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 

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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 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: DICHLOBENIL

OFFICE OF PESTICIDE PROGRAMS 

OFFICE OF SCIENCE COORDINATION AND POLICY 

U.S. ENVIRONMENTAL PROTECTION AGENCY 

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DICHLOBENIL FINAL 

ii 

TABLE OF CONTENTS 

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

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

I. Introduction.......................................................................................4 

II. Sources of Scientific Data and Technical Information......................................5 

A. EDSP Tier 1 Screening Assays............................................................5 

B. Other Scientifically Relevant Information (OSRI)......................................5 

III. Weight of Evidence (WoE) Evaluation.......................................................5 

A. EDSP Tier 1 Screening Assays.............................................................8 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9 

1. Effects on Estrogen Pathway.......................................................9 

2. Effects on Androgen Pathway....................................................14 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................19 

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

A. Systemic/Overt Toxicity in the in vivo Tier 1 assays and OSRI .....................22 

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

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

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

C. Androgen Pathway.........................................................................24 

D. Thyroid Pathway...........................................................................24 

E. Conclusions.................................................................................25 

V. EDSP Tier 2 Testing Recommendations....................................................25 

VI. References.......................................................................................26 

LIST OF TABLES: 

Table 1. Tier 1 Assay for Dichlobenil.....................................................................8 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Dichlobenil.......................................11 

Table 3. Androgenic/Anti-Androgenic Pathway for Dichlobenil.....................................16 

Table 4. Thyroid Pathway for Dichlobenil..............................................................20 

APPENDIX 1. EDSP Tier 1 Screening Assays.................................................................29 

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

APPENDIX 3. References Not Utilized in the Dichlobenil WoE Analysis.............................52 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen 

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol 

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

F2 Second filial generation 

Fcd Fecundity

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iv 

Abbreviation Terminology 

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/M 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 

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

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

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Abbreviation Terminology 

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

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

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Abbreviation Terminology 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

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Page 1 of 52 

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-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In addition 

to the available Tier 1 assay 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 dichlobenil 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 January 15, 2014, the Tier 1 Assay Weight of Evidence Review Committee (T1WoERC) of the 

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

conducted a weight-of evidence (WOE) analysis of the potential interaction of dichlobenil with the 

E, A or T hormone pathways. The T1WoERC conclusions from the WoE evaluation in this report 

are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in vitro assays 

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

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

For the estrogen pathway, there was no convincing evidence supporting potential estrogenic or 

anti-estrogenic effects for dichlobenil. The Tier 1 ER binding, ERTA, and aromatase assays as 

well as the uterotrophic assay were negative, whereas the results of the steroidogenesis assay were 

equivocal (non-concentration dependent decreases in estradiol production). Decreased uterine 

weights (wet and blotted) were noted in the female pubertal study, but there was no effect on 

uterine weights after adjustment for PND 21 body weights. In the FSTRA, reduced fecundity and 

changes in ovary histopathology (↑oocyte atresia and a shift to more mature stages) were observed 

at the mid and high test concentration (0.21 and 2.1 mg/L). In the high treatment discoloration 

(i.e., lighter) in the fish was reported, which may be suggestive of stress. There were no effects on 

fertilization, GSI or VTG in this study. In the OSRI, estrogen-related findings in the absence of 

overt toxicity included a decrease in uterus and ovary weights in the Part 158 chronic dog study 

(which occurred in the presence of increased liver weights). Decreases in egg laying and hatchling 

success (survival and/or weight) were observed in the mallard duck and bobwhite quail 

reproduction study (decreases in male body weight observed in the bobwhite quail study). 

Decreases in fecundity (fish and birds) in of themselves are limited in their diagnostic specificity 

for the estrogen-pathway. A slight 4.2% decrease in eggshell thinning was observed in the 

bobwhite quail study, however, the Tier 1 in vitro and in vivo uterotrophic assays were negative or 

equivocal (steroidogenesis, non-concentration decreases in estradiol production), and no effect on 

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Page 2 of 52 

eggshell thinning was observed in the mallard duck study with no or limited estrogen-related 

effects observed in the other in vivo studies (sometimes observed in the presence of systemic 

toxicity). 

For the androgen pathway, dichlobenil was a binder in the Tier 1 AR binding assay and was 

equivocal in the steroidogenesis assay. No androgen-related effects were observed in the male 

pubertal assay and effects in the Hershberger assay occurred in the presence of overt toxicity. In 

the FSTRA dose-dependent reductions in median male tubercle scores at all three dose levels (21, 

14, and 12 in low, mid and high treatment, respectively vs. 26 negative control); however, it is 

noted that there were no other androgen-related effects observed. It is noted that observation of 

discoloration in the fish was reported for the high treatment group. In the mice and rat OSRI 

studies, changes in androgen-related endpoints occurred concomitantly with increases in liver 

weights. However, in hamsters, changes in prostate weights and histopathology of the prostrate 

and seminal vesicles occurred in the absence of overt toxicity or liver effects. Effects on hatchling 

success (numbers and survival) was observed in both the bobwhite quail and mallard duck study, 

however, these endpoints are limited in informing potential androgen-related interactions. 

The weight of evidence suggests that Dichlobenil may potentially interact with the androgen 

pathway based on: (i) positive AR binding; (ii) decreases in testosterone production in the 

steroidogenesis assay; (iii) tubercle score reductions in FSTRA; and (iv) decreases in hamster 

testes and prostate weights and changes in prostate histopathology in the Part 158 studies. 

There is no convincing evidence for a potential effect of dichlobenil on the thyroid pathway. No 

thyroid-related effects were observed in the male or female pubertal assays. In the AMA, there was 

no effect when compared to solvent control for the developmental endpoints. In the OSRI, there 

were no thyroid-related effects in the absence of overt toxicity except for an increase in thyroid 

weight without any corresponding changes in thyroid histopathology in dogs in the chronic study. 

For dichlobenil, the point of departure (POD) is 1 mg/kg/day used for human health risk 

assessment is based on liver toxicity (changes in liver enzymes, increased liver weights, and 

periportal hepatocytes hypertrophy) in male dogs at 6 mg/kg/day (LOAEL) in a chronic toxicity 

study. This POD is used to derive the chronic Reference Dose (RfD) for dietary exposure risk 

assessments. The POD of 3.0 mg/kg/day used for calculating Margin of Exposure (MOEs) for nondietary exposure risk assessments is based on decreased pup body weight at weaning at 17.5 

mg/kg/day (LOAEL) in a two-generation reproduction study. Accordingly, the PODs (1 and 3 

mg/kg/day) used for the overall risk assessments are 20 to 60-fold lower than the lowest dose (60 

mg/kg/day) where a potentially androgen related effect was observed in the male pubertal assay. 

Consequently, the current RfD and MOEs are protective of potentially endocrine-related effects 

observed in the pubertal assay. 

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

for dichlobenil since additional testing will not impact the regulatory endpoints used for human 

health risk assessments. 

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EDSP Tier 2, Medaka Extended One Generation Reproduction Test (MEOGRT) is recommended 

due to the potential interaction with the androgen pathway and the effects observed in the FSTRA 

assay. Currently, the chronic NOAEC endpoint for fish is <0.33 mg/L, based on growth effects at 

all concentrations tested in an early life-cycle study with rainbow trout. Effects on male tubercle 

score in the FSTRA were observed at all concentrations tested (≥0.021 mg/L); additionally, 

fecundity was decreased at 0.21 and 2.1 mg/L. Therefore, a Tier 2 MEOGRT is requested to 

provide data to understand potential effects of dichlobenil on fish reproduction and development. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard duck 

are available for dichlobenil. In both species, effects on reproductive parameters were observed. 

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 dichlobenil 

exposure. Relative to EDSP, additional testing is not recommended. 

. 

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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-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) axes. In addition 

to the available Tier 1 assay 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 a pesticide chemical 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 January 15, 2014, the 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 dichlobenil with the 

E, A or T hormone 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).

Dichlobenil has a water solubility of 21.2 mg/L (at 25°C), a vapor pressure of 0.00101 mm Hg 

(at 25°C) and a partition coefficient (log Kow) of 2.74. Dichlobenil dissipates in the environment 

principally by volatilization, although it may persist under conditions that reduce the potential for 

volatilization. It is stable to hydrolysis, photodegradation in soil and metabolic degradative 

processes. Bioconcentration factors in fish are 32X, 63X and 110X for fillet, whole fish and 

viscera, respectively, with a high depuration rate (within 1 day of the 14 day depuration period). 

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

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

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

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

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

Tier 2 studies recommendations are presented in Section V. 

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II. Sources of Scientific Data and Technical Information 

A. EDSP Tier 1 Screening Assays 

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

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

1. 

B. Other Scientifically Relevant Information (OSRI) 

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

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 dichlobenil (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. 

Summaries of the Tier 1 assays and the studies cited as other scientifically relevant information 

(OSRI) are presented in Appendix 1 and 2, respectively. 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. 

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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 

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

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

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

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

interactions with the E, A, or T pathway. Additionally, the consistency of the responses in the 

individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured 

in each assay. 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

organization can indirectly provide information on potential compensatory capabilities of an 

individual organism. 

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

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

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

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

biological organization were the responses observed and whether organisms exhibit compensatory 

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

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

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

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the ability to distinguish effects specifically related to an endocrine-related effect from a nonendocrine toxic response. 

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

the 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 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 Part 158 studies for both human and ecological risk 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

IV.A. 

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

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

modes of action in its overall characterization of a chemical’s potential to interact with the E, A or 

T pathway. Adequately addressing these three main components described above is fundamental 

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

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

conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) 

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

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

took all available toxicity data into account. 

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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 in regards 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. 

A. EDSP T1 Screening Assays 

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

in Table 1. 

Table 1: Tier 1 Screening Assays for Dichlobenil.

Tier 1 Assays Test Guideline Test Order Status 

Estrogen Receptor (ER) Binding Assay 

(Rat uterine cytosol) OSCPP 890.1250 Requirement Satisfied 

(MRID No. 48616104) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OSCPP 

890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48616105) 

Androgen Receptor (AR) Binding Assay 

(Rat prostate cytosol) OSCPP 890.1150 Requirement Satisfied 

(MRID No. 48616102)

Steroidogenesis Assay 

(Human cell line H295R) 

OSCPP 

890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48616110) 

Aromatase Assay 

(Human recombinant microsomes) OSCPP 890.1200 Requirement Satisfied 

(MRID No. 48616103) 

Uterotrophic Assay (Rat) 

OSCPP 

890.1600; 

OECD 440 

Requirement Satisfied 

(MRID No. 48616111) 

Hershberger Assay (Rat) 

OSCPP 

890.1400; OECD

441 

Requirement Satisfied 

(MRID No. 48616107) 

Pubertal Female Assay (Rat) OSCPP 890.1450 Requirement Satisfied 

(MRID No. 48616108) 

Pubertal Male Assay (Rat) OSCPP 890.1500 Requirement Satisfied 

(MRID No. 48616109 ) 

Fish Short-term Reproduction Assay 

OSCPP

890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48616106) 

Amphibian Metamorphosis Assay (Frog) 

OSCPP 

890.1100; 

OECD 231 

Requirement Satisfied 

(MRID No. 48616101) 

Page 16 of 60
DICHLOBENIL FINAL 

Page 9 of 52 

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

1. Effects on Estrogen Pathway 

The data utilized to assess the potential for dichlobenil to interact with the estrogen pathway are 

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. 

Dichlobenil did not produce responses indicative of estrogen agonism or antagonism in the ER 

binding assay, the ERTA assay, the aromatase assay, or the in vivo uterotrophic assay. However, the 

results of the Tier 1 steroidogenesis assay indicated that no changes in estradiol concentration were 

observed during Runs 1 and 2, but estradiol was decreased (p≤0.05) in Run 3 at all dichlobenil 

concentrations except 100 and 0.1 μM. Therefore, the results of the steriodogenesis assay were 

considered equivocal. In the female pubertal assay, there was a dose-related decrease in mean 

absolute blotted uterus weight at doses of 20 mg/kg/day [(↓12%; not significant [NS])] and 60 

mg/kg/day (21%; p<0.05) and a decrease (NS) in mean absolute wet uterus weight at 60 mg/kg/day 

(↓26%). Overt toxicity was not noted in the female pubertal assay; however, liver weights were 

increased by 18-19% at the high dose. In the FSTRA, fecundity was significantly (p<0.05) reduced 

by 53% and 90% at the 0.21 and 2.1 mg a.i./L treatment levels, respectively, compared to the 

negative control. Also in the FSTRA, there was an increased incidence and/or severity (minimal to 

moderate) of oocyte atresia and a shift to more mature stages in the ovaries in the 0.21 and 2.1 mg 

a.i./L treatments. Fish at the high dose in the FSTRA were reported to be discolored (i.e. lighter) 

compared to fish in other treatment groups including controls which may suggest signs of stress in 

this treatment group, however, the incidence of this effect was not reported. 

In a Part 158 chronic toxicity study in dogs, absolute (↓50%) and relative (↓54%) ovarian weights 

and absolute (↓22%) and relative (↓24%) uterine weights were decreased at the high dose. No overt 

toxicity was noted in this study, but both relative and absolute liver weights were increased by 

≥18%. At the high dose (36 mg/kg/day), decreased uterine weights were accompanied by “early” 

maturation of the uterus (5/5). In the 2-generation reproduction toxicity study in rats, a decrease in 

implantations/dam (12.3 treated vs. 14.6 controls) was observed at 2000 ppm; however, this effect 

occurred in conjunction with overt toxicity based on decreased body weights. There were no other 

effects observed in the 158 mammalian data. 

In the Part 158 quail reproduction studies, decreases in egg laying were observed at the mid and 

highest dose tested (289 and 600 mg/kg-diet) as well as decreases in eggshell thinning (4.2%), 

number of hatchings and offspring weight at 600 ppm. Additionally, 14-day old survivors were 

affected at all concentrations (≥146 mg/kg-diet). In this study, reductions in male body weight gain 

were observed at all concentration (25-50% non-treatment responsive), however, no other clinical 

signs of toxicity were observed. In the mallard duck reproduction study, decreases in eggs laid, 

Page 17 of 60
DICHLOBENIL FINAL 

Page 10 of 52 

number of hatchlings, and 14 day old survivors were also observed at 600 mg/kg-diet and signs of 

toxicity were not observed in the mallard duck study. In the Part 158 early life-stage study with 

rainbow trout, effects on length were observed at ≥0.33 mg/L. 

Page 18 of 60
DICHLOBENIL 

Page 11 of 52 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Dichlobenil 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Dichlobenil1

Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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) 

EDSP Tier 1 Assays 

ER Binding (MRID 48616104) NERTA (MRID 48616105) NAromatase (MRID 48616103) NSteroidogenesis (MRID 48616110) E Uterotrophic (MRID 48616111) N Female Pubertal Rat (MRID 48616108) 

↓21-

26%

(H) 

N N N N N 

FSTRA (MRID 48616106) NE N P4 (M, H) N

Fcd: ↓53%, 

↓90%

(M, H)

OSRI

Subchronic toxicity (Rats; MRID 00107106) N N N N 
DICHLOBENIL 

Page 12 of 52 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Dichlobenil1

Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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) 

Subchronic toxicity (Hamsters; MRID 40600701) 

N N N N 

Subchronic toxicity (Mice; MRID 48043302) N N N N Chronic toxicity (Dogs; MRID 43969701) 

↓78%

(H)

↓50%

(H)

P6

(H)

N 

Chronic toxicity / carcinogenicity (Rats; MRIDs 00147438, 40401101) 

N N N N 

High-dose carcinogenicity (Hamsters; MRID 42221201) 

N N N N 

Low-dose carcinogenicity (Hamsters; MRID 41988301, 42015101)

N N N N

Two-generation reproduction toxicity (Rats; MRID 41257303, 42239101) 

N N N N 

Developmental toxicity (Rabbits; MRID 41257302) 

N N 
DICHLOBENIL 

Page 13 of 52 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Dichlobenil1

Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Sex Steroid 

Hormones 

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) 

Developmental toxicity (Rats; MRID 00147437) N N Avian Reproduction (Quail; MRID 45280301) 

P7 

(L,M,H) 

Avian Reproduction (Mallard; MRID 45280302) 

P9 

(H) 

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

or ↑) indicate the direction of a given response. A shaded cell indicates that is parameter is not routinely evaluated or is not appLiver Weight Changes 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 

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 

details see Section IV. A 

4. Increased incidence and/or severity (minimal to moderate) of oocyte atresia and a shift to more mature stages in the ovaries in 

treatments. 

5. Fish were discolored (i.e. lighter) compared to fish in other treatment groups including controls, however, the incidence of this 6. At the high dose (36 mg/kg/day), decreased uterine weights were accompanied by “early” maturation of the uterus (5/5). 7. Decreases in egg laid at the mid and highest dose tested (289 and 600 mg/kg-diet) as well as decreases in eggshell thinning (4.

and offspring weight at 600 ppm. Additionally, 14-day old survivors were affected at all concentrations (≥146 mg/kg-diet). 

8. Reductions in male body weight gain were observed at all concentration (25-50% non-treatment responsive), however, no other 

were observed. 

9. Decreases in eggs laid, number of hatchlings and 14-d offspring survival at 600 mg/kg-diet. P Positive findings N Negative findings (in vitro) / No effect (in vivo) 

E Equivocal findings 
DICHLOBENIL FINAL

Page 14 of 52 

2. Effects on Androgen Pathway 

The data utilized to assess the potential for dichlobenil to interact with the androgen pathway are 

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. 

Dichlobenil was a binder in the AR assay, and gave equivocal results in the steroidogenesis 

assay based on a non-dose-dependent decrease in testosterone levels in two of three runs. In the 

Hershberger assay decreased seminal vesicle, ventral prostate, LABC, and Cowper’s gland 

weights at the high dose of 180 mg/kg/day; however the effects occurred in conjunction with 

overt toxicity (≥10% decrease in body weight). At the mid dose (60 mg/kg/day), a decrease in 

seminal vesicle weight was observed; however, this dose was considered to be negative since 

only one of the five androgen-responsive tissue weights was decreased (a positive response 

requires changes in two or more of the five AST weights). Additionally, liver weights were 

increased (32-40%) at the mid and high dose. In the FSTRA, dose-dependent reductions in 

median male tubercle scores were observed at all three dose levels (21, 14 and 12 in low, mid 

and high-concentration vs. 26 in negative control). There were no androgen-related effects 

observed in the male pubertal assay. 

In the Part 158 studies, testes (with epididymides) weights were decreased 12-13% at 117 and 277 

mg/kg/day, and pigmented macrophages and dilated seminiferous tubules were observed at 277 

mg/kg/day in a carcinogenicity study in hamsters. In another carcinogenicity study evaluating 

lower doses in hamsters, reduced secretion of the prostate and seminal vesicles was noted at 9.4 

and 46 mg/kg/day. In a subchronic study in hamsters, testes weights were decreased 33% and 

seminal vesicle weights were decreased 31%, both at 263 mg/kg/day; prostate weights were 

decreased 19-36% at 16, 79, and 263 mg/kg/day. In addition, and small testes, decreased number 

of spermatocytes, prostatic mineralization, tubular degeneration in the testes, small epididymides, 

and slight hemorrhagic area in the epididymides were noted at 263 mg/kg/day. In a chronic 

toxicity study in dogs, testes weights were increased by 33% at the high dose (36 mg/kg/day) and 

there were dose-dependent decreases (↓14-37%) in prostate weights, which attained significance 

(p<0.05) at the high dose. Finally, in a subchronic study in mice, absolute and relative testes 

weights were increased 7-13% at 19, 95, and 473 mg/kg/day. Overt toxicity was noted in each of 

these studies at the high dose and three of these studies reported increases (≥13%) in liver weights. 

No androgen-related effects were seen in the remaining Part 158 studies. 

In the Part 158 avian reproduction studies, effects on offspring survival were observed ≥146 

mg/kg-diet and number of hatchlings/offspring weight at 600 mg/kg-diet in the bobwhite quail 

study; decreases in number of hatchlings/offspring survival at 600 mg/kg-diet was observed in 

the mallard duck study. In the bobwhite quail study, reductions in male body weight gain were 

Page 22 of 60
DICHLOBENIL FINAL

Page 15 of 52 

observed at all concentration (25-50% non-treatment responsive), however, no other clinical signs 

of toxicity were observed. 

Page 23 of 60
DICHLOBENIL 

Page 16 of 52 

Table 3: Androgenic/Anti-Androgenic Pathway for Dichlobenil

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Dichlobenil1

Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

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 

EDSP Tier 1 Assays 

AR Binding (MRID 48616102) PSteroidogenesis (MRID 48616110) PHershberger (MRID 48616107) N Male Pubertal Rat (MRID 48616109) N N N N N N N N FSTRA (MRID 48616106) N N N

Tub

sc

(L, 

OSRI

Subchronic toxicity (Rats; MRID 00107106) N N N N N N Subchronic toxicity (Hamsters; MRID 40600701) 

 N N N N N

Prostate:

↓19 - 

36%; 

(ML, 

MH) 
DICHLOBENIL 

Page 17 of 52 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Dichlobenil1

Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

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 

Subchronic toxicity (Mice; MRID 48043302) 

↑7-13%

(ML, 

MH)

N N N N N

Chronic toxicity (Dogs; MRID 43969701) 

N N N N N N 

Chronic toxicity / carcinogenicity (Rats; MRIDs 00147438, 40401101) 

N N N N N N 

High-dose carcinogenicity (Hamsters; MRID 42221201) 

N N N N N N 

Low-dose carcinogenicity (Hamsters; MRID 41988301, 42015101) 

N P (MH) N N N N 

Two-generation reproduction toxicity (Rats; MRID 41257303, 42239101) 

N N N N N N 
DICHLOBENIL 

Page 18 of 52 

Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Dichlobenil1

Study Type/ Literature Citation 

AR Binding 

Steroidogenesis 

Testosterone 

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 

Avian Reproduction (Quail; MRID 45280301) 

P5 

(L,M,H) 

Avian Reproduction (Mallard; MRID 45280302) 

P7 

(H) 

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

(↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not appl

LW= Liver Weight. Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Ventral prostate (VP), Dorsal prostate (DP), Levator ani- bulbocavernosus (LABC), Epididymides (EP). 

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

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 a

details see Section IV. A 

4. Fish were discolored (i.e. lighter) compared to fish in other treatment groups including controls, however, the incidence of this e

5. Decreases in number of hatchlings/offspring weight at 600 mg/kg-diet. Additionally, 14-day old survivors were affected at all 

mg/kg-diet). 

6. Reductions in male body weight gain were observed at all concentration (25-50% non-treatment responsive), however, no other 

were observed. 

7. Decreases in number of hatchlings and 14-d offspring survival at 600 mg/kg-diet P Positive findings N Negative findings (in vitro) / No effect (in vivo) 
DICHLOBENIL FINAL

Page 19 of 52 

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

(frog). 

The data utilized to assess the potential for dichlobenil to interact with thyroid regulation are 

summarized in Table 4. The various targets of the T 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. 

No effects on thyroid weights, hormones or histology were noted in the male pubertal assay. 

In the female pubertal assay, serum TSH levels were increased by 18-22% (20 and 60 

mg/kg/day, respectively), but these changes did not attain statistical significance and were 

not accompanied by any changes in thyroid weight or histology. Substantial increases in 

liver weights were also noted at the high dose in both the male (↑35-36%) and female (↑18-

19%) pubertal assays. In the AMA, the 21-day normalized HLL was increased by 7-11% at 

all concentrations (0.02, 0.2, and 2.0 mg/L, respectively), and a lower incidence and severity 

of follicular cell hypertrophy was noted at 2.0 mg/L compared to the negative control. 

However there were no significant differences for the 21-day normalized HLL endpoint, 

when treatment groups were compared to the solvent control. 

In a Part 158 chronic toxicity study in dogs (MRID 00067649), increased absolute and 

relative thyroid weights were observed in both sexes. No other thyroid-related effects were 

reported in the Part 158 studies. 

 

Page 27 of 60
DICHLOBENIL FINAL

Page 20 of 52 

Table 4: Thyroid Pathway for Dichlobenil

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Dichlobenil1

Study Type / Literature 

Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels Serum TSH levels Pituitary Weight Developmental stage or asynchronous & NHLL Growth (BW, SVL) Systemic Toxicity

Observed2 

Overt Systemic Toxicity 3

EDSP Tier 1 Assays

Male Pubertal 

(Rat) N N N N N N N ↑LW 

 (H) N

Female Pubertal 

(Rat) N N N N N N N ↑LW 

(H) N

AMA (Frog) N

Norm.

HLL: ↑7-

11% (21-d; 

L, M, H)4

N N

OSRI

Subchronic toxicity 

(Rats; MRID 00107106) N N X 

(M,H)

X 

(H) 

Subchronic toxicity 

(Hamsters; MRID 

40600701) 

N N

X 

(ML, MH, 

H)

X 

(H) 

Subchronic toxicity 

(Mice; MRID 48043302) N N X 

(ML, MH, H)

X 

(H) 

Chronic toxicity 

(Dog; MRID 00067649) 

P 

(H) N X (H) N

Chronic toxicity 

(Dogs; MRID 43969701) 

↑33-

56% 

(L, M; 

females)

N X 

(M, H) 

X 

(H)

Combined chronic toxicity / 

carcinogenicity 

(Rats; MRIDs 00147438,

N N N X 

(M,H) 

X 

(H) 

High-dose carcinogenicity 

(Hamsters; MRID 

42221201)

N N N X 

(L,M,H) 

X 

(L,M,H)

Low-dose carcinogenicity 

(Hamsters; MRID 41988301,

42015101)

N N N X 

(MH,H) 

X 

(H) 

Page 28 of 60
DICHLOBENIL FINAL

Page 21 of 52 

1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Mediumhigh treatment, H=High treatment. 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. LW = Liver Weight. 

Changes 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 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. No effect on HLL was observed when treatment groups were compared to solvent control. 

P Positive finding 

N Negative finding 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of dichlobenil 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 evoke 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 non-endocrine organs or overall disruptions in homeostasis” (USEPA, 2013). 

Page 29 of 60
DICHLOBENIL FINAL

Page 22 of 52 

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. In addition to the endocrine-related effects described above 

for the in vivo Tier 1 assays and OSRI, other effects observed which may be considered 

overt/systemic toxicity are also describe 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., organ weights, blood chemistry). 

1. Tier 1 in vivo Assays 

In the uterotrophic assay, the high dose (180 mg/kg/day) caused significant body weight losses 

(8-9%) on Days 3 and 4. Overt toxicity was not observed in the female pubertal assay; 

however, liver weights (18-19%) and cholesterol (18%) were increased at the high dose (60 

mg/kg/day). In the Hershberger assay at the high dose (180 mg/kg/day), overt toxicity, 

manifested as decreased body weights (>9-11%) and body weight gains in both the androgenic 

(>44%) and anti-androgenic (>40%) of the assay. Although overt toxicity was not identified in 

the male pubertal assay, liver weights were increased by >35 – 36%, however no clinical 

chemistry changes were observed. 

Discoloration [(i.e., lighter, more yellowish color compared to the fish in the controls and other 

treatment levels (incidence rate not reported)] was observed in the FSTRA in the high treatment 

group. 

2. OSRI 

The major target organs for dichlobenil-induced toxicity are the liver and the nose in mice and 

rats and the male reproductive system in the hamsters. In a 90-day study with mice, absolute (7-

13%) and relative (7-11%) testes weight were increased at >19 mg/kg/day. In a 90 day oral 

toxicity study in rats, inflammation and necrosis were observed in the liver of males at 45 

mg/kg/day, and increased liver weight and liver histopathology (swelling and vacuolation of 

hepatocytes) were observed in females at 135 mg/kg/day. In a 90 day oral toxicity study with 

hamsters, when administered in the diet at 395 mg/kg/day, there was an excessive body weight 

loss (>13-25%) in both sexes after 2 weeks of treatment. Therefore, the dose was reduced to 263 

mg/kg/day from weeks 3 until week 13. Mean body weight in males at the high dose remained 

decreased (>21%) at the end of treatment and mean food consumption was decreased (>11%) 

after lowering the dose. In males at the high dose, there was increase in absolute liver weights 

(>13%) and enlarged liver at necropsy; histopathology revealed calculi in the gall bladder and 

hepatocytes in the liver. Mean absolute prostate weight was reduced (>19%) 16 mg/kg/day 

while mean absolute seminal vesicle and testis weights were reduced at 16 mg/kg/day (>16%) 

Page 30 of 60
DICHLOBENIL FINAL

Page 23 of 52 

and at 263 mg/kg/day (33%). Histopathology revealed a marked decrease in the number of 

spermatocytes at the high dose. Marginal to moderate prostatic mineralization was seen at 16 

mg/kg/day. Marginal to marked tubular degeneration in the testes was seen at the high dose. In 

females, increased liver weight, enlarged liver and swollen hepatocytes were seen at 79 

mg/kg/day. In the chronic toxicity study in dogs, lower mean relative body weights (12-16%) 

were seen throughout the study in males only at 36 mg/kg/day. At doses >6 mg/kg/day, both 

sexes of dogs exhibited increases in several hepatic enzymes and increased liver weights, males 

revealed periportal hypertrophy of the liver. Male dogs also exhibited dose-dependent decreases 

in absolute weight of the prostate, increases in absolute testes weight at 36 mg/kg/day and 

increases in relative testes weight at 6 and 36 mg/kg/day. None of these weight increases were 

corroborated with histopathological lesions in these organs. In the combined chronic 

toxicity/carcinogenicity study with rats, at the high dose, (173 mg/kg/day), there were 

significant decreases in body weight in males (33%) and females (34%), changes in multiple 

hepatic enzymes, increases in absolute and relative liver weights, enlarged livers at necropsy, 

and histopathological lesions of the liver at 18.9 mg/kg/day In a carcinogenicity study with 

hamsters, at 51 mg/kg/day, there were decreases in body weight, enlarged liver, increases in 

absolute and relative liver weights, histopathological lesions changes, and increases in liver 

tumors 

In the Part 158 bobwhite quail reproduction study, reductions in male body weight gain were 

observed at all concentration (25-50% non-treatment responsive), however, no other clinical 

signs of toxicity were observed. No clinical signs of toxicity were observed in the mallard duck 

reproduction study. 

B. Estrogen Pathway 

There was no convincing evidence supporting potential estrogenic or anti-estrogenic effects for 

dichlobenil. The Tier 1 ER binding, ERTA, and aromatase assays as well as the uterotrophic 

assay were negative, whereas the results of the steroidogenesis assay were equivocal (nonconcentration dependent decreases in estradiol production). Decreased uterine weights (wet 

and blotted) were noted in the female pubertal study, but there was no effect on uterine weights 

after adjustment for PND 21 body weights. In the FSTRA, reduced fecundity and changes in 

ovary histopathology (↑oocyte atresia and a shift to more mature stages) were observed at the 

mid and high test concentration (0.21 and 2.1 mg/L). In the high treatment discoloration (i.e., 

lighter) in the fish was reported, which may be suggestive of stress. There were no effects on 

fertilization, GSI or VTG in this study. In the OSRI, estrogen-related findings in the absence 

of overt toxicity included a decrease in uterus and ovary weights in the Part 158 chronic dog 

study (which occurred in the presence of increased liver weights). Decreases in egg laying and 

hatchling success (survival and/or weight) were observed in the mallard duck and bobwhite 

quail reproduction study (decreases in male body weight observed in the bobwhite quail study). 

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Decreases in fecundity (fish and birds) in of themselves are limited in their diagnostic 

specificity for the estrogen-pathway. A slight 4.2% decrease in eggshell thinning was observed 

in the bobwhite quail study, however, the Tier 1 in vitro and in vivo uterotrophic assays were 

negative or equivocal (steroidogenesis, non-concentration decreases in estradiol production), 

and no effect on eggshell thinning was observed in the mallard duck study with no or limited 

estrogen-related effects observed in the other in vivo studies (sometimes observed in the 

presence of systemic toxicity). 

C. Androgen Pathway 

Dichlobenil was a binder in the Tier 1 AR binding assay and was equivocal in the 

steroidogenesis assay. No androgen-related effects were observed in the male pubertal assay 

and effects in the Hershberger assay occurred in the presence of overt toxicity. In the FSTRA 

dose-dependent reductions in median male tubercle scores at all three dose levels (21, 14, and 

12 in low, mid and high treatment, respectively vs. 26 negative control); however, it is noted 

that there were no other androgen-related effects observed. It is noted that observation of 

discoloration in the fish was reported for the high treatment group. In the mice and rat OSRI 

studies, changes in androgen-related endpoints occurred concomitantly with increases in liver 

weights. However, in hamsters, changes in prostate weights and histopathology of the 

prostrate and seminal vesicles occurred in the absence of overt toxicity or liver effects. Effects 

on hatchling success (numbers and survival) was observed in both the bobwhite quail and 

mallard duck study, however, these endpoints are limited in informing potential androgenrelated interactions. 

The weight of evidence suggests that Dichlobenil may potentially interact with the androgen 

pathway based on: (i) positive AR binding; (ii) decreases in testosterone production in the 

steroidogenesis assay; (iii) tubercle score reductions in FSTRA; and (iv) decreases in hamster 

testes and prostate weights and changes in prostate histopathology in the Part 158 studies. 

D. Thyroid Pathway 

There is no convincing evidence for a potential effect of dichlobenil on the thyroid pathway. 

No thyroid-related effects were observed in the male or female pubertal assays. In the AMA, 

there was no effect when compared to solvent control for the developmental endpoints. In the 

OSRI, there were no thyroid-related effects in the absence of overt toxicity except for an 

increase in thyroid weight without any corresponding changes in thyroid histopathology in dogs 

in the chronic study. 

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E. Conclusions 

There is no convincing evidence for interaction with the estrogen pathway. The observed 

estrogen-related effects in the absence of overt toxicity occurred in isolation without pathway 

concordance or redundancy. However, there is evidence for potential for interaction with the 

androgen pathway. Dichlobenil did not show convincing evidence for a potential interaction 

with the thyroid pathway. 

V. EDSP Tier 2 Testing Recommendations 

For dichlobenil, the point of departure (POD) is 1 mg/kg/day used for human health risk 

assessment is based on liver toxicity (changes in liver enzymes, increased liver weights, and 

periportal hepatocytes hypertrophy) in male dogs at 6 mg/kg/day (LOAEL) in a chronic toxicity 

study. This POD is used to derive the chronic Reference Dose (RfD) for dietary exposure risk 

assessments. The POD of 3.0 mg/kg/day used for calculating Margin of Exposure (MOEs) for 

non-dietary exposure risk assessments is based on decreased pup body weight at weaning at 

17.5 mg/kg/day (LOAEL) in a two-generation reproduction study. Accordingly, the PODs (1 

and 3 mg/kg/day) used for the overall risk assessments are 20 to 60-fold lower than the lowest 

dose (60 mg/kg/day) where a potentially androgen related effect was observed in the male 

pubertal assay. Consequently, the current RfD and MOEs are protective of potentially 

endocrine-related effects observed in the pubertal assay. 

Based on weight of evidence considerations, mammalian EDSP Tier 2 testing is NOT 

recommended for dichlobenil since additional testing will not impact the regulatory endpoints 

used for human health risk assessments. 

EDSP Tier 2, Medaka Extended One Generation Reproduction Test (MEOGRT) is 

recommended due to the potential interaction with the androgen pathway and the effects 

observed in the FSTRA assay. Currently, the chronic NOAEC endpoint for fish is <0.33 mg/L, 

based on growth effects at all concentrations tested in an early life-cycle study with rainbow 

trout. Effects on male tubercle score in the FSTRA were observed at all concentrations tested 

(≥0.021 mg/L); additionally, fecundity was decreased at 0.21 and 2.1 mg/L. Therefore, a Tier 2 

MEOGRT is requested to provide data to understand potential effects of dichlobenil on fish 

reproduction and development. 

For birds, Part 158 avian reproduction studies with both northern bobwhite quail and mallard 

duck are available for dichlobenil. In both species, effects on reproductive parameters were 

observed. 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 

dichlobenil exposure. Relative to EDSP, additional testing is not recommended. 

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VI. References 

Barker L. (1989). Dichlobenil: oral (gavage) teratology study in the rabbit (experiment II), 

repeat study. Unpublished study prepared by Hazleton UK (England). Report No. 56645/21/89. 

(MRID 41257302). 

Barker, L. (1992). Dichlobenil: 2-generation oral (dietary administration) reproduction study in 

the rat; Addendum 1 – Pathology report. Unpublished study prepared by Hazleton UK 

(England). Report No. PDR 5798-65/18 Addendum 1, January 1992. (MRID 42239101). 

Chambers, PR, Hornby, SB, Gibson, WA, et al. (1992). Dichlobenil: potential tumorigenic 

effects in prolonged dietary administration to hamsters. Unpublished study prepared by 

Huntingdon Research Centre, Ltd. (England). Report No. PDR 511/9178, February 10, 1992. 

(MRID 42221201). 

Chambers, PR, Brown, R, Gibson, WA, et al. (1992). Background tumor incidence in hamsters 

(Background data study). Unpublished study prepared by Huntingdon Research Centre, Ltd. 

(England). Report No. PDR 540/920478, September 22, 1992. (MRID 42563601). 

Cooke, A (1977). Effects of field applications of the herbicides diquat and dichlobenil on 

amphibians. Published. Envir. Pollut. 12: 43-50. 

Dijkstra, A. (1988). Chronic feeding study on dichlobenil in the rat (Duphar Report No. 

56645/41/84): Addendum 2. (MRID 40823801). 

Hodge, HC. (1961). Ninety day feeding study of casoron in rats. Unpublished study prepared 

by Dept. of Pharmacology, Univ. of Rochester School of Medicine and Dentistry, Nov. 25, 

1961. (MRID 00107106). 

Hooks, WN, Cary, RJ, Colman, K, et al. (1991). Dichlobenil: potential tumorigenic effects in 

prolonged dietary administration to hamsters. Unpublished study prepared by Huntingdon 

Research Centre, Ltd. (England). Report No. PDR 478/90177, May 3, 1991. (MRID 

41988301). 

Hooks, WN, Cary, RJ, Colman, K, et al. (1991). Dichlobenil: potential tumorigenic effects in 

prolonged dietary administration to hamsters (Supplemental data). Unpublished study prepared 

by Huntingdon Research Centre, Ltd. (England). Report No. PDR 478/91486, June 27, 1991. 

(MRID 42015101). 

Inoue, H, and Enomoto, M. (1983). Chronic feeding study on dichlobenil in the rat. 

Unpublished study prepared by Biosafety Research Center, Foods, Drugs, and Pesticides 

(Japan). Report No. 56645/41/84, November 30, 1983. (MRID 00147438). 

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Page 27 of 52 

Kemp, A., DeHaan, M, and Dawes, R (1987). A 13-week (dietary) toxicity study with 

dichlobenil in male and female mice. Unpublished study prepared by Duphar B.V. Project ID 

DT 87/01, April 1987. (MRID 48043302). 

Kemp, A, and Dawes, R. (1987). Chronic feeding study on dichlobenil in the rat: Addendum to 

Duphar Report No. 56645/41/84. (MRID 40401101). 

Kemp, A, de Haan, M, and Jager, FC. (1987). A 13-week toxicity study with dichlobenil in 

male and female hamster followed by a 4-week recovery period. Unpublished study prepared 

by Duphar B.V., Dept. of Toxicology (Netherlands). Laboratory study number 56645/04/87, 

June 10, 1987. (MRID 40600701). 

Koëter, HB. (1984). Oral embryotoxicity/teratogenicity study with dichlobenil in rats. 

Unpublished study prepared by Netherlands Organization for Applied Scientific Research. 

Report No. V 84.026/231045, January 1984. (MRID 00147437). 

Leeming, NM. (1989). Dichlobenil: 2-generation oral (dietary administration) reproduction 

study in the rat. Unpublished study prepared by Hazleton UK (England). Report No. PDR 

5798-65/18, August 15, 1989. (MRID 41257303). 

Meerts, I.A.T.M. (2011). Dichlobenil Technical: Estrogenic Receptor Transcriptional 

Activation Assay (Human Cell Line (HeLa-9903)). Unpublished study prepared by NOTOX 

B.V, Hertogenbosch, The Netherlands. Laboratory Report No.: 495782, December 9, 2011. 

(MRID 48616105). 

Pickersgill, N. (1995). Dichlobenil — 52 week oral (capsule) toxicity study in the Beagle dog. 

Unpublished study prepared by Pharmakon Europe (France). Study No. 65/510, December 20, 

1995. (MRID 43969701). 

McVey, E. A (2012) Weight of evidence summary of Tier 1 and OSRI data from dichlobenil 

Technical. Chemtura Corporation. Middlebury, CT. NOTOX Project 498767. MRID No. 

48670501. 

Til, HP, Feron, VJ, Huismans, JW, et al. (1969). Chronic (Two-year) Toxicity Study with 

Dichlobenin (H133) in Beagle Dogs. Unpublished study prepared by Thompson-Hayward 

Chemical Co. Report No. R2813. (MRID 00067649). 

USEPA. (2010). Dichlobenil — Report of the OPP Endocrine Disruptor Review Team; Test 

Order #: EDSP-027401-120. December 7, 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. U.S. Environmental Protection Agency, Washington, DC. 

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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. U.S. Environmental Protection Agency, Washington, DC

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APPENDIX 1. EDSP T1 Screening Assays

Amphibian Metamorphosis Assay (Frog); OSCPP 890.1100; OECD 231 

The 21-day assay (MRID No. 48616101) of Dichlobenil (99.3% purity) on amphibian metamorphosis 

of African clawed frog (Xenopus laevis) was conducted under flow-through conditions. Amphibian 

larvae at Nieuwkoop-Faber (NF) stage 51 (80 per control and treatment group) were exposed to a 

negative control and a solvent control (0.1 mL/L; acetone) and nominal dichlobenil concentrations of 

0.02, 0.2, and 2.0 mg a.i./L. Mean-measured concentrations were <0.001 (<LOQ; negative and 

solvent), 0.019, 0.19, and 1.9 mg a.i./L. The test system was maintained at 21.0 to 22.5°C and a pH of 

7.3 to 8.2. 

No mortalities were observed in the solvent control, or low or mid treatment groups. One mortality 

each was recorded in the negative control and high treatment group. Clinical signs (i.e., behavioral and 

other sublethal effects) including curved spine were observed in tadpoles in the solvent control and 

0.019 and 0.19 mg a.i./L treatment groups. This effect did not appear to be related to the test substance. 

No significant acceleration or delay of body weight, median NF developmental stage, snout-to-vent 

length (SVL), hind-limb length (HLL), or normalized (for SVL) HLL was observed at Day 7 

compared to the negative control. Further, no asynchronous development was observed. 

At Day 21, >20% of amphibians were at NF stage >60. When excluding organisms with NF stages 

greater than 60, dichlobenil significantly increased Day 21 normalized HLL at all test concentrations 

(7-10% greater than negative control for non-late stage amphibians, p<0.05). No significant treatment 

effects were detected for Day 21 median developmental stage, body weight, or SVL for non-late stage 

amphibians compared to the negative control. Additionally, no significant effects on development or 

growth were observed when comparing treatment and negative control late-stage amphibians (NF>60). 

Normalized HLL at Day 21 was also significantly greater (10%, p<0.05) in the solvent control than in 

the negative control, as was NF development stage. 

There was a lower incidence and severity of follicular cell hypertrophy in the high treatment group 

compared to the negative or solvent control. No treatment-related effects were observed for thyroid 

gland hypertrophy or atrophy, or follicular cell hyperplasia, and there were no differences in the 

amount or quality of follicular colloids. 

AR Binding Assay (rat prostate cytosol); OSCPP 890.1150 

In an androgen receptor (AR) binding assay (MRID 48616102), 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 

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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 conducting the 

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 

(10−10 to 10−3

 M) of dichlobenil (99.3%, Lot# AM9/K12A222). Dimethyl sulfoxide (DMSO) was used 

as the solvent vehicle at a final assay concentration of 3.3%. A total of three independent runs were 

performed, and the assay included dexamethasone as a weak positive control, and R1881 as the ligand 

reference standard in each run. 

Only one run was considered acceptable in the saturation binding experiment, but the estimates for the 

dissociation constant (Kd), Bmax, and specific binding generally met the acceptance criteria, and the 

Scatchard plot was linear (r2 = 0.994). For the successful run, the dissociation constant (Kd) was 0.65 

nM, and the Bmax was 2.58 fmol/100μg protein. The specific binding reached a plateau, and nonspecific binding was less than 20% of total binding (4.2%). 

In the competitive binding experiment, dichlobenil reduced the mean specific binding of the 

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

[

3

H]-R1881 binding occurred at the highest concentration of dichlobenil tested (10−3 M), with specific 

binding being reduced to 40-46%. Specific binding was also reduced at 10−4 M (52-56%), but not at 

concentrations ≤10−5

 M (86-103% binding). Dichlobenil is classified as a binder in the AR assay as 

specific [3H]-R1881 binding to the AR was reduced to <50% at the highest concentration tested in all 

three runs. 

The estimated mean log IC50 was −3.85 M for dichlobenil, −8.73 M for R1881 and −4.58 M for 

dexamethasone (weak positive control). Compared to R1881, the mean Relative Binding Affinity 

(RBA) was 0.0013% for dichlobenil and 0.0071% for dexamethasone. All performance criteria were 

met except that the ligand depletion was greater (~18%) than the ≤10-15% criteria, and the bottom 

percent binding for R1881 was low in one of the runs. 

Although only one acceptable run was conducted for the saturation binding assay, and the Kd was low 

for that run, the androgen receptor was present in a reasonable concentration and functioning with 

appropriate affinity for the ligand in the competitive binding assays based on the performance criteria 

and estimated IC50s for R1881 and the weak positive control. It is unlikely that information from 

additional saturation binding runs would change the interpretation of this study. 

Based on the results from the three runs, dichlobenil is classified as a Binder in the Androgen Receptor 

Binding Assay. 

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Aromatase Assay (Human recombinant microsomes); OSCPP 890.1200 

In an in vitro aromatase (CYP 19) assay (MRID 48616103), dichlobenil (99.3%, Lot # AM9K12A222) 

was incubated with human recombinant aromatase and tritiated androstenedione (1-β [3

H(N)]-androst4-ene-3,17-dione; [3

H]ASDN) in ethanol at log concentrations of 10-10 to 10-3 M for 15 minutes to 

assess the potential of dichlobenil 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 chemical. Tritiated water was quantified with liquid 

scintillation counting (LSC). Three runs were conducted, and each run included a full activity control, 

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

 M) using a known inhibitor (4-

hydroxyandrostenedione; 4-OH ASDN), and a dichlobenil series (10−10 to 10−3 M) with three 

repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.4156 to 0.7358 nmol·mg-protein−1

·min−1

for the three test runs, with a mean and standard deviation of 0.5750 ± 0.1208 nmol·mgprotein−1

·min−1. The response of each full activity control within a run was between 90 to 110% of the 

mean. Activity in the background controls ranged from 0.06% to 0.39% and averaged 0.21% of the 

full activity controls. For the positive control substance (4-OH ASDN), aromatase activity results 

were within the recommended ranges for the performance criteria. The estimated log IC50 for 4-OH 

ASDN averaged −7.34 M and the Hill slope was −0.94. 

Aromatase activity at the lowest and highest successfully tested concentrations of dichlobenil (10-10

and 10−4

 M) were 99.6% and 87.5% of control. The highest tested concentration of dichlobenil (10−3

M) showed precipitation during the 15-minute incubation period; therefore, the results of the highest 

test concentration were not used for interpretation. 

Based on the data from the average response curve, dichlobenil is classified as a Non-inhibitor of 

aromatase activity in this assay. There is no indication in the study report that half-log concentrations 

(i.e., 10-3.5 M) were tested for solubility as recommended by the Test Guideline. At concentrations up 

to 10-4 M, dichlobenil would be classified as a non-inhibitor. However, as there is no indication in the 

study report that 10–3.5 M was evaluated for solubility, there is less confidence in the non-inhibitor 

classification. It should be noted that based on the shape of the response curve, if a soluble 10–3.5 M 

concentration had been tested, it is possible that the lowest portion of the curve would fall within the 

50-75% activity range resulting in an equivocal classification for this chemical. 

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ER Binding Assay (Rat uterine cytosol); OSCPP 890.1250 

In an estrogen receptor (ER) binding assay (MRID 48616104), uterine cytosol from Sprague Dawley 

rats was used as the source of ER to conduct saturation binding and competitive binding experiments. 

The saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol 

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

reference estrogen prior to routinely 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 (10-10 to 10-3 M) of dichlobenil 

(99.3% a.i., Batch #AM9K12822). DMSO was used as the solvent vehicle at a final concentration of 

2%. Three independent runs were performed, and each run included 19-norethindrone as a weak 

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

reference material. 

For the saturation binding experiment, the appropriate protein concentration for the saturation binding 

assay was determined to be 40 μg/tube, as it bound 30.2% of the [3

H]-17β-estradiol. The mean Kd for 

[

3

H]-17β-estradiol was 0.12 nM and the mean Bmax was 57.19 fmol/100 μg protein for the prepared rat 

uterine cytosol. The data produced consistent and linear Scatchard plots (r2

 >0.98). In the competitive 

binding experiments, the reference standard (E2), the weak positive control (19-nonethindrone), and 

the negative control (octyltriethoxysilane) each exhibited the expected responses. The estimated mean 

log IC50s were −9.08 and −5.87 M for E2 and 19-norethindrone, respectively, and the relative binding 

affinity (RBA) was 0.062% for 19-norethindrone, compared to the natural ligand. 

Precipitation of dichlobenil was not observed at any concentration. In all three runs, dichlobenil 

showed minimal displacement (93-104% binding) of radiolabeled estradiol at dichlobenil 

concentrations up to 10−3 M; therefore an IC50 and RBA could not be determined for dichlobenil. 

Based on the results from the three competitive runs, dichlobenil is classified as Not Interactive in the 

Estrogen Receptor Binding Assay. 

ERα Transcriptional Activation Assay (Human cell line HeLa 9903); OSCPP 890.1300; OECD 

455 

In an estrogen receptor transcriptional activation assay (MRID 48616105), hERα-HeLa-9903 cells 

cultured in vitro were exposed to dichlobenil (99.3% a.i., Batch # AM9K128222) at logarithmically 

increasing concentrations from 1 × 10-9 to 2.5 × 10-4 M in DMSO (0.1%). The experiments were 

performed using 96-well plates and each dichlobenil concentration was tested in triplicate (3 

wells/plate). Cells were exposed to the test agent for 20-24 hours to determine if reporter (luciferase) 

gene products were induced. Luciferase expression in response to activation of the estrogen receptor 

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was measured upon addition of a luciferase substrate and detection with a luminometer with acceptable 

sensitivity. A total of four independent runs of the assay were conducted. Laboratory proficiency was 

demonstrated by testing the 10 chemicals listed in the test guideline. 

Dichlobenil was tested up to the limit of solubility, 2.5 × 10-4 M. The mean RPCMax for dichlobenil 

was 0.9% to 5.1%, and the associated PCMax was 10-8 or 10-6 M. Performance criteria were met for 

17β-estradiol, 17α-estradiol and corticosterone. For 17α-methyltestosterone, the Log PC50 was not 

calculable for Run 1, Run 2 and Run 4, and the Log PC10 was higher than the expected range for all 

four Runs. The responsiveness of the cells to the very weak positive control 17α-methyltestosterone 

was lower than the expected values, indicating a decreased sensitivity of the assay to very weak 

agonists. Although the conditions of this assay were not optimal to detect very weak activity, 

dichlobenil responses were similar to those of the negative control corticosterone and not comparable 

to the responses of 17α-methyltestosterone, which was able to reach a maximum of 34-51% PC. 

Dichlobenil was only able to reach a maximum of 0.9-5.1% PC when tested up to the highest 

concentration possible based on cytotoxicity. Because the RPCMax < PC10 in both assay runs, 

dichlobenil was considered negative for estrogen receptor transcriptional activation in this test system. 

Fish Short-term Reproduction Assay; OSCPP 890.1350; OECD 229 

The 21-day short-term reproduction assay (MRID No. 48616106) of dichlobenil with fathead minnows 

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

(2 males and 4 females in each group; 6-7 months old) were exposed to dichlobenil (99.3% purity) at 

nominal concentrations of 0 (negative control), 0 (solvent control; 0.1 mL/L acetone), 0.021, 0.20, and 

2.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.001; both controls), 0.021, 0.21, and 2.1 

mg a.i./L. The test system was maintained at 24.3 to 26.2o

C and a pH of 7.3 to 7.9. Unless otherwise 

indicated, the treatment groups were compared to the negative control. 

There were no statistically significant differences (p>0.05) in mean male or female body weights or 

lengths at any test level compared to the control. Overall mean survival values were 100, 96, 100, 100 

and 92% in the negative control, solvent control, and low, mid, and high treatment levels, respectively. 

Fish exposed to the high treatment level were observed to have a lighter, more yellowish color 

compared to the fish in the controls and other treatment levels; however, the incidence of this effect 

was not reported. No other changes in behavior or appearance were noted. 

Spawning frequency in the negative and solvent control was at least every 4 days in 3 and 2 of the 4 

replicates, respectively; the mean fecundity in the negative and solvent controls was 12.1 (range 6.6-

15.8) and 6.5 (range 4.3-11.6) eggs/female/day/replicate. Fecundity was significantly (p<0.05; 

Jonckheere-Terpstra) reduced by 53% and 90% at the mid and high treatment levels, respectively, 

compared to the negative control. The study author did not provide fertility data, but reported fertility 

was “more than 99.9% in all test groups” and fertility of eggs from control animals was at least 95%. 

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Median male tubercle score showed a statistically significant reduction from 26 in the negative control 

to 21, 14, and 12 in the low, mid and high treatment groups, respectively (p<0.05; JonckheereTerpstra). No tubercles were observed for females, except for one female with one tubercle in the low 

treatment group which was reported not to be treatment related. There were no significant differences 

in male or female gonadal stage index (GSI) or vitellogenin (VTG). 

In males, no treatment-related histopathological gonadal findings were reported. In females, there was 

an increased incidence and/or severity (minimal to moderate) of oocyte atresia and a shift to more 

mature stages in the ovaries in the mid and high treatment groups. 

Sex steroids (testosterone and 17β-estradiol) were not measured. No dorsal nape pad measurements or 

other dorsal nape pad observations were reported. 

While not statistically significant (p=0.078), the fecundity in the solvent control group was reduced by 

46% compared to the negative control. However, large variability was observed between the preexposure and exposure fecundity rate for both the negative and solvent control replicates, suggesting 

this difference may not be solvent related. Additionally, this was the only endpoint for which a 

notable difference between the negative and solvent control was observed. 

Hershberger Assay (Rat); OSCPP 890.1400; OECD 441 

In a Hershberger assay (MRID 48616107) screening for androgenic activity, dichlobenil (99.3% a.i., 

Lot# AM9K12A222) in 1.0% gum tragacanth was administered daily via oral gavage to groups of 55-

day old, castrated male Sprague Dawley rats (6/group) at dose levels of 0 (vehicle), 20, 60, or 

180 mg/kg/day. An androgenic positive control group consisting of six castrated rats exposed to 0.2 

mg/kg/day testosterone propionate (TP) by subcutaneous (s.c.) injection was also included as a 

positive control. 

To screen for potential anti-androgenic activity, dichlobenil in 1.0% gum tragacanth was also 

administered daily via oral gavage to 55-day old, castrated male Sprague Dawley rats (6/group) at dose 

levels of 20, 60, or 180 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.2 

mg/kg/day by s.c. injection. The vehicle control groups consisted of six castrated male rats dosed 

daily with TP (0.2 mg/kg/day) by s.c. injection in corn oil, and the anti-androgenic positive control 

group consisted of six castrated male rats dosed orally with flutamide (FT) in corn oil at 3 mg/kg/day 

in conjugation with a daily s.c. dose of TP (0.2 mg/kg/day). 

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For both components of the assay, the animals were dosed for 10 consecutive days and necropsied 

approximately 24 hours after the final dose administration to determine weights of liver, kidneys and 

the five androgen-dependent tissues. 

All animals survived until scheduled termination. There were no treatment-related clinical signs of 

toxicity. 

In the androgen agonist assay, body weights and overall body weight gains for the 20 and 60 

mg/kg/day treatment groups were comparable to controls throughout the assay. At 180 mg/kg/day, 

body weights were decreased (p<0.05) by 9-10% on Days 2-7, and 9. The overall (Days 0-10) body 

weight gains for 180 mg/kg/day were decreased (p<0.05) by 44%. Liver weights were not affected in 

the low-dose and positive control groups, but were increased (p<0.05) in the 60 mg/kg/day (↑24%) and 

180 mg/kg/day (↑32%) groups. Kidney weights were unaffected, except in the TP group, in which 

kidney weights increased (p<0.05) by 8%. There were no significant changes in weight of the 

accessory sex organs for dichlobenil-dosed animals. Animals in the TP group (positive control) had 

increases (p<0.05) in accessory sex organ weights as follows: 339% in seminal vesicles, 638% in 

ventral prostate, 165% in levator ani-bulbocavernosus (LABC), 513% in Cowper’s gland, and 72% in 

glans penis. All %CV values were less than the maximum recommended guideline values. 

In the anti-androgen assay, body weights and overall body weight gains for the 20 and 60 mg/kg/day 

treatment groups were comparable to controls throughout the assay. At 180 mg/kg/day, body weights 

were decreased (p<0.05) by 9-11% on Days 1-10. The overall (Days 0-10) body weight gains for 180 

mg/kg/day were decreased (p<0.05) by 40%. For the positive control group (TP + FT), overall body 

weight gains were decreased (p<0.05) by 13%. No changes were noted in liver weight for any dose 

group and kidneys were only affected in the positive control (TP + FT) group, in which kidney weights 

significantly decreased (↓10%). 

Statistically significant decreases in 4 of the 5 target accessory sex organ weights were observed at the 

high dose (180 mg/kg/day). The following decreases (p<0.05) in accessory sex organ weights were 

noted at 180 mg/kg/day: 36% in seminal vesicles, 35% in ventral prostate, 27% in LABC, and 49% in 

Cowper’s gland. In addition, a decrease (21%; p<0.05) in seminal vesicle weight was observed at 60 

mg/kg/day. Animals dosed with TP + FT (positive control) had decreased (p<0.05) accessory sex 

organ weights as follows: 73% in seminal vesicles, 75% in ventral prostate, 48% in LABC, 71% in 

Cowper’s gland, and 20% in glans penis. The maximum guideline-specified %CV was exceeded for 

the glans penis (20% vs. 17%) in the TP +FT (positive control) group. All other %CV values were 

less than the maximum recommended values. 

Dichlobenil was negative for androgenic activity in the Hershberger assay as no statistically significant 

increases in organ weights were seen in two or more of the five androgen sensitive tissues in the 

androgen agonist assay. Dichlobenil was positive for anti-androgenicity in the Hershberger assay as 

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statistically significant decreases in organ weights were seen in four of the five androgen sensitive 

tissues in the androgen antagonist assay. However, bodyweights were decreased ≥10% at the dose that 

resulted in the changes in androgen-sensitive tissue weights indicating overt toxicity. 

Pubertal Female Assay (Rat); OSCPP 890.1450 

In a Female Pubertal Assay (MRID 48616108), 15 Sprague Dawley rats/dose group were treated daily 

via oral gavage (5 mL/kg) with dichlobenil (99.3% a.i., lot/batch #: AM9K12A222) in aqueous 1.0% 

gum tragacanth at doses of 0 (vehicle), 20, or 60 mg/kg/day from post-natal day (PND) 22 to 42. 

Animals were examined for vaginal opening (VO) daily beginning on PND 22, and age and weight at 

day of attainment was recorded. Blood samples for chemical chemistry analyses were taken following 

sacrifice on PND 42. Total serum thyroxine (T4) and thyroid stimulating hormone (TSH) levels were 

determined using electrochemiluminescent immunoassays and radioimmunoassays. The weights of 

adrenals, kidneys, liver, pituitary, thyroid, and urogenital organs were recorded and microscopic 

examination of the ovaries, uterus, thyroid, liver, and kidneys were performed. 

There were no dichlobenil-related effects observed on mortality, clinical signs of toxicity, body 

weights, body weight gains, age and weight at attainment of VO, mean age at first vaginal estrus, mean 

cycle length, or percent cycling and regularly cycling. Organ weights were similar between the control 

and dichlobenil treated groups with the exceptions of liver and uterus weights. Unadjusted, adjusted 

and related (to body) liver weight was increased (p<0.05) by 18–19% at 60 mg/kg/day, and relative 

liver weight was increased by 9% at 20 mg/kg/day. Blotted uterus weight was decreased by 12% (NS) 

and 21% (p<0.05) at 20 and 60 mg/kg/day, respectively, and wet uterus weight was decreased by 26% 

(NS) at 60 mg/kg/day. However, no histological changes were noted in the liver, uterus or ovaries. 

 

Serum T4 and TSH levels were altered in a dose dependent manner, but the changes did not attain 

significance. At 20 and 60 mg/kg/day, T4 levels were decreased by 4% and 7%, respectively, and TSH 

levels were increased by 18 and 22%, respectively. The changes in thyroid hormone levels were not 

corroborated by changes in thyroid weight or histology. 

The dose levels tested were determined to be adequate based on the dose dependent changes were seen 

in the uterus and the thyroid in this assay, and the results of the dose range-finding study. 

Pubertal Male Assay (Rat); OSCPP 890.1500 

In a Male Pubertal Assay (MRID 48616109), 15 Sprague-Dawley (Crl:CD(SD)) rats/dose group were 

treated daily via oral gavage (5 mL/kg) with dichlobenil (99.3% a.i., Lot #: AM9K12A222) in aqueous 

1.0% gum tragacanth at doses of 0, 20, or 60 mg/kg/day from post-natal day (PND) 23 to 53. The 

animals were examined for preputial separation (PPS) daily beginning on PND 30, and age and body 

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weight at day of attainment was recorded. Following sacrifice on PND 53, total serum testosterone, 

thyroxine (T4), and thyroid stimulating hormone (TSH) levels were analyzed using 

electrochemiluminescent immunoassays and radioimmunoassays. The weights of adrenals, liver, 

pituitary, thyroid, and urogenital organs were recorded and microscopic examinations of the testes, 

epididymides, thyroid, liver, and kidneys were performed. 

No adverse treatment-related effects were observed in mortality, clinical signs of toxicity, body 

weight, body weight gain, age or weight at PPS, clinical chemistry, gross pathology, or microscopic 

examination of the testes, epididymides, thyroid, liver, or kidney. 

The age and weight at attainment of PPS at all doses was within the acceptable range of the 

performance criteria. The unadjusted values for organ weights and hormone levels in the control 

group were within the acceptable range of the performance criteria provided in the Guideline with the 

exception of a minor departure in the thyroid weight of 13.44 mg compared to Guideline criterion of 

14-26 mg. All thyroid glands examined in all dose groups were considered within normal limits of 

control tissues. 

Increased (p≤0.05) unadjusted, adjusted (covariate PND 23 body weight), and organ-to-body weight 

ratio liver (↑35–36%) and kidney (↑9–10%) weights were observed at 60 mg/kg/day. Increased 

(p≤0.05) organ-to-body weight ratio liver (↑16%) and kidney (↑7%) weights were observed at 20 

mg/kg/day. Increased (p≤0.05) adjusted adrenal weights were also noted at 60 mg/kg/day (↑13%). 

Except for the liver weights at 60 mg/kg/day, these differences were considered minor. Furthermore, 

in the absence of corroborating evidence of hepatotoxicity from gross and microscopic examination of 

the liver and clinical chemistry, the increased weight was considered an adaptive response. 

No biologically significant effect was noted on clinical chemistry parameters in the treated groups; 

however several clinical chemistry parameters in the 60 mg/kg/day group differed statistically 

(p≤0.05) from controls. 

Although no treatment-related effects were noted on pubertal development, the thyroid, or signs of 

systemic toxicity, the dosing levels utilized in the current study are considered appropriate based on 

the results of the dose range-finding study. 

Steroidogenesis Assay (Human cell line H295R); OSCPP 890.1550; OECD 456

In a steroidogenesis assay (MRID 48616110), H295R cells cultured in vitro in 24-well plates were 

incubated with dichlobenil (99.3% a.i., Batch # AM9K12A222) at serial log concentrations of 100 to 

0.0001 μM for 48 hours in triplicate for four independent experiments. Dimethyl sulfoxide (DMSO) 

was used as a vehicle, at a final concentration of 0.1%. 

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Testosterone and estradiol levels were measured using ELISA. A Quality Control (QC) plate was run 

concurrently with each independent run of a test chemical plate to demonstrate that the assay 

responded properly to positive control agents at two concentration levels. The positive controls 

included a known inhibitor (prochloraz) and inducer (forskolin) of estradiol and testosterone 

production. 

Guideline acceptability recommendations and requirements were generally met, including adequate 

production of testosterone and estradiol, acceptable reproducibility (low %CV), and appropriate 

induction and inhibition with positive controls. Precipitation of the test compound was not reported, 

and cytotoxicity was not observed. 

Treatment-related effect from dichlobenil observed on testosterone or estradiol levels were not 

consistent across all runs. In Run 1, there were significant reductions in testosterone levels at 0.0001, 

0.001, 0.01, 1 and 100 μM of dichlobenil, with the greatest reduction (0.71-fold) occurring at 0.01 μM. 

During Run 2, decreased (p≤0.05) testosterone levels were also noted at 0.0001 and 0.01 μM. 

However, no reductions in testosterone were noted in Run 3. No changes in estradiol concentration 

were observed during Runs 1 and 2, but estradiol was decreased (p≤0.05) in Run 3 at all dichlobenil 

concentrations except 100 and 0.1 μM. 

Due to the inconsistencies and variability in the hormone alterations across the three runs, the results 

for dichlobenil in this assay were considered to be equivocal. 

Uterotrophic Assay (Rat)

In a Uterotrophic Assay (MRID 48616111) conducted to screen for potential estrogenic activity, 

dichlobenil (99.3% a.i., Lot #AM9K12A222) in 1.0% gum tragacanth in deionized water was 

administered daily via oral gavage to groups of six ovariectomized Sprague Dawley rats at dose levels 

of 0 (vehicle), 20, 60 or 180 mg/kg/day on post-natal days (PND) 63-65. A positive control group was 

treated with a daily dose of 17α-ethynyl estradiol (EE) at 0.003 mg/kg/day by subcutaneous (s.c.) 

injection. Body weights were determined daily. All animals were terminated and necropsied on PND 

66 approximately 24 hours after the final dose administration to determine wet and blotted uterine 

weights. 

All animals survived until scheduled termination and no clinical findings were observed in animals 

dosed with dichlobenil at any level. Body weights were decreased (p<0.05) by 8% on Day 3 and 9% 

on Day 4 (termination) at 180 mg/kg/day compared to controls, resulting in a decrease (p<0.001) of 

129% in overall body weight gain. Body weights in the 20 and 60 mg/kg/day dose groups were 

comparable to controls throughout the duration of the assay. Uterine weights in the dichlobenil treated 

groups were comparable to the vehicle controls.

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The body weights for the positive control (EE) group were decreased (p<0.05) by 8% on Day 4 and 

overall body weight gain for the EE animals was decreased (p<0.001) by 104% in compared with the 

controls. Absolute wet and blotted uterus weights for the positive control group were increased 

(p<0.001) by 766% and 234%, respectively, as expected.

The highest dose tested (180 mg/kg/day) was determined to be adequate based on the decrease in body 

weight gain (129%) at this dose. 

No statistically significant changes were seen in uterine weight in this assay. Dichlobenil was negative 

in the uterotrophic assay. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) Subchronic Oral Subchronic 

Oral Toxicity Study (Rat)

In a 90-day oral toxicity study (MRID 00107106), dichlobenil technical (90.5% a.i.) was 

administered in the diet to albino rats (12/sex/dose) at daily dose levels of 0, 100, 1000, or 3000 ppm 

(equivalent to 0, 4.5, 45, or 135 mg/kg/day; adjusted for purity) for 3 months. An additional 6 males 

were treated with 10,000 ppm (453 mg/kg/day) for 3 months. Five (of 6) males treated with 10,000 

ppm dichlobenil died by Week 7 of the study (causes undetermined). Mortality, due either to 

respiratory infection or “other causes”, was evenly distributed across males in all other groups (2/12, 

2/12, 1/12, and 2/12 at 0, 100, 1000, and 3000 ppm, respectively); 2/12 females died on study at 1000 

ppm only. Mean body weights were decreased in males (≥14%) by the end of the study at ≥ 3000 

ppm. There were no treatment-related effects on urinary protein or glucose up to 3000 ppm. In 

addition, no adverse effects were observed on hematological parameters. Mean absolute liver weights 

were increased in males (39%) and females (52%) at 3000 ppm. The effect was dose-dependent and is 

considered adverse since swelling and vacuolation of the hepatocytes with or without necrosis was 

observed in all rats/sex at ≥ 3000 ppm. An inflammatory, necrotic focus was also observed in the 

liver of 1 male at 1000 ppm, while vacuolar swelling was observed in a second animal at the same 

dose. 

No treatment-related changes were observed in absolute or relative testes weight, and no histological 

lesions were noted in the testes, ovaries, thyroid and adrenal glands. 

The LOAEL is 1000 ppm (45 mg/kg/day) in males, based on hepatocytic inflammation and necrosis; 

and 3000 ppm (135 mg/kg/day) in females, based on increased liver weight and liver histopathology 

(swelling and vacuolation of hepatocytes). The NOAEL is 100 (4.5 mg/kg/day) in males and 1000 

ppm (45 mg/kg/day) in females. 

Subchronic Oral Toxicity Study (Hamster)

In a 90-day oral toxicity study (MRID 40600701), dichlobenil technical (98.5% a.i., batch # FUX 

0100000) was administered to 10 Bio F1 Alexander hamsters/sex/dose in the diet at dose levels of 

41, 209, 1289, or 7500/4648 ppm (adjusted due to evaporation; equivalent to 3, 16, 79, or 395/263 

mg/kg/day). Twenty animals/sex were tested as a control group. Ten hamsters/sex from the 

control and high dose groups were kept on study for an additional 4-week recovery period during 

which the animals received diet without dichlobenil. Due to excessive weight loss in both sexes 

after 2 weeks (13%-25%) at 7500 ppm, all high- dose animals received 4648 ppm dichlobenil from 

Week 3 until Week 13. 

There were no treatment-related effects on survival, and no adverse clinical signs or ophthalmological 

changes were observed. Body weight gains in high-dose males did not rebound to control levels until 

Week 10; however, mean body weights in high-dose males remained decreased (21%) at the end of 

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treatment. Mean food consumption was decreased (≥11%) in high-dose males after dose lowering. 

Mean serum cholesterol and phospholipids were increased by 55% each (p<0.01) in females at 4648 

ppm. Concentrations of these clinical chemistry parameters, body weight gain, and food consumption 

returned to control levels during the recovery period. Other changes in hematological or clinical 

chemistry parameters were slight and not considered toxicologically significant. Mean absolute liver 

weights were increased by ≥13% (p<0.01) in males at ≥1289 ppm and by 16% (p<0.01), 34% 

(p<0.01), and 85% (p<0.01) in females at 209, 1289, and 4648 ppm, respectively. Mean absolute 

prostate weight was reduced by ≥19% (p<0.05) at ≥209 ppm, while mean absolute seminal vesicle 

and testicular weights were reduced by 16% at ≥209 ppm and 33% (p<0.01) at 4648 ppm, 

respectively. Statistically significant changes in other mean absolute and/or relative organ weights 

(e.g., kidney, spleen, brain, uterus) were observed at the highest dose tested; however, in the absence 

of histopathological correlates, these changes were not considered toxicologically significant. 

At necropsy, livers of 5/10 (p<0.01) males were enlarged at 4648 ppm, while 5/10 or 10/10 (p<0.01) 

females had enlarged livers at 1289 or 4648 ppm, respectively. Rough surface of the liver was also 

noted for 3/10 and 8/10 (p<0.01) females at 1289 or 4648 ppm, respectively. Black grit was found in 

the gallbladders of 2/10 (p<0.01) females at 4648 ppm at the end of treatment. Control incidences for 

each of these hepatobiliary observations were 0/20. At 4648 ppm, small testis was observed in 3/9 

males (p<0.01), while small epididymis was seen in 2/9 males (p<0.01); the control incidences for 

each of these observations was 0/20. Slight hemorrhagic area in the epididymis and seminal vesicle 

was also observed in 1/9 males (vs. 0/20 controls) at 4648 ppm. Bile granules in the gall bladder 

were observed in 2/10 males and 3/10 females in the high-dose recovery group (vs. 0/10 and 1/10, 

respectively, in controls). 

Histopathological observation revealed a marked decrease in the number of spermatocytes at the end 

of treatment in 5/9 high-dose males (vs. 1/20 in controls). Marginal to moderate prostatic 

mineralization was observed in 3 or 4 (of 9 or 10) males at ≥209 ppm after 13 weeks (vs. 1/20 in 

controls). Marginal to marked tubular degeneration in the testes was observed in 7/9 (p<0.01) males 

at 4648 ppm (vs. 2/20 in controls). Calculi in the gall bladder were observed at the end of treatment 

in 7/10 males (p<0.01) and 10/10 (p<0.01) females at 4648 ppm (vs. 0/20 in each control group). 

Swollen hepatocytes were noted in 2/9 males at 4648 ppm and in 2/10 females at 209 (marginal), 4/10 

females at 1289 (marginal to slight; p<0.01), and 4/10 females at 4648 ppm (marginal to moderate; 

p<0.01). Control incidences for swollen hepatocytes were 1/20 for males and 0/20 for females. 

The LOAEL for males is 209 ppm (16 mg/kg/day), based on decreased weight and mineralization of 

the prostate and decreased mean absolute seminal vesicle and testicular weights; and 1289 ppm (79 

mg/kg/day) for females, based on increased liver weight, enlarged liver (with rough surface), and 

swollen hepatocytes. The NOAEL for males is 41 ppm (3 mg/kg/day) and 209 ppm (16 mg/kg/day) 

for females. 

Subchronic Oral Toxicity Study (Mouse)

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In a 90-day oral toxicity study (MRID 48043302), dichlobenil was administered to 22-day old male 

and female CD-1 mice (number not specified) in the diet at 0, 25, 125, 625, or 3125 ppm ( 0, 4, 19, 

95, or 473 mg/kg/day). Adverse effects in the liver (specific effects were not reported) were observed 

in female mice at the 95 mg/kg/day. Absolute (7 -13%) and relative (7 -11%) testes weights were 

increased at ≥19 mg/kg/day. No treatment-related histopathological lesions were seen in the testes, 

epididymides, seminal vesicle, prostate, ovaries, uterus, vagina, thyroid, adrenal or pituitary glands. 

Chronic Toxicity (Dog)

In a non-guideline chronic toxicity study (MRID 00067649), groups of beagle dogs (4/sex/dose) were 

given dichlobenil in the diet for 2 years at dosing levels of 0, 20, 50, or 350 ppm (0, 0.5, 1.25, or 8.75 

mg/kg/day). The NOEL for systemic toxicity was 1.25 mg/kg/day. The LOEL for systemic toxicity 

was 8.75 mg/kg/day based on (1) an increase in absolute and relative liver and thyroid weights in both 

sexes; (2) an increase in serum alanine aminotransferase in females, and serum alkaline phosphatase 

in both sexes; (3) an increase in liver enzyme glucose-6- phosphatase and glucose-6-phosphatase 

dehydrogenase activity in both sexes; and (4) leukocytic infiltration and fibrinoid degeneration 

around the central hepatic veins of both sexes. 

Chronic Toxicity (Dog)

In a chronic toxicity study (MRID 43969701), dichlobenil (98.8% a.i.; Batch FUX 011000/FUN 

87E08A) was administered by capsule to 5 beagle dogs per sex at doses of 0, 1, 6, or 36 mg/kg/day 

for 52 weeks. 

All animals survived to the scheduled termination date. No treatment-related clinical signs of toxicity 

were observed in any dogs, and no compound-related effects were noted on food or water 

consumption, ophthalmoscopic examination, urinalysis, or gross pathology. Lower mean body 

weights (12-16%) and body weight gains (36-44%; p<0.05) were observed throughout the study in 

males at 36 mg/kg/day (relative to controls), whereas no biologically significant changes in body 

weight or body weight gain were observed in females. 

Increased serum cholesterol, triglyceride, phospholipid, and alkaline phosphatase levels were 

observed in males and females at ≥6 mg/kg/day. Gamma-glutamyltransferase was also increased in 

males only. Increased (≥18%) absolute and relative liver weights were observed in mid- and highdose males and females. This effect corresponded to periportal hepatocytic hypertrophy in males at 

≥6 mg/kg/day and at 36 mg/kg/day in females. 

Absolute thyroid weights were significantly (p <0.01) increased at the low (34%), mid (33%) and 

high (50%) dose groups for females and relative thyroid weight was increased (63%) at the high dose. 

For males, both absolute (56%) and relative (84%) thyroid weights were increased at the high dose. 

However, no thyroid lesions were seen in either sex at any dose level. 

In the males, there was a dose-dependent decrease in absolute weight of the prostate glands with the 

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decrease reaching statistical significance (p <0.05) at the high dose; the decreases were 14%, 22% 

and 37%, at the low, mid and high dose, respectively. The absolute weights of the testes were 

increased (33%) at the high dose; whereas, the relative testes weight was significantly increased at 

both the mid (18%) and high (56%) doses. None of these weight increases were corroborated with 

histopathological lesions in these organs. 

In the females, absolute (50%; p<0.01) and relative (46%) ovarian weights and absolute (78%; p 

<0.05) and relative (76%; p<0.05) uterine weights were decreased at the high dose. No 

histopathological lesions were seen in the ovaries, but “early” maturity of the uterus was reported in 

all females (5/5) at the high dose. 

No treatment-related histopathological lesions were seen in the testes, epididymides, prostate, ovaries, 

mammary glands, thyroid or pituitary glands. Cortical vacuolation of the adrenal glands were seen in 

5/5 males and compared to 0/15 control males; incidences in females were comparable to the controls. 

The LOAEL is 6 mg/kg/day, based on increased liver weights and increased serum cholesterol, 

triglycerides, phospholipids, and alkaline phosphatase in males and females and increased serum γGT and periportal hypertrophy of the hepatocytes in males. The NOAEL is 1 mg/kg/day. 

Combined Chronic Toxicity/Carcinogenicity (Rat)

In a combined chronic toxicity/carcinogenicity study (non-guideline; MRIDs 00147438 and 

40401101), dichlobenil (96.7% a.i.; lot# FUN80C25A) was administered in the diet for 110 weeks to 

Fisher 344 rats (50/sex/dose) at daily dose levels of 0, 50, 400, or 3200 ppm (equal to 0, 2.3, 18.9, or 

173.1 mg/kg/day). Interim sacrifices of 10 additional animals/sex/dose took place at Weeks 52 and 

78. Mortality was 100% in males by Week 98 at 3200 ppm. Survival in males was similar to 

controls at the mid and low doses and in females at all doses tested. No treatment-related clinical 

signs of toxicity were observed upon re-examination of the data (MRID 40823801). 

At 3200 ppm, body weight was decreased by 33% (p<0.001) by Week 88 in males and 34% 

(p<0.001) by Week 104 in females. Decrements were proportional to time on study in both sexes. 

Similarly, mean body weight gain in males was decreased at 3200 ppm by 19% from Week 1-13 and 

by 160% from Week 52-66. In females, mean body weight gain was decreased at 3200 ppm by 24% 

from Week 1-13 and by 40% from Week 52-66. No differences were observed in food consumption 

when corrected for body weight, whereas food efficiency was decreased by 35% in males and 29% in 

females at 3200 ppm. Water consumption (corrected for body weight) in high-dose animals was 

increased by 30% and 55% in males and 22% and 39% in females at Weeks 5 and 15, respectively. 

Urinary volume was also over 2-fold higher in high-dose males at Week 78 (p<0.01). 

No toxicologically significant effects were observed on hematological parameters. Mean blood urea 

nitrogen (BUN) was 3-fold higher (p<0.01) at Week 78 in males treated with 3200 ppm; in males 

treated with 400 ppm, mean BUN was increased by 75% (p<0.05) at termination. Mean serum uric 

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acid levels were also increased by 35% (p<0.01) at 3200 ppm in males. Mean cholesterol in males 

treated with 3200 ppm was increased by 91% (p<0.05) at Week 52 and by 176% (p<0.01) at Week 

78; in males treated with 400 ppm, mean cholesterol was increased by 136% (p<0.01) at termination. 

In females treated with 3200 ppm, mean BUN was over 3-fold higher (p<0.05) at Week 110. Mean 

cholesterol in females treated with 3200 ppm was increased by 168%, 157%, and 83% (all p<0.01) at 

Weeks 52, 78, and 110, respectively. No treatment-related changes in mean serum uric acid levels 

were observed in females. 

Mean absolute and relative liver weights were increased in males by 48% (p<0.01) and 118% 

(p<0.01), respectively, after 78 weeks at 3200 ppm (changes at 78 weeks were similar to those at 52 

weeks and termination); however, the increased relative liver weight was likely due to a combination 

of increased absolute liver and decreased body weight. In females at week 78, mean absolute and 

relative liver weights were increased (p<0.01) by 75% and 139%, respectively, at 3200 ppm, and by 

16% and 23%, respectively, at 400 ppm. Absolute kidney weights were increased by 16% (p<0.05) 

in both males and females at 3200 ppm, as was the absolute weight of the adrenals in males (22%, 

p<0.01) at the same dose. 

At terminal necropsy, dose-dependent increases in the incidences of enlarged liver and kidneys were 

observed in males and females. The incidences of enlarged liver in males were 3/50, 9/50, 15/50, and 

45/50 at 0, 50, 400, and 3200 ppm, respectively, while enlarged liver was observed in 4/50, 3/50, 

11/50, and 44/50 females, respectively. The incidence of enlarged kidney was 2/50, 5/50, 8/50, and 

40/50 in males and 0/50, 0/50, 2/50, and 13/50 in females at 0, 50, 400, and 3200 ppm, respectively. 

Incidences of fatty metamorphosis of the liver was increased above controls at ≥ 50 ppm at Week 52 

in males and females; however, dose-dependence was lacking for the effect in both sexes at Week 78. 

At terminal sacrifice, increased incidences of fatty metamorphosis (with increased severity) of the 

liver were clearly observed in males (45/50) and females (39/40) at 3200 ppm. Liver necrosis was 

increased in males at 3200 ppm at Week 52 only and slightly increased in females at Week 78. 

Cytologic alterations (polyploidy with hepatocytic swelling) were increased in females at ≥ 400 ppm 

at terminal sacrifice, whereas increased incidences of “hepatic nodules” (hyperplasia) were observed 

in males and females at 3200 ppm at terminal sacrifice. Clear increases in calcification of the kidney 

were observed at 3200 ppm in both males (24/50) and females (46/47) at terminal sacrifice, whereas 

nephrosis (kidney damage) was observed in 100% males at ≥ 400 ppm (increased severity at high 

dose) and 98% females at 3200 ppm at terminal sacrifice. Nephrosis was also increased in both sexes 

at both interim sacrifice times at ≥ 400 ppm. Last, parathyroid hyperplasia was increased at terminal 

sacrifice only at ≥ 50 ppm in males and at 3200 ppm in females. Parathyroid hyperplasia was likely a 

response to the reported kidney damage (nephrosis), in order to maintain normal serum calcium 

concentrations. 

The LOAEL is 400 ppm (18.9 mg/kg/day), based on changes in clinical chemistry (increased blood 

urea nitrogen, cholesterol), gross pathology (enlarged liver, enlarged kidney), and histopathology 

(nephrosis, parathyroid hyperplasia) in males; and increased liver weight, enlarged liver, and 

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cytologic alterations (polyploidy with hepatocytic swelling) in the liver in females. The NOAEL is 

50 ppm (2.3 mg/kg/day). 

According to the Health Effects Division 2nd Carcinogenicity Peer Review (1995), a treatmentrelated increase in the incidence of hepatocellular adenomas and combined adenomas/carcinomas was 

observed in females only at 3200 ppm. Dosing in females was considered adequate at 3200 ppm, 

based on decreased body weight and body weight gain, changes in clinical chemistry and organ 

weights, gross pathology, and histopathology. Dosing in males was considered excessive at 3200 

ppm, but adequate at 400 ppm, based on changes in clinical chemistry, gross pathology, and 

histopathology. 

Carcinogenicity Study (Hamster)

In a high dose carcinogenicity study (MRID 42221201), dichlobenil (99.4% a.i.; batch# 

FUN87EO8A/FUX011000) was administered in the diet for 91 (males) or 78 (females) weeks to Bio 

F1D Alexander Syrian hamsters (50/sex/dose) at daily dose levels of 675, 1500, or 3375 ppm (equal 

to 51/55, 117/121, or 277/277 mg/kg/day for males/females). Control groups (0 ppm) consisted of 

100 animals/sex. The criterion for study termination was 50% mortality in the control groups of each 

sex. There was no difference in survival across groups in males after 91 weeks, while survival was 

higher in females at the high dose than in controls after 78 weeks (78% vs. 50%). Submitted 

historical control data (MRID 42563601) indicated that the lifespan of untreated females of this strain 

of hamster is less than that of untreated males (88-91 weeks vs. 77-78 weeks). There were no clinical 

signs of toxicity or ophthalmic abnormalities after treatment with dichlobenil. 

Body weight was decreased (>10%) in males at 3375 ppm from Week 2 of the study until 

termination, while overall body weight gain in males was decreased (p<0.05) by 68%, 136%, and 

166% at 675, 1500, and 3375 ppm, respectively, from Week 0-91. Overall body weight gain in 

females was decreased (p<0.01) by 34%, 37%, and 30% at 675, 1500, and 3375 ppm, respectively, 

from Week 0- 78. Food consumption was decreased (9-11%; p<0.01) at 3375 ppm in males 

throughout the treatment period. No adverse effects were observed on differential leukocyte counts in 

males. 

In males, enlarged liver (11/50; p<0.05) and liver masses (6/50; p<0.05) were observed at 3375 ppm 

(vs. 1/100 and 3/100, respectively, in controls), whereas in females, the incidence of enlarged liver 

was 3/50 and 30/50 (p<0.05) at 1500 and 3375 ppm, respectively (vs. 1/00 in controls). Pale liver 

was also observed in males at incidences of 5/100, 7/50, 8/50 (p<0.05), and 13/50 (p<0.05) at 0, 675, 

1500, and 3375 ppm, respectively. Incidences of minimal adipose tissue in males, which correlated 

with decreased body weight gains, were also decreased at 675, 1500, and 3375 ppm [19/50, 23/50 

(p<0.01), and 27/50 (p<0.01), respectively vs. 18/100 in controls]. Mean relative liver weights in 

males were increased by 13% (p<0.01) and 53% (p<0.01) at 1500 and 3375 ppm. This is likely 

explained by the dose-dependent decreases in body weight in males, since absolute liver weights were 

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not significantly different across dose. In females, a 50% increase in absolute liver weight was 

observed at 3375 ppm, while relative liver weights were increased by 9% (p<0.05), 23% (p<0.01) and 

59% (p<0.01) at 675, 1500, and 3375 ppm, respectively. Mean relative testes/epididymis weights 

were decreased by 12-13% at ≥1500 ppm. Decreases in mean absolute heart weights in either males 

or females were also observed, but without histopathological correlates. 

Dose-dependent increases in microscopic effects indicative of liver toxicity at ≥675 ppm in males 

included finely vacuolated hepatocytes, hepatitis, and brown pigment in the hepatocytes. In females, 

dose-dependent increases in hepatitis and pigmented giant cells were observed at ≥1500 ppm. 

Histopathology of the testes (pigmented macrophages, dilated seminiferous tubules) in males and of 

the urinary bladder (urothelial hyperplasia) and pituitary (microcysts) in females was observed at the 

high dose only. 

The LOAEL is 675 ppm (51/55 mg/kg/day in males/females), based on decreased body weight gain 

in males and females and liver histopathology (finely vacuolated hepatocytes, hepatitis, and brown 

pigment in the hepatocytes) in males. The NOAEL was not identified. 

According to the Health Effects Division 2nd Carcinogenicity Peer Review (1995), there was a 

treatment- related increase in liver adenomas and combined adenomas/carcinomas in males only at 

3375 ppm, when compared to controls. Dosing was considered excessive at ≥1500 ppm in both 

sexes, based on decreased body weight gains and severe hepatotoxicity. 

Carcinogenicity Study (Hamster)

In a low dose carcinogenicity study (MRIDs 41988301 and 42015101), dichlobenil (99.4% a.i.; batch# 

FUN87EO8A/FUX011000) was administered in the diet for 88 (males) or 80 (females) weeks to Bio 

F1D Alexander Syrian hamsters (50/sex/dose) at daily dose levels of 5, 26, 132, or 675 ppm (equal to 

0.34/0.35, 1.69/1.78, 9.39/9.20, or 45.64/48.85 mg/kg/day for males/females). Control groups (0 ppm) 

consisted of 100 animals/sex. The criterion for study termination in males was 50% mortality in 

controls. Mortality in control females at termination was 42%. There were no dose-dependent 

differences in survival in males or females at termination. Submitted historical control data (MRID 

42563601) indicated that the lifespan of untreated females of this strain of hamster is less than that of 

untreated males (88-91 weeks vs. 77-78 weeks). 

Mean body weight was decreased (10-11%) in males at 675 ppm from Weeks 80-88 only of the study. 

Mean overall body weight gain was decreased at 675 ppm by 96% (p<0.01) in males from week 0-88 

and by 14% in females from Week 0-80. No differences in food consumption or efficiency were 

observed across dose. No adverse effects were observed on differential leukocyte counts or 

ophthalmological endpoints or at necropsy. 

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Mean relative liver weights were increased in females by 15% (p<0.01) at 675 ppm; however, the 

effect showed no relationship to dosing. Increased incidences (p<0.05) of reduced secretion of the 

prostate and seminal vesicles were observed in males at ≥ 132 ppm, at which the dose-response curve 

plateaued for both endpoints. The dose-response curve also plateaued at ≥26 ppm, at which an 

increased incidence (p<0.05) of hyperplasia of the non-glandular region of the stomach was observed 

in males. The toxicological relevance of this endpoint is questionable, however, since humans lack a 

homologue for the rodent non-glandular stomach. Increased incidences (p<0.05) of hyperplasia of the 

non-glandular region of the stomach were also observed in females; however, the effect was not dosedependent. Increased incidences of hyperplasia (p<0.05) were also observed at several other sites at 

675 ppm, including pancreatic islet cells and skin (acanthosis) in males and adrenal cortex, small 

intestine, and bone marrow (sternum) in females. Hypertrophy of the skin (hyperkeratosis) and 

hepatocytes was also observed at the high dose in females. 

The LOAEL is 132 ppm (9.39 mg/kg/day) in males, based on reduced secretion of the prostate and 

seminal vesicles; and 675 ppm (48.85 mg/kg/day) in females, based on decreased body weight gain, 

peritonitis, and hyperplasia of the adrenal cortex, small intestine, and bone marrow (sternum). The 

NOAEL is 26 ppm (1.69 mg/kg/day) in males and 132 ppm (9.20 mg/kg/day) in females. 

According to the Health Effects Division 2nd Carcinogenicity Peer Review (1995), there was no 

treatment- related increase in the incidence of any tumor type in this study. Dosing was considered 

adequate, based on decreased body weight gains and hyperplasia in various tissues in both sexes. 

Reproduction and Fertility Effects (Rat)

In a 2-generation reproduction study (MRIDs 41257303 and 42239101), dichlobenil technical (99.4% 

a.i.; lot #s 4 and 5; batch# F6N87EO8A/FUX011000) was administered in the diet to Crl:CD(SD)BR 

rats (30/sex/dose in the P generation; 25/sex/dose in the F1 generation) at daily dose levels of 0, 60, 

350, or 2000 ppm (equivalent to 0, 3, 17.5, or 100 mg/kg/day) for 2 consecutive generations. One (of 

30) high-dose P males was found dead during week 14 of the study. The cause of death was liver 

necrosis and hemorrhage (MRID 42239101). One (of 25) F1 high-dose males was sacrificed in 

extremis during week 12 of the study due after an accidental injury to the snout. Both deaths were 

considered incidental to treatment. There were no treatment-related clinical signs of toxicity in the 

study. 

During the 10-week premating period for P animals, mean cumulative body weight gains were 

decreased by 25-26% (p<0.05) at 2000 ppm in both males and females. Similarly, mean overall body 

weight gains for F1 males were decreased by 25% during the 10-week premating period and by 18% 

for F1 females at 2000 ppm. During gestation, mean body weight gains were decreased by 13-14% in 

both P and F1 females at 2000 ppm. Decrements in body weight gains were not observed in adult 

females of either generation during lactation. Mean food consumption at 2000 ppm was decreased by 

11-33% (p<0.001) in P males and by 17-28% (p<0.001) in P females during premating. In F1 males, 

mean food consumption during premating was decreased by 16-21% (p<0.001) and by 16-20% 

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(p<0.001) in F1 females at 2000 ppm. Mean food consumption for P and F1 females during gestation 

and lactation was not reported. 

No treatment-related effects were observed on fertility index, fecundity index, gestation index, or 

mean gestation length in either P or F1 females. The mean number of implantations/dam was 

unreported for P females and decreased at 2000 ppm in F1 females (12.3 treated vs. 14.6 in controls). 

The parental systemic LOAEL is 2000 ppm (100 mg/kg/day), based on decreased body weight gains 

during premating (males and females) and gestation (females) in both generations, decreased food 

consumption during premating in both generations (males and females), and decreased number of 

implantations/dam in F1 females. The parental systemic NOAEL is 350 ppm (17.5 mg/kg/day). 

The reproductive LOAEL is 2000 ppm (100 mg/kg/day), based on decreased number of 

implantations/dam in F1 females. The reproductive NOAEL is 350 ppm (17.5 mg/kg/day). 

No treatment-related effects were observed on live birth index, viability index, lactation index, or sex 

ratio in either F1 or F2 offspring. In addition, the mean number of pups born live/litter was 

unaffected in F1 offspring but decreased (p<0.001) at 2000 ppm in F2 offspring (11.0 vs. 13.7 in 

controls). This was likely due to the decreased number of implantations/F1 dam at 2000 ppm. At 

≥350 ppm, the mean pup body weight of F1 offspring was decreased by 16-23% (p<0.05) from 

postnatal day (PND) 4 (precull)-21. The effect was dose-dependent. In F2 offspring, mean pup body 

weight was also dose-dependently decreased by 19-22% (p<0.05) from PND 14-21. An increased 

incidence of pelvic cavitation of the kidney at 2000 ppm (3% vs. 0%) was observed during necropsy 

in weanling F2 offspring; however, the incidence was similar to the sporadic incidence observed in P 

and F1 animals and was not considered toxicologically significant. 

The offspring LOAEL is 350 ppm (17.5 mg/kg/day), based on decreased body weight during weaning 

in both generations. The offspring NOAEL is 60 ppm (3 mg/kg/day). 

Teratogenicity Study (Rabbit)

In a prenatal developmental toxicity study (MRID 41257302), dichlobenil technical (98.5% a.i.; lot#s 

3 and 6; batch# FUX010000) was administered by gavage in 1% gum tragacanth to 18 pregnant New 

Zealand white rabbits/sex/dose from gestation day (GD) 7-19 inclusive at daily dose levels of 0, 15, 

45, or 135 mg/kg/day. No treatment-related mortality, clinical signs of toxicity (including abortions), 

or gross pathology were observed in the study. 

A decrease (129%; p<0.05) in body weight gain was observed during the dosing period only (GD 7-

19) in does treated at 135 mg/kg/day. This effect was accompanied by a 30% (p<0.01) decrease in 

food consumption at the high dose during the dosing period only. Body weight gain and food 

consumption in high-dose animals rebounded during the post-dosing period (GD 19-29). 

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The maternal LOAEL is 135 mg/kg/day, based on decreased body weight gain and food consumption 

during dosing. The maternal NOAEL is 45 mg/kg/day. 

Increases in total resorptions/dam (1.3) and post-implantation loss (17.9%) were observed at 135 

mg/kg/day. Although the effects were not dose-dependent, the incidences were outside the historical 

control range and considered treatment-related. Although generally occurring at very low incidences 

(1-3 fetuses), several external, visceral, and skeletal defects anomalies were reported at 135 

mg/kg/day. These effects were not observed in either concurrent or historical controls or were 

observed at incidences outside historical control ranges and were therefore considered toxicologically 

significant. External anomalies included bilateral open eye (3/115 fetuses; 3/14 litters), cleft palate, 

and adactyly. High-dose visceral anomalies included abnormal cystic gallbladder and distended 

ureter with bilateral severe hydronephrosis. Skeletal defects at 135 mg/kg/day were composed of 

malformed and malpositioned right scapula, right radius absent with malpositioned ulna and humerus, 

fused cervical vertebral arches, asymmetrically ossified and fused cervical vertebra centra, 

abnormally shaped cranium with enlarged and misshapen fontanelle, enlarged fontanelle (19/115 

fetuses; 13/14 litters), misshapen frontals (2/115 fetuses; 2/14 litters), skull and frontals foreshortened 

and nasal malpositioned, and major fusion of sternebrae (3/115 fetuses; 3/14 litters). 

The developmental LOAEL is 135 mg/kg/day, based on increased number of total resorptions/dam 

and post-implantation loss; and increased incidences of external (cleft palate, adactyly, bilateral open 

eye), visceral (abnormal cystic gallbladder, distended ureter with bilateral severe hydronephrosis), 

and skeletal (malformed and malpositioned right scapula, right radius absent with malpositioned ulna 

and humerus, fused cervical vertebral arches, asymmetrically ossified and fused cervical vertebra 

centra, abnormally shaped cranium with enlarged and misshapen fontanelle, enlarged fontanelle, 

misshapen frontals, skull and frontals foreshortened and nasal malpositioned, and major fusion of 

sternebrae) anomalies. The developmental NOAEL is 45 mg/kg/day. 

Teratogenicity Study (Rat)

In a prenatal developmental toxicity study (MRID 00147437), dichlobenil technical (purity not 

reported; batch# FUN82B07A/FUX003000) was administered by gavage in 1% gum tragacanth to 25 

pregnant Wistar Cpb:WU rats/sex/dose from gestation day (GD) 6-15 inclusive at daily dose levels of 

0, 20, 60, or 180 mg/kg/day. No treatment-related mortality or clinical signs of toxicity were 

observed in pregnant does in the study. 

A 29% (p<0.01) and 36% (p<0.01) decrease in mean body weight gain was observed during the 

dosing period in does treated at 60 and 180 mg/kg/day, respectively. This effect was accompanied by 

a 15% (p<0.01) and 21% (p<0.01) decrease in food consumption at 60 and 180 mg/kg/day, 

respectively, as well as a respective 19% (p<0.05) and 24% (p<0.01) decrease in food efficiency. 

During the post-dosing period (GD 16-21), body weight gain, food consumption, and food efficiency 

values in mid- and high-dose animals rebounded to levels that were similar to or greater than those of 

controls. 

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The maternal LOAEL is 60 mg/kg/day, based on decreased body weight gain, food consumption, and 

food efficiency during dosing. The maternal NOAEL is 20 mg/kg/day. 

No treatment-related changes were observed in cesarean section parameters (death, altered growth) 

for either embryos or fetuses. It is unclear from the study report whether statistical analysis was 

performed for litter incidences of external, skeletal, or visceral observations. With regard to external 

anomalies, a shallow dose-dependent increase in the incidence of small subcutaneous hemorrhage or 

petechia was observed (0/23, 1/22, 2/22, and 3/21 litters at 0, 20, 60, and 180 mg/kg/day); however, 

the increase in the number of fetuses affected (0-3) was only slightly increased across dose and not 

statistically significant. Historical control incidences for this specific observation were not reported. 

However, the percentage of litters affected at all doses was within the historical control range (0-

20%) of litter incidences of externally visible alterations. 

An increased litter (13.6-14.3%) and fetal (3.1-3.5%) incidence of unilateral supernumerary rib (14th) 

was observed at ≥60 mg/kg/day. The fetal incidences were not statistically significantly different 

from concurrent controls. The litter incidence (19%) of bilateral supernumerary rib (14th) was also 

increased at 180 mg/kg/day, as was the fetal incidence (4.7%; p<0.05). However, both the litter and 

fetal incidences of supernumerary (14th) rib were below the historical control incidences (32.26% and 

10.86%, respectively). An increase (p<0.01) in the “degree” of absence of ossification of the 

sternebrae (expressed as transformed ossification values per litter) was also observed at 180 

mg/kg/day (7.03 vs. 0.59 in controls). The increase was outside the historical control range (0-0.31). 

However, given that the concurrent control value (0.59) as above the upper limit of the historical 

control range and dose-response was lacking in the effect, the calculated value is not considered 

toxicologically significant. 

With respect to visceral anomalies, a very slight increase in the malformations, unilateral 

microphthalmia and intestinal alteration of the situs viscerum (combined with focal fibrosis of the 

peritoneum and mesentery) was observed at the high dose in one animal in 1/21 litters only. The 

malformation, soft consistency of the lens/unilateral folded retina, was observed in a different animal 

in 1/21 litters only. The incidence of each effect was above that of the historical controls (1/2691 

fetuses; 1/401 litters); however, fetal incidences were not statistically significant and a dose-response 

was lacking at the doses tested. 

The developmental LOAEL was not identified. The developmental NOAEL is 180 mg/kg/day. 

Avian Reproduction Study (Bobwhite Quail)

In a one-generation reproductive study (MRID 45280301) the toxicity of dichlobenil to groups (16 

pens/treatment level) of 1 male and 1 female, 20-week-old Northern Bobwhite quail was assessed 

over approximately 21weeks. Dichlobenil was administered to the birds in the diet nominal 

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concentrations of 0 (negative control), 150,300, and 600 mg/kg-diet. Mean-measured concentrations 

were -30.8 (<LOD, control), 146, 289, and 600 ppm a.i. 

The most sensitive parameters was 14-day old survivors to number of hatchlings and adult male body 

weight, with the reviewer's analysis detecting significant reductions at all treatment levels. Treatmentrelated effects at the 289 ppm treatment level included reductions in eggs laid (effects on eggs set, 

viable embryos, live 3-week embryos, number of hatchlings, and hatchling survival were also reduced 

compared to per pen, but not when compared to eggs set). There were adverse effects at the 600 ppm 

treatment level on the number of eggs laid, ratios of number of hatchlings to eggs set and to live 3-

week embryos, as well as on the ratio of hatchling survival to eggs set (effects on eggs set, viable 

embryos, live 3-week embryos, number of hatchlings, and hatchling survival were also reduced 

compared to per pen, but not when compared to eggs set)). Furthermore, eggshell thickness (↓4.2%), 

hatchling (↓18%) and survivor weight (↓26%), were all adversely affected at the 600 ppm treatment 

level. Adult male weight gain was significantly reduced 42, 25 and 50% compared to control at 146, 

300 and 600 ppm; no effect on female body weight gain. There were no significant treatment-related 

effects on adult mortality, clinical effects, food consumption, or necropsy findings at any test level. 

Avian Reproduction Study (Mallard Duck)

In a one-generation reproduction study (MRID 45280302), Mallard ducks were fed diets containing 

nominal concentrations of 0, 150, 300, and 600 ppm a.i. Dichlobenil technical for 21 weeks. Mean 

measured dietary concentrations of the test substance were 0,146, 289, and 600 ppm a.i. 

There were no clear toxic-induced mortalities, pathological responses, or adverse effects on food 

consumption by adults or adult bodyweight. Significant reductions were detected for number of eggs 

laid and ratios involving egg production, number of hatchlings, and 14 day old survivors. As a result, 

the NOAEC was 289 ppm and the LOAEC was 600 ppm a.i. 

Early-Life Stage - Fish (Rainbow Trout)

An early-life stage study with dichlobenil on rainbow trout demonstrated a statistically significant 

effect on fry length at 34 days post-hatch at all concentrations tested (mean measured values of 0.33, 

0.66, 1.2, 2.4, and 5.1 mg a.i./L) (MRID 42224202). Therefore, the NOAEC is <0.33 mg/L and the 

LOEC is 0.33 mg/L. 

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APPENDIX 3: References Not Utilized in the Dichlobenil 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_rereg

istration_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 

Dichlobenil 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. 

Barker, L. (1989) Dichlobenil: Oral (Gavage) Teratology Study in the Rabbits (Experiment II): Duphar 

Report No. 56645/85/87: Laboratory Project ID 562665/17B. Unpublished study prepared by Hazleton 

UK. 139 p. (MRID 41257301) 

Cooke, A.S. (1977) Effects of Field Applications of the Herbicides Diquat and Dichlobenil on 

Amphibians. Published in Environ. Pollut. (12) 1977, p. 43- 50. 

Til, H.P., Feron, V.J., and de Groot, A.P. (1968) Short-term (90-day) Toxicity Study with Dichlobenil 

(H133) in Beagle Dogs. Study conducted by Central lnstituut Voor Voedingsonderzoek for PhilipsDuphar N.V. Weesp, The Netherlands, Report No. R2362. 35 p. (MRID 48043301) 

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