were euthanized or found dead prior to study termination: one animal 

was found dead, four were euthanized due to abnormal breathing and body weight loss, and the 

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remaining three animals were humanely euthanized prior to the potential onset of breathing 

difficulties and severe weight loss. All animals in the negative control group survived to 

scheduled euthanasia with none showing signs of moribundity. One animal in the positive 

control group (FT and TP) was humanely euthanized due to a dosing error. 

It was noted that moribund animals all exhibited abnormal breathing that was severe enough to 

result in rapid weight loss and at necropsy, the stomach and/or intestines were severely distended 

with air; similar but less severe symptoms were observed in the range finding study. The oral 

LD50 in rat is >5000 mg/kg (material data safety sheet for captan). It was stated that the increase 

in moribundity may have been due to gavage-related reflux, but no explanation was given as to 

why similar moribundity was not observed at the same dose levels in the range finding study. 

In the androgen agonist assay, clinical signs of abnormal breathing were observed in two rats in 

the 200 mg/kg/day group, the only treatment group which contained animals that survived until 

termination. In that group, body weights were decreased (not statistically analyzed) by 9-10% on 

Days 4 and 7 and decreased (p<0.05) by 12% on Day 11 compared to controls, resulting in an 

overall body weight loss (p<0.05) of 6.7 g (compared to gain of 37.7 g in the vehicle control). 

Final body weights in the positive control group were comparable to the vehicle controls; 

however, body weight gains in this group were increased (p<0.05) by 61%. There were no 

treatment-related effects in accessory sex organs for the 200 mg/kg/day group. Animals in the 

positive control group had increased (p<0.05) accessory sex organ weights as follows: 1235% in 

seminal vesicles; 905% in ventral prostate; 183% in levator ani-bulbocavernosus muscle 

complex (LABC); 458% in Cowper’s gland; and 57% in glans penis. 

In the anti-androgen assay, one animal in the 100 mg/kg group, the only treatment group which 

contained animals that survived until termination, exhibited clinical signs of toxicity including 

lethargy and decreased movement, abnormal breathing, and thin appearance. One additional 

animal in this group was found to have rales and another found to be lethargic. In the 100 

mg/kg/day group, final body weights were comparable to the negative control; however, body 

weight gains were decreased (NS) by 49%. Final body weights and body weights gains in the 

positive control group were comparable to the negative control. There were no treatment-related 

effects in accessory sex organs for the 100 mg/kg/day group. It was noted that ventral prostate 

weight in the 100 mg/kg/day group was increased (NS; p>0.05 and <0.1) compared to the 

negative control (↑19%). Animals dosed with FT + TP (positive control) had decreased (p<0.01) 

accessory sex organ weights as follows: 79% in seminal vesicles; 73% in ventral prostate; 60% 

in LABC; 64% in Cowper’s gland; and 24% in glans penis. 

In summary, in the Tier 1 Hershberger assay, no androgen-related effects were observed in the 

agonist phase (200 and 400 mg/kg/day) or in the antagonist phase (100, 200 and 400 mg/kg/day). 

No overt toxicity was seen at 100 mg/kg/day. Animals at the higher doses (≥ 200 mg/kg/day) 

died or were sacrificed due to severe clinical signs and body weight loss. Overall this assay was 

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considered unacceptable due to the excess mortality and clinical signs seen in both phases of the 

assay 

Percent CVs for the negative control and the treatment groups were compared to the performance 

criteria in the Guidelines. All %CV values were less than the maximum recommended values, 

with the exception of the %CV for LABC for the vehicle control group in the androgen agonist 

assay (CV = 37%; maximum permissible CV = 30%). 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 48669502), 16 Sprague Dawley rats/dose group were treated 

daily by oral gavage with captan (94.9%, Lot/Batch No. 91138103) in aqueous 1% 

carboxymethylcellulose (CMC) at doses of 0 (vehicle), 300, or 600 mg/kg/day from post-natal 

day (PND) 22 to 42/43. Animals were examined for vaginal opening (VO) daily beginning on 

PND 22, and weight at day of attainment was recorded. Following sacrifice on PND 42 or 43, 

total serum levels of thyroxine (T4) and thyroid stimulating hormone (TSH) levels were 

determined by radioimmunoassay; serum was also analyzed for concentrations of selected 

clinical chemistry parameters. Adrenal, liver, pituitary, thyroid, and urogenital organs were 

weighed, and microscopic examinations were conducted on the ovaries, uterus, thyroid, and 

kidneys. 

Two animals in the 600 mg/kg/day group were found moribund and euthanized on Study Days 5 

and 17; all other rats survived until scheduled sacrifice. Cage-side and clinical observations 

included decreased movement on one occasion (300 mg/kg/day) and abnormal breathing in 

several animals in both dose groups. 

The mean age at VO was increased (p<0.05) by 4.0 and 5.3 days at 300 and 600 mg/kg/day, 

respectively, compared to the control group (VO at PND 31.8). The weight at VO was increased 

(P<0.05) by 15% in the 600 mg/kg/day group, suggesting that the delay in attaining VO was not 

related to reductions in body weight. However, final body weights were decreased (p<0.01) by 9 

and 10% in the 300 and 600 mg/kg/day dose groups, respectively, yielding decreased (p<0.001) 

overall body weight gains of 14% and 15%, respectively. All animals achieved VO prior to 

termination with the exception of one animal in the 600 mg/kg/day group euthanized prior to 

VO. 

Relative (to body weight) kidney weights were increased (p<0.05) by 5 and 8% at 300 and 600 

mg/kg/day, respectively. There were also dose-dependent decreases in pituitary and ovary 

weights. Absolute and adjusted pituitary weights were decreased (p<0.01) by 18% at 300 

mg/kg/day, and absolute, adjusted, and relative pituitary weights were decreased (p<0.01) by 19-

28% at 600 mg/kg/day. Absolute and adjusted ovary weights were decreased (p<0.01) by 18 and 

23% at 300 and 600 mg/kg/day, respectively. Adjusted wet uterus weights were decreased 

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(p<0.05) by 35% and absolute blotted uterus weights were decreased (p<0.01) by 34% at 600 

mg/kg/day. At 300 mg/kg/day, liver weights were decreased (p<0.05) by 12%, but liver weights 

at 600 mg/kg/day were comparable to controls. There were no treatment-related effects on 

adrenal gland or thyroid weights. 

The mean age at first vaginal estrus was delayed by 2.5 days at 300 mg/kg/day (p<0.05; PND 

35.7) and by 5.2 days at 600 mg/kg/day (p<0.0001; PND 38.4) compared to controls (PND 33.2). 

No other treatment-related effects on estrus cycle parameters were observed. 

Serum T4 concentrations were decreased in a dose-dependent manner by 27% (p<0.001) at 300 

mg/kg/day and by 42% (p<0.0001) at 600 mg/kg/day. At 600 mg/kg/day, increased (p<0.05) 

concentrations of sodium (↑3%) and chloride (↑6%) were observed. Sorbital dehydrogenase 

concentration also was increased (p<0.001) by 7% in the 600 mg/kg/day group. There were no 

dose-related effects on any other serum chemistry parameters. 

In the thyroid, follicular cell height was increased (p<0.05) compared to control in the 600 

mg/kg/day animals, and there was a corresponding decrease (p<0.05) in the colloid area of 

thyroid follicles. There was a similar, though reduced, trend in increased follicular cell height at 

300 mg/kg/day. No histopathological findings were observed in the ovaries, and any findings 

noted in the uterus and kidneys were generally minimal or mild, if present. The number of 

follicles (small, medium, antral, and antretic) and follicular cysts were not significantly different 

between control and treated females, but the number of corpora lutea were decreased (p<0.05) by 

30% and 29% at 300 and 600 mg/kg/day, respectively. 

Based on the 10% decrease in final body weight, clinical signs of toxicity and death in the 600 

mg/kg/day dose group, the high dose level was considered overtly toxic. At the low dose (300 

mg/kg/day), clinical signs of toxicity were observed that were at the cusp of overt toxicity (9% 

decreased body weight and abnormal breathing and decreased movement). 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 48669503), 16 Sprague Dawley rats/dose group were treated 

daily by oral gavage with captan (94.9%, Lot/Batch No. 91138103) in aqueous 1% 

carboxymethylcellulose (CMC) at doses of 0 (vehicle), 100, or 200 mg/kg/day from post-natal 

day (PND) 23 to 53/54. Animals were examined for preputial separation (PPS) daily beginning 

on PND 30, and weight at day of attainment was recorded. Following sacrifice on PND 53 or 

54, total serum concentrations of thyroxine (T4), thyroid stimulating hormone (TSH), and 

testosterone were determined by radioimmunoassay; serum was also analyzed for concentrations 

of selected clinical chemistry parameters. Adrenal, liver, pituitary, thyroid, and urogenital 

organs were weighed, and microscopic examinations were conducted on the testes, 

epididymides, thyroid, and kidneys. 

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At 200 mg/kg/day, two animals were found moribund with abnormal breathing (gasping) and 

were euthanized on Study Days 18 and 27. All other rats survived until scheduled sacrifice. 

Cage-side and clinical observations included one animal with alopecia (100 mg/kg/day), one 

ungroomed animal (200 mg/kg/day), and two animals with rales (one each at 100 and 200 

mg/kg/day). 

At 100 and 200 mg/kg/day, mean unadjusted and adjusted weights at day of PPS were decreased 

(p<0.05) by 8 and 13%; however, mean age at PPS and partial PPS were unaffected by 

treatment. Additionally at 100 and 200 mg/kg/day, mean unadjusted and adjusted final body 

weights were decreased (p<0.01) by 9 and 13%, resulting in decreased (p<0.01) unadjusted and 

adjusted overall body weight gains of 11 and 16%, respectively. At termination, the number of 

animals that had attained PPS was 16/16 in the vehicle control and 100 mg/kg/day groups, and 

15/16 in the 200 mg/kg/day group (one animal euthanized prior to attainment of PPS). 

At 100 and 200 mg/kg/day, relative (to body weights) kidney weights were increased (p<0.01) 

by 8 and 14%, relative adrenal weights were increased (p<0.05) by 13 and 14%, and unadjusted 

and adjusted ventral prostate weights were decreased (p<0.05) by 15 and 20%, respectively. 

Additionally at 200 mg/kg/day, unadjusted and adjusted LABC weights were decreased (p<0.01) 

by 14 and 15%, respectively. There were no dose-related effects on liver or pituitary weights, or 

on other accessory sex organ weights. Additionally, at 200 mg/kg/day, serum T4 and 

testosterone concentrations were decreased (p<0.05) by 18% and 42%, respectively. 

Administration of captan at 100 or 200 mg/kg/day was not associated with histologic changes in 

the thyroid gland. Findings in the testes, epididymides, and kidneys were only observed in a 

single animal and/or were generally minimal or mild in severity, if present. 

Based on the >10% decrease in final body weight, clinical signs of toxicity and death in the 200 

mg/kg/day dose group, the high dose level was considered overtly toxic. At the low dose (100 

mg/kg/day), clinical signs of toxicity were observed that were at the cusp of overt toxicity (9% 

decreased body weight and rales and alopecia). 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48615106), H295R cells cultured in vitro in 24-well plates 

were incubated with captan (93% purity, Batch # 91138103) at serial log concentrations of 

0.0001 to 100 μM for 48 hours in triplicate for four independent experiments. Dimethyl 

sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.05%. Precipitation was 

noted microscopically at ≥0.1 μM captan in Run 4, but was not observed at concentrations up to 

100 μM captan in the other runs. The precipitation observed in Run 4 was probably the result of 

a technical error. Cytotoxicity greater than 20% was observed at 100 μM in Runs 2 and 3. 

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Samples with precipitation or cytotoxicity were not analyzed. Run 1 was not analyzed because 

one of the forskolin concentrations on the quality control plate was incorrect (3.33 μM was used 

instead of 1 μM). 

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) 

plate was run concurrently with each independent run of a test chemical plate to demonstrate that 

the assay responded properly to positive control agents at two concentration levels; positive 

controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and 

testosterone production. 

Guideline acceptability recommendations and requirements were met, including adequate 

production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate 

induction and inhibition with positive controls. 

Statistically significant inhibition of testosterone (9%) was noted at 10 μM in Run 3, and 

inhibition of estradiol levels was observed in Run 2 at 0.001 (12%), 1 (9%), and 10 (25%) μM. 

These results were considered equivocal for several reasons. Differences were observed in a 

single run each for testosterone and estradiol, and only two runs were evaluated at 1 and 10 μM. 

The apparent effect on testosterone was minor (0.91-fold of solvent control). All other values of 

testosterone and estradiol in the treated cells were similar to the solvent control (SC). 

Based on the hormone responses in at least three independent runs, captan treatment did not 

result in statistically significant and reproducible alterations in testosterone or estradiol 

production. 

Uterotrophic Assay (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48615107) conducted to screen for potential estrogenic activity, 

captan (93% a.i., Batch/lot# 91138103) in 1% carboxymethylcellulose (CMC) was administered 

daily via oral gavage to groups of eight ovariectomized female Sprague Dawley rats at dose 

levels of 0 (vehicle), 313, or 1,000 mg/kg/day on post-natal days (PND) 58-60. A positive 

control group was treated with a daily dose of 17α-ethynyl estradiol (EE) in corn oil at 0.05 

mg/kg/day by oral gavage. Body weights were determined daily. All animals were terminated 

and necropsied on PND 61 approximately 24 hours after the final dose administration to 

determine wet and blotted uterine weights. 

All animals survived until scheduled termination, with the exception of one animal in the 1,000 

mg/kg/day group that was euthanized moribund on Day 3. There were no treatment-related 

clinical findings in the surviving rats. 

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Final body weights were decreased (p<0.05) by 15% on Day 3 in the 1,000 mg/kg/day group 

compared to vehicle controls, resulting in an overall (Days 1-4) body weight loss (p<0.05) of 

18.3 g (compared to a gain of 15.8 g in the controls). Final body weights in the 313 mg/kg/day 

group were decreased (not significant; NS) by 11%, with an overall body weight loss (p<0.05) of 

10.2 g. The decreased body weights indicate that the dose levels tested were adequate. Uterine 

weights in the captan treatment groups were comparable to the vehicle controls. 

Comparative statistical analyses were not conducted for the final body weights and body weight 

gain of animals in the positive control (EE) group. However, final body weights in the EE group 

were decreased by 10%, with an overall body weight loss of 3.2 g. Absolute wet and blotted 

uterus weights for the EE group were increased (p<0.05) by 375% and 194%, respectively, as 

expected. 

No statistically significant changes were seen in uterine weight in this assay. Captan is negative 

in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

Receptor Transactivation Assays 

In a published study by Kojima et al. (2004), two hundred pesticides covering nine chemical 

classes were screened for their potential to interact with two human ER subtypes (hERα and 

hERβ) and one human AR (hAR) in transactivation assays in Chinese hamster ovary cells (CHOK1 cells). Cells were grown in DMEM/F-12 (Dulbecco’s modified Eagle medium plus Ham’s F12 nutrient mixture) supplemented with 10% fetal bovine serum (FBS) and antibiotics at a 

temperature of 37°C in an atmosphere of 5% CO2 under saturating humidity. Captan was 

dissolved in DMSO to a final concentration of 10 mM and diluted to the desired concentration(s) 

in DMEM/F12. The final solvent concentration in the culture medium did not exceed 0.1% and 

did not affect cell yields. 

The hERα and hAR expression vectors (pcDNAERα and pZeoSV2AR, respectively) were 

constructed as described previously (Kojima et al., 2003). The hERβ expression vector was 

constructed by cloning the hERβ cDNA with reverse transcriptase-polymerase chain reaction 

from human placental RNA. The sequence was verified and inserted into the mammalian 

expression vector pcDNA3.1Zeo(-) creating pcDNAERβ. The construction of the estrogen or 

androgen responsive elements containing reporter plasmid (pGL3-tkERE and pIND-ARE, 

respectively) was described previously (Kojima et al., 2003). pRL-SV40 containing the Renilla

luciferase gene was used as an internal control for transfection efficiency. 

Host CHO cells were plated into 96-well microtiter plates at a density of 8,400 cells/well in 

phenol red-free DMEM/F12 containing 5% CD-FBS (complete medium) one day before 

transfection. For detection of hERα or hERβ activity, cells were transfected with 5 ng 

pcDNAERα or pcDNAERβ, 50 ng pGL3-tkERE, and 5 ng pRL-SV40. For detection of hAR 

activity, cells were transfected with 2.5 ng pZeoSV2AR, 50 ng pIND-ARE and 5 ng pRL-SV40. 

After a 3-hour transfection period, cells were dosed with various concentrations of test 

compounds (95-100% purity) or with 0.1% DMSO (vehicle control) in complete medium. For 

measurement of antagonistic activity to hERα, hERβ and hAR, either 10−11 or 10−10 M E2 (17βestradiol, > 97% purity) or 10−10 M DHT (5α-dihydrotestosterone, 95% purity) was added to the 

cell cultures along with the test compound, respectively. Cells were incubated for 24 hours, 

rinsed with phosphate buffered saline (pH 7.4), and lysed with passive lysis buffer (50 μL/well). 

Luciferase activity was measured with a MiniLumat LB 9506 luminometer and was normalized 

based on the activity of co-transfected pRL-SV40. At least three independent experiments were 

conducted for each compound. 

Agonistic activities were evaluated by relative activity expressed as REC20 (20% relative 

effective concentration). The REC20 is the concentration of the compound showing 20% of the 

activity of 10−10 M E2, 10−9 M E2, or 10−9 M DHT for ERα, ERβ and AR, respectively. 

Antagonistic activities were evaluated by relative activity expressed as RIC20 (20% relative 

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inhibitory concentration). The RIC20 is the concentration of the compound showing 20% of the 

inhibition of the activity of 10−10 M E2, 10−9 M E2 or 10−9 M DHT for ERα, ERβ and AR, 

respectively. The range of concentrations tested was approximately 10−8 to 10−5 M; all pesticides 

were tested at concentrations ≤ 10−5

 M to avoid cytotoxicity. When the activity of the test 

compound was higher than the REC20 the pesticide was judged to be positive for activity. When 

the activity of the test compound was higher than the RIC20 the pesticide was judged to be 

positive for inhibitory activity. The statistical significance of differences was evaluated with a 

two-tailed Student’s t test with a nominal level of significance of p<0.05. 

Captan exhibited no activity in these screening studies. 

In a published study by Kojima et al. (2005), the response of an in vitro ER–related reporter 

gene assay was investigated after exposure to thirty two pesticides either alone or in 

combination. The compounds tested included 8 pyrethroids, 3 organochlorines (including 

captan), 7 organophosphates, and 14 other pesticides, as well as combinations of two types of 

pesticides. The source of the test compounds was not reported; however, captan had a reported 

purity of 98%. The test compounds were prepared in DMSO at 10−2

 M. 

The E-CALUX Assay (Xenobiotic Detection Systems, Inc., NC, USA) uses a human ovarian 

carcinoma cell line (BG1) transfected with a luciferase reporter gene under the control of a 

mouse mammary tumor virus (MMTV) promoter. Estrogen-responsive concatemers (repetitive 

DNA sequences), inserted directly above the promoter, confer estrogen responsiveness to the 

viral promoter and reporter gene. The transfected cells are made stable by an antibiotic resistance 

gene resulting in the cell line used in the assay (BG1Luc4E2). Compounds that interact with 

estrogen-responsive DNA are identified by the luciferase gene response and quantified in terms 

of Relative Light Units (RLU). 

The BG1Luc4E2 cells were grown in medium for four days, then the assay medium was 

replaced, and cells were transferred (2 × 105 cells/mL) into 96-well plates and incubated at 37 ̊C 

with 5% CO2 for 24 hours. The medium was removed, and the test compound or estrogen 

standard (E2) was added. After incubation at 37 ̊C with 5% CO2 for another 24-hour period, the 

medium was removed and the cells were lysed with Promega lyse buffer. Luciferase activity was 

measured with a luminometer in order to compare treatment RLU to the E2 standard. For 

comparative purposes, the EC10 (Effective Concentration at which 10% of the maximum 

response was observed) was used. The data were not sufficient to allow calculation of an EC50. 

Although the article does not specifically identify the E2 standard, the reviewers assumed that E2 

referred to 17β-estradiol. 

The EC10 of E2 was 3.2 ×10-12 M, which is within the expected range, and the dose-response 

curve in the paper indicates that the assay performed as expected for chemicals that have positive 

estrogenic activity. Captan also tested negative for ER activity in this assessment. 

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In a published study by Okubo et al. (2004), the effects of selected pesticides on the 

proliferation of an estrogen-responsive cell line was investigated with captan and other 

pesticides. Captan (purity not reported) was obtained from Wake Pure Chemical, MCF-7 cells 

were obtained from Drs. A.M. Soto and C. Sonneschein (Tufts University School of Medicine, 

Boston MA), 17β-estradiol and diethylstilvestrol (DES) were obtained from Sigma (purity not 

reported), and ICI 182,780 (anti-estrogen) was obtained from Tocris Cookson (purity not 

reported). MCF-7 cells, an estrogen-responsive cell line, were grown and maintained in DMEM 

supplemented with 5% fetal bovine serum growth medium. Cells were inoculated into 96-well 

culture plates (density not reported), and after 24 hours, the medium was replaced with 

experimental medium (phenol red-free DMEM supplemented with 5% charcoal-dextran treated 

human serum). Captan, dissolved in ethanol and diluted with phenol red-free DMEM, was added 

to the wells at final concentrations of approximately 0, 1, 10, and 100 μM (concentrations were 

not reported, but were estimated by reviewer from a graph of study results). The cultures were 

grown for an additional 6 days and the cell number was estimated with a commercial cell 

proliferation assay kit. Experiments were conducted 3-5 times, n=8 per assay run. Cell numbers 

were expressed as percentages of control cultures (0.1% ethanol alone). Statistical analysis was 

conducted with the two-tailed Student’s t-test (p ≤ 0.1% was considered significant). 

To examine anti-estrogenic activity, 30 pM 17β-estradiol was added to the growth medium, and 

cells were exposed to the same concentrations of captan. To examine the effect of a pure antiestrogen on cell proliferation induced by captan, ICI 182,780 (10 nM) was added to cells 

exposed to the concentration of captan with the highest cell yield in the cell proliferation 

experiment (100 μM). Cytotoxicity was determined by neutral red uptake assay; captan was not 

found to be cytotoxic to cells at the concentration range examined. Captan was reported to bind 

to the ER with an IC50 of 142±17 μM. Captan did not induce estrogen-related cell proliferation, 

but was anti-estrogenic in the cell proliferation assay at 10 and 100 μM.

One-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a one-generation reproduction toxicity study (MRID 00120315), captan (technical; purity not 

reported) was administered to COBS CD rats (15 male and 30 female /dose group) in the diet at 

concentrations equivalent to doses of 0, 6, 12.5, and 25 mg/kg/day during the pre-mating period 

of 102 days; exposure in the P generation dams continued through gestation, lactation and 

weaning of the Fl litters. Pup survival was recorded at birth (LD 0) and at LDs 1, 4, 7, 14, and 21. 

Litters were randomly reduced to 10 pups on LD 4; and litters were weighed as a group on LDs 

1, 4, 7, and 14. Individual pup weights and physical observations were recorded on LD 21. 

There were no treatment-related effects on parental mortality, parental or litter body weights, 

food consumption, or clinical signs. Litter weights were decreased (NS) by 5% at LD 4 and by 

6% on LDs 7 and 14, but no differences in pup body weights were present at LD 21. No 

treatment-related effects were observed in male and female fertility index, gestation length, pup 

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viability at birth, pup survival to weaning, or fetal sex ratio. The organ weights were not 

determined and histopathology was not conducted. 

The NOAELs for parental and litter toxicity are 25 mg/kg/day, and the LOAELs could not be 

established (>25 mg/kg/day). This study is supplemental to the following three-generation rat 

study (MRID 00125293). 

Three-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a three-generation reproduction toxicity study (MRID 00125293), captan (89% purity) was 

administered to COBS CD rats (15 males and 30 females/dose group) in the diet at 

concentrations nominally equivalent to doses of 0, 25, 100, 250, and 500 mg/kg/day. P 

generation males were treated during the initial pre-mating period (102 days) with exposure 

continuing until completion of the second mating. Exposure in the P generation dams occurred 

during the initial pre-mating period (102 days) and a 10-day rest period prior to the second 

mating, as well as through gestation, lactation and weaning of the Fla and F1b litters. After 

weaning, 15 male and 30 female F1b pups per dose group were selected to comprise the Fl

generation parental animals, and were mated three times to produce the F2a, F2b, and F2c litters. 

Further pre-mating and rest periods were not stated explicitly, but were presumed to be similar to 

the P generation schedule. After weaning, 15 male and 30 female F2b pups were selected to 

comprise the F2 generation parental animals, and were mated to produce the F3a and F3b litters. 

Pup survival was recorded at birth (LD 0) and at LDs 1, 4, 7, 14, and 21. Litters were randomly 

reduced to 10 pups on LD 4; and litters were weighed as a group on LDs 1, 4, 7, and 14. 

Individual pup weights and physical observations were recorded on LD 21. Gross examination 

was done of two thirds of the pups (after litter reduction) from each litter except the F2c. Soft 

tissues, eyes, brain, reproductive organs, and the heart were examined. Tissues and organs from 

male and female F3b pups (10/sex), and from parents dying during the study were preserved, but 

no histopathology examinations were conducted. All fetuses from the F2c generation were 

removed by cesarean section on GD 19, weighed, and examined externally. Approximately one 

half the fetuses were fixed in Bouin's solution for visceral sectioning, and the other half were 

fixed, cleared, and stained with Alizarin Red S for skeletal examination. Organ weights were not 

evaluated. 

There were no reported treatment-related effects on mortality in the P, F1, or F2 parental animals. 

For the medium-high dose pups, increases (significance not reported) in mortality were observed 

in the F2a litter at LDs 1 and 4, and in the F3a litter at LD 4. For the high-dose pups, increases 

(significance not reported) in mortality were observed in the F2a and F3a litters at LDs 1 and 4, 

and in the F2b litter at LD 7. Decreased parental body weights were observed in all groups, 

except the low-dose animals. Decreases (significance not reported) in the medium-low dose 

group were occasional, and ranged from 15-18% in males and 5-9% in females. Body weight 

decreases (significance not reported) in the medium-high and high-dose parental groups ranged 

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from 8-19% and 12-37%, respectively. Decreases (significance not reported) in mean litter 

weights were observed in all treatment groups, with decreases ≤44% in the high-dose pups. Food 

consumption was decreased in all treatment groups except for the low-dose F1 males and the 

low- and medium-low dose F2 females. 

Litter size was decreased (significance not reported) in the F1b litter at the high dose. Fertility 

was decreased (significance not reported) in medium-high dose Fla males and in high-dose Fla

females. No treatment-related effects were observed on the duration of gestation, or in other 

reproductive performance parameters (male or female fertility indices, gestation index, live birth 

index, lactation index, parturition, or fetal sex ratio) in the other generations. No treatmentrelated effects were observed in the number of corpora lutea, number of total implantations, early 

or late resorptions, and in the number and location of viable and non-viable fetuses at the 

cesarean section on GD 19 of the F2c animals. In addition, no effects of developmental toxicity 

were present in the soft tissue and skeletal examinations. No treatment-related histopathological 

lesions were observed in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, 

cervix, thyroid, adrenal, and pituitary glands of F3b offspring. 

Based on these study results, the NOAEL could not be established for offspring based on 

decreased litter pup weights in all treatment groups (≥25 mg/kg/day). Consideration of these 

results and the results from the supplemental one-generation reproduction study (MRID 

00120315) yield NOAEL and LOAEL estimates of 12.5 and 25 mg/kg/day, respectively. 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 48126603), pregnant CD(SD) rats (22/dose 

group) were administered captan (91% purity) in 0.5% CMC containing 0.05% acetic acid by 

oral gavage at doses of 0, 18, 90, or 450 mg/kg/day during GDs 6-15, instead of through GD 20 

(the day before expected parturition). All dams were sacrificed on GD 20, and the number of 

corpora lutea, implantations, and resorptions were recorded. Fetuses were examined for external 

abnormalities, sexed, and weighed. Visceral examination was performed on one-half of the 

fetuses in each litter. The remaining fetuses were fixed in ethanol, stained with alizarin red S, 

and examined for skeletal abnormalities. 

No mortalities occurred during the study, and clinical signs were limited to increased (p<0.05) 

hair loss and lack of grooming (NS) in some high-dose dams. Maternal body weights in the highdose dams were decreased throughout treatment (GDs 7-15) and on GD 17 by 5-10% (p<0.001 

or 0.01; generally <10%). Body weight gains were decreased (p<0.05) during GDs 0-20 and 6-20 

by 8 and 10%, respectively. During GDs 7-9, food consumption was decreased (p<0.001) by 

47%, with decreases (p<0.001) of 17% during GDs 10-13 and 14-16. Recovery was evident posttreatment based on an increase (p<0.001) in food consumption of 17% during GDs 17-20. 

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The maternal LOAEL is 450 mg/kg/day, based on decreased body weights, body weight gains, 

and food consumption, and the maternal NOAEL is 90 mg/kg/day. 

No treatment-related changes were observed in pregnancy rate, number of corpora lutea, mean 

number of implantations/litter, pre-or post-implantation loss, early or late resorptions, number of 

live fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose. At the high dose, fetal 

weights (combined sexes) were decreased (p<0.01) by 6%, and there was an increase (p<0.01) in 

the number of fetuses that weighed less than 3.0 g (runts; 53 fetuses from 19 litters) compared to 

control (24 fetuses from 12 litters). Observed skeletal findings at the high dose included: one or 

two thoracic vertebral hemicentra incompletely fused (21 treated vs. 16 control); reduced 

ossification of the pubis (8 treated vs. 1 control); and 14th rib present unilaterally (8 treated vs. 2 

control) or bilaterally (5 treated vs. 0 control). 

The developmental LOAEL is 450 mg/kg/day, based on decreased fetal weight, increased 

number of runts, and skeletal variations, and the developmental NOAEL is 90 mg/kg/day. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 00120316), CD rats (70/sex/dose 

group) were administered captan (89% purity) in the diet at concentrations equivalent to doses of 

0, 25, 100, and 250 mg/kg/day for 24 months. Ten rats/sex/group were randomly allocated for 

12- and 18-month interim sacrifices. Organs that were weighed at interim and final sacrifice 

included the liver, adrenals, kidneys, pituitary, thyroid/parathyroid, ovaries, and testes. Gross and 

microscopic pathological examinations were conducted on these tissues, as well as the mammary 

gland, prostate, urinary bladder, uterus, vagina, and epididymides (with testes), for the control 

and all dose groups. 

Survival was generally similar between the control and treatment groups until Week 70 for the 

high-dose animals; further decreases in survival were noted in the medium-dose males and 

females at Weeks 86 and 94, respectively, and at Weeks 94 and 82, respectively, in the low-dose 

males and females. No treatment related effects were observed for clinical signs, ophthalmology, 

hematology, blood chemistry, or urinalyses. Dose-related decreases in mean body weights for 

both sexes were observed at the medium- (↓6-20%) and high-doses (↓8-19%) throughout the 

study. Feed consumption was only slightly decreased (↓≤5%) in the medium- and high-dose 

animals. No treatment-related changes were seen in absolute or relative weights of the testes, 

ovaries, and pituitary gland. Increases in organ weights in the medium- and high-dose animals 

(generally relative-to-body, although occasional increases in absolute weights) were noted at 

interim and terminal sacrifices, including adrenals, kidneys, liver, and thyroids. Absolute weights 

were generally not affected, and the increases in relative weights were attributed primarily due to 

body weight decreases. Hepatocellular hypertrophy was noted at the high-dose for both sexes at 

the 18-month sacrifice, and in medium- and high-dose males at study termination. No treatmentrelated histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, 

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ovaries, uterus, vagina, cervix, mammary, thyroid, adrenal and pituitary glands. In males, a 

possible dose-related trend in kidney adenomas and carcinomas was observed.

The NOAEL is 25 mg/kg/day, and the LOAEL is 100 mg/kg/day based on decreased mean body 

weight, hepatocellular hypertrophy, and increases in relative organ weights. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 00161230), SPF Wistar Cpb:WU 

rats (50/sex/dose group) were administered captan (92% purity) in the diet at nominal 

concentrations of 0, 125, 500, or 2000 ppm; actual average daily concentrations were reported to 

be 100, 475, and 1950 ppm with standard conversion equivalency to doses of 5, 24, and 98 

mg/kg/day, respectively) for 104 weeks (animals were actually dosed for 30 months). Organs 

that were weighed at final sacrifice included the liver, adrenals, kidneys, pituitary, 

thyroid/parathyroid, ovaries, and testes. For animals in the low- and medium-dose groups, gross 

and microscopic pathological examinations were conducted only on the liver, pituitary, thyroid, 

and adrenals. Gross and microscopic pathological examinations were conducted on these tissues, 

as well as other tissues (forty in total, but the tissues were not specified), for the control and the 

high-dose groups. 

No treatment related effects were observed for mortality, clinical signs, or hematology. In the 

high-dose animals, mean body weight decreases ranged from 12-14% in males and 6-8% in 

females through Week 16, with similar decreases throughout the study and final decreases of 8 

and 10% in males and females, respectively, after treatment for 24 months. Food consumption 

was reported to be slightly lower in the high-dose animals, but the extent and significance were 

not reported. No treatment-related changes were seen in absolute or relative weights of the testes, 

ovaries, thyroid, adrenal and pituitary glands. Liver weight (relative to body weight) was 

increased (significance not reported) in the high-dose males, but this was not considered to be 

related to treatment as absolute weight effects and histological changes were not present. An 

increased incidence of kidneys with a granular appearance also was observed in the high-dose 

males. No treatment-related histopathological lesions were seen in the testes, epididymides, 

prostate, ovaries, mammary, thyroid, or pituitary glands in any dose group. High-dose females 

exhibited an increased (p<0.05) incidence of uterus sarcomas (8%) compared to control (0%). 

The medium-dose males exhibited an increased (p<0.05) incidence of benign adrenal 

pheochromocytomas, but the incidence in the high-dose animals was similar to control.

The NOAEL was 5 mg/kg/day (100 ppm), and the LOAEL was 24 mg/kg/day (475 ppm) based 

on increased incidences of benign adrenal pheochromocytomas in males. 

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Chronic Toxicity (Dog, OCSPP 870.4300) 

In a chronic toxicity study (MRID 40893604), Beagle dogs (5/sex/dose group) were orally 

administered gelatin capsules (2/day) containing captan (90.4% purity) at doses of 0, 12.5, 60, or 

300 mg/kg/day for 12 months. Organs that were weighed included the liver, adrenals, kidneys, 

thyroids with parathyroids, ovaries, prostate, and testes. Gross and microscopic pathological 

examinations were conducted on these tissues as well as the pituitary, uterus, urinary bladder, 

epididymides, vagina, and mammary gland. 

No treatment-related effects were observed for mortality, body weight, food consumption, 

ophthalmology, hematology, clinical chemistry, or urinalysis. All dogs survived, but increased 

incidences of emesis and soft/mucoid stools were observed in the high-dose animals. Non-dosedependent changes in feed consumption were observed in all treatment groups, with increases in 

males and decreases in females. No treatment-related changes were seen in absolute or relative 

weights of the testes, prostate, ovaries, thyroid, and adrenal glands. No treatment-related gross or 

histopathological lesions were seen in the testes, epididymides, prostate, ovaries, uterus, vagina, 

or the mammary, thyroid, adrenal, and pituitary glands. 

The LOAEL is 300 mg/kg/day based on increased clinical signs of emesis and soft/mucoid 

stools; the NOAEL is 60 mg/kg/day. 

Avian Reproduction Toxicity (Quail, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 48126601/00098295), Bobwhite quail (Colinus 

virginianus; 5 months old) were administered captan (89% purity) in the diet at concentrations of 

0 (corn oil), 100, 300, or 1000 mg/kg. The birds were fed the diets ad libitum during the duration 

of the study (ten weeks prior to egg laying and eight weeks during egg collection; eighteen 

weeks total). The birds were housed in twelve pens/dose group, with one male and two females 

per pen. A progressive photo period was employed in this study with 8 hours/day through Week 

6 and 17 hours/day for the remainder of the study. Mean temperature was 76°F (approximately 

24.4°C) throughout the study; the mean humidity was not reported. Individual adult body 

weights were recorded at study initiation, biweekly through Week 8, and at study termination. 

Feed consumption in each replicate was measured weekly throughout the study. Adult birds were 

observed routinely for mortality and clinical signs. The following reproductive parameters were 

observed and recorded: eggs laid, eggs cracked, eggs set, viable embryos, live 3-week embryos, 

hatchlings, 14-day-old survivors, body weight of 14-day-old survivors, and egg shell thickness. 

No treatment -related mortalities were observed; one mortality each occurred in the medium- and 

high-treatment groups due to physical injury. Evidence of stress including lacerated scalps, head 

lesions, and signs of head and toe-picking were observed in the control and all treatment groups. 

No treatment-related effects on body weight were observed. Feed consumption was reported to 

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be decreased in the 300 mg/kg (p<0.05) and 1000 mg/kg (p=0.01) treatment groups, although the 

decreases were not apparent at each measurement interval. 

The parental toxicity LOAEC was 300 mg/kg in the diet, based on reduced food consumption, 

and the NOAEC was 100 mg/kg. 

No treatment-related effects were observed on the number of eggs laid, eggs cracked, viable 

embryos, live 3-week embryos, hatchlings normal, 14-day old survivors, and hatchling body 

weight and eggshell thickness. Although a decrease (p=0.01) in mean egg weight was noted in 

the 100 mg/kg treatment group, the effect was minimal (9.6 g vs. 10.0 g control) and no effects 

on this parameter occurred in the medium and high-treatment groups. Therefore, this effect was 

not considered to be related to treatment. The reproductive toxicity LOAEC is not determined 

(>1000 mg/kg), and the NOAEC is 1000 mg/kg in the diet. 

Avian Reproduction Toxicity (Duck, OCSPP 850.2300) 

In an avian reproduction toxicity study (MRID 48126602/00098296), Mallard ducks (Anas 

platyrhyncos; six months old) were administered captan (purity not reported; presumed to be 

89%) in the diet at concentrations of 0 (corn oil), 100, 300, or 1000 mg/kg. The birds were fed 

the diets ad libitum during the duration of the study. The total duration of the study was nineteen 

weeks (eleven weeks prior to egg laying and eight weeks of egg collection). The birds were 

housed in five pens/dose group, with two males and five females per pen. A progressive photo 

period was employed in this study with 8 hours/day through Week 6 and 17 hours/day for the 

remainder of the study. Temperature and relative humidity were allowed to fluctuate with 

ambient conditions; temperature limits were 1.7 to 32.2°C. Individual adult body weights were 

recorded at study initiation, biweekly through Week 8, and at study termination. Feed 

consumption in each replicate was measured weekly throughout the study. Adult birds were 

observed routinely for mortality and clinical signs. The following reproductive parameters were 

observed and recorded: eggs laid, eggs cracked, eggs set, viable embryos, live 3-week embryos, 

hatchlings, 14-day-old survivors, body weight of 14-day-old survivors, and egg shell thickness. 

No treatment -related effects on mortality, body weight, or feed consumption were observed. 

One hen in the low-treatment group died during the study, but the death was attributed to the 

stress of egg production. Average hatchling body weight was increased (p<0.01; ↑6%) in the 

high-concentration group. Although the number of eggs cracked/eggs laid was increased (p=0.01 

and 0.02, respectively) in the medium- and high-concentration groups, this effect was not 

concentration dependent and the difference between the control and high-concentration value 

was only 0.5%. Because there was no effect on eggshell thickness, the effect on eggs 

cracked/eggs laid was not considered a treatment-related effect. In addition, no other effects were 

determined for eggs laid, egg weight, viable embryos, live 3-week embryos, 14-day survivor 

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weight, normal hatchlings, and 14-day survivors. The reproductive toxicity LOAEC is not 

determined (>1000 mg/kg), and the NOAEC is 1000 mg/kg in the diet. 

Fish Full Life Cycle Toxicity (Fathead minnow, OCSPP 850.1500) 

In a published study by Hermanutz et al. (1973; MRID 00057846), groups of 9-day old of 

Fathead minnow (Pimephales promelas) embryos (25 embryos/aquaria; 2 aquaria/dose group) 

were exposed to captan (88.4% purity) at nominal concentrations of 0 (negative control), 12.5-

15.6, 25-31.3, 50-62.5, 100-125, or 200-250 μg/L under flow-through conditions for 45 weeks. 

Increasing amounts of Triton X-100 and acetone were used as vehicles for captan, and 

concentrations of these components never exceeded 0.0007 and 1.5%, respectively. After Week 

3, nominal concentrations of captan were increased by 100% because no difference in survival or 

growth of the young fish was apparent, and after Week 17, nominal concentrations were again 

increased by 25% because of a decrease in all measured concentrations starting at Week 4. 

Mean-measured concentrations (Weeks 3-45) of captan were 3.3, 7.4, 16.8, 39.5, and 63.5 μg/L 

(25.4, 31.6, 26.9, 23.6, and 21.2% of the highest nominal values). After subtraction of the 12% 

captan that precipitated at the outlet of the needle, the measured concentrations averaged about 

30% of the adjusted nominal concentrations. On Day 51, the larvae were randomly culled to 15 

larvae/aquaria. Growth and survival were determined for 40 one-day old F1 fish reared in larval 

chambers for 30 days. The measured parameters for the parental fish included growth on Day 51, 

and the sex gonadal condition, length, and weight at Week 45. The Agency noted that the ability 

to detect statistically significant changes in study endpoints is limited by the few number of 

spawning replicates (n=2). 

For the chronic exposure of Fathead minnows, survival of parental fish was decreased (p<0.05) 

in the mean-measured 63.5 μg/L treatment group (↓98%). The parental fish length and the F1 fish 

survival and length were also decreased (p<0.05) in the mean-measured 39.5 μg/L treatment 

group. At the medium and medium-high concentrations (16.8 and 39.5 μg/L), the mean number 

of spawnings/female were decreased (NS) by ca. 82 and 96%, respectively, compared to the 

control, and the mean number of eggs spawned/female were decreased (NS),by ca. 77 and 98%, 

respectively. The mean number of eggs/spawning was also decreased (NS) at the medium-high 

concentration by ca. 42%, compared to the control. Percent hatchability of embryos from 

unexposed parents incubated at 63.5 μg/L captan was similar to that of control embryos, but all 

larvae died 5-8 days after hatching. There was 100% mortality of 1-day-old larvae from 

unexposed parents incubated at 63.5 μg/L captan within 24 hours. Survival of 3-day-old 

hatchlings in the medium-high concentration group was decreased (p=0.05) approximately 50%. 

The results of the post-mortem examination of gonads was not discussed in detail; no difference 

in gonadal development was noted for fish in one of the medium-low (7.4 μg/L) dose chambers 

where spawning was absent. The LOAEC is 39.5 μg/L based on decreased growth and survival, 

and the NOEAC is 16.8 μg/L. 

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APPENDIX 3: References Not Utilized in the Captan WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Captan but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used in the study; 3) the 

test material was not adequately described; 4) a formulated end-use product or mixture of 

chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 

6) the experimental conditions were not adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Arndt, T. and Dohn, D. (2004) Measurement of the Half-Life of Thiophosgene in Human Blood. 

PTRL West, Inc., Hercules, CA; Captan Task Force. Report no. 1146W-1. Research and 

Development Corporation for ICI Americas Inc. Project No. IRDC 153-198. (MRID 46296101) 

Band, P.R., Abanto, Z., Bert, J., Lang, B., Fang, R., Gallagher, R.P., and Le, N.D. (2011) 

Prostate cancer risk and exposure to pesticides in British Columbia farmers. The Prostate

71:168-183. 

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Gordon, E.B., Ehrlich, T., Mobely, S., and Williams, M. (2000) Measurement of the reaction 

between the fungicides captan or folpet and blood thiols. Makhteshim-Agan of North America, 

Inc., New York, NY. Laboratory Project ID MANA 00-01. (MRID 45745103) 

Hari, S., Nishikawa, J-I., Horiguchi, K., Iida, M., and Nishihara, T. (2006) Anti-androgenic 

activity of N-nitrosodibenzylamine, N- nitrosodiphenylamine and N-nitrosodicyclohexylamine. J. 

Health Sci. 52(5), 522-531. 

Nishihara, T., Nishikawa, J., Kanayama, T., Dakeyama, F., Saito, K., Imagawa, M., Takatori, S., 

Kitagawa, Y., Hori, S., and Utsumi, H. (2000) Estrogenic activities of 517 chemicals by yeast 

two-hybrid assay. J. Health Sci. 46:282-298.

Reyna, M.S. (1973) Two-year carcinogenic study with captan technical in albino rats. IndustrialBiotest Labs Inc. for Chevron Chemical Company. IBT No. 89267. (MRID 00069243) 

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