UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

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

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

Page 1 of 70
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 70
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: CARBARYL 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 70
CARBARYL 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 Relevant Information (OSRI)......................................4 

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

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

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

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

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

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................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-Estrogenic Pathway for Carbaryl....................................10 

 Table 3. Androgenic/Anti-Androgenic 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...........................60 

Page 4 of 70
CARBARYL 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 Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

Page 5 of 70
CARBARYL FINAL 

iv 

Abbreviation Terminology 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods

Kd Equilibrium Dissociation Constant 

KOW Octanol/Water Partition Coefficient 

L Low dose 

LABC Levator Ani-Bulbocavernosus

LAGDA Larval Amphibian Growth and Development Assay

LC50 Lethal Concentration in 50% of test organisms 

LD Lactation Day 

LH Luteinizing hormone 

LOAEC Lowest Observed Adverse Effect Concentration 

LOAEL Lowest Observed Adverse Effect Level 

LOQ Limit of Quantitation 

M Male 

MDL Minimum Detection Level

MEOGRT Medaka Extended One Generation Reproduction Test

MH Medium high 

ML Medium low 

MoA Mode of Action 

MOE Margin of Exposure

MRID Master Record Identifier 

MROD Methoxyresorufin-O-dealkylase 

MTC Maximum Tolerated Concentration 

MTD Maximum Tolerated Dose

Page 6 of 70
CARBARYL FINAL 

v 

Abbreviation Terminology 

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

TP Testosterone Propionate

Page 7 of 70
CARBARYL FINAL 

vi 

Abbreviation Terminology 

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

Page 8 of 70
CARBARYL FINAL 

Page 1 of 62 

Executive Summary 

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

necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), 

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

at the estrogen and androgen receptors, altered steroidogenesis, as well as 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 considered 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 responses observed across studies, taxa, and sexes 

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

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

general systemic toxicity or overt toxicity. 

On October 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) 

conducted a weight-of-evidence (WoE) analysis of the potential interaction of 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) beginning with the results of the Tier 1 in vitro assays 

followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for 

mammalian and wildlife studies (40 CFR Part 158 and literature). 

For carbaryl, there is no convincing evidence for an interaction with the estrogen pathway. While 

there was equivocal or conflicting responses in the guideline and non-guideline estrogen receptor 

(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 studies in mallard ducks whereas 

decreases in female body weight gain were also observed, no estrogen-related effects were noted in 

the in vivo studies with carbaryl.

For the androgen pathway, carbaryl was 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 perturbations 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 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 significant female weight decreases) were observed. However, 

Page 9 of 70
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 interaction 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 presence 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 convincing evidence for interaction of carbaryl with the 

androgen pathway in mammals. However the evidence suggest potential interaction with the 

androgen pathway in fish. 

Based on weight of evidence conclusions, mammalian EDSP Tier 2 testing is not recommended for 

carbaryl since there is no evidence for potential interaction with the estrogen, androgen or thyroid 

pathways. 

Effects were observed in males in the FSTRA at 0.82 mg/L which 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 group at 0.68 mg/L with a NOAEC of 0.21 mg/L. 

Effects on F1 survival were observed 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, 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 considered sufficient for evaluating potential reproductive effects to 

birds from carbaryl exposure. Relative to the EDSP, additional testing is not recommended. 

 

Page 10 of 70
CARBARYL FINAL 

Page 3 of 62 

I. Introduction 

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

necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), 

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

at the estrogen and androgen receptors, altered steroidogenesis, as well as 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 considered 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 responses observed across studies, taxa, and sexes 

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

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

general systemic toxicity or overt toxicity. 

On October 8, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of 

the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) 

conducted a weight-of-evidence (WOE) analysis of the potential interaction of 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) beginning with the results of the Tier 1 in vitro assays 

followed by in vivo mammalian and wildlife results, then the results of the cited OSRI for 

mammalian and wildlife studies (40 CFR Part 158 and literature). 

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 phases on release to the atmosphere. 

Vapor-phase carbaryl is degraded by reaction with photochemically-produced hydroxyl radicals in 

the atmosphere, with an estimated half-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 determining 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 studies and/or other published articles. These data are summarized in 

Sections III.A through III.C. An analysis of the data submitted to the agency, using the WoE 

approach outlined by the Agency (USEPA, 2011), is presented in Section IV. The EDSP Tier 2 

studies recommendations are presented in Section V. 

Page 11 of 70
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 below in Table 1. Executive Summaries are presented in 

Appendix 1. 

B. Other Scientifically Relevant Information (OSRI) 

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

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

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

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

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

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

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

literature/studies considered but not utilized for the WoE analysis are listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

the WoE guidance document (USEPA, 2011) and presented at the 2013 FIFRA Scientific Advisory 

Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how the individual lines of 

evidence are assembled and integrated along two concepts (i.e., complementarity and redundancy) 

within the conceptual framework of an adverse outcome pathway). Broadly, there are four main 

steps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to 

evaluate the individual studies for their scientific quality and relevance in evaluating potential 

endocrine interaction(s). The second step is to integrate the data along different levels of biological 

organization while examining the extent of complementarity (i.e., the concordance of endpoints 

within an assay that measures multiple endpoints) and redundancy (i.e., the concordance of 

endpoints/responses across assays) in the observed responses across these different levels of 

biological organization. The third step is to characterize the main lines of evidence as well any 

conclusions. Finally, the last step is to evaluate whether additional testing is needed based on the 

evidence and conclusions described above. 

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

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

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

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

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

Page 12 of 70
CARBARYL FINAL 

Page 5 of 62 

binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the 

HPG and HPT axes. OSRI may include published literature studies as well as studies conducted 

under USEPA (often referred to as Part 158 data) or OECD guidelines submitted in support of 

pesticide registrations. Each study is evaluated for scientific quality and relevance for informing 

interactions with the E, A or T pathway. Additionally, the concordance or consistency 

(complementarity) of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, 

often multiple endpoints are measured in each assay. 

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

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

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

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

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

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

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

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

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

These two concepts provide a basis for considering the plausibility, coherence, strength, and 

consistency of the body of evidence. The current EDSP Tier 1 screening assays are meant to 

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

biological organization from a molecular initiating event such as receptor binding through potential 

adverse effects in apical endpoints such as sexual development and fecundity at the whole organism 

level. The extent of expression of responses at higher levels of biological organization can indirectly 

provide information on potential compensatory capabilities of an individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit compensatory 

responses at higher-levels of biological organization? Finally, under what conditions did responses 

occur including consideration of whether the responses were observed in the presence of overt or 

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

ability to distinguish effects specifically related to an endocrine-related effect from a non-endocrine 

toxic response. 

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

2013 FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the 

EDSP Tier 1 screening process. In October, 2013, the SAP stated that , “In summary, the Panel 

agreed that little, if any, weight should be placed on signs of endocrine disruption in the presence of 

Page 13 of 70
CARBARYL FINAL 

Page 6 of 62 

overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms of primary 

interactions with the endocrine system vs. secondary effects related to toxicity in non-endocrine 

organs or overall disruptions in homeostasis” (USEPA, 2013). 

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

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

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

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

and abnormal swimming (fish and aquatic-phase amphibians); and body weight decreases of ≥10% 

in mammals. Additionally, other effects including morphological (e.g., organ 

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

were also considered when evaluating overt toxicity, especially if the effects were extreme. In some 

instances, one parameter (e.g., death or >10% decrease in mammalian body weight) was sufficient to 

consider a dose/concentration to be overtly toxic. However, in other instances, more than one 

parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body 

weight decreases were considered along with other responses when assessing potential overt 

toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also 

captured and these effects were generally considered as less severe forms of toxicity (e.g., changes in 

organ weights or blood chemistry). The circumstances for which a dose/concentration was 

considered overtly toxic for a particular study are described in Section IV.A. 

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

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

modes of action in its overall characterization of a chemical’s potential to interact with the E, A or T 

pathway. Adequately addressing the three components described above is fundamental to the WoE 

process and in determining whether additional data are needed. In addition to characterizing the 

WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) 

discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of 

evidence supporting a conclusion; and, 5) what, if any, additional data are needed and 

why. Assessing the need for additional data is based on a case-by-case analysis which took all 

available toxicity data into account. .

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

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

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

supports a conclusion of whether a substance has the potential to interact with the E, A or T 

pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each 

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

Page 14 of 70
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) 

ERα 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) 

Page 15 of 70
CARBARYL FINAL 

Page 8 of 62 

B. Effects on Hypothalamic-Pituitary-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 assays are delineated so as to 

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

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

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

considered further in the WOE assessment. 

The mean specific binding of carbaryl 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 independent 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 IC50 of >10-4 M in a nonguideline rat uterine cytosol binding assay while Klotz et al., (1997) reported carbaryl as positive for 

ER binding in a MCF-7 whole cell binding assay with an IC50 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 and 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 antagonists in transiently transfected ER reporter gene assays in breast 

cancer and endometrial cancer cell lines. Lemaire et al., (2006), reported negative findings for ERα

and ERβ agonism using a reporter gene in stably transfected HeLa cells; however carbaryl treated 

cells showed antagonistic activities toward both ERα and ERβ in the presence of estradiol in these 

cells. The authors also reported that carbaryl inhibited [3H]-estradiol binding by 45 % in these cell 

lines at 10-5 M. 

In the Tier 1 aromatase assay, carbaryl exhibited a concentration-dependent inhibition of aromatase 

activity, decreasing activity by more than 50% with a Log IC50 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 negative in the steroidogenesis assay for 

estradiol production.

Carbaryl was classified as negative for estrogenic activity in the uterotrophic assay. The requirement 

for the female pubertal assay was considered satisfied by OSRI, including a two-generation 

reproduction study (MRID 45448101) conducted according 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). 

Page 16 of 70
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 F1 females at the high dose (approximately 135 mg/kg/day) 

which co-occurred with decreased body weights (11%) in the pups on PND 4-21. There were no 

other effects on estrogen-related endpoints, 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 study 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 observed 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 toxicity. 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 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 dose (MRID 49312801). No treatment-related effects were noted on 

reproduction in the avian toxicity study in bobwhite quail up to 3000 mg/kg-diet. 

Page 17 of 70
CARBARYL FINAL 

Page 10 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Carbaryl 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Carbaryl1Study Type/ Literature Citation 

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) 

EDSP Tier 1 Assay 

ER Binding (MRID 48615203) E ERTA (MRID 48615204) N Aromatase (MRID 48615202) E Steroidogenesis (MRID 48615205) N Uterotrophic (MRID 47987702) N Female Pubertal Rat Requirement satisfied by OSRI (Two-generation reproduction study, MRID 45448101; see beFSTRA (MRID 48669601) NE N N N N N 

OSRI 

ER Transactivation (Kojima et al., 2004) N ER Binding (Blair et al., 2000) N ER Binding (Klotz et al., 1997) P PERTA (Lemaire et al., 

2006) P P 
CARBARYL FINAL 

Page 11 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Carbaryl 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Carbaryl1Study Type/ Literature Citation 

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) 

Two-generation reproduction (Rat; MRID 45448101) 

 N N N N N N N 

Developmental neurotoxicity (Rat; MRID 44393701) 

 N N 

Developmental toxicity (Rat; MRID 44732901) 

 N N 

Developmental toxicity (Rabbit; MRID 44904202) 

 N N 

Chronic toxicity/ carcinogenicity (Rat; MRID 42918801) 

 NE N N NE 

Carcinogenicity (Mouse; MRID 42786901) 

 NE N N NE 

Chronic toxicity (Dog; MRIDs 40166701 and 42022801) 

 NE N N N 

Avian reproduction (Duck; MRID 00161500) 

 P4 

Avian reproduction (Quail; MRID 00160044) 

 N 
CARBARYL FINAL 

Page 12 of 62 

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

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

weight 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 O

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 

details see Section IV. A 

4. Decrease in the number of eggs laid at mid and high treatment (924 and 2932 mg/kg-diet); hatchling weight was significantly redu

During a recovery period, viable embryos and number and survival of hatchling (based on eggs set) were reduced at the top dose, significantly 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 

body weight gain were also observed. There were no other signs of toxicity. 

E Equivocal P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined 
CARBARYL 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 androgenic, anti-androgenic, or HPG axis effects. This 

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

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

considered further in the WOE assessment. 

In an evaluation of androgenic/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 noted in testosterone production 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 agonist or antagonist activity in an AR transcriptional activation 

assay in CHO-K1 cells transfected with hAR (Kojima et al., 2004). 

Carbaryl was negative for anti-androgenic activity 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 F1 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 observed 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 testicular 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 decreased pituitary weights at the mid- and highdoses were associated with overt toxicity (i.e., significant decreases in AChE activities). No 

androgen-related effects were seen in the other Part 158 mammalian studies. 

Page 21 of 70
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 top 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 study which reported decreases in offspring body 

weight). Decreases in offspring body weight were 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 and adult Drucker albino rats received 25, 50, or 

100 mg/kg/day (5 days/week) for 60 days. The results of the first study indicated 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-related effects also were 

apparent. Sperm count and motility were decreased in the 50 and 100 mg/kg/day dose groups, 

with greater decreases (↓50-100%) in the juvenile rats compared to adults. Increases in abnormal 

sperm also occurred, with a greater percentage present in young rats. Finally, reproductive organ 

weights were decreased in the high-dose juvenile rats only, and body weight gain was decreased 

in the 50 and 100 mg/kg/day dose groups, with a greater effect in the juvenile rats. 

Page 22 of 70
CARBARYL FINAL 

Page 15 of 62 

Table 3: Androgenic/Anti-Androgenic Pathway for Carbaryl Lines 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) 

EDSP Tier 1 Assay 

AR Binding (MRID 48615201) P Steroidogenesis (MRID 48615205) P Hershberger (MRID 47987701) NE N Male Pubertal Rat Requirement satisfied by OSRI (MRIDs +, 42918801, and 44402501; Pant et al., 1995 and Pant et al., 1996FSTRA (MRID 48669601) NE N P (H)5 P (H)6 N N 

OSRI

ARTA (Kojima et al., 2004) N AR Binding (Fang et al., 2003) P Two-generation reproduction (Rat; MRID 45448101) 

NE N N N N N N N 

Developmental neurotoxicity (Rat; MRID 44393701) 

N 

Chronic toxicity/ carcinogenicity (Rat; MRID 42918801) 

N N N N NE NE 

Carcinogenicity (Mouse; MRID 42786901) 

N N N N NE NE 
CARBARYL FINAL 

Page 16 of 62 

Table 3: Androgenic/Anti-Androgenic Pathway for Carbaryl Lines 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) 

Chronic toxicity (Dog; MRIDs 40166701 and 42022801) 

N N N N N NE 

Subchronic toxicity/ Metabolism (Rat; MRID 44402501) 

NE NE NE NE NE NE 

Avian reproduction (Duck; MRID 00161500) 

P7 

Avian reproduction (Quail; MRID 00160044) 

N 

Male Reproductive Toxicity-Rat  (Pant et al., 1995) 

 N P (L,H)9 N P ↓ (L,H)10 N 

Spermatoxicity-Rat (Pant et al., 1996)9 ↓ (H) NE ↓(H) P ↓ (L,H)12 ↓ SV, VP (H)13 

1. Key to responses: ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High. Arrows (↓ or ↑) indicate 

response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay. Changes in weight are abs

indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate (PRbulbocavernosus (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 

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 th

Section IV. A 

4. 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 
CARBARYL FINAL 

Page 17 of 62 

6. Male nuptial tubercle score was significantly decreased by in the high-concentration group (0.82 mg/L); (median value of 30 vs. 33 cont

7. In the exposure period hatchling weight was significantly reduced at the highest dose. During a recovery period, viable embryos and num

hatchling (based on eggs set) were reduced at the top dose, and hatchling weight was significantly reduced at the mid and high dose. 

8 Reductions (NS) in adult male body weight gain were observed at the low (15%), mid (44%) and high (72%) test concentration levels. D

weights also observed. There were no other signs of toxicity 

9. Dose-dependent increases in congestion and edema; decreases in spermatogenesis; and atrophy of seminiferous tubules. 10. Decreases were observed in total epididymal sperm count (↓40 and 60%) and percent sperm motility (↓25 and 63%) in the low- and high

mg/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 s

presented 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%; adu

percent sperm motility (young: ↓24 and 43%; adult: ↓15 and 26%), respectively. 

13. Study tested both immature and mature male rats; decreases in androgen sensitive organs were observed only in immature rats at the hig

P Positive findings N Negative findings (in vitro)/No effect (in vivo) NE Not examined 
CARBARYL FINAL 

Page 18 of 62 

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

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

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

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

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

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

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

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

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

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

further in the WOE assessment. 

The requirements for the male pubertal and female 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-generation reproduction study (MRID 45448101), a 

subchronic metabolism study (MRID 44402501) and a thyroid receptor transactivation assay by 

Sun et al., (2008). 

In the chronic toxicity/carcinogenicity study in rats, an increased incidence 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 follicular 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 

relative (↑39% liver and ↑54% spleen) weights. 

In the chronic toxicity study, relative thyroid weight were decreased (27%) at the high-dose male 

without microscopic changes (terminal body weight was increased by 15%). In addition, 

decreased pituitary weights were observed in parental F1 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.

Page 26 of 70
CARBARYL FINAL 

Page 19 of 62 

Sun et al. (2008) studied carbaryl in a thyroid transcriptional activation assay in HepG2 cell. The 

results indicated that carbaryl had no agonist activity, but inhibited T3 activity with an IC50 of 

8.40×10−5 M. A modified Frog Embryo Developmental 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.

Page 27 of 70
CARBARYL FINAL 

Page 20 of 62 

Table 4. Thyroid Pathway for Carbaryl.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Carbaryl1

Study Type/ 

Literature Citation 

TR Activation Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

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) 

P4

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 shaded 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. Carbaryl had no agonist activity, but inhibited T3 activity with an IC50 of 8.40×10−5

 M.

P Positive finding 

N Negative findings 

NE Not examined 

Page 28 of 70
CARBARYL FINAL 

Page 21 of 62 

IV. Committee’s Assessment of Weight of Evidence 

This section of the document describes the weight 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 recommendations regarding Tier 2 testing. The results of the Tier 1 assays 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated 

with weight of evidence evaluation of the results of the Tier 1 assays stated that “In summary, the 

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

presence of overt toxicity. All effects in endocrine sensitive tissues should be evaluated in terms 

of primary interactions with the endocrine system vs. secondary effects related to toxicity in 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 below. Generally, one parameter (i.e., death or >10% decrease in 

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

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

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

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

Page 29 of 70
CARBARYL FINAL 

Page 22 of 62 

1. Tier 1 in vivo Assays 

In the Hershberger assay, (50 mg/kg/day + TP), treatment-related clinical signs consisting of 

increased salivation, reduced motor activity, tremors, and piloerection 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 salivation and piloerection in one animal. In addition, there was a 12% 

decrease in final body weight and a 49% decrease 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 also noted in 4/10 animals on Day 1 or Day 2 

(post-dosing), and one animal presented many clinical signs during the dosing period including 

reduced motor activity, prostration, wasted appearance, genital discharge, and soiled anogenital 

region. Clinical signs were reduced in the 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 decreased 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 reproduction toxicity study, no treatment-related 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 decreased by 5-6% and 4-5%, in the high-dose F0 males 

and females, respectively, and by 10 and 8% in the high-dose F1 males and females. Overall 

body weight gains (Days 0-70) were decreased by 7-9% in the F0 generation and by ≤8% in the 

F1 generation. Mean body weights also were decreased in high-dose F0 and F1 females during 

gestation and lactation, with decreases of ≥10% in the F1 females (↓7-14%) and ≤10% in the 

F0 females. No treatment-related effects were observed on body weights or weight gain of adults 

at the low and mid doses. The most notable effects of carbaryl were on the F1 and F2 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 F1 and ↓13-23% for F2

pups). 

In a rat developmental neurotoxicity study, there were no carbaryl-associated deaths and no 

treatment-related clinical signs of toxicity in the F0 generation dams or F1 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 control during all other periods. During the FOB 

measurements, the incidence of females in the high-dose group with decreased pupil size 

(pinpoint pupils) was increased on all occasions during the dosing period. An increased 

Page 30 of 70
CARBARYL FINAL 

Page 23 of 62 

incidence of dams with slight tremors affecting the head, body and/or limbs was noted on the 

majority of assessment occasions in the high-dose animals. There were also occasional 

occurrences of ataxic gait/overall gait incapacity (dose-dependent 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 animals 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 the high-dose group (30 mg/kg/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 were <10%, and overall body weight gain in the 

high-dose group was only decreased 27%. AChE activity was not determined in the rat 

developmental study. 

In the prenatal developmental toxicity study in rabbits, mortalities that occurred on study were 

not considered to be related to treatment, and no clinical toxicity was reported at doses up to 150 

mg/kg/day. Body weights were not affected by treatment, but overall 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/carcinogenicity study, clinical observations included thin appearance, 

alopecia and urine staining in high-dose males and/or females (349.5/484.6 mg/kg/day [M/F]). 

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 were decreased by 

23 and 24%, respectively, and by Week 13 and were 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 recovery animals demonstrated continued, although 

attenuated, decreases in males and females between Weeks 53 (↓32 and 36%, respectively) and 

57 (↓23 and 27%, respectively). Overall body weight gains also were decreased in the high-dose 

animals (↓53 and 69% in males and females, respectively), with an overall body weight gain 

decrease of 18% observed in the mid-dose females. Determination of AChE activities at 6-month 

intervals indicated decreases in plasma AChE (↓17-57%), RBC AChE (↓19-38%), and brain 

AChE (↓10-31%), with a return to control levels demonstrated in recovery animals by Week 56. 

Page 31 of 70
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 toxicity in the high-dose (1249/1441 

mg/kg/day [M/F]) animals was noted as thin appearance and hunched posture; these signs were 

present early in the study, disappeared by Week 6, and re-appeared during the 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 decreased 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 AChE 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 was initially decreased by 50% in the high-dose females during 

Weeks 0-5, with lesser decreases 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 all 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 overall 12 and 16% decreases body weights. Body 

weight gains were also decreased by 39% at the mid dose, and there were actual losses in body 

weight at the high dose. In addition, liver and spleen weights were increased by 18 and 30%, 

respectively, at the high dose. 

Page 32 of 70
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 signs 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, mallard 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 the 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 second reproduction toxicity study in 

ducks, with no effects on mortality or clinical signs. In a reproduction toxicity study in quail, 

bobwhite quail displayed no treatment-related effects on survival or growth parameters. 

B. Estrogen Pathway 

There is no convincing evidence for an interaction with the estrogen pathway. While there was 

equivocal or conflicting responses in the 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 studies in mallard ducks whereas decreases in 

female body weight gain were also observed, no estrogen-related effects 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 Tier 1 AR binding assay and altered testosterone 

production in the Tier 1 steroidogenesis 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 supported by the available mammalian in vivo studies. Although 

perturbations 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 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 significant female weight decreases) were observed. However, 

effects in the FSTRA (secondary sex characteristics and gonadal histopathology) are suggestive 

of a potential interaction with the androgen pathway. Cholinesterase activity was not measured 

in the FSTRA. Together, results in the FSTRA and the positive in vitro assays provide 

redundancy for a potential interaction with the androgen pathway in male fish. 

Page 33 of 70
CARBARYL FINAL 

Page 26 of 62 

D. Thyroid Pathway 

There is no convincing evidence for an interaction 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 presence of overt toxicity. 

E. Conclusions 

There is no convincing evidence for interaction of carbaryl with the estrogen or thyroid 

pathways, in mammals or wildlife. There is also no convincing evidence for interaction of 

carbaryl with the androgen pathway in mammals. However the evidence suggest potential 

interaction with the androgen pathway in fish. 

V. EDSP Tier 2 Testing Recommendations 

Based on weight of evidence conclusions, mammalian EDSP Tier 2 testing is not recommended 

for carbaryl since there is no evidence for potential interaction with the estrogen, androgen or 

thyroid pathways. 

Effects were observed in males in the FSTRA at 0.82 mg/L which 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 group at 0.68 mg/L with a NOAEC of 

0.21 mg/L. Effects on F1 survival were observed at all concentrations tested (≥0.008 mg/L). 

Since the responses were observed in the FSTRA at similar concentrations as the current chronic 

endpoint, the EDSP Tier 2 Medaka Extended One-Generation Reproduction Test is 

recommended. 

For birds, Part 158 avian reproduction studies with 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 considered sufficient for evaluating potential 

reproductive effects to birds from carbaryl exposure. Relative to the EDSP, additional testing is 

not recommended.

Page 34 of 70
CARBARYL FINAL 

Page 27 of 62 

VI. References

Bacchetta, R., Mantecca, P., Andrioletti, M., Vismara, 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 the Potential Estrogenic Activity of Carbaryl in 

the Rat. Unpublished study conducted by Bayer CropScience, Sophia Antipolis, France. SA 

02282, January 16, 2003. (MRID 47987702) 

Blanck, O. (2011) Carbaryl: Evaluation in the 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 and reproduction study with carbaryl technical in 

Bobwhite quail. Neillsville, Wisconsin, Unpublished study conducted by Bio-Life Associate Ltd 

(Report No. 85-QR-15). Rhone-Poulenc Agro. Lyon, France. (MRID 00160044) 

Fletcher DW, Leonard MI. (1986b). 28-day dietary toxicity and reproduction 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 Study 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 Carbaryl 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) 

Page 35 of 70
CARBARYL FINAL 

Page 28 of 62 

Kennel, P. (2003) Hershberger Assay to Assess the Potential Antiandrogenic Activity of 

Carbaryl in Rat. Unpublished study conducted by Bayer CropScience, 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 breast 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 derivative on androgen and estrogen 

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

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

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

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

Lemaire, G., Mnif, W., Mauvais, P., Balaguer, P, and Rahmani, R. (2006) Activation of a- and Pestrogen receptors by persistent pesticides 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) Carbaryl 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 Developmental Neurotoxicity Study of Orally 

Administered Carbaryl, Technical Grade, in the Rat. Unpublished study conducted by ClinTrials 

BioResearch, Ltd. Quebec, Canada. Laboratory 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, Sophia 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, Sophia Antipolis Cedex, France. Laboratory Study 

No.: SA 11209, October 26, 2011. (MRID 48615203) 

Page 36 of 70
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 Antipolis Cedex, France. Laboratory Report No.: 

SA 11060, October 20, 2011. (MRID 48615202) 

Tinwell, H. (2011) Evaluation of carbaryl in the H295R steroidogenesis assay. Unpublished 

study conducted by Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. 

Laboratory Study No.: SA 11066, October 18, 2011. (MRID 48615205) 

Totis M. (1997) Investigation of the metabolism of 14C-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 Administered by Gavage to New Zealand White 

Rabbits. Unpublished study conducted by Reproductive & Developmental Toxicology 

Laboratory, Center for Life Sciences and Toxicology, Research 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, Center for Life Sciences and 

Toxicology, Chemistry and Life Sciences, Research 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 Disruptor Screening Program – Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. Office of Chemical 

Safety and Pollution Prevention Science Coordination and Policy. 

U.S. Environmental Protection Agency, Washington, DC. York, D.O. (2012). Carbaryl - ShortTerm Reproduction Assay with Fathead Minnow (Pimephales promelas) Following OPPTS 

890.1350 and OECD 229 Guidelines. Unpublished study conducted by Smithers Viscient, 

Wareham, MA. Laboratory report number: 13798.6275. (MRID 48669601) 

Page 37 of 70
CARBARYL FINAL 

Page 30 of 62 

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

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

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

Agency, Washington, DC. 

Page 38 of 70
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) Binding 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 saturation binding experiment was conducted to 

demonstrate that the AR in the rat prostate cytosol was present in reasonable numbers and was 

functioning with appropriate affinity for the radio-labeled reference androgen (R1881) prior to 

routinely conducting AR competitive binding experiments. 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 carbaryl (logarithmic increase from 10−10 to 10−3

 M). DMSO was 

used as the vehicle at a final assay concentration of ≤3.3%. The assay included dexamethasone 

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

In the saturation binding experiment, the mean maximum binding capacity (Bmax) was 14 

fmol/100 μg protein and the mean dissociation constant (Kd) was 1.01 nM. The Kd obtained 

from Saturation Assay #2 was slightly low (0.59 nM), but the Kds 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 binding 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, carbaryl reduced the mean specific binding of the 

radiolabeled ligand in a concentration dependent manner in all three runs. The maximum 

reduction in [3

H]-R1881binding occurred at the highest 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 IC50 was −3.5 M for carbaryl, −4.4 M for the weak positive 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 positive control. The solvent control responses 

indicated no drift in the study assay. All performance criteria were met, with the exception that 

Page 39 of 70
CARBARYL FINAL 

Page 32 of 62 

ligand depletion and non-specific binding (NSB) were not reported, but the IC50s for R1881 and 

the weak positive control were within the expected ranges. These deviations are considered 

minor; the performance criteria were met. 

Based on the results from the three 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 [3H(N)]-androst-4-ene-3,17-dione; [3H]ASDN) in dimethyl sulfoxide (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 concentration of carbaryl. Tritiated water was quantified 

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

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

) using a 

known inhibitor (4-hydroxyandrostenedione; 4-OH ASDN; formestane), and the test carbaryl 

series (10−10 to 10−3

) with three repetitions per concentration. 

Aromatase activity in the full activity 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 averaged 7.4% of the full control activity. The 

response of each full activity control 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), aromatase 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 IC50. 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 between 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 IC50 averaged −6.83 

M (outside of recommended −7.3 to −7.0 M range). 

Page 40 of 70
CARBARYL FINAL 

Page 33 of 62 

For carbaryl, aromatase activity 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 activity compared to controls, on average, 

to approximately 65% at 10−4

 M and 42% at 10−3 M. 

Although carbaryl exhibited a concentration-dependent inhibition of aromatase activity and 

decreased activity by more than 50% (log IC50 = −3.83 M), the average slope of the inhibition 

curve was steeper than –2 (-2.92). Therefore, carbaryl is classified as Equivocal for aromatase 

inhibition in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID 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]-17β-estradiol (1 nM) in the 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 17β-estradiol as the natural ligand 

reference material. 

In the saturation binding experiment, the appropriate protein concentration was determined to be 

0.5 mg/mL as it bound 25-35% of the [3

H]-17β-estradiol. The Kd for [3H]-17β-estradiol was 

0.070 nM, and the estimated Bmax was 34.3 fmol/100 μg protein for the prepared uterine cytosol. 

The Kd for each run was within the expected range of 0.03 to 1.5 nM. 

In the competitive binding experiment, the reference compounds generally performed as 

expected. The negative control, octyltriethoxysilane, had no effect on binding, and there were 

concentration dependent decreases in binding for the natural ligand and the weak positive 

control. The mean IC50 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 was outside the acceptable 

range, at −2.0 to −1.7, indicating that the receptors were behaving as though there was more than 

one binding site for 17β-estradiol. Carbaryl was tested over a concentration range that fully 

defined the top of the curve. The maximum percent binding 109.0-117.9% was within 

approximately 25 percentage points of the lowest concentration of the estradiol standard 83.5-

116.2%. 

Page 41 of 70
CARBARYL FINAL 

Page 34 of 62 

An estimated mean IC50 was not calculated for carbaryl as it did not achieve 50% binding 

inhibition. In two assay runs, carbaryl reduced the specific binding of the radiolabeled estradiol 

at the highest concentration tested (10−3 M) to 62-71%; in Run 3 percent binding was ≥75% at 

10−3 M. 

Based on the results from the three runs, carbaryl is classified as Equivocal in the Estrogen 

Receptor Binding Assay. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48615204), hERα-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. Luciferase 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 using the required reference compounds adequately 

demonstrated that the assay was functioning properly. The mean RPCMax for carbaryl was 2.7% 

for the first run and 0.5% for the second run, and the associated PCMax was 10−4M for both runs. 

Because the RPCMax < PC10 in both assay runs, carbaryl was considered negative for estrogen 

receptor transcriptional activation in this test system. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48669601) of carbaryl with fathead minnow 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (21 weeks old; 

2 males and 4 females in each group; 4 groups per treatment) were exposed to carbaryl (99.2% 

purity) at nominal concentrations of 0 (negative control), 0.032, 0.16, and 0.80 mg a.i./L; meanmeasured concentrations were <0.0038 (<LOQ; 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, respectively, for which there were 

Page 42 of 70
CARBARYL FINAL 

Page 35 of 62 

no observed significant differences. There were no notable observations with regards to behavior 

or changes in appearance (specifically color/banding, ovipositor appearance, or size of dorsal 

nape pad) in the control or treated groups and no reported clinical signs of toxicity. Male body 

length was 9% greater (p<0.05) in the 0.82 mg a.i./L treatment group. Neither female body 

length nor body weight for either sex was affected by carbaryl exposure. 

Carbaryl had no effect on fecundity or fertility in any treatment group. Additionally, plasma 

vitellogenin (VTG) concentrations were unaffected in males and females and there were no 

treatment-related effects on GSI in either sex. However, 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 minimal testicular degeneration 

and the percentage of spermatogonia at the mean-measured 0.82 mg a.i./L treatment level. No 

remarkable histopathological observations 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) screening for potential anti-androgenic activity, 

carbaryl (99.1% a.i., Batch #21114920) in 0.5% aqueous 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.) injection. 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 androgenic activity of TP. The animals were dosed for 10 consecutive 

days and terminated approximately 24 hours after the final dose administration. At necropsy, the 

five androgen-dependent tissues and liver were collected and weighed. 

All animals survived until scheduled termination. 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 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. In the 15 mg/kg/day + TP group, treatment-related clinical signs were noted between 

Days 1 and 4 and consisted of reduced motor activity and/or tremors in 5/6 animals, associated 

with increased salivation and piloerection in one animal. No clinical signs of toxicity were noted 

in animals in the 5 mg/kg/day+TP group, or any of the control groups. 

Page 43 of 70
CARBARYL FINAL 

Page 36 of 62 

In the 50 mg/kg/day +TP group, there was a 12% decrease 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 decreased (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 were comparable to the TP control. There 

were no treatment-related effects on the weights of seminal vesicles, levator ani-bulbocavernosus 

muscle complex (LABC), Cowper’s glands, ventral 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 prostate; 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, final 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 group. Animals in the TP control group had the 

expected increases (p<0.05) in weights of accessory sex organ weights as follows: 796% in 

seminal vesicles; 832% in ventral prostate; 252% in LABC; 600% in Cowper’s gland; and 14% 

in glans penis. In addition, liver weight was increased (p<0.05) by 14%. 

Percent CVs for the vehicle control, TP control, and 50 mg/kg/day +TP groups were compared to 

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

permissible values, with the exception of ventral prostate (CV = 57%; maximum permissible CV 

= 45%) and the glans penis (CV = 23%; maximum permissible 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 observed at this dose level. 

No statistically significant decreases were seen in the weights of the five androgen sensitive 

tissues. Carbaryl was negative for anti-androgenicity in the Hershberger assay. 

Page 44 of 70
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 (MRID 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 for three independent experiments. 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 estradiol using EIA kits in the presence of 10–4 and 

10–5 M carbaryl; therefore, these concentrations 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 positive control agents at two 

concentration levels. The positive controls included a known inhibitor (prochloraz) and a known 

inducer (forskolin) of estradiol and testosterone production. 

In the QC plate, the minimum basal hormone production 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 production, induction, and inhibition were met for 

testosterone and estradiol, 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 exceeded the Guideline criteria (≤30%). 

Precipitation and cytotoxicity were not reported at any concentration tested. No effects of 

carbaryl on testosterone production 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 testosterone 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, although there was a 1.35-fold 

increase (not significant) in estradiol at the highest concentration successfully tested, 10-6 M. 

Page 45 of 70
CARBARYL FINAL 

Page 38 of 62 

Based on the hormone responses in each of three independent runs, carbaryl treatment resulted in 

statistically significant and reproducible 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 rats 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 17α-ethynyl estradiol (EE) at 0.003 mg/kg/day by oral gavage and a negative control 

group was treated with a daily dose of dibutylphthalate (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 scheduled termination. At 30 mg/kg/day, tremors were noted in 7/10 

animals and chewing in 9/10 animals. Piloerection was also noted in 4/10 animals on Day 1 or 

Day 2 (post-dosing) and one animal presented many clinical signs during the dosing period: 

reduced motor activity, prostration, wasted appearance, genital discharge and soiled anogenital 

region. At 10 mg/kg/day, piloerection was noted 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 decreased (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 weights in the 3 and 10 mg/kg/day groups were comparable to the 

control throughout the study; however, overall body weight gain was decreased (p<0.05) by 11% 

in the 3 mg/kg/day group. The decreased body weights indicate that the dose levels tested were 

adequate. Uterine weights in the carbaryl treatment groups were comparable to the vehicle 

controls. 

Body weights and overall body weight gain in the positive control (EE) group were comparable 

to the controls. In the negative control (DBP) group, final body 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 were comparable to controls. 

Page 46 of 70
CARBARYL FINAL 

Page 39 of 62 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 general toxicity studies considered relevant for determining the 

potential interaction of carbaryl with the endocrine system are summarized below. Given the 

breadth of toxicity data available on carbaryl, the only Part 158 studies 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 chemicals (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 Kd = 2.03 ± 0.15 nM (SD; n = 4) for R1881 

(methyltrienolone), the standard 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 [3H]-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 concentration was 1.84 nM. In addition to the 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 supernatant 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 IC50 (50% 

inhibition of [3

H]-R1881 binding) for each competitor was determined. 

The AR binding affinity was expressed as either RBA or log RBA in this study. The mean IC50

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 replications. The RBA values 

of active chemicals are the means of the replicate values. Chemicals that failed to compete with 

[

3

H]-R1881 in binding were designated as “non-binders” (NB). Chemicals that showed binding 

but did not reach 50% inhibition at maximum concentration were designated as “slight binders” 

(SB). A less than IC50 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 > 

Page 47 of 70
CARBARYL FINAL 

Page 40 of 62 

0.01), or weak binders (0.01 > RBA > 0.0001), and inactive chemicals. The inactive group 

includes both NB and SB chemicals. 

Results obtained relevant to the evaluation of carbaryl are as follows: (i) carbaryl had a logP of 

2.35, an IC50 of 4.00×10−4

 M, an RBA of 0.0008, and a logRBA of −3.12; (ii) dexamethasone (a 

weak positive reported for comparative purposes) had a logP of 1.72, an IC50 of 8.00×10−5

 M, an 

RBA of 0.0038, and a logRBA of −2.42; and (iii) DHT (natural ligand reported for comparative 

purposes) had a logP of 3.07, an IC50 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 β1 thyroid hormone receptor-related 

transcription in vitro. 

The study used a luciferase reporter gene assay system to measure potential 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 T3 (triiodothyronine) and 

T4 (thyroxine). T3 (purity ≥98%) was used in the present evaluation of compounds for TR 

antagonism. The TR selected for this assay system was TRβ1 (c-erb-A beta). pGal4-L-TRβ (the 

expression vector coding for the ligand binding domain of TRβ fused to the DNA binding 

domain of Gal4) and Gal4 (the responsive luciferase 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 transfection 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 carcinoma) cell line was maintained in Dulbecco’s modified Eagle 

medium (DMEM) with 5% fetal bovine serum (FBS) at 37°C and 5% CO2. Host cells were 

plated into 48-well microplates at a density of 0.5×105

 cells per well in phenol red-free DMEM 

containing charcoal-dextran stripped FBS and incubated. The cells were transfected with 0.25 μg 

pUAS-tk-luc, 0.1 μg Gal4-TRβ, and 0.05 μg phRL-tk including 2.5 μg SofastTM transfection 

reagent per well. After incubation, the transfection medium was removed and replaced with 

medium containing various concentrations of the test chemicals for measurement of agonist 

activity. For measurement of antagonist activity, the cells were treated with test chemicals and 5 

nM T3. Medium with 0.1% ethanol or 0.1% DMSO were used as solvent controls. 

After exposure to the test chemicals for 24 hours, the cells were harvested, rinsed three times 

with phosphate-buffered saline, lysed with an equal volume of passive lysis buffer, and analyzed 

with a 96-well luminometer. The amount of luciferase and Renilla luciferase were measured with 

Page 48 of 70
CARBARYL FINAL 

Page 41 of 62 

a Dual-luciferase reporter assay system kit. The relative transcriptional activity was converted to 

fold induction above the corresponding vehicle control value (n-fold). The values presented were 

means and standard deviations from three independent experiments. The median inhibitory 

concentration (IC50) or median effective concentration (EC50) was calculated. For antagonists, 

treatments were compared to the 5 nM T3 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. The HepG2 reporter system showed the appropriate 

response to the natural ligands T3 and T4 and to the known antagonist, BPA. For T3, the maximal 

induction (7.85-fold of vehicle control) was observed at 10−8 M, with an EC50 of 0.46 nM. For 

T4, the maximal induction (8.20-fold of vehicle control) was observed at 10−7

 M, with an EC50 of 

25.53 nM. BPA did not induce the expression of luciferase, but demonstrated antagonist activity. 

After incubation with T3 at 5 nM, BPA inhibited (p<0.05 or 0.01) luciferase activity at 

concentrations of ≥2.5×10−5 M, with an IC50 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 T3 at 5 nM, carbaryl 

demonstrated antagonist activity (p<0.01) with an IC50 of 8.40×10−5

 M in this assay. This assay 

is not required in the Tier 1 battery, but the results 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 (hERα and hERβ) and one human androgen 

receptor (hAR) in transactivation assays with Chinese hamster ovary cells (CHO-K1 cells) was 

investigated. For detection of hERα or hERβ activity, cells were transfected with pcDNAERα or 

pcDNAERβ, pGL3-tkERE, and pRL-SV40. For detection of hAR activity, cells were transfected 

with pZeoSV2AR, pIND-ARE, and pRL-SV40. Flutolanil was tested over a concentration range 

from 10−8

 to 10−5 M, and the responses were compared to the activity of 17β-estradiol (E2; purity 

>97%) or 5α-dihydrotestosterone (DHT; purity 95%) for the ER or AR assays, respectively. For 

measurement of antagonistic activity to hERα, hERβ and hAR, either 10−11 or 10−10 M E2 or 

10−10 M DHT was added to the cell cultures along with the test compound, respectively. 

Agonistic activities were evaluated by the relative activity expressed as 20% relative effective 

concentration (REC20), the concentration of the compound demonstrating 20% of the activity of 

10−10 M E2, 10−9 M E2, or 10−9

 M DHT for ERα, ERβ, and AR, respectively. Antagonistic 

activities were evaluated similarly by the relative activity expressed as 20% relative inhibitory 

concentration (RIC20), the concentration of the compound showing 20% inhibition of the activity 

of E2 or DHT against ERα, ERβ, and AR, respectively. Carbaryl (10−8

 to 10−5 M) did not show 

agonist or antagonist activity for ERα and ERβ, or agonist activity for AR in this study. 

In another published, non-Guideline study, Blair et al. (2000) evaluated 188 pesticides 

(including carbaryl; purity 99%) with an ER competitive binding assay. Uteri from 

Page 49 of 70
CARBARYL 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 concentrations to determine whether a chemical competed with [3H]-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 characterize the binding curve and to determine the 

IC50 and RBA values. The mean IC50 for carbaryl was >1.00 x 10−4

 M in this screening 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 conducted in transiently transfected MCF-7 human 

breast and Ishikawa human endometrial cell lines using an estrogen-responsive luciferase 

reporter gene. The test chemicals were evaluated 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) estradiol-induced luciferase 

activity 70-75% in MCF-7 cells and 65-70% in Ishikawa cells. Cell 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 medium (serum-free, phenol red-free DMEM containing 15 mM 

HEPES) and resuspended in binding medium. 1×106

 cells were transferred to microcentrifuge 

tubes, and the volume of binding medium was increased to l mL. A volume of 500 pM [3

H]-17βestradiol was added to the MCF-7 cells in the presence or absence of vehicle, 100-, 1000-, or 

10,000-fold molar excess radioinert 17β-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. Carbaryl inhibited binding of [3

H]-17β-estradiol to hER in a dosedependent manner, with decreased (p<0.01) binding at carbaryl concentrations of 1 and 10 μM, 

with a calculated IC50 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 ERα and ERβ activation or 

inhibition in stable reporter cell lines, HELN ERα and ERβ (HeLa cells stably transfected with 

an ERE-driven luciferase plasmid). 

To create the stably-transfected cell lines, HeLa cells were first transfected with the GAL4REERE-βGlob-Luc-SVNeo plasmid. HELN cells expressing ERα or ERβ were obtained by a 

second transfection of the corresponding pSG5 puro plasmids (pSG5-ERα puro or pSG5-ERβ

puro, respectively). HeLa-derived cell lines were cultured in phenol red-free DMEM 

supplemented with 6% DCC-FBS for the strain culture. 

Page 50 of 70
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 added, 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 ERα and ERβ competitive-binding assay, ERα and ERβ cells were seeded at a density of 

1.5×105

 cells per well in 24-well tissue culture plates. Cells were labeled with 0.1 nM [3H]-E2

([3

H]-17β-estradiol, 41.3 Ci/mmol specific activity) in the presence or absence of increasing 

concentrations of cold E2 (as control) or each of the tested compounds in 400 μl of DMEM 

supplemented with 6% DCC-FBS. Each dilution was performed in duplicate. Plates were put on 

ice for 15 minutes, and the labeling medium was removed. Cells were washed with cold 

phosphate-buffered saline and harvested in ethanol. 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 assay. The plates were read by absorbance at 

540 nm. All results are expressed as the average of three wells. The control 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 potential of pesticides, HELN ERα and HELN ERβ were used to 

identify the active compounds and discriminate between ERα and ERβ activities. To determine 

ERα and ERβ agonistic activity of pesticides, the relative transactivation activity on HELN ER 

cells was computed as the ratio of luciferase reporter gene induction values of each compound at 

a concentration of 10 μM to the luciferase reporter gene induction value of E2 at 10 nM. As the 

ERTA assay was conducted at only a single concentration, it is not discussed further. 

Antagonistic effects of pesticides (including carbaryl) in the ERα and ERβ transactivation assay 

were determined over a range of five concentrations from 10−7

 to 10−5. In addition, antiestrogenic competitive inhibition was examined against ERα and ERβ at carbaryl concentrations 

of 10 μM, 3 μM, and 1 μM. 

To confirm the ER antagonist effects in the transactivation assays reflected an inhibition of E2 

binding to ERα and/or ERβ, whole cell competitive binding assays were conducted with the 

HELN ERα and ERβ cells. The binding affinity for ER of anti-estrogenic pesticides was 

assessed with 0.1 nM [3

H]-E2 as a tracer. As expected, non-radiolabelled E2 displaced [3H]-E2 by 

50% at a concentration of 0.12 nM for ERα and 0.18 nM for ERβ, and ICI 164,384 inhibited 

[

3

H]-E2 binding by 50% at the concentration of 2.5±0.5 nM for ERα and 3.3±1.0 nM for ERβ. 

The authors reported that carbaryl inhibited (p<0.001) [3

H]-E2 binding (ERα and ERβ) by 

approximately 45 and 60%, respectively, at a concentration of 10 μM, showing anti-estrogenic 

activity. 

Page 51 of 70
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 the respective premating periods, the dietary 

intakes of carbaryl corresponded to doses of 0, 4.67, 31.34, and 92.43 mg/kg/day for F0 males; 0, 

5.56, 36.32, and 110.78 mg/kg/day for F0 females; 0, 5.79, 23.49, and 124.33 mg/kg/day for F1

males; and 0, 6.41, 26.91, and 135.54 mg/kg/day for F1 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. Mean carbaryl intakes for F0 and F1 parental females during gestation 

were slightly lower compared to premating intakes, and carbaryl intakes during lactation was 

increased approximately two-fold compared to premating intakes in each dose group. F1 pups 

selected as parents to produce the F2 generation were weaned onto the same food as their parents 

(PND 21). Parental males were sacrificed after delivery of their litters and parental females were 

sacrificed after weaning of their litters (PND 21).

No treatment-related deaths or clinical signs were observed at any dose level in either generation. 

With a few exceptions at the high dose, there were no treatment-related effects on organ weight 

changes (absolute or relative weights of the testes, epididymides, seminal vesicle with 

coagulating glands, prostate, uterus [with oviducts and cervix], vagina, ovaries, adrenal, or 

pituitary glands). However at the high-dose, the relative kidney weight increased by 7% (p<0.01) 

in F0 males, and there were decreases in absolute pituitary (↓9%, p<0.05) and epididymides 

(↓5%, p<0.05) weights and relative weights of the liver, kidneys, and brain F1 males. For highdose females, the absolute and relative liver weights were elevated by 10% (p<0.01) and 14%, 

respectively, the absolute adrenal weight was decreased by 11% (p<0.05), and the relative kidney 

weight was increased by 6% (p<0.05) for F0 females. Also in high-dose F1 females, the absolute 

brain weight was decreased by 4% (p<0.05). No gross or microscopic lesions were observed in 

adult rats of either generation. 

At the low- and mid-dose, no treatment-related effects were observed on body weights, weight 

gain, feed consumption, or food efficiency in F0 or F1 male or female rats at any time during the 

study, including during gestation and lactation periods of the females. 

For the high-dose group, body weights and body weight gains in F0 and F1 male and female rats 

were decreased (p<0.001, 0.01, or 0.05) during the premating period. Body weights in the F0

males and females were decreased 5-6% and 4-5%, respectively. Body weight gains were 

maximally decreased during premating Days 0-7 (23 and 27%, respectively) in F0 males and 

females, with overall body weight gain decreases (Days 0-70) of 9 and 7%, respectively. Mean 

body weight in F1 males was decreased 19% on PND 22 (Day 0 of premating), decreased by 

Page 52 of 70
CARBARYL FINAL 

Page 45 of 62 

12% on PND 56, but was only decreased by 10% on PND 91. Similarly, mean body weight in F1

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 F1 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 F1 females was initially decreased on Days 0-7 

by 9%, with no effect on overall body weight gain. Food consumption and food efficiency for F0

and F1 rats followed patterns similar to that of body weight and body weight gain; the largest 

difference between the high-dose animals and control occurred during the early part of the 

premating period. When averaged over the entire premating period, F0 and F1 males consumed 6-

7% less food compared to control and had food efficiency values similar to control. Feed 

consumption and food efficiency for the F0 females were similar to those of the control group, 

but food consumption in F1 females was decreased (p<0.01) by 9% with a food efficiency value 

increased (p<0.01) by 10% compared to control. Mean body weights were decreased (p<0.001, 

0.01, or 0.05) in high-dose F0 and F1 females during gestation and lactation, with the effect being 

greater in the F1 females (↓7-14%) compared to F0 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 F0 and F1 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 decreased 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 observed on estrous cyclicity in F0 or F1 females, or on percent 

motile sperm, sperm count, percent progressively motile sperm, epididymal sperm count, 

spermatid head count, daily sperm production, or efficiency of daily sperm production in F0 or F1

males. There was a dose-related increase (NS) in the percentage of abnormal sperm in the treated 

males. No treatment-related gross or microscopic effects were observed 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, gestation index, pregnancy 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, clinical signs, or organ 

weight or necropsy findings in pups surviving to 21 days. Pup survival was slightly decreased 

(NS) at the mid- and high-dose for both generations. An increased number of deaths in the F2

generation pups resulted in an 18-19% decrease in mean litter size on PND 4 (p<0.01 or <0.05), 

Page 53 of 70
CARBARYL FINAL 

Page 46 of 62 

and decreased viability and lactation indexes at the high dose. A large number of pups that died 

had no milk in their stomachs. In addition, pup weight/litter in the high-dose pups was decreased 

(p<0.001 or <0.01) for both generations during PNDs 4-21 (↓11-15% for F1 and ↓13-23% for F2

pups). Body weight gain in pups was decreased (NS) throughout lactation, with the greatest 

effect (↓≥20%) occurring during LDs 0-7 for F1 pups and LDs 0-14 for F2 pups. Sexual 

maturation was delayed in the high-dose F1 offspring as evidenced by delayed PPS in the males 

(+2.1 days) and vaginal patency (VO) in the females (+1.4 days). The differences remained 

statistically significant after adjustment for body weight decreases. Anogenital distance was 

decreased (p<0.05) in F2 male pups in the high-dose group, but 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) based on increased numbers of F2 pups with no milk in the 

stomach and decreased pup survival. The NOAEL 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 administered 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 females (6/dose group) were administered the 

same doses of carbaryl for the AChE determinations. AChE determinations 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 Battery (FOB) measurements were conducted at 

approximately 0.5 and 2 hours post-dosing on the same 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 for body weight, physical development landmarks 

(tooth eruption and eye opening), FOB assessments (LDs 4, 7, 11, 13, 17, and 21), and motor 

activity (LDs 13, 17, and 21). On LD 11, one animal/sex/litter was sacrificed for determination 

of brain weights (of these, six/sex were randomly selected for neuropathological evaluation). 

After LD 21, three animals/sex/litter were separated from the dams and constituted the F1 adult 

generation. These animals were evaluated for body weight, physical development (VO and PPS), 

motor activity (Day 60), startle habituation response (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-associated deaths in the F0 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 period, but body weight gains were similar during 

Page 54 of 70
CARBARYL 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 with decreased pupil size 

(pinpoint pupils) was increased on all occasions during the dosing period. An increased 

incidence of dams with slight tremors affecting the head, body and/or limbs was noted on the 

majority of assessment occasions in the high-dose animals. There were also occasional 

occurrences of ataxic gait/overall gait incapacity (dose-dependent increased incidences or 

occurrences in the high-dose animals only) that were considered to be of toxicological 

significance due to other 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. There were no treatment-related effects on gross 

necropsy findings for the F0 generation animals. There were no effects observed on maternal 

performance parameters of pregnancy rate, gestation index and duration, numbers of live pups, 

dead or malformed pups, implantation scars, sex ratio or post-implantation loss. 

For the F1 generation pups, there were no treatment-related effects on pup weight, pup survival 

indices, developmental landmarks (tooth eruption and eye opening), clinical toxicity, FOB 

measurements, or motor activity assessments. At sacrifice on LD 11, there were no treatmentrelated effects on brain weight and gross or microscopic pathology. Noted differences in some 

morphometric measurements are not pertinent to the EDSP WoE determination. 

For the F1 generation adults, there were no treatment-related effects on clinical condition, body 

weight, physical development (VO and PPS), motor activity, auditory startle response, passive 

avoidance and water maze measurements. At sacrifice, there were no gross or microscopic 

neuropathological lesions observed for animals examined 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 inhibition of RBC, plasma, whole blood, and brain AChE 

activities. The maternal NOAEL was 1.0 mg/kg/day. 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 because the morphometric analyses at the lower 

doses were not conducted. The developmental effect persisted from F1 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 administered by gavage at 0, 1, 4, and 30 mg/kg/day to pregnant 

Page 55 of 70
CARBARYL 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-dosing salivation occurred in 18/25 dams (vs 0/25 control) in the 

high-dose group. This clinical sign appeared within 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, respectively; the corrected (for gravid uterine 

weight) final body weight and body weight 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 gross pathologic findings noted in any of the dams. There were 

no differences of toxicological concern in mortality, pregnancy rate, number of corpora lutea, 

implantations, viable fetuses, pre- and post-implantation losses, placental weights, and sex ratio. 

There were no differences of toxicological concern observed in the mid- and low-dose groups. 

The maternal LOAEL is 30 mg/kg/day based on clinical signs of toxicity, decreased body weight 

gains, and food consumption. The maternal NOAEL is 4 mg/kg/day. 

In the high-dose fetuses, mean fetal 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 metatarsal. No effects on fetal viability were observed. 

There were no treatment-related effects in developmental 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 administered 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-dosing 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. 

Page 56 of 70
CARBARYL FINAL 

Page 49 of 62 

Four does died on study (one each in the control 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 affected by treatment, but body weight 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 group 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 was no treatment-related effect 

on food consumption. Decreases (p<0.01) in plasma (↓46 and 68%, respectively) and RBC (↓19 

and 27%, respectively) AChE activities were observed in the mid- and high-dose groups. Lesser 

decreases (NS) were noted in both AChE 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 maternal 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/implantations, 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 fetuses or individually for males and females) in the 

high-dose group. There were no treatment-related developmental effects observed in the lowand mid-dose groups. 

The developmental LOAEL is 150 mg/kg/day based on decreased fetal weight. The 

developmental NOAEL is 50 mg/kg/day. 

Page 57 of 70
CARBARYL FINAL 

Page 50 of 62 

Chronic Toxicity/Carcinogenicity (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-dose 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). Ten animals/sex/dose group were sacrificed after 

53 weeks of treatment, and ten animals/sex in the control and high-dose 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 maintained on the treatment diets until study 

termination at Week 105. At each scheduled sacrifice, surviving rats were sacrificed for gross 

and histopathological evaluation. The organs weighed at each sacrifice 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 pituitary, thyroid/parathyroid, 

urinary bladder, prostate, seminal vesicles, mammary gland, uterus, and vagina with cervix. 

Clinical observations included thin appearance (high-dose males), alopecia (high-dose females), 

and urine staining in high-dose males and females. 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 significance (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, decreases in males and females between Weeks 53 

(↓32 and 36%, respectively) and 57 (↓23 and 27%, respectively). Overall 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 included decreased food 

consumption/efficiency, ophthalmologic abnormalities, and altered hematological and clinical 

chemistry parameters (increased cholesterol and decreased AST, ALT, and CK concentrations). 

Determination of AChE activities at 6-month intervals indicated 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 levels 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 increases (p<0.05) for liver and kidney in high-dose 

males and females at Week 53. At study termination, 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 relative (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 adrenal glands (except for decreased [p<0.05, ↓45%] 

Page 58 of 70
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, ovaries, uterus, vagina, cervix, and 

adrenal or pituitary glands at any dose. Histopathological effects at the high dose that were 

observed after unscheduled deaths and/or in surviving animals at termination included thyroid 

hypertrophy (males, 11% vs. 1% control and females, 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 findings 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, urinary bladder transitional cell adenoma and carcinoma 

in males and females, and thyroid follicular cell adenoma and carcinoma in males. 

The high dose (7500 ppm; 349.5/484.6 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 chemistry parameters. The LOAEL is 1500 ppm 

(60.2/78.6 mg/kg/day in males and females, respectively) due to decreased brain and RBC 

activity, alterations in clinical chemistry parameters, 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 diets containing carbaryl (99.3% purity) 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 histopathological evaluation. The organs weighed at 

sacrifice included the adrenals, liver, kidneys, testes with epididymides, and ovaries. Gross and 

microscopic pathological examinations were conducted on these organs as well as the pituitary, 

thyroid/parathyroids, urinary bladder, prostate, seminal vesicles, mammary gland, and uterus. 

Four males in the high-dose group died or were sacrificed moribund during Week 1 and were 

replaced. Systemic toxicity in the high-dose animals 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 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. 

Page 59 of 70
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 decreased (p<0.05) in the mid- and 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 groups by 13 and 13% at Week 53 and by 20 and 24% 

at Week 105. Additionally, in females, brain AChE activities were decreased (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, increased (p<0.05) absolute liver weight (↑24%) and decreased (p<0.05) 

absolute ovary weight (↓33%) was observed in the high-dose females. Relative weight increases 

(p<0.05) were present in the mid- and high-dose males, but absolute 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 the testes with epididymides or adrenal glands. 

Non-neoplastic histopathological lesions included an increased incidence of intra-cytoplasmic 

protein-like droplets in the superficial epithelium of the urinary bladder (mid- and high-dose 

animals), cataracts (high-dose animals; an increased incidence of opaque eyes was noted in highdose females during gross necropsy), and an increased severity of extramedullary hematopoiesis 

and pigment in the spleen in high-dose animals. No treatment-related histopathological findings 

were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uterus, mammary, 

thyroid, adrenal or pituitary glands. 

An increased incidence of neoplasms (hemangiomas 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 incidence 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/day in males and females, respectively) 

was demonstrated as the MTD based on clinical signs, decreases in body weight gain, alterations 

in clinical pathology parameters, and histopathology changes. The systemic LOAEL was 

1000 ppm (145.99 and 180.86 mg/kg/day in males and females, respectively) based on decreases 

in RBC and brain AChE activities, increased kidney weight (interim sacrifice), and bladder 

pathology (males) and on a decrease in brain activities and bladder pathology (females). The 

systemic NOAEL was 100 ppm (14.73 and 18.11 mg/kg/day, respectively). 

Page 60 of 70
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/day) for 52 weeks. At study termination, all 

surviving dogs were sacrificed for gross and histopathological evaluation. The organs weighed at 

sacrifice included the adrenals, thyroid, pituitary, liver, kidneys, testes, epididymides, and 

ovaries. Gross and microscopic pathological examinations were conducted on these organs as 

well as the urinary bladder, prostate, mammary gland, and uterus. 

The occurrence of clinical signs (e.g., emesis, lacrimation, salivation, 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 during Weeks 0-5 compared to control 

(body weight data were not provided in review). Lesser decreases (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 mortality, food consumption, ophthalmology, urinalysis, or 

macroscopic pathology. No treatment-related effects 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 the 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 pituitary weights (NS) in the low, 

mid- and high-dose males (↓11, 22, and 33%, respectively). Relative thyroid weight was 

decreased (p≤0.05) in the high-dose males by 27%. No treatment-related histopathological 

findings were seen in the testes, epididymides, prostate, ovaries, uterus, mammary gland, 

thyroid, adrenal, or pituitary at any dose, as well as the liver or kidneys. 

Plasma and RBC AChE activities were determined 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; males (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) resulted in AChE inhibition in the females, a 

five-week feeding study in dogs (MRID 42022801) was conducted to provide supplementary 

Page 61 of 70
CARBARYL FINAL 

Page 54 of 62 

data to establish a NOAEL/LOAEL. Based on the results of the five-week study, the LOAEL is 

125 ppm (3.37 and 3.73 mg/kg/day in males and females, 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/Metabolism (Rat, OCSPP 870.7485) 

In a rat metabolism study (MRID 44402501), 1-naphthyl-14C-labeled carbaryl (100% purity) 

was administered to 15-month-old male Iffa Credo CD (SD-derived) 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 feeding study in the rat at a carbaryl 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 carbaryl 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, indicating 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 glucuronide) of carbaryl metabolites (5,6-dihydro-5,6-

dihydroxy-1-naphthyl N-methylcarbamate 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 weights were observed in 

the 1500 and 7500 ppm groups compared to control. Absolute and relative liver weights were 

increased 18% and 39%, respectively, in the 7500-ppm group. Absolute thyroid weight was 

increased (non-dose-dependent) by 63% and 69% in the 7500 and 1500 ppm dose groups, 

respectively, and relative thyroid weights were increased 103% and 121%, respectively. 

There were no treatment-related increases in the incidence of hepatocellular adenoma (benign), 

C-cell adenoma in the thyroid gland, or follicular cell adenoma (benign). However, treatmentrelated changes were noted in liver, thyroid glands, and kidneys in the 7500 ppm group. The 

Page 62 of 70
CARBARYL 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 incidences of follicular cell hypertrophy of the thyroid 

glands were 0/5, 3/5, 5/5, and 5/5 and the incidences of transitional cell hyperplasia of the renal 

pelvis were 0/5, 0/5, 1/5 and 2/5 in the 0, 250, 1500, and 7500 ppm 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 nominal 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 consumption 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-related. 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 and 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 comparison to eggs set); hatchling 

weight were significantly reduced at only the highest 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 significant differences. Viable embryos and number 

and survival of hatchling (based on eggs set) were reduced, with significant reductions at the top 

dose. Hatchling weight was significantly reduced at the mid and high dose. The appearance of 

these effects during the recovery period suggests that adverse worsen, 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). 

Page 63 of 70
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 significantly 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 control vs. 31.1 at 1000 mg/kg-diet); 

percent of eggs set of eggs laid also significantly reduced at this test 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 effects 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 (purity 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 euthanized on Day 91, and testes, epididymides, seminal 

vesicles, ventral prostate, and coagulating glands were excised and weighed. Sperm count, sperm 

motility and morphology were determined with sperm obtained from the epididymis. One testis 

from each animal was preserved for histology in 10% neutral buffered formalin, dehydrated, 

infiltrated and embedded in paraffin, sectioned, 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 measurements of sorbitol dehydrogenase (SDH), lactate 

dehydrogenase (LDH), glucose-6-P-dehydrogenase (G6PDH), and γ-glutamyl transpeptidase 

(γGT). Protein content was determined with the Lowry method. 

The only treatment-related sign of toxicity was 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-dependent 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 vesicle, ventral prostate and coagulating gland (data 

not provided). 

Enzyme specific activities (nmol 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 γGT 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%, respectively, 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 were also decreased by 25 and 63%, respectively. In addition, the 

percentages of morphological abnormalities in sperm were increased to a similar degree in both 

Page 64 of 70
CARBARYL 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 congestion and edema in the peripheral region, and 

seminiferous tubules with depressed 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 regions, and seminiferous tubules with disturbed 

spermatogenesis and acellular homogeneous masses. 

The authors concluded that exposure to carbaryl in male rats (up to 100 mg/kg/day) results in 

testicular atrophy and affects spermatogenesis, yielding reduced sperm production and an 

increase in the percentage of abnormal spermatozoa. 

Mechanistic Study: Spermatotoxic Effects

In another published, non-Guideline study, Pant et al. (1996) evaluated the spermatotoxic 

effects of the insecticide carbaryl in male rats. Carbaryl (purity 99.2%) was administered daily 

by oral gavage 5 days/week for 60 days at 0 (vehicle), 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 pelleted diet and water ad libitum, and were housed under standard animal 

care conditions. Body weights were determined at initiation and completion of the study. After 

the final dose, the rats were fasted overnight and sacrificed by cervical dislocation on Day 61. 

Testes, epididymides, seminal vesicles, ventral prostate, and coagulating 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 treatment groups. Effects on body 

weight gain were reported in the young and adult males at 50 and 100 mg/kg/day, with greater 

effects the young males (data, and direction of effect, were not reported). For the young rats, 

absolute weights of testes, epididymides, seminal vesicles, ventral prostate, and coagulating 

gland were decreased (p<0.05) in the 100 mg/kg/day dose group only (data not reported), but no 

effects were observed in the relative weights of reproductive organs. No effects were observed 

on absolute or relative organ weights in the adult males (data not reported). 

For young and adult rats, sperm motility and count 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 reduced 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 males, 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 decreases 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 all sperm from young males, respectively, and 

16% and 23% of all sperm from adult males, respectively. Abnormalities in control males (young 

Page 65 of 70
CARBARYL 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 the 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 carbaryl had spermatotoxic effects in young and adult rats at doses of 

50 and 100 mg/kg/day. The LOAEL and NOAEL for spermatotoxic 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 embryotoxic effects of carbaryl 

(99.8% purity) on the African Clawed frog (Xenopus laevis) during conduct of a modified Frog 

Embryo Teratogenesis Assay-Xenopus (FETAX), coupled with a histopathological 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 contained 4 mL of control or carbaryl test solutions. Plates were 

duplicated or triplicated for each female if well-cleaved embryos were available. All the plates 

were incubated in a thermostatic chamber at 23±0.5°C until the embryos reached stage 47 (freeswimming larva). The number of dead larvae 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. Larvae were fixed or frozen for the successive 

histological and biochemical analyses, respectively. The assay was repeated three times under 

the same experimental conditions. 

Histological examinations were conducted on control 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 independent experiments from five pools of five whole 

body larvae. Each assay was performed in triplicate 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 larvae, and the results were expressed as the 

percentage of AChE inhibition 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 percentage of overall malformations from 6.8% at 

1 mg/L to 100% at 24 mg/L, with no malformations observed in the control embryos. The 

calculated teratogenic index of 2.41 (calculated as the ratio of the LC50 and the TC50

[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. 

Page 66 of 70
CARBARYL FINAL 

Page 59 of 62 

All larvae at the mid-high concentration had pathological lesions; none of the larvae exposed to 

the high concentration were processed due to severe external malformations. Histopathological 

examination indicated that the tail musculature and the notochord were the main targets for 

carbaryl effects. Inhibition of AChE activity was investigated in larvae exposed to carbaryl 

concentrations lower and higher than the calculated TC50 (8.43 mg/L). A well-defined 

concentration response was observed between the higher carbaryl concentrations and percentages 

of AChE inhibition observed at these carbaryl exposures. In the 8 mg/L carbaryl-treatment group 

(approximate TC50), the AChE was decreased by 61.6%. Mean AChE concentrations were 

decreased by 35.6% in the 4 mg/L concentration group and decreased by 77% in the 16 mg/L 

group. This study did not expose Xenopus laevis) to carbaryl during the thyroid-critical period of 

metamorphosis and development (stages 51-57), but the inhibition of AChE is implicated in the 

treatment-related malformations and histopathological effects. 

Page 67 of 70
CARBARYL FINAL 

Page 60 of 62 

APPENDIX 3: References Not Utilized in the Carbaryl WoE Analysis 

In 2009, after public review and comment, a final list of 67 chemicals and schedule for issuing 

Test Orders for the EDSP Tier 1 screening battery was made available in a Federal Register 

Notice issued October 21, 2009 (74 FR 54422). The agency’s review of the initial data submitted 

as “other scientifically relevant information (OSRI) was provided in the Report of the Endocrine 

Disruptor Review Team (USEPA, 2010). 

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies 

previously submitted to the agency for registration/reregistration, published literature articles 

and/or Tier 1 assays. The agency also conducted a more recent search (2009 to 2014) of 

available scientific literature for any additional relevant information for their weight of evidence 

(WoE) evaluations. These articles were evaluated in accordance with the agencies Evaluation 

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

(http://www.epa.gov/pesticides/science/efed/policy_guidance/team_authors/endangered_species

_reregistration_workgroup/PDF_rot/esa_evaluation_open_literature.pdf) and the 2012 Guidance 

for considering and Using Open Literature Toxicity Studies to Support Human Health Risk 

Assessment (http://www.epa.gov/pesticides/science/lit-studies.pdf). 

The following published and unpublished references were considered for use in the WoE 

analysis for Carbaryl but were not utilized due to one or more of the following reasons: 1) the 

article was not available in English; 2) the compound of interest was not used in the study; 3) the 

test material was not adequately described; 4) a formulated end-use product or mixture of 

chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 

6) the experimental conditions were not adequately described; 7) only an abstract of the study 

was available; 8) the reference is a review article or book chapter and does not contain primary 

study data; 9) insufficient information was available to adequately assess the validity of the study 

results; 10) the 40 CFR Part 158 guideline study was classified as unacceptable/inadequate; 11) 

the study dealt only with non-EDSP assay development; 12) no specific endocrine-related 

endpoints were assessed in the study; and 13) the study contained only data on invertebrates. 

Beauvais, S.L., Jones, S.B., Parris J.T., Brewer, S.K. and Little, E.E. (2001) Cholinergic and 

Behavioral Neurotoxicity of Carbaryl and Cadmium to Larval Rainbow Trout (Oncorhynchus 

mykiss). Ecotoxicol. Environ. Saf. 49: 84-90. 

Beyers, D.W. and Sikoski, P.J. (1994) Acetylcholinesterase 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 interactive effects of three insecticides on 

amphibian metamorphosis. Environmental Toxicology and Chemistry, Vol. 27 No.7: 1561-1568. 

Page 68 of 70
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) Interactions 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 ovarian 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, Damodaran AC. (1995). Simple spectrophotometric 

method for the determination of carbaryl in soil and insecticide 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. 

Page 69 of 70
CARBARYL FINAL 

Page 62 of 62 

Peterson, H.G., Boutin, C., Martin, P.A., Freemark, K.E., Ruecker, N.J., and Moody, M.J. (1994) 

Aquatic phytotoxicity of 23 pesticides applied 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. 

Page 70 of 70