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 Assess ment T y pe: 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 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

## ~ S

## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 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

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: CAPTAN

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYCAPTAN FINAL

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 Rele vant 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-Estr ogenic Pathway for Captan........................................10
Table 3. Androgenic/Anti-Andr ogenic 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.................................50CAPTAN FINAL

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 Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r 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

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationCAPTAN FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS High Pressure Liquid Chroma tography/Mass Spectroscopy

HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods

K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y 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 Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record IdentifierCAPTAN FINAL

v Abbreviation Terminology

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration
MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 TriiodothyronineCAPTAN FINAL

vi Abbreviation Terminology

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceCAPTAN FINAL

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 poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as perturbations of the hypothalamic-pituitary-gon adal (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 conditi ons under which effects occur were considered, in particular, whether or not endoc rine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On December 3, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of captan with the E, A or T signaling path ways. 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 develo pment (increase in age at VO) and decreased ovarian weights were observed at the low dose, it is noted that a 9% de crease in body weight and clinical signs of toxici ty were observed suggesting that these effects were due to a dose that was on the cusp of being overtly toxic. Additionall y, there were no other corroborating evidence in the Tier 1 assays or OSRI to s uggest that captan pote ntially interacts with the estrogen pathway.

There was no convincing evidence of potential inte raction of captan with the androgen pathway. Captan was negative in the AR binding and ster iodogenesis assays. No androgen-related effects were observed in the antagonist portion of the as say, the agonist portion of the assay could not be evaluated due to overt toxicity and/or excess mortality. There we re no treatment-related effects in males in the FSTRA, except for an increa se in body weight and length at the high concentration. In the male pubertal assay, ther e was an isolated finding observed (decreased ventral prostate weight); however , there were no complementary res ponses in this assay at a doseCAPTAN FINAL

Page 2 of 51 that was at the cusp of overt toxicity. In addi tion, there were no androgen -related effects seen in any of the mammalian and w ildlife Part 158 studies.

In the AMA, there were no thyroi d-related effects observed. In th e male pubertal assay and Part 158 mammalian studies, there were no thyroid-related effects obser ved in the absence of overt toxicity. In the female pubertal assay, while a decrease in T 4 was observed, there were no other effects observed at this dose (which was on the cu sp of overt toxicity). Collectively, there is no convincing evidence of a poten tial thyroid interaction in mammals or wildlife.

Based on the weight of evidence evaluation, ma mmalian and wildlife EDSP Tier 2 studies are not recommended for captan since there was no convincing evidence of potent ial interaction with the estrogen, androgen or thyroid pathways.CAPTAN FINAL

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 poten tial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interaction includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as perturbations of the hypothalamic-pituitary-gon adal (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 c onsidered. Additionally, the conditi ons under which effects occur were considered, in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in general syst emic toxicity or overt toxicity.

On December 3, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of captan with the E, A or T signaling path ways. 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 solubili ty of 5.1 mg/L at 25°C, with a log K ow 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 di rect photolysis. Captan is expe cted to have high to moderate mobility in soil but volatilization from moist or dr y 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 ha lf-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 determin ing 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 stud ies 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 (U SEPA, 2011), is presented in Section IV. The
EDSP Tier 2 studies r ecommendations are presen ted in Section V.CAPTAN FINAL

Page 4 of 51 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 believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Orde r 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 re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on captan (USEPA, 20 10). 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 c onsidered but not util ized 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 presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual 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 endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting 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 assaysCAPTAN FINAL

Page 5 of 51 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 in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach 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 speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate 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 suppor t a hypothesis that a chemical has the potential to interact E, A or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, 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 ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment 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 releva nt, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal 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 co nsideration 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 eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.CAPTAN FINAL

Page 6 of 51 This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting in July 2013 that evaluated scie ntific issues associ ated with the WoE evaluation of the EDSP Tier 1 screening proce ss. In October 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All effects in endocrine se nsitive 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 gene rally 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 include d, but were not limited to: mortal ity; 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, es pecially 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/concentra tion 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 decr eases 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 ef fects were generally cons idered 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/chemi cal properties and its
known modes of action in its overal l 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 determ ining 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 eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble 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 bi ologically relevant with regard to a treatment-related effectCAPTAN FINAL

Page 7 of 51 which supports a conclusion of whether or not a s ubstance 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 ar e 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)

ER α 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 Satisfied 1 (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 e ffects were seen in the other Tier 1 assays and will not impact the risk assessment.CAPTAN FINAL

Page 8 of 51 B. Effects on Hypothalamic-P ituitary-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 a ssays 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 reporte d 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 te stosterone concentration was observed in one of two runs at 10 and 100 μM, respectively. Wh ereas 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 act ivity at the highest tested concentration (10

− 5 M). In addition, captan was demonstrat ed 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 μ M]. Captan was negative in the Tier 1 uterotrophic assay.

In the female pubertal assay, ove rt 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 animal s). Also, at the lo w dose (300 mg/kg/day), clinical signs of toxici ty were observed that were at the cu sp of overt toxicity (9% decreased body weight, abnormal breathing and decreased movement). In th is 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 firs t 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 repr oductive organ weight included: decreased ovarian weights in the lo w 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 atre tic) 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 hi gh-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 av ian reproduction toxicity studies in bobwhite quail and mallard
ducks.CAPTAN FINAL

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 fec undity at the medium and medium-high concentrations (16.8 and 39.5 μ g/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 obs erved. However, there were se veral limitations in this study including: use of a surfactant in addition to the acetone co-solve nt; test concentrations were increased several times during th e course of the study; and, low statistical power to detect differences between test groups.CAPTANFINALPage 10 of 51Table 2: Estrogenic/Anti-Estrogenic Pathway for CaptanLines 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 Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER Binding(MRID 48615103)NERTA(MRID 48615104)NAromatase(MRID 48615102)ESteroidogenesis(MRID 48615106)N4Uterotrophic(MRID 48615107)NNX5(L, H)Female Pubertal Rat(MRID 48669502)N↓18%(L)N↓18%(L)↑2.5days(L)Age:↑4.0days (L)BW:↓9%(L)CS(L, H)X6(H)FSTRA(MRID 48669501)NENNNNNNOSRIReceptorTransactivation(Kojimaet al.,2004)NReceptorTransactivation(Kojimaet al.,2005)NReceptorTransactivation(Okuboet al.,2004)NCAPTANFINALPage 11 of 51Table 2: Estrogenic/Anti-Estrogenic Pathway for CaptanLines 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 Systemic Toxicity Observed 2 Overt Toxicity Observed 3One-generationreproduction (Rat;MRID 00120315)NENENENENNNThree-generationreproduction (Rat;MRID 00125293)NENENENENNX (MH,H)Developmentaltoxicity (Rat; MRID48126603)NENNX(H)Chronic toxicity/carcinogenicity (Rat;MRID 00120316)NENNNNX(M, H)Chronic toxicity/carcinogenicity (Rat;MRID 00161230)NENNNNX(H)Chronic toxicity(Dog; MRID40893604)NENNNEX(H)NAvian reproduction(Quail; MRID00098295/48126601)NN NAvian reproduction(Duck; MRID00098296/48126602)NN N1.Key to responses: Low treatment (L), Medium-low treatment (ML), Medium treatment (M), Medium-high treatment (MH), 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. Changes inweight are absolute unless otherwise indicated. KW= Kidney weightCAPTANFINALPage 12 of 512. 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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4.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.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 animalsN Negative findings (in vitro)/No effect (in vivo)E Equivocal assay responseNE Not examinedCAPTAN FINAL

Page 13 of 51 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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e 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 additio nal third run, precipitatio n was observed at this concentration. In addition, no ot her significant changes in testos terone production was observed in the remaining test concentrations up to 100 μM.

In the Tier 1 Hershberger assay, in the agonist po rtion of the assay both doses were overtly toxic (exhibiting severe clinical signs of toxicity, death or were sacrif iced due to clinical signs). In the antagonist phase of the assay, no effects on AS T 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 toxici ty were observed that were at the cu sp of overt toxicity (9% decreased body weight, rales and alopecia). In this st udy, 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 decrease d 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.CAPTANFINALPage 14 of 51Table 3: Androgenic/Anti-Androgenic Pathway for CaptanLines 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 Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48615101)NSteroidogenesis(MRID 48615106)N4Hershberger(MRID 48615105)N5N5CS(L, M, H)X5(M, H)Male Pubertal Rat(MRID 48669503)NNNNNNVP:↓15%(L)NCS( L, H)X6(H)FSTRA(MRID 48669501)NENNNNNNOSRIReceptorTransactivation(Kojimaet al.,2004)NOne-generationreproduction (Rat;MRID 00120315)NENENENENENENN NThree-generationreproduction (Rat;MRID 00125293)NENENENENENENNX(ML,MH,H)CAPTANFINALPage 15 of 51Table 3: Androgenic/Anti-Androgenic Pathway for CaptanLines 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 Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity/carcinogenicity(Rat; MRID00120316)NNNENNNEX(M, H)X(M, H)Chronic toxicity/carcinogenicity(Rat; MRID00161230)NNNENNNEX(H)X(H)Chronic toxicity/carcinogenicity(Dog; MRID40893604)NNNENNENX(H)NAvianreproduction(Quail; MRID00098295/48126601)NN NAvianreproduction(Duck; MRID00098296/48126602)NN N1.Key to responses: Low treatment (L), Medium-Low treatment (ML), Medium treatment (M), Medium high treatment (MH), High treatment (H). Arrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weightare absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate(VP), Dorsal prostate (DP),Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). CS= clinical signs2. 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 inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. ACAPTANFINALPage 16 of 514. A 9% decrease in testosterone concentration was observed inone of two runs at 10 μM, whereas in an additional third run, precipitation was observed at thisconcentration. In addition, no other significant changes in testosterone production was observed in the remaining test concentrations up to 100 μM.5. In the agonist phase, both doses were overtly toxic (exhibiting severe clinical signs of toxicity, death or were sacrificeddue to clinical signs). In the antagonistphase 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 the higher doses (200 and400 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, two high-dose animals wereeuthanized after being found moribund.N Negative findings(in vitro)/No effect(in vivo)NE Not examinedCAPTAN FINAL

Page 17 of 51 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry 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 de tect changes in the HPT axis, i.e ., the pubertal female and male (rat) assays and the AMA (frog).

The potential for captan to interact wi th thyroid regulation is summarized in Table 4. The various targets of the th yroid 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 ar e not reported in the ta ble 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 th at at the high dose, there was an 18% decrease in serum T4 in the presence of ove rt 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, clinic al signs and significant decreases in body weight). Serum T 4 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) concentra tions were decreased in a non-dose-dependent manner. In the thyroid, follicular cell height wa s increased in the high-dose animals, and there was a corresponding decrease in the colloid area of thyroid folli cles. 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 e ffects of captan in the AMA. No thyroid-related effects were
observed in the mammalia n Part 158 studies.CAPTAN FINAL

Page 18 of 51 Table 4. Thyroid Pathway for Captan.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Captan 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Female Pubertal Rat (MRID 48669502) N N ↓ 27% (L) N ↓ 18% (L) BW: ↓ 9% CS ( L, H) BW 4 (H)

Male Pubertal Rat (MRID 48669503) N N N N N CS (L, H) BW 5 (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 as say. 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 examinedCAPTAN FINAL

Page 19 of 51 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of captan to interact w ith endocrine related processes ( i.e. , E, A, or T signaling pathways) as well as recommendati ons 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 pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (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 consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, 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 evalua tion in helping elucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e 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 ar e 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 Sc ientific Advisory Panel (SAP) that evaluated scientific issues associated with weight of eviden ce 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 presen ce of overt toxicity. All effect s in endocrine se nsitive tissues should be evaluated in terms of primary intera ctions with the endocri ne 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/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).CAPTAN FINAL

Page 20 of 51 1. Tier 1 in vivo Assays

In the 21-day AMA, Day 7 mortality was not re ported 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 re maining 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 treatme nt groups. An increase in male body weight at the high-treatment concentrati on, and increases in male body length at the medium- and high-treatment levels were observe d 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 a gonist portion: one animal was f ound dead (suspected dosing error)
and one animal was euthanized because of breat hing difficulties. All animals in the high-dose group (400 mg/kg/day) were euthani zed prior to study termination: four animals were euthanized due to abnormal breathing and body weight loss a nd the remainder were humanely euthanized prior to the potential onset of br eathing 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 hum anely 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 obs erved 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 an imal 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 anim al in this group was found to have rales and another exhibited lethargy. Fi nal body weights were comparab le 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 severa l low- and high-dose animals (including the euthanized animals). In addition, final body weig hts were decreased by 9 and 10% in the lowand high-dose groups, respectively, yielding decreased overall body weight gains of 14% and 15%, respectively.CAPTAN FINAL

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 St udy 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 decrease d 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 un til scheduled termination, with the exception of one animal in the 1000 mg/kg/day group that wa s euthanized moribund on Day 3. There were no
treatment-related clinical findi ngs in the surviving rats. Fina l 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 anoverall 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 reprodu ction toxicity study in rats up to the highest dose (25 mg/kg/day).

There were no reported treatment-relate d effects on mortality in the P, F 1 or F 2 parental animals in the three-generation reproducti on toxicity study in rats. Di fferences in mortality were
observed in the F 2a litter at LDs 1 and 4, and in the F 3 a litter at LD 4, for the medium-high dose pups (250 mg/kg/day); and in the F 2a and F 3a litters at LDs 1 and 4, and in the F 2b 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 group s, 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%, re spectively. Decreases in mean litter weights were observed in all treatment groups, with decr eases 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 F 1 males and the low- and medium-low dose F 2 females.

In the prenatal developmental toxicity study in rats, no mortal ities occurred dur ing 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 ofCAPTAN FINAL

Page 22 of 51 17% during GD 10-13 and 14-16. Food consumption r ecovered with an increase of 17% during

GD 17-20.

In the combined chronic toxic ity/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 tr eatment-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/carcinogeni city study in rats, no tr eatment-related effects were observed for mortality or clinical signs . In the high-dose animals, mean body weight decreases in the high-dose gr oup (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 sligh tly lower in the high-dose animals.

In the chronic toxicity study in dogs, no treatmen t-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 th e high-dose animal s (300 mg/kg/day).

In the avian reproduction toxicity study in bobwhite quail, no trea tment-related mortalities were observed; one mortality each occurred in the medium- and high-treatment groups due to physical injury. Evidence of stress including lacerated s calps, 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 toxic ity 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 attr ibuted 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 μ g/L (high concentration) group ( ↓ 98%). The parental fish length and the F 1 fish survival and length were decreased in the mean-measured

39.5 μ g/L (medium-high concentration) group. Percen t hatchability of embryos from unexposed parents incubated at 63.5 μ g/L captan was similar to that of c ontrol embryos, but all larvae died 5-8 days after hatching. There also was 100% mortality of 1- day-old larvae from unexposedCAPTAN FINAL

Page 23 of 51 parents incubated at 63.5 μ g/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 ster iodogenesis 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 wi ldlife 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 potenti ally interacts with the estrogen pathway.

C. Androgen Pathway

There was no convincing evidence of potential inte raction of captan with the androgen pathway. Captan was negative in the AR binding and ster iodogenesis assays. No androgen-related effects were observed in the antagonist portion of the as say, the agonist portion of the assay could not be evaluated due to overt toxicity and/or excess mortality. There we re no treatment-related effects in males in the FSTRA, except for an increa se in body weight and length at the high concentration. In the male pubertal assay, ther e was an isolated finding observed (decreased ventral prostate weight); however , there were no complementary res ponses in this assay at a dose that was at the cusp of overt toxicity. In addi tion, there were no androgen -related effects seen in any of the mammalian and w ildlife Part 158 studies.

D. Thyroid Pathway

In the AMA, there were no thyroi d-related effects observed. In th e male pubertal assay and Part 158 mammalian studies, there were no thyroid-related effects obser ved in the absence of overt toxicity. In the female pubertal assay, while a decrease in T 4 was observed, there were no other effects observed at this dose (which was on the cu sp of overt toxicity). Collectively, there is no convincing evidence of a poten tial thyroid interaction in mammals or wildlife.

E. Conclusions

Captan demonstrates no convinci ng evidence of potential interac tion with the estrogen, androgen or thyroid pathways in mammals or wildlife.CAPTAN FINAL

Page 24 of 51 V. EDSP Tier 2 Testing Recommendations

Based on the weight of evidence evaluation, ma mmalian and wildlife EDSP Tier 2 studies are not recommended for captan since there was no convincing evidence of potent ial interaction with the estrogen, androgen or thyroid pathways.CAPTAN FINAL

Page 25 of 51 VI. References

Beavers, J.B. (1980a) One-generation reproduc tion study-- Bobwhite quail: Captan technical (SX-1086). Report No. S-1717. Unpublished study c onducted by Wildlife Inte rnational Ltd. for Chevron Chemical Company. (MRID 00098295/48126601)

Beavers, J.B. (1980b) One-generation reproduc tion study-- Mallard duck: Captan technical (SX-1086). Report No. S-1716. Unpublished study c onducted by Wildlife Inte rnational 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 Deve lopment 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, In c., Durham, NC. Laboratory Project Study No.: C198-100, December 20, 2011. (MRID 48615107)

Davis, J. P. (2012) The Hershberger Bi oassay (OPPTS 890.1400); Captan. Unpublished study conducted by Integrated Laboratory Systems, In c., Durham, NC. Laboratory Project Study No.: C198-200, January 5, 2012. (MRID 48615105)

Davis, J.P. (2012) Pubertal development and t hyroid function in intact juvenile/peripubertal female rats (OPPTS 80.1450); Captan. Unpublis hed 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 thyroi d function in intact juve nile/peripubertal male rats (OPPTS 890.1500); Captan. Unpublished st udy 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 toxici ty/carcinogenicity study of captan in rats. Unpublished study conducted by International Re search and Development Corporation for Stauffer Chemical Company. (MRID 00120316)

Gordon, E. (2013). Captan: A weight of eviden ce evaluation of the U.S. EPA’s endocrine screening program Tier 1 screening batter y. 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 toxi city to Fathead minnows ( Pimephales promelas ), Bluegills ( Lepomis macrochirus ) and Brook trout ( Salvelinus fontinalis ). J. Fish. Res. Board Can. 30: 1811-1817. (MRID 00057846).CAPTAN FINAL

Page 26 of 51 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 de rivative 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 activi ties in 200 pesticides by in vitr o 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 us ing an in vitro reporter gene assay. Int. J. Environ. Health

Res

. 15: 271-280.

Lee, M.R. (2013) Captan- Amphibian Metamo rphosis Assay with African Clawed Frog ( Xenopus laevis ) Following OPPTS Test Guideline 890.1100 and OECD Test Guideline No. 231. Unpublished study conducted by Smithers Visc ient, 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 pest icides by MCF-7 cell proliferation assay. Arch. Environ.
Contam. Toxicol. 46:445-453.

Rubin, Y. (1987) Captan: tera tology study in the rat. Unpub lished 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 repr oduction study in rats. Unpublished study conducted by International Research and Deve lopment Corporation for Stauffer Chemical Company. (MRID 00125293)

Schardein, J.L. (1982b) One-gene ration reproduction study in rats with captan. Unpublished study conducted by International Research and De velopment Corporation for Stauffer Chemical Company. (MRID 00120315)

Til, H.P., Kuper, C.F., and Folke, H.E. (1983) Li fe-span oral carcinogenic ity study of Merpan in rats. Unpublished study conducted by CNO Institu te TNO for Makhteshim Chemical Works Ltd. (MRID 00161230)

USEPA (2010) CAPTAN - Report of the Endocrine Di sruptor Review Team- Test Order#: EDSP-081301-163 and 164. December 9, 2010. Office of Chemical Safety and Pollution
Prevention. U.S. Environmental Pr otection Agency, Washington, D.C.CAPTAN FINAL

Page 27 of 51 USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. 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 Recomb inant 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, In c., Kalamazoo, MI. July19, 2012. (MRID 48843501)

Willoughby, J.A. (2012) Captan: Estrogen Recepto r Transcriptional Activation (Human Cell Line HeLa-9903)). Unpublished study conduc ted 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)CAPTAN FINAL

Page 28 of 51 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 49136701) of captan on amphi bian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through c onditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80/control and treatment group; 20/replicate) were exposed to nominal concentrations of captan (93% purit y) at 0 (negative a nd 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; c ontrols), 0.000064, 0.00077, and 0.0073 mg a.i./L; reviewer-calculated time-weighted average (T WA) 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 23 o C and a pH of 6.9 to 7.8.

There were no significant differenc es (p>0.05) between the negative control and solvent control for any of the endpoints. Unless otherwise indica ted, all effects are repo rted 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 w ith 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 effect s (p>0.05) on normalized (for snout -vent length) hind limb length (HLL) on Day 7 or 21 at any of the treatment le vels. There was no signifi cant (p>0.05) acceleration or delay of median NF developm ental stage at 7 or 21 days at any treatment level; no asynchronous development was observed. Late-stage tadpoles we re found in both c ontrol groups and all treatment groups with no apparent pattern in their occurrence; ther e were 3, 4, 4, 2, and 1 individuals at NF 61 in the nega tive control, solvent control, and low, mid, and high treatment groups, respectively. Thyroid gland histopathology findings were obs erved 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 patte rns in the histopat hological findings.

Androgen Receptor (AR) Bi nding 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 bi nding 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 captanCAPTAN FINAL

Page 29 of 51 (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 wa s 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 (K d ) for [ 3 H]-R1881
was 0.613±0.041 nM and the estimated B max was 0.817±0.049 fmol/100 μg protein for the single batch of prostate cytosol that was prepared. The mean and individual K d and B max values were below the expected ranges repor ted in the EPA validation program (0.685 to 1.57 nM and 7 to 16 fmol/100 μg protein, respectively). However, c onfidence in these numbers is high based on the goodness of fit (R 2 = 0.957-0.984) and the small variation among runs.

In the competitive binding experiment, the estimated mean log IC 50 s for R1881 (

− 9.0 M) and the weak positive control, dexamethasone (

− 4.6 M), were within the expe cted 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 [ 3 H]-ligand over the conc entration range of 10

− 11 to 10

− 4 M; therefore, a log IC 50 and RBA could not be calculated fo r 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 arom atase and tritiated androstenedione ([1 β

3 H(N)]- androst-4-ene-3,17-dione; [ 3 H]ASDN) in dimethyl sulfoxide (D MSO) at log concentrations of

10 -10 to 10 -3 M or 10 -11 to 10 -5 M for 15 minutes to assess the pot ential of captan to inhibit aromatase activity.CAPTAN FINAL

Page 30 of 51 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). Th ree runs were conducted, and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) with a known inhibitor (4-hydroxya ndrostenedione; 4-OH ASDN), and the test chemical series (10

− 11 to 10

− 5 M) with three repetitions per concen tration. 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 fu ll activity controls averag ed 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 contro ls ranged 0.25 to 0.37% an d 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 IC 50 , and coefficient of variation for replicates of each concen tration within runs. The estimated log IC 50 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 be tween 50 and 75% activity (60.3% at 10 -5 M), indicating an equivocal response.

Based on the data from the average response cu rve, captan is classified as Equivocal for aromatase inhibition in this assay.

Estrogen Receptor (ER) Bi nding Assay (OCSPP 890.1250)

In an estrogen receptor (ER) binding a ssay (MRID 48615103) for captan (93% purity, Batch #91138103), uterine cytosol from Sprague Dawley ra ts 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]-17 β -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 17 β -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.CAPTAN FINAL

Page 31 of 51 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 pr ovided in the study re port; however, summarized saturation binding data (MRID 48843501) from th e 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 approxima tely 3- to 6-fold greater than recommended (160 to 320 μg versus 50±10 μg). The mean dissociation constant (K d ) for [ 3 H]-17 β -estradiol
was 0.331±0.061 nM and the estimated B max was 74.55±3.03 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 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 IC 50 was

− 9.0 M for 17 β -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 gene rally met; however, octylt riethoxysilane displaced more than 25% of [ 3 H]-17 β -estradiol from the ER at the highest concentration tested (10 -3 M) in two of the three assay runs. Total [ 3 H]-17 β -estradiol binding was >75% at all soluble concentrations ( ≤ 10 -4 M) of captan tested in all three valid competitive binding runs. A log IC 50 and RBA could not be determined for captan sinc e 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.

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

In an estrogen receptor transcript ional activation assay (MRID 48615104), hER α -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 experi ments 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 es trogen receptor by captan was measured using a proprietary luciferase assay.

Captan was tested up to the limit of cytoto xicity, which was observe d at concentrations ≥ 10 -5 M 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 un der the conditions of this assay.

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

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

Page 32 of 51 the assay to very weak agonists. Furthermore, 17 α -methyltestosterone responses reached only
6.3-7.9% PC and were similar to those of the ne gative 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, 17 α -estradiol (117-124% PC) and the

RPC Max < PC 10 in both assay runs.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 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 replic ates/treatment; ~21 weeks old) were exposed to captan (94.9% purity) at nomina l concentrations of 0 (neg ative 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; c ontrols), 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 e ffects are reported based on comparison to the negative (clean water) control.

Overall survival in the negative and solvent contro ls was 88 and 96%, respectiv ely; overall survival was 100% in all captan treatment groups and was not significantly different (p>0.05) from the negative control. Signifi cant 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-re lated effects on secondary sex char acteristics or clinical signs ( i.e. , behavioral and other subletha l effects including (discolorati on, changes in dorsal nape pad, lethargy, loss of equilibrium).

In the negative and solvent contro ls, spawning occurred at least ever y 4 days in three out of four replicates in the negative contro l 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 su ccess was 96 and 97%, respectively. Fecundity and fertilization success was not signifi cantly different (p>0.05) in a ny treatment group relative to the negative control.

Plasma vitellogenin (VTG) was significantly incr eased (p<0.05; Mann-Whitney U test) in males at the mid and high treatment levels relative to th e negative control; howev er, the increase was not concentration-responsive. There was no significant effect (p>0.05) on female plasma VTG at any
treatment level.CAPTAN FINAL

Page 33 of 51 Treatment-related effects on gona dal histopathology were not obser ved 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 betwee n the negative control and treatment groups for males; no nuptial tubercles were observed in female s. 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% carboxy methylcellulose (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 androgeni c positive control grou p consisted of eight castrated rats exposed to the vehicle by oral gavage plus 0.4 mg/kg/day of testosterone propionate (TP) by subcutan eous (s.c.) injection.

To screen for potential anti-andr ogenic activity, captan in 1% CM C 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 cont rol 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 da ily. The animals were dosed for 10 consecutive days and terminated a pproximately 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 euthan ized 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 anim als in the 200 mg/kg/day group were humanely euthanized prior to study termination: two an imals were euthanized due to abnormal breathing and body weight loss, and the remaining six an imals were humanely euthanized due to body weight loss or prior to the potenti al 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 theCAPTAN FINAL

Page 34 of 51 remaining three animals were humanely euthanized prior to the potential onset of breathing difficulties and severe weight lo ss. All animals in the negati ve 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 exhibite d abnormal breathing that was severe enough to result in rapid weight loss and at necropsy, the st omach and/or intestines were severely distended with air; similar but less severe symptoms were observed in the range finding study. The oral

LD 50 in rat is >5000 mg/kg (material data safety sheet for captan). It was st ated that the increase in moribundity may have been due to gavage-rel ated reflux, but no explan ation 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 obs erved 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 decr eased (not statistically analyzed) by 9-10% on Days 4 and 7 and decreased (p<0.05) by 12% on Da y 11 compared to controls, resulting in an overall body weight loss (p<0.05) of 6.7 g (compare d to gain of 37.7 g in the vehicle control). Final body weights in the positiv e control group were comparab le 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 acce ssory 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-bul bocavernosus muscle complex (LABC); 458% in Cowper’s gland; and 57% in glans penis.

In the anti-androgen assay, one animal in th e 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 breat hing, and thin appearance. One additional animal in this group was found to have rales a nd another found to be lethargic. In the 100 mg/kg/day group, final body weight s 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 nega tive 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 increa sed (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 gla nd; and 24% in glans penis. In summary, in the Tier 1 Hershberger assay, no androgen-related effect s were observed in the agonist phase (200 and 400 mg/kg/ day) or in the antagonist ph ase (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 wasCAPTAN FINAL

Page 35 of 51 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 trea tment 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 pe rmissible 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 do ses 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 (T 4 ) 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 sa crifice. 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 de lay in attaining VO was not related to reductions in body wei ght. However, final body weight s 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%, resp ectively. 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 in creased (p<0.05) by 5 and 8% at 300 and 600 mg/kg/day, respectively. There were also dose-dependent decr eases in pituitary and ovary weights. Absolute and adjusted pituitary weights were decreased (p<0.01) by 18% at 300 mg/kg/day, and absolute, adjuste d, and relative pituitary weights were decreased (p<0.01) by 19- 28% at 600 mg/kg/day. Absolute and adjusted ov ary weights were decreased (p<0.01) by 18 and 23% at 300 and 600 mg/kg/day, respectively. Adju sted wet uterus weights were decreasedCAPTAN FINAL

Page 36 of 51 (p<0.05) by 35% and absolute bl otted uterus weights were d ecreased (p<0.01) by 34% at 600 mg/kg/day. At 300 mg/kg/day, li ver weights were decreased (p <0.05) by 12%, but liver weights at 600 mg/kg/day were comparable to controls . There were no treatm ent-related effects on adrenal gland or thyroid weights.

The mean age at first vaginal estrus was de layed 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 es trus cycle parameters were observed.

Serum T 4 concentrations were decreased in a dos e-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 increas ed (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 incr eased (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 redu ced, trend in increased follicular cell height at 300 mg/kg/day. No histopatholog ical findings were observed in the ovaries, and any findings noted in the uterus and kidneys were generally minimal or mil d, 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, c linical signs of toxic ity and death in the 600 mg/kg/day dose group, the high dose level was cons idered 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 d ecreased movement).

Male Pubertal Assay (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48669503), 16 Sp rague Dawley rats/dose group were treated daily by oral gavage with captan (94. 9%, Lot/Batch No. 91138103) in aqueous 1% carboxymethylcellulose (CMC) at do ses of 0 (vehicle), 100, or 200 mg/kg/day from post-natal day (PND) 23 to 53/54. Animals were examined for preputial separati on (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 (T 4 ), thyroid stimulati ng hormone (TSH), and
testosterone were determined by radioimmunoassay; serum was also analyzed for concentrations of selected clinical chemistry parameters. Ad renal, liver, pituitar y, thyroid, and urogenital organs were weighed, and microscopic ex aminations were conducted on the testes, epididymides, thyroid, and kidneys.CAPTAN FINAL

Page 37 of 51 At 200 mg/kg/day, two animals were found mo ribund with abnormal breathing (gasping) and were euthanized on Study Days 18 and 27. All other rats survived until scheduled sacrifice. Cage-side and clinical observat ions included one animal with alopecia (100 mg/kg/day), one ungroomed animal (200 mg/kg/day), and two an imals with rales (one each at 100 and 200 mg/kg/day).

At 100 and 200 mg/kg/day, mean unadjusted and adju sted 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 d ecreased (p<0.01) unadjusted and adjusted overall body weight gains of 11 and 16%, respectively. At termin ation, 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 in creased (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 ad justed LABC weights were decreased (p<0.01) by 14 and 15%, respectively. There were no dose-rela ted effects on liver or pituitary weights, or on other accessory sex organ weights. Additionally, at 200 mg/kg/day, serum T 4 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, epid idymides, and kidneys were only observed in a single animal and/or were generally mini mal or mild in severity, if present.

Based on the >10% decrease in final body weight, c linical signs of toxic ity and death in the 200 mg/kg/day dose group, the high dose level was cons idered 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 ra les and alopecia).

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48615106), H295R cells cultured in vitro in 24-well plates were incubated with captan (93% purity, Batch # 91138103) at se rial log concentrations of 0.0001 to 100 μM for 48 hours in triplicate for f our 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 precipitati on observed in Run 4 was probably the result of a technical error. Cytotoxici ty greater than 20% was observe d at 100 μM in Runs 2 and 3.CAPTAN FINAL

Page 38 of 51 Samples with precipitation or cytotoxicity were not analyzed. Run 1 was not analyzed because
one of the forskolin concentrations on th e quality control plate was incorrect (3.33 μ M was used
instead of 1 μ M).

Testosterone and estradiol levels were measured using HPLC-MS/MS. A Quality Control (QC) plate was run concurrently with ea ch independent run of a test chem ical plate to demonstrate that the assay responded properly to positive contro l agents at two concentration levels; positive
controls included the known inhi bitor (prochloraz) and inducer (forskolin) of estradiol and testosterone production.

Guideline acceptability recommendations and requirements were met, including adequate production of testosterone and estradiol, acceptabl e reproducibility (low %CV), and appropriate
induction and inhibition with positive controls.

Statistically significant inhibi tion 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 th ree independent runs, captan treatment did not result in statistically signifi cant 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% ca rboxymethylcellulose (C MC) 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/da y on post-natal days (PND) 58-60. A positive control group was treated with a daily dose of 17 α -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 dos e administration to
determine wet and blo tted uterine weights.

All animals survived until sche duled termination, with the exce ption 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.CAPTAN FINAL

Page 39 of 51 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 we ight loss (p<0.05) of 18.3 g (compared to a gain of 15.8 g in the contro ls). Final body weights in the 313 mg/kg/day group were decreased (not signifi cant; NS) by 11%, with an over all body weight loss (p<0.05) of 10.2 g. The decreased body weights i ndicate that the dose levels te sted were adequate. Uterine weights in the captan treatment groups were comparable to the vehicle controls.

Comparative statistical analyses were not conducted for the fi nal 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 wei ght loss of 3.2 g. Absolute wet and blotted uterus weights for the EE group were increas ed (p<0.05) by 375% and 194%, respectively, as expected.

No statistically significant changes were seen in uterine weight in this as say. Captan is negative in the uterotrophic assay.CAPTAN FINAL

Page 40 of 51 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 (hER α and
hER β ) 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% fe tal bovine serum (FBS) and antibiotics at a temperature of 37°C in an atmosphere of 5% CO 2 under saturating humidity. Captan was
dissolved in DMSO to a final c oncentration of 10 mM and diluted to the desired concentration(s)
in DMEM/F12. The final solvent co ncentration in the culture medi um did not exceed 0.1% and did not affect cell yields.

The hER α and hAR expression vectors (pcDNAER α and pZeoSV2AR, respectively) were constructed as described previously (Kojima et al. , 2003). The hER β expression vector was constructed by cloning the hER β cDNA with reverse transcript ase-polymerase chain reaction from human placental RNA. The sequence was verified and inserted into the mammalian expression vector pcDNA 3.1Zeo(-) creating pcDNAER β . The construction of the estrogen or androgen responsive elements containing re porter 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 intern al control for transfection efficiency.

Host CHO cells were plated into 96-well microt iter plates at a dens ity of 8,400 cells/well in phenol red-free DMEM/F12 containing 5% CD-F BS (complete medium) one day before
transfection. For detection of hER α or hER β activity, cells were transfected with 5 ng pcDNAER α or pcDNAER β , 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 con centrations of test compounds (95-100% purity) or with 0.1% DMSO (v ehicle control) in complete medium. For measurement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 (17 β

estradiol, > 97% purity) or 10

− 10 M DHT (5 α -dihydrotestosterone, 95% purity) was added to the cell cultures along with the test compound, resp ectively. 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 Mini Lumat LB 9506 luminometer and was normalized based on the activity of co-transfected pRL-SV4 0. At least three independent experiments were conducted for each compound.

Agonistic activities were evaluated by relative ac tivity expressed as REC 20 (20% relative effective concentration). The REC 20 is the concentration of the compound showing 20% of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 , or 10

− 9 M DHT for ER α , ER β and AR, respectively. Antagonistic activities were evaluated by relative activity expressed as RIC 20 (20% relativeCAPTAN FINAL

Page 41 of 51 inhibitory concentration). The RIC 20 is the concentration of the compound showing 20% of the inhibition of the activity of 10

− 10 M E 2 , 10

− 9 M E 2 or 10

− 9 M DHT for ER α , ER β 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. Wh en the activity of the test compound was higher than the REC 20 the pesticide was judged to be positive for activity. When the activity of the test co mpound was higher than the RIC 20 the pesticide was judged to be positive for inhibitory activity. The statistical si gnificance 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 af ter exposure to thirty two pest icides 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 wa s 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 pr omoter, confer estrogen responsiveness to the viral promoter and reporter gene. The transfected cells are made st able by an antibiotic resistance gene resulting in the cell lin e used in the assay (BG1Luc4E 2 ). Compounds that interact with estrogen-responsive DNA are identif ied by the luciferase gene re sponse and quantified in terms of Relative Light Units (RLU).

The BG1Luc4E 2 cells were grown in medium for f our days, then the assay medium was replaced, and cells were transferred (2 × 10 5 cells/mL) into 96-well pl ates and incubated at 37  ̊ C
with 5% CO 2 for 24 hours. The medium was removed, and the test compound or estrogen standard (E2) was added. After incubation at 37  ̊ C with 5% CO 2 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 EC 10 (Effective Concentration at which 10% of the maximum response was observed) was used. The data were not sufficient to allow calculation of an EC 50

. Although the article does not sp ecifically identify the E 2 standard, the reviewers assumed that E2
referred to 17 β -estradiol.

The EC 10 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 perfor med as expected for chemicals that have positive estrogenic activity. Captan also tested nega tive for ER activity in this assessment.CAPTAN FINAL

Page 42 of 51 In a published study by Okubo et al . (2004) , the effects of selected pesticides on the proliferation of an estrogen -responsive cell line was inves tigated with captan and other pesticides. Captan (purity not reported) was obt ained from Wake Pure Chemical, MCF-7 cells
were obtained from Drs. A.M. Soto and C. Sonne schein (Tufts Universi ty School of Medicine, Boston MA), 17 β -estradiol and diethylstilv estrol (DES) were obtaine d from Sigma (purity not reported), and ICI 182,780 (anti-estrogen) was obt ained from Tocris Cookson (purity not reported). MCF-7 cells, an estroge n-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, th e medium was replaced with experimental medium (phenol red-free DMEM s upplemented with 5% charcoal-dextran treated human serum). Captan, dissolved in ethanol and diluted with phe nol red-free DMEM, was added
to the wells at final concentrati ons of approximately 0, 1, 10, and 100 μ M (concentrations were not reported, but were estimated by reviewer fr om a graph of study resu lts). 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-estr ogenic activity, 30 pM 17 β -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 cap tan, ICI 182,780 (10 nM) was added to cells exposed to the concentration of captan with th e highest cell yield in the cell proliferation experiment (100 μ M). Cytotoxicity was determined by neut ral 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 IC 50 of 142±17 μ M

. Captan did not induce estroge n-related cell proliferation, but was anti-estrogenic in the cell proliferation assay at 10 and 100 μ M

.

One-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a one-generation repr oduction 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 F l 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 physi cal observations were recorded on LD 21.

There were no treatment-related effects on parent al mortality, parental or litter body weights, food consumption, or clinical signs. Litter weight s 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 i ndex, gestation length, pupCAPTAN FINAL

Page 43 of 51 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 li tter toxicity are 25 mg/kg/day, and the LOAELs could not be established (>25 mg/kg/day). This study is suppl emental to the following three-generation rat study (MRID 00125293).

Three-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a three-generation re production 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 pe riod (102 days) with exposure continuing until completion of the second mating. Exposure in the P generation dams occurred during the initial pre-mating pe riod (102 days) and a 10-day rest period prior to the second mating, as well as through gestati on, lactation and w eaning of the F la and F 1b litters. After weaning, 15 male and 30 female F 1b pups per dose group were selected to comprise the F l generation parental animals, and were mated three times to produce the F 2a , F 2b , and F 2c 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 F 2 b pups were selected to comprise the F 2 generation parental animals, and were mated to produce the F 3a and F 3b litters.

Pup survival was recorded at birth (LD 0) a nd 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 phys ical observations were record ed on LD 21. Gross examination was done of two thirds of the pups (after litte r reduction) from each litter except the F 2c . Soft tissues, eyes, brain, reproductive organs, and the h eart were examined. Tissues and organs from
male and female F 3b pups (10/sex), and from parents dyi ng during the study we re preserved, but no histopathology examinations were conducted. All fetuses from the F 2c generation were removed by cesarean section on GD 19, weighed, a nd 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 fo r skeletal examination. Organ weights were not
evaluated.

There were no reported treatment-relate d effects on mortality in the P, F 1 , or F 2 parental animals. For the medium-high dose pups, increases (signifi cance not reported) in mo rtality were observed
in the F 2a litter at LDs 1 and 4, and in the F 3a litter at LD 4. For th e high-dose pups, increases (significance not reported) in mo rtality were observed in the F 2a and F 3a litters at LDs 1 and 4,
and in the F 2b litter at LD 7. Decreased parental b ody weights were observed in all groups, except the low-dose animals. Decreases (signi ficance 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 me dium-high and high-dose parental groups rangedCAPTAN FINAL

Page 44 of 51 from 8-19% and 12-37%, respectively. Decreases (significance not reported) in mean litter weights were observed in all tr eatment groups, with decreases ≤ 44% in the high-dose pups. Food consumption was decreased in all treat ment groups except for the low-dose F 1 males and the
low- and medium-low dose F 2 females.

Litter size was decreased (significance not reported) in the F 1b litter at the high dose. Fertility was decreased (significance not reported) in medium-high dose F l a males and in high-dose F la females. No treatment-related effects were obs erved 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 th e number of corpora lutea, numb er of total implantations, early or late resorptions, and in the number and loca tion of viable and non-viable fetuses at the
cesarean section on GD 19 of the F 2c animals. In addition, no effect s of developmental toxicity were present in the soft tissue and skeletal ex aminations. No treatment-related histopathological
lesions were observed in the te stes, epididymides, seminal vesicle, prostate, ovaries, uterus, cervix, thyroid, adrenal, a nd pituitary glands of F 3b 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 supplem ental 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 developm ental toxicity study ( MRID 48126603 ), pregnant CD(SD) rats (22/dose group) were administered capta n (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 partur ition). All dams were sacrifi ced on GD 20, and the number of corpora lutea, implantations, and resorptions were recorded. Fetuses were examined for external abnormalities, sexed, and weighed. Visceral exam ination 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 clin ical signs were limited to increased (p<0.05) hair loss and lack of grooming (NS) in some hi gh-dose dams. Maternal body weights in the highdose dams were decreased th roughout 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. Du ring GDs 7-9, food consumption was decreased (p<0.001) by 47%, with decreases (p<0.001) of17% during GDs 10-13 and 14-16. Recovery was evident posttreatment based on an increas e (p<0.001) in food consumption of 17% during GDs 17-20.CAPTAN FINAL

Page 45 of 51 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<0.0 1) 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 skel etal findings at the hi gh 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 presen t unilaterally (8 treated vs. 2 control) or bilaterally (5 treated vs. 0 control).

The developmental LOAEL is 450 mg/kg/day, ba sed on decreased fetal weight, increased number of runts, and skeletal variations, and the developmental NOAEL is 90 mg/kg/day.

Chronic Toxicity/Carcinoge nicity (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 concentr ations equivalent to doses of 0, 25, 100, and 250 mg/kg/day for 24 months. Ten ra ts/sex/group were ra ndomly allocated for 12- and 18-month interim sacrifices. Organs thatwere weighed at interim and final sacrifice included the liver, adrenals, kidneys, pituitary, th yroid/parathyroid, ovaries , and testes. Gross and microscopic pathological examinations were co nducted 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 surviv al 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 effect s were observed for clin ical 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 we re seen in absolute or rela tive weights of the testes, ovaries, and pituitary gland. Incr eases in organ weights in th e medium- and high-dose animals (generally relative-to-body, although occasional in creases in absolute weights) were noted at interim and terminal sacrifices, including adrena ls, kidneys, liver, and thyroids. Absolute weights were generally not affected, and th e increases in relative weights we re 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 hi gh-dose males at study termination. No treatmentrelated histopathological lesions we re seen in the testes, epididym ides, seminal vesicle, prostate,CAPTAN FINAL

Page 46 of 51 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 hype rtrophy, and increases in re lative organ weights.

Chronic Toxicity/Carcinoge nicity (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% purit y) in the diet at nominal concentrations of 0, 125, 500, or 2000 ppm; actual av erage daily concentrations were reported to be 100, 475, and 1950 ppm with standard convers ion 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 incl uded the liver, adrenals, kidneys, pituitary, thyroid/parathyroid, ovaries, and testes. For animals in the low- and medium-dose groups, gross and microscopic pathological ex aminations were conducted only on the liver, pituitary, thyroid,
and adrenals. Gross and microsc opic 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, af ter treatment for 24 months. Food consumption was reported to be slightly lowe r in the high-dose animals, but the extent and significance were not reported. No treatment-related changes were seen in ab solute or relative weights of the testes, ovaries, thyroid, adrenal and p ituitary 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 lesi ons were seen in the testes, epididymides, prostate, ovaries, mammary, thyroid, or pituita ry glands in any dose group. High-dose females exhibited an increased (p<0.05) incidence of ut erus sarcomas (8%) compared to control (0%).
The medium-dose males exhibited an increa sed (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 th e LOAEL was 24 mg/kg/day (475 ppm) based

on increased incidences of benign ad renal pheochromocytomas in males

.CAPTAN FINAL

Page 47 of 51 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) containi ng captan (90.4% purity) at doses of 0, 12.5, 60, or 300 mg/kg/day for 12 months. Orga ns that were weighed include d the liver, adrenals, kidneys, thyroids with parathyroids, ova ries, prostate, and testes. Gros s 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 su rvived, but increased
incidences of emesis and soft/mucoid stools we re observed in the high-dose animals. Non-dosedependent changes in feed consumption were obser ved in all treatment gro ups, 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 tr eatment-related gross or histopathological lesions were seen in the testes, epididymides , prostate, ovaries, uterus, vagina, or the mammary, thyroid, adre nal, 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 a nd 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 peri od was employed in this stu dy with 8 hours/day through Week 6 and 17 hours/day for the remainder of the st udy. 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, biw eekly through Week 8, and at study termination. Feed consumption in each replicate was measur ed weekly throughout the study. Adult birds were observed routinely for mortality and clinical signs. The followi ng reproductive parameters were observed and recorded: eggs lai d, eggs cracked, eggs set, viab le 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 in cluding lacerated scalps, head lesions, and signs of head and to e-picking were observed in the control and all treatment groups. No treatment-related effects on body weight were observed. Feed consumption was reported toCAPTAN FINAL

Page 48 of 51 be decreased in the 300 mg/kg (p<0.05) and 1000 mg/kg (p=0.01) trea tment 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 th e number of eggs laid, eggs cracked, viable embryos, live 3-week embryos, hatchlings norma l, 14-day old survivors, and hatchling body weight and eggshell thickness. Although a decreas e (p=0.01) in mean egg weight was noted in the 100 mg/kg treatment group, the effect was mini mal (9.6 g vs. 10.0 g control) and no effects on this parameter occurred in the medium and hi gh-treatment groups. Therefore, this effect was
not considered to be related to treatment. The reproductive toxi city 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 capta n (purity not reported ; presumed to be 89%) in the diet at concentra tions 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 to tal duration of the study was nineteen weeks (eleven weeks prior to egg laying and ei ght weeks of egg collec tion). 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 w eekly throughout the study. Adult birds were observed routinely for mortality and clinical signs. The followi ng reproductive parameters were observed and recorded: eggs lai d, eggs cracked, eggs set, viab le 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 wa s 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 betw een the control and high-concentration value was only 0.5%. Because there was no effect on e ggshell 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 survivorCAPTAN FINAL

Page 49 of 51 weight, normal hatchlings, and 14-day survivor s. 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/aquari a; 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 μ g/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 ex ceeded 0.0007 and 1.5%, respectively. After Week 3, nominal concentrations of captan were increas ed by 100% because no difference in survival or growth of the young fish was apparent, and afte r Week 17, nominal concentrations were again increased by 25% because of a decrease in a ll 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 μ g/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 ou tlet 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 F 1 fish reared in larval chambers for 30 days. The measured parameters fo r the parental fish in cluded growth on Day 51, and the sex gonadal condition, length, and weight at Week 45. The Agency noted that the ability to detect statistically signif icant 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 μ g/L treatment group ( ↓ 98%). The parental fish length and the F 1 fish survival and length were also decreas ed (p<0.05) in the mean-measured 39.5 μ g/L treatment group. At the medium and medium-h igh concentrations (16.8 and 39.5 μ g/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. Pe rcent hatchability of embryos from unexposed parents incubated at 63.5 μ g/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 μ g/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 gona ds was not discussed in detail; no difference in gonadal development was noted for fish in one of the medium-low (7.4 μ g/L) dose chambers where spawning was absent. The LOAEC is 39.5 μ g/L based on decreased growth and survival,
and the NOEAC is 16.8 μ g/L.CAPTAN FINAL

Page 50 of 51 APPENDIX 3: References Not Utilized in the Captan WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt 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 registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t 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 com pound of interest was not us ed 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 adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s 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) th e study contained only da ta on invertebrates.

Arndt, T. and Dohn, D. (2004) Measurement of the Half-Life of Thiophosgene in Human Blood. PTRL West, Inc., Hercules, CA; Captan Ta sk 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., Fa ng, R., Gallagher, R.P., and Le, N.D. (2011) Prostate cancer risk and exposure to pe sticides in British Columbia farmers. The Prostate

71:168-183.CAPTAN FINAL

Page 51 of 51 Gordon, E.B., Ehrlich, T., Mobely, S., and Willia ms, M. (2000) Measurement of the reaction between the fungicides captan or folpet and bl ood 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., Dake yama, 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 carci nogenic study with captan techni cal in albino rats. IndustrialBiotest Labs Inc. for Chevron Chem ical Company. IBT No. 89267. (MRID 00069243)