UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

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

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

Page 1 of 69
Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

Page 2 of 69
EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: CHLOROTHALONIL 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

Page 3 of 69
CHLOROTHALONIL FINAL 

ii 

TABLE OF CONTENTS 

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

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

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

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

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

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

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

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

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

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

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

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

IV. Committee’s Assessment of Weight of Evidence..........................................20 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20 

1. Tier 1 in vivo Assays.............................................................21 

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

B. Estrogen Pathway...........................................................................21 

C. Androgen Pathway.........................................................................22 

D. Thyroid Pathway...........................................................................22 

E. Conclusions.................................................................................22 

V. EDSP Tier 2 Testing Recommendations....................................................22 

VI. References.......................................................................................24 

LIST OF TABLES: 

Table 1. Tier 1 Assays for Chlorothalonil...........................................................7 

Table 2. Estrogenic/Anti-Estrogenic Pathway for Chlorothalonil ...............................9 

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

Table 4. Thyroid Pathway for Chlorothalonil ....................................................18 

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

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

APPENDIX 3. References Not Utilized in the Chlorothalonil WoE Analysis...................60 

Page 4 of 69
CHLOROTHALONIL FINAL 

iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

Page 5 of 69
CHLOROTHALONIL FINAL 

iv 

Abbreviation Terminology 

F2 Second filial generation 

Fcd Fecundity

FIFRA Federal Insecticide, Fungicide and Rodenticide Act 

FOB Field Observation Battery

FQPA Food Quality Protection Act

Frt Fertility

FSH Follicle Stimulating Hormone

FSTRA Fish Short-Term Reproduction Assay

FT Flutamide 

GD Gestation Day

GGT Gamma-glutamyl Transpeptidase

GnRH Gonadotropin-releasing hormone

GP Glans Penis 

GSI Gonado-Somatic Index

H High 

HLL Hind Limb Length

HPG Hypothalamic-Pituitary-Gonadal Axis

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

HPT Hypothalamic-Pituitary-Thyroidal Axis

I Inadequate 

IC50 Inhibitory Concentration at 50% of response

ICCVAM Interagency Coordinating Committee on the Validation of Alternative 

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 

Page 6 of 69
CHLOROTHALONIL FINAL 

v 

Abbreviation Terminology 

MTD Maximum Tolerated Dose

N Negative 

NE Not examined/evaluated

NF stage Nieuwkoop and Faber’s Staging Atlas

NOAEC No Observed Adverse Effect Concentration

NOAEL No Observed Adverse Effect Level

NS Not Statistically Significant

NR Not Reported

OCSPP Office of Chemical Safety Pollution and Prevention

OECD Organization for Economic Co-Operation and Development 

OPP Office of Pesticide Programs 

ORD Office of Research and Development

OSCP Office of Science Coordination and Policy

OSRI Other Scientifically Relevant Information

P Positive 

P Parental generation

PC Positive Control 

PC10 Positive Control at 10% of response 

PC50 Positive Control at 50% of response 

PND Post-Natal Day

POD Point of Departure

PPS Preputial Separation 

PROD Pentaoxyresorufin-O-dealkylase (or depentylase)

PXR Pregnane X receptor

QC Quality Control 

RBA Relative Binding Affinity 

RBC Red Blood Cells 

RfD Reference Dose 

RPCmax Relative to Positive Control at maximum 

SAP Scientific Advisory Panel

SC Solvent Control 

s.c Subcutaneous 

SDH Sorbitol dehydrogenase

SDWA Safe Drinking Water Act 

SEP Standard Evaluation Procedure

SD Standard Deviation or Sprague-Dawley 

SVL Snout-to-Vent Length

SV Seminal Vesicles

T Thyroid (hormonal pathway)

T1WoERC EDSP Tier 1 Weight of Evidence Review Committee

T3 Triiodothyronine 

T4 Thyroxine (tetraiodothyronine) 

Page 7 of 69
CHLOROTHALONIL FINAL 

vi 

Abbreviation Terminology 

TP Testosterone Propionate

TR Thyroid Receptor

TSH Thyroid Stimulating Hormone 

UDPGT Uridine Diphosphate Glucuronyltransferase (also known as UGT) 

VC Vehicle Control 

VO Vaginal Opening 

VP Ventral Prostate 

VTG Vitellogenin

WoE Weight-of-Evidence

Page 8 of 69
CHLOROTHALONIL FINAL 

Page 1 of 61 

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 chlorothalonil 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 August 20th, 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 analysis of the potential interaction of chlorothalonil 

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).

There was no convincing evidence of potential interaction with the estrogen pathway in either 

the in vitro or in the in vivo Tier 1 assays. Chlorothalonil gave negative results for estrogen 

binding (ER) binding, ERα transcriptional activation assay (ERTA), aromatase, and steroidogenesis. 

There was no increase in uterine weight in the uterotrophic assay. In the female pubertal assay, 

there were no estrogen-related effects observed. In the fish short term reproduction assay 

(FSTRA), effects (decreased fecundity) was only in the presence of overt toxicity (decreased 

survival in males 63% and 88% in females). There were estrogen-related effects were observed 

in the mammalian Part 158 toxicity studies in the absence of overt toxicity. Decreased egg 

production was seen in the Part 158 avian reproduction and fish life-cycle study in the absence of 

overt toxicity. However, these effects on this endpoint alone is limited in diagnostic specificity 

for a potential interaction with the estrogen pathway. Furthermore there is a lack of redundancy 

across the assays. 

There was no convincing evidence of potential interaction with the androgen pathway either in 

the in vitro or in the in vivo Tier 1 assays. Chlorothalonil was negative for androgen binding 

(AR) binding and steroidogenesis. In the Hershberger assay, there were no changes in accessory 

Page 9 of 69
CHLOROTHALONIL FINAL 

Page 2 of 61 

sex tissue weights in the androgenic or antiandrogenic portion of the assay. In the male pubertal 

assay and FSTRA, there were no androgen-related effects observed. There were no androgenrelated effects in the mammalian or wildlife Part 158 studies in the absence of overt toxicity. 

There was also no convincing evidence of interaction with the thyroid pathway in mammals. 

While there was a lack of redundancy across the battery (i.e., no convincing evidence of 

interaction in mammals), the complementary evidence of effects observed in the amphibian 

metamorphosis assay (AMA) (described below) indicate potential interaction with the thyroid 

pathway. No thyroid-related effects were seen in the male pubertal assay. In the female pubertal 

assay, there was an isolated finding of a 14% increase in T4 level at the low dose which was not 

complemented by any treatment-related changes in thyroid stimulating hormone (TSH) level, 

thyroid weight or histopathology. There were no thyroid related effects observed in the absence 

of overt toxicity in the mammalian Part 158 toxicity studies to support a thyroid interaction in 

mammals. 

In the AMA study, there were complementarity of effects consistent with potential interaction 

with the thyroid pathway in amphibians, including: increased frequency and severity of thyroid 

gland hypertrophy and follicular cell hypertrophy and hyperplasia at the high dose. Furthermore, 

there was a significant decrease (25%) in 21-day normalized hind limb length (HLL) at the high 

dose as well as an indication that the developmental stage was delayed at the mid and high doses 

based on a slight, but significant decrease in NF stage and dose-dependent decrease in the 

number of late stage frogs; no late stage tadpoles were seen at the high dose. 

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

mammals or fish since there was no convincing evidence of potential interaction with the 

estrogen, androgen or thyroid pathways in mammals or fish. 

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

duck are available for chlorothalonil. In quail, 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 chlorothalonil 

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

As the results of the AMA suggest a potential interaction with the thyroid pathway in amphibians 

at a concentration of 5.0 μg a.i./L which is comparable to the current chronic endpoint value of 

1.4 μg a.i./L for fish [which are surrogates for aquatic-phase amphibians; value from fathead 

minnow life-cycle study (MRID 00030391]an EDSP Tier 2 Larval Amphibian Growth and 

Development Assay (LAGDA) is recommended. 

Page 10 of 69
CHLOROTHALONIL FINAL 

Page 3 of 61 

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 chlorothalonil 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 August 20th, 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 WoE analysis of the potential interaction of chlorothalonil 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).

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) has a water solubility at 25°C of 0.8-0.96 

mg/L. Chlorothalonil has an octanol/water partition coefficient (log Kow) of 3.8. It has a vapor 

pressure of 5.72 × 10-7 mm Hg at 25°C and a Henry’s law constant of 2.6 × 10-7 atm-m3

/mol, and 

some degree of volatility from both soil and water (semi-volatile) could be anticipated. Studies 

indicate chlorothalonil will transform primarily through aqueous photolysis in clear, shallow 

water, and is susceptible to microbial-mediated transformation, and is slightly to hardly mobile 

in soil systems. Chlorothalonil is anticipated to be rapidly transformed in water/sediment 

systems under both aerobic and anaerobic conditions. Field dissipation studies show that 

chlorothalonil dissipates (e.g., transformation or relocation) with half-lives less than 100 days; 

however, in a few field dissipation studies, chlorothalonil was observed in soil samples taken 

after one year. 

The available information considered in determining the potential interaction of chlorothalonil 

with the E, A and/or T pathways include submitted EDSP Tier 1 assays and/or other 

scientifically relevant information (OSRI) such as general toxicity studies and other published 

articles. These data are summarized in Sections III.A through III.C. An analysis of the data 

Page 11 of 69
CHLOROTHALONIL FINAL 

Page 4 of 61 

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. 

II. Sources of Scientific Data and Technical Information 

A. EDSP T1 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 chlorothalonil (USEPA, 

2010). Since then, the Agency has also conducted a more recent search of available scientific 

literature for any additional relevant information. Summaries of the available OSRI are 

presented in Appendix 2. Additionally, literature/studies considered but not utilized for the WoE 

analysis are listed in Appendix 3. 

III. Weight of Evidence (WoE) Evaluation 

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

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

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

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

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

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

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

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

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

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

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

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

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

is to then 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. 

Page 12 of 69
CHLOROTHALONIL FINAL 

Page 5 of 61 

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, and 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 did the responses occur including discussions regarding whether the responses were 

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

Page 13 of 69
CHLOROTHALONIL FINAL 

Page 6 of 61 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

forms of toxicity (e.g., changes in organ weights or blood chemistry). The circumstances for 

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

Section IV.A. 

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

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

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

the E, A or T pathway. Adequately addressing these three main questions 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. 

Page 14 of 69
CHLOROTHALONIL FINAL 

Page 7 of 61 

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

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

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

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

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

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

 A. EDSP T1 Screening Assays 

The Tier 1 studies submitted in response to the agency’s test order for chlorothalonil are shown 

below in Table 1. 

Table 1. Tier 1 Screening Assays for Chlorothalonil 

Tier 1 Assays Test 

Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OSCPP 

890.1250 

Requirement Satisfied 

(MRID No. 48615401) 

ERα Transcriptional Activation Assay (Human 

cell line HeLa 9903) 

OSCPP 

890.1300; 

OECD 455 

Requirement Satisfied 

(MRID No. 48615402) 

AR Binding Assay (Rat prostate cytosol) OSCPP 

890.1150 

Requirement Satisfied 

 (MRID No. 48676301) 

Steroidogenesis Assay (Human cell line H295R) 

OSCPP 

890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No. 48669903) 

Aromatase Assay (Human recombinant 

microsomes) 

OSCPP 

890.1200 

Requirement Satisfied 

(MRID No. 48615403) 

Uterotrophic Assay (Rat) 

OSCPP 

890.1600; 

OECD 440 

Requirement Satisfied 

 (MRID No. 48669904) 

Hershberger Assay (Rat) 

OSCPP 

890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No. 48669902) 

Pubertal Female Assay (Rat) OSCPP 

890.1450 

Requirement Satisfied 

(MRID No. 48699301) 

Pubertal Male Assay (Rat) OSCPP 

890.1500 

Requirement Satisfied 

(MRID No. 48699302)

Fish Short-term Reproduction Assay 

OSCPP 

890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No. 48950801)

Amphibian Metamorphosis Assay (Frog) 

OSCPP 

890.1100; 

OECD 231 

Requirement Satisfied (MRID 

No. 49002801) 

Page 15 of 69
CHLOROTHALONIL FINAL 

Page 8 of 61 

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

1. Effects on Estrogen Pathway 

The potential interactions of chlorothalonil 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. 

In the Tier 1 in vitro studies, chlorothalonil was negative in the ER binding, ERTA, aromatase, and 

steroidogenesis assays. Furthermore in a literature study considered as OSRI, Kojima et al., 2004 

reported negative results for ER transcriptional activation. Additionally there were no estrogenrelated effects in the in vivo uterotrophic or female pubertal assays. In the fish short-term 

reproduction assay (FSTRA), there was a 48% decrease in fecundity at the highest tested 

concentration (9.9 μg a.i./L); however, survival was also decreased to 79% at this concentration. 

In a Part 158 2-generation mammalian reproduction study, vaginal opening was delayed by 3.4 

days at the high dose although body weight at vaginal opening was comparable to the control body 

weight. This finding was observed in the presence of both systemic (kidney and fore-stomach 

lesions were observed in parental animals and offspring of all generations) and overt (decreased 

parental and pup body weights) toxicity. In a developmental toxicity study in rats, increased 

resorptions and post implantation loss were observed at the high dose. This finding was again 

observed in the presence of both systemic (clinical signs of toxicity) and overt (parental mortality) 

toxicity. In addition, post implantation loss and resorptions were not observed in a second 

developmental toxicity study in rats. No other estrogen-related effects were observed in the other 

Part 158 mammalian toxicity studies. 

In an available Part 158 avian reproduction study on bobwhite quail, biologically relevant 

reductions in the number of eggs laid/hen at the high dose (624 mg/kg-diet) occurred in the 

absence of overt toxicity. In a set of reproduction studies with bobwhite quail and mallard ducks, 

effects on reproductive parameters in the presence of overt toxicity were observed at 5,000 mg/kgdiet in the bobwhite and no reproductive effects were reported at concentrations of 10,000 mg/kgdiet in the mallard. There were no effects on mortality or any reproductive parameters determined 

in two more avian reproductive toxicity studies (one each for mallard duck and bobwhite quail) up 

to and including the highest treatment concentration of 50 mg/kg-diet). In addition, reductions in 

egg production were also observed in a fathead minnow life-cycle toxicity study at 3.0 μg/L in the 

absence of overt toxicity. Chlorothalonil significantly reduced hatching success and survivability 

of fathead minnows at concentrations at 6.5 μg/L (F1 reduced hatching success). 

Page 16 of 69
CHLOROTHALONIL FINAL 

Page 9 of 61 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Chlorothalonil 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chlorothalonil1Study 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) 

Vitellogenin 

SiTii

EDSP Tier 1 Assay 

ER Binding (MRID 48615401) N ERTA (MRID 48615402) N Aromatase (MRID 48615403) N Steroidogenesis (MRID 48669903) N Uterotrophic (MRID 48669904) N Female Pubertal Rat (MRID 48699301) N N N N N N FSTRA (MRID 48950801) NE N N N N N 

OSRI

ER Binding (Kojima, et al., 2004) N Two-generation reproduction (Rat; MRID 41706201) 

NR NR N N NE N 

(L

Two-generation reproduction (Rat; MRID 45710209) 

NR N N N N N N 

(L
CHLOROTHALONIL FINAL 

Page 10 of 61 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Chlorothalonil 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chlorothalonil1Study 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) 

Vitellogenin 

SiTii

Developmental toxicity (Rat; MRID 00130733) NR N Developmental toxicity (Rat; MRID 45710207) NR N Developmental toxicity (Rabbit; MRID 00047944) NR N Developmental toxicity (Rabbit; MRID 45710208) NR N Chronic toxicity/ carcinogenicity (Rat; MRID 00146945) 

NR N N NR 

(L

Chronic toxicity/ carcinogenicity (Rat; MRID 45710212) 

NE N N N 

(L, 

Carcinogenicity (Mouse; MRID 00127858) NE N N NE 

(L

Carcinogenicity (Mouse; MRID 45710211) N N N NE 

(ML

Chronic toxicity (Dog; MRID 43653603) NE NE N NE Chronic toxicity (Dog; MRID 45710210) N N N N Subchronic toxicity (Rat; MRID 45710205) N N N N 

(ML

Subchronic toxicity (Dog; MRID 45710206) N N N N 
CHLOROTHALONIL FINAL 

Page 11 of 61 

Table 2: Estrogenic/Anti-Estrogenic Pathway for Chlorothalonil 

Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for Chlorothalonil1Study 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) 

Vitellogenin 

SiTii

Avian reproduction (Quail; MRID 00044140 

 N 

Avian reproduction (Quail; MRID 45710218) 

P6

(H) 

Avian reproduction (Quail; MRID 40964104) 

 N 

Avian reproduction (Duck; MRID 00041441; 40964102) 

 N 

Fish Life Cycle Toxicity (Fathead Minnow, MRID 00030391) 

P7 

(M) 

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

Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is no

assay. 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 O

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 

For details see Section IV. A 

4. Decreased final body weight of 11% at the high dose. 5. Survival was decreased to 88% for females at the highest concentration (9.9 μg a.i./L). 6. Reduced egg production. 6. Reduction in egg production. F1 hatching success also reduced at mid-high 6.5 μg/L. P Positive findings N Negative findings (in vitro)/No effect (in vivo) 
CHLOROTHALONIL FINAL 

Page 12 of 61 

NE Not examined NR Not reported 
CHLOROTHALONIL FINAL 

Page 13 of 61 

2. Effects on Androgen Pathway 

The potential interactions of chlorothalonil 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. 

Chlorothalonil was classified as negative in the in vitro AR binding and steroidogenesis assays. 

Furthermore, in a literature study considered as OSRI, Kojima et al. (2004) reported negative results 

for an AR transcriptional activation. 

Additionally, the mammalian in vivo assays (Hershberger and Male Pubertal) were also negative for 

all of the androgen-sensitive parameters evaluated. In addition, no effects on androgen-related 

endpoints were noted in the FSTRA. 

In the mammalian Part 158 data, the only finding related to the androgen pathway was a delay in 

preputial separation (PPS) of 3.2 days at the high dose in a 2-generation reproduction study in rats. 

The body weight at PPS was comparable to the control body weight. The finding of delayed PPS 

was observed in the presence of both systemic (kidney and fore-stomach lesions in parental animals) 

and overt (decreased body weight in F1 males and F1/2 pups) toxicity. There were no androgen 

related effects in the wildlife Part 158 data. 

Page 21 of 69
CHLOROTHALONIL FINAL 

Page 14 of 61 

Table 3: Androgenic/Anti-Androgenic Pathway for Chlorothalonil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chlorothalonil1Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging and 

 Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

 Weights/ 

2° Sex Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at PPS 

Vitellogenin 

EDSP Tier 1 Assay 

AR Binding (MRID 48676301) N Steroidogenesis (MRID 48669903) N Hershberger (MRID 48669902) N N Male Pubertal Rat (MRID 48699302) N N N N N N N N FSTRA (MRID 48950801) NE N N N N N 

OSRI

AR Binding (Kojima, 2004) N Two-Generation reproduction (Rat; MRID 41706201)

NR NE N NE N NE NE 

Two-Generation reproduction (Rat; MRID 45710209)

NE N N N N N N N N X 

Chronic toxicity / Carcinogenicity (Rat; MRID 00146945) 

N N NR N NR NR 

Chronic toxicity / Carcinogenicity (Rat; MRID 45710212) 

N N NE NR N NE 
CHLOROTHALONIL FINAL 

Page 15 of 61 

Table 3: Androgenic/Anti-Androgenic Pathway for Chlorothalonil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chlorothalonil1Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging and 

 Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

 Weights/ 

2° Sex Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at PPS 

Vitellogenin 

Carcinogenicity (Mouse; MRID 00127858) 

N N NE NE NE NE 

Carcinogenicity (Mouse; MRID 45710211) 

N N NE N NE NE 

Chronic toxicity (DogMRID 43653603) N N N N NE NE Chronic toxicity (DogMRID 45710210) N N N N N N Subchronic toxicity (Rat; MRID 45710205) 

N N NE N N N 

Subchronic toxicity (Dog; MRID 45710206) 

N N NE N N N 

Avian reproduction (Quail; MRID 00044140 

 N 

Avian reproduction (Quail; MRID 00044140 

 N 

Avian reproduction (Duck; MRID 00041441) 

 N 

Avian reproduction (Quail; MRID 45710218) 

 N 
CHLOROTHALONIL FINAL 

Page 16 of 61 

Table 3: Androgenic/Anti-Androgenic Pathway for Chlorothalonil Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for Chlorothalonil1Study Type/ Literature Citation 

AR Binding/ARTA 

Steroidogenesis 

Sex Steroid Hormones 

Testes Weight/GSI 

Gonad Staging and 

 Histopathology 

Epididymides Weight 

Epididymides 

Histopathology 

Pituitary Weight 

Accessory Sex Organ 

 Weights/ 

2° Sex Characteristics 

Fertility (Frt)/ 

Fecundity (Fcd) 

Age and Weight at PPS 

Vitellogenin 

Fish Life Cycle Toxicity (Fathead Minnow, MRID 00030391) 

 N 

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

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

absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Do(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). 

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

this column. For details see Section IV. A 

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

see Section IV. A 

4. Survival of males was decreased to 63% at highest concentration (9.9 μg a.i./L). P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined NR Not reported 
CHLOROTHALONIL FINAL 

Page 17 of 61 

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

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

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

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

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

The potential interactions of chlorothalonil with thyroid regulation are summarized in Table 4. 

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

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

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

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

further in the WOE assessment. 

There were no effects of chlorothalonil noted on serum TSH levels, pituitary or thyroid weights, or 

thyroid histopathology in either the male pubertal or female pubertal assays. Although treatmentrelated increases in serum T4 levels were noted in the female pubertal assay, these changes were 

not supported by changes in TSH levels or in thyroid weight and histology. In the AMA, no 

asynchronous development was observed, but median NF developmental stage was delayed 

slightly at the high concentration on Day 7 (53.5 vs. 54), and at the mid and high concentrations on 

Day 21 (59 and 58.5 vs. 59.5 in control). Additionally, there was an observed dose dependent 

decrease in the number of late stage frogs with increasing chlorothalonil concentration (control 

(15.5%), low (15%), mid (8.8%), and high (0%) treatment groups). In addition, thyroid gland 

hypertrophy and follicular cell hypertrophy and hyperplasia were observed at the highest 

concentration (5 μg/L), along with a decrease in normalized HLL on Day 21. Decreases in body 

weights (↓42, 23%) and reductions in SVL (↓18, 7%) were also observed at the high concentration 

on Days 7 and 21. 

In the Part 158 chronic rat study, the transient increase in adjusted thyroid weight (19%) seen in 

females at the high dose was determined to be biologically not significant since it was seen only at 

week 52 (not week 104) and was not seen in males at any dose or time period. Also, the increase 

was seen in the presence of stomach irritation and inflammation and nephrotoxicity. In the chronic 

dog study, increased thyroid weight was observed at the highest dose tested but this was in the 

presence of both systemic (macroscopic and microscopic effects on the stomach and hypertrophy 

of cells in the zona fasciculata of the adrenal glands) and overt (decreased body weight in males) 

toxicity. 

Page 25 of 69
CHLOROTHALONIL FINAL 

Page 18 of 61 

Table 4. Thyroid Pathway for Chlorothalonil.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for Chlorothalonil1

Study Type/ 

Literature Citation 

Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

Serum TSH levels Pituitary Weight Developmental stage (±) or asynchronous or HLL Growth (BW, SVL) Systemic Toxicity2 

Overt Toxicity Observed3

Male Pubertal Rat 

(MRID 48699302) N N N N N N N 

Female Pubertal Rat

(MRID 48699301) N N ↑14%(L) N N N X4(H) 

AMA 

(MRID 49002801) 

P5

(H)

P6 

(M, H)

Days 7 and 21: 

BW: ↓42, 23%; 

SVL: ↓18, 7% 

(H) 

 N

OSRI

Two-Generation 

reproduction (Rat; 

MRID 45710209)

NE N N X 

(L, M, H)

X 

(H)

Chronic toxicity/ 

Carcinogenicity 

(Rat; MRID 

00146945) 

NR N NR X 

(L, M, H)

X 

(M,H)

Chronic toxicity/ 

Carcinogenicity 

(Rat; MRID 

45710212) 

N N N 

X 

(L, ML, MH,

H)

N 

Carcinogenicity 

(Mouse; MRID 

00127858) 

NE N NE X

(L, M, H)

X 

(H)

Carcinogenicity 

(Mouse; MRID 

45710211) 

NE N NE 

X 

(L, ML, MH,

H)

N 

Chronic toxicity 

(Dog, MRID 

43653603) 

N N N X 

(H)

X 

(M,H)

Chronic toxicity 

(Dog, MRID 

45710210) 

N N N X 

(H)

X 

(H) 

Subchronic toxicity 

(Rat; MRID 

45710205) 

N N N 

X 

(L, ML, MH,

H)

N 

Subchronic toxicity 

(Dog; MRID 

45710206) 

N N N X

(M, H)

X 

(H)

Page 26 of 69
CHLOROTHALONIL FINAL 

Page 19 of 61 

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

treatment, H=High treatment. Arrows (↓ or ↑) indicate the direction of the response. A shaded cell indicates 

that is parameter is not routinely evaluated or is not applicable in this assay. 

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

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

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

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

4. Decreased final body weight of 11% at the high dose. 

5. Thyroid gland hypertrophy, and follicular cell hypertrophy and hyperplasia were noted at the high concentration. 

6. Normalized HLL decreased by 25% on Day 21 at the high concentration; NF developmental stage was delayed 

slightly on Day 7 in the high concentration, and at the mid and high concentrations on Day 2. Also there was an 

observed dose dependent decrease in the number of late stage frogs with increasing chlorothalonil concentration. 

No asynchronous development. 

P Positive findings 

N Negative findings 

NE Not examined 

NR Not reported 

 

Page 27 of 69
CHLOROTHALONIL FINAL 

Page 20 of 61 

IV. Committee’s Assessment of Weight of Evidence 

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

potential of chlorothalonil 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 case-by-case 

basis by first assessing the different lines of evidence (i.e., specific Tier 1 assays and OSRI), then 

performing an integrated analysis of those lines of evidence. 

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

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

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

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

in the Tier 1 assay are related to perturbations of the endocrine system per se or alternatively a 

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

consequences other than those evaluated in the Tier 1 assays. 

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

or not the responses are mediated 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 Tire 1 assays stated that “In summary, the Panel agreed 

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

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

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

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

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

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

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

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

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

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

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

Page 28 of 69
CHLOROTHALONIL FINAL 

Page 21 of 61 

1. Tier 1 in vivo Assays 

No signs of overt or systemic toxicity were noted in the Hershberger Uterotrophic, or male 

pubertal assays at dose levels up to 1000 mg/kg/day. Overt toxicity was observed at the highest 

dose tested (175 mg/kg) in the female pubertal assay. In the FSTRA, survival was reduced to 

79% at the highest test concentration (males 63%, females 88%). 

2. OSRI 

Systemic and/or overt toxicity observed in nearly all the mammalian toxicity studies were changes 

in body weight, and histopathological changes in the gastro-intestinal tract and kidneys. In the two 

2-generation reproduction studies, kidney and fore stomach lesions were observed at all dose 

levels. In the development toxicity studies, systemic toxicity was evident based on reductions in 

body weight or body weight gains with increases in mortality at the highest doses. Nephrotoxicity 

and gastro-intestinal irritation and inflammation were also seen in the two chronic rat studies; these 

were observed both macro- and microscopically. In two mouse carcinogenicity studies, 

histological changes were noted in the kidneys and stomach. In two chronic dog studies, macroand microscopic lesions were reported in the stomach with concomitant reduced body weight gain 

and mortality at the high dose. The subchronic rat studies demonstrated reductions in body weight, 

stomach lesions and effects on kidneys. In the dog subchronic study, reduction in body weight and 

body weight gain were seen, along with effects on the liver. 

No systemic or overt toxicity was seen in the quail avian reproductive study at fed levels up to 642 

ppm. In the minnow life-cycle study, overt toxicity was noted at the highest test concentration 

(16.0 μg/L), based on reductions in survival. 

B. Estrogen Pathway 

There was no convincing evidence of potential interaction with the estrogen pathway in either the 

in vitro or in the in vivo Tier 1 assays. Chlorothalonil was negative for ER binding, ERTA, and 

steroidogenesis. There was no increase in uterine weight in the uterotrophic assay. In the female 

pubertal assay, there were no estrogen-related effects observed. In the FSTRA, there was a 

significant decrease (48%) in fecundity in the presence of overt toxicity (decreased survival in 

males 63% and 88% in females); no other effects were noted. There were estrogen-related effects 

were observed in the mammalian Part 158 toxicity studies in the absence of overt toxicity. 

Decreased egg production was seen in the Part 158 avian reproduction and fish life-cycle study in 

the absence of overt toxicity. However, these effects on this endpoint alone is limited in diagnostic 

specificity for a potential interaction with the estrogen pathway. Furthermore there is a lack of 

redundancy across the assays. 

Page 29 of 69
CHLOROTHALONIL FINAL 

Page 22 of 61 

C. Androgen Pathway 

There was no convincing evidence of potential interaction with the androgen pathway in either the 

in vitro or in the in vivo Tier 1 assays. Chlorothalonil was negative for AR binding and 

steroidogenesis. In the Hershberger assay, there were no changes in accessory sex tissue weights in 

the androgenic or antiandrogenic portion of the assay. In the male pubertal assay and FSTRA, 

there were no androgen-related effects observed. There were no androgen-related effects in the 

mammalian or wildlife Part 158 studies in the absence of overt toxicity. 

D. Thyroid Pathway 

There was also no convincing evidence of interaction with the thyroid pathway in mammals. While 

there was a lack of redundancy across the battery (i.e., no convincing evidence of interaction in 

mammals), the complementary evidence of effects observed in the amphibian metamorphosis 

assay (AMA) (described below) indicate potential interaction with the thyroid pathway. No 

thyroid-related effects were seen in the male pubertal assay. In the female pubertal assay, there was 

an isolated finding of a 14% increase in T4 level at the low dose which was not complemented by 

any treatment-related changes in thyroid stimulating hormone (TSH) level, thyroid weight or 

histopathology. There were no thyroid related effects observed in the absence of overt toxicity in 

the mammalian Part 158 toxicity studies to support a thyroid interaction in mammals. In the AMA 

study, there were complementarity of effects consistent with potential interaction with the thyroid 

pathway in amphibians, including: increased frequency and severity of thyroid gland hypertrophy 

and follicular cell hypertrophy and hyperplasia at the high dose. Furthermore, there was a 

significant decrease (25%) in 21-day normalized hind limb length (HLL) at the high dose as well 

as an indication that the developmental stage was delayed at the mid and high doses based on a 

slight, but significant decrease in NF stage and dose-dependent decrease in the number of late 

stage frogs; no late stage tadpoles were seen at the high dose. 

E. Conclusions 

There was no convincing evidence of potential interaction with the estrogen pathway or androgen 

pathway in mammals and wildlife. There was also no convincing evidence of interaction with the 

thyroid pathway in mammals. While there was a lack of redundancy across the battery (i.e., no 

convincing evidence of interaction in mammals), the complementary evidence of effects observed 

in the AMA indicate potential interaction with the thyroid pathway. 

V. EDSP Tier 2 Testing Recommendations 

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

mammals or fish since there was no convincing evidence of potential interaction with the estrogen, 

androgen or thyroid pathways in mammals or fish. 

Page 30 of 69
CHLOROTHALONIL FINAL 

Page 23 of 61 

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

are available for chlorothalonil. In quail, 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 chlorothalonil exposure. 

Relative to the EDSP, additional testing is not recommended 

As the results of the AMA suggest a potential interaction with the thyroid pathway in amphibians 

at a concentration of 5.0 μg a.i./L which is comparable to the current chronic endpoint value of 1.4 

μg a.i./L for fish [which are surrogates for aquatic-phase amphibians; value from fathead minnow 

life-cycle study (MRID 00030391]an EDSP Tier 2 Larval Amphibian Growth and Development 

Assay (LAGDA) is recommended. 

 

Page 31 of 69
CHLOROTHALONIL FINAL 

Page 24 of 61 

VI. References

Cary, George A., 1980. A Chronic Study on the Fathead Minnow (Pimephales promelas) with 

Technical Chlorothalonil. Unpublished report prepared by EG & G, Bionomics for Diamond 

Shamrock Corporation. Received 2/19/80. MRID 00030391. Unpublished. 

Diamond Shamrock Corp. (1983) A Chronic Dietary Study in Mice with Technical Chlorothalonil: 

Document No. 108-5TX-79-0102-004. (submitted by Diamond Shamrock Agricultural Chemicals, 

Cleveland, OH; CDL:071541-E; 071542; 071543; 071544; 071545; 071546), April 21, 1983. 

MRID 00127858. Unpublished. 

Diamond Shamrock Corp. (1983) A Teratology Study in Rats with Technical Chlorothalonil: 

Document No. 517- STX-82-0011-003. (submitted by Diamond Shamrock Agricultural 

Chemicals, Cleveland, OH; CDL:251069-A), July 7, 1983. MRID 00130733. Unpublished. 

Huntingdon Research Centre, Inc. (1993) Chlorothalonil: A study of the effect on pregnancy of the 

rat. Huntingdon Research Centre, Inc. Laboratory Study No.: VCM 22/930637. MRID 45710207. 

Unpublished. 

Huntingdon Research Centre, Inc. (1994) Chlorothalonil: A study of the effect on pregnancy of the 

rabbit Huntingdon Research Centre, Inc. Laboratory Study No.: VCM 22/930638. MRID 

45710208. Unpublished. 

Huntingdon Research Centre, Inc. (1995) Chlorothalonil: A study of the effect on reproductive 

function of two generations in the rat. Huntingdon Research Centre, Inc. Laboratory Study No. 

VCM 211942505. MRID 45710209. Unpublished. 

Huntingdon Research Centre, Inc. (1995) Chlorothalonil: Potential tumorigenic effects in 

prolonged dietary administration in mice. Huntingdon Research Centre, Inc. Laboratory Study No.: 

VCM 16/943065. MRID 45710211. Unpublished. 

Huntingdon Research Centre, Inc. (1995) Chlorothalonil: Toxicity to dogs by repeated dietary 

administration for 52 weeks. Huntingdon Research Centre, Inc. Laboratory Study No.: VCM 

14/943124. MRID 45710210. Unpublished. 

Huntingdon Research Centre, Inc. (1996) Chlorothalonil: Potential tumorigenic effects in 

prolonged dietary administration in rats. Huntingdon Research Centre, Inc. Laboratory Study No.: 

VCM 15/943286. MRID 45710212. Unpublished. 

International Research and Development Corporation. (1976) Teratology Study in Rabbits: IRDC 

No. 293-032a. (submitted by Diamond Shamrock Agricultural Chemicals, Cleveland, OH; 

CDL:095783-K). MRID 00047944. Unpublished. 

Page 32 of 69
CHLOROTHALONIL FINAL 

Page 25 of 61 

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

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

Chinese hamster ovary cells. Environmental Health Perspectives 112:524-531. 

Lee, M.R. (2012) Chlorothalonil- Amphibian Metamorphosis Assay with African Clawed Frog 

(Xenopus laevis). Smithers Viscient, Wareham, MA. Laboratory report number: 1781.6774, 

November 29, 2012. MRID 49002801. Unpublished. 

Lucas, F.; Benz, G. (1990) A Two Generation Reproduction Study in Rats with Technical 

Chlorothalonil. Ricerca, Inc., and Experimental Pathology Labs, Inc. Lab Project Number: 87-

0121: 1722-87-0121-TX-003. MRID 41706201. Unpublished. 

Mizns, M., Laveglia, J (1994) A chronic (12 month) oral toxicity study in dogs with technical 

chlorothalonil. Pharmaco LSR Inc. and Experimental Pathology Labs, Inc. Lab Project Number: 

92-135: 92-0457: 5211-92-0457-TX-003. MRID 43653603. Unpublished. 

Redgrave, V. (1993) Chlorothalonil: Bobwhite Quail Dietary Reproduction and Tolerance Studies: 

Lab Project Number: VCM 11/930496. Unpublished study prepared by Huntingdon Research 

Centre Ltd. 314 p. MRID 45710218. 

Sawhney Coder, P. (2012) Chlorothalonil – Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Female Rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Study No.: WIL-639143, March 21, 2012. MRID 48699301. Unpublished. 

Sawhney Coder, P. (2012) Chlorothalonil – Pubertal Development and Thyroid Function in Intact 

Juvenile/Peripubertal Male Rats. WIL Research Laboratories, LLC, Ashland, OH. Laboratory 

Study No.: WIL-639142, March 21, 2012. MRID 48699302. Unpublished. 

Sawhney Coder, P. (2012) Chlorothalonil – Hershberger Bioassay in Orchidoepidiymectomized 

Rats Final Report. WIL Research Laboratories, LLC, Ashland, Ohio. Laboratory Project ID: WIL639141, January 26, 2012. MRID 48669902. Unpublished. 

Sawhney Coder, P. (2012) Chlorothalonil – Uterotrophic Assay in Ovariectomized Rats Final 

Report. WIL Research Laboratories, LLC, Ashland, Ohio. Laboratory Project ID: WIL-639140, 

January 26, 2012. MRID 48669904. Unpublished. 

SDS Biotech Corp. (1985) A Tumorigenicity Study of Technical Chlorothalonil in Rats:. SDS 

Biotech Corp. Document No. 099-STX- 80-0234-008. MRID 00146945. Unpublished. 

Spencer-Briggs, D. (1994) Chlorothalonil: Toxicity to rats by dietary administration for 13 weeks. 

Huntington Research Centre Ltd. Lab Project Number: VCM/9/90339. MRID 45710205. 

Unpublished. 

Page 33 of 69
CHLOROTHALONIL FINAL 

Page 26 of 61 

Spencer-Briggs, D. (1994) Chlorothalonil: Toxicity to dogs by dietary administration for 13 weeks. 

Huntington Research Centre Ltd. Lab Project Number: VCM/12/92413. MRID 45710206. 

Unpublished. 

USEPA (2010) CHLOROTHALONIL - Report of the Endocrine Disruptor Review Team - Test 

Order #: EDSP-081901-113 to EDSP-081901-118. December 8, 2010. Office of Chemical Safety 

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

USEPA. (2011) Endocrine Disruptor Screening Program – Weight of Evidence: Evaluating 

Results of EDSP Tier 1 Screening to Identify the Need for Tier 2 Testing. September 14, 2011. 

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

Washington, DC. 

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

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

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

Agency, Washington, DC. 

Wagner, H. (2012) Chlorothalonil - H295R Steroidogenesis Assay. CeeTox, Inc., Kalamazoo, MI. 

Laboratory Study No.: R9047V-100331STER, February 1, 2012. MRID 48669903. Unpublished. 

Wilga, P.C. (2011) Chlorothalonil - Human Recombinant Aromatase Assay. CeeTox, Inc., 

Kalamazoo, MI. Study No. :9047V-100331AROM, November 30, 2011. MRID 48615403. 

Unpublished. 

Willoughby, J.A., (2011). Chlorothalonil - Estrogen receptor binding (rat uterine cytosol). CeeTox, 

Inc., Kalamazoo, MI. Laboratory Study No.: 9047V-100331ERB, November 30, 2011. MRID 

48615401. Unpublished. 

Willoughby, J.A. (2011) Chlorothalonil - Estrogen receptor transcriptional activation (Human Cell 

Line (HeLa-9903)) final report. CeeTox, Inc., Kalamazoo, MI. Laboratory Report No.: 9047V100331ERTA, November, 30 2011. MRID 48615402. Unpublished. 

Willoughby, J.A. (2012) Chlorothalonil: Androgen Receptor Binding (Rat Prostate Cytosol). 

CeeTox, Inc., Kalamazoo, MI. Laboratory Project ID No.: 9047V-100331ARB, February 2, 2012. 

MRID 48676301. Unpublished. 

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

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

CeeTox, Inc., Kalamazoo, MI. July 10, 2011. MRID 48843501. Unpublished. 

York, D.O. (2012) Chlorothalonil – Fish Short-Term Reproduction Assay with Fathead Minnow 

(Pimephales promelas). Smithers Viscient, Wareham, MA. Laboratory report number: 1781.6775. 

Page 34 of 69
CHLOROTHALONIL FINAL 

Page 27 of 61 

Study submitted by Syngenta Crop Protection, LLC, Greensboro, NC, October 1, 2012. MRID 

48950801. Unpublished. 

 

Page 35 of 69
CHLOROTHALONIL FINAL 

Page 28 of 61 

APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 49002801) of chlorothalonil (99.0% purity) on amphibian 

metamorphosis of African clawed frog (Xenopus laevis) was conducted under flow-through 

conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80 larvae per 

group) were exposed to nominal concentrations of 0 (negative control), 0.000090, 0.00060, and 

0.0040 mg a.i./L. The reviewer-calculated time-weighted average (TWA) concentrations were 

<0.000027 (<LOQ; control), 0.000093, 0.00061, 0.0050 mg a.i./L. The test system was maintained 

at 22 to 24°C and a pH of 7.4 to 8.1. 

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

water) control. Mortality was observed in the control (2.5%), mid (3.75%), and high (2.5%) 

treatment groups; no deaths occurred in the low-dose treatment group. Spinal deformities were 

observed in the control and all treatment groups; 18%, 19%, 25%, and 3% in the 0 (negative 

control), low, mid, and high treatment groups, respectively, and were not considered treatmentrelated. 

There was a slight but significant delay of median NF developmental stage after 7 days (53.5 vs. 

54 in control) in the high treatment group, and on Day 21, again, slight but significant (p<0.05) 

delays were noted in the mid and high treatment groups (58.5 and 59 in mid and high-treatment vs. 

59.5 in control). Additionally, it is noted that late stage tadpoles (NF>60) were observed in the 

control (15.5%), low (15%), and mid (8.8%) treatment groups, but the high treatment group did 

not contain any late stage tadpoles, further suggesting a development delay. Late stage tadpoles 

(NF>60) were excluded from statistical analysis of the growth parameters (HLL, normalized HLL, 

wet weight, and SVL). No asynchronous development was observed. Chlorothalonil had no effect 

on Day 7 normalized hind-limb length (HLL) at any treatment level relative to the negative control 

(p>0.05), whereas Day 21 HLL was significantly (p>0.05) reduced by 24.5% in the high treatment 

group. Significant decreases in body weights (↓42, 23%) and reductions in snout-vent length 

(SVL) (↓18, 7%) were also observed at the high concentration on Days 7 and 21. Thyroid gland 

histopathology observations included increased frequency and severity of thyroid gland 

hypertrophy, and/or follicular cell hypertrophy and hyperplasia in the control and treatment 

groups. Treatment-related thyroid gland histopathology effects were observed at the high treatment 

group (thyroid gland hypertrophy and follicular cell hypertrophy and hyperplasia). 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID # 48676301) ventral prostate cytosol from 

Sprague Dawley rats was used as the source of AR to examine the ability of chlorothalonil to 

Page 36 of 69
CHLOROTHALONIL FINAL 

Page 29 of 61 

bind to the AR receptor. Competitive binding experiments were performed with chlorothalonil 

(99.0% a.i., Batch# 617642). 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 (logarithmic increase from 10−11 to 10−3

 M) of chlorothalonil. DMSO was used 

as the solvent vehicle at a final assay concentration of approximately 3.2%. A total of three runs 

were performed, and each run included dexamethasone as a weak positive control, and R1881 as 

the ligand reference standard. 

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

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

radiolabeled reference androgen (R1881) prior to routinely conducting AR competitive binding 

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

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

submitted following a request by the Agency. Separate cytosolic preparations, evaluated for 

binding activity at different dates, were used for each of the three competitive binding runs, so 

data from the three saturation binding experiments were compiled. For the first cytosolic 

preparation evaluated on March 30, 2011, the mean dissociation constant (Kd) for [3

H]-R1881 

was 0.614±0.065 nM, and the estimated Bmax was 1.080±0.109 fmol/100 μg protein for the 

single batch of prostate cytosol that was prepared. For the second cytosolic preparation 

evaluated on July 23, 2011, the Kd for [3

H]-R1881 was 1.080 ± 0.690 nM, and the estimated Bmax

was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol that was 

prepared. For the third cytosolic preparation evaluated on September 24, 2011, the dissociation 

constant (Kd) for [3H]-R1881 was 0.613 ± 0.041 nM, and the estimated Bmax was 0.817 fmol/100 

μg protein (±0.049) for the single batch of prostate cytosol that was prepared. The mean and 

individual Kd values for the three experiments were generally below the range reported in the 

EPA validation program (0.685 to 1.57 nM), although two individual values were within the 

range and one value was above the range. Confidence in these numbers is generally high 

according to the goodness of fit (R2

 = 0.880-0.992) and the small variation in R2 and Kd

estimates among runs. 

In the competitive binding experiment, precipitation of chlorothalonil was visually observed at 

the highest tested concentration (10−3 M) in the first run. Therefore, the highest concentration 

used for data evaluation in Run 1 was 10−4

 M, and the concentration range in Runs 2 and 3 was 

10−11 to 10−4

 M. Specific binding was >75% (i.e. <25% displacement of radioligand) at soluble 

concentrations ≤10−4 M in all three runs. An IC50 and relative binding affinity (RBA) could not 

be calculated for chlorothalonil. 

The estimated average log IC50 was −9.3 M for R1881 and −4.4 M for the weak positive control 

(dexamethasone). Compared to R1881, the mean relative binding affinity (RBA) for 

dexamethasone was 0.0022%. Confidence in these numbers is high for dexamethasone due to 

Page 37 of 69
CHLOROTHALONIL FINAL 

Page 30 of 61 

the generally small variation, but there was greater than a 1-log difference for the IC50 of R1881 

in the second run compared to Runs 1 and 3. However, this variance did not appear to affect the 

study results as all performance criteria were met in the three runs.

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

Androgen Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP 19) assay (MRID 48615403), chlorothalonil (99% a.i., Lot # 

617642) in dimethyl sulfoxide (DMSO) at logarithmic concentrations from 10–10 to 10−3

 M was 

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

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

H]-ASDN) for 15 minutes at 37°C to assess the potential of chlorothalonil to 

inhibit aromatase activity. 

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

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

quantified using liquid scintillation counting (LSC). Three independent 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 

the test chemical series (10−10 to 10−3

) with 3 repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.166 to 0.293 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.218 ± 0.050 

nmol·mg-protein−1

·min−1. Activity in the background controls ranged from 0.47% to 0.80% and 

averaged 0.67% of the full activity controls. The response of the full activity controls and 

background controls was acceptable for each run. 

Results for the positive control were generally within the recommended ranges for the top of the 

curve, bottom curve, Hill slope, log IC50, and coefficient of variation for replicates of each 

concentration within runs. Also, the coefficients of variations (%CVs) for replicates of each 

concentration of chlorothalonil within a run were generally within the 15% guideline. For 4-OH 

ASDN, the estimated log IC50 averaged -7.17, and the slope was -0.94. 

For chlorothalonil, aromatase activity averaged 0.205 ± 0.052 nmol·mg-protein−1

·min−1 at the 

lowest tested concentration of 10-10 M and 0.194 ± 0.045 nmol·mg-protein-1·min−1

 at the highest 

successfully tested concentration of 10−4

 M (highest non-precipitating dose in all 3 runs). 

Precipitation was observed in Run 1 at 10−3 M and in Run 2 at 10-3.5 M. The data for 

chlorothalonil were not suitable for modeling. The average lowest portion of the response curve 

across runs was >75% activity (88.75% at 10–4 M chlorothalonil). 

Page 38 of 69
CHLOROTHALONIL FINAL 

Page 31 of 61 

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

of aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

In an estrogen receptor (ER) binding assay (MRID# 48615401), uterine cytosol from SpragueDawley rats was used as the source of ER to conduct binding experiments. A competitive 

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

H]-17βestradiol (1 nM) in the presence of increasing concentrations of chlorothalonil (logarithmic 

increase from 10−10 to 10−3 M or 10−11 to 10− 4 M). DMSO was used as the vehicle at a final 

concentration of 2%. The assay included 19-norethindrone as a weak positive control, 

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

material. 

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

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

following a request by the Agency. Data from two separate saturation binding assays were 

reported due to the use of two separate cytosol preparations during this ER assay for 

chlorothalonil. For the first batch of cytosol evaluated on the September 27, 2010, the Kd for 

[

3

H]-17β-estradiol was 0.081±0.006 nM, and the estimated Bmax was 52.70±1.05 fmol/100 μg 

protein. The second batch evaluated on July 10, 2011 resulted in a Kd for [3H]-17β-estradiol of 

0.303±0.090 nM, and the estimated Bmax was 33.59±0.70 fmol/100 μg protein. The Kd for each 

run was within the expected range of 0.03 to 1.5 nM. Confidence in these numbers is high due to 

the goodness of fit and the small variation among runs. 

The reference compounds performed as expected in the assay. Octyltriethoxysilane had no effect 

on binding, and 17β-estradiol and 19-norethindrone inhibited binding at the expected 

concentrations. The mean log IC50 was −5.6 M for 19-norethindrone and −9.2 M for 

17β-estradiol. The relative binding affinity (RBA) was 0.026% for 19-norethindrone when 

compared to the natural ligand. 

In the competitive binding experiment, chlorothalonil had no effect on specific binding at 

concentrations of ≤10-4 M in Runs 1 and 2, and ≤10-5 in Run 3. Precipitation occurred at 10−3

 M 

in Runs 1 and 2, and at 10−4

 M in Run 3; therefore, data from these chlorothalonil concentrations 

were not used. Chlorothalonil displaced <25% of the radiolabeled estradiol at the highest 

concentrations successfully tested (10−4 or 10−5 M) in the three independent runs. 

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

Estrogen Receptor Binding Assay. 

Page 39 of 69
CHLOROTHALONIL FINAL 

Page 32 of 61 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

In an estrogen receptor transcriptional activation assay (MRID 48615402), hERα-HeLa-9903 

cells cultured in vitro were exposed to chlorothalonil (99.0% a.i., Batch # 617642) at 

logarithmically increasing concentrations from 10−11 to 10−4 M in cell culture media containing 

0.1% DMSO for 24 hours in two independent tests. The experiments were performed using 96-

well plates and each chlorothalonil concentration was tested in 6 replicates/plate. Cells were 

exposed to the test agent for 24±2 hours to induce reporter (luciferase) gene products. Luciferase 

expression in response to activation of the estrogen receptor by chlorothalonil was measured 

using a proprietary luciferase assay. 

Chlorothalonil was tested up to the limit of solubility (10–4 M); however, chlorothalonil was 

cytotoxic at concentrations ≥10–6 M in the first run and at ≥10–5 M in the second run. 

Cytotoxicity was also observed in the first run for 17β-estradiol (10–13, 10–8 and 10–9 M), 

17α-estradiol (10–6 M) and corticosterone (10–4 M) and in the second run for corticosterone 

(10-4 M). The study also included supplemental data evaluating the response of chlorothalonil at 

each concentration (2 replicates/concentration) in the presence of the hERα antagonist ICI 

182,780 (4-hydroxytamoxifen, 1 μM) to identify non-specific luciferase induction. Performance 

criteria for reference chemicals were generally not met, especially in the first run. However in 

most cases, values were lower than validated ranges and suggested increased sensitivity of the 

assay to weak ligands. In the absence of cytotoxicity, the mean RPCMax was 3.0% for the first 

run and 7.6% for the second run, and the associated PCMax was 10–7 M for the first run and 10–5.5

M for the second run. In run 2, a higher RPCMax was achieved (12.5% at 10-5 M) in the presence 

of cytotoxicity. However, both RPCMax were within the variability range for each assay. 

Because the RPCMax < PC10 in at least two assay runs, chlorothalonil was considered negative for 

estrogen receptor transcriptional activation in this test system. The data seems to indicate that 

chlorothalonil is not an ERα agonist under the conditions of this study; however, confidence in 

the results is lessened due to the excess cytotoxicity observed with the reference chemicals. 

Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 48950801) of chlorothalonil with fathead 

minnow (Pimephales promelas) was conducted under flow-through conditions. Groups of adult 

(ca. 6 months old) fish (16 spawning groups of 2 males and 4 females per replicate tank with 4 

replicates per test concentration) were exposed to chlorothalonil (99.0% purity) at nominal 

concentrations of 0 (negative control), 0.000070, 0.00070, and 0.0070 mg a.i./L. The reviewercalculated time-weighted average (TWA) concentrations were below the limit of concentration 

Page 40 of 69
CHLOROTHALONIL FINAL 

Page 33 of 61 

(<LOQ; <0.000028 mg a.i./L, control), 0.000074, 0.00077, and 0.0099 mg a.i./L). The test system 

was maintained at 24 to 26°C and a pH of 6.8 to 7.9. 

Survival at test termination was 100% in the control and 96% in both the low and mid treatment 

groups. Overall survival was decreased (p<0.05) to 79% at the high concentration (males - 63%; 

females - 88%), compared to the control. At test termination, a single bloated female was observed 

in the high treatment group. Chlorothalonil exposure was associated with an increase (4%; p<0.05) 

in male length at the high concentration, but wet weight was not significantly different between the 

control and treatment groups. 

Spawning in controls occurred at least every three days and the mean fecundity in the control was 

24.7 eggs/female/day/replicate (range: 19.8-27.8); fertilization success in controls was 98%. At the 

high concentration, fecundity was significantly decreased (p<0.01) by 48%, and fertility was 

significantly increased (p<0.05) although the difference was minor (99% versus 98% for control). 

There were no significant differences in plasma vitellogenin (VTG) or gonado-somatic index 

(GSI) between control and treated fish for both males and females. Similarly, nuptial tubercle 

score was not significantly different between control and treated males; no nuptial tubercles were 

detected in females. 

Histopathological examinations showed increased testicular generation in male fish in the control 

and low and mid treatment groups. For females, minimal to moderate increased oocyte atresia was 

noted in the control and all treatment groups, though moderate cases were restricted to the mid and 

high concentrations. Oocyte atresia was associated with minimal to mild multifocal granulomatous 

inflammation 53% of the time (9 of the 17 cases); protozoal spores (presumptive microsporidia) 

were observed in two fish. Minimal egg debris in the oviduct was found in two females each in the 

control and high concentration groups. Minimal granulomatous inflammation was observed in the 

control and all treatment groups and ranged in prevalence from 7 to 29% with the highest 

occurrence in the high treatment group. Overall, the study author reported that there were no 

apparent treatment-related patterns in histopathological findings for males or females and that the 

findings were considered background or incidental. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger Assay (MRID 48669902) screening for androgenic activity, chlorothalonil 

(99.0% a.i., Batch # 617642) in aqueous 1% carboxymethylcellulose (CMC) was administered 

daily via oral gavage to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at 

dose levels of 0 (vehicle), 300 or 1000 mg/kg/day (limit dose). An androgenic positive control 

group consisted of six castrated rats exposed to the vehicle (1% CMC by oral gavage) plus 0.2 

mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) injection. 

Page 41 of 69
CHLOROTHALONIL FINAL 

Page 34 of 61 

To screen for potential anti-androgenic activity, chlorothalonil in 1% CMC was administered 

daily via oral gavage to groups of six 54- to 56-day old, castrated male Sprague Dawley rats at 

dose levels of 0 (vehicle), 100, 300 or 1000 mg/kg/day (limit dose) in conjunction with a daily 

dose of reference androgen TP at 0.2 mg/kg/day by s.c. injection. The anti-androgenic positive 

control group consisted of six castrated rats exposed to 3 mg/kg/day flutamide (FT) in corn oil 

via oral gavage and 0.2 mg/kg/day TP (s.c. injection). The positive control animals for the 

androgen agonist assay served as the anti-androgenic negative controls. 

For both components of the assay, body weights and food consumption were determined daily. 

The animals were dosed for 10 consecutive days and terminated approximately 24 hours after the 

final dose administration. At necropsy, the five androgen-dependent tissues and liver were 

collected and weighed. 

One animal in the androgen agonist assay 1000 mg/kg/day group was euthanized in extremis on 

Day 4 following a body weight loss of 33.1 g and essentially no food consumed on Days 3-4. 

The death of this animal was not considered to be test substance related, and all other rats in the 

study survived until scheduled sacrifice. Clinical findings noted for surviving rats in the 

chlorothalonil-treated groups were limited to soft stool, which infrequently occurred (one animal 

from the 1000 mg/kg/day chlorothalonil group and one animal from the 1000 mg/kg/day 

chlorothalonil + TP group). 

In the androgen agonist assay, body weights throughout the study and overall body weight gains 

in the chlorothalonil treated groups were comparable to the vehicle controls. Body weights 

throughout the study in the positive control (vehicle + TP) group were similar to the vehicle 

controls, but overall body weight gains were increased (p<0.05) by 19% compared to the vehicle 

controls. Food consumption was unaffected by the administration of chlorothalonil or TP. There 

were no treatment-related effects in any of the accessory sex organs of chlorothalonil-dosed 

animals. Animals in the positive control (vehicle + TP) group had accessory sex organ weight 

increases (p<0.05) as follows: 351% in seminal vesicles; 628% in ventral prostate; 191% in 

levator ani-bulbocavernosus (LABC); 451% in Cowper’s glands; and 60% in glans penis. 

In the anti-androgen assay, body weights throughout the study and overall body weight gains in 

the chlorothalonil treated groups were comparable to the negative (vehicle + TP) controls. Food 

consumption was unaffected by the administration of chlorothalonil (+ TP). However, the 

overall mean food consumption in the positive control (FT +TP) group was decreased (p<0.05) 

by 9% compared to the negative controls; no corresponding effects on mean body weight gains 

were noted. There were no treatment-related decreases in accessory sex organ weights for all 

treatment groups in the anti-androgen assay. Animals in the positive control (FT + TP) group 

had accessory sex organ weight decreases (p<0.05) as follows: 70% in seminal vesicles; 77% in 

ventral prostate; 45% in LABC; 70% in Cowper’s glands; and 28% in glans penis. 

Page 42 of 69
CHLOROTHALONIL FINAL 

Page 35 of 61 

The high dose of chlorothalonil (1000 mg/kg/day) used in the assays represents the maximum 

dose level recommended in the guidelines. Although one animal was euthanized in extremis on 

Day 4, this was not considered to be treatment related, and no extreme signs of toxicity were 

observed in the surviving animals. Liver weights in the chlorothalonil treated groups were 

comparable to the vehicle/negative controls. The performance criteria indicated that the assays 

were performing as expected. 

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Chlorothalonil was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRID# 48699301), 15 Sprague-Dawley rats/dose group were 

treated daily via oral gavage with chlorothalonil (99.0% a.i., Batch #: 617642) in aqueous 1.0% 

carboxymethylcellulose at doses of 0, 90 or 175 mg/kg/day from post-natal day (PND) 22 to 42 

or 43. The animals were examined for vaginal opening (VO) daily beginning on PND 22, and 

age and body weight at day of attainment were recorded. Following sacrifice on PND 42 or 43, 

blood was collected for clinical chemistry analyses, including thyroxine (T4) and thyroid 

stimulating hormone (TSH) which were analyzed using electrochemiluminescent immunoassays 

or radioimmunoassays, respectively. Liver, kidney, pituitary, adrenals, thyroid, ovaries, and 

uterus were weighed and examined macroscopically. The thyroid, ovaries, uterus, and kidneys 

were examined microscopically. 

No treatment-related effect was observed on survival, clinical signs, age of attainment of VO, 

body weight at VO, or gross pathology. No significant differences were observed in liver, 

thyroid, uterus, or ovary weights of the treated groups compared to controls. No treatmentrelated effect was noted on cycle status at necropsy, mean age at first estrus, mean cycle length, 

percent cycling, or percent regularly cycling. No treatment-related lesions were observed on the 

thyroid, ovary, uterus, or kidney. 

At 175 mg/kg/day, the final body weight was decreased by 11% (p<0.01), and overall 

bodyweight gain was decreased (p<0.05) by 15%. Mean body weights were decreased (p<0.05) 

by 10-16% during PND 41-43. Mean body weight gains were generally comparable to the 

control group from PND 22-40. However, a mean body weight loss (-3.2 g) was noted in this 

group compared to controls (6.5 g) during PND 40-41, followed by lower mean body weight 

gains during PND 41-42 (↓61%) and 42-43 (↓32%). Mean food consumption (g/cage/day) was 

generally similar to the control group during PND 22-40, but decreased mean cage food 

Page 43 of 69
CHLOROTHALONIL FINAL 

Page 36 of 61 

consumption was noted during PND 40-43 (corresponding to lower body weight gains). Body 

weight, body weight gain, and mean food consumption were similar to controls at 90 mg/kg/day. 

Also at 175 mg/kg/day, organ-to-body weight ratios were increased (p<0.01) by 26% in the 

adrenal gland, 19% in kidney, and 15% in the pituitary. Additionally, increased (p<0.01) 

kidney-to-body weight ratio was increased by 10% in the 90 mg/kg/day group. However, 

adjusted (and unadjusted) values in the treated groups did not differ statistically from controls. 

As there were no significant changes in unadjusted and adjusted organ weights, the slight 

changes in relative organ weights are not considered to be biologically significant. 

Serum TSH levels in the treated groups were similar to controls. Serum T4 was increased 

(p<0.05) by 14% at 90 mg/kg/day and by 27% at 175 mg/kg/day. The changes in T4 levels were 

not corroborated by changes in TSH levels, thyroid gland weight, or gross or histological 

changes in the thyroid. 

 

A gas-filled stomach and intestine was noted in one 175 mg/kg/day females, but no other gross 

findings were observed. This finding was considered incidental. 

The doses tested were adequate based on the observed decreases in body weight and body weight 

gain at the high dose. 

Male Pubertal (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID# 48699302), 15 Sprague-Dawley rats/dose group were treated 

daily via oral gavage with chlorothalonil (99.0% a.i., Batch #: 617642) in aqueous 1.0% 

carboxymethylcellulose at doses of 0, 90 or 175 mg/kg/day from post-natal day (PND) 22 to 53 

or 54. Animals were examined for preputial separation (PPS) daily beginning on PND 30, and 

age and body weight at day of attainment were recorded. Following sacrifice on PND 53 or 54, 

blood was collected for clinical chemistry analyses, including selected hormones. Total serum 

testosterone and thyroxine (T4) levels were determined using electrochemiluminescent 

immunoassays, and thyroid stimulating hormone (TSH) levels were determined using a 

radioimmunoassay. Liver, adrenals, pituitary, and urogenital organ were weighed, and 

microscopic examinations were performed on the testes, epididymides, thyroid, and kidneys. 

No treatment-related effects were observed on mortality, clinical signs, body weights, body 

weight gain, food consumption, organ weights, or gross pathology. Age at incomplete PPS in 

the treated groups was similar to controls. Thyroid hormone levels, colloid area and follicular 

cell height scores were similar in the treated groups to the control group. Serum testosterone 

levels were similar in the treated groups to the control group. No treatment-related findings were 

noted in the epididymis, testis, and kidneys. 

Page 44 of 69
CHLOROTHALONIL FINAL 

Page 37 of 61 

At 175 mg/kg/day, age at PPS was decreased (p<0.05) by 1.7 days (43.7 days vs. 45.4 days). 

When a second analysis was performed, adjusting the day of attainment for those males with 3 or 

more days of incomplete separation (persistent threads), there were no statistically significant 

differences between the 175 mg/kg/day group (43.2 days) and the control group (41.8 days). The 

significant effect on complete PPS for the 175 mg/kg/day group was the result of a greater 

number of control animals with incomplete separation (persistent threads). Unadjusted and 

adjusted body weight at PPS was decreased (p<0.05) by 11%. The unadjusted mean body 

weight at PPS (203.8 g) was similar to the minimum mean value in the WIL historical control 

data (203.7 g) and is not considered biologically significant. 

In the kidneys, unadjusted, adjusted, and relative (to body) organ weights were increased 

(p<0.05) at 90 mg/kg/day by 10-12%, and relative weight was increased (p<0.001) by 10% at 

175 mg/kg/day. Relative (to body) liver weights were increased (p<0.05) by 8% at 90 

mg/kg/day and by 7% at 175 mg/kg/day. These effects were considered minor and without dosedependency, and adjusted organ weights were similar to controls in the high dose group. 

Furthermore, no additional evidence of toxicity was observed on the kidney and liver in this 

study. 

Although there was no evidence of an adverse effect in this study, the high dose was considered 

adequate based on decreased terminal body weights of 11% in a 14-Day range finding study 

(MRID 48699303; see Appendix) and the 11% decrease in body weight observed in the 

concurrent female pubertal study (MRID 48699301). Mean terminal body weight at the high 

dose in this study (male pubertal) was decreased by only 3% compared to controls; no 

explanation was provided for this discrepancy with the other stated results. 

Steroidogenesis (OCSPP 890.1550) 

In a steroidogenesis assay (MRID# 48669903), H295R cells cultured in vitro in 24-well plates 

were incubated with chlorothalonil (99.0% purity, Batch # 617642) at concentrations of 100, 10, 

1, 0.1, 0.01, 0.001, and 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 0.05%. 

Precipitation of the test compound was observed microscopically at 100 μM in Runs 2 and 4.

Cytotoxicity greater than 20% was also observed at 10 and 100 μM chlorothalonil in all three 

valid runs; therefore, the results from these wells were not evaluated statistically. Run 1 was not 

analyzed because a forskolin concentration on the quality control (QC) plate was incorrect (3.33 

μM was used instead of 1 μM). 

Testosterone and estradiol levels were measured using HPLC-MS/MS with internal standards. A 

QC plate was run concurrently with each independent run of a test chemical plate to demonstrate 

Page 45 of 69
CHLOROTHALONIL FINAL 

Page 38 of 61 

that the assay responded properly to positive control agents at two concentration levels; positive 

controls included the known inhibitor (prochloraz) and inducer (forskolin) of estradiol and 

testosterone production. 

Guideline acceptability recommendations and requirements were met, including adequate 

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

induction and inhibition with positive controls. 

Chlorothalonil treatment to H295R cells did not affect testosterone or estradiol production. 

Inhibition (p≤0.05) of testosterone production was observed in Run 4 at 0.01 μM (0.89-fold) and 

Run 3 at 1 μM (0.82-fold). Induction (p≤0.05) of estradiol production was noted in Run 2 at 1 

μM (1.09-fold). However, these changes were minor and not reproducible in this study. All 

other values of testosterone and estradiol in the treated cells were similar to the solvent control 

group. 

Based on hormone responses in three independent runs, chlorothalonil treatment did not result in 

statistically significant and reproducible alterations in testosterone or estradiol production. 

Uterotrophic (OCSPP 890.1600) 

In a Uterotrophic Assay (MRID 48669904) conducted to screen for potential estrogenic activity, 

chlorothalonil (99.0% a.i., Batch# 617642) in aqueous 1% carboxymethylcellulose (CMC) was 

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

levels of 0 (vehicle), 300 or 1000 (limit dose) mg/kg/day on post-natal days (PND) 65-67 or 66-

68. A positive control group was treated with a daily dose of 17α-ethynyl estradiol (EE) in corn 

oil at 0.3 mg/kg/day by oral gavage. Body weights and food consumption were determined 

daily. All animals were terminated and necropsied on PND 68 or 69 approximately 24 hours 

after the final dose administration to determine wet and blotted uterine weights. 

All animals survived until scheduled termination and no significant clinical observations were 

noted in any of the animals during the study. Body weights throughout the duration of the study 

and overall (Day 1-4) body weight gains in the chlorothalonil-dosed groups were comparable to 

the vehicle controls. Food consumption was unaffected by the administration of chlorothalonil. 

The overall average food consumption was decreased (37%) in the positive control (EE) group 

and corresponded to the decrease in overall body weight gain; body weights for the EE group 

were decreased (p≤0.01) by 9% on Day 4, and overall body weight gain was decreased (p≤0.001) 

by 208% in comparison with the vehicle controls. 

Page 46 of 69
CHLOROTHALONIL FINAL 

Page 39 of 61 

Uterine weights in the chlorothalonil treated groups (300 and 1000 mg/kg/day) were comparable 

to the vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) 

group were increased (p≤0.01) by 752% and 221%, respectively, as expected. 

No statistically significant changes were seen in uterine weight in this assay. Chlorothalonil is 

negative in the uterotrophic assay. 

Page 47 of 69
CHLOROTHALONIL FINAL 

Page 40 of 61 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)

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

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

breadth of toxicity data available on chlorothalonil, the only Part 158 studies discussed in this 

appendix are those that fulfill current guideline requirements and are directly relevant to assessing 

potential endocrine effects (i.e., multiple-generation rodent reproductive studies, development 

studies, chronic toxicity studies, etc.). 

ER and AR Transcriptional Activation Assays (ERTA & ARTA) 

In a published, non-Guideline study (Kojima et al., 2004), examined 200 pesticides, including 

chlorothalonil (>95-100% purity), for their potential to interact with two human ER subtypes 

(hERα and hERβ) and one human androgen receptor (hAR) in transactivation assays with Chinese 

hamster ovary cells (CHO-K1 cells). The human ER (hERα) and AR (hAR) expression vectors 

(pcDNAERα and pZeoSV2AR, respectively) were constructed as described previously (Kojima et 

al., 2003). The hERβ expression vector was constructed by cloning the hERβ cDNA with reverse 

transcriptase-polymerase chain reaction from human placental RNA. The sequence was verified 

and inserted into the mammalian expression vector pcDNA3.1Zeo(-) creating pcDNAERβ. The 

construction of the estrogen or androgen responsive elements containing reporter plasmid (pGL3-

tkERE and pIND-ARE, respectively) was described previously (Kojima et al., 2003). pRL-SV40 

containing the Renilla luciferase gene was used as an internal control for transfection efficiency. 

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

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

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

10−8

 to 10−5 M, and the responses were compared to the activity of 17β-estradiol (E2, >97% purity) 

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

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

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

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

(REC20), the concentration of the compound demonstrating 20% of the activity of 10−10 M E2, 10−9 

M E2, or 10−9 M DHT for ERα, ERβ, and AR, respectively. Antagonistic activities were evaluated 

similarly by the relative activity expressed as 20% relative inhibitory concentration (RIC20), the 

concentration of the compound showing 20% inhibition of the activity of E2 or DHT against ERα, 

ERβ, and AR, respectively. The range of concentrations tested was ≈10-8 to 10-5 M; all pesticides 

were tested at concentrations ≤ 10-5 M to avoid cytotoxicity. When the activity of the test 

compound was higher than the REC20 the pesticide was judged to be positive for activity. When 

the activity of the test compound was higher than the RIC20 the pesticide was judged to be positive 

for inhibitory activity. 

Page 48 of 69
CHLOROTHALONIL FINAL 

Page 41 of 61 

Chlorothalonil was negative for agonist or antagonist activity against ERα and ERβ, and negative 

for agonist or antagonist activity against AR in this transcriptional activation test system. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction toxicity study (MRID 41706201, 1990), chlorothalonil (98.1% 

purity) was administered to 35 Sprague Dawley rats/sex/dose via the diet at dose levels equivalent 

to 0, 38, 115, and 234 mg/kg/day during the initial pre-mating period of 10 or 14 weeks (P or F1

generations, respectively); with exposure continuing in dams through gestation, lactation and 

weaning. Both the P and F1 generation rats were mated twice to produce the F1a and F1b litters and 

F2a and F2b litters respectively. The parent animals were rested for two weeks between mating 

periods. The standard reproductive study parameters were evaluated. Organ weights were not 

measured. 

There were no treatment related effects on mortality, food consumption, or clinical signs in P or F1

adult animals. In the males, body weights were decreased (p<0.01, ↓7-11%) in the 234 mg/kg/day 

group during all intervals in the P and F1 generations. In the females, there were significant 

(p≤0.05) lower group mean weights (↓5-8%) in the 234 mg/kg/day group during the 10 weeks 

prior to mating (F1a). During gestation for F1a, group mean body weights were decreased by 5-7% 

and mean body weight gains were essentially the same for all 4 groups (117-119 g). For the F1a

lactation period (3 weeks), group mean gains were -1, -3, 13, and 23 g in the control, 38, 115, and 

234 mg/kg/day groups, respectively, so that the group mean final lactation body weights for all 4 

groups was essentially the same (307-317 g). Group mean body weight gains during the 2 week 

rest period between F1a and F1b were decreased (p<0.01) by 7-8% at 234 mg/kg/day compared to 

the controls. 

During the 14 weeks pre-mating for F2a, group mean body weights were decreased (p<0.05) by 6-

10%, 4-9%, and 6-13% in the 38, 115, and 234 mg/kg/day groups, respectively, compared to 

controls. Body weight gains for this period were 209, 190, 186, and 178 g in the control, 38, 115, 

and 234 mg/kg/day groups, respectively. During the F2a gestation period, body weights were 

decreased (p<0.01) by 9-14% in the 234 mg/kg/day group; however, the all treated groups gained 

118-121 g (vs 116 g control). During the F2a lactation period, body weights were decreased 

(p<0.01) by 6-9% in the 234 mg/kg/day animals. The control, 38, and 115 mg/kg/day groups lost 

3-10 g; whereas, the 234 mg/kg/day group gained 12 g. During the rest period, body weights 

remained decreased (p<0.01) in the high-dose group (↓10-11%) compared to controls, and all 

treated groups gained 2 g each (vs 6 g control). Although there were significantly (p<0.01) lower 

body weights in the 234 mg/kg/day group (↓9-13%) during the F2b gestation period compared to 

controls, the weight gained during this 3 week period was 117-124 g for all treated groups (vs 116 

g control). The 234 mg/kg/day group gained 8 g during lactation compared with losses of 16, 12 

and 4 g in the control, 38, and 115 mg/kg/day groups. 

Page 49 of 69
CHLOROTHALONIL FINAL 

Page 42 of 61 

No treatment-related effects were observed in the reproductive parameters (male or female fertility 

indices, gestation index, gestation length, live birth index, viability index, lactation index, 

parturition, or fetal sex ratio). No treatment-related changes were seen in the testes, epididymides, 

seminal vesicle, prostate, ovaries, uterus, or vagina at any dose in either generation. 

The NOAEL for parental toxicity was not attained. The LOAEL for parental toxicity was 38 

mg/kg/day based on kidney and forestomach lesions. The LOAEL for reproductive effects was 

234 mg/kg/day based on decreased neonatal body weights on LD 21. The NOAEL for 

reproductive effects was 115 mg/kg/day. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800) 

In a two-generation reproduction toxicity study (MRID 45710209, 1995), chlorothalonil (99.16% 

purity) was administered continuously in the diet to Crl: CD®BR VF/Plus rats (32 

animals/sex/dose) at dose levels of 0, 400, 1200, or 3000 ppm (equivalent to 0/0, 30.8/34.3, 

92.5/106.0, and 247.5/270.0 mg/kg/day [M/F]). The P and F1 parents were dosed for 10 weeks and 

12 weeks, respectively, before they were mated to produce the F1 and F2 litters. The F1 pups were 

weaned on postnatal day (PND) 21, and 28 pups/sex/group (1 pup/sex/litter as nearly as possible) 

were randomly selected as parents of the F2 generation. 

 

In the parental animals, no treatment-related effects were observed on survival, or food or water 

consumption. In the 3000 ppm P animals, body weight gains during Weeks 0-1 were decreased, 

and food consumption for Week 1 was decreased. Body weight gains were decreased during 

Week 1-10 in the males in this generation, but cumulative food consumption was comparable for 

Weeks 2-10 in both sexes in this generation. In the 3000 ppm F1 males, body weights were 

decreased throughout treatment (Weeks 4-26), resulting in decreased body weight gains during 

pre-mating (Weeks 4-16), that continued to termination (Weeks 17-26). Similarly in the 3000 ppm 

F1 females, body weights were decreased during premating (Weeks 4-16), resulting in decreased 

body weight gains during this period. In the males, food consumption was not affected, while food 

consumption was slightly decreased during Week 1 in the females. 

Treatment-related effects were observed on the kidney at 1200 and 3000 ppm. Adjusted (to 

terminal body weight) kidney weights were increased in both sexes in both generations. Enlarged 

kidney was observed in the P and F1 males compared to controls, and the following microscopic 

pathological findings were noted: i) trace to moderate dilated/basophilic cortical tubules containing 

colloidal material in the P males compared to controls; ii) trace to moderate 

hyperplasia/hypertrophy of the inner cortical tubules in both sexes in both generations compared to 

controls; and iii) trace to minimal dystrophic mineralization at the corticomedullary junction in the 

P females compared to controls. Bright yellow/orange urinary staining and wetter than normal 

feces on the cage tray paper under the cages of the P and F1 parents; and cage sawdust yellow 

stained and wet during lactation phase in the P and F1 females was observed. 

Page 50 of 69
CHLOROTHALONIL FINAL 

Page 43 of 61 

In the 400 ppm and above P and F1 animals, the forestomach was observed to be thickened and 

roughened, compared to controls, with depressions in the epithelial aspect compared to controls. 

At 1200 ppm and above, white areas in the forestomach were noted in the P animals, and the 

limiting ridge of the forestomach was prominent in the F1 animals both compared to controls. In 

the 400 ppm and above P and F1 males and females, trace to moderate hyperplasia and 

hyperkeratosis of the non-glandular epithelium of the stomach was observed compared to controls. 

At 3000 ppm, minimal focal ulceration of the non-glandular epithelium of the stomach was 

observed in the P females, and trace to moderate erosion of the non-glandular epithelium of the 

stomach was noted in the F1 females, both compared to controls. Increases were observed in 

relative kidney weights (↑9-13%) in the 400 ppm P and F1 males. The kidney was enlarged in 3 

males in the P generation and in 7 males in the F1 generation. Microscopically, there was an 

increased incidence of trace to minimal dystrophic mineralization at the corticomedullary junction 

of the kidneys in the P females (34%) when compared to controls (13%). 

No treatment-related changes in absolute or relative weights were seen for parental animals in the 

testes, epididymides, prostate (weighted with seminal vesicle and coagulating gland), ovaries, 

pituitary or adrenal glands of the parental animals. In addition, No treatment-related 

histopathological changes were seen in the testes, epididymides, seminal vesicle, prostate, 

coagulating gland, ovaries, uterus, cervix, vagina, mammary, thyroid, adrenal and the pituitary 

glands at any dose in either generation. 

The LOAEL for parental toxicity was 400 ppm (equivalent to 30.8/34.3 mg/kg/day male/female), 

based on macroscopic and microscopic pathological findings in the stomach- including: thickened, 

roughened, and white areas in forestomach with depressions in the epithelial aspect, and 

hyperplasia and hyperkeratosis of non-glandular epithelium in the stomach, and kidney effects, i.e., 

enlargement, relative weight increases and dystrophic mineralization at the corticomedullary 

junction. The NOAEL was not established. 

In the offspring, no treatment-related effects were observed on post-implantation survival, live 

birth, viability, or lactation indices, on the sex ratio, clinical signs, gross pathology, or microscopic 

pathology. 

At 3000 ppm, pup body weights were decreased during PND 12-21 in the F1 and F2 pups, resulting 

in decreased overall (PND 0-21) pup body weight gains. Litter weights were decreased in the F1

pups during PND 16-21 and in the F2 pups during PND 8-21, resulting in decreased overall litter 

weight gains. Additionally, balano-preputial skinfold cleavage was delayed and vaginal opening 

was delayed compared to controls. However, since no differences were observed in the 

subsequent mating and reproductive performance of these animals, these effects were considered 

equivocal and were attributed to the reduction in body weight gain observed during lactation. This 

Page 51 of 69
CHLOROTHALONIL FINAL 

Page 44 of 61 

assertion is supported by the comparable body weight at attainment of criterion in all treatment 

groups. 

At ≥400 ppm, a dose-dependent increase in the incidence of thickening and/or roughening of the 

forestomach was observed in both generations, and increased incidence and severity of trace to 

moderate hyperplasia and hyperkeratosis of the non-glandular epithelium of the stomach and trace 

to marked sub-epithelial edema in the non-glandular region of the stomach were observed 

compared to controls. Additionally, in the F1 group, trace to moderate inflammatory cells in the 

non-glandular region of the stomach was noted compared to controls. 

The LOAEL for offspring toxicity was 400 ppm (equivalent to 30.8/34.3 mg/kg/day male/female), 

based on thickening and/or roughening of the forestomach with depressions in the epithelial 

aspect, and hyperplasia and hyperkeratosis of the non-glandular epithelium of the stomach. The 

NOAEL was not established. 

No treatment-related effects were observed on estrous cycle length or periodicity, pre-coital 

interval, duration of gestation, or on mating, gestation, or parturition indices. 

The LOAEL for reproductive performance was not observed. The NOAEL for reproductive 

performance was 3000 ppm (equivalent to 247.5/270.0 mg/kg/day males/females). 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In an oral developmental toxicity study (MRID 00130733, 1983), chlorothalonil (98% purity) in 

0.5% methylcellulose was administered daily by oral gavage at a dose volume of 10 mL/kg bw to 

25 female Sprague-Dawley rats/group at dose levels of 0, 25, 100, or 400 mg/kg/day on gestation 

days (GD) 6 through 15. All surviving dams were killed on GD 20; their fetuses were removed by 

cesarean section and examined. 

There were no treatment-related effects on mortality, clinical signs, body weight gain, food 

consumption, or food efficiency observed in the 25 or 100 mg/kg/day groups. In the 400 

mg/kg/day group, there were 3 mortalities; however, the cause of death was not determined. 

Clinical signs of toxicity in this group included: mucoid, loose or white discoloration of the feces; 

brown material around the nose and mouth; matting of the urogenital fur; slight increase in the 

number of animals with hair loss; and 2 animals with a red vaginal discharge occurring between 

GD 12 and 18. During the dosing period, body weight gains were decreased (p<0.01) by 52%, 

food consumption was decreased by 29% and food efficiency was decreased by 33% compared to 

the control. 

There were no effects of treatment on gross pathology at any dose. 

Page 52 of 69
CHLOROTHALONIL FINAL 

Page 45 of 61 

The maternal LOAEL was 400 mg/kg/day based on mortality, clinical signs, decreased body 

weight gains, reduced food consumption, and lower food efficiency during the dosing period. The 

maternal NOAEL was 100 mg/kg/day. 

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation 

sites, implantations/dam, late resorptions, number of live fetuses/litter, fetal sex ratio, or soft tissue 

abnormalities at any dose. Increases in total resorptions and resorptions/dam were observed in the 

400 mg/kg/day dose group with a related increase in post-implantation loss (13.4% treated vs. 

5.2% controls). No effects of treatment were observed on fetal growth. There were no treatmentrelated external, visceral, or skeletal malformations or variations. 

The developmental LOAEL was 400 mg/kg/day based on an increase in total resorptions and 

resorptions/dam with a related increase in post-implantation loss. The developmental NOAEL was 

100 mg/kg/day. 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In an oral developmental toxicity study (MRID 45710207, 1993), chlorothalonil (99.15% purity) 

in 1.0% methylcellulose was administered daily by gavage at a dose volume of 15 mL/kg bw to 25 

female (Crl: CD®[SD] BR VAF/Plus) rats/group at dose levels of 0, 80, 200, or 500 mg/kg/day on 

GD 6 through 15. All dams were killed on GD 20; their fetuses were removed by cesarean section 

and examined. 

There were no effects of treatment on gross pathology. 

At 500 mg/kg/day, one dam was found dead on GD 9. This animal exhibited noisy respiration 

prior to dosing on the previous day, and post-mortem examination revealed red/brown staining of 

the perinasal and perioral regions, enlarged cervical lymph nodes, firm lungs with patchy 

congestion, and gaseous distension of the gastrointestinal tract. Also at the above dose level, two 

dams were killed for humane reasons on GD 12. These dams exhibited loss of body tone, 

noisy/irregular respiration, piloerection, and brown perinasal staining immediately prior to death, 

as well as lethargy, distended abdomen, and yellow stained urogenital region. Post-mortem 

examination revealed enlarged cervical lymph nodes and severe gaseous distension of the 

gastrointestinal tract in both animals; one animal was also found to have a roughened forestomach. 

All other animals survived to study termination. Wet feces were noted on GD 7 or 8 to GD 16, 

post-dosing salivation was observed in 6/22 dams on GD 11/12, and noisy/irregular respiration 

was noted in 2/22 dams on GD 10-16 and 13-19. Body weight gains were decreased (p≤0.05) 

during treatment on GD 6-8 and 6-14 (decreased 19-41%). Body weight gains continued to be 

decreased (p≤0.05) to termination (GD 6-20; decreased 9%) and for the overall (GD 2-20) study 

(decreased 7%; not significant [NS]). Food consumption was decreased (p≤0.05) during GD 6-7 

(decreased 13%), and water consumption was increased (p≤0.05) during GD 8-9, 12-13, 14-15, 

and 18-19 (increased 21-33%). 

Page 53 of 69
CHLOROTHALONIL FINAL 

Page 46 of 61 

The maternal LOAEL was 500 mg/kg/day, based on increased mortality, clinical signs of toxicity, 

decreased body weight gains and food consumption, and increased water consumption. The 

maternal NOAEL was 200 mg/kg/day. 

There were no abortions, premature deliveries, or dead fetuses. No effects of treatment were noted 

on numbers of litters, live fetuses, resorptions (early, late, or complete litter), or on sex ratio or 

post-implantation losses. No effects of treatment were observed on fetal growth. There were no 

treatment-related external, visceral, or skeletal malformations or variations. 

The developmental LOAEL was not observed. The developmental NOAEL was the highest dose 

of 500 mg/kg/day. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In an oral developmental toxicity study (MRID 00047944, 1976), chlorothalonil (≥99% purity) 

was administered daily by gavage (vehicle and dose volume were not reported) to 10 female Dutch 

Belted rabbits/group at dose levels of 0, 1, 2.5, or 5.0 mg/kg/day on gestation days (GD) 6 through 

18. All dams were killed on GD 28; their fetuses were removed by cesarean section and examined. 

No significant adverse effects were evident at 1.0 mg/kg/day. Dose dependent maternal toxicity 

was evident at ≥2.5 mg/kg/day in the form of one death and one abort at 2.5 mg/kg/day and 2 

deaths and four aborts at 5 mg/kg/day. 

The maternal LOAEL was 2.5 mg/kg/day, based on mortality and abortion. The maternal NOAEL 

is 1.0 mg/kg/day. 

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation 

sites, implantations/dam, early or late resorptions, number of live fetuses/litter, fetal sex ratio, or 

soft tissue abnormalities at any dose. 

The developmental LOAEL was not observed. The developmental NOAEL was 5.0 mg/kg/day. 

Developmental Toxicity (Rabbit, OCSPP 870.3700)

In an oral developmental toxicity study (MRID 45710208, 1994), chlorothalonil (99.15% purity) 

in 1.0% (w/v) aqueous methylcellulose was administered daily by gavage at a dose volume of 5 

mL/kg bw to 16 female New Zealand White rabbits/group at dose levels of 0, 5, 10 or 20 

mg/kg/day on gestation days (GD) 6 through 18. All does were killed on GD 29; their fetuses 

were removed by cesarean section and examined. 

No effects of treatment were observed on mortality, clinical signs, body weights, body weight 

gains, food consumption, or gross pathology. 

Page 54 of 69
CHLOROTHALONIL FINAL 

Page 47 of 61 

At 5 mg/kg/day, one doe died prior to dosing on GD 8. One 10 mg/kg/day female aborted her 

litter on GD 29; this animal exhibited cold ears during dosing, and anorexia/few feces, cold ears, 

red material/blood on tray paper, and no feces post-dosing. One 20 mg/kg/day aborted her litter on 

GD 25-26; this animal exhibited anorexia/few feces prior to dosing, anorexia/few feces, cold ears, 

and no feces during dosing, and anorexia/few feces, cold ears, dark eyes, no feces, fouled 

anogenital region, nasal exudate, soft/liquid feces, and thin appearance post-dosing. It was stated 

that similar findings were not observed in a preliminary developmental toxicity study in rabbits, 

and in view of the low incidence of animals aborting prior to terminal sacrifice, these findings are 

considered to be incidental to treatment. 

The maternal LOAEL was not observed. The maternal NOAEL was 20 mg/kg/day. 

No effects of treatment were noted on numbers of litters, live fetuses, resorptions (early, late, or 

complete litter), or post-implantation losses. Fetal growth was unaffected by treatment. 

There were no treatment-related external, visceral, or skeletal malformations. 

At 20 mg/kg/day, increased (p≤0.05) incidence of thirteen ribs, a variant, was observed (75.0% 

fetuses; 100% litters) compared to concurrent controls (49.2% fetuses; 93.3% litters), and reduced 

sternebrae, a variant, was observed (17.8% fetuses; 76.9% litters) compared to concurrent controls 

(8.3% fetuses; 40.0% litters). In the absence of historical control data, these findings are 

considered treatment-related. 

The developmental LOAEL was 20 mg/kg/day based on increased incidence of 13th ribs and 

reduced sternum. The developmental NOAEL was 10 mg/kg/day. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 00146945, 1985), groups of 60 

Fischer 344 rats/sex were fed diets containing chlorothalonil (98.1% purity) at levels equivalent to 

doses of 0, 40, 80, or 175 mg/kg/day for 116 (males) or 129 (females) weeks. 

Survival was comparable in all groups, both sexes, for the first 24 months. Continuation on study 

decreased survival in the 178 mg/kg/day males resulting in all males sacrificed at 27 months. 

Females were terminated on schedule at 30 months. The major cage-side clinical observation 

included dark yellow urine in the 175 mg/kg/day males and females from Weeks 27-91. An 

increased brown, red staining around the anogenital region of the ≥80 mg/kg/day females was also 

observed. There was a significant body weight decrease (↓10-29%) in both sexes at 175 

mg/kg/day throughout study, as well as a 5-12% body weight decrease in both sexes at 80 

mg/kg/day. There was no body weight reduction in either sex at 40 mg/kg/day. Food 

consumption was unaffected, except for an increase in high dose animals, generally towards the 

last half of the study. 

Page 55 of 69
CHLOROTHALONIL FINAL 

Page 48 of 61 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries or 

adrenal glands at any dose level. No treatment-related histopathological lesions were seen in the 

testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thyroid, adrenal and the pituitary 

glands at any dose level. 

Parameters measured which were compound related and associated with the effects on the kidneys 

included increased BUN and serum creatinine in 175 mg/kg/day males and females, decreased 

serum albumin and serum glucose in 175 mg/kg/day males and females, increased urine volume 

and decreased specific gravity in all treated males throughout the study, and in all treated females 

initially (first year), but in 175 mg/kg/day females only, after the first year. Relative kidney 

weights were significantly increased in all treated males and in the ≥80 mg/kg/day females. 

Relative liver weight was affected in the same groups, being significantly increased in all treated 

males, and the ≥80 mg/kg/day females. Gross necropsy of' all animals demonstrated a compound 

related effect on the kidneys and stomach. In all treated male groups and the 175 mg/kg/day 

female group there were kidney masses and/or nodules as well as increased granularity of' the 

surface of' the kidneys (the latter observed in all dose groups). There were increased incidences of' 

erosions and ulcerations in the non-glandular stomach of all treated rats as well as a significant 

increase in discoloration of' the mucosa in 175 mg/kg/day males. 

 

Histologically there was evidence of compound related effects on the kidneys, esophagus, stomach 

and duodenum. Non-neoplastic changes in the kidney included: chronic glomerulonephritis which 

increased in severity in a dose-related manner in all groups; dose related increase in cortical 

tubular hyperplasia in all dosed rats; increased incidence of' tubular cysts in all dosed rats; and 

increased incidence in dosed males only of' hyperplasia of the papillary/pelvic epithelium. Other 

changes included increased hyperplasia/hyperkeratosis of' the squamous mucosa of the esophagus 

(all dose groups); increased mucosal hypertrophy of the duodenum (all dose groups); 

hyperplasia/hyperkeratosis of the parathyroid (all dosed male and 175 mg/kg/day females, 

considered a secondary lesion as a result of severe chronic renal disease); increased 

hyperplasia/hyperkeratosis of the squamous mucosa of all dose groups; increased incidences of 

foci of necrosis or ulcers in the glandular stomach of all dose groups; increased incidence of 

suppurative prostatitis in all male dose groups (considered associated with treatment related renal 

lesions); complete involution of the thymus was increased in 175 mg/kg/day males and all female 

dose groups. Interesting inverse dose related changes included: chronic interstitial prostatitis 

(increased in control and 40 mg/kg/day males); increased incidence of medullary tumors of the 

adrenal (control and 40 mg/kg/day females); increased incidences of' osteosclerosis of the femur 

and sternum (control females); and an increased incidence of basophilic cell focus/foci of the liver 

(control females, a common finding in aging Fischer 344 rats). 

Neoplastic changes associated with treatment were observed in kidneys and stomach 

(forestomach). Tubular adenomas and carcinomas, aneplastic renal carcinomas and transitional 

cell carcinomas were observed in the kidney of treated rats only, being statistically significant in 

Page 56 of 69
CHLOROTHALONIL FINAL 

Page 49 of 61 

all treated rats except the 40 mg/kg/day females. There was also a possible decrease in time to 

tumor in 175 mg/kg/day rats for renal adenomas and carcinomas. There was no evidence however, 

that the occurrence of' cortical tubular hyperplasia or tubular cysts predisposed animals to such 

tumors since only 1/11 tumor bearings low dose rats also had a tubular cyst. This was also true at 

the higher doses as well. 

Papillomas and carcinomas of' the squamous mucosa or the stomach were present in treated rats 

only, but statistically significant in the 175 mg/kg/day females only. There was no evidence that 

hyperplasia or hyperkeratosis predisposed the animals to such tumors as the degree or severity or 

hyperplasia/hyperkeratosis varied from slight to marked in these animals, and was equally 

prevalent in non-tumor bearing rats, although the severity of response increased in a dose related 

manner. 

The LOAEL was 40 mg/kg/day based on renal adenomas and carcinomas in both sexes. The 

NOAEL was <40 mg/kg/day. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300) 

In a combined chronic toxicity/carcinogenicity study (MRID 45710212, 1996), Chlorothalonil 

(99.28% purity) was administered in the diet for 2 years to 50 Crl:CD (SD) BR rats/sex/dose at 

doses of 0, 15, 60, 240, and 1200 ppm; these dietary levels were equivalent to 0, 0.7, 2.7, 10.6 and 

54 mg/kg/day in males and 0, 0.9, 3.3, 13.9 and 70 mg/kg/day in females). Additionally, 20 

rats/sex/dose were treated similarly and sacrificed at 12 months. 

No treatment-related effects were observed on mortality, food and water consumption, food 

efficiency, ophthalmoscopic examinations, hematology, and non-protein thiol concentration in 

liver. 

At 1200 ppm, an increased incidence of generalized yellow staining of the fur was observed in the 

interim and terminal groups (66-100% treated vs 0% controls). This sign was observed generally 

from Week 16 in males and Week 19 in females. Body weight gain was decreased (p<0.05) by 15-

17% in the females during Weeks 0-1, 13-26, and 26-52; however, overall (Weeks 0-104) body 

weight gain was similar to controls, indicating recovery during the second year. 

At 1200 ppm, nephrotoxicity was evident. Urinary protein concentration was increased (p<0.05) 

by 137-279% in the males at Weeks 52, 78, and 104. Adjusted (for terminal body weight) kidney 

weights were increased (p<0.05) in the males at Weeks 52 and 104 (↑21-28%). Necropsy of the 

males, revealed uniform cortical scarring of the kidneys at the interim (15% treated vs 0% 

controls) and terminal (44% treated vs 14% controls) sacrifices. Kidney enlargement in the males 

(44% vs 20%) was also observed at the terminal necropsy. At the terminal sacrifice, there was an 

increased incidence of minimal to marked progressive glomerulonephrosis in the males (70% vs 

52%) and the females (40% vs 26%). 

Page 57 of 69
CHLOROTHALONIL FINAL 

Page 50 of 61 

At necropsy following the interim sacrifice, the forestomach in the ≥240 ppm groups was 

thickened, roughened, and white. Trace to moderate epithelial hyperplasia and hyperkeratosis in 

the non-glandular region and in the limiting ridge of the stomach was observed in these animals. 

At necropsy following the terminal sacrifice, the forestomach had depressions in the ≥60 ppm 

groups; and was thickened, roughened, and white in the ≥240 ppm groups; and limiting ridge 

thickened or prominent in the 1200 ppm males. There was an increased incidence in the following 

lesions in these animals: (i) epithelial hyperplasia and hyperkeratosis in the nonglandular region of 

the stomach in all treated animals; (ii) epithelial hyperplasia and hyperkeratosis in the limiting 

ridge of the stomach in ≥240 ppm males or the 1200 ppm females; (iii) stomach ulceration in the 

nonglandular region in the ≥60 ppm females; and (iv) submucosal fibrosis and inflammatory cells 

in the nonglandular region of the stomach in ≥60 ppm females. 

No treatment-related changes were seen in absolute or relative weights of the testes, ovaries, 

adrenal and pituitary glands at any dose level. At Week 52, adjusted thyroid weight was increased 

(p<0.05) by 19% in females at the high dose; however, no difference in thyroid weight was seen at 

Week 104, or in males at any dose or interval. 

No treatment-related histopathological lesions were seen in the testes, epididymides, seminal 

vesicle, prostate, ovaries, uterus, vagina, mammary, adrenal or pituitary glands at any dose level. 

In male rats, there was a non-statistically increase in the incidences of thyroid C-cell adenomas at 

2.7 mg/kg/day (2%), 10.6 mg/kg/day (2%) and 54 mg/kg/day (4%) compared to control males (0% 

). Thyroid C-cell carcinomas were increased only at the 10.6 mg/kg/day (4%) compared to control 

males (2%). There were no corroborative non-neoplastic lesions or changes in the absolute or 

relative weight of the thyroid in males. It is important to note that C-cell tumors arise from 

parafollicular cells that secrete calcitonin and not involved in thyroid hormone regulation. No 

treatment-related lesions were seen in females. 

The LOAEL was 15 ppm (0.9 mg/kg/day) in females based on an increased in the incidence and 

severity of epithelial hyperplasia, hyperkeratosis and ulceration of the nonglandular region of the 

stomach females, and 60 ppm (2.7 mg/kg/day) based on an increase in the incidence and severity 

of epithelial hyperplasia and hyperkeratosis of the nonglandular region of the stomach in males. 

The NOAEL was <15 ppm (0.9 mg/kg/day) in females and 15 ppm (0.7 mg/kg/day) in males.

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 00127858, 1983), groups of 60 CD-1 mice/sex were fed diets 

containing chlorothalonil (97.7% purity) at 0, 750, 1500, or 3000 ppm for approximately 24 

months, equivalent to doses of approximately 113, 225, and 450 mg/kg/day, respectively. 

Page 58 of 69
CHLOROTHALONIL FINAL 

Page 51 of 61 

No effects of treatment were observed on clinical signs, body weights, or food consumption. The 

number of mortalities was increased in the 450 mg/kg/day males (39/60 treated vs 29/60 controls). 

Mortality in all other groups was similar to controls. 

At 450 mg/kg/day, decreased hemoglobin, hematocrit and red cell values were reported in the 

males at 24 months and in the females at 12 and 24 months. Hyperplastic bone marrow was 

reported in all treatment group males and females. Hyperplasia of the splenic red pulp was noted 

in the male treatment groups. Hemosiderosis was not a prominent finding in this study. 

Relative spleen weights were reported in the high-dose females; however, no significant pathology 

was reported. Spleen enlargement occurred in the ≥225 mg/kg/day males. Ovarian weight ratios 

were decreased in all treatment groups; however, no histopathological findings were associated 

with this. The same was true for the relative testes weights in the 450 mg/kg/day males. Kidney 

weights were significantly increased (p<0.01) in all treatment groups. This finding was considered 

to be treatment-related. No treatment-related changes were seen in absolute or relative weight of 

adrenal glands at any dose level. No treatment-related histopathological lesions were seen in the 

testes, seminal vesicle, prostate, preputial gland, ovaries, uterus, mammary, thyroid, adrenal, and 

pituitary glands at any dose level. 

Compound related effects in the kidney were described as renal enlargement, discoloration, surface 

irregularities, pelvic dilation, cysts, nodules and masses in all treatment groups. 

In the stomach, the incidence and severity of hyperplasia and hyperkeratosis of the esophageal 

squamous mucosa in treated males and females was significant and was considered to be doserelated. This was not seen control animals. There was a significant increase in gastric squamous 

cell tumors in the 450 mg/kg/day females but this was not dose related. Glandular epithelial 

tumors were present in the treated groups but not in statistically significant numbers. 

In the kidney, chronic glomerulonephritis was seen in all groups, but the incidence was not 

significantly different among them, although it was higher in the 450 mg/kg/day males. Increased 

tubular degeneration was noted in the ≥225 mg/kg/day males and in the 450 mg/kg/day females. 

Increased incidences of cortical cysts were seen in all treated males and in the 450 mg/kg/day 

females. 

Adenomas and carcinomas of the cortical tubules were increased in all treated males but not in 

females. The only neoplasm seen in the females was one renal hemangiosarcoma in a 113 

mg/kg/day female. 

The LOAEL was 113 mg/kg/day based on increased incidence of adenomas and carcinomas of the 

cortical tubules in the kidneys of males. The NOAEL was not established. 

Page 59 of 69
CHLOROTHALONIL FINAL 

Page 52 of 61 

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 45710211, 1995), chlorothalonil (99.28% purity) was 

administered in the diet to Crl:CD(SD)BR mice (50/sex/dose) at concentrations of 0, 15, 60, 240, 

or 960 ppm for up to 18 months. These dietary concentrations were equivalent to 0, 1.9, 7.8, 30.4 

and 130 mg/kg/day in males and 0, 2.5, 9.9, 40.6 and 157 mg/kg/day in females. 

No treatment-related effects were observed on mortality, clinical signs, body weights, body weight 

gains, food consumption, food utilization, or hematology. No treatment-related changes were seen 

in absolute or relative weights of the testes, uterus or ovaries at any dose level. No treatmentrelated histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, 

ovaries, uterus, vagina, mammary glands, thyroid, or pituitary glands at any dose level. 

Minimal to moderate epithelial hyperplasia of the nonglandular region of the stomach in all treated 

male groups (23-42 vs 10 control) and epithelial hyperplasia (limiting ridge) of the stomach in all 

treated male groups (13-32 vs 3 control) was observed. Minimal to moderate epithelial 

hyperplasia and of the nonglandular region of the stomach was observed in ≥60 ppm females (27-

42 vs 9 control). Folding of the epithelium of the nonglandular region of the stomach was 

observed in the ≥60 ppm males (16-18 vs 3 control). Hyperkeratosis of the nonglandular region of 

the stomach was observed in the ≥240 ppm males (39-45 vs 23 control) and ≥240 ppm females 

(45-47 vs 26 control). At ≥60 ppm in the kidney, increased (p≤0.05) incidences (# affected/50 in 

treated vs controls) of the following histological lesions were observed in males: (i) minimal to 

moderate basophilic cortical tubules vs minimal in the controls (23-32 vs 12 control); (ii) cystic 

atrophic glomerulus with hypertrophic parietal epithelium (13-22 vs 4 control); and (iii) foci of 

mineralization in the cortex (17-19 vs 9 control; not statistically significant at 240 ppm). The 

increase in this spectrum of lesions indicates a treatment-related effect that is considered adverse 

because it suggests an acceleration of the natural, age-related degeneration of the kidney. 

Urinalysis or PAS staining (evaluating the integrity of the basement membrane) could have 

provided further information regarding the impact of these lesions on renal function. 

At 960 ppm, eosinophilic droplets in keratin was observed in the stomach (7 vs 0) of males; and an 

increased incidence of enlarged kidneys were observed in males, and in uniform cortical scarring 

of the kidneys in both sexes. Additionally, the adjusted kidney weight was increased (p≤0.01) in 

both sexes. In the 960 ppm males, increased incidences of sinus histiocytosis and 

erythrophagocytosis were observed (p≤0.05) in the mesenteric lymph node. Focal epithelial 

hyperplasia of the nonglandular region of the stomach (7 vs 0) and papillomatous hyperplasia 

(nonglandular region and limiting ridge) was observed in the 960 ppm females (5 vs 0, each 

region). 

The LOAEL was 15 ppm (equivalent to 1.9 mg/kg/day) in males, based on epithelial hyperplasia 

of the nonglandular region of the stomach and epithelial hyperplasia (limiting ridge) of the 

Page 60 of 69
CHLOROTHALONIL FINAL 

Page 53 of 61 

stomach. The NOAEL was < 15 ppm (equivalent to 1.9 mg/kg/day) in males. The LOAEL was 

60 ppm (equivalent to 9.9 mg/kg/day) in females based on epithelial hyperplasia and of the 

nonglandular region of the stomach. The NOAEL was 15 ppm (equivalent to 2.5 mg/kg/day) in 

females.

Chronic Toxicity (Dog, OCSPP 870.4100) 

In a chronic toxicity study (MRID 43653603, 1994), chlorothalonil (98.3% purity) was 

administered orally by gelatin capsules to Beagle dogs (5/sex/group) at doses of 0, 15, 150 or 500 

mg/kg/day for 52 weeks. 

There were no effects on mortality, clinical signs, food consumption, ophthalmology, hematology, 

urinalysis, or macroscopic pathology. No treatment-related effects were observed on organ 

weights, including absolute or relative testes weight at any dose level. No treatment-related 

histopathological lesions were observed in the testes, epididymides, prostate, ovaries, uterus, 

mammary, thyroid, adrenal and pituitary glands at any dose level. 

There were decreases in body weight gains in males and females at 500 mg/kg/day. Effects on 

clinical chemistry were limited to decreased alanine aminotransferase levels (↓90%) in all dosed 

animals compared with controls as well as with pretreatment levels. 

The LOAEL was 500 mg/kg/day based on decreased body weight gains in both sexes. The 

NOAEL was 150 mg/kg/day. 

Chronic Toxicity (Dog, OCSPP 870.4100) 

In a chronic oral toxicity study (MRID 45710210, 1995), 4 beagle dogs/sex/dose were given 

chlorothalonil (99.28% purity) in the diet at nominal concentrations of 0, 160, 1280, or 10,240 

ppm for 52 weeks. These dietary concentrations were equivalent to doses of 0, 5.10, 43.26 and 

374 mg/kg/day in males and 0, 5.92, 45.30 and 354 mg/kg/day in females. 

There were no effects of treatment on ophthalmoscopy, hematology, or urinalysis. No treatmentrelated changes were seen in absolute or relative weights of the testes, epididymides, prostate, 

ovaries, uterus, thyroid (except in males), adrenal or pituitary glands at any dose level. No 

treatment-related histopathological lesions were seen in the testes, epididymides, prostate, ovaries, 

uterus, vagina, mammary, thyroid, and pituitary glands at any dose level. 

Vasodilatation of the gums and/or ears was noted in all of the 10,240 ppm males and females 

compared to 0 controls, with males affected for 33-84 days and females affected for 30-191 days. 

With the exception of a single female, this effect tapered off after 26 weeks. 

One 10,240 ppm female (#370) was killed for humane reasons on the first day of Week 31, and the 

death was considered to be treatment-related. The following were noted in this animal prior to 

Page 61 of 69
CHLOROTHALONIL FINAL 

Page 54 of 61 

death: vasodilatation of the gums later followed by paleness of gums; decreased body weight, food 

consumption, red cell indices, and albumin; reticulocytosis; and increased total globulin. Postmortem examination revealed microscopic changes in the heart, spleen, bone marrow, stomach, 

liver, and kidneys. 

At 10,240 ppm, body weights were decreased (decreased 4-11%) in the males beginning at Week 

2, resulting in significantly decreased (p<=0.05) cumulative body weight gains beginning at Week 

8 (decreased 36-47%) and an overall (Weeks 0-52) body weight gain decrease of 44% compared to 

controls. In the females at this dose, cumulative body weight gains were decreased (p≤0.01) early 

in treatment, with decreases of 50% compared to controls at Week 4 and decreases of 38% at 

Week 8. However, overall body weight gains were comparable to controls. 

At 10,240 ppm, weekly food consumption was decreased during the first six months of the study in 

the males and generally throughout treatment in the females, with greater decreases in the females 

(decreased ≤18% compared to controls) than in the males (decreased ≤8%). Average food 

consumption for the overall study was decreased in both sexes by 2% compared to controls, with 

the difference attaining significance (p≤0.05) in the females. 

Cholesterol was increased (increased 7-48%) in the ≥1280 ppm males throughout treatment. Total 

protein was decreased (decreased 8-14%; p≤0.05) at Weeks 39 and 52 in the ≥1280 ppm males and 

in the 10,240 ppm females. Albumin was decreased (decreased 7-20%; p≤0.05) at 10,240 ppm in 

both sexes throughout treatment. Alkaline phosphatase was increased (increased 39-68%) 

throughout treatment in the 10,240 ppm males. 

Alanine aminotransferase (ALT) was severely decreased (p≤0.01) in all treated groups of both 

sexes, with average values of <1 to 3 mU/mL in the treated groups compared to 18-27 mU/mL in 

the controls. It was stated that normal ALT levels were attained when a pre-incubation stage was 

incorporated into the assay using pyridoxal-5'-phosphate (IFCC reagent) and that the initial 

decreases were attributed to a decrease in pyridoxal-5'-phosphate, a cofactor necessary for the full 

expression of ALT in addition to the metabolism of cysteine conjugates of β-lyase. The reduction 

in ALT activity was therefore considered not to be an indication of toxicity but a consequence of 

pyridoxal-5'-phosphate depletion by β-lyase during the metabolism of chlorothalonil. However, no 

data were provided for the levels measured from the assay incorporating this pre-incubation stage 

with pyridoxal-5'-phosphate. 

Non-protein thiol concentration was increased (p≤0.05) compared to controls in the: (i) liver and 

kidneys of the 10,240 ppm males (increased 30-40%); (ii) kidneys in the ≥160 ppm females 

(increased 30-80%); (iii) liver in the >=1280 ppm females (increased 60%); and (iv) stomach in 

the 10,240 ppm females (increased 20%). It was stated that these increases are likely due to the 

conjugation of chlorothalonil with glutathione in these organs. 

Page 62 of 69
CHLOROTHALONIL FINAL 

Page 55 of 61 

At ≥1280 ppm, absolute liver weights in the males and absolute and adjusted liver weights in the 

females were increased (increased 9-21%), with the adjusted values in the females attaining 

significance. Increased absolute (increased 27%) and adjusted (increased 47%; p<=0.05) thyroid 

weights were noted in the 10,240 ppm males. However, there were no macroscopic or 

microscopic findings to corroborate an effect of treatment in either of these organs. 

In males, an increased incidence of minimal hypertrophy of the cells of the zona fasciculata of the 

adrenals was seen at the low (2/4) and mid (2/4) dose group dogs compared to controls males 

(114). All high dose males (4/4) had moderate hypertrophy of the cells of the zona fasciculata. 

Adrenal lesions were not seen in females. 

The Pathology Working Group concluded that changes were limited to the dogs fed at 10,240 ppm, 

with the exception of a slight increase in brown pigment in cortical tubules of the kidney in dogs at 

1,240 ppm. All treatment-related changes were minor in severity considering the magnitude of the 

dose and length of exposure. They consisted of a small increase in the incidence and severity of 

brown pigment in the tubular epithelium of the renal cortex, a slight increase in severity (but not in 

incidence) of intraepithelial nuclear pyknosis in the mucosal epithelium of the antrum of the 

stomach, and a very slight increase in the incidence and severity of hypertrophy of the cells in the 

zona fasciculata of the adrenal glands. Changes considered to be the result of treatment were not 

observed in the stomach or adrenals of male and female dogs receiving 1,280 or 160 ppm 

chlorothalonil in the diet for 52 weeks. 

The LOAEL was 10,240 ppm (equivalent to 374/354 mg/kg/day in M/F) based on death in a 

female, decreased body weight gain and food consumption, macroscopic and microscopic 

pathological findings in the stomach including: thickened appearance of the stomach, often with a 

catarrhal adhesion to the mucosal surface, and intra-epithelial nuclear pyknosis in the mucosal 

epithelium of the antrum of the stomach and a very slight hypertrophy of the cells in the zona 

fasciculata of the adrenal glands. The NOAEL was 1280 ppm (equivalent to 43.3/45.3 mg/kg/day 

in M/F). 

Subchronic Toxicity (Rat, OCSPP 870.3100) 

In a subchronic oral toxicity study (MRID 45710205, 1994), 10 Crl:CD (SD) BR rats/sex/dose 

were exposed to Chlorothalonil (99.18% purity) in the diet at nominal concentrations of 0, 30, 60, 

300, or 1500 ppm (equivalent to 0/0, 2.3/2.7, 4.7/5.5, 23.6/28.8, and 117/130 mg/kg/day in 

males/females) for up to 3 months. 

No treatment-related adverse effects were observed on mortality, clinical signs, body weight, food 

or water consumption, food efficiency, ophthalmoscopic examination, hematology, or clinical 

chemistry. No treatment-related changes were seen in absolute or relative weights of the testes, 

ovaries, uterus, thyroid, adrenal and pituitary glands. No treatment-related histopathological 

Page 63 of 69
CHLOROTHALONIL FINAL 

Page 56 of 61 

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

vagina, mammary, thyroid, adrenal and pituitary glands. 

Body weight gain was decreased (p<0.05) by 27-36% in the 1500 ppm group during the first week 

of treatment which was attributed to a palatability problem. The forestomach was roughened in all 

animals in the ≥300 ppm groups. The forestomach was also thickened in the 1500 ppm males (10 

treated vs 0 controls) and females (4 treated vs 0 controls). The incidences of minimal to moderate 

epithelial hyperplasia and hyperkeratosis observed at the limiting ridge and the non-glandular 

region of the forestomach were increased in the ≥300 ppm groups (7-10/10 treated vs 0/10 

controls). The incidence of minimal to moderate hyperplasia of the duodenum was increased in 

the 1500 ppm group (3/10 each treated vs 0/10 controls). The incidence of minimal epithelial 

hyperplasia and hyperkeratosis observed at the limiting region of the forestomach was also 

increased in the 30 and 60 ppm males and the 60 ppm females (4-5/10 treated). 

The LOAEL was 30 ppm (equivalent to 2.3/2.7 mg/kg/day [M/F]) based on minimal epithelial 

hyperplasia and hyperkeratosis observed at the limiting ridge and/or non-glandular region of the 

forestomach in males and females. The NOAEL was < 30 ppm (equivalent to < 2.3/2.7 mg/kg/day 

[M/F]). 

Subchronic Toxicity (Dog; OCSPP 870.3100) 

In a subchronic oral study (MRID 45710206, 1994), chlorothalonil (99.18% purity) was 

administered to 4 beagle dogs/sex/dose in the diet at nominal concentrations of 0, 160, 1600, or 

16,000 ppm for13 weeks. These dietary levels were equivalent to 0, 5.6, 56.5 and 597 mg/kg/day 

in males and 0, 6.1, 61.0 and 570 mg/kg/day in females. 

There were no treatment-related effects on survival, hematology, ophthalmoscopy, or gross 

pathology. No treatment-related changes were seen in absolute or relative weights of the testes, 

prostate, ovaries, uterus, thyroid or pituitary glands. No treatment-related histopathological lesions 

were seen in the testes, epididymides, prostate, ovaries, uterus, mammary, vagina, thyroid and 

pituitary glands. 

At 16,000 ppm, body weights were decreased throughout treatment in both sexes, resulting in a 

decrease of 167-188% (p≤0.01) in overall body weight gains. Weekly food consumption was 

decreased by 3-29% in the females throughout treatment, resulting in decreased mean overall food 

consumption of 14%. 

At ≥1600 ppm, in the liver, minimal foci of necrotic hepatocytes with inflammatory cell 

infiltration was observed in the males (1-2/4 vs. 0 controls). Also, in the liver at ≥1600 ppm, 

minimal parenchymal foci of inflammatory cells was noted in both sexes (1-3/4). Adjusted liver 

weights were increased by 23-25% (p≤0.05) in the females at these doses. Albumin was decreased 

Page 64 of 69
CHLOROTHALONIL FINAL 

Page 57 of 61 

(p<=0.05) by 5% in both sexes at 1600 ppm at Week 13 and additionally by 13-21% at 16,000 

ppm at Weeks 6 and 13. 

At ≥1600 ppm, trace to minimal brown pigment was observed (vs 0 controls) in the epithelium of 

the cortical tubules of the kidneys in both sexes (1 to 3 of 4 dogs). Thiol concentration was 

decreased prior to alkali hydrolysis in the males at this dose (decreased 64-100%; p≤0.05). 

Additionally at 16,000 ppm, urinalysis showed increased protein in the females (incr. 91%; p≤

0.01), and thioether concentration (per μmole creatinine) was increased in the males for the 0-8 

hour urine sample (increased 69%; p≤ 0.01). 

In the adrenals at 16,000 ppm, increased width of the zona glomerulosa was noted in the females 

(3 of 4 dogs vs 0 controls), and absolute adrenal weights were increased (increased 21%; p ≤ 0.05) 

in the males. 

The LOAEL for this study was 1600 ppm (equivalent to 56.5/61.0 mg/kg/day in M/F) based on 

minimal foci of necrotic hepatocytes with inflammatory cell infiltration in males, and minimal 

parenchymal foci of inflammatory cells in both sexes. The NOAEL was 160 ppm (equivalent to 

5.6/6.1 mg/kg/day). 

Avian Reproductive Toxicity (OCSPP 850.2300) 

In an avian reproduction study with the mallard duck (Anas platyrhynchos), (MRID 00041441 

1976), birds of unspecified age were administered chlorothalonil (99.6% purity) in the feed at 

nominal concentration of 0, 5, or 50 mg/kg-diet. Five pens were used at each test level with 2 

males and 5 females per pen. There were no significant effects (p>0.05) to any reproductive 

parameters examined in the study up to and including the 50 ppm concentration.. It was not noted 

whether any mortality occurred in the study. 

In an avian reproduction study with the mallard duck (Anas platyrhynchos), (MRID 4096402, 

1988), birds were administered chlorothalonil (98.3%) in the feed at nominal concentration of 

1000, 5000 and 10000 mg/kg-diet. There were no significant effects (p>0.05) to any reproductive 

parameters examined in the study up to and including the 10000 ppm concentration. No clinical 

signs were reported the study. 

In an avian reproduction study with the bobwhite quail (Colinus virginianus) (MRID 00041440, 

1976), birds of unspecified age were administered chlorothalonil (99.6% purity) in the feed at 

nominal concentration of 0, 5, or 50 mg/kg-diet. Twelve pens were used, with 1 male and 2 

females per pen. There was no mortality or effect on any reproductive parameter tested up to and 

including the 50 ppm concentration. 

In an avian reproduction study with the bobwhite quail (Colinus virginianus), (MRID 4096404, 

1988), birds were administered chlorothalonil (98.3%) in the feed at nominal concentration of 

Page 65 of 69
CHLOROTHALONIL FINAL 

Page 58 of 61 

1000, 5000 and 10000 mg/kg-diet. Significant effects on reproductive parameters and clinical 

signs of toxicity were reported at 5000 and 10000 ppm. 

In another avian reproduction study (MRID 45710218, 1993), 6-month old Bobwhite quail were 

administered chlorothalonil (99.18% purity) in the feed at nominal concentrations of 0, 40, 160, or 

640 mg/kg diet for 22 weeks (10 weeks prior to the start of egg production and 12 weeks of egg 

production). Mean-measured concentrations were 1.5 (<LOD, control), 41, 153, and 624 ppm, 

respectively. Each treatment group was comprised of 24 mating pairs, with one pair per pen. A 

progressive photo period was employed in this study: 7 hours/day through Week 8 and 17 

hours/day until the end of the study. Temperature was maintained at 23-27°C throughout the study 

and humidity was maintained at ca 55%. Individual adult body weights were recorded at Week 0, 

2, 4, 6, 8 and 22, and body weight changes were determined for each interim period. Food 

consumption in each replicate was measured weekly throughout the pre-treatment and treatment 

periods of the adult phase. Individual chick body weights were recorded after hatching and on Day 

14 after hatching; egg shell thickness was measured every two weeks during egg production. 

Adult birds and chicks were observed routinely for mortality and clinical signs. 

There was no reported treatment-related effect on adult mortality. There were 1, 2, and 5 deaths 

during the study in the low-, medium-, and high-dose groups, respectively, but the deaths were 

generally attributed to pen-mate aggression. Adult body weight changes and food consumption 

during the treatment period were generally similar in all groups, and there was no evidence of a 

treatment-related effect. No treatment-related effects were observed on egg quality, fertility, 

embryonic development, hatchability, or survival of hatchlings. Although not statisticallysignificant, a treatment-related reduction in the number of eggs laid/hen was observed at 640 ppm, 

which also resulted in a reduction of the number of 14-day old survivors/hen. The number of eggs 

laid/hen were in the control, low-, mid- and high-dose groups were 62.0, 62.4, 68.9, and 51.0 

respectively, and the resulting number of 14-day old survivors/hen were 37.2, 42.8, 42.2, and 30.4, 

respectively. These findings are considered to be biologically significant. Therefore, reproductive 

LOAEC is 624 ppm based on decreased egg production, and the NOAEC is 153 ppm. 

Life-cycle Toxicity in Freshwater Fish (OPPTS 850.1500) 

!

In a life-cycle toxicity study (MRID 00030391, 1980). Fathead minnows (Pimphales promelas) 

were exposed to chlorothalonil (96% purity) under flow-through conditions, beginning as embryos 

(F0) through to spawning and hatching of the subsequent generation (F1). Mean-measure 

concentrations of chlorothalonil were 0 (control, <LOD), 0.6, 1.4, 3.0, 6.5 and 16 μg a.i./L. The 

following parameters were assessed: hatching success of F0 generation eggs, survival of F0

generation fry at 35 and 64 days, number of spawns of F0 generation fish, number of F1 eggs per 

spawn of F0 fish, hatching success of F1 generation fry at 34 days. 

Chlorothalonil significantly reduced hatching success and survivability of fathead minnows at 

concentrations as low as 6.5 ppb, with most deaths occurred before Day 35 at all concentrations. 

Page 66 of 69
CHLOROTHALONIL FINAL 

Page 59 of 61 

The hatching success of F1 generation eggs spawned from exposed F0 parents at the 6.5 ppb test 

level was significantly less than the control in the F1 generation, less than the F0 generation eggs at 

the 6.5 ppb test level, and less than the F1 generation eggs exposed to 6.5 ppb chlorothalonil but 

spawned from unexposed F0 parents. The growth of fish exposed to chlorothalonil was not reduced 

significantly compared to the controls. Although reductions in the number of eggs per spawn and 

hatchability of F1 eggs only attained significance at 6.5 μg/L, a decrease in the total number of 

spawns and eggs was observed in the 3.0 μg/L compared to the control. The number of eggs per 

female in the negative control was 494 whereas it was 103 in the 3.0 μg/L group; however, 

because there is inherent variability in egg production and the use of only two replicates, this 

decrease (79% difference) was not significant. However, because this decrease is thought to be 

biologically significant and treatment-related, the LOAEC is 3.0 μg/L and the NOAEC is 1.4 μg/L 

where the number of eggs per female was 459. 

! !

Page 67 of 69
CHLOROTHALONIL FINAL 

Page 60 of 61 

APPENDIX 3: References Not Utilized in the Chlorothalonil WoE Analysis 

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

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

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

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

Disruptor Review Team (USEPA, 2010). 

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

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

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

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

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

Guidelines for Ecological Toxicity Data in Open Literature, May 2011 

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

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

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

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

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

analysis for Chlorothalonil 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. 

Andersen, H.R., A.M. Vinggaard, T.H. Rasmussen, I.M. Gjermandsen, and E.C. BonefeldJorgensen. (2002) Effects of currently used pesticides in assays for estrogenicity, androgenicity, 

and aromatase activity in vitro. Toxicology and Applied Pharmacology 179:1-12. 

Diamond Shamrock Corp. (1982b) A Chronic Dietary Study in Mice with DS-3701: Document 

No. 098-STX-78- 0024-001. (submitted by Diamond Shamrock Agricultural Chemicals, 

Cleveland, OH; CDL: 071531-A; 071532; 071533; 071534; 071548; 071530), April 21, 1983. 

MRID 00127848. Unpublished. 

Page 68 of 69
CHLOROTHALONIL FINAL 

Page 61 of 61 

Diamond Shamrock Corp. (1981) A 90-day Toxicity Study of Technical Chlorothalonil in Rats: 

Document No. 099-5TX-80-0200-006. (submitted by Diamond Shamrock Agricultural Chemicals, 

Cleveland, OH; CDL:071535-E; 071536), April 21, 1983. MRID 00127850. Unpublished. 

Hastings, T.F.; Stemmer, K.L. (1975) 120-Day Dietary Toxicity Study--Rats: DAC-3701: Project 

No. 24-051. Final report. Bio/Tox Research Laboratories, Inc., submitted by Diamond Shamrock 

Agricultural Chemicals, Cleveland, OH; CDL:095783-A). MRID 00047936. Unpublished. 

Nishihara, T., J. Nishikawa, T. Kanayama, F. Dakeyama, K. Saito, M. Imagawa, S. Takatori, T. 

Kitagawa, S. Hori, and H. Utsumi. (2000) Estrogenic activities of 517 chemicals by yeast twohybrid assay. Journal of Health Science 46:282-298. 

Petit, F. P. Le Go, J.-P. Cravedi, Y. Valotaire, and F. Pakdel. (1997). Two complementary 

bioassays for screening the estrogenic potency of xenobiotics: Recombinant yeast for trout 

estrogen receptor and trout hepatocyte cultures. Journal of Molecular Endocrinology 19:321-335.

Serrone, D.M. and F.C. Killeen, Jr. (1990) A 90-day oral toxicity study in dogs with 3-carbamyl2,4,5 trichlorobenzoic acid. Biodynamics. Document No.: 3080-88-0082-TX-003. MRID 

41564805. 

Soto, A.M., C. Sonnenschein, K.L. Chung, M.F. Fernandez, N. Olea, and F.O. Serrano. (1995) The 

ESCREEN assay as a tool to identify estrogens: An update on estrogenic environmental pollutants. 

Environmental Health Perspectives 103 (suppl. 7):113-122. 

Yu, S., Wages, M. R., Cobb, G. P., and Maul, J. D. (2013). Effects of chlorothalonil on 

development and growth of amphibian embryos and larvae. Environ Pollut. 181:329-334. 

Page 69 of 69