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: CARBARYL

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYCARBARYL 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...........................18

IV. Committee’s Assessment of Weight of Evidence..........................................21

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................21

1. Tier 1 in vivo Assays.............................................................22

2. OSRI...............................................................................22

B. Estrogen Pathway...........................................................................25

C. Androgen Pathway.........................................................................25

D. Thyroid Pathway...........................................................................26

E. Conclusions.................................................................................26

V. EDSP Tier 2 Testing Recommendations....................................................26

VI. References.......................................................................................27

LIST OF TABLES: Table 1. Tier 1 Assays for Carbaryl.................................................................7
Table 2. Estrogenic/Anti-Estr ogenic Pathway for Carbaryl....................................10
Table 3. Androgenic/Anti-Andr ogenic Pathway for Carbaryl..................................15
Table 4. Thyroid Pathway for Carbaryl..........................................................20

APPENDIX 1. EDSP Tier 1 Screening Assays.........................................................31
APPENDIX 2. Other Scientifically Relevant Information (OSRI).................................39
APPENDIX 3. References Not Utilized in the Carbaryl WoE Analysis...........................60CARBARYL 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 eneration F2 Second filial g enerationCARBARYL FINAL

iv Abbreviation Terminology

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 Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py 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 Intera g enc y Coordinatin g 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 Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration
MTD Maximum Tolerated DoseCARBARYL FINAL

v Abbreviation Terminology

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 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionateCARBARYL FINAL

vi Abbreviation Terminology

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-EvidenceCARBARYL FINAL

Page 1 of 62 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is de signed to provide the necessary empirical data to evalua te the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. Th is interaction includes agonism and antagonism at the estrogen and androgen receptors, altered st eroidogenesis, as well as hypothalamic-pituitarygonadal (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 consider ed in this weight of evidence (WoE) assessment.

In determining whether carbaryl interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of re sponses observed across studies, taxa, and sexes were considered. Additionally, th e conditions under which effect s occur were considered, in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On October 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the O ffice of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction of car baryl with the E, A or T signaling pathways. The T1Wo ERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) be ginning 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 carbaryl, there is no convincing evidence for an interaction with the estrogen pathway. While there was equivocal or conflicti ng responses in the guideline a nd non-guideline estrogen receptor (ER) binding and transcript ional activation assays, the in vitro findings were not supported by the
available in vivo studies. With the exception of the reprodu ctive studies in mallard ducks whereas decreases in female body weight gain were also ob served, no estrogen-related effects were noted in
the in vivo studies with carbaryl.

For the androgen pathway, carbaryl wa s classified as a binder in the in vitro Tier 1 androgen receptor (AR) binding assay and altered testosterone production in the Tier 1 steroidogenesis assay. However, carbaryl did not alter AR transcriptional activity in an AR transactivation assay (ARTA) considered as OSRI. The positive Tier 1 in vitro findings were generally not supported by the
available mammalian in vivo studies. Although perturbati ons of spermatozoa, as well as reproductive organ weights and histopathology were observed in the studies by Pant et al. (1995 and 1996), these effect occurred at doses that would be expected to inhibit cholinesterase activity, and may have cooccurred with overt toxicity. Similar results were not demonstrated in the in vivo mammalian Part 158 studies in the absence of systemic or overt to xicity. Decreases in embr yo viability and offspring weight effects were observed in the mallard duck studies where decreases in m ale body weight gain (not significantly, but al ong with significant female weight d ecreases) were observed. However,CARBARYL FINAL

Page 2 of 62 effects in the fish short-term reproduction assay (FSTRA, secondary sex characteristics and gonadal histopathology) are suggestive of a potential interaction with the androgen pathway. Together, results in the FSTRA and the positive in vitro assays provide redundancy for a potential interaction with the androgen pathway in male fish.

There is no convincing evidence for an interacti on of carbaryl with the thyroid pathway. Although
the results from Sun et al. (2008) submitted as OSRI suggest that carbaryl may alter thyroid receptor-related transcription in vitro , with the exception of an isolated effect in one metabolism study, the only thyroid effects observed in vivo occurred in the presen ce of overt toxicity.

There is no convincing evidence for interaction of carbaryl with the estrogen or thyroid pathways, in
mammals or wildlife. There is also no convinci ng evidence for interacti on of carbaryl with the androgen pathway in mammals. However the evidence suggest potential in teraction with the androgen pathway in fish.

Based on weight of evidence conclusions, mammalia n EDSP Tier 2 testing is not recommended for carbaryl since there is no evidence for potential inte raction with the estrogen, androgen or thyroid pathways.

Effects were observed in males in the FSTRA at 0.82 mg/L which s uggests potential interaction with the androgen pathway. A fish life-cycle study fo r carbaryl using a formulated product indicated effects on fecundity and survival in the parent al group at 0.68 mg/L w ith a NOAEC of 0.21 mg/L.
Effects on F1 survival were observe d at all concentrations tested ( ≥ 0.008 mg/L). Since responses were observed in the FSTRA in at similar concentrations as the current chronic endpoint. Therefore, the EDSP Tier 2 Medaka Extended One-Generation Reproduction Test (MEGORT) is
recommended.

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard duck are available for carbaryl. In mallard ducks, eff ects on reproductive parameters were observed; no effects observed in bobwhite quail. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are consider ed sufficient for evaluating po tential reproductive effects to birds from carbaryl exposure. Relative to the EDSP, additional testing is not recommended.CARBARYL FINAL

Page 3 of 62 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay battery is de signed to provide the necessary empirical data to evalua te the potential of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pathways. Th is interaction includes agonism and antagonism at the estrogen and androgen receptors, altered st eroidogenesis, as well as hypothalamic-pituitarygonadal (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 consider ed in this weight of evidence (WoE) assessment.

In determining whether carbaryl interacts with E, A or T hormone pathways, the number and type of effects induced, the magnitude and pattern of re sponses observed across studies, taxa, and sexes were considered. Additionally, th e conditions under which effect s occur were considered, in particular, whether or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On October 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the O ffice of Science Coordination and Policy (OSCP) conducted a weight-of-evidence (WOE) analysis of th e potential interaction of carbaryl with the E, A or T signaling pathways. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) be ginning 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).

Carbaryl has a water solubility of 40 mg/L and a vapor pressure of 1.36×10

− 6 mm Hg at 25°C, therefore, carbaryl will exist in both vapor and particulate phas es on release to the atmosphere. Vapor-phase carbaryl is degraded by reaction with photochemically -produced hydroxyl radicals in the atmosphere, with an estimated ha lf-life of 15 hours. If released to soil, carbaryl is expected to have moderate mobility, with little contribution due to volatilization from moist soil. Carbaryl’s
bioconcentration factor in fish is 9-34.

The available information considered in determini ng the potential interaction of carbaryl with the E, A, T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity st udies and/or other published articles . These data are summarized in Sections III.A through III.C. An analysis of th e data submitted to the agency, using the WoE approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 studies recommendations are pr esented in Section V.CARBARYL FINAL

Page 4 of 62 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 for carbaryl are shown belo w 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 Screening assays were submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration ( e.g. , Part 158 guideline studies). The Agency’s review of the initial OSRI is provided in the Report of the Endocrine Disruptor Review Team on carbaryl (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific lite rature for any additional
relevant information. Summaries of the availabl e OSRI are presented in Appendix 2. Additionally,
literature/studies considered but not utilized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in th e WoE determination on the potential of a substance
to interact with endoc rine-related processes ( i.e., E, A, or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and pres ented at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how the individual lines of
evidence are assembled and inte grated along two concepts ( i.e ., complementarity and redundancy) within the conceptual framework of an adverse outcome pathway) . Broadly, there are four main steps outlined in the guidance whic h provide the foundation for WoE eval uations. The first step is to
evaluate the individual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data al ong different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoints/responses across assays) in the observed responses acro ss these different levels of biological organization. The third st ep is to characterize the main lines of evidence as well any conclusions. Finally, the la st step is to evaluate whether addi tional testing is needed based on the
evidence and conclusions described above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays 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 receptorCARBARYL FINAL

Page 5 of 62 binding (estrogen and androgen agon ist and antagonist), steroidogene sis, and other effects on the HPG and HPT axes. OSRI may include published li terature studies as we ll as studies conducted under USEPA (often referred to as Part 158 data ) or OECD guidelines submitted in support of pesticide registrations. Each st udy is evaluated for scientific qu ality and relevance for informing interactions with the E, A or T pathway. Additionally, the concordance or consistency (complementarity) of the responses in the in dividual study is eval uated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study, as well as the relative degree of diagnostic utility of a specific endpoint for discerning wh ether or not the chemical has interacted with the endocrine system, are consid ered. The collective resp onse of the individual endpoints, as well as the condi tions under which they were e xpressed, are considered when evaluating an overall indication 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 support a hypothesis that a chemical has the potential to interact E, A or T pathways, ar e the concepts of comple mentarity and redundancy. These two concepts provide a basis for consider ing the plausibility, c oherence, 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 c onsisting of different levels of biological organization from a molecular initiating event such as receptor bindi ng through potential adverse effects in apical endpoint s such as sexual development and fecundity at the whole organism level. The extent of expression of responses at hi gher levels of biological or ganization can indirectly provide information on potential compensatory capabilities of an i ndividual organism.

After the data have been assembled and integrated, th e third step is to charact erize the main lines of evidence along with the conclusi ons; this characterization involv es three components. The first component is whether the data provide relevant , 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 bi ological organization? Finally, under what conditions did responses occur including considera tion of whether the responses were obs erved in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish effects spec ifically related to an endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishing whether the responses were endocri ne-related was discussed at the 2013 FIFRA SAP meeting that evaluate d scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In Oc tober, 2013, the SAP stated that , “ In summary, the Panel agreed that little, if any, weight should be placed on signs of endocrine disruption in the presence ofCARBARYL FINAL

Page 6 of 62 overt toxicity. All effects in e ndocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. second ary 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 Pa rt 158 studies for both human and ecological risk assessments. Specifically, in these analyses, the eff ects that EPA considered to be potential evidence of overt toxicity included, but were not limited to: mortality; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g., organ weights/histopathology), biochemical ( e.g., blood chemistry), and other clinical signs ( e.g., lethargy)
were also considered when evaluatin g overt toxicity, especially if the effects were extreme. In some instances, one parameter ( e.g., death or >10% decrease in mamma lian body weight) was sufficient to
consider a dose/concentration to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxic ity. For example, in the FSTRA, generally, body weight decreases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these effects were generally cons idered as less severe forms of toxicity ( e.g., changes in organ weights or blood chemistry). The circum stances for which a dose/concentration was considered overtly toxic for a particul ar 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 ch emical’s physical/chemical properties and its known
modes of action in its overall charac terization of a chemical’s potential to interact with the E, A or T pathway. Adequately addressing th e three components described a bove is fundamental to the WoE process and in determining whether additional data are needed. In additi on to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) desc ription of inconsistent or conf licting data; 4) overall strength of evidence supporting a conclusion ; and, 5) what, if any, additional data are needed and why. Assessing the need for additional data is based on a case-by-case analysis which took all available toxicity da ta 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 biol ogically relevant with regard to a treatment-related effect which supports a conclusion of whether a substance has the potential to interact with the E, A or T pathway. Effects that occurred in th e presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but are not re ported in the table for E, A or T.CARBARYL FINAL

Page 7 of 62 A. EDSP Tier 1 Screening Assays

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

.

Table 1. Tier 1 Screening Assays for Carbaryl

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48615203)

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied (MRID No. 48615204)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No.48615201) and by OSRI (Fang et al ., 2003)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No.48615205)

Aromatase Assay (human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No.48615202)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No.47987702)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No.47987701)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement satisfied by OSRI (MRID 45448101)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied by OSRI (MRID No.45448101, 42918801, 44402501, 48161803, 48067701) Pang et al., 1995; 1996

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No.48669601)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied by OSRI (MRID No. 45448101, 42918801, 44402501, 48161801)CARBARYL FINAL

Page 8 of 62 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for carbaryl to interact with the estrogen 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.

The mean specific binding of carba ryl to the ER in the Tier 1 in vitro ER binding assay was between 50 and 75% at concentrations up to 10 –3 M in two of the three indepe ndent runs, and >75% in the remaining run. This resulted in an equivocal classification for carbaryl in this assay. In literature studies considered as OSRI, Blair et al ., (2000) reported a carbaryl ER IC 50 of >10 -4 M in a nonguideline rat uterine cytoso l binding assay while Klotz et al ., (1997) reported carbaryl as positive for ER binding in a MCF-7 whole cell binding assay with an IC 50 of 6 x 10 -6 M. In the in vitro Tier 1 ERTA assay, carbaryl was negative for ER agonist activity. This finding was corroborated by the negative results reported by Kojima et al. (2004) for both hER agonist an d antagonist activities in a reporter gene assay in CHO cells up to 10 -4 M. However, Klotz et al ., (1997) reported that carbaryl acted as a weak agonist and antagon ists in transiently transfected ER reporter gene assays in breast
cancer and endometrial cancer cell lines. Lemaire et al ., (2006), reported ne gative findings for ER α and ER β agonism using a reporter gene in stably tr ansfected HeLa cells; how ever carbaryl treated cells showed antagonistic activities toward both ER α and ER β in the presence of estradiol in these cells. The authors also repor ted that carbaryl inhibited [ 3 H]-estradiol binding by 45 % in these cell
lines at 10 -5 M.

In the Tier 1 aromatase assay, carbaryl exhibited a concentration-dependent i nhibition of aromatase activity, decreasing activity by mo re than 50% with a Log IC 50 of

− 3.83 M but the average slope of the inhibition curves was steeper than

− 2 (

− 2.92); therefore, carbaryl is classified as an equivocal inhibitor of aromatase. Finally, carbaryl was classified as negativ e in the steroidogenesis assay for estradiol production.

Carbaryl was classified as negative for es trogenic activity in th e uterotrophic assay. The requirement for the female pubertal assay was considered satisfied by OSRI, including a two-generation reproduction study (MRID 45448101) conducted accordi ng to the revised 1998 test guidelines, which is also the EDSP Tier 2 study for mammals. The results of this study demonstrated no treatment-related changes in any of the estrogen-related endpoints. In the FSTRA, while it is noted that female fish (including controls) had findings of oocyte atresia (13-36%) sometimes associated with inflammation and presence of protozoal spores , fecundity was adequate in the assay, and there

were no treatment-related effects observed in females for any measured endpoint up to the highest concentration tested (0.82 mg/L).CARBARYL FINAL

Page 9 of 62 In the two-generation reproduction study in rats, there was a non-significant delay (1.4 days) in vaginal opening (VO) observed in the F 1 females at the high dose (a pproximately 135 mg/kg/day) which co-occurred with decreased body weight s (11%) in the pups on PND 4-21. There were no other effects on estrogen-related end points, including estrous cyclicity. Although ovarian weight was decreased by 33% in the high-dose females in the carcinogenicity study in mice, there were no accompanying histopathological changes. No estrogen- related effects were seen the other Part 158
mammalian studies.

In the Part 158 avian toxicity st udy in mallard ducks, the number of eggs laid was decreased in the high and mid test concentration (924 and 2932 mg/ kg-diet); hatchling weight was significantly reduced at the highest dose. These effects were obs erved in the presence of reduced adult female body weight gain [(53 (NS), 73 and 85% at the low, mid and high test concentration levels)]. There were however, no other signs of t oxicity. During a recovery peri od, viable embryos and number and survival of hatchling (based on eggs set) were reduced at the top dose, and hatchling weight was significantly reduced at the mid and high dose. While another mallard reproduction study is available, only a preliminary review of the study was conducted at this time. However, the reported affected endpoints are similar to the other mallard study in that the number of eggs laid were decreased and female body weight gain was also decreased 62.5% (at 1000 mg/kg-diet); eggshell thickness (5.5%) and hatchling weights were also reduced as well as increase in number of regressing ovaries at this dos e (MRID 49312801). No treatment-re lated effects were noted on reproduction in the avian toxicity study in bobwhite quail up to 3000 mg/kg-diet.CARBARYLFINALPage 10 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for CarbarylLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Carbaryl1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity OBserved 3EDSP Tier 1 AssayER Binding(MRID 48615203)EERTA(MRID 48615204)NAromatase(MRID 48615202)ESteroidogenesis(MRID 48615205)NUterotrophic(MRID 47987702)NNX(H)Female Pubertal RatRequirement satisfied by OSRI (Two-generation reproduction study, MRID 45448101; see below)FSTRA(MRID 48669601)NEN NN N NNOSRIER Transactivation(Kojimaet al., 2004)NER Binding(Blairet al., 2000)NER Binding(Klotzet al., 1997)P PERTA (Lemaireet al.,2006)P PCARBARYLFINALPage 11 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for CarbarylLines of Evidence Indicating Potential Interaction withthe Estrogenic/Anti-Estrogenic Pathway for Carbaryl1Study Type/ LiteratureCitation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity OBserved 3Two-generationreproduction (Rat;MRID 45448101)N N N N N NNX(H)X(H)Developmentalneurotoxicity (Rat;MRID 44393701)NNNX(H)Developmentaltoxicity (Rat; MRID44732901)NNX(H)NDevelopmentaltoxicity (Rabbit;MRID 44904202)NNX(H)X(H)Chronic toxicity/carcinogenicity (Rat;MRID 42918801)NE N N NEX(H)X(M, H)Carcinogenicity(Mouse; MRID42786901)NE N N NEX;(H)X(M,H)Chronic toxicity (Dog;MRIDs 40166701 and42022801)NE N N NX(H)X(L, M,H)Avian reproduction(Duck; MRID00161500)P4NN5(M, H)Avian reproduction(Quail; MRID00160044)NN NCARBARYLFINALPage 12 of 621. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment.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 inweight are absolute unless otherwise indicated.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 Xin this 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. Decrease in the number of eggs laid at mid and high treatment (924 and 2932 mg/kg-diet); hatchling weight was significantlyreduced at the highest dose.During a recovery period, viable embryos and number and survival of hatchling (based on eggs set) were reduced at the top dose,and hatchling weight wassignificantly reduced at the mid and high dose.5. Reduced adult female body weight gain (53 (NS), 73 and 85% at the low, mid and high test concentration levels). Reductions,but not significant, in malebody weight gain were also observed. There were no other signs of toxicity.E EquivocalP Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedCARBARYL FINAL

Page 13 of 62 2. Effects on Androgen Pathway

The potential for carbaryl 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 androgeni c, 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.

In an evaluation of androgeni c/anti-androgenic activity from in vitro Tier 1 assays, carbaryl was classified as positive in the in vitro androgen receptor (AR) binding (rat prostate cytosol) assay, as mean specific binding was reduced to 28-34% and 60-85% at concentrations of 10 -3 and 10 -4 M, respectively; no effect on AR binding was noted at ≤ 10

− 5 M. In the steroidogenesis assay, decreases of 52-59% (0.48- to 0.41-fold) were no ted in testosterone pr oduction at the highest carbaryl concentration (10

− 4 M) in each assay run. In two published studies considered as OSRI, carbaryl was negative for competitive binding in a recombinant rat AR binding assay (Fang et al ., 2003) and did not show AR agonis t or antagonist activity in an AR transcrip tional activation assay in CHO-K1 cells transfected with hAR (Kojima et al ., 2004).

Carbaryl was negative for anti-a ndrogenic activit y in the Hershberger assay (the agonist portion was not conducted). The requirement for the male pubertal assay was considered satisfied by OSRI, including a two-generation reproduction study conducted according to the revised 1998 guideline, which is also the EDSP Tier 2 study for mammals. In the F 1 males the absolute weights of the pituitary and epididymides were decreased in the high-dose group ( ↓ 9 and 5%, respectively), and PPS was delayed by 2.1 days, but these effects occurred in the presence of overt toxicity. In addition, there were treatment-related effects obs erved in high-dose level (0.82 mg/L) male fish in the FSTRA. The male nuptial tubercle score was significantly decreased in the high-dose group (median tubercle score of 33 in control vs. 30 in 0.82 mg/L), alterations in gonadal histopathology (increases in testicul ar degeneration and the percentage of spermatogonia) were observed and a slight increase ( ↑ 9%; p=0.04) in male body length was also
noted.

In other mammalian Part 158 studies, non-significant decreases in male pituitary weights in were noted in the chronic toxicity study in dog, but there were no accompanying histopathological changes. In addition, the decrease d pituitary weights at the mid- and highdoses were associated with overt toxicity (i.e., significant decreas es in AChE activities). No androgen-related effects were seen in the other Part 158 mammalian studies.CARBARYL FINAL

Page 14 of 62 In the Part 158 mallard duck reproduction study, embryos viability and number and survival of hatchling were reduced at the t op dose, and hatchling weight was significantly reduced at the mid and high dose. As with the females, reductions (NS) in adult male body weight gain were observed at the low (15%), mid (44%) and high ( 72%) test concentration levels in one of the mallard duck studies (no effect in second st udy which reported decreases in offspring body weight). Decreases in offspring body weight we re also observed in the additional mallard duck study (preliminary review). No androgen-related effects were noted in the bobwhite quail study at the highest concentration tested (3000 mg/kg-diet).

Finally, two published studies (Pant et al. , 1995 and Pant et al. , 1996) were conducted that examined the effects of carbaryl on the male reproductive system in rats. In the first study, carbaryl was administered to adult Wistar albino rats at 50 or 100 mg/kg/day (5 days/week) for 90 days, and in the second study, juvenile a nd adult Drucker albino rats received 25, 50, or 100 mg/kg/day (5 days/week) for 60 days. The resu lts of the first study in dicated decreases in sperm count and motility at both doses, as well as histopathological effects, with no effect on reproductive organ weights. Body weight gain also was decreased at the high dose by Day 60.
Similar results were observed in the second study, although age-relate d effects also were apparent. Sperm count and motility were decr eased in the 50 and 100 mg/kg/day dose groups, with greater decreases ( ↓ 50-100%) in the juven ile rats compared to adul ts. Increases in abnormal sperm also occurred, with a greater percentage present in young rats. Finally, reproductive organ weights were decreased in the hi gh-dose juvenile rats only, an d body weight gain was decreased in the 50 and 100 mg/kg/day dose groups, with a greater effect in th e juvenile rats.CARBARYLFINALPage 15 of 62Table 3: Androgenic/Anti-Androgenic Pathway for CarbarylLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Carbaryl1Study 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48615201)PSteroidogenesis(MRID 48615205)PHershberger(MRID 47987701)NENNX4(H)Male Pubertal RatRequirement satisfied by OSRI (MRIDs +, 42918801, and 44402501; Pantet al., 1995 and Pantet al., 1996; see below)FSTRA(MRID 48669601)NENP(H)5P (H)6NN N NOSRIARTA (Kojimaetal., 2004)NAR Binding (Fanget al., 2003)PTwo-generationreproduction (Rat;MRID 45448101)NENNNNNN NX(H)X(H)Developmentalneurotoxicity (Rat;MRID 44393701)NNX(H)Chronic toxicity/carcinogenicity(Rat; MRID42918801)NNNNNENEX(H)X(M,H)Carcinogenicity(Mouse; MRID42786901)NNNNNENEX(H)X(M, H)CARBARYLFINALPage 16 of 62Table 3: Androgenic/Anti-Androgenic Pathway for CarbarylLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Carbaryl1Study 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 (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity(Dog; MRIDs40166701 and42022801)NNNNN NEX(H)X(M, H)Subchronictoxicity/Metabolism (Rat;MRID 44402501)NENENENENENEX(H)X(M, H)Avian reproduction(Duck; MRID00161500)P7NN8(M,H)Avian reproduction(Quail; MRID00160044)NN NMale ReproductiveToxicity-Rat(Pantet al., 1995)NP(L,H)9NP↓(L,H)10NX(L, H)N11Spermatoxicity-Rat(Pantet al., 1996)9↓(H) NE↓(H)P↓(L,H)12↓SV, VP(H)13X↓(M,H)N1. Key to responses: ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High. Arrows (↓or↑) indicate the direction of theresponse. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are absolute unless otherwiseindicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PR), Levator anibulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP).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 thiscolumn. 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 details seeSection IV. A4. Based on clinical signs of neurotoxicity and a 12% decrease in body weight at the high dose (50 mg/kg/day).5. Increase in minimal testicular degeneration and a minimal increase in the percentage of spermatogonia at the highest concentration (0.82 mg/L).CARBARYLFINALPage 17 of 626. Male nuptial tubercle score was significantly decreased by inthe high-concentration group (0.82 mg/L); (median value of 30vs. 33 control).7. In the exposure period hatchling weight was significantly reduced at the highest dose. During a recovery period, viable embryos and number and survival ofhatchling (based on eggs set) were reduced at the top dose, and hatchling weight was significantly reduced at the mid and highdose.8 Reductions (NS) in adult male body weight gain were observedat the low (15%), mid (44%) and high (72%) test concentration levels. Decreases in female bodyweights also observed. There were no other signs of toxicity9. Dose-dependent increases in congestion and edema; decreases in spermatogenesis; and atrophy of seminiferous tubules.10. Decreases were observed intotal epididymal sperm count (↓40 and 60%) and percent sperm motility (↓25 and 63%) in the low- and high-dose groups (50 and 100mg/kg/day), respectively.11. Degree/magnitude/significance of body weight decreases seen at the two doses (50 and 100 mg/kg/day) tested could not be determined since the data waspresented graphically.12. At the mid and high doses (50 and 100 mg/kg/day), decreases were observed in total epididymal sperm count (young:↓25 and 38%; adult:↓13 and 25%) andpercent sperm motility (young:↓24 and 43%; adult:↓15 and 26%), respectively.13. Study tested both immature and mature male rats; decreasesin androgen sensitive organs were observed only in immature ratsat the high dose (100 mg/kg/day)P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedCARBARYL FINAL

Page 18 of 62 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 carbaryl to interact with t hyroid regulation is summa rized 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.

The requirements for the male pubertal and fe male pubertal assays, and the AMA, were considered to be satisfied by submitted OSRI, including the chronic toxicity/carcinogenicity study in rats (MRID 42918801), the two-ge neration reproduction study (MRID 45448101), a subchronic metabolism study (MRID 44402501) and a th yroid receptor tran sactivation assay by
Sun et al. , (2008).

In the chronic toxicity/carcinogenicity study in rats, an increased in cidence of thyroid follicular cell hypertrophy was observed in the high-dose animals at both the interim and terminal sacrifices. The incidences of thyroid follicular cell adenomas (8 vs. 0 control) and combined adenomas /carcinomas (9 vs. 0 control) were increased in high-dose male rats. The thyroid follicular cell hypertrophy was observed at an overtly toxic dose that manifested as >10% decrease in body weight and decreases in cholinesterase activity (plasma >17%; RBC > ↓ 19%; and brain >10%).

In a metabolism study conducted in male rats after subchronic exposure to carbaryl, absolute and relative thyroid weights were increased in a non-dose-dependent manner in the mid- and highdose groups. In addition, the incidences of follic ular cell hypertrophy were increased in all dose groups compared to the control in this study. However, the mid- and high doses were associated with overt toxicity characterized as increases in absolute ( ↑ 18% liver and ↑ 30% spleen) and

r elative ( ↑ 39% liver and ↑ 54% spleen) weights.

In the chronic toxicity study, rela tive thyroid weight were decreased (27%) at the high-dose male without microscopic changes (terminal body we ight was increased by 15%). In addition, decreased pituitary weights we re observed in parental F 1 high-dose male rats ( ↓ 9%) in the twogeneration reproduction study and all treated males ( ↓ 8, 15, and 22%) in the chronic toxicity study in dogs. There were no thyroid-related effects in the mouse carcinogenicity study.CARBARYL FINAL

Page 19 of 62 Sun et al. (2008) studied carbaryl in a thyroid transcriptional activ ation assay in HepG2 cell. The results indicated that carbaryl had no agonist activit y, but inhibited T 3 activity with an IC 50 of
8.40×10

− 5 M. A modified Frog Embryo Developmenta l Assay (FETAX) in the African clawed frog ( Xenopus laevis ; Bacchetta et al., 2008) demonstrated increases in malformations, as well as stimulation of development and metamorphosis after carbaryl treatment. Mortality was 15 and 76% in the two highest test concentrations (16 and 24 mg/L), respectively. However, X. laevis embryos/larvae in this study (NF stage 8-47) were not exposed during the thyroid- critical period
of metamorphosis and development (in the AMA, exposure starts at NF Stage 51 through at least Stage 57). Therefore, this study does not directly contribute to the evaluation of effects on the T signaling pathway.CARBARYL FINAL

Page 20 of 62 Table 4. Thyroid Pathway for Carbaryl.

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

Study Type/ Literature CitationTR ActivationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmentalstage (± orasynchronous,Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assay

Male Pubertal Rat Requirement satisfied by OSRI (MRIDs 45448101, 42918801, and 44402501; Sun et al. , 2008; see below) Female Pubertal
Rat Requirement satisfied by OSRI (MRIDs 45448101, 42918801, and 44402501; Sun et al. , 2008; see below)

AMA Requirement satisfied by OSRI (MRIDs 45448101, 42918801, and 44402501; Sun et al. , 2008; see below)

OSRI Thyroid Receptor Transactivation (Sun et al ., 2008) P 4

Two-Generation Reproduction (Rat; MRID 45448101) N N N X (H) X (H)

Chronic toxicity/ carcinogenicity (Rat; MRID 42918801) NE N NE X (M, H) X (M, H)

Carcinogenicity (Mouse; MRID 42786901) NE N NE X (H) X (M, H)
Chronic toxicity (Dog; MRIDs 40166701 and 42022801) N N N X (L, M, H) X (L, M,

H)

Subchronic Toxicity/ Metabolism (Rat; MRID 44402501) N P NE X (M, H) X (M, H)

1. Key to responses: ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shad ed cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. 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. C arbaryl had no agonist ac tivity, but inhibited T 3 activity with an IC 50 of 8.40×10

− 5 M. P Positive finding N Negative findings NE Not examinedCARBARYL FINAL

Page 21 of 62 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 carbaryl to interact with endocrine related processes ( i.e. , E, A or T hormonal 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 SAP that evaluated scientific issues associated with weight of evidence evalua tion of the results of the Ti er 1 assays 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 e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine 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 assays and
OSRI studies are described belo w. 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).CARBARYL FINAL

Page 22 of 62 1. Tier 1 in vivo Assays

In the Hershberger assay, (50 mg/kg/day + TP), treatment-related clini cal signs consisting of increased salivation, reduced moto r activity, tremors, and piloerec tion were found in all animals throughout the study. Chewing was observed in 4/ 6 animals between Days 8 and 10 and soiled nose was observed in 1/6 animals between Days 3 and 6. Fewer treatment-related clinical signs were noted in the mid-dose group, consisting of reduced motor activity and/or tremors in 5/6 animals, with increased saliva tion and piloerection in one anim al. In addition, there was a 12% decrease in final body weight and a 49% decreas e in overall body weight gain in the high-dose

group.

In the uterotrophic assay, tremors were noted in 7/10 animals, and chewing in 9/10 animals, at the high dose (30 mg/kg/day). Piloerection was al so noted in 4/10 animals on Day 1 or Day 2 (post-dosing), and one animal presented many c linical signs during the dosing period including reduced motor activity, prostration, wasted appear ance, genital discharge, and soiled anogenital region. Clinical signs were reduced in th e mid-dose group, with piloerection noted in 2/10 animals on Day 1 and chewing observed in 3/10 animals on Days 2 and 3, and minimal in the low-dose group. Final body weight was decreas ed by 10% in the high-dose group, with a decrease of 39% in the overall body weight gain.

2. OSRI

In the two-generation rat reproductio n toxicity study, no treatment-rela ted deaths or clinical signs were observed at dietary concentrations up to 1500 ppm (92.4-135.5 mg/kg/day). Body weight at the end of the premating periods were decr eased by 5-6% and 4-5% , in the high-dose F 0 males and females, respectively, and by 10 and 8% in the high-dose F 1 males and females. Overall body weight gains (Days 0-70) were decreased by 7-9% in the F 0 generation and by ≤ 8% in the

F 1 generation. Mean body weights also were decreased in high-dose F 0 and F 1 females during gestation and lactati on, with decreases of ≥ 10% in the F 1 females ( ↓ 7-14%) and ≤ 10% in the

F 0 females. No treatment-related effects were obse rved on body weights or weight gain of adults
at the low and mid doses. The most notab le effects of carbaryl were on the F 1 and F 2 pups. Pup survival was slightly decreased (NS) at the mid and high dose for both generations, with a 18-
19% decreased in mean litter size on PND 4. In addition, pup weight/litter in the high-dose pups was decreased for both generations during PNDs 4-21 ( ↓ 11-15% for F 1 and ↓ 13-23% for F 2 pups).

In a rat developmental neurotoxicity study, ther e were no carbaryl-associated deaths and no treatment-related clinical signs of toxicity in the F 0 generation dams or F 1 offspring. Body weight gain was decreased by 92% in females in the high-dose group (10 mg/kg/day) during GDs 6-9, but body weight gains were similar to contro l during all other peri ods. During the FOB measurements, the incidence of females in the high-dose group w ith decreased pupil size (pinpoint pupils) was increased on all occas ions during the dosing period. An increasedCARBARYL FINAL

Page 23 of 62 incidence of dams with slight tremors affecting the head, bod y and/or limbs was noted on the majority of assessment occasions in the high -dose animals. There were also occasional occurrences of ataxic gait/ove rall gait incapacity (dose-depend ent increased incidences or occurrences in the high-dose animals only). In the high-dose group, RBC and whole blood AChE activities were decreased in RBC ( ↓ 28%), whole blood (34 and 32%, respectively), and plasma (39 and 36%, respectively) on GD 20 and LD 10. In addition, brain AChE activity was decreased by 42% in the high-dose anim als at study termination.

In the prenatal developmental toxicity studies in rats, at least one occurrence of salivation occurred in 18/25 dams (vs 0/25 control) in th e high-dose group (30 mg/k g/day). This clinical sign appeared post-dosing (within 20 minutes), resolved in approximately one hour, and was
observed from GDs l3 to 20. There were no deaths and no other treatment-related clinical signs. Body weight decreases in the high-dose dams we re <10%, and overall body weight gain in the high-dose group was only decreased 27%. AChE activity was not dete rmined in the rat developmental study.

In the prenatal developm ental toxicity study in rabbits, mort alities that occurred on study were
not considered to be related to treatment, and no clinical toxici ty was reported at doses up to 150 mg/kg/day. Body weights were not affected by treatment, but ove rall body weight gain was decreased in the mid-dose ( ↓ 14%) and the high-dose ( ↓ 38%) does. AChE activities were decreased in plasma ( ↓ 46 and 68%, respectively) and RBC ( ↓ 19 and 27%, respectively) in the mid- and high-dose groups, with minor decreases noted in both AChE activities in the low-dose
animals.

In the rat chronic toxicity/carci nogenicity study, clinical observations incl uded thin appearance, alopecia and urine staining in high-dose male s and/or females (349.5/ 484.6 mg/kg/day [M/F]). Decreased body weights and body weight gains we re observed in high-dose males and females over the two-year study period. Body weight decreases in males and females were decreased by 23 and 24%, respectively, and by Week 13 and we re further decreased by 35 and 45% by Week 105. Females in the mid-dose group (78.6 mg/kg/day) also had body weight decreases of 12% by study termination. Body weights in the recove ry animals demonstrat ed continued, although attenuated, decreases in males and females between Weeks 53 ( ↓ 32 and 36%, respectively) and 57 ( ↓ 23 and 27%, respectively). Overall body weight ga ins also were decreased in the high-dose animals ( ↓ 53 and 69% in males and females, respec tively), with an overall body weight gain
decrease of 18% observed in the mid-dose females. Determination of AChE activities at 6-month
intervals indicated decrea ses in plasma AChE ( ↓ 17-57%), RBC AChE ( ↓ 19-38%), and brain AChE ( ↓ 10-31%), with a return to c ontrol levels dem onstrated in recovery animals by Week 56.CARBARYL FINAL

Page 24 of 62 In the mouse carcinogenicity study, mortalities in high-dose males that occurred on study were
not considered to be related to treatment. Systemic toxic ity in the high-dose (1249/1441 mg/kg/day [M/F]) animals was noted as thin a ppearance and hunched posture; these signs were present early in the study, disappeared by Week 6, and re-appeared during th e last six months of the study. Body weights were decreased in the high-dose animals (up to 13 and 14% in males and females, respectively), with similar decreases persisting at study termination ( ↓ 12 and 13%, respectively, in males and females). Body weight gain decreases for the cumulative measurement periods in the high-dose males and females ranged from 23-35% in males and 10-32% in females, with overall decreases of 38 and 32%, respectively. Decreases of ≤ 10% were observed in the high-dose females throughout the study.

In male mice, RBC AChE activities were decreased in the mid- and high-dose groups by 23 and 30% at Week 53, with only a 13% decrease at the high dose at Week 105. Additionally, in males,
brain AChE activities were decr eased in the mid- and high-dose groups by 18 and 57% at Week 53, and by 13 and 40% at Week 105. In females, RBC AChE activities were decreased in the mid- and high-dose groups by 13 and 13% at Week 53 and by 20 and 24% at Week 105. Additionally, in females, brain AC hE activities were decreased in the mid- and high-dose groups by 13 and 47% at Week 53, and by 11 and 40% at Week 105.

In the chronic toxicity study in dogs, the occurrence of clinical signs ( e.g. , emesis, lacrimation, salivation, and tremors) was increased in the high-dose females (31.25 mg/kg/day) during the 52- week study. Body weight gain wa s initially decrease d by 50% in the high-dose females during Weeks 0-5, with lesser d ecreases in body weight gain during Weeks 6-9 ( ↓ 4%) and Weeks 28-36 ( ↓ 6%). Terminal body weights in the low-, mid- , and high-dose males were increased by 8, 9, and 15%, respectively.

Decreases in AChE activity were observed at al l doses in the chronic dog study. In the low-dose females, decreased AChE activity was seen in plasma ( ↓ 19-23%) and brain ( ↓ 20%). In the middose males, decreased AChE activity was seen in plasma ( ↓ 30-36%), RBC ( ↓ 23-28%) and brain ( ↓ 32%), with decreased AChE activity in the plasma ( ↓ 26-31%), RBC ( ↓ 29-34%) and brain ( ↓ 22%) of mid-dose females. Finally, in the high- dose males, decreased AChE activity was seen in plasma ( ↓ 57-66%), RBC ( ↓ 46-56%) and brain ( ↓ 25%), with decreased AChE activity in the plasma ( ↓ 47-60%), RBC ( ↓ 29-38%) and brain ( ↓ 36%) of mid-dose females.

In the subchronic toxicity/metabolism study on male rats, overt toxicity was noted at the mid and high doses (1500 and 7500 ppm) based on the over all 12 and 16% decreases body weights. Body weight gains were also decrea sed by 39% at the m id dose, and there were actual losses in body weight at the high dose. In a ddition, liver and spleen weight s were increased by 18 and 30%, respectively, at the high dose.CARBARYL FINAL

Page 25 of 62 Finally, in the two published subchronic studies by Pant et al . (1995 and 1996), which examined potential reproductive effects in male rats, no overt toxicity was noted at doses up to 100 mg/kg/day. However, clinical sign s of toxicity (lethargy) were noted at the high dose, along with dose-dependent decreases in body weight and body weight gains at 50 and 100 mg/kg/day.

In an avian reproduction toxicity study, malla rd ducks treated with carbaryl up to 2932 mg a.i./kg-diet showed no symptoms of toxicity or behavioral abnormalities for the duration of the study. There were two mortalities on study in th e high-dose group (one/sex), but neither death
was considered to be related to treatment. Body weight gains were decreased in a dosedependent manner in all treatment groups, with decreases (NS) in males of 15, 44, and 72%, and decreases in females of 53 (NS), 73, and 85% , respectively. Body weight gain also was decreased at 1023 mg a.i./kg-diet (high-dose females) in a sec ond reproduction toxicity study in ducks, with no effects on mortalit y or clinical signs. In a repr oduction toxicity study in quail, bobwhite quail displayed no treatment-related e ffects on survival or growth parameters.

B. Estrogen Pathway

There is no convincing evidence for an interact ion with the estrogen pathway. While there was equivocal or conflicting responses in th e guideline and non-guideline ER binding and transcriptional activation assays, the in vitro findings were not supported by the available in vivo studies. With the exception of the reproductive st udies in mallard ducks whereas decreases in female body weight gain were also observed, no estrogen-related effect s were noted in the in

vivo studies with carbaryl, no estrogen -related effects were noted in the in vivo studies with carbaryl.

C. Androgen Pathway

Carbaryl was classified as a binder in the Ti er 1 AR binding assay and altered testosterone production in the Tier 1 ster oidogenesis assay. However, carbaryl did not alter AR transcriptional activity in an ARTA study considered as OSRI. The positive Tier 1 in vitro findings were generally not suppor ted by the available mammalian in vivo studies. Although perturbations of spermatozoa, as well as re productive organ weights and histopathology were observed in the studies by Pant et al. (1995 and 1996), these effect occurred at doses that would be expected to inhibit cholinesterase activity, and may have co-occurred with overt toxicity.
Similar results were not demonstrated in the in vivo mammalian Part 158 studies in the absence of systemic or overt toxicity. Decreases in embryo viability and offspring weight effects were
observed in the mallard duck studies where decreases in male body weight gain (not significantly, but along with signi ficant female weight decreas es) were observed. However, effects in the FSTRA (secondary sex characteri stics and gonadal histopa thology) are suggestive of a potential interaction with the androgen pathway. Cholineste rase activity was not measured in the FSTRA. Together, results in the FSTRA and the positive in vitro assays provide redundancy for a potential inte raction with the androgen pathway in male fish.CARBARYL FINAL

Page 26 of 62 D. Thyroid Pathway

There is no convincing evidence for an interact ion of carbaryl with the thyroid pathway. Although the results from Sun et al. (2008) suggest that carbaryl may alter in vitro thyroid receptor-related transcription in vitro , with the exception of an isolated effect in one metabolism study, the only thyroid effects observed in vivo occurred in the presen ce of overt toxicity.

E. Conclusions

There is no convincing evidence for interacti on of carbaryl with th e estrogen or thyroid pathways, in mammals or wildlife. There is also no convincing evidence for interaction of carbaryl with the androgen pathway in mamm als. However the evidence suggest potential interaction with the androgen pathway in fish.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence conclusions, mamma lian EDSP Tier 2 testing is not recommended for carbaryl since there is no evidence for poten tial interaction with th e estrogen, androgen or thyroid pathways.

Effects were observed in males in the FSTRA at 0.82 mg/L whic h suggests potential interaction with the androgen pathway. A fish life-cycle study for carbaryl using a formulated product indicated effects on fecundity and survival in the parental gro up at 0.68 mg/L with a NOAEC of 0.21 mg/L. Effects on F1 survival were obs erved at all concen trations tested ( ≥ 0.008 mg/L). Since the responses were observed in the FSTRA at sim ilar concentrations as the current chronic endpoint, the EDSP Tier 2 Medaka Extende d One-Generation Reproduction Test is
recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are available for carbaryl. In mallard ducks, effects on reproductive parameters were observed; no effects observed in bobwhite quail. The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are consid ered sufficient for evaluating potential reproductive effects to birds from carbaryl exposur e. Relative to the EDSP, additional testing is
not recommended.CARBARYL FINAL

Page 27 of 62 VI. References

Bacchetta, R., Mantecca, P., Andrioletti, M., Vism ara, C., and Vailati, G. (2008) Axial-skeletal defects caused by Carbaryl in Xenopus laevis embryos. Sci Total Environ . 392(1):110-8.

Blanck, O. (2003) Uterotrophic Assay to Assess th e Potential Estrogenic Ac tivity of Carbaryl in the Rat. Unpublished study conducted by Baye r CropScience, Sophia Antipolis, France. SA 02282, January 16, 2003. (MRID 47987702)

Blanck, O. (2011) Carbaryl: Evaluation in th e in vitro (HeLa-9903) estrogen receptor transcriptional activation assay. Unpublished study conducted by Bayer SAS, Sophia Antipolis Cedex, France. Laboratory Report No.: SA 11155, October 17, 2011. (MRID 48615204)

Fang, H., Tong, W., Branham, W.S., Mass, B.S., Moland, C.L., Dial, S.L, Hong, H., Xie, Q., Perkins, R., Owens, W., and Sheehan, D.M. (2003) Study of 202 natural, synthetic, and environmental chemicals for binding to the androgen receptor. Chern. Res. Toxicol . 16: 1338- 1358 (MRID 48074110)

Fletcher DW, Leonard MI. (1986a). Toxicity an d reproduction study with carbaryl technical in Bobwhite quail. Neillsville, Wisconsin, Unpublished study conducted by Bio-Life Associate Ltd (Report No. 85-QR-15). Rhone-Poulen c Agro. Lyon, France. (MRID 00160044)

Fletcher DW, Leonard MI. (1986b). 28-day dietar y toxicity and reprodu ction pilot study with carbaryl technical in Mallard ducks. Neillsville, Wisconsin, Unpublished study conducted by Bio-Life Associate Ltd (Report No. 85 DR P14). Rhone-Poulenc Agro. Lyon, France. (MRID 00161500)

Hamada, N. (1987) One-Year Oral Toxicity Stud y in Beagle Dogs. Unpublished study conducted by Hazleton Laboratories. HLA 400-715, March 18, 1987. (MRID 40166701)

Hamada, N. (1991) Subchronic Toxicity Study in Dogs with Carbaryl Technical: Lab Project Number: 656-152. Unpublished study prepared by Hazleton Laboratories America, Inc. 181 p. (MRID 42022801)

Hamada, N. (1993a) Oncogenicity Study with Ca rbaryl Technical in CD-1®Mice. Unpublished study conducted by Hazleton Washington, Inc., Vienna, VA. HWA 656-138., May 20, 1993. (MRID 42786901)

Hamada, N. (1993b) Combined Chronic Toxicity and Oncogenicity Study with Carbaryl Technical in Sprague-Dawley Rats. Unpublished study conducted by Hazleton Washington, Inc., Vienna, VA. HW A 656-139, September 7, 1993. (MRID 42918801)CARBARYL FINAL

Page 28 of 62 Kennel, P. (2003) Hershberger Assay to A ssess the Potential Antia ndrogenic Activity of Carbaryl in Rat. Unpublished study conducted by Bayer CropScien ce, Sophia Antipolis, France. SA 02261, January 15, 2003. (MRID 47987701)

Klotz, D.M., Arnold, S.F., and McLachlan, J.A. (1997) Inhibition of 17 beta-estradiol and progesterone activity in human br east and endometrial cancer cells by carbamate insecticides. Life Sciences 60: 1467-1475 (MRID 48067701)

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.

Lemaire, G., Mnif, W., Mauvais, P., Balaguer, P, and Rahmani, R. (2006) Activation of a- and Pestrogen receptors by persistent pes ticides in reporter cell lines. Life Sciences 79: 1160- 1169 (MRID 48074114)

Pant, N., Srivastava, S.C., Prasad, A.K., Shanka , R. and Srivastava, S.P. (1995) Effects of carbaryl on the rat's male reproductive system. Vet. Human Toxicol . 37: 421-5 (MRID 48161803)

Pant, N., Shankar, R. and Srivastava, S.P. ( 1996) Spermatotoxic effects of carbaryl in rats. Hum. Exp. Toxicol. 15: 736-8 (MRID 48067701)

Repetto-Larsay, M. (1998) Carb aryl Developmental Toxicity Study in the Rat by Gavage. Unpublished study conducted by Rhone-Poulenc Agro Centre de Recherche, Sophia Antipolis, France. Study SA 98070, October 21, 1998. (MRID 44732901)

Robinson, K. and Broxup, B. (1997) A Devel opmental Neurotoxicity Study of Orally Administered Carbaryl, Technica l Grade, in the Rat. Unpublishe d study conducted by ClinTrials BioResearch, Ltd. Quebec, Canada. Laborator y Project I.D. 97391, September 23, 1997. (MRID 44393701), Amended June 1, 1999 (MRID 44904204)

Rouquie, D. (2011) Evaluation of Carbaryl in the Androgen Receptor Binding Assay. Unpublished study conducted by Bayer SAS, Sophi a Antipolis Cedex, France. Laboratory Study No.: SA 11189, October 26, 2011. (MRID 48615201)

Rouquie, D. (2011) Evaluation of Carbaryl in the Estrogen Receptor Binding Assay. Unpublished study conducted by Bayer SAS, Sophi a Antipolis Cedex, France. Laboratory Study No.: SA 11209, October 26, 2011. (MRID 48615203)CARBARYL FINAL

Page 29 of 62 Sun, H., Shen O.X., Xu, X.L., Song, L., and Wang, X.R. (2008) Carbaryl, 1-naphthol and 2- naphthol inhibit the beta-1 thyroid hormone receptor-related transcription in vitro. Toxicology 249: 238-242 (MRID 48161801)

Tinwell, H. (2011) Evaluation of carbaryl in the aromatase assay. Unpublished study conducted by Bayer S.A.S., Bayer CropScience, Sophia Anti polis Cedex, France. Laboratory Report No.: SA 11060, October 20, 2011. (MRID 48615202)

Tinwell, H. (2011) Evaluation of carbaryl in the H295R st eroidogenesis assay. Unpublished study conducted by Bayer S.A.S., Bayer CropS cience, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 11066, October 18, 2011. (MRID 48615205)

Totis M. (1997) Investiga tion of the metabolism of 14 C-carbaryl in the 15-month-old rat following dietary administration. Unpublished study conducted by Rhone-Poulenc Ag Co. North Carolina, USA. Report No. SA 95288. (MRID 44402501)

Tyl, R.W., Marr, M.C., and Myers, C.B. ( 1999) Developmental Toxicity Evaluation (with Cholinesterase Assessment) of Carbaryl Ad ministered by Gavage to New Zealand White Rabbits. Unpublished study conducted by Reproductive & Developmental Toxicology Laboratory, Center for Life Sc iences and Toxicology, Resear ch Triangle Park, NC. RTI Identification No. 65C- 7297- 200/100, June 3, 1999. (MRID 44904202)

Tyl, R., Myers, C. and Marr, M. (2001) Two- generation reproductive toxicity evaluation of carbaryl (RPA007744) administered in the feed to CD® (Sprague-Dawley) rats. Unpublished study conducted by Reproductive and Developmental Laboratory, Cent er for Life Sciences and Toxicology, Chemistry and Life Sciences, Resear ch Triangle Institute, Life Sciences and Toxicology, Research Triangle Park, NC 27709. Laboratory report number 65C-07407-400, May 24, 2001. (MRID 45448101)

USEPA (2010) Carbaryl: Report of the Endocrine Disruptor Review Team; Test Order #: EDSP056801-57, October 28, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Sc ience Coordination and Policy.

U.S. Environmental Protection Agency, Washi ngton, DC. York, D.O. (2012). Carbaryl - ShortTerm Reproduction Assay with Fathead Mi nnow (Pimephales promelas) Following OPPTS 890.1350 and OECD 229 Guidelines. Unpublished study conducted by Smithers Viscient, Wareham, MA. Laboratory repo rt number: 13798.6275. (MRID 48669601)CARBARYL FINAL

Page 30 of 62 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.CARBARYL FINAL

Page 31 of 62 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

Requirement satisfied by OSRI (MRID Nos. 45448101, 42918801, 44402501, 48161801); see Appendix 2.

Androgen Receptor (AR) Bi nding Assay (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48615201), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a saturation binding experiment and a competitive binding experiment. The sa turation binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affi nity for the radio-labeled refe rence androgen (R1881) prior to routinely conducting AR competitive binding experiments. The competitive binding assay was conducted to measure the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations of car baryl (logarithmic increase from 10

− 10 to 10

− 3 M). DMSO was
used as the vehicle at a fi nal assay concentration of ≤ 3.3%. The assay included dexamethasone as a weak positive control, and R 1881 as the ligand reference standard.

In the saturation binding experiment , the mean maximum binding capacity (B max ) was 14 fmol/100 μg protein and the m ean dissociation constant (K d ) was 1.01 nM. The K d obtained from Saturation Assay #2 was slightly low (0.59 nM), but the K d s from the other two runs were within the recommended range (0.685 to 1.57 nM ) and the Scatchard plots for the three saturation assays were linear. The specific bi nding reached a plateau, and non-specific binding was less than 20% of total binding (4.6- 12.6%). The saturation binding experiment demonstrated that the AR was present in a reasonable concentration and functioning with appropriate affinity for the R1881 ligand.

In the competitive binding experiment, carbary l reduced the mean specific binding of the radiolabeled ligand in a con centration dependent manner in all three runs. The maximum reduction in [ 3 H]-R1881binding occurred at the highe st concentration tested (10

− 3 M), with specific binding being reduced to 28-34%. Specific binding was also reduced at 10

− 4 M (60- 85%), but not at concentrations ≤ 10

− 5 M (87-111% binding). Carbaryl was classified as a binder in all three competitive binding runs.

The estimated mean log IC 50 was

− 3.5 M for carbaryl,

− 4.4 M for the weak pos itive control and

− 9.1 M for R1881. Compared to the R1881 ligand, the mean relative binding affinity (RBA) was 0.00029% for carbaryl and 0.0020% for the positiv e control. The solvent control responses indicated no drift in the study a ssay. All performance criteria were met, with the exception thatCARBARYL FINAL

Page 32 of 62 ligand depletion and non-specific bindi ng (NSB) were not reported, but the IC 50 s for R1881 and the weak positive control were within the expect ed ranges. These deviations are considered minor; the performance criteria were met.

Based on the results from the th ree runs, carbaryl is classified as a Binder in the Androgen Receptor Binding Assay.

Aromatase Assay (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48615202), Carbaryl (100% a.i., Batch # NNCAAX0025) was incubated with human recombinant aromatase and tritiated androstenedione (1- β [ 3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in dimethyl sulfoxi de (DMSO) at increasing logarithmic concentrations from 10 –10 M to 10

− 3 M for 15 minutes to assess the potential of carbaryl to inhibit aromatase activity.

Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of a 15-minute incubation for each concentrati on of carbaryl. Tritiat ed 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 ) using a known inhibitor (4-hydroxyandrostenedione; 4-OH AS DN; formestane), and the test carbaryl series (10

− 10 to 10

− 3 ) with three repetitions per concentration.

Aromatase activity in the full act ivity controls ranged from 0.078 to 0.106 nmol/mg-protein/min for the 3 test runs, with a mean (± SD) of 0.091 ± 0.009 nmol/mg-protein/min. Activity in the background controls ranged from 6.8 to 8.2% and av eraged 7.4% of the fu ll control activity. The response of each full activity c ontrol within a run was 89 to 122% of the average full activity. The response of the full activity controls and background controls was acceptable, although activity in the full controls was marginally acceptable.

For the positive control (4-OH ASDN), arom atase activity averaged 0.0849 ± 0.0216 nmol/mgprotein/min at the lowest tested concentration (10

− 10 M) and 0.0027 ± 0.0008 nmol/mgprotein/min at the highest tested concentration (10

− 5 M). These results were generally within the recommended ranges for the top of the curve, bottom curve, Hill slope, and log IC 50 . The overall
%CV exceeded the recommended 15% at the 10

− 10 to 10

− 6 M test concentrations (18-27%), primarily due to the large difference in activity be tween the third run and the first two runs. The top curve (% activity) was 104.3% (Run 1), 100.7% (Run 2), and 65.5% (Run 3) compared to the specified guideline range of 90-110%. For 4-OH ASDN, the estimated log IC 50 averaged

− 6.83 M (outside of recommended

− 7.3 to

− 7.0 M range).CARBARYL FINAL

Page 33 of 62 For carbaryl, aromatase activ ity averaged 0.0785 ± 0.0190 nmol/mg-protein/min at the lowest tested concentration (10

− 10 M) and 0.0382 ± 0.0024 nmol/mg-protein/min at the highest tested concentration (10

− 3 M). Carbaryl reduced aromatase activit y compared to controls, on average, to approximately 65% at 10

− 4 M and 42% at 10

− 3 M.

Although carbaryl exhibited a concentration-de pendent inhibition of aromatase activity and decreased activity by mo re than 50% (log IC 50 = − 3.83 M), the average slope of the inhibition curve was steeper than –2 (-2.92). Therefore, carb aryl 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 assa y (MRID 48615203), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct saturation and competitive binding experiments. 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. The competitive binding experiment was conducted to measure the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in th e presence of increasing concentrations of carbaryl (logarithmic increase from 10

− 10 to 10

− 3 M). DMSO was used as the vehicle at a final concentration of <2%. The assay included 19-norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17 β -estradiol as the natural ligand
reference material.

In the saturation binding experiment, the appropriate protein concen tration was determined to be 0.5 mg/mL as it bound 25-35% of the [ 3 H]-17 β -estradiol. The K d for [ 3 H]-17 β -estradiol was 0.070 nM, and the estimated B max was 34.3 fmol/100 μ g protein for the prepared uterine cytosol.
The K d for each run was within the exp ected range of 0.03 to 1.5 nM.

In the competitive binding experiment, the re ference compounds generally performed as expected. The negative control, octyltriethoxy silane, had no effect on binding, and there were concentration dependent decrea ses in binding for the natural ligand and the weak positive
control. The mean IC 50 was 1.8 × 10

− 9 M for the natural ligand and 9.0 × 10

− 6 M for the weak positive control. The relative binding affinity (RBA) for the weak positive control was 0.019%, compared to the natural ligand. The Hill Slope for the natural ligand wa s outside the acceptable
range, at

− 2.0 to

− 1.7, indicating that the receptors were be having as though there was more than one binding site for 17 β -estradiol. Carbaryl was tested ove r a concentration range that fully defined the top of the curve. The ma ximum percent binding 109.0-117.9% was within approximately 25 percentage points of the lowest concentration of the estradiol standard 83.5-
116.2%.CARBARYL FINAL

Page 34 of 62 An estimated mean IC 50 was not calculated for carbaryl as it did not achieve 50% binding inhibition. In two assay runs, car baryl reduced the specific binding of the radiolabeled estradiol at the highest concentration tested (10

− 3 M) to 62-71%; in R un 3 percent binding was ≥ 75% at
10

− 3 M.

Based on the results from the th ree runs, carbaryl is classified as Equivocal 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 48615204), hER α -HeLa-9903
cells cultured in vitro were exposed to carbaryl (100% a.i ., Batch # NNCAAX0025 ) at serial log
concentrations of 10

− 10 M to 10

− 4 M in DMSO (0.1% v/v) for approximately 24 hours. The experiments were performed using 96-well plates and each carbaryl concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test agent for 23 ±1 hr to induce reporter (luciferase) gene products. Lu ciferase expression in response to activation of the estrogen receptor by carbaryl was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable sensitivity. Carbaryl was tested up to the limit of solubility, 10

− 4 M. The laboratory proficiency assays usi ng the required reference compounds adequately demonstrated that the assay was functioning properly. The mean RPC Max for carbaryl was 2.7%
for the first run and 0.5% for the second run, and the associated PC Max was 10

− 4 M for both runs.
Because the RPC Max < PC 10 in both assay runs, carbaryl was considered negative for estrogen receptor transcriptional activa tion in this test system.

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 48669601) of carbaryl with fathead minnow ( Pimephales promelas ) was conducted under flow-through conditi ons. Adult fish (21 weeks old; 2 males and 4 females in each group; 4 groups pe r treatment) were exposed to carbaryl (99.2% purity) at nominal concentrati ons of 0 (negative control), 0.032, 0.16, and 0.80 mg a.i./L; meanmeasured concentrations were <0.0038 (<L OQ; control), 0.031, 0.15, and 0.82 mg a.i./L. The test system was maintained at 24 to 26°C and a pH of 6.1 to 7.6.

All effects are reported based on comparison to the negative control. Mean survival in males was 100% in the control, 0.31, and 0.82 mg a.i./L groups; survival in the 0.15 mg a.i./L group was 88% (7/8). Female survival was 94% (15/16), 84% (15/16), 88% (14/16), and 100% (16/16) in the control, 0.031, 0.15, and 0.82 mg a.i./L groups, respectively. Combined sex survival was 96, 96, 88 and 100% in the 0.031, 0.15, and 0.82 mg a.i./L groups, respec tively, for which there wereCARBARYL FINAL

Page 35 of 62 no observed significant differences. Th ere were no notable observations with regards to behavior or changes in appearance (specifically color/ banding, ovipositor appearan ce, or size of dorsal nape pad) in the control or tr eated groups and no reported clini cal signs of toxicity. Male body length was 9% greater (p<0.05) in the 0.82 mg a.i./L treat ment group. Neither female body length nor body weight for either se x was affected by carbaryl exposure.

Carbaryl had no effect on fecundity or fertil ity in any treatment group. Additionally, plasma vitellogenin (VTG) concentrati ons were unaffected in males and females and there were no
treatment-related effects on GSI in either sex. Ho wever, median male nuptial tubercle score was lower in the 0.82 m a.i./L treatment group ( 30) than in control fish (33) (p<0.05). Histopathological observations in males included increases in mi nimal testicular degeneration and the percentage of spermatogonia at the mean -measured 0.82 mg a.i./L treatment level. No remarkable histopathological obse rvations were noted in males at other treatment levels or in
females. Plasma sex steroids were not measured.

Hershberger Assay (OCSPP 890.1400)

In a Hershberger Assay (MRID 47987701) screen ing for potential anti -androgenic activity, carbaryl (99.1% a.i., Batch #21114920) in 0.5% aque ous methyl cellulose 400 was administered daily via oral gavage to groups of six 8- to 9-week old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 5, 15, or 50 mg/kg/day in conjunction with a daily dose of testosterone propionate (TP) at 0.3 mg/kg/day by subcutaneous (s.c.) inject ion. The negative control group consisted of six castrated rats dosed daily for 10 days with TP at 0.3 mg/kg/day with the aqueous solution of methylcellulose 400 at 0.5% daily by gavage. The anti-androgenic positive control group consisted of six castrated rats exposed to 0.3 mg/kg/day TP (s.c . injection) plus 25 mg/kg/day flutamide (FT, via oral gavage). A final group of six castrated rats was dosed with control vehicles (methylcellulose and corn oil) via gavage and by the subcutaneous route to serve as a control check for androgeni c activity of TP. The animal s were dosed for 10 consecutive days and terminated approximately 24 hours after the final dose administration. At necropsy, the five androgen-dependent tissues and liver were collected and weighed.

All animals survived until scheduled termina tion. In the 50 mg/kg/day + TP group, treatmentrelated clinical signs consisted of increased salivation, reduced motor activity, tremors and piloerection were found in all animals thr oughout the study; chewing was observed in 4/6 animals between Days 8 and 10 and soiled nose was observed in 1/6 animals between Days 3 and 6. In the 15 mg/kg/day + TP group, treatmen t-related clinical signs were noted between Days 1 and 4 and consisted of reduced motor activ ity and/or tremors in 5/6 animals, associated with increased salivation and pilo erection in one animal. No clini cal signs of toxicity were noted in animals in the 5 mg/kg/day+TP gr oup, or any of the control groups.CARBARYL FINAL

Page 36 of 62 In the 50 mg/kg/day +TP group, there was a 12% d ecrease in final body weights, a 49% decrease in overall body weight gains, and a 20-32% decrease in food consumption (each p<0.01) compared to the TP control; body weights in this group were also decreas ed (not statistically analyzed) by 11% and 13% on Days 4 and 7, respectively. In the 15 mg/kg/day +TP group, decreases (p<0.05) of 6%, 20%, and 15-18% were observed in the final body weights, overall body weight gains, and food consumption, respectively. Final body weights, body weights gains, and food consumption in the 5 mg/kg/day +TP group were comparable to the TP control. Body weight gains in the positive control (FT + TP ) group were decreased (p<0.05) by 13%, but final body weights and food consumption in this group we re comparable to the TP control. There
were no treatment-related effects on the weights of seminal vesicl es, levator ani-bulbocavernosus muscle complex (LABC), Cowper’s glands, ventra l prostate or glans penis for any carbaryl treatment group. Liver weights were decreased (p<0.05) by 12% at 15 mg/kg/day and by 18% at 50 mg/kg/day compared to the TP control. Liver weights in the 5 m/kg/day and positive (TP+FT) control groups were comparable to the TP control. Animals in the positive (TP+FT) control group had the expected decreases (p<0.01) in weights of accessory sex organs as follows: 93% in seminal vesicles; 94% in ventral prosta te; 75% in LABC; 86% in Cowper’s gland; and 38% in glans penis.

The androgen agonist activity of carbaryl was not evaluated in this study. However, the androgenic activity of TP was validated. In the TP control group, fina l body weight, overall body weight gains, and food consumption were increased (p<0.05) by 9%, 57%, and 18-23%, respectively, compared to the vehicle control gr oup. Animals in the TP control group had the expected increases (p<0.05) in weights of acce ssory sex organ weights as follows: 796% in seminal vesicles; 832% in ventral prostate; 252% in LABC; 600% in Cowp er’s gland; and 14% in glans penis. In addition, liver we ight was increased (p<0.05) by 14%.

Percent CVs for the vehicle control, TP control, and 50 mg/kg/day +TP gr oups were compared to the performance criteria in the Guidelines. All %CV values were less than the maximum permissible values, with the ex ception of ventral pros tate (CV = 57%; maximum permissible CV = 45%) and the glans penis (CV = 23%; maximum pe rmissible CV = 22%) in the vehicle control

group.

The dosing for this study (high dose of 50 mg/kg/ day) was considered adequate based on adverse clinical signs (increased salivation, reduced motor activity, tremors and piloerection) and the 12% decrease final body weights obser ved at this dose level.

No statistically significant decreases were seen in the weights of the five androgen sensitive tissues. Carbaryl was negative for anti-a ndrogenicity in the Hershberger assay.CARBARYL FINAL

Page 37 of 62 Female Pubertal Assay (OCSPP 890.1450)

Requirement satisfied by OSRI (MRID 45448101); see Appendix 2.

Male Pubertal Assay (OCSPP 890.1500)

Requirement satisfied by OSRI (MRID No.45448101, 42918801, 44402501, 48161803, 48067701); see Appendix 2.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48615205), H295R cells cultured in vitro in 24-well plates were incubated with carbaryl (100% purity, Lot # NNCAAX0025) at serial log concentrations of
10

− 10 to 10

− 4 M for 48 hours in triplicate fo r three independent experime nts. Dimethyl sulfoxide (DMSO) was used as the vehicle, at a final concentration of 0.1%.

Testosterone and estradiol levels were measured using EIA. The hormone assay interference test demonstrated interference with measuring estr adiol using EIA kits in the presence of 10 –4 and
10 –5 M carbaryl; therefore, these co ncentrations were not evaluated for estradiol in the assays. A Quality Control (QC) plate was run concurrently with each independent run of the test chemical plate to demonstrate that the assay responded properly to pos itive control agents at two concentration levels. The posit ive controls included a known i nhibitor (prochloraz) and a known inducer (forskolin) of estradiol and test osterone production.

In the QC plate, the minimum basal hormone pr oduction levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both blank and solvent control (SC) wells for testosterone. Guideline requirements for basal hormone pr oduction, induction, and inhibition were met for
testosterone and estradio l, except for induction of testosterone with 10 μM forskolin in Runs 1 and 3 (1.66 to 1.79–fold) which was below the Guideline criteria of ≥ 2-fold induction. Evaluation of the intra- and interplate variability (%CV) for the SC in the test and QC plates indicated consistent performance of the assays, except that the between plate %CV for estradiol (33%) in the QC plates exceed ed the Guideline criteria ( ≤ 30%).

Precipitation and cytotoxicity were not reported at any concentration tested. No effects of carbaryl on testosterone pr oduction were noted from 10 -10 to 10 -5 M with the % changes for the individual evaluations varying between +12% (1.12-fold) and -33% (0.67-fold). In contrast, marked decreases of 52-59% (0.48- to 0.41-fold) were noted in testoste rone production at 10 -4 M in each run, for an overall significant reduction of 55% (p ≤ 0.01; 0.45-fold). No concentration dependent effects of carbaryl were noted on estradiol production, althoug h there was a 1.35-fold increase (not significant) in estradiol at th e highest concentration successfully tested, 10 -6 M.CARBARYL FINAL

Page 38 of 62 Based on the hormone responses in each of three i ndependent runs, carbaryl treatment resulted in statistically significant and repr oducible decreases in testosterone production, but did not result in statistically significant and reproducible alterations in estradiol production.

Uterotrophic Assay (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 47987702) conducted to screen for potential estrogenic activity, carbaryl (99.1% a.i., Batch #21114920) in corn oil was administered daily via oral gavage to groups of 10 immature female Sprague Dawley ra ts at dose levels of 0 (vehicle), 3, 10, or 30 mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with a daily
dose of 17 α -ethynyl estradiol (EE) at 0.003 mg/kg/day by oral gavage and a negative control group was treated with a daily dose of dibut ylphthalate (DBP) at 1,000 mg/kg/day by oral gavage. Body weights were determined daily. All animals were terminated and necropsied on PND 22 approximately 24 hours after the final dose administration to determine wet and blotted uterine weights.

All animals survived until sche duled termination. At 30 mg/kg/ day, tremors were noted in 7/10 animals and chewing in 9/10 animals. Piloerecti on was also noted in 4/10 animals on Day 1 or Day 2 (post-dosing) and one animal presented many clinical signs durin g the dosing period: reduced motor activity, prostration, wasted app earance, genital discharg e and soiled anogenital region. At 10 mg/kg/day, piloerection was not ed in 2/10 animals on Day 1, and chewing was observed in 3/10 animals on Days 2 and 3. At 3 mg/kg/day, 1/10 animals had piloerection on Day 1 only.

Final body weights were decrease d (not statistically analyzed ) by 10% in the 30 mg/kg/day group compared to vehicle controls, resulting in a decrease (p<0.01) of 39% in the overall (Days 1-4) body weight gain. Body wei ghts in the 3 and 10 mg/kg/day gr oups were comparable to the control throughout the study; howev er, overall body weight gain was decreased (p<0.05) by 11% in the 3 mg/kg/day group. The decreased body weight s indicate that the dose levels tested were adequate. Uterine weights in th e carbaryl treatment groups were comparable to the vehicle
controls.

Body weights and overall body weight gain in th e positive control (EE) group were comparable to the controls. In the nega tive control (DBP) group, final bo dy weight was decreased by 10%, and overall body weight gain was decreased (p<0.01) by 40%. Absolute wet and blotted uterus weights for the EE group were increased (p<0.01) by 518% and 394%, respectively, as expected. Uterine weights in the DBP group we re comparable to controls.CARBARYL FINAL

Page 39 of 62 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of carbar yl with the endocrine system are summarized below. Given the breadth of toxicity data available on carbaryl , the only Part 158 studi es discussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

AR Binding

In a published, non-Guideline study, Fang et al. (2003) reported development of a recombinant AR competitive binding assay. A panel of 202 ch emicals (including carbaryl; purity not reported) from a broad range of structural classes was evaluated. The AR protein used in the assay was a recombinant rat protein expressed in Escherichia coli . The amino acid sequence of the AR ligand binding domain (LBD ) is identical to that of the human AR LBD. Scatchard analysis of binding data provided a K d = 2.03 ± 0.15 nM (SD; n = 4) for R1881 (methyltrienolone), the standa rd AR ligand in the assay.

The Competitive Binding Experiment was conducted as follows. [ 3 H]-R1881, the test chemical, and AR protein were combined in test tubes on ice. The [ 3 H]-R1881 concentration was 1 nM, the competitor concentration ranged from 4.28 × 10

− 9 to 4.28 × 10

− 4 M by one log unit concentration intervals, and the AR concentra tion was 1.84 nM. In addition to th e radio-inert competitor, each assay included a zero tube (total binding of [ 3 H]-R1881) and a R1881 standard curve. The
1×10

− 6 M R1881 tube contained radio- inert R1881 that was at a 1000-fold molar excess over the [ 3 H]-R1881 (1 nM) to determine NSB. The radioactivity in the ethanolic s upernatant containing the AR plus bound radioactivity was determined by liquid scintillation counting. Radioactivity counts (dpm) of the NSB tubes were subtracted from all tubes prior to calculation of percent [ 3 H]-R1881 bound. Data for each competitor and the R1881 standard curve were plotted as percent [ 3 H]-R1881 bound (relative to the standard) vs. molar concentration, and the IC 50 (50% inhibition of [ 3 H]-R1881 binding) for each competitor was determined.

The AR binding affinity was expressed as eith er RBA or log RBA in this study. The mean IC 50 was 3.07×10

− 9 ± 9.12×10

− 10 M ( n = 82) for R1881. The RBA for R1881 was set to 100% (log RBA = 2). All assays were run in duplicate tubes with at least two repli cations. The RBA values of active chemicals are the means of the replicat e values. Chemicals that failed to compete with [ 3 H]-R1881 in binding were designated as “non-bi nders” (NB). Chemicals that showed binding
but did not reach 50% inhibition at maximum concentration were designated as “slight binders” (SB). A less than IC 50 value was assigned for both NB and SB compounds to indicate the
maximum concentration used. The AR data set in this study was classified into four activity groups with respect to their RBA ranges, designated as strong ( RBA > 1), moderate (1 > RBA >CARBARYL FINAL

Page 40 of 62 0.01), or weak binders (0.01 > RBA > 0.0001), a nd inactive chemicals. The inactive group
includes both NB and SB chemicals.

Results obtained relevant to th e evaluation of carbaryl are as fo llows: (i) carbaryl had a logP of 2.35, an IC 50 of 4.00×10

− 4 M, an RBA of 0.0008, and a logRBA of

− 3.12; (ii) dexamethasone (a weak positive reported for comparativ e purposes) had a logP of 1.72, an IC 50 of 8.00×10

− 5 M, an RBA of 0.0038, and a logRBA of

− 2.42; and (iii) DHT (natural lig and reported for comparative purposes) had a logP of 3.07, an IC 50 of 2.23×10

− 9 M, an RBA of 137.9008, and a logRBA of 2.14. Accordingly, carbaryl was determined to be a weak binder to AR in this assay.

Thyroid Receptor Activation

In a published, non-Guideline study, Sun et al. (2008) evaluated the potential of three chemicals (carbaryl, 1-naphthol and 2-naphthol) to inhibit β 1 thyroid hormone receptor-related transcription in vitro

.
The study used a luciferase reporter gene assay sy stem to measure potenti al chemical activation of the thyroid receptor (TR). There are four known isoforms of the TR derived from two genes; three have been shown to be activated by the thyroid hormones T 3 (triiodothyronine) and

T 4 (thyroxine). T 3 (purity ≥ 98%) was used in the present evaluation of compounds for TR antagonism. The TR selected fo r this assay system was TR β 1 (c-erb-A beta). pGal4-L-TR β (the expression vector coding for the ligand binding domain of TR β fused to the DNA binding domain of Gal4) and Gal4 (the responsive lucife rase reporter gene pUAS -tk-luc containing four copies of the Gal4 binding site) were obtained. The plasmid phRL-tk ( Renilla luciferase reporter gene) was used as an internal control for tran sfection efficiency and cytotoxicity. Carbaryl (purity 99.5%), 1-napthol (purity ≥ 99%), and 2-napthol (purity ≥ 99%) were tested for TR agonist and antagonist activity, and bisphenol-A (BPA; purity ≥ 99%) was included as a known TR antagonist.

The HepG2 (hepatocellular carci noma) cell line was maintained in Dulbecco’s modified Eagle medium (DMEM) with 5% fetal bovine serum (FBS) at 37°C and 5% CO 2 . Host cells were plated into 48-well microplat es at a density of 0.5×10 5 cells per well in phenol red-free DMEM containing charcoal-dextran stri pped FBS and incubated. The cells were transfected with 0.25 μg pUAS-tk-luc, 0.1 μg Gal4-TR β , and 0.05 μg phRL-tk including 2.5 μg SofastTM transfection reagent per well. After incuba tion, the transfection medium wa s removed and replaced with medium containing various concentrations of th e test chemicals for measurement of agonist activity. For measurement of antagonist activity, the cells were treated with test chemicals and 5

nM T 3 . Medium with 0.1% ethanol or 0.1% DM SO were used as solvent controls.

After exposure to the test chemicals for 24 hours, the cells were harves ted, rinsed three times with phosphate-buffered saline, lyse d with an equal volume of passive lysis buffer, and analyzed
with a 96-well luminometer. Th e amount of luciferase and Renilla luciferase were measured withCARBARYL FINAL

Page 41 of 62 a Dual-luciferase reporter assay system kit. The relative transcrip tional activity was converted to fold induction above the corresponding vehicle cont rol value (n-fold). The values presented were
means and standard deviations from three inde pendent experiments. The median inhibitory concentration (IC 50 ) or median effective concentration (EC 50 ) was calculated. For antagonists, treatments were compared to the 5 nM T 3 positive control. Data were analyzed by 1-way ANOVA followed by Duncan’s multiple comparison test. The level of significance was set at p<0.05.

No cytotoxicity was detected in the assay. Th e HepG2 reporter system showed the appropriate response to the natural ligands T 3 and T 4 and to the known antagonist, BPA. For T 3 , the maximal induction (7.85-fold of vehicl e control) was observed at 10

− 8 M, with an EC 50 of 0.46 nM. For

T 4 , the maximal induction (8.20-fold of vehicle control) was observed at 10

− 7 M, with an EC 50 of 25.53 nM. BPA did not induce the expression of luci ferase, but demonstrat ed antagonist activity.
After incubation with T 3 at 5 nM, BPA inhibited (p<0.05 or 0.01) luciferase activity at
concentrations of ≥ 2.5×10

− 5 M, with an IC 50 of 6.45×10

− 5 M. Carbaryl also had no agonist activity in the assay at up to 10

− 4 M; however, after incubation with T 3 at 5 nM, carbaryl demonstrated antagonist ac tivity (p<0.01) with an IC 50 of 8.40×10

− 5 M in this assay. This assay is not required in the Tier 1 battery, but the resu lts suggest that carbaryl could inhibit the action of thyroid hormones in vivo

.
ER Binding/AR Binding or Activation

In a published, non-Guideline study ( Kojima et al ., 2004) , the potential of carbaryl (purity 95- 100%) to interact with two human ER subtypes (hER α and hER β ) and one human androgen receptor (hAR) in transactivati on assays with Chinese hamster ovary cells (CHO-K1 cells) was investigated. For detection of hER α or hER β activity, cells were tr ansfected with pcDNAER α or pcDNAER β , pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flut olanil was tested over a concentration range
from 10

− 8 to 10

− 5 M, and the responses were compared to the activity of 17 β -estradiol (E 2 ; purity >97%) or 5 α -dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For measurement of antagonistic activity to hER α , hER β and hAR, either 10

− 11 or 10

− 10 M E 2 or 10

− 10 M DHT was added to the cell cultures al ong with the test compound, respectively. Agonistic activities were evaluated by the relative activity ex pressed as 20% relative effective concentration (REC 20 ), the concentration of the compound de monstrating 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 similarly by the relative activity expressed as 20 % relative inhibitory concentration (RIC 20 ), the concentration of the compound showing 20% in hibition of the activity
of E 2 or DHT against ER α , ER β , and AR, respectively. Carbaryl (10

− 8 to 10

− 5 M) did not show agonist or antagonist activity for ER α and ER β , or agonist activity for AR in this study.

In another publishe d, non-Guideline study, Blair et al . (2000) evaluated 188 pesticides (including carbaryl; purity 99%) with an ER competitive binding assay. Uteri fromCARBARYL FINAL

Page 42 of 62 ovariectomized SD rats were used as the source of ER for the assay. Initially, test chemicals were screened at high concentrati ons to determine whether a chemical competed with [ 3 H]-estradiol for the ER. Test chemicals that exhibited affinity for the ER in the first tier were subsequently assayed with a range of concentrations to char acterize the binding curv e and to determine the
IC 50 and RBA values. The mean IC 50 for carbaryl was >1.00 x 10

− 4 M in this sc reening study.

In a published, non-Guideline study, Klotz et al. (1997) evaluated carbamate insecticides (including carbaryl; purity 99-100%) for activity in ERTA and whole-cell competitive ER binding assays. The ERTA assays were conducte d in transiently transfected MCF-7 human
breast and Ishikawa human endom etrial cell lines using an es trogen-responsive luciferase reporter gene. The test chemicals were evaluate d at a single concentration (100 nM). In MCF-7 cells the authors reported that the carbaryl significantly (p<0 .01) increased estrogen-responsive luciferase activity above control levels. Comparable results were observed in Ishikawa cells. The authors also reported that 100 nM carbamate decreased (p<0.01) estrad iol-induced luciferase activity 70-75% in MCF-7 cells a nd 65-70% in Ishikawa cells. Ce ll viability was not reported in this study.

For the whole-cell Competitive Binding Experiment, MCF-7 cells were transferred to phenol red-free DMEM supplemented with 5% DCC-FBS for 72 hours prior to the assay. Cells were rinsed once with binding medi um (serum-free, phenol red- free DMEM containing 15 mM HEPES) and resuspended in binding medium. 1×10 6 cells were transferre d to microcentrifuge tubes, and the volume of binding medium wa s increased to l mL. A volume of 500 pM [ 3 H]-17 β

estradiol was added to the MCF- 7 cells in the presence or absence of vehicle, 100-, 1000-, or 10,000-fold molar excess radioinert 17 β -estradiol or carbaryl. Samples were incubated at 37°C with gentle shaking for 1 hour. After incubation, samples were centrifuged, supernatant discarded, and the cells were washed three times in ice-cold PBS. Final cell pellets were resuspended in 100 μl PBS, and bound radiolabeled hormone was determined by liquid scintillation counting. Carbar yl inhibited binding of [ 3 H]-17 β -estradiol to hER in a dosedependent manner, with decreased (p<0.01) bindi ng at carbaryl concentrations of 1 and 10 μM,
with a calculated IC 50 for carbaryl of 6 μM.

In a published, non-Guideline study, Lemaire et al. (2006) evaluated 49 pesticides (including carbaryl; purity 95-100%) covering nine chemical classes, for ER α and ER β activation or inhibition in stable repo rter cell lines, HELN ER α and ER β (HeLa cells stably transfected with an ERE-driven luciferase plasmid).

To create the stably-transfected cell lines, He La cells were first transfected with the GAL 4 REERE- β Glob-Luc-SVNeo plasmid. HELN cells expressing ER α or ER β were obtained by a
second transfection of the corresponding pSG 5 puro plasmids (pSG 5 -ER α puro or pSG 5 -ER β puro, respectively). HeLa-derived cell line s were cultured in phenol red-free DMEM supplemented with 6% DCC-FBS for the strain culture.CARBARYL FINAL

Page 43 of 62 For the stable gene expression assay, cells were plated into 96-well plates at 30,000 cells per well in phenol red-free DMEM, supplemented with 6% DCC-FBS. Twenty-four hours after seeding, the test compounds (95-100% purity) were adde d, and cells were incubated for 16 hours. The vehicle (DMSO) concentration never exceeded 0. 1% of the culture medium. At the end of incubation, luciferase activity was measured.

For the ER α and ER β competitive-binding assay, ER α and ER β cells were seeded at a density of
1.5×10 5 cells per well in 24-well tissue culture pl ates. Cells were labeled with 0.1 nM [ 3 H]-E 2 ([ 3 H]-17 β -estradiol, 41.3 Ci/mmol specific activity) in the presence or absence of increasing
concentrations of cold E 2 (as control) or each of the tested compounds in 400 μ l of DMEM supplemented with 6% DCC-FBS. Each dilution wa s performed in duplicate. Plates were put on ice for 15 minutes, and the labeling medium wa s removed. Cells were washed with cold phosphate-buffered saline and harvested in etha nol. Total cell lysate was counted by liquid scintillation counting.

The effect of pesticides on cell viability was assessed with a 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl-tetrazolium bromide (MTT) toxicity a ssay. The plates were read by absorbance at 540 nm. All results are expressed as the average of three wells. The contro l consisted of medium alone with no cells. Treatment with carbaryl, as well as all other pesticides, did not result in cytotoxicity in the study.

To evaluate the estrogenic pot ential of pesticides, HELN ER α and HELN ER β were used to identify the active compounds and discriminate between ER α and ER β activities. To determine

ER α and ER β agonistic activity of pes ticides, the relative transact ivation activity on HELN ER cells was computed as the ratio of luciferase reporter gene induction values of each compound at
a concentration of 10 μ M to the luciferase reporter gene induction value of E 2 at 10 nM. As the ERTA assay was conducted at only a single c oncentration, it is not discussed further. Antagonistic effects of pesticides (including carbaryl) in the ER α and ER β transactivation assay were determined over a range of five concentrations from 10

− 7 to 10

− 5 . In addition, antiestrogenic competitive inhibition was examined against ER α and ER β at carbaryl concentrations of 10 μM, 3 μM, and 1 μM.

To confirm the ER antagonist effects in the tran sactivation assays reflected an inhibition of E2 binding to ER α and/or ER β , whole cell competitive binding assays were conducted with the

HELN ER α and ER β cells. The binding affinity for ER of anti-estrogenic pesticides was assessed with 0.1 nM [ 3 H]-E 2 as a tracer. As expected, non-radiolabelled E 2 displaced [ 3 H]-E 2 by
50% at a concentra tion of 0.12 nM for ER α and 0.18 nM for ER β , and ICI 164,384 inhibited [ 3 H]-E 2 binding by 50% at the concen tration of 2.5±0.5 nM for ER α and 3.3±1.0 nM for ER β

. The authors reported that carbaryl inhibited (p<0.001) [ 3 H]-E 2 binding (ER α and ER β ) by approximately 45 and 60%, respecti vely, at a concentration of 10 μ M, showing anti-estrogenic activity.CARBARYL FINAL

Page 44 of 62 Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation reproduction study ( MRID 45448101 ), carbaryl (99. 1% purity) was administered to groups of CD[SD] IGS BR rats (30/sex/group) at nominal concentrations of 0, 75, 300, or 1500 ppm in the diet. Averaged over th e respective premating periods, the dietary intakes of carbaryl corresponded to do ses of 0, 4.67, 31.34, and 92.43 mg/kg/day for F 0 males; 0, 5.56, 36.32, and 110.78 mg/kg/day for F 0 females; 0, 5.79, 23.49, and 124.33 mg/kg/day for F 1 males; and 0, 6.41, 26.91, and 135.54 mg/kg/day for F 1 females. Each group received treated or control diet continuously for 70 days prior to mating and during mating, gestation, and lactation of one litter per generation. M ean carbaryl intakes for F 0 and F 1 parental females during gestation were slightly lower compared to premating inta kes, and carbaryl intakes during lactation was increased approximately two-fold compared to premating intakes in each dose group. F 1 pups selected as parents to produce the F 2 generation were weaned onto th e same food as their parents (PND 21). Parental males were sacrificed after deliv ery of their litters and parental females were sacrificed after weaning of their litters (PND 21).

No treatment-related deaths or c linical signs were observed at a ny dose level in either generation. With a few exceptions at the high dose, there we re no treatment-related effects on organ weight changes (absolute or relative weights of the testes, epididymides, seminal vesicle with coagulating glands, prostate, uterus [with oviduc ts and cervix], vagina, ovaries, adrenal, or pituitary glands). However at the high-dose, the relative kidney weight increased by 7% (p<0.01)
in F 0 males, and there were decreases in absolute pituitary ( ↓ 9%, p<0.05) and epididymides ( ↓ 5%, p<0.05) weights and relative weight s of the liver, kidneys, and brain F 1 males. For highdose females, the absolute and relative liver weights were elevated by 10% (p<0.01) and 14%, respectively, the absolute adre nal weight was decreased by 11% (p<0.05), and the relative kidney weight was increased by 6% (p<0.05) for F 0 females. Also in high-dose F 1 females, the absolute brain weight was decreased by 4% (p<0.05). No gr oss or microscopic lesions were observed in adult rats of either generation.

At the low- and mid-dose, no treatment-relate d effects were observed on body weights, weight gain, feed consumption, or food efficiency in F 0 or F 1 male or female rats at any time during the study, including during gestation and l actation periods of the females.

For the high-dose group, body wei ghts and body weight gains in F 0 and F 1 male and female rats were decreased (p<0.001, 0.01, or 0.05) during th e premating period. Body weights in the F 0 males and females were decreased 5-6% and 4-5%, respectively. B ody weight gains were maximally decreased during premating Days 0-7 (23 and 27%, respectively) in F 0 males and females, with overall body weight gain decreases (Days 0-70) of 9 and 7%, respectively. Mean body weight in F 1 males was decreased 19% on PND 22 (Day 0 of premating), decreased byCARBARYL FINAL

Page 45 of 62 12% on PND 56, but was only decreased by 10% on PND 91. Similarly, mean body weight in F 1 females was decreased 22% on PND 22 (Day 0 of premating), decreased by 12% on PND 56, and decreased by 8% on PND 91. Body weight gain in the F 1 males was decreased during premating Days 0-35 by 11-20%, with an overall body weight gain decrease of 8% by PND 91 (Premating Days 0-70). Body weight gain in the F 1 females was initially decreased on Days 0-7 by 9%, with no effect on overall body weight ga in. Food consumption and food efficiency for F 0 and F 1 rats followed patterns similar to that of body weight and body weight gain; the largest difference between the high-dose animals and c ontrol occurred during th e early part of the premating period. When averaged ov er the entire premating period, F 0 and F 1 males consumed 6- 7% less food compared to control and had food efficiency values similar to control. Feed consumption and food efficiency for the F 0 females were similar to those of the control group, but food consumption in F 1 females was decreased (p<0.01) by 9% with a food efficiency value increased (p<0.01) by 10% compared to contro l. Mean body weights were decreased (p<0.001, 0.01, or 0.05) in high-dose F 0 and F 1 females during gestation and la ctation, with the effect being greater in the F 1 females ( ↓ 7-14%) compared to F 0 females ( ↓ 4-7%). Body weight gains also were decreased (p<0.001, 0.01, or 0.05) during gestation ( ↓≤ 25%), but overall body weight gains during lactation were increased (NS) for F 0 and F 1 females ( ↑ 55 and 86%, respectively).

The LOAEL for parental systemic toxicity is 1500 ppm (92.43-124.33 mg/kg/day for males and 110.78-135.54 mg/kg/day for females) based on decr eased body weight, body weight gain, and feed consumption. The NOAEL level is 300 ppm (23.49-31.34 mg/kg/day for males and 26.91- 36.32 mg/kg/day for females).

No treatment-related effects were ob served on estrous cyclicity in F 0 or F 1 females, or on percent motile sperm, sperm count, percent progressive ly motile sperm, epididymal sperm count, spermatid head count, daily sperm production, or efficiency of daily sperm production in F 0 or F 1 males. There was a dose-related in crease (NS) in the percentage of abnormal sperm in the treated males. No treatment-related gross or microscopic effects were observe d in male or female rats of either generation. No treatment-related effects were observed on any parameter of reproductive performance including, mating and fertility indexes, ge station index, pregna ncy index, pre-coital duration, gestation length, or number of females producing live litters.

The LOAEL for reproductive toxicity could not be established because no effects were observed at any dose; therefore, the NOAEL is ≥ 1500 ppm (92.43-124.33 mg/kg/day for males and 110.78-135.54 mg/kg/day for females).

No treatment-related effects were observed on implantation sites/litter , number of live pups born/litter, number of dead pups born/litter, live birth index, sex ratio, cl inical signs, or organ weight or necropsy findings in pups surviving to 21 days. Pup su rvival was slightly decreased (NS) at the mid- and high-dose for both genera tions. An increased number of deaths in the F 2 generation pups resulted in an 18-19% decrease in mean litter size on PND 4 (p<0.01 or <0.05),CARBARYL FINAL

Page 46 of 62 and decreased viability and lactat ion indexes at the high dose. A la rge number of pups that died had no milk in their stomachs. In addition, pup we ight/litter in the high -dose pups was decreased (p<0.001 or <0.01) for both gene rations during PNDs 4-21 ( ↓ 11-15% for F 1 and ↓ 13-23% for F 2 pups). Body weight gain in pups was decreased (NS) throughout lactation, with the greatest effect ( ↓≥ 20%) occurring duri ng LDs 0-7 for F 1 pups and LDs 0-14 for F 2 pups. Sexual maturation was delayed in the high-dose F 1 offspring as evidenced by delayed PPS in the males (+2.1 days) and vaginal patency (VO) in the fe males (+1.4 days). The differences remained statistically significant after adjustment for body weight decreases. Anogenital distance was decreased (p<0.05) in F 2 male pups in the high-dose group, bu t this difference was not observed after adjustment for body weight.

The LOAEL for offspring toxicity was 300 ppm (23.49-31.34 mg/kg/day for males and 26.91- 36.32 mg/kg/day for females) base d on increased numbers of F 2 pups with no milk in the stomach and decreased pup survival. The NOAE L is 75 ppm (4.67-5.79 mg/kg/day for males and 5.56-6.41 mg/kg/day for females).

Developmental Neurotoxicity (Rat, OCSPP 870.6300)

In a developmental neurotoxicity study in rats ( MRID 44393701 and 44904204 ), pregnant female SD rats (26/dose group) were admi nistered carbaryl (99.1% purity) in 0.5% carboxymethyl cellulose and 0.1% Tween 80 by gavage from GD 6 through LD 10 at doses of 0, 0.1, 1.0, or 10 mg/kg/day. Additional pregnant fe males (6/dose group) were administered the same doses of carbaryl for the AChE determinat ions. AChE determinatio ns were conducted predosing (GD 6) and post-dosing at time of peak effect (1 hour post-dosing) on GDs 6, 15, and 20
and LDs 4 and 10. Functional Observational Batt ery (FOB) measurements were conducted at approximately 0.5 and 2 hours post-dosing on the sa me days as body weight measurements (GDs 0, 6, 9, 12, 15, 18 and 20 and LDs 4, 7, 11, 13 and 21). Measures of reproductive performance were evaluated. Offspring were examined fo r body weight, physical development landmarks (tooth eruption and eye opening), FOB assessm ents (LDs 4, 7, 11, 13, 17, and 21), and motor activity (LDs 13, 17, and 21). On LD 11, one anima l/sex/litter was sacrifi ced for determination of brain weights (of these, six/sex were random ly selected for neuropathological evaluation). After LD 21, three animals/sex/litter were se parated from the dams and constituted the F 1 adult generation. These animals were evaluated for bo dy weight, physical development (VO and PPS), motor activity (Day 60), startle habituation resp onse (Days 22 and 60), passive avoidance (Day 23), and water maze behavior (Day 60). After completion of the behavior test period (at approximately ten weeks of age), twelve animals/sex/group were anesthetized and perfused for post-mortem examination.

There were no carbaryl-asso ciated deaths in the F 0 generation dams, and no treatment-related clinical signs of toxicity were observed. Body weight gain was decreased (p<0.01, ↓ 92%) in females in the high-dose group for the GD 6-9 peri od, but body weight gains were similar duringCARBARYL FINAL

Page 47 of 62 all other periods. Food consumption was not measured during the study. During the FOB measurements, the incidence of females in the high-dose group w ith decreased pupil size (pinpoint pupils) was increased on all occas ions during the dosing period. An increased incidence of dams with slight tremors affecting the head, bod y and/or limbs was noted on the majority of assessment occasions in the high -dose animals. There were also occasional occurrences of ataxic gait/ove rall gait incapacity (dose-depend ent increased incidences or occurrences in the high-dose animals only) that were considered to be of toxicological significance due to ot her effects upon gait.

In the high-dose group, RBC and whole blood AChE activities were decreased in RBC (p<0.01, ↓ 28%) and whole blood (p<0.05, 34 and 32%, respectively) on GD 20 and LD 10. Plasma AChE activities were decreased (NS) up to 39%. Brain AChE activity was decreased (p<0.01, 42%) in the high-dose animals at study termination. Ther e were no treatment-re lated effects on gross necropsy findings for the F 0 generation animals. There were no effects observed on maternal performance parameters of pregnancy rate, gest ation index and duration, numbers of live pups, dead or malformed pups, implantation scars, sex ratio or post-implantation loss.

For the F 1 generation pups, there were no treatment-r elated effects on pup weight, pup survival indices, developmental landmarks (tooth eruptio n and eye opening), c linical toxicity, FOB measurements, or motor activity assessments. At sacrifice on LD 11, there were no treatmentrelated effects on brain weight and gross or microscopic pathol ogy. Noted differences in some morphometric measurements are not pert inent to the EDSP WoE determination.

For the F 1 generation adults, there were no treatmen t-related effects on clinical condition, body weight, physical development ( VO and PPS), motor activity, audito ry startle response, passive
avoidance and water maze measurements. At sacr ifice, there were no gross or microscopic neuropathological lesions observed for animals examin ed in this study that were attributable to treatment with carbaryl. Noted differences in some morphometric measurements are not pertinent to the EDSP WoE determination.

The maternal toxicity LOAEL was 10 mg/kg/ day based on decreased body weight gain,
alterations in FOB measurements and inhibiti on of RBC, plasma, whol e blood, and brain AChE activities. The maternal NOAEL was 1.0 mg/kg/da y. The tentative developmental neurotoxicity LOAEL was 10 mg/kg/day based on alterations in the morphometric measurements at Days 11 and 60; the tentative NOAEL was 1 mg/kg/day becau se the morphometric analyses at the lower
doses were not conducted. The devel opmental effect persisted from F 1 pups to adults, indicating that this developmental effect was not transitional.

Developmental Toxicity (Rat, OCSPP 870.3700)

In an oral developmental toxicity study in rats ( MRID 44732901 ), carbaryl (99% purity) in 0.5% aqueous methylcellulose was admi nistered by gavage at 0, 1, 4, and 30 mg/kg/day to pregnantCARBARYL FINAL

Page 48 of 62 Crl:CD (SD) BR rats (25/dose group) during GDs 6 through 20. At GD 21, surviving dams were sacrificed and necropsied.

At least one occurrence of post-dos ing salivation occurred in 18/25 dams (vs 0/25 control) in the high-dose group. This clinical sign appeared wi thin 20 minutes of treatment, resolved in approximately one hour, and was observed from GDs l3 to 20. There were no deaths and no
other treatment-related clinical signs. Body weight in the high-dose dams was decreased (NS) 5 and 8% compared to control on GDs 16 and 21, re spectively; the corrected (for gravid uterine weight) final body weight and body we ight gain were decreased (p ≤ 0.01) by 7 and 38%, respectively. Body weight gain in the high-dose group was decreased (p ≤ 0.01) after initiation of dosing (GDs 6-9, ↓ 108%) and decreased (p ≤ 0.01) for the overall treatment period (GDs 6-21, ↓ 27%). Food consumption (g/animal/day) was decreased (p ≤ 0.01) throughout the treatment period ( ↓ 10-17%).

There were no treatment-related gr oss pathologic findings noted in any of the dams. There were no differences of toxicological concern in mort ality, pregnancy rate, number of corpora lutea, implantations, viable fetuses, pre- and post-impl antation losses, placental weights, and sex ratio.

There were no differences of t oxicological concern observed in the mid- and low-dose groups. The maternal LOAEL is 30 mg/kg/day based on clin ical signs of toxicity, decreased body weight gains, and food consumption. The maternal NOAEL is 4 mg/kg/day.

In the high-dose fetuses, mean fe tal body weights were decreased (p ≤ 0.01) by 7-8%. Additional effects observed in the high-dose fetuses included: (i) an increase in incomplete ossification of the 5th sternebrae, (ii) unossified 7th cervical centrum, (iii) incomplete ossification of 7th cervical centrum, and (iv) unossified 1st metatars al. No effects on fetal viability were observed.

There were no treatment-related effects in deve lopmental parameters observed in the mid- and low dose groups. The developmental LOAEL is 30 mg/kg/day based on decreased fetal body weights and increased incomplete ossification of multiple bones. The developmental NOAEL is 4 mg/kg/day.

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a developmental toxicity study in rabbits ( MRID 44904202 ), carbaryl (99% purity) in 0.5% aqueous methylcellulose was administ ered by gavage at doses of 0, 5, 50, or 150 mg/kg/day to pregnant New Zealand White rabbits (22/dose group) during GDs 6-29. On GD 25, blood was collected at 1 hour post-dos ing for plasma and RBC AChE activity determinations. At GD 30, surviving dams were sacrificed and necropsied, and fetuses were examined for evidence of developmental effects.CARBARYL FINAL

Page 49 of 62 Four does died on study (one each in the cont rol and low-dose group, and two in the mid-dose group); none of the deaths were considered to be related to treatment. No clinical toxicity was reported. Body weights were not a ffected by treatment, but body we ight gain was decreased at the mid dose (NS) and the high dose (p<0.01 for GDs 6-29, 3-30, and overall gestation). Decreased body weight gains in the mid-dose gr oup ranged from 55% (GDs 6-9) to 14% (GDs 3- 30) with an overall body weight gain decrease during gestation of 14%. Decreased body weight gains in the high-dose group ranged from 208% (GDs 6-9) to 33% (GDs 3-30) with an overall body weight gain decrease during gestation of 38%. Corrected body weight change was also decreased at the high dose (-219.73 g vs -81.86 g control). There wa s no treatment-related effect on food consumption. Decrease s (p<0.01) in plasma ( ↓ 46 and 68%, respectively) and RBC ( ↓ 19 and 27%, respectively) AChE activ ities were observed in the mi d- and high-dose groups. Lesser decreases (NS) were noted in both AC hE activities in the low-dose animals.

The maternal LOAEL is 50 mg/kg/day based on decreased body weight gain and decreased plasma and RBC AChE activity. The ma ternal NOAEL is 5 mg/kg/day.

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation sites, number of live fetuses/litter , the number of resorptions/implant ations, fetal sex ratio, or soft tissue abnormalities at any dose. The only evidence of developmental toxicity was a decrease in fetal body weights of 10% (calculated for all fetuse s or individually for males and females) in the high-dose group. There were no trea tment-related developmental e ffects observed in the lowand mid-dose groups.

The developmental LOAEL is 150 mg/kg/da y based on decreased fetal weight. The developmental NOAEL is 50 mg/kg/day.CARBARYL FINAL

Page 50 of 62 Chronic Toxicity/Carcinoge nicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study in rats ( MRID 42918801 ), carbaryl (99% purity) was administered to SD rats (90/ sex/dose in the control and high-dose groups and
80/sex/dose in the low- and mid-dos e groups) in the diet for either 53 or 104 weeks at doses of 0, 250, 1500, or 7500 ppm (mean-measured intakes of 10.0/12.6, 60.2/78.6, and 349.5/484.6 mg/kg/day in males and females, respectively). Te n animals/sex/dose group were sacrificed after 53 weeks of treatment, and ten animals/sex in the control and high-dos e groups were fed the control diet for an additional four weeks (Weeks 53 to 57) prior to sacrifice for the recovery assessment. The remaining animals were main tained on the treatment diets until study
termination at Week 105. At each scheduled sacrif ice, surviving rats were sacrificed for gross and histopathological evaluation. The organs weighed at each sa crifice included the adrenals, brain, liver, kidneys, testes with epididymides, and ovaries. Gross and microscopic pathological examinations were conducted on these organs as well as the pituitar y, thyroid/parathyroid, urinary bladder, prostate, seminal vesicles, mammary gland, uterus, and vagina with cervix.

Clinical observations included th in appearance (high-dose males) , alopecia (high-dose females), and urine staining in high-dose males and fe males. Decreased body weights and body weight gains were observed in high-dose males and females over the two-year study period. Body weight decreases in males and females increased from Week 13 ( ↓ 23 and 24%, respectively) to Week 105 ( ↓ 35 and 45%, respectively), although signi ficance (p<0.05) only occurred at Weeks 23, 53, and 105. Females in the mid-dose group also had decreased (p<0.05) body weights at Weeks 13 ( ↓ 4%), 53 ( ↓ 5%), and 105 ( ↓ 12%). Body weights in the recovery animals demonstrated continued, although attenuated, decr eases in males and females between Weeks 53 ( ↓ 32 and 36%, respectively) and 57 ( ↓ 23 and 27%, respectively). Ov erall body weight gains also were decreased in the high-dose males ( ↓ 53) and females ( ↓ 69%) and in mid-dose females ( ↓ 18%). Other evidence of systemic toxicity at the high dose in cluded decreased food consumption/efficiency, ophthalmologic abnormalitie s, and altered hemato logical and clinical chemistry parameters (increased cholesterol and decreased AST, ALT, a nd CK concentrations).
Determination of AChE activitie s at 6-month intervals indicate d dose-dependent decreases (NS or p<0.05) in plasma AChE ( ↓ 17-57%), RBC AChE ( ↓ 19-38%), and brain AChE ( ↓ 10-31%) in males and females at the mid and high doses, with a return to control le vels demonstrated in high-dose recovery animals by Week 56.

Absolute liver and kidney weights were decreased (p<0.05) in high-dose females ( ↓ 16 and 13%, respectively), with relative (to body) weight incr eases (p<0.05) for liver and kidney in high-dose

m ales and females at Week 53. At study termin ation, decreased (p<0.05) absolute liver and kidney weights were observed in high-dose males ( ↓ 14 and 14%, respectively) and females ( ↓ 12 and 16%, respectively), with rela tive (to body) weight increases (p<0.05) for liver and kidney in all high-dose animals. No treatment-related changes were seen in the absolute or relative weights of the testes, epididymides, ovaries, or ad renal glands (except for decreased [p<0.05, ↓ 45%]CARBARYL FINAL

Page 51 of 62 absolute adrenal weight in high-dose males at study termination). No treatment-related histopathological findings were seen in the epididymides, ovari es, uterus, vagina, cervix, and adrenal or pituitary glands at any dose. Histopa thological effects at th e high dose that were
observed after unscheduled deaths and/or in su rviving animals at termination included thyroid hypertrophy (males, 11% vs. 1% control and fe males, 48% vs. 6%), hepatocyte hypertrophy (males, 49% vs. 6% control and females, 53% vs . 13%), renal transitional epithelium hyperplasia (males, 42% vs. 17% control), and hyperplasia in the urinary bladder (males, 76% vs. 11% control and females, 81% vs. 9%). Additional fi ndings in males included bilateral tubular atrophy, decreased secretion in seminal vesicles , and suppurative inflammation in the prostate.

Tumorigenicity was observed in both sexes at the high dose; these included increases in the incidences of liver adenoma in females, urinar y bladder transitional ce ll adenoma and carcinoma in males and females, and thyroid follicul ar cell adenoma and carcinoma in males.

The high dose (7500 ppm; 349.5/48 4.6 mg/kg/day in males and females, respectively) was demonstrated as the MTD due to decreased body weight gain, decreased food efficiency, and alterations in hematology and clinical ch emistry parameters. The LOAEL is 1500 ppm (60.2/78.6 mg/kg/day in males and females, re spectively) due to decreased brain and RBC activity, alterations in clinical chemistry paramete rs, decreased adrenal weight in male rats at Week 104, and decreased body weights and body weight gains as early as Week 13. The NOAEL is 250 ppm (10.0/12.6 mg/kg/day in males and females, respectively).

Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study in mice ( MRID 42786901 ), Crl:CD-1®(ICR)BR mice (80/sex/dose group) were administered diet s containing carbaryl (99.3% pu rity) at 0, 100, 1000, or 8000 ppm (mean-measured intakes of 14.73/18.11, 145.99/180.86, and 1248.93/1440.62 mg/kg/day in males and females respectively) for 104 weeks. At study termination, all surviving mice in all groups were sacrificed for gross and histopa thological evaluation. Th e organs weighed at sacrifice included the adrenals, liver, kidneys, testes with epid idymides, and ovaries. Gross and microscopic pathological examinations were con ducted on these organs as well as the pituitary, thyroid/parathyroids, urinary bl adder, prostate, seminal vesicl es, mammary gland, and uterus.

Four males in the high-dose group died or we re sacrificed moribund dur ing Week 1 and were replaced. Systemic toxicity in the high-dose an imals was noted as thin appearance and hunched posture; these signs appeared early, disappeared by Week 6, and re-appeared during the last six months of the study. Body weights were decreased (p<0.05) in the high-dose animals at Weeks 1, 13, 26, 52, and 78 (7-13% in males and 7-14% in females), with similar decreases (NS) persisting at Week 104 ( ↓ 12 and 13%, respectively, in males and females). Body weight gain decreases (significance not reported) for the cumu lative measurement periods in the high-dose males and females ranged from 23-35% in males and 10-32% in females, with overall decreases of 38 and 32%, respectively.CARBARYL FINAL

Page 52 of 62 Brain and RBC AChE activities generally were decreased RBC at Weeks 53 and 105. In males,
RBC AChE activities were decreas ed (p<0.05) in the mid- a nd high-dose groups by 23 and 30% at Week 53, with only a 13% decrease (NS) at the high dose at Week 105. Additionally, in males, brain AChE activities were decreased (p<0.05) in the mid- and high-dose groups by 18 and 57% at Week 53 and by 13 and 40% at Week 105. In females, RBC AChE activities were decreased (NS) in the mid- and high-dose grou ps by 13 and 13% at Week 53 and by 20 and 24% at Week 105. Additionally, in females, brain AC hE activities were decr eased (p<0.05) in the mid- and high-dose groups by 13 and 47% at Week 53 and by 11 (NS) and 40% at Week 105.

At the interim sacrifice, increase d (p<0.05) absolute liver weight ( ↑ 24%) and decreased (p<0.05) absolute ovary weight ( ↓ 33%) was observed in the high-dose fe males. Relative weight increases (p<0.05) were present in the mid- and high-dose males, but absolu te weights were not affected. Relative weight changes also were observed for the liver (males and females) and ovary. Although no treatment-related effects were noted on absolute liver and kidney weights at the terminal sacrifice, relative weight increases (p<0.05) were noted in the high-dose animals. Effects on ovary weight were not noted at study termination. No treatment-related changes were seen in the absolute or relative weights of th e testes with epididymid es or adrenal glands.

Non-neoplastic histopathol ogical lesions included an increased incidence of intra-cytoplasmic protein-like droplets in the superficial epithelium of th e urinary bladder (mid- and high-dose animals), cataracts (high-dose animals; an increa sed incidence of opaque eyes was noted in highdose females during gross necropsy), and an increas ed severity of extramedullary hematopoiesis and pigment in the spleen in high-dose animals. No treatment-related hi stopathological findings were seen in the testes, epididymides, semina l vesicle, prostate, ovaries, uterus, mammary, thyroid, adrenal or pituitary glands.

An increased incidence of neoplasms (hemangi omas and hemangiosarcomas) of the vascular system was observed in all treated males and in the high-dose females. In addition, an increased incidence of renal neoplasms (adenomas, multiple adenomas, and carcinomas) was observed in the high-dose males, and an increased incide nce of hepatic neoplasms (adenomas, carcinomas and one hepatoblastoma) was observed in the high -dose females. The study demonstrated that carbaryl is carcinogenic in mice at doses of 100 ppm (14.73 mg/kg/day) or higher in males and 8000 ppm (1440.62 mg/kg/day) in females.

The high dose (8000 ppm; 1248.93 and 1440.62 mg/kg/da y in males and females, respectively)
was demonstrated as the MTD ba sed on clinical signs, decreases in body weight gain, alterations in clinical pathology parameters, and hist opathology changes. The systemic LOAEL was 1000 ppm (145.99 and 180.86 mg/kg/day in males and fe males, respectively) based on decreases in RBC and brain AChE activities, increased kidney weight (int erim sacrifice), and bladder pathology (males) and on a decrease in brain ac tivities and bladder pathology (females). The systemic NOAEL was 100 ppm (14.73 a nd 18.11 mg/kg/day, respectively).CARBARYL FINAL

Page 53 of 62 Chronic Toxicity (Dog, OCSPP 870.4100)

In a chronic toxicity study ( MRID 40166701 ), carbaryl (99% purity) was administered to Beagle dogs (6/sex/group) in the diet at doses of 0, 125, 400, and 1250 ppm (calculated doses based on standard conversion of 3.12, 10, or 31.25 mg/kg/da y) for 52 weeks. At study termination, all surviving dogs were sacrificed for gross and hist opathological evaluation. The organs weighed at
sacrifice included the adrenals , thyroid, pituitary, liver, kidne ys, testes, epididymides, and ovaries. Gross and microscopic pathological exam inations were conducted on these organs as well as the urinary bladder, pros tate, mammary gland, and uterus.

The occurrence of clinical signs ( e.g. , emesis, lacrimation, salivati on, and tremors) was increased in the high-dose females during the 52-week study. In addition, body weight gains were decreased (p ≤ 0.05) by 50% in the high-dose females dur ing Weeks 0-5 compared to control (body weight data were not provi ded in review). Lesser decrease s (significance not reported) in body weight gain during were noted in females during Weeks 6-9 ( ↓ 4%) and Weeks 28-36 ( ↓ 6%). Terminal body weights in the low-, mid-, and high-dose males were increased (significance not reported) by 8, 9, and 15%, respectively.

There were no effects on mo rtality, food consumption, opht halmology, urinalysis, or macroscopic pathology. No treatment-related eff ects were observed on absolute or relative weights of the testes, epididymides, ovaries, or adrenal gland at any dose. Increased (p ≤ 0.05) absolute liver weight was observed in the high-dose males ( ↑ 24%), and absolute pituitary weights were decreased (NS) in th e low, mid- and high-dose males ( ↓ 8, 15, and 22%, respectively). Similar changes were seen in the relative weights with increased (NS) relative liver weight in the high-dose males ( ↑ 8%), and decreased relative pitu itary weights (NS) in the low, mid- and high-dose males ( ↓ 11, 22, and 33%, respectively). Re lative thyroid weight was decreased (p ≤ 0.05) in the high-dose males by 27%. No treatment-related histopathological findings were seen in the testes, epididymid es, prostate, ovaries, uterus, mammary gland, thyroid, adrenal, or pituita ry at any dose, as well as the liver or kidneys.

Plasma and RBC AChE activities were de termined at Weeks 5, 13, 26, and 52 (study termination). Brain AChE activity also was determined at Week 52. Decreases (p ≤ 0.05) in AChE activity were as follows: (i) low dose; females (plasma, ↓ 19-23% at Weeks 5, 13, and 26 and brain, ↓ 20% at Week 52); (ii) mid dose; males (plasma, ↓ 30-36% throughout the study and RBC, ↓ 23-28% at Weeks 5 and 13) and females (plasma, ↓ 26-31% at Weeks 5, 13, and 26; RBC, ↓ 29- 34% at Weeks 5, 13, and 26; and brain, ↓ 22% at Week 52); and (iii) high dose; m ales (plasma, ↓ 57-66% all intervals and RBC, ↓ 46-56% all intervals) and females (plasma, ↓ 47-60% all intervals; RBC, ↓ 29-38% all intervals; and brain, ↓ 36% at Week 52). Brain AChE activities were inhibited (NS) by 14-32% in the treated males.

Because the lowest dose tested (3.12 mg/kg/day) re sulted in AChE inhibition in the females, a five-week feeding study in dogs ( MRID 42022801 ) was conducted to provide supplementaryCARBARYL FINAL

Page 54 of 62 data to establish a NOAEL/LOA EL. Based on the results of th e five-week study, the LOAEL is 125 ppm (3.37 and 3.73 mg/kg/day in males and fe males, respectively) based on decreased plasma and brain AChE activity in females. The NOAEL is 45 ppm (1.43 and 1.54 mg/kg/day in males and females, respectively).

Subchronic Toxicity/Metab olism (Rat, OCSPP 870.7485)

In a rat metabolism study ( MRID 44402501 ), 1-naphthyl- 14 C-labeled carbaryl (100% purity)
was administered to 15-month-ol d male Iffa Credo CD (SD-deri ved) rats (5/dose group) as a single oral gavage dose of 50 mg/kg (group A) or as a daily oral dose of 2 mg/kg for 7 days after an 83-day dietary administration with unlabeled carbaryl (98. 4 and 99.8% purity; 25/dose group) at 0 (group B), 250 (Group C), 1500 (Group E), or 7500 ppm (Group D). The purpose of this study was to investigate the mechanism(s) that resulted in an increased incidence of tumors during the final year of a chronic dietary feedi ng study in the rat at a ca rbaryl dose of 7500 ppm.

In all dietary dosing regimens, urinary and fecal excretion totaled 96-103% of the administered dose. Most of the radioactivity was eliminated in the urine and feces within 24 hours after dosing. Further discussion of carbary l excretion is not relevant to the EDSP WoE, and, therefore, is not included for purposes of this discussion. Tissue distribution for single- or multiple-dose administration of carbaryl was minimal, indicati ng that the potential for bioaccumulation of carbaryl in rats is low.

HPLC analysis of carbaryl metabolites in urine, fecal, and tissue samples indicated the presence of approximately twenty components. Several of the major components identified by LC/MS are the Phase II conjugates (sulfate and glucur onide) of carbaryl metabolites (5,6-dihydro-5,6- dihydroxy-1-naphthyl N-methyl carbamate and naphthyl).

Body weight was decreased (p<0.05 or p<0.01) by 9-20% compared to control in Group D (7500 ppm) as early as Study Day 14, with decreases sustained throughout the remainder of the study. Terminal body weights were decreased by 12 and 16% in the 1500 ppm and 7500 ppm dose groups. Decreases in body weight gain and food consumption also were observed throughout the study.

Increases (statistical analyses were not conducted) in liver and thyroid we ights were observed in the 1500 and 7500 ppm groups compared to control. Absolute and relative liver weights were increased 18% and 39%, respectiv ely, in the 7500-ppm group. Absolute thyroid weight was increased (non-dose-dependent) by 63% a nd 69% in the 7500 and 1500 ppm dose groups, respectively, and relative thyroid weights were increased 103% a nd 121%, respectively.

There were no treatment-related increases in th e incidence of hepatoce llular adenoma (benign), C-cell adenoma in the thyroid gland, or follicul ar cell adenoma (benign). However, treatmentrelated changes were noted in liver, thyroi d glands, and kidneys in the 7500 ppm group. TheCARBARYL FINAL

Page 55 of 62 incidences of hepatic centrilobular hypertrophy, pericholangitis (an inflammatory cell infiltrate around biliary ducts), and bile duct hyperplasia were increased non-dose-dependently (5/5, 3/5, and 3/5, respectively). In addition, the incidenc es of follicular cell hypertrophy of the thyroid glands were 0/5, 3/5, 5/5, and 5/ 5 and the incidences of transiti onal cell hyperplasia of the renal pelvis were 0/5, 0/5, 1/5 a nd 2/5 in the 0, 250, 1500, and 7500 pp m dose groups, respectively.

Avian Reproduction (OCSPP 850.2300)

In an avian reproduction study ( MRID 00161500 ), mallard ducks ( Anas platyrhynchos ) were administered carbaryl (99.9% purity) at nom inal concentrations of 0, 300, 1000, or 3000 ppm (mg/kg) in the diet for 24 weeks (twelve weeks prior to the start of egg production and twelve weeks of egg production). After the completion of dose administration at Week 24, there was a three-week recovery period for surviving birds. Mean-measured carbaryl concentrations were 0, 280, 924, and 2932 mg/kg diet. Each treatment group was comprised of sixteen pens, with one male and one female per pen. Body weights were recorded at Weeks 0, 2, 4, 6, 8, and 10, and at termination of the study Week 24). Food cons umption was recorded biweekly. Egg shell thickness was determined on all eggs once every other week after egg production had reached approximately 20%.

There were two mortalities on study in the high-dose group (one/sex ) at Weeks 13 and 19.
Neither death was considered to be treatment-re lated. Body weight gains were decreased in a dose-dependent manner in all treatment groups, with decreases (NS) in males of 15, 44, and 72%, and decreases (NS at the low dose and p<0. 05) in females of 53, 73, and 85%, respectively.

The reviewer's analysis detected significant adverse effects on several reproductive endpoints during the 24-week exposure period. Eggs laid a nd eggs set were adversely affected at the 924 and 2932 mg/kg diet doses (while embryo viability /hatchlings was significantly reduced on a per pen basis, these endpoints were not significant based on compar ison to eggs se t); hatchling weight were significantly reduced at only the hi ghest dose. Based on these effects, the 24-week exposure period NOAEC and LOAEC are 280 and 924 mg /kg diet, respectively. Analysis of the three-week recovery period data revealed signi ficant differences. Viab le embryos and number and survival of hatchling (based on eggs set) we re reduced, with significant reductions at the top dose. Hatchling weight was signi ficantly reduced at the mid a nd high dose. The appearance of these effects during the recovery period suggests that adverse worse n, even after the test material
is removed.

In another avian reproduction study ( MRID 00160044 ), bobwhite quail ( Colinus virginianus ) were administered carbaryl (99.9% purity) at nominal concentrations of 0, 300, 1000, or 3000 ppm (mg/kg) in the diet for 22 weeks. No treatment-related effects were observed on survival, growth, or reproductive parameters in this study. The NOAEC is 3000 ppm (mg/kgdiet) and the LOAEC is >3000 ppm (mg/kg-diet).CARBARYL FINAL

Page 56 of 62 In another mallard reproduction study ( MRID 49312801 ), which was preliminarily reviewed, carbaryl (99.2%) was administered in diet at nominal concentrations of 111, 333, and 1000 mg/kg-diet for 22 weeks. There was one adult mortality at the 1000 mg/kg-diet, and no reported treatment-related clinical symptoms. There was no reported effect on food consumption or male body weight gain, but female body weight gain was significan tly decreased (p<0.05) at 1000 mg/kg-diet (195.5 gr in control vs . 73.2 gr in 1000 mg/kg-diet). The number of eggs laid per hen was significantly reduced at 1000 mg/kg-diet (45.8 in contro l vs. 31.1 at 1000 mg/kg-diet); percent of eggs set of eggs laid also significantly reduced at this te st level. A significant decrease in egg strength and eggshell thickness (5.5%) reported at 1000 mg/kg- diet. Initial and 14-d hatchling weights were also significantly reduced. No other e ffects were noted.

Mechanistic Study: Male Reproduction Toxicity

In a published, non-Guideline study, Pant et al. (1995) evaluated the toxicity of carbaryl on the reproductive system of male rats. Carbaryl (pur ity 99.2%) was administered five days/week for 90 days by oral gavage to male Wistar albino rats (8/dose group) at doses of 0 (vehicle), 50, and 100 mg/kg/day in peanut oil. Rats were weighed approximately every fifteen days. After the final dose, rats were fasted overnight and euthani zed on Day 91, and testes, epididymides, seminal vesicles, ventral prostate, and coagulating gla nds were excised and weighed. Sperm count, sperm motility and morphology were determined with sp erm obtained from the epididymis. One testis from each animal was preserved for histology in 10% neutral buffere d formalin, dehydrated, infiltrated and embedded in paraffin, sectione d, and stained with hematoxylin and eosin for examination by light microscopy. Sections of the other testicle were homogenized for testicular enzyme assays. The assays included measurem ents of sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH), glucose-6- P-dehydrogenase (G6PDH), and γ -glutamyl transpeptidase ( γ GT). Protein content was determined with the Lowry method.

The only treatment-related sign of toxicity wa s lethargy in the carbaryl-treated rats. Body weight/body weight gain was decreased in the 100 mg/kg/day dose group by Day 60 (data presented graphically), and there was a dose-de pendent decrease in body weight by Day 90 in both treatment groups (significance not reported) . There were no treatment-related effects on weights of the testes, epididymides, seminal vesicl e, ventral prostate and coagulating gland (data not provided).

Enzyme specific activities (nmo l product/min/mg protein) in the testes were decreased (p<0.05) for SDH ( ↓ 49%) and G6PDH ( ↓ 71%) in the 100 mg/kg group. In contrast, the activities of LDH
and γ GT in the testes were increased (p<0.05) by 60% and 61%, respectively, in the 50 mg/kg dose group, and by 105% and 153%, respectiv ely, in the 100 mg/kg dose group.

Total sperm counts were decreased in the low ( ↓ 40%) and high ( ↓ 60%) dose groups, and the percentages of sperm motility we re also decreased by 25 and 63%, respectively. In addition, the percentages of morphological abnormalities in sperm were increased to a similar degree in bothCARBARYL FINAL

Page 57 of 62 treatment groups (data illegible). Histopathological examinations of the testes from the 50 mg/kg dose group found slight to moderate congesti on and edema in the peripheral region, and seminiferous tubules with depr essed spermatogenesis, loss of sperm, and moderate atrophy. In the 100 mg/kg dose group, examination of the testes showed an increased intensity of congestion and edema in the peripheral and central regi ons, and seminiferous tubules with disturbed spermatogenesis and acellular homogeneous masses.

The authors concluded that exposur e to carbaryl in male rats ( up to 100 mg/kg/day) results in testicular atrophy and affects spermatogenesi s, yielding reduced sperm production and an increase in the percentage of abnormal spermatozoa.

Mechanistic Study: Spermatotoxic Effects

In another publishe d, non-Guideline study, Pant et al. (1996) evaluated the spermatotoxic effects of the insecticide carbary l in male rats. Carbaryl (pur ity 99.2%) was administered daily by oral gavage 5 days/week for 60 days at 0 (veh icle), 25, 50, and 100 mg/kg/ day in peanut oil to both immature and mature male Druckery albino rats (6 young and 6 old rats/dose group). The rats were provided pell eted diet and water ad libitum , and were housed under standard animal care conditions. Body weights were determined at initiation and comple tion of the study. After the final dose, the rats were fasted overnight and sacrificed by cervi cal dislocation on Day 61. Testes, epididymides, seminal vesicles, ventral prostate, and coagulati ng glands were excised and weighed at necropsy. Sperm count, sperm motility, and morphology were determined from epididymal sperm.

No mortality or signs of toxicity occurred in the control or treatme nt groups. Effects on body weight gain were reported in the young and a dult males at 50 and 100 mg/kg/day, with greater effects the young males (data, a nd direction of effect, were not reported). For the young rats, absolute weights of testes, epididymides, semi nal vesicles, ventral prostate, and coagulating gland were decreased (p<0.05) in the 100 mg/kg/day dose group onl y (data not reported), but no
effects were observed in the rela tive weights of reproductive organs. No e ffects were observed on absolute or relative organ weights in the adult males (data not reported).

For young and adult rats, sperm motility and coun t were decreased (p<0.05) in the 50 and 100 mg/kg/day dose groups in a dose- and age- dependent manner. For the young males, sperm motility and sperm count were respectively reduc ed by 24% and 25% at the 50 mg/kg/day dose, and by 43% and 38% at the 100 mg /kg/day dose. For the adult ma les, sperm motility and sperm count were respectively reduced by 15% and 13% at the 50 mg/kg/day dose, and by 26% and 25% at the 100 mg/kg/day dose. The decrease s in sperm motility and count were 50 to 100% greater in young vs. old male rats. The percentage of abnormal sperm were increased (p<0.05) at 50 and 100 mg/kg/day, also in a dose- and age-dependent manner. At the mid- and high-doses, abnormal sperm accounted for 20% and 34% of a ll sperm from young males, respectively, and 16% and 23% of all sperm from adult males, respectively. Abnormalities in control males (youngCARBARYL FINAL

Page 58 of 62 and adult) accounted for 10% of all sperm, and a similar level of abnormalities (11%) was observed in the low-dose group. Abnormalities in th e head, neck and tail of the sperm were observed for both young and adult males, but bent up or down acrosomes were observed only in young males.

The authors concluded that carbar yl had spermatotoxic effects in young and adult rats at doses of 50 and 100 mg/kg/day. The LOAEL and NOAEL for sper matotoxic effects of carbaryl in rats were 50 and 25 mg/kg/day, respectively.

Mechanistic Study: Teratogenicity (Frogs)

In a published study, Bacchetta et al . (2008) evaluated the embryotoxi c effects of carbaryl (99.8% purity) on the African Clawed frog ( Xenopus laevis ) during conduct of a modified Frog Embryo Teratogenesis Assay-Xenopus (FETAX), coupl ed with a histopathol ogical screening of the surviving larvae. Embryos were exposed to 0 (FETAX solution), 1, 2, 4, 8, 16, and 24 mg/L carbaryl from stage 8 to stage 47. Blastulae from the same female were placed one per well in a twelve-well plate; each plate cont ained 4 mL of control or carbar yl test solutions. Plates were duplicated or triplicated for each female if well-c leaved embryos were available. All the plates
were incubated in a thermosta tic chamber at 23±0.5°C until the embryos reached stage 47 (freeswimming larva). The number of dead larv ae was recorded for each treatment group, and survivors were anaesthetized with MS-222 (100 mg/L) and evaluated for single malformations by examination under a dissecting microscope. Larv ae were fixed or frozen for the successive histological and biochemical an alyses, respectively. The assay was repeated three times under the same experimental conditions.

Histological examinations were conducted on co ntrol and treated larvae (35/group), except for the 24 mg/L group due to gross malformations in the surviving larvae in this group. Stage 47 control larvae, and those exposed to carbaryl at 4, 8, and 16 mg/L, were used for AChE activity evaluation that was determined in three independ ent experiments from five pools of five whole body larvae. Each assay was performed in trip licate with a modified procedure with acetylthiocholine iodide (0.075 M) as a substrate. AChE activity was calculated as nmol of acetylthiocholine iodide hydrolyzed/min/g larv ae, and the results were expressed as the percentage of AChE i nhibition vs. control.

Very low mortality was observed at carbaryl concentrations of 1-8 mg/L; mortality was increased (p<0.001) at 16 and 24 mg/L (15 and 76%, respectively). There was a concentrationdependent increase (p<0.01 or 0.001) in percenta ge of overall malformations from 6.8% at 1 mg/L to 100% at 24 mg/L, w ith no malformations observed in the control embryos. The calculated teratogenic in dex of 2.41 (calculated as the ratio of the LC 50 and the TC 50 [concentration that yields 50% teratogenicity]) indicates that carbaryl is teratogenic to Xenopus embryos. Abnormal tail flexure (dorsal flexure only) and abnormal gut coiling were the most
often observed malformations.CARBARYL FINAL

Page 59 of 62 All larvae at the mid-high concentration had path ological lesions; none of the larvae exposed to the high concentration were processed due to se vere external malforma tions. Histopathological
examination indicated that the tail musculatur e and the notochord were the main targets for carbaryl effects. Inhibition of AC hE activity was investigated in larvae exposed to carbaryl concentrations lower and higher than the calculated TC 50 (8.43 mg/L). A well-defined concentration response was observe d between the higher carbaryl co ncentrations and percentages
of AChE inhibition observed at these carbaryl exposures. In th e 8 mg/L carbaryl-treatment group (approximate TC 50 ), the AChE was decreased by 61.6%. Mean AChE concentrations were decreased by 35.6% in the 4 mg/L concentratio n group and decreased by 77% in the 16 mg/L group. This study did not expose Xenopus laevis ) to carbaryl during the th yroid-critical period of metamorphosis and development (stages 51-57), but the inhibition of AChE is implicated in the
treatment-related malformations a nd histopathological effects.CARBARYL FINAL

Page 60 of 62 APPENDIX 3: References Not Utilized in the Carbaryl 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 Carbaryl 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.

Beauvais, S.L., Jones, S.B., Parris J.T., Brewer, S.K. and Little, E.E. (2001) Cholinergic and
Behavioral Neurotoxi city of Carbaryl and Cadmium to Larval Rainbow Trout ( Oncorhynchus mykiss ). Ecotoxicol. Environ. Saf . 49: 84-90.

Beyers, D.W. and Sikoski, P.J. (1994) Acetylc holinesterase inhibition in federally endangered Colorado squawfish exposed to carbaryl and malathion. Environ. Toxicol. Chern . 13:935-939.

Boone, M.D. (2007). Examining the single and in teractive effects of three insecticides on amphibian metamorphosis. Environmental Toxicology and Chemistry , Vol. 27 No.7: 1561-1568.CARBARYL FINAL

Page 61 of 62 Boone, M.D. and Bridges, C.M. (2003) Effects of carbaryl on green frog ( Rana clamitans ) tadpoles: timing of exposure versus multiple exposures. Environmental Toxicology and Chemistry , Vol. 22 No. 11: 2695-2702.

Boone, M.D., Bridges, C.M. and Rothermel, B. B. (2001) Growth and development of larval green frogs ( Rana clamitans ) exposed to multiple doses of an insecticide. Oecologia 129: 518-
524.

Boone, M.D. and Semlitsch, R.D. (2003) Intera ctions of bullfrog tadpole predators and an insecticide: predation release and facilitation. Oecologia 137: 610-616.

Carlson, A.R. (1971) Effects of long-term exposure to carbaryl (Sevin), on survival, growth, and reproduction of the fathead minnow (Pimephales promelas). J. Fish. Res. Board Can . 29(5):583- 587. (MRID 40644801)

Choudhury, C., Ray, A.K., Bhattacharya, S., and Bhattacharya, S. (1993) Nonlethal concentrations of pesticide impair ovari an function in the freshwater perch, Anabas testudineus

. Environ. Biol. Fishes . 36: 319-324.

Ghosh, P. and Bhattacharya, S. (1990). Impairment of the regulation of gonadal function in Channa punctatus by Metacid-50 and Carbaryl under laboratory and field conditions. Biomed.
Environ. Sci . 3:106-112.

Klimisch, H.-J., Andreae, M, and Tillman, U. ( 1997) A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data. Regulatory Tox. and Pharm

. 25: 1-5.

Kulshrestha S.K. and Arora N. (1984) Impairments induced by sublethal doses of two pesticides
in the ovaries of a freshwater teleost Channa striatus Bloch. Toxicol. Lett . 20 93-98.

Mathew L, Reddy MLP, Rao TP, lyer CSP, Da modaran AC. (1995). Simple spectrophotometric method for the determination of carbaryl in soil and insec ticide formulations. Analyst. 120:1799-
801.

Meeker, J.D., Ryan, L., Barr, D.B., Herrick, R.F., Bennett, D.H., Bravo, R., and Hauser, R. (2004) The relationship of urinary metabolites of carbaryl/naphthalene and chlorpyrifos with human semen quality. Environ. Health Perspect . 112(17): 1665-70.

Meeker, J.D., Barr, D.B., Serdar, B., Rappaport, S. M., and Hauser, R. (2007) Utility of urinary 1- naphthol and 2-naphthol levels to assess environmental carbaryl and naphthalene exposure in an epidemiology study. J. Expo. Sci. Environ. Epidemiol . 17(4):314-20.CARBARYL FINAL

Page 62 of 62 Peterson, H.G., Boutin, C., Martin, P.A., Freema rk, K.E., Ruecker, N.J., and Moody, M.J. (1994) Aquatic phytotoxicity of 23 pesticides applie d at expected environmental concentrations. Aquat.
Toxicol . 28:275-92.

Sinha, N., Lai, B., and Singh, T.P. (1991) Carbaryl -induced thyroid dysfunction in the freshwater Catfish, Clarias batrachus

. Ecotoxicol. Environ. Saf . 21: 240-247.

Sinha, N., Lai, B., Singh, T.P. (1991) Pesticides induced changes in circulating thyroid hormones in the freshwater catfish, Clarias batrachus

. Comp. Biochem. Physiol. C . 100:107-110.

Todd, N.E. and Van Leeuwen, M. (2002) Effects of Sevin (carbaryl insecticide) on early life stages of zebrafish ( Danio rerio ). Ecotoxicol. Environ. Saf . 53:267-72.

Zinkl, J.G., Shea P.J., Nakamoto R.J., and Callman J. (1987) Brain cholinesterase activity of rainbow trout poisoned by carbaryl. Bull Environ Contam. Toxicol . 38: 29-35.