UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: June 29, 2015 

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

List 1 Chemicals 

PC Code: See table, Attachment A 

Decision No.: NA 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: See table, Attachment A 

MRIDNo.:NA 

DP Barcode: NA 

Registration No.: NA 

Regulatory Action: NA 

Case No.: NA 

CAS No.: NA 

40CFR: NA 

FROM: Greg Akerman & 

Health Effects Divison 

::~:E£'~:::e f]g:ams d f 'v--

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

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

Health Effects Division 

Office of Pesticide Programs 

TO: Jolene Trujillo 

Chemical Review Manager 

Pesticide Re-Evaluation Division 

Office of Pesticide Programs 

Ver.Apr. 2010 

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

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

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

and report identifiers. 

Page I of2 

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Page 2 of 2 

Attachment A. EDSP List 1 Chemicals 

Chemical Name PC Code TXR Number 

2,4-D 030001 0057151 

Abamectin 122804 0057152 

Acephate 103301 0057153 

Acetone 044101 0057154 

Atrazine 080803 0057155 

Benfluralin 084301 0057156 

Bifenthrin 128825 0057157 

Captan 081301 0057158 

Carbaryl 056801 0057159 

Carbofuran 090601 0057160 

Chlorothalonil 081901 0057161 

Chlorpyrifos 059101 0057162 

Cyfluthrin 128831 0057163 

Cypermethrin 109702 0057164 

DCPA 078701 0057165 

Diazinon 057801 0057166 

Dichlobenil 027401 0057167 

Dimethoate 035001 0057168 

EPTC 041401 0057169 

Esfenvalerate 109303 0057170 

Ethoprop 041101 0057171 

Fenbutatin-Oxide 104601 0057172 

Flutolanil 128975 0057173 

Folpet 081601 0057174 

Glyphosate 417300 0057175 

Imidacloprid 129099 0057176 

Iprodione 109801 0057177 

Isophorone 847401 0057178 

Linuron 035506 0057179 

Malathion 057701 0057180 

Metalaxyl 113501 0057181 

Methomyl 090301 0057182 

Metolachlor 108801 0057183 

Metribuzin 101101 0057184 

MGK-264 057001 0057185 

Myclobutanil 128857 0057186 

Norflurazon 105801 0057150 

o-Phenylphenol 064103 0057146 

Oxamyl 103801 0057142 

PCNB 056502 0057138 

Permethrin 109701 0057149 

Phosmet 059201 0057145 

Piperonyl Butoxide 067501 0057141 

Pronamide 101701 0057137 

Propargite 097601 0057148 

Propiconazole 122101 0057144 

Pyriproxyfen 129032 0057140 

Simazine 080807 0057136 

Tebuconazole 128997 0057143 

Tetrachlorvinphos (TCVP) 083701 0057147 

Triadimefon 109901 0057139 

Trifluralin 036101 0057135 

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EDSP: WEIGHT OF EVIDENCE ANALYSIS OF POTENTIAL 

INTERACTION WITH THE ESTROGEN, ANDROGEN OR THYROID 

PATHWAYS 

CHEMICAL: PENTACHLORONITROBENZENE (PCNB) 

OFFICE OF PESTICIDE PROGRAMS

OFFICE OF SCIENCE COORDINATION AND POLICY

U.S. ENVIRONMENTAL PROTECTION AGENCY

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ii 

TABLE OF CONTENTS 

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

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

I. Introduction.......................................................................................5 

II. Sources of Scientific Data and Technical Information......................................6 

A. EDSP Tier 1 Screening Assays............................................................6 

B. Other Scientifically Relevant Information (OSRI)......................................6 

III. Weight of Evidence (WoE) Evaluation.......................................................6 

A. EDSP Tier 1 Screening Assays.............................................................9 

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis.............................10 

1. Effects on Estrogen Pathway...................................................10 

2. Effects on Androgen Pathway..................................................17 

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................21 

IV. Committee’s Assessment of Weight of Evidence..........................................25 

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................25 

1. Tier 1 in vivo Assays.............................................................26 

2. OSRI...............................................................................26 

B. Estrogen Pathway...........................................................................27 

C. Androgen Pathway.........................................................................27 

D. Thyroid Pathway...........................................................................28 

E. Conclusions.................................................................................28 

V. EDSP Tier 2 Testing Recommendations....................................................29 

VI. References.......................................................................................31 

LIST OF TABLES: 

 Table 1. Tier 1 Assays for PCNB......................................................................9 

 Table 2. Estrogenic/Anti-Estrogenic Pathway for PCNB ..........................................12 

 Table 3. Androgenic/Anti-Androgenic Pathway for PCNB .......................................18 

 Table 4. Thyroid Pathway for PCNB ...............................................................23 

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................35 

APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................44 

APPENDIX 3. References Not Utilized in the PCNB WoE Analysis.................................62 

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iii 

Abbreviations 

Abbreviation Terminology 

A Androgen (hormonal pathway) 

ADME Absorption, Distribution, Metabolism, Excretion 

ALP Alkaline Phosphatase 

ALT Alanine Aminotransferase 

AMA Amphibian Metamorphosis Assay 

ARTA Androgen Receptor Transcriptional Activation 

AST Aspartate Aminotransferase 

ANOVA Analysis of Variance 

AOP Adverse Outcome Pathway

AR Androgen Receptor

Bmax Binding at maximum

BROD Benzyloxyresorufin-O-dealkylase 

BUN Blood Urea Nitrogen

CAR Constitutive Androstane Receptor

CFR Code of Federal Regulations 

CG Cowper’s Gland 

ChE Cholinesterase 

ChEI Cholinesterase inhibition 

CMC Carboxymethyl cellulose 

CTA Comparative Thyroid Assay 

CV Coefficient of Variation 

CYP Cytochrome 450 

DER Data Evaluation Record

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

DO Dissolved Oxygen

DP Dorsolateral Prostrate

E Estrogen hormonal pathway

EDSTAC Endocrine Disruptor Screening and Testing Advisory Committee 

EDRT Endocrine Disruptor Review Team

EDSP Endocrine Disruptor Screening Program

EE Ethinyl Estradiol

ELISA Enzyme Linked Immunosorbent Assay

EOGRTS Extended One-Generation Reproductive Toxicity Study (Rat)

ER Estrogen Receptor

EROD Ethoxyresorufin-O-dealkylase (or deethylase)

ERTA Estrogen Receptor Transcriptional Activation

EtOH Ethanol 

F Female 

F1 First filial generation 

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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 

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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) 

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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

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Page 1 of 62 

Executive Summary 

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

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

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

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

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

addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this weight of evidence (WoE) assessment. 

In determining whether a pesticide chemical interacts with E, A or T hormone pathways, the 

number and type of effects induced, the magnitude and pattern of responses observed across 

studies, taxa and sexes were considered. Additionally, the conditions under which effects occur 

were considered, in particular, whether or not endocrine-related responses occurred at dose(s) 

that also resulted in general systemic toxicity or overt toxicity. 

On October 22, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of PCNB with the E, A or T hormone pathways. The T1WoERC conclusions from the WoE 

evaluation in this report are presented by pathway (E, A and then T) beginning with the results of 

the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of 

the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For PCNB, there was no convincing evidence of potential interaction of PCNB with the estrogen 

pathway. The Tier 1 or OSRI in vitro assays were negative with the exception of the 

steroidogenesis assay where there was a slight increase of estradiol production (<1.4 fold) at the 

10-20 mM concentrations. Additionally, PCNB showed no estrogen-related effects in the Tier 1 

in vivo mammalian assays or the Part 158 mammalian toxicity studies. In the Part 158 avian 

reproduction studies, decreases in offspring survival and or weights were observed in the mallard 

duck, however, theses endpoints are limited in informing potential estrogen (and androgen) 

related effects and no effects on fecundity was observed; no reproductive effects observed in the 

bobwhite quail. In the FSTRA, fecundity at the highest concentration tested (0.074 mg/L) was 

decreased (67%) and GSI was increased by 35% and histological changes were noted in the 

ovaries, including decreased post-ovulatory follicles and increased oocyte atresia. However, it is 

noted that there were no effects on fertilization or serum vitellogenin (VTG) levels. While there 

is limited evidence of redundancy across the battery (all assays negative except for the Tier 1 

steroidogenesis assay), there is complementarity of evidence within the FSTRA which suggests 

potential interaction with the estrogen pathway in fish. 

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There was no evidence of interaction of PCNB with the androgen pathway in the Tier 1 in vitro

(i.e, AR binding and steriodogenesis assays were negative) or Tier 1 in vivo mammalian assays 

(i.e., Hershberger and male pubertal assays were negative in the absence of overt toxicity). In 

addition, PCNB was negative in an OSRI AR transactivation assay (Kojima et al., 2004). There 

were no treatment-related effects on male reproductive parameters in the Part 158 mammalian 

studies. In the Part 158 avian studies, while reduction in number/weight of offspring were 

observed, these effects lack diagnostic specificity with regards to the androgen pathway. While 

there was a significant increase in male GSI in the FSTRA at the mid and high concentrations, 

there was a lack of complementarity evidence as there were no other effects noted in the males. 

Therefore, the Tier 1 battery data do not support a conclusion of potential interaction with the 

androgen pathway. 

For the thyroid pathway, there is evidence of potential interaction of PCNB in mammals based 

on alterations on thyroid hormone levels and subsequent changes in thyroid histopathology. In 

both the Tier 1 male and female pubertal assays, there were significant decreases in T4 levels and 

increases in thyroid stimulating hormone (TSH) levels, but there were no significant changes in 

thyroid weight or histology. Although there was a slight, dose-related trend toward decreased 

colloid area and increased follicular cell height in the female pubertal study. It is noted that the 

thyroid effects were seen in the presence of significant (p <0.05) increases in liver and kidney 

weights were noted in both studies at both dose levels along with changes in clinical chemistry 

parameters (↑alkaline phosphatase and ↑bilirubin) that are indicative of altered liver function. 

In the amphibian metamorphosis assay (AMA), while there was a slight but significant delay in 

the median developmental stage at Day 21 at all PCNB levels compared to the negative control, 

no treatment-related effects were seen in thyroid histopathology, asynchronous development or 

normalized hind limb length (HLL). A decrease in body weight and snout ventral length (SVL) 

were observed on Day 21 at the high treatment group. Overall, these findings do not suggest that 

the delay in development or decreased growth is thyroid-related. 

In the mammalian Part 158 studies, alterations in thyroid hormones, thyroid weights and thyroid 

lesions were seen in the subchronic, chronic and two-generation reproduction studies in rats. 

Additionally, alterations in T3, T4 and TSH levels were observed in a special non-guideline 

subchronic study with male rats, conducted specifically to examine potential thyroid effects of 

PCNB. Across these studies, the observed effects were concurrent with alteration in liver weight 

and liver histopathology. 

In summary, there is no evidence of potential interaction of PCNB with the estrogen pathway in 

the mammals. However, PCNB may potentially interact with the estrogen pathway in wildlife 

based on the complementarity effects observed within the FSTRA along with the results of the 

Tier 1 steroidogenesis assay. For the androgen pathway, there no convincing evidence of 

potential interaction of PCNB with the mammals or wildlife. Additionally, there is no evidence 

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of potential interaction of PCNB with the thyroid pathway in amphibians. However, there is 

evidence of potential interaction of PCNB with the thyroid pathway in mammals, although it is 

noted that the observed thyroid effects were consistently associated with alterations in the liver. 

In the EDSP Tier 1 assays and OSRI, there was evidence for potential interaction with the 

thyroid pathway in studies conducted with adult animals, but no such data exists for its effects in 

the young animals. 

For PCNB, the current point of departure (POD) of 1 mg/kg/day for human health risk 

assessment is based on hepatocellular hypertrophy and hyperplasia seen at 150 mg/kg /day 

(LOAEL) in a combined chronic toxicity/carcinogenicity study in rats. Thyroid hypertrophy and 

hyperplasia were seen at the next higher dose (357 mg/kg/day). This POD is used for deriving 

the chronic Reference Dose (RfD) and for calculating the Margins of Exposure (MOEs) used for 

non-dietary risk assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. However in the case of PCNB, although no data exists for potential 

thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended since: 1) 

the POD is approximately 300-fold lower than the dose that caused thyroid lesions seen at 357 

mg/kg/day; 2) in subchronic studies conducted with young rats changes in thyroid weights and/or 

thyroid hormone levels were seen at only doses greater than 300 mg/kg/day; 3) the thyroid 

lesions were seen in aged rats after chronic (104 weeks) dietary exposures (i.e, geriatric effects); 

4) available mechanistic studies show that the thyroid effects are related through the liver and 

liver lesions are used as the toxicity endpoint of concern for establishing the POD; 5) the CTA 

would be tested at higher doses and would be expected to yield a POD lower than the level 

currently used for risk assessment; and 6) the RfD and the MOEs are protective of the thyroid 

effects seen in the pubertal assays and the OSRI. Therefore, mammalian EDSP Tier 2 testing is 

not recommended for PCNB since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

In the FSTRA, fecundity was decreased, GSI was increased and there were histological changes 

in the ovaries at 74 μg a.i./L. Currently, the chronic fish risk assessments are based on a NOAEC 

of 13 μg a.i./L and a LOAEC of 32 μg a.i./L established in the fish early-life cycle (ELS) toxicity 

study with the rainbow trout. Based on the concentrations at which responses observed in the 

FSTRA occurred in at similar concentrations as the current chronic endpoint, the EDSP Tier 2 

Medaka Extended One-Generation Reproduction Test (MEGORT) is recommended. 

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

duck are available for PCNB. In mallard ducks, effects on reproductive parameters were 

observed; no reproductive effects were observed in bobwhite quail. The type of data obtained 

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from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for 

evaluating potential reproductive effects to birds from PCNB exposure. Relative to the EDSP, 

additional testing is not recommended.

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I. Introduction 

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

the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen 

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

antagonism at the estrogen and androgen receptors, altered steroidogenesis, as well as 

hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary thyroid (HPT) perturbations. 

In addition to the available Tier 1 data, other scientifically relevant information (OSRI), 

including general toxicity data and open literature studies of sufficient quality, were considered 

in this assessment. 

In determining whether PCNB interacts with E, A or T hormone pathways, the number and type 

of effects induced, the magnitude and pattern of responses observed across studies, taxa, and 

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

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

general systemic toxicity or overt toxicity. 

On October 22, 2014, the EDSP Tier 1 Assay Weight of Evidence Review Committee 

(T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination 

and Policy (OSCP) conducted a weight-of-evidence (WoE) analysis of the potential interaction 

of pentachlorobenzene 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).

Pentachloronitrobenzene is a contact organochlorine fungicide with a water solubility of 0.44 

mg/L at 25° C and a vapor pressure of 1.13 x 10-4 mmHg at 25°C. PCNB is not likely to be very 

mobile in most soils given the chemical’s tendency to sorb to organic matter. However, on sandy 

soils low in organic matter, PCNB may leach to groundwater. Once in the atmosphere, PCNB is 

not expected to degrade by photo-oxidation. The primary route of degradation for PCNB is 

through aqueous photolysis.

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

E, A, 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 submitted to the agency, 

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

EDSP Tier 2 study recommendations are presented in Section V. 

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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 recipient(s) 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 PCNB (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 (AOP). Broadly, there are four main steps outlined in the guidance which provide the 

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

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

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

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

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

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

is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to 

evaluate whether additional testing is needed based on the evidence and conclusions described 

above. 

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

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

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

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

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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 consideration of whether the responses were 

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

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

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

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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 the three components described above is 

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

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

potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or 

conflicting data; 4) overall strength of evidence supporting a conclusion; and, 5) what, if any, 

additional data are needed and why. Assessing the need for additional data is based on a caseby-case analysis which took all available toxicity data into account. 

 

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

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

considered scientifically and biologically relevant 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 assays submitted in response to the agency’s test order for PCNB are shown below in 

Table 1. 

Table 1: Tier Screening Assays for PCNB 

Tier 1 Assays Test Guideline Test Order Status 

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied by OSRI 

(Ashby et al., 2005) 

ERα Transcriptional Activation Assay 

(Human cell line HeLa 9903) 

OCSPP 890.1300; 

OECD 455 Requirement Satisfied by OSRI 

(Ashby et al., 2005) 

AR Binding Assay (Rat prostate 

cytosol) OCSPP 890.1150 Requirement Satisfied 

(MRID No.48946801) 

Steroidogenesis Assay (Human cell line 

H295R) 

OCSPP 890.1550; 

OECD 456 

Requirement Satisfied 

(MRID No.48916704) 

Aromatase Assay (human recombinant 

microsomes) OCSPP 890.1200 Requirement Satisfied 

(MRID No.48916701) 

Uterotrophic Assay (Rat) OCSPP 890.1600; 

OECD 440 

Requirement Satisfied by OSRI 

 (Ashby et al., 2005) 

Hershberger Assay (Rat) OCSPP 890.1400; 

OECD 441 

Requirement Satisfied 

(MRID No.48916702) 

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied 

(MRID No. 49042103) 

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied 

 (MRID No.49042103) 

Fish Short-term Reproduction Assay OCSPP 890.1350; 

OECD 229 

Requirement Satisfied 

(MRID No.49042102) 

Amphibian Metamorphosis Assay 

(Frog) 

OCSPP 890.1100; 

OECD 231 

Requirement Satisfied 

 (MRID No.49042101) 

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Page 10 of 62 

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

1. Effects on Estrogen Pathway 

The potential interactions of PCNB 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 an evaluation of estrogenic/anti-estrogenic activity from in vitro Tier 1 testing results, PCNB 

was classified as negative in the aromatase assay and equivocal in the steroidogenesis assay, 

based on a slight induction of estradiol production (≤1.4-fold increase) at the highest achieved 

test concentration (10-20 μM). The test order requirements for the estrogen receptor (ER) 

binding and ER transactivation assays (ERTA) were satisfied by OSRI (Ashby et al. 2005), in 

which PCNB was negative for competitive binding in assays using rat uterine cytosolic ER, 

hERα, or hERβ. Additionally, PCNB was negative in a yeast based ER transactivation assay 

using hERα. These results are further support by the in vitro ER binding/ERTA assays by 

Kojima et al. 2004, Vinggaard et al.1999 and Zou et al. 2002, in which PCNB showed no 

estrogenic or anti-estrogenic activity. 

The test order requirements for the uterotrophic assay was satisfied by OSRI (Ashby et al. 2005) 

in which PCNB had no estrogenic activity when administered alone or anti-estrogenic activity 

when administered with estradiol (E2). However, PCNB did appear to enhance the estrogenic 

activity of E2 (1μg/kg) when added exogenously at doses down to 100 mg/kg/day, but it did not 

enhance the activity of the synthetic estrogen diethylstilbestrol. However, in the Tier 1 female 

pubertal rat assay, no estrogenic/anti-estrogenic activity was observed at doses up to 1000 

mg/kg/day. These findings are further supported by the female pubertal assay conducted by 

Ashby et al. (2005), where no anti-estrogenic activity was seen when dosed with PCNB at doses 

of 800 mg/kg/day in conjunction E2. 

In the FSTRA, fecundity was decreased by 67% at the highest test concentration (0.074 mg/L), 

but no effects were noted on fertilization or serum vitellogenin levels. Also at the high test 

concentration, GSI was also increased by 35% and histological changes were noted in the 

ovaries, including decreased post-ovulatory follicles and increased oocyte atresia. There was a 

dose-dependent increase in body weight in female fish that attained significance at the high 

treatment (↑10%). 

No estrogen-related effects were observed in the Part 158 mammalian studies. 

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Page 11 of 62 

In the Part 158 bobwhite quail reproduction studies, decreased survival of 14-day old chicks 

were observed at the respective high concentrations of 1200 and 2,500 mg/kg-diet. In the latter 

study, 14-day old survivor body weights were also decreased at doses ≥ 2500 mg/kg-diet as well 

as decreases in offspring viability (normal eggs/hatchlings) at 5000 mg/kg-diet. In two Part 158 

mallard studies, there were no effects on reproductive parameters. In the Part 158 rainbow trout 

fish early life-cycle study, hatchability was decreased along with the number of surviving fry at 

the highest test concentration dose of 52 μg/L, and there were reductions in body length and wet 

weight at concentration ≥32 μg/L. In a fathead minnow early life-cycle study, larval growth was 

significantly reduced (20%) at the 54 μg/L.

Page 19 of 70
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Page 12 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNB Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen levels 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

EDSP Tier 1 Assay 

ER Binding Requirement satisfied by OSRI (Ashby et al, 2005; see below) ERTA Requirement satisfied by OSRI (Ashby et al, 2005; see below) Aromatase (MRID 48916701) N Steroidogenesis (MRID 48916704) P Uterotrophic Requirement satisfied by OSRI (Ashby et al, 2005; see below) Female Pubertal Rat (MRID 49042103) N N N N N N FSTRA (MRID 49042102) NE ↑35% (H) P4 (H) N Frt: N Fcd: ↓67% (H) 

OSRI 

ER Binding, Transactivation (Ashby et al, 2005) 

N N 

Receptor Transactivation (Kojima et al., 2004)

N N 
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Page 13 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNB Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen levels 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Receptor Transactivation (Vinggaard et al., 

1999)

 N 

Receptor Transactivation (Zou et al., 2002)

 N 

Uterotrophic assay (Ashby et al, 2005) N Female Pubertal assay (Ashby et al., 2005) N N Three-generation reproduction (Rat; MRIDs 00001666 and 40865903) 

 NR NR N NR N 

Two-generation reproduction (Rat; MRID 41918701) 

NR NR N NR N 

Two-generation reproduction (Rat; MRID 43469301) 

NE N N NR N 

Developmental toxicity (Rat; MRID 40588601) 

NR N 

Developmental toxicity (Rat; MRID 41361201) 

NE N 
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Page 14 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNB Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen levels 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Developmental toxicity (Rabbit; MRID 41361301) 

 NR N 

Developmental toxicity (Rabbit; MRID 40717102) 

 NR N 

Chronic toxicity/ carcinogenicity (Rat; MRID 41987301) 

NE N N NE 

Chronic toxicity/ carcinogenicity (Rat; MRID 43015801) 

N N N NE 

Carcinogenicity (Mouse; MRID 00114224) 

NE NE N NR 

Carcinogenicity (Mouse; MRID 45609101) 

NE NE N NR 

Chronic toxicity (Dog; MRID 00001667) NE NE N NE Chronic toxicity (Dog; MRID 41718601) NE N N N Subchronic oral toxicity (Rat dietary; MRID 40636701) 

N N N NE 
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Page 15 of 62 

Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNB Lines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ Literature Citation 

ER Binding 

ER Activation 

Steroidogenesis 

Estrogen levels 

Uterine Weight 

Ovarian 

Weight/GSI 

Ovarian/Gonad 

Staging and 

Histopathology 

Pituitary Weight 

Estrous Cyclicity 

Age & Weight at 

VO 

2° Sex 

Characteristics 

Fertility (Frt)/ 

 Fecundity (Fcd) 

Subchronic oral toxicity (Rat gavage; MRID 42416001) 

 NE N N NE 

Subchronic toxicity (Rat dermal; MRID 42413901) 

NE NE N NE 

Subchronic toxicity (Rat dermal; MRID 42416002) 

NE N N NE 

Avian reproduction (Quail; MRID 41332801) 

P5 (H) 

Avian reproduction (Quail; MRID 43980301) 

P6

(ML,MH

, H) 

Avian reproduction (Duck; MRID 41332802) 

N 

Avian reproduction (Duck; MRID 43903301) 

N 

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

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this absolute unless otherwise indicated. LW: Liver weight; KW: Kidney weight 

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

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 

see Section IV. A 

4. At the high concentration (0.074 mg/L), the incidences of severe oocyte atresia were increased and decreases in post-ovulatory foll
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Page 16 of 62 

5. Reductions in 14-d offspring survival at 1200 mg/kg-diet 6. Reductions in 14-d offspring survivors and body weight at 2500 mg/kg-diet; effects on embryo viability/hatchling success at 5000 

7. Decreases in male body weight at 1200 mg/kg-diet. P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examine NR Not reported 
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Page 17 of 62 

2. Effects on Androgen Pathway 

The potential interactions of PCNB 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. 

The in vitro Tier 1 AR binding and steroidogenesis assays were both negative, as was an AR 

transactivation assay conducted by Kojima et al. (2004). PCNB was also negative for androgenic 

or anti-androgenic activity in the in vivo Hershberger and male pubertal assays. In the FSTRA, 

GSI was increased by 42 and 59% in male fish at the mid and high concentrations (0.0069 and 

0.074 mg/L), but no effects were noted on staging or histology of the testes, fertilization success, 

secondary sexual characteristics or plasma vitellogenin levels. 

There was no evidence of androgen-related effects in the Part 158 mammalian toxicity studies. In 

the Part 158 mallard duck reproduction studies, decreases in offspring survival and/or hatch were 

observed; no effect in the bobwhite quail studies. 

Page 25 of 70
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Page 18 of 62 

Table 3: Androgenic/Anti-Androgenic Pathway for PCNB Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for PCNB1Study Type/ Literature Citation 

AR Binding 

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 48946801) N Steroidogenesis (MRID 48916704) N Hershberger (MRID 48916702) NE N LMale Pubertal Rat (MRID 49042103) N N N N N N N N FSTRA (MRID 49042102) NE 

↑42, 

59% 

(M, H) 

N N N N 

OSRI 

Receptor Transactivation (Kojima et al., 2004)

N 

Three-generation reproduction (Rat; MRIDs 00001666 and 40865903)

NR N NR N NR NR 

Two-generation reproduction (Rat; MRID 41918701) 

NR N NR N NR N N 
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Page 19 of 62 

Table 3: Androgenic/Anti-Androgenic Pathway for PCNB Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for PCNB1Study Type/ Literature Citation 

AR Binding 

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 

Two-generation reproduction (Rat; MRID 43469301) 

N N N N NR N N 

Chronic toxicity/ carcinogenicity (Rat; MRID 41987301) 

N N NE N NE NE 

Chronic toxicity/ carcinogenicity (Rat; MRID 43015801) 

N N NE N NE NE 

Carcinogenicity (Mouse; MRID 00114224) 

NE N NE NE NE NE 

Carcinogenicity (Mouse; MRID 45609101) 

NR N NE N NR NR 

Chronic toxicity (Dog; MRID 00001667) N N NE NE NE NE Chronic toxicity (Dog; MRID 41718601) N N NE N N NE Subchronic oral toxicity (Rat dietary; MRID 40636701) 

N N NE N NE NE 

Subchronic oral toxicity (Rat gavage; MRID 42416001) 

N N NE NE NE NE 

Subchronic toxicity (Rat dermal; MRID 42413901) 

N N NE N NE NE 
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Page 20 of 62 

Table 3: Androgenic/Anti-Androgenic Pathway for PCNB Lines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway for PCNB1Study Type/ Literature Citation 

AR Binding 

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 

Subchronic toxicity (Rat dermal; MRID 42416002) 

N N NE NE NE NE 

Avian reproduction (Quail; MRID 41332801) 

P4 (H) 

Avian reproduction (Quail; MRID 43980301) 

P5 

(ML,

MH, 

H) 

 

Avian reproduction (Duck; MRID 41332802) 

N 

Avian reproduction (Duck; MRID 43903301) 

N 

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

indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assayabsolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal p(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). LW: Liver weight; KW: Kidney 

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

column. For details see Section IV. A 

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

Section IV. A 

4 Reductions in 14-d offspring survival at 1200 mg/kg-diet 5. Reductions in 14-d offspring survivors and body weight at 2500 mg/kg-diet; effects on embryo viability/hatchling success at 5000 mg/k

6. Decreases in male body weight at 1200 mg/kg-diet. P Positive findings N Negative findings (in vitro)/No effect (in vivo) 

NE Not examined NR Not reported 
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Page 21 of 62 

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

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

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

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

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

The potential interactions of PCNB 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. Three Tier 1 studies were 

evaluated for potential effects of PCNB. 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. 

In both the Tier 1 male and female pubertal assays, there were significant decreases in T4 levels 

and increases in TSH levels in both the low- and high-dose groups (500 and 1000 mg/kg/day). 

Although the increased TSH levels in males (39 and 25% at the low and high dose, respectively) 

were not statistically significant, the effect was considered biologically meaningful. There were 

no significant changes in thyroid weight or histology, although there was a slight, dose-related 

trend toward decreased colloid area and increased follicular cell height in the female pubertal 

study. No effects were observed on pituitary weight. 

In the AMA, there was a slight but significant one stage delay in the median Nieuwkoop-Faber 

(NF) developmental stage at Day 21 at all PCNB levels compared to the negative control, with 

no effect at Day 7. No asynchronous development was noted and there was no effect on 

normalized hind limb length (HLL). Mild thyroid atrophy was noted at the highest test 

concentration (0.016 mg/L) compared to the control but were not statistically significant; and, 

the histopathologist report stated these effects were not a result of treatment. The high 

concentration also resulted in an observed 19% decrease in tadpole body weight. 

In the Part 158 two-generation rat reproductive study that included analyses of thyroid 

histopathology, incidences of thyroid follicular cell hypertrophy/hyperplasia were increased in 

adult males in both generations at PCNB doses of 100 and 1000 mg/kg/day and in adult females 

at doses of 1000 mg/kg/day. In the two chronic/carcinogenicity studies in the rat, thyroid 

follicular cell hypertrophy and hyperplasia, and increased follicular cell adenomas. For thyroids, 

the absolute and/or relative weights were significantly (p <0.01) increased in both sexes at the 

mid and high dose groups at the terminal sacrifice. Absolute thyroid weights were increased in 

males and females by 26 and 21%, respectively, at the mid dose, and by 45 and 32% at the high 

dose. In addition, thyroid glands were enlarged in males at the high dose (3/49; 6%) compared to 

control males (0/49). Non-neoplastic lesions in the high-dose males and females included colloid 

Page 29 of 70
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Page 22 of 62 

cysts, hypertrophy, cystic follicular cell and/or hyperplasia. The neoplastic lesions mid- and 

high-dose males and females included thyroid follicular cell adenomas, carcinomas and/or 

adenomas plus carcinomas combined. In the other carcinogenicity study with rats, absolute 

thyroid weights were increased (22%, not significant) only in males at 1000 mg/kg/day There 

were no thyroid effects in the mouse carcinogenicity or the chronic dog studies. No effects on 

thyroid histology were noted in the dietary subchronic rat study at doses up to 381/455 

mg/kg/day (M/F). However in the other two subchronic rat studies (oral gavage and dermal), 

follicular cell hypertrophy and hyperplasia were observed at the high dose (985-1000 

mg/kg/day). When examined, none of the Part 158 mammalian toxicity studies showed effects of 

PCNB on pituitary weight or histopathology. 

To specifically examine potential thyroid effects of PCNB, a special, non-guideline subchronic 

study was also conducted with male rats at two dietary dose levels (equivalent to 1 or 300 

mg/kg/day). At the high-dose, T3 levels were decreased by 19-26%, T4 was decreased by 48-

54%, and TSH levels were increased by 33-132%, each at multiple sampling intervals (Days 7, 

14, 30 and 90). In addition, follicular epithelial hypertrophy was observed in all high-dose rats at 

14, 30 and 90 days and in the low-dose rats by Day 90. Hepatocellular hypertrophy was also 

observed in the low- and high-dose groups, with the severity increasing with dose. 

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Table 4. Thyroid Pathway for PCNB.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for PCNB1

Study Type/ 

Literature Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

EDSP Tier 1 Assay 

Male Pubertal Rat 

(MRID 49042103) N N ↓≥78% 

(L, H)4

↑39, 25% 

[NS] 

(L, H) 

N 

LW:↑8, 

10% 

KW: 

↑8, 6% 

(L, H) 

N 

Female Pubertal Rat 

(MRID 49042103) N N 

↓70, 

72%, 

 (L, H) 

↑53, 59% 

 (L, H) N 

LW: 

↑8, 6% 

KW: 

↑8% 

(L, H) 

N 

AMA 

(MRID 49042101) N 

↓1 NF 

stage 

(L, M, 

H)

21-

Day 

BW:↓

19% 

SVL: 

↓8% 

(H) 

 N

OSRI

Two-generation 

reproduction (Rat; 

MRID 43469301) 

 NR 

P5

 

(M, 

H) 

NE X

(H) N 

Chronic toxicity/ 

carcinogenicity 

(Rat; MRID 

41987301) 

↑21-

26% 

(M) 

P5 

(M) NE X 

 (M, H)

X 

Chronic toxicity/ 

carcinogenicity 

(Rat; MRID 

43015801) 

 N P5 

(H) NE 

X 

(H, 

males) 

N 

Carcinogenicity 

(Mouse; MRID 

00114224) 

 NE NR NE 

X 

(MH, 

H) N 

Carcinogenicity 

(Mouse; MRID 

45609101) 

 NR N NR N X 

(H)

Chronic toxicity 

(Dog; MRID 

00001667) 

 NE N NE 

X 

(MH, 

H) 

N 

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Table 4. Thyroid Pathway for PCNB.

Lines of Evidence Indicating Potential Interaction with the Thyroid Pathway for PCNB1

Study Type/ 

Literature Citation 

Thyroid receptors Thyroid Weight Thyroid: Gross and Histopathology Serum T4 Levels 

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

Overt Toxicity Observed3

Chronic toxicity 

(Dog; MRID 

41718601) 

 N N N 

X 

(H) N 

Subchronic toxicity 

(Rat dietary; MRID 

40636701) 

 NE N NE X 

(M, H) N 

Subchronic oral 

toxicity (Rat 

gavage; MRID 

42416001) 

 NE P5 

(H) NE X 

(H) N 

Subchronic toxicity 

(Rat dermal; MRID 

42416002) 

 NE P5 

(H) NE N N 

Special Subchronic 

toxicity (Rat; MRID 

42630801) 

↓ (H) P5 

(L, H)

T3: 

↓19-26% 

T4: 

↓48-54% 

(H) 

↑33-

132% 

(H) 

N X 

(H) N 

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. Compared to the controls, serum T4 levels were decreased (p<0.001) by ≥78% at 500 and 1000 mg/kg/day, 

with T4 levels being below the method limit of detection (<LOD, 0.82μg/dL) in 6/15 males at 500 mg/kg/day 

and 12/15 males at 1000 mg/kg/day. 

5. Alterations in thyroid histopathology were observed and included increased incidences of follicular cell 

hypertrophy and/or hyperplasia 

P Positive findings 

N Negative findings 

NE Not examined 

NR Not reported 

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IV. Committee’s Assessment of Weight of Evidence 

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

potential of PCNB to interact with endocrine related processes (i.e., E, A, or T hormonal 

pathways) as well as recommendations regarding Tier 2 testing.. The results of the Tier 1 assays 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

with weight of evidence evaluation of the results of the 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 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).

 

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1. Tier 1 in vivo Assays 

No overt toxicity was noted in the male or female pubertal assays or in the Hershberger assay at 

doses up to 1000 mg/kg/day. However, systemic toxicity was noted at the high dose in the 

Hershberger assay and at both the low (500 mg/kg/day) and high doses in the pubertal assays. In 

these assays, toxicity was characterized by increases liver (6-10%) and kidney (6-8%) weights 

and changes in liver enzymes. No signs of overt toxicity were observed in the FSTRA. 

2. OSRI 

No overt or systemic toxicity was observed in the uterotrophic and female pubertal assays 

conducted by Ashby et al. (2005) at PCNB doses up to 800 mg/kg/day; however, animals in 

these studies were dosed by subcutaneous injection, as opposed to oral dosing in the above Tier 1 

assays. 

In part 158 mammalian studies, toxicity was typically characterized by increases in liver weight, 

changes in liver histopathology and enzyme activities, and decreases in body weight and/or body 

weight gain. 

No overt toxicity was noted in any of the multi-generation reproductive studies in rats, but 

decreases in body weight were reported at the mid and high dose groups in one study and 

hepatocellular hypertrophy was noted in females at the high-dose (1000 mg/kg/day) in another 

study. No overt or systemic toxicity was observed in the two rat development studies and one of 

the rabbit developmental studies, but clinical signs of toxicity were noted in the other rabbit 

development study at the high dose (900 mg/kg/day). 

No overt toxicity was noted in the chronic studies in rat, mouse or dogs, with the exception of 

one of the mouse carcinogenicity studies, in which increased mortality was observed in females 

at the high dose (750 mg/kg/day). Systemic toxicity was observed at the high-dose (300 

mg/kg/day) in one chronic rat study and consisted of increases in liver weight (11-20%), 

decreases in body weight (11%) and body weight gain (≤10%), and microscopic changes in the 

liver (hypertrophy). Increased liver weight was also noted in high-dose males in the other 

chronic rat study, along with increased incidences of hepatocellular hypertrophy in males at ≥36 

mg/kg/day and females at ≥357 mg/kg/day. Dose-dependent increases in liver weights in both 

sexes were also observed in one of the chronic mouse studies at doses of 60 mg/kg/day (↑10-

11%) and 180 mg/kg/day (↑40-54%). Increases in liver weight were also observed in both 

chronic dog studies at doses ≥27 mg/kg/day. No overt or systemic toxicity were noted in the two 

rat subchronic dermal studies, but systemic toxicity findings in the subchronic oral studies were 

similar to the findings in the chronic studies. Body weights and/or body weight gains were 

decreased at the high dose in several subchronic studies, and liver weights were increased in all 

the studies at the highest doses tested (300-985 mg/kg/day). 

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In the avian reproduction studies, no overt or systemic toxicity were noted in the quail or duck 

studies at dietary concentration of PCNB up to 5500 ppm, although transient decreases in body 

weight (Week 8) were observed in one of the duck studies at 1200 ppm. In the early fish lifecycle study, overt toxicity was observed at the highest test concentration (52 μg/L) based on the 

reduction in fry survival (75%), and systemic effects were also observed at 32 μg/L, based on 

reductions in body length and weight. In a fathead minnow fish early life-cycle study, larval 

growth was significantly reduced (20%) at 54 μg/L. 

B. Estrogen Pathway 

There was no convincing evidence of potential interaction of PCNB with the estrogen pathway. 

The Tier 1 or OSRI in vitro assays were negative with the exception of the steroidogenesis assay 

where there was a slight increase of estradiol production (<1.4 fold) at the 10-20 mM 

concentrations. Additionally, PCNB showed no estrogen-related effects in the Tier 1 in vivo

mammalian assays or the Part 158 mammalian toxicity studies. In the Part 158 avian 

reproduction studies, decreases in offspring survival and or weights were observed in the mallard 

duck, however, theses endpoints are limited in informing potential estrogen (and androgen) 

related effects and no effects on fecundity was observed; no reproductive effects observed in the 

bobwhite quail. In the FSTRA, fecundity at the highest concentration tested (0.074 mg/L) was 

decreased (67%) and GSI was increased by 35% and histological changes were noted in the 

ovaries, including decreased post-ovulatory follicles and increased oocyte atresia. However, it is 

noted that there were no effects on fertilization or serum vitellogenin (VTG) levels. While there 

is limited evidence of redundancy across the battery (all assays negative except for the Tier 1 

steroidogenesis assay), there is complementarity of evidence within the FSTRA which suggests 

potential interaction with the estrogen pathway in fish. 

C. Androgen Pathway 

There was no evidence of interaction of PCNB with the androgen pathway in the Tier 1 in vitro

(i.e, AR binding and steriodogenesis assays were negative) or Tier 1 in vivo mammalian assays 

(i.e., Hershberger and male pubertal assays were negative in the absence of overt toxicity). In 

addition, PCNB was negative in an OSRI AR transactivation assay (Kojima et al., 2004). There 

were no treatment-related effects on male reproductive parameters in the Part 158 mammalian 

studies. In the Part 158 avian studies, while reduction in number/weight of offspring were 

observed, these effects lack diagnostic specificity with regards to the androgen pathway. While 

there was a significant increase in male GSI in the FSTRA at the mid and high concentrations, 

there was a lack of complementarity evidence as there were no other effects noted in the males. 

Therefore, the Tier 1 battery data do not support a conclusion of potential interaction with the 

androgen pathway. 

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D. Thyroid Pathway 

There is evidence of potential interaction of PCNB with the thyroid pathway in mammals based 

on alterations on thyroid hormone levels and subsequent changes in thyroid histopathology. In 

both the Tier 1 male and female pubertal assays, there were significant decreases in T4 levels and 

increases in TSH levels, but there were no significant changes in thyroid weight or histology. 

Although there was a slight, dose-related trend toward decreased colloid area and increased 

follicular cell height in the female pubertal study. It is noted that the thyroid effects were seen in 

the presence of significant (p <0.05) increases in liver and kidney weights, were noted in both 

studies at both dose levels along with changes in clinical chemistry parameters (↑ALP and 

↑bilirubin) that are indicative of altered liver function. 

In the AMA, while there was a slight but significant delay in the median developmental stage at 

Day 21 at all PCNB levels compared to the negative control, no treatment-related effects were 

seen in thyroid histopathology, asynchronous development or normalized hind limb length 

(HLL). A decrease in body weight and SVL were observed on Day 21 at the high treatment 

group. Overall, these findings do not suggest that the delay in development or decreased growth 

is thyroid-related. 

 

In the mammalian Part 158 studies, alterations in thyroid hormones, thyroid weights and thyroid 

lesions were seen in the subchronic, chronic and two-generation reproduction studies in rats. 

Additionally, alterations in T3, T4 and TSH levels were observed in a special non-guideline 

subchronic study with male rats, conducted specifically to examine potential thyroid effects of 

PCNB. Across these studies, the observed effects were concurrent with alteration in liver weight 

and liver histopathology. 

E. Conclusions 

There is no evidence of potential interaction of PCNB with the estrogen pathway in the 

mammals. However, PCNB may potentially interact with the estrogen pathway in wildlife based 

on the complementarity effects observed within the FSTRA along with the results of the Tier 1 

steroidogenesis assay. For the androgen pathway, there no convincing evidence of potential 

interaction of PCNB with the mammals or wildlife. Additionally, there is no evidence of 

potential interaction of PCNB with the thyroid pathway in amphibians. However, there is 

evidence of potential interaction of PCNB with the thyroid pathway in mammals, although it is 

noted that the observed thyroid effects were consistently associated with alterations in the liver. 

 

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V. EDSP Tier 2 Testing Recommendations 

In the EDSP Tier 1 assays and OSRI, there was evidence for potential interaction with the 

thyroid pathway in studies conducted with adult animals, but no such data exists for its effects in 

the young animals. 

For PCNB, the current point of departure (POD) of 1 mg/kg/day is based on hepatocellular 

hypertrophy and hyperplasia seen at 150 mg/kg /day (LOAEL) in a combined chronic 

toxicity/carcinogenicity study in rats. Thyroid hypertrophy and thyroid hyperplasia were seen at 

the next higher dose (357 mg/kg/day). This POD is used for deriving the chronic Reference Dose 

(RfD) and for calculating the Margins of Exposure (MOEs) used for non-dietary risk 

assessments. 

In general, since the POD and the toxicity endpoints of concern are based on data obtained from 

adult animals, there usually would be a concern that the POD may not be protective of potential 

thyroid toxicity in the young. However in the case of PCNB, although no data exists for potential 

thyroid toxicity in the young, a comparative thyroid assay (CTA) is not recommended since: 1) 

the POD is approximately 300-fold lower than the dose that caused thyroid lesions at 357 

mg/kg/day; 2) in subchronic studies conducted with young rats changes in thyroid weights and/or 

thyroid hormone levels were seen at only doses greater than 300 mg/kg/day; 3) the thyroid 

lesions were seen in aged rats after chronic (104 weeks) dietary exposures (i.e, geriatric effects); 

4) available mechanistic studies show that the thyroid effects are related through the liver and 

liver lesions are used as the toxicity endpoint of concern for establishing the POD; 5) the CTA 

would be tested at higher doses and would not be expected to yield a POD lower than the level 

currently used for risk assessment; and 6) the RfD and the MOEs are protective of the thyroid 

effects seen in the pubertal assays and the OSRI. Therefore, mammalian EDSP Tier 2 testing is 

not recommended for PCNB since additional testing is not expected to impact EPA’s current 

regulatory point of departures and endpoints for human health risk assessments. 

In the FSTRA, fecundity was decreased, GSI was increased and there were histological changes 

in the ovaries at 74 μg a.i./L. Currently, the chronic fish risk assessments are based on a NOAEC 

of 13 μg a.i./L and a LOAEC of 32 μg a.i./L established in the Fish Early Life Cycle toxicity 

study with the rainbow trout. Based on the concentrations at which responses observed in the 

FSTRA occurred in at similar concentrations as the current chronic endpoint, the EDSP Tier 2 

Medaka Extended One-Generation Reproduction Test (MEDGORT is recommended. 

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

duck are available for PCNB. In mallard ducks, effects on reproductive parameters were 

observed; no reproductive effects were observed in bobwhite quail. The type of data obtained 

from Part 158 avian reproduction studies (OCSPP 850.2300) are considered sufficient for 

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Page 30 of 62 

evaluating potential reproductive effects to birds from PCNB exposure. Relative to the EDSP, 

additional testing is not recommended. 

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Page 31 of 62 

VI. References 

Akhurst, L. C. (2012) PCNB Technical H295R Steroidogenesis Assay. Huntingdon Life 

Sciences Ltd., Cambridgeshire, England. Laboratory Study No. BDG0126, July 4, 2012. 

Unpublished. (MRID 48916704) 

Ashby, J., Odum, J., Burns, A., and Lefevre, P. (2005) The report in vitro anti-estrogen 

pentachloronitrobenzene enhances the estrogenic activity of estradiol in vivo in the rat. 

Environmental Toxicology and Pharmacology 20(2Q05):199-208. (MRID 48233802) 

Baxter, G.E. (2012) PCNB: Assessment of Pubertal Development and Thyroid Function in 

Juvenile Male and Female Rats. Huntingdon Life Sciences, East Millstone, NJ. Laboratory Study 

No.: 12-4389, December 19, 2012. Unpublished. (MRID 49042103) 

Beavers, J.B., J.W. Foster, A.S. Lattin, and M.J. Jaber. (1989) Pentachlorobenzene (PCNB): A

one-generation reproduction study with the bobwhite (Colinus virginianus). Lab Project No.

117-161. Wildlife International, Ltd., Easton, MD. Unpublished. (MRID 41332801)

Beavers, J.B., J.W. Foster, AS. Lattin, and M.J. Jaber. (1989) Pentachlorobenzene (PCNB): A

one-generation reproduction study with the mallard (Anas platyrhhynchos). Lab Project No. 117-

162. Wildlife International, Ltd., Easton, MD. Unpublished. (MRID 41332802) 

Cohle, P., and R. Herzig. (1990) Early-life stage toxicity of PCNB to rainbow trout 

(Oncorhynchus mykiss) in a flow-through system. ABC Lab Project No. 36871. Analytical BioChemistry Laboratories, Inc., Columbia, MO. (MRID 41663401) 

Donington, S. (2012). PCNB Technical: Hershberger bioassay in the castrated rat (oral 

administration). Huntingdon Life Sciences, Cambridgeshire, England. Laboratory Project Study 

ID: BDG0121, June 19, 2012. Unpublished. (MRID 48916702) 

Foster, J.R. (2012) PCNB Technical: Aromatase (Human Recombinant) Assay. Huntingdon Life 

Sciences Ltd., Huntingdon, Cambridgeshire, England. Laboratory Report No.: BDG0124, June 

15, 2012. Unpublished. (MRID 48916701) 

Goldenthal, E.I. (1990) One year chronic dietary study in dogs. International Research and 

Development Corporation. Study #399-087. [900-CHR-013]. (MRID 41718601) 

Goldenthal, E.I. (1991) Two year dietary toxicity and carcinogenicity study in rats. International 

Research and Development Corporation. Study #399-072. [900-CHR-014]. (MRID 41987301) 

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Page 32 of 62 

Goldenthal, E.I. (1992) 21-day dermal toxicity study in rats (PCNB). Laboratory Project 

Number: 399-125. International Research and Development Corporation. Unpublished. (MRID 

42413901) 

Goldenthal, E.I. (1993) 90-day Dietary toxicity study in rats. International Research and

Development Corporation. Study #399-122. [900-SCH-003]. (MRID 42630801) 

Hoberman, A.M. (1989a) Developmental toxicity (embryo-fetal toxicity and teratogenic

potential) study of pentachloronitrobenzene (PCNB) administered orally via gavage to CRL:

CDTM(SD)BR presumed pregnant rats. Argus Research Laboratories, Inc. Protocol# 310-005.

[900-CHR-002]. (MRID 41361201) 

Hoberman, A.M. (1989b) Developmental toxicity (embryo-fetal toxicity and teratogenic 

potential) study of pentachloronitrobenzene (PCNB) administered orally (stomach tube) to New 

Zealand white rabbits. Argus Research Laboratories, Inc. Protocols 310-006. [900-CHR004]. (MRID 41361301) 

Keefe, R.T. (1992) 90-Day subchronic oral toxicity study in rats with pentachloronitrobenzene 

(MRD-89505). EXXON Biomedical Sciences, Inc. Project #250570A. [900-SCH-001]. (MRID 

42416001) 

Keller, K. (1988a) Developmental toxicity study in rats: Laboratory Project ID 399-068. 

International Research and Development Corporation. Unpublished. (MRID 40588601) 

Keller, K (1988b) Developmental toxicity study in New Zealand White rabbits: 

Pentachloronitrobenzene (PCNB): Laboratory Project ID 399-070. International Research and 

Development Corporation. Unpublished. (MRID 40717102) 

Larson, P.S and Borzelleca, J.F (1968a) Three generation reproduction study on rats receiving 

Terraclor in their diet. Medical College of Virginia, Dept. of Pharmacology. CDL:091391-B. 

Unpublished. (MRIDs 00001666 and 40865903) 

Larson, P.S and Borzelleca, J.F (1968b) Toxicological study on the effect of adding Terraclor to 

the diet of beagle dogs for a period of two years. Medical College of Virginia, Dept. of 

Pharmacology. CDL:091391-C. Unpublished. (MRID 00001667) 

Matthews, A. (2012) PCNB Technical: Androgen Receptor Binding (Rat Prostate Cytosol). 

Huntingdon Life Sciences, Huntingdon, Cambridgeshire, UK. Laboratory Study No.: BDG0128, 

July 26, 2012. Unpublished. (MRID 48946801) 

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Page 33 of 62 

McGee, D.H. (1988) 13 Week Dietary toxicity study in rats with PCNB 

(pentachloronitrobenzene). International Research and Development Corporation. Study #399- 

071. [900-CHR-008]. Unpublished. (MRID 40636701) 

National Toxicology Program (1987) Toxicology and Carcinogenesis Studies of 1,4- 

Dichlorobenzene in F344/N Rats and B6C3F1 Mice. NTP TR 319. US Dept. of Human Services: 

US Government Printing Office. (MRID 45609101) 

Odum, J and Croudace, C (2013). Pentachloronitrobenzene (PCNB): Weight of Evidence 

Evaluation of US EPA EDSP Tier 1 Studies AMVAC Chemical Corporation, 2110 Davie 

Avenue, Commerce, CA90040. (MRID No. 49079601)

Pederson, C.A., and A.M. Solatycki. (1995) Toxicity and reproduction study with PCNB 

technical in mallard ducks. Lab Report ID 146-002-008. Bio-Life Associates, Ltd., Neillsville, 

WI. (MRID 43903301) 

Pederson, C.A., and A.M. Solatycki. (1995) Toxicity and reproduction study with PCNB 

technical in bobwhite quail. Lab Report ID 146-001-007. Bio-Life Associates, Ltd., Neillsville, 

WI. (MRID 43980301) 

Phillips R.D. (1994) Two generation reproduction toxicity study in rats with 

pentachloronitrobenzene (PCNB) (MRD-89-505). EXXON Biomedical Sciences, Inc. Project 

#1S0535. [900-REP-001]. (MRID 43469301) 

Plutnick, R.T. (1993) 2-year Chronic toxicity/oncogenicity study in rats with 

pentachloronitrobenzene (PCNB) (MRD-89-505). EXXON Biomedical Sciences, Inc. Project 

#2SOS70A. [900-CHR-005]. (MRID 43015801) 

Schneider, S.Z., T. Z. Kendall, and H.O. Krueger. (2013) PCNB Technical: Amphibian 

Metamorphosis Assay for the Detection of Thyroid Active Substances. Wildlife International, 

Ltd., Easton, Maryland. Laboratory project number 246A-111. Unpublished. (MRID 49042101) 

Schneider, S.Z., K.H. Martin, T.Z. Kendall, and H.O. Krueger. (2013) PCNB Technical: Fish 

Short-Term Reproduction Assay with the Fathead Minnow (Pimephales promelas). Wildlife 

International, Ltd., Easton, Maryland. Laboratory report number 246A-110A. Unpublished. 

(MRID 49042102) 

Trimmer, G. (1992) 21-day repeated dose dermal toxicity study in rats with 

pentachloronitrobenzene (Final Report). Laboratory Project Number: 150509. Exxon Biomedical 

Sciences, Inc. (MRID 42416002) 

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Page 34 of 62 

USEPA (2009) Reregistration Eligibility Decision for Pentachloronitrobeneze. EPA-OPP-2006-

XX. June 2006. Office of Pesticide Programs. U.S. Environmental Protection Agency, 

Washington, DC. 

USEPA (2010). PCNB - Report of the Endocrine Disruptor Review Team- Test Order#: EDSP056502-105. December 9, 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. Office of Chemical 

Safety and Pollution Prevention Science Coordination and Policy. 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. 

van der Heijden, C.A. and Til, H.P. (1974) Pentachloronitrobenzene (PCNB) carcinogenicity 

study in mice. Central Institute for Nutrition and Research. Rapport# 436S. [900-CHR-010]. 

(MRID 00114224) 

Wirth, M.J. and Schardien, J.L. (1991) Two generation reproduction study in rats. Amendment to 

the Final Report issued February 1, 1991.International Research and Development Corporation. 

Study #399-086. 900-DAR-008. (MRID 41918701) 

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APPENDIX 1. EDSP Tier 1 Screening Assays 

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100) 

The 21-day assay (MRID 49042101) of PCNB technical (95.6% purity) on amphibian 

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

conditions. Amphibian larvae at Nieuwkoop-Faber (NF) stage 51 (80 tadpoles/group, 20 

tadpoles/replicate) were exposed to a negative control and nominal concentrations of 0.00022, 

0.0022, and 0.022 mg a.i./L; the %CV of measured concentrations exceeded 20.0% over the 

course of the study (i.e., 28-52%, reviewer-calculated). The time-weighted average (TWA) 

concentrations (calculated by the reviewer) were <0.00012 (<LOQ; control), 0.00018, 0.0013, 

and 0.017 mg a.i./L The test system was maintained at 21.6 to 22.0oC and a pH of 7.9 to 8.1. 

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

water) control. By test termination, mean survival was 98% in both the negative control and the 

mid treatment group, 97% in the low treatment group, and 100% in the high treatment group. 

Clinical signs (i.e., behavioral and other sublethal effects) including tail curvature and small size 

were observed in all treatment groups, with no apparent pattern. Tail curvature was seen in 54% 

(32/59) of tadpoles in the negative control and in 28% (16/58), 32% (19/59), and 22% (13/60) 

tadpoles the low, mid, and high treatment levels, respectively. Small size was seen in one 

tadpole each in the negative control and mid treatment groups. 

Day 21 median developmental stage was reduced at all treated levels, (NF 56; p<0.05, 

Jonckheere-Terpstra) relative to the negative control (NF 57). Furthermore, PCNB significantly 

decreased Day 21 snout-to-vent (SVL; ↓8%) and wet weight (↓19%) at the high treatment level, 

relative to the negative control (p<0.05, Dunnett’s). No asynchronous development was 

observed. There were no significant effects on normalized (for SVL) hind limb length (HLL) on 

Day 7 or 21 at any treatment level. The one late stage (NF > 60) tadpole in the negative control 

was excluded from analysis for continuous endpoints (SVL, weight, and normalized HLL). 

The incidence and severity of thyroid gross pathology and histopathology (follicle size) findings 

was visually observed to be greater with increasing test concentrations. The study author 

analyzed the incidence and severity counts using the Jonckheere-Terpstra step-down test and 

determined that the differences were not statistically significant (p>0.05). 

Androgen Receptor (AR) Binding Assay (OCSPP 890.1150) 

In an androgen receptor (AR) binding assay (MRID 48946801), prostate cytosol from SpragueDawley rats was used as the source of AR to conduct binding experiments. Prior to conducting 

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the competitive binding experiments, 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 ([3

H]-R1881). The 

competitive binding experiment 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 

10−10 to 10−3.5 M) of PCNB (95.6% a.i.). Ethanol was used as the solvent vehicle at a final assay 

concentration of 3%. The highest soluble concentration for PNCB was 10−3.5 M. Three valid 

independent runs were performed, and each run included dexamethasone as a weak positive 

control, and R1881 as the ligand reference standard. 

For the saturation binding assay, the appropriate protein concentration was determined to be 4.67 

mg/mL as it bound 24% of the [3

H]-R1881. The mean Kd for [3H]-R1881 was 1.6246±0.3283 

nM, and the mean estimated Bmax was 0.1805±0.0199 nM for the prepared rat prostate cytosol. 

The Kd was within the guideline recommended range of 0.03 to 1.5 nM for two of the three runs, 

but the Kd for Run 2 (2.2810±0.2936 nM) was above the recommended range. 

The protein concentration for the competitive binding assay was determined to be 2.0 mg/mL as 

it bound 13% of the [3H]-R1881 at 1 nM. Across all three valid runs, the estimated mean IC50

was 1.95 × 10−9 M for R1881 and 1.01 × 10−4 M for the weak positive control, dexamethasone. 

The corresponding log IC50 values for R1881 and dexamethasone were −8.72 and −4.00 M, 

respectively. The mean relative binding affinity (RBA) for dexamethasone compared to R1881 

was 0.0019%, for a log RBA of −4.72%. Three valid assay runs were completed out of a total of 

eight runs; five of the runs were appropriately rejected as they failed to meet various 

performance or acceptance criteria. 

For PCNB, the minimum mean specific binding of [3H]-R1881 to the AR was 64, 83 and 80% in 

the three valid runs, which corresponded to an equivocal classification (50-75% [3

H]-R1881 

binding) for the first valid run and non-binder (>75% [3H]-R1881 binding) in the other two valid 

runs. 

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

Receptor Binding Assay. 

Aromatase Assay (OCSPP 890.1200) 

In an in vitro aromatase (CYP19) assay (MRID 48916701), PCNB (95.6% a.i. Lot # 

107030L052) was incubated with human recombinant aromatase and tritiated androstenedione 

([1β3

H(N)]-androst-4-ene-3,17-dione; [3H]-ASDN) in ≤1% ethanol at log concentrations from 

10−10 to 10−4

 M for 15 minutes to assess the potential of PCNB to inhibit aromatase activity. 

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

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

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

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

 M) 

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

chemical series (10−10 to 10−4

 M) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.251 to 0.340 nmol·mgprotein−1

·min−1 for the three test runs, with a mean and standard deviation of 0.297 ± 0.044 

nmol·mg-protein−1·min−1. Activity in the background controls ranged from 0.38 to 1.78% and 

averaged 0.78% of the full control activity. The response of the full activity controls and 

background controls was generally acceptable for each run. 

For the positive control substance (4-OH ASDN), aromatase results were generally within the 

recommended ranges for the top of the curve, bottom curve, Hill slope, log IC50, and %CV for 

replicates of each concentration within runs. The estimated mean log IC50 for 4-OH ASDN was 

−7.39 M and the mean Hill slope was −0.955. 

For PCNB, aromatase activity averaged 0.286 ± 0.051 nmol·mg-protein−1

·min−1 at the lowest 

tested concentration of 10−10 M and 0.275 ± 0.038 nmol·mg-protein−1

·min−1 at the highest tested 

concentration of 10−4 M. Aromatase activity ranged from 92.5 to 98.9% of the full activity 

control at PCNB concentrations from 10−10 to 10−4 M. Although the study author used the 4-

parameter nonlinear regression model to extrapolate possible log IC50 and Hill slope values for 

PCNB (−1.23 M and −0.465, respectively), these values are not supported by the experimental 

data and are well outside the bounds of the tested concentrations. 

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

aromatase activity in this assay. 

Estrogen Receptor (ER) Binding Assay (OCSPP 890.1250) 

Requirement satisfied by OSRI (Ashby et al, 2005); see Appendix 2. 

ERα Transcriptional Activation (ERTA) Assay (OCSPP 890.1300) 

Requirement satisfied by OSRI (Ashby et al, 2005); see Appendix 2. 

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Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350) 

The 21-day short-term reproduction assay (MRID 49042102) of PCNB with fathead minnow 

(Pimephales promelas) was conducted under flow-through conditions. Adult fish (2 males and 4 

females/group; 4 groups/treatment; ~5.5 months old) were exposed to nominal concentrations of 

PCNB (95.6% purity) of 0 (negative control), 0.0010, 0.010, and 0.100 mg a.i./L.; meanmeasured concentrations were <0.000600 (<LOQ; control), 0.00066, 0.0071, and 0.076 mg 

a.i./L. Reviewer-calculated time-weighted average (TWA) concentrations were 0.00063, 0.0069, 

and 0.074 mg a.i./L. The test system was maintained at 24.1 to 25.6°C and a pH of 7.6 to 8.3. 

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

water) control. Survival was 100% in all treatment groups, and all fish appeared normal and 

healthy throughout the exposure period, with the exception of two females in the high treatment 

group who appeared to be eggbound. One female was observed to be eggbound from Days 7 to 

21, while the other female was observed to be eggbound on Days 18 and 19. PCNB exposure 

was associated with a concentration-dependent increase in body weight in female fish, with 

significant promotion in the high treatment group, relative to the negative control. 

Fecundity exceeded 15 eggs/female/day in each of the control replicates (mean 18.7 

eggs/female/day/replicate; range 16.4-23.8), although each replicate included 4- or 5-day periods 

without spawning. Fertilization success in controls was 97.4%. PCNB significantly decreased 

fecundity by 67% (p<0.01) at the high treatment concentration; fertilization success was 

comparable between the control and treatment groups. Plasma vitellogenin (VTG) levels in male 

and female fish were unaffected at all treatment levels relative to the negative control. No 

treatment-related effects on gonadal histopathology were observed in male fish at any treatment 

level; however, treatment-related effects were noted in females at the high treatment level. There 

was an increase in median gonadal staging (3.3 vs. 2.5 in control), and histopathological effects 

included decreased post-ovulatory follicles and increased oocyte atresia. A significant 

concentration-dependent increase in gonado-somatic index (GSI) was observed in male fish in 

the mid and high treatment groups (↑42 and 59%; p<0.05 and <0.01) and in female fish in the 

high treatment group (↑35%; p<0.05; Jonckheere-Terpstra). Nuptial tubercle score was 

unaffected by PCNB exposure in males; no tubercles were observed on females. 

Hershberger Assay (OCSPP 890.1400) 

In a Hershberger assay (MRID 48916702) screening for androgenic activity, PCNB (95.6% a.i., 

Lot# 107030L052) in aqueous sodium carboxymethyl cellulose (1% SCMC) was administered 

daily via oral gavage to groups of 55-day old, castrated male Sprague Dawley (Crl:CD[SD]) rats 

(10/group) at dose levels of 0 (vehicle), 300, or 1000 mg/kg/day. The androgenic assay included 

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a positive control group consisting of 10 castrated rats exposed to 0.4 mg/kg/day of testosterone 

propionate (TP) by subcutaneous (s.c.) injection. 

To screen for potential anti-androgenic activity, PCNB in 1% SCMC was administered daily via 

oral gavage to 54-day old, castrated male Sprague Dawley rats (10/group) at dose levels of 100, 

300, or 1000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 

mg/kg/day by s.c. injection. The control group for the anti-androgenic assay consisted of ten 

castrated male rats dosed daily with TP (0.4 mg/kg/day) in corn oil by s.c. injection, and the 

positive control group consisted of ten castrated male rats dosed orally with flutamide (FT) in 

corn oil at 3 mg/kg/day in conjugation with a daily s.c. dose of TP (0.4 mg/kg/day). 

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

necropsied approximately 24 hours after the final dose administration to determine weights of 

the five androgen-dependent tissues. 

In the androgen agonist assay, body weights and body weight gains for the 300 and 1000 

mg/kg/day treatment groups were comparable to controls throughout the assay. Animals dosed 

with TP had increased (p≤0.05) body weights on Days 8-11 (↑5-8%) and overall body weight 

gains (↑48%) compared to controls. There were no treatment-related weight increases in any of 

the accessory sex organs of animals dosed with PCNB. At 1000 mg/kg/day, the adjusted liver 

weights were increased by 8% (p≤0.05) and the unadjusted liver weights were increased by 7% 

(not significant, NS). Animals in the TP group had increases (p≤0.01) in unadjusted accessory 

sex organ weights as follows: 750% in seminal vesicles, 1316% in ventral prostate, 179% in 

levator ani-bulbocavernosus (LABC), 518% in Cowper’s gland, and 50% in glans penis. The 

unadjusted liver weights in the TP group were increased by 15% (p≤0.01) and the adjusted liver 

weights were increased by 8% (p≤0.05). All %CV values were less than or equal to the 

maximum recommended values. 

In the anti-androgen assay, body weights and body weight gains for the 100, 300, and 1000 

mg/kg/day dose groups were comparable to controls throughout the assay. No treatment-related 

decreases in weights of androgen sensitive tissues were observed in any PCNB dose group, 

although there was a 15% increase (p≤0.05) in Cowper’s gland weights at 1000 mg/kg/day. No 

treatment-related decreases were observed in any of the accessory sex organs of PCNB-dosed 

animals. The unadjusted liver weights were increased by 8% (NS) at 1000 mg/kg/day. Animals 

dosed with TP + FT (positive control) had decreases (p≤0.01) in unadjusted accessory sex organ 

weights as follows: 76% in seminal vesicles, 72% in ventral prostate, 53% in LABC, 58% in 

Cowper’s gland, and 27% in glans penis. The unadjusted liver weights in the TP + FT group 

were increased by 11% (p≤0.05) and the adjusted liver weights were increased by 10% (p≤0.01). 

All %CV values were less than or equal to the maximum recommended values. 

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The dosing levels selected for the study were acceptable as animals were dosed up to limit dose 

(1000 mg/kg/day) for the assay. 

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

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

assay. 

Female Pubertal Assay (OCSPP 890.1450) 

In a Female Pubertal Assay (MRID 49042103A), 15 Sprague Dawley [Crl:CD® (SD) IGS BR] 

rats/dose group were treated daily via oral gavage with PCNB (95.6% a.i.; Lot # 107030L052) in 

aqueous 1% sodium carboxymethyl cellulose at doses of 0 (vehicle), 500, or 1000 (limit dose) 

mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined for vaginal opening 

(VO) daily beginning on PND 22, and age and weight at day of attainment were recorded. 

Following sacrifice on PND 42, blood was collected for clinical chemistry and hormone 

analyses. Total serum thyroxine (T4) was determined by radioimmuno-assay, but the method 

used for determination of thyroid stimulating hormone (TSH) levels was not reported. Adrenal, 

liver, pituitary, thyroid, and urogenital organs were weighed, and microscopic examinations were 

performed on the ovaries, uterus, thyroid, liver, and kidneys. 

There were no effects of treatment on mortality, clinical signs of toxicity, final body weights or 

body weight gains, food consumption, or gross pathology. Compared to the control group, 

absolute, adjusted, and relative (to body) liver weights were increased (p<0.05) by 6-8% at 500 

mg/kg/day and by 6% at 1000 mg/kg/day, and absolute, adjusted and relative kidney weights 

were increased (p<0.01) by 6-8% at500 mg/kg/day and by 8% at 1000 mg/kg/day. The 

following dose-dependent changes in clinical chemistry parameters were observed at 500 and 

1000 mg/kg/day, respectively: alkaline phosphatase (ALP) activity was increased (p<0.05) by 

20 and 26%; alanine aminotransferase (ALT) activity was decreased (p<0.001) by 42 and 51%; 

total bilirubin was increased (p<0.01) by 44 and 56%; and urea nitrogen was increased (p<0.05) 

by 8 and 15%. Additionally at 1000 mg/kg/day, albumin was decreased (p<0.05) by 3% and 

total protein was decreased (p<0.01) by 4%. 

There were no effects of treatment on age and body weight at attainment of VO. All females 

achieved complete VO. There were no effects of treatment on mean age at first vaginal estrus, 

mean cycle length, % cycling or regularly cycling, or cycle status at necropsy. Antral and 

tertiary follicles, corpora lutea, and atretic follicles were present in the ovary of all individuals, 

and the numbers of primordial and primary follicles showed no biologically relevant differences 

across groups. The number of 1000 mg/kg/day females classified in metestrus by examination of 

the uterus (with cervix) was increased (6 treated vs. 1 control; p<0.05); however, the biological 

significance of this finding is unclear. 

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Serum T4 levels were decreased (p<0.001) by 70% at 500 mg/kg/day and by 72% at 1000 

mg/kg/day. Concomitantly, serum TSH levels were increased (p<0.001) by 53% at 500 

mg/kg/day and by 59% at 1000 mg/kg/day. No significant changes were observed in thyroid 

histology; however, there was a slight, dose-related trend toward decreased colloid area and 

increase follicular cell height. 

The dose levels tested in assay are adequate since the high dose was the Limit Dose (1000 

mg/kg/day). 

Male Pubertal Assay (OCSPP 890.1500) 

In a Male Pubertal Assay (MRID 49042103), 15 Sprague Dawley [Crl:CD® (SD) IGS BR] 

rats/dose group were treated daily via oral gavage with PCNB (95.6% a.i.; Lot # 107030L052) in 

aqueous 1% sodium carboxymethyl cellulose at doses of 0 (vehicle), 500, or 1000 (limit dose) 

mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined for preputial separation 

(PPS) daily beginning on PND 30, and age and weight at day of attainment were recorded. 

Following sacrifice on PND 53, blood was collected for clinical chemistry and hormone 

analyses. Total thyroxine (T4) and testosterone serum levels were determined by 

radioimmunoassay, but the method used for determination of thyroid stimulating hormone (TSH) 

levels was not reported. Adrenal, liver, pituitary, thyroid, and urogenital organs were weighed, 

and microscopic examinations were performed on the epididymis, testis, thyroid, liver, and 

kidneys. 

There were no treatment-related effects on mortality, clinical signs of toxicity, body weights or 

body weight gains, food consumption, or gross pathology. Compared to the control, absolute, 

adjusted, and relative (to body) liver weights were increased (p<0.05) by 8-10% at 500 

mg/kg/day and by 10-11% at 1000 mg/kg/day, and relative kidney weights were increased 

(p<0.05) by 8 and 6% at 500 and 1000 mg/kg/day, respectively. Alanine aminotransferase 

(ALT) was decreased (p<0.001) by 38% at 500 mg/kg/day and by 52% at 1000 mg/kg/day and 

total bilirubin was increased (p<0.001) by 100% at 500 and 1000 mg/kg/day. Additionally at 

1000 mg/kg/day, alkaline phosphatase (ALP) was increased (p<0.05) by 15%. Minimal 

increased inflammatory foci with/without single cell necrosis was observed in livers of 5/15 

males at 1000 mg/kg/day (NS) compared to 2/15 controls. 

There were no effects of treatment on age and body weight at attainment of PPS. All males 

achieved complete PPS, and serum testosterone levels were unaffected by treatment. There were 

also no treatment-related changes in weights or microscopic findings for the male reproductive 

tissues. 

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Compared to the controls, serum T4 levels were decreased (p<0.001) by ≥78% at 500 and 1000 

mg/kg/day, with T4 levels being below the method limit of detection (<LOD, 0.82μg/dL) in 6/15 

males at 500 mg/kg/day and 12/15 males at 1000 mg/kg/day. Concomitantly, serum TSH levels 

were increased (not significant [NS]) by 39 and 25% at 500 and 1000 mg/kg/day, respectively. 

However, no treatment-related changes were noted in thyroid histology. 

The high dose (1000 mg/kg/day) was adequate based on increased liver and kidney weights, and 

changes in clinical chemistry parameters in the study, and considering that 1000 mg/kg/day is the 

limit dose for the assay. 

Steroidogenesis Assay (OCSPP 890.1550) 

In a steroidogenesis assay (MRID 48916704), H295R cells cultured in vitro in 24-well plates 

were incubated with PCNB (95.6% purity, Lot # 107030L052 at log concentrations of 0.0001 to 

100 μM for 48 hours in triplicate for three independent experiments. Dimethyl sulfoxide 

(DMSO) was used as the vehicle, at a final concentration of 0.1%. 

Testosterone and estradiol levels were measured with specific EIA kits. A Quality Control (QC) 

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

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

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

and testosterone production. 

Precipitation and/or cell cytotoxicity was not observed at PCNB concentrations of ≤ 20 μM. 

However, cell viability was reduced to 78% at 30 μM in Run 2 and 31% at 100 μM in Run 1, 

and precipitation was also noted at 100 μM in Run 1. Therefore, the highest PCNB 

concentrations evaluated were 10 μM in Runs 1 and 2 and 20 μM in Run 3. 

For the test plates, the coefficients of variation (% CVs) ranged from 4.8–5.8% for absolute 

testosterone concentrations and 1.7–3.2% for absolute estradiol concentrations in the solvent 

control (SC) replicate wells within the test plates. The between-plate CVs for hormone 

concentrations in the SC wells were 13.6% for testosterone and 50.3% for estradiol. The withinplate CVs were low and met the performance criteria (≤30%), as did the between-plate CV for 

testosterone. Although the between plate CVs for estradiol exceeded the guideline performance 

criteria, the estradiol data is considered acceptable in light of the low within-plate CV values. 

Basal hormone production in the SC wells was acceptable both in terms of the absolute 

concentrations and relative to the method’s lower limit of quantitation (LLQ). Compared to SC 

wells, 10 μM forskolin induced testosterone production on average by 2.26-fold, but induced 

estradiol by an unknown factor. For estradiol, the fold induction resulting from forskolin could 

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not be confirmed to be above the guideline specified >7.5-fold for the first two assay runs 

because the estradiol levels were above the maximum limit of quantitation (MLQ); however, 

based on the MLQ, the minimum levels of induction for estradiol production were greater than 

5.12-, 3.12- and 7.70-fold in Runs 1, 2 and 3, respectively. Compared to the SC, 1 μM 

prochloraz inhibited the synthesis of testosterone on average to 0.38-fold and estradiol to 0.09-

fold. The range of %CV for hormone concentrations for the SC replicates within the QC plates 

was 1.5–4.9% for testosterone and 2.5–5.2% for estradiol. The between plate CVs for the SC 

wells was 15.9% for testosterone and 45.0% for estradiol. Similar to the test plates, the withinplate CVs for hormone concentrations on the QC plates were low and met the performance 

criteria. The performance criteria (≤ 30%) for between-plate CVs for the QC plates was also met 

for testosterone, but exceeded for estradiol; as with the between-plate value for estradiol on the 

test plates, this value is considered acceptable in light of the within-plate CV values. 

There were no significant differences in testosterone concentrations in the PCNB-treated cells 

relative to the SC, or in estradiol concentrations at PCNB levels ≤1 μM. PCNB caused small, 

but statistically significant (p ≤ 0.05), increases in estradiol concentrations in the culture medium 

at 10 μM in all three runs (1.09- to 1.21-fold) and at 20 μM in the third run (1.36-fold). 

However, all significant increases were <1.5-fold relative to the SC. 

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

statistically significant and reproducible increases in estradiol production, but did not result in 

statistically significant and reproducible alterations in testosterone production.

Uterotrophic Assay (OCSPP 890.1600) 

Requirement satisfied by OSRI (Ashby et al, 2005); see Appendix 2. 

. 

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APPENDIX 2. Other Scientifically Relevant Information (OSRI) 

ER Transactivation and Binding Assays 

In a multi-component, published study by Ashby et al. (2005), PCNB was tested using several 

assays for estrogenic/anti-estrogenic activity including in vitro competitive binding assays to 

recombinant human α and β estrogen receptors (ERs; hERα and hERβ) or rat uterine ER and an 

in vitro yeast-based hER assay. PCNB (99% purity) was obtained from Sigma Chemicals (Poole, 

Dorset, UK) and was prepared as required for each assay. 

Competitive binding assays with immature rat uterine cytosolic ER and hERα and hERβ were 

performed according to procedures that were previously described for the rat assay. Briefly, 

recombinant hERα and hERβ were diluted in hER buffer, and incubated for 17 h at 4°C with 

DMSO or 10-fold dilutions of either 17β-estradiol (E2; 5 x 10-10 to 5 x 10-4 M) or PCNB (5 x 10-

10 to 5 x 10-4 M; both prepared in DMSO) in hER buffer containing [2,4,6,7-3

H]E2 (~0.22 mCi, 

88 Ci/mmol) at a final concentration of 5 x 10-9 M E2. Receptor-ligand complexes were 

precipitated with 0.25 mL high resolution hydroxylapatite in hER buffer, centrifuged, and 

washed with hER buffer for determination of radioactivity by liquid scintillation counting. 

For the yeast hER assay, yeast cells transfected with the hERα gene together with expression 

plasmids containing an estrogen responsive element and the lac-Z reporter gene (encoding the 

enzyme β-galactosidase), were incubated in medium containing the test chemical and the 

chromogenic substrate, CPRG. Active ligands (which bind to the receptor) induce βgalactosidase expression, causing CPRG (initially yellow) to change into a red product that can 

be measured by absorbance. PCNB was serially diluted in ethanol and transferred to a 96-well 

flat bottom microtiter plate and allowed to evaporate to dryness. To test for anti-estrogenic 

activity, E2 was added at 5 nM (a concentration determined to produce a sub-optimal assay 

response). Following addition of the assay medium containing recombinant yeast and CPRG, the 

plates were sealed, shaken for 2 minutes, and incubated at 32°C for 3 days. Color development 

was measured at 540 nm using a plate reader. Niacinamid and 4-(2-hydroxyethyl)-1-piperazine 

ethanesulfonic acid (Hepes) were used as negative controls, and hydroxytamoxifen was used as a 

positive control anti-estrogen. 

The ER binding assays provided equivocal evidence that PCNB competed with E2 in binding to 

hERα. Although the binding curve was reproducible, no more than ~25% displacement could be 

achieved. The authors noted that this apparent displacement could be an artifact of the 

diminished aqueous solubility of PCNB. No binding of PCNB to hERβ or rat uterine ER was 

observed. Overall, it was concluded that PNCB does not show biologically relevant binding to 

ER. 

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In the yeast hER assay, PCNB did not show any anti-estrogenic activity. The anti-estrogen 

hydroxytamoxifen gave the expected inhibition of E2-induced activation of β-galactosidase, and 

the negative controls niacinamide and Hepes showed no effect on E2-induced activation of 

β-galactosidase. 

Based on these results, PCNB is neither an estrogen nor an anti-estrogen in vitro. 

ER Transactivation and Binding Assays

In a published study by Kojima et al. (2004), 200 pesticides (including PCNB; purity >99%) 

were evaluated in transactivation assays for hERα, hERβ, or hAR in CHO cells. PCNB was 

tested over a concentration range from 10−8 to 10−5 M, and the responses were compared to the 

activity of 17β-estradiol or dihydrotestosterone (DHT) for the ER or AR assays, respectively. In 

addition, for measurement of antagonist activity, PCNB was co-administered with 17β-estradiol 

(10−11 M for ERα; 10−10 M for ERβ) or DHT (10−10 M) for the ER and AR transactivation assays, 

respectively. It was reported that PCNB had neither estrogenic nor anti-estrogenic activity using 

transfected Chinese Hamster Ovary-K1 cells. 

ER Transactivation Assays 

In a published study by Vinggaard et al. (1999), a total of 20 pesticides, including PCNB (purity 

97-99%), were tested in an MCF-7 cell proliferation assay and in a yeast estrogen screening 

(YES) assay. MCF-7 cells were incubated at 0.001, 0.1, 1,0 and 10 μM with PCNB, and 

proliferation was determined by measuring DNA content. After 6 days of exposure, PCNB did 

not have any effect on cell proliferation. PCNB was negative in the YES screen, but no data were 

provided.

ER Transactivation Assays 

In a published study by Zou et al. (2002), a cell proliferation assay with MCF-7 cells was used to 

determine the effects of PCNB (purity 99%) on the ER and ligand independent c-Neu signaling 

pathways. PCNB tested at 100 nM exerted an inhibitory effect on the formation of MCF-7 cell 

foci formation through a non-ER binding pathway. 

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Uterotrophic Assay 

In a multi-component, published study by Ashby et al. (2005, MRID 48233802), PCNB was 

tested using several assays for estrogenic/anti-estrogenic activity including in vivo weanling rat 

uterotrophic assays assessing both estrogenic and anti-estrogenic activity. PCNB (99% purity) 

was obtained from Sigma Chemicals (Poole, Dorset, UK) and was prepared as required for each 

assay. For in vivo testing, Alpk:APfSD (Wistar derived) female rats were obtained from the 

AstraZeneca breeding unit (Alderley Park, UK). Rats used for the weanling assays were 18-19 

day old (37-45 g). The animals were acclimated for 1 day for the weanling assay; food and water 

were provided ad libitum. 

In the weanling rat uterotrophic assays (Experiments 2-6), PCNB was tested for estrogenic and 

anti-estrogenic activity. Test compounds were administered by subcutaneous injection in peanut 

oil (dose volume 5 mL/kg) once daily, beginning on PND 19 or 20, for 3 days. Compounds that 

were co-administered were injected at different sites within 5 min of each other. In Experiments 

2 and 3, PCNB was administered at 800 mg/kg/day in the presence and absence of estradiol (E2) 

over a dose range of 1-10 μg/kg/day; Faslodex (positive control) was administered at 0.5 

mg/kg/day in the presence and absence of E2 at 10 μg/kg/day. In Experiment 4, a dose response 

study for PCNB was conducted with doses of 100 to 800 mg/kg/day in the presence and absence 

of E2 (1 μg/kg/day). Experiments 5 and 6 used PCNB at 100 mg/kg/day, a concentration at 

which a clear inhibitory effect was observed and a dose at which PCNB remained soluble in 

peanut oil. PCNB was administered in the presence of either E2 at 0.1-5 μg/kg/day (Expt. 5) or 

diethylstilbestrol (DES), a synthetic estrogen, at 0.01-0.5 μg/kg/day (Expt. 6). Animals were 

killed as described above 24 h after the final dose. Uteri were removed and weighed. In 

Experiment 5, blood was collected in heparinized tubes by cardiac puncture, and plasma was 

prepared by centrifugation for use in various biochemical assays which measured the following: 

alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), and 

glutamate dehydrogenase (GLDH). Livers from animals in Experiment 5 were removed, 

weighed, and used to prepare microsomes and cytosol; liver preparations were stored at -70C 

until analysis for determination of protein content of liver microsomes and cytosol, cytochrome 

P450 (CYP) content of microsomes (including total CYP and CYP2B1/2 isoenzyme profiles), 

and estrogen sulfotransferase (EST) activity in liver cytosol. 

In the uterotrophic assays, PCNB failed to antagonize the uterotrophic activity of E2 in 

Experiments 2 and 3, while Faslodex abolished it. Unexpectedly, PCNB potentiated the 

uterotrophic activity of E2 at all dose levels of E2 evaluated. This potentiation by 800 mg/kg 

PCNB was maintained down to 100 mg/kg PCNB when evaluated against a fixed dose of 1 

μg/kg E2. In contrast, PCNB (100 mg/kg) had no effect on the uterotrophic activity of DES over 

the dose range of 0.01-0.5 μg/kg. The absence of a potentiating effect of PCNB on the 

uterotrophic activity of DES indicated that the potentiation by PCNB of the uterotrophic activity 

of E2 was specific to E2. 

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PCNB had no effect on liver weight or the plasma markers of hepatotoxicity ALP, AST, and 

GLDH. A small reduction in ALT (~75% of controls) was consistently observed in all groups 

receiving PCNB; this effect was considered to be biologically insignificant. PCNB in the 

presence and absence of E2 had no effect on total cytochrome P450 levels, and no changes in 

CYP2B1/2 levels were observed. PCNB treatment (in the presence and absence of E2) did not 

inhibit EST activity. The authors concluded that PCNB is neither an estrogen nor an antiestrogen in vivo. The enhancement of estradiol uterotrophic activity by PCNB was attributed to 

possible alteration of the metabolism of estradiol. 

Female Pubertal Assay

In a multi-component published study by Ashby et al. (2005), PCNB was tested using several 

assays for estrogenic/anti-estrogenic activity including an in vivo female pubertal rat assay for 

anti-estrogens. PCNB (99% purity) was obtained from Sigma Chemicals (Poole, Dorset, UK) 

and was prepared as required for each assay. For in vivo testing, Alpk:APfSD (Wistar derived) 

female rats were obtained from the AstraZeneca breeding unit (Alderley Park, UK). Rats used 

for the pubertal assay were 22-23 day old (up to 62 g). The animals were acclimated for 3 days 

prior to dosing for the pubertal assay; food and water were provided ad libitum. 

For the pubertal rat anti-estrogen assay (designated Experiment 1), all compounds were 

administered by subcutaneous injection in peanut oil (dose volume 5 mL/kg). PCNB was 

administered at 800 mg/kg/day, and the ER-antagonist Faslodex was administered 

subcutaneously at 0.5 mg/kg/day and served as a positive control anti-estrogen; control animals 

received peanut oil only. Compounds were administered once daily, beginning on post-natal day 

(PND) 25 or 26, until >95% of the rats had open vaginas (12 days of dosing). Vaginal opening 

was monitored daily. Animals were killed 24 h following final dosing (PND 37 or 38) by overexposure to halothane followed by cervical dislocation. Uteri were removed and weighed. A 

vaginal smear was taken at the time of death to determine the stage of estrous cycle. 

In the pubertal rat anti-estrogen assay, PCNB at 800 mg/kg had no effect on peri-pubertal uterine 

growth in female rats. PCNB had no effect on body weight, or on age and weight at vaginal 

opening, and increases in uterine weight were not related to treatment with PCNB. The positive 

control Faslodex inhibited uterine growth and delayed vaginal opening. 

The authors concluded that PCNB is neither an estrogen nor an anti-estrogen in vivo. 

Three-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a three-generation reproduction toxicity study (MRIDs 00001666 and 40865903), PCNB 

(98.2% purity) was administered to 25 CD Sprague Dawley rats/sex/dose via the diet at dose 

levels equivalent to 0, 5, 50, or 500 ppm (0, 0.25, 2.5, or 25 mg/kg/day) for 3 consecutive 

generations; with exposure continuing in dams through gestation, lactation and weaning. The P 

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generations received the test diet for 11 weeks prior to mating to produce the F1a offspring. After 

the F1a offspring were weaned and sacrificed, the P animals were mated again to produce the F1b

offspring. The F1 parental animals were mated when they reached 105 days of age to produce the 

F2a and F2b litters; parents for the third generation were selected from the F2b offspring. 

Significant differences in body weights were observed for the F2a females at weaning and the F2b

females at mating in the 5 ppm group (7.7% less than control), the F2b at mating and F3a at 

weaning in the 50 ppm group (9.7 and 7.9% less than control), and the F2b at the second mating 

(6.3% less than control). 

No treatment-related effects were seen in the male or female fertility index, viability index, 

lactation index, or mean litter size. No treatment-related gross pathological changes were seen in 

the offspring from the first litter in each generation (F1a, F2a and F3a). No treatment-related gross 

pathological changes were seen in the gonads, thyroid, adrenal and pituitary glands of the F3b

litters. 

The NOAEL is 500 ppm (25 mg/kg/day) for reproduction effects, and the LOAEL was not 

determined. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation reproduction toxicity study (MRID 41918701), PCNB (>99% purity) was 

administered to 26 Sprague Dawley rats/sex/dose via the diet at dose levels equivalent to 0, 20, 

3000, and 6000 ppm (0, 1.2, 169, or 344 mg/kg/day for males and 0, 1.5, 218, or 455 mg/kg/day 

for females) during the pre-mating period of 81-90 days (P or F1 generations, respectively); with 

exposure continuing in dams through gestation, lactation and weaning. The P generation rats 

were mated once to produce F1 litters, and the F1 generation rats were mated twice to produce F2a

and F2b litters. 

There were no treatment-related effects on mortality in either P or F1 animals. Clinical signs 

included small size in high-dose F1 males (21/26) and females (24/26) from study week 4 to 

week 15 (males) or 17 (females). Emaciation was observed in a few high-dose females early in 

the first F2a gestation period. In the 3000 ppm group, some small size males and one emaciated 

female was observed. Significant bodyweight reductions were observed in the mid- and highdose P generation males and females during the pre-mating periods and in the high-dose females 

during the first mating (gestation and lactation) periods. Food consumption was consistently 

reduced in the high-dose group with effects observed early during the study periods for both 

generations. No treatment-related effects were observed in the 20 ppm group. 

For the P parental animals, no treatment related reproductive effects were observed for male or 

female mating or fertility indices, gestation index, mean gestation length in either mating, mean 

number of live newborns, sex ratio and offspring survival. For the F1 parental animals, no 

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treatment-related effects were seen in male or female mating, fertility and gestation indices, and 

mean gestation length. Significant (p<0.01) decreases in mean number of male newborns in F2a

litters were seen at the mid-(6.1) and high (5.3) dose groups compared to controls (8.6). There 

were no changes in numbers of dead or stillborn pups at birth or in any survival indices for males 

throughout lactation. No changes were seen in number of F2a newborn females or the litter 

indices for F2b mating. 

No treatment-related histopathological changes were seen in the testes, epididymides, seminal 

vesicle, prostate, coagulating gland, ovaries, uterus, vagina, cervix, or pituitary glands of the P 

parental animals. 

The NOAEL and LOAEL for parental toxicity are 20 and 3000 ppm, respectively, based on 

systematic effects. The NOAEL and LOAEL for neonatal effects are 20 and 3000 ppm, 

respectively, based on decreased number of males in F2a litters. 

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation reproduction toxicity study (MRID 43469301), PCNB (98% purity) was 

administered to 35 Sprague Dawley rats/sex/dose by gavage at dose levels equivalent to 0, 10, 

100, or 1000 mg/kg/day in 0.1% carboxymethylcellulose/Tween. The parental animals (P) 

received the test material for 10 weeks prior to mating and the females continued to be dosed 

through weaning of the F1 generation. The F1 males and females were treated from weaning for 

at least 10 weeks prior to mating and through the mating period for the F2 litters. The females 

were treated until the weaning of the F2 offspring. 

There were no treatment-related effects on mortality, body weight, food consumption, or clinical 

signs in the parental animals. 

No treatment-related effects were seen in the reproductive parameters such as male or female 

mating, fertility and gestation indices, gestation length, mean litter size, mean number of live 

offspring or sex ratio in either generation. 

In the F1 generation, there were no treatment-related effects on the absolute or relative weights of 

the testes, epididymides, seminal vesicle, prostate, or ovaries. At 1000 mg/kg/day in the F1

generation, the absolute (0.128 g) ovarian weight was significantly (p<0.05) increased when 

compared to controls (0.109 g). There was no effect on the mean relative ovary weight or any 

corroborative histopathological lesions in the ovaries. In addition, one female had an ovarian 

weight that was 65% greater than the mean for the groups which may have skewed the results. 

Therefore, the ovarian weight increase is not considered to be toxicologically significant. 

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No treatment-related histopathological lesions were seen in the testes, epididymides, seminal 

vesicle, prostate, ovaries, uterus, vagina, cervix and pituitary glands of the P and F1 animals. 

Hepatocellular hypertrophy was observed in P males and females at 1000 mg/kg/day and F1 

males at 1000 mg/kg/day, and thyroid follicular cell hypertrophy/hyperplasia was observed in 

100 and 1000 mg/kg/day males and 1000 mg/kg/day females of the P and F1 generations. In the 

P generation adults, the incidences of thyroid follicular cell hypertrophy/hyperplasia were 

increased in males at 100 and 1000 mg/kg/day (31 and 74% vs. 16% in controls) and in females 

at 1000 mg/kg/day (67% vs. 20% in controls). In F1 generation adults, the incidences of thyroid 

follicular cell hypertrophy/hyperplasia were also increased in males at 100 and 1000 mg/kg/day 

(38 and 91% vs. 15% in controls) and in females at 1000 mg/kg/day (54% vs. 14% in controls). 

The NOAEL values are 10 mg/kg/day in males and 100 mg/kg/day in females based on thyroid 

follicular cell hypertrophy/hyperplasia. The LOAEL values are 100 mg/kg/day in males and 

1000 mg/kg/day in females. 

Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40588601) groups of 25 pregnant Sprague 

Dawley COBS CD rats were administered PCNB (96.0% purity) in 0.2% 

carboxymethylcellulose by oral gavage at dose levels of 0, 30, 600, or 1200 mg/kg/day during 

GD 6-15. Surviving rats were euthanized on GD 20. 

There were no deaths and no treatment related clinical signs were observed in any group. Body 

weights were slightly reduced (not significant) in the high dose group during the entire gestation 

period. Food consumption was significantly decreased in the high dose group on days 6-9. No 

other significant differences in food consumption were observed. The maternal NOAEL is 1200 

mg/kg/day, and the LOAEL was not determined. 

No treatment-related changes were observed in the pregnancy rate, number of corpora lutea, 

implantation sites, post implantation loss, early or late resorptions/dam, number of live 

fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose. The developmental NOAEL 

is 1200 mg/kg/day, and the LOAEL was not determined. 

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Developmental Toxicity (Rat, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 41361201) groups of 25 pregnant CRL:

CDTM(SD)BR Sprague Dawley rats were administered PCNB (98.5% purity) in 1% 

carboxymethylcellulose by oral gavage at dose levels of 0, 250, 750, or 1500 mg/kg/day during 

GD 6-15. Surviving rats were euthanized on GD 21. 

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

group. The decrease in bodyweight gain between days 6 and 7 was not considered 

toxicologically significant. Food consumption was decreased (not significant) in the mid- and 

high-dose groups during the first 3 days of treatment. The maternal NOAEL is 1500 mg/kg/day, 

and the LOAEL was not determined. 

No treatment-related changes were observed for pregnancy rate, number of corpora lutea, 

implantation sites, post implantation loss, early or late resorptions/dam, number of live 

fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose. Significant decreases in the 

number of fetuses with an incompletely ossified pubis were observed in all dose groups. For the 

mid- and high-dose groups, significant decreases or increases were observed in the number of 

ossification sites in the lumbar vertebrae, in the thoracic vertebrae, and pairs of thoracic ribs. The 

developmental NOAEL is 250 mg/kg/day, and the LOAEL is 750 mg/kg/day. 

Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 41361301), groups of 20 pregnant New 

Zealand White rabbits were administered PCNB (98.5% purity) in 1% aqueous 

carboxymethylcellulose by oral gavage at dose levels of 0, 100, 300, and 900 mg/kg/day during 

GD 6-18. Surviving rabbits were euthanized on GD 29. 

There were no deaths observed in any group. In the high dose group, two doses were aborted 

with clinical signs of toxicity including soft or liquid feces, dried feces, weight loss, decreased 

food consumption, gastric ulceration and/or litter with six conceptuses. One high dose doe 

delivered prematurely on day 28 and exhibited the clinical signs of weight loss, decreased food 

consumption, soft or liquid feces, decreased motor activity, and fluid-filled caecum. The mean 

bodyweight decreases and reduced food consumption observed in the high dose group were not 

statistically significant. The maternal NOAEL is 300 mg/kg/day, and the LOAEL is 900 

mg/kg/day based on clinical signs of toxicity. 

No treatment-related changes were observed for pregnancy rate, number of corpora lutea, 

implantation sites, post implantation loss, early or late resorptions/doe, number of live 

fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose. The developmental NOAEL 

is 900 mg/kg/day, and the LOAEL was not determined.

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Developmental Toxicity (Rabbit, OCSPP 870.3700) 

In a prenatal developmental toxicity study (MRID 40717102), groups of 20 pregnant New 

Zealand White rabbits were administered PCNB in 1% aqueous carboxymethylcellulose by oral 

gavage at dose levels of 0, 6.25, 12.5, 125, or 250 mg/kg/day during GD 7-19. Surviving rabbits 

were euthanized on GD 29.

No treatment-related changes were seen in pregnancy rate, number of corpora lutea, implantation 

sites, post implantation loss, early or late resorptions/doe, number of live fetuses/litter, fetal sex 

ratio, or soft tissue abnormalities at any dose. 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study (MRID 41987301, 1991), groups of 50 CD 

rats/sex were fed diets containing PCNB (99.4% purity) at levels equivalent to doses of 0, 1, 150, 

or 300 mg/kg/day (0, 20, 3000, or 6000 ppm) for 104 weeks. Additional rats were randomly 

allocated for 12-month (10 rats/sex/group) interim sacrifice. For clinical chemistry examinations, 

additional groups of 10 rats/sex were sacrificed at the 6-, 12-, 18- and 24-month intervals. 

Organs that were weighed included the liver, kidneys, testes, ovaries, brain, adrenals, and 

thyroid/parathyroids. Gross and microscopic pathological examinations were conducted on these 

tissues as well as the prostate, seminal vesicles, uterus, and mammary gland. 

No treatment related clinical signs or differences in mortality were observed during the study. In 

the high-dose male and female rats, body weight gains were decreased (p<0.05) up to 10% 

compared to control throughout the study. The occasionally significant body weight differences 

observed in the mid-dose males and females were not considered biologically significant; 

however, mean body weights in high-dose males and females were decreased by 11% by study 

termination (Week 104). Food consumption, as g/animal/day, was significantly lower for highdose males during the first 10 weeks of the study. Food consumption, as g/kg/day, was 

significantly higher in the high-dose males and females. 

No treatment-related effects were observed for hematological or urinalysis parameters. 

Significant increases in cholesterol were observed in high-dose females. No other clear 

treatment-related responses were observed in significant clinical chemistry results (within 

historical ranges). No treatment related changes in absolute or relative weights were seen in the 

testes, ovaries or adrenal glands in either sex at any dose level. 

In the liver, absolute and relative weights were increased in males at the high dose by 20 and 

24%, respectively, and relative weights were increased by 20% in high-dose females. Histologic 

findings in the liver included increased hepatocellular hypertrophy at the mid and high doses in 

both sexes. For thyroids, the absolute and/or relative weights were significantly (p <0.01) 

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increased in both sexes at the mid and high dose groups at the terminal sacrifice. Absolute 

thyroid weights were increased in males and females by 26 and 21%, respectively, at the mid 

dose, and by 45 and 32% at the high dose. In addition, thyroid glands were enlarged in males at 

the high dose (3/49; 6%) compared to control males (0/49). 

Non-neoplastic lesions in the high-dose males and females included colloid cysts, hypertrophy, 

cystic follicular cell, and/or hyperplasia. The neoplastic lesions mid- and high-dose males and 

females included thyroid follicular cell adenomas, carcinomas and/or adenomas plus carcinomas 

combined. No treatment-related histopathological lesions were observed in the testes, prostate, 

ovaries, uterus, and cervix, mammary and adrenal glands. 

PCNB is considered oncogenic in both sexes of rats dosed at 150 mg/kg/day (≥3000 ppm) based 

on thyroid follicular cell adenomas. The NOAEL is 1 mg/kg/day (20 ppm), and the LOAEL is 

150 mg/kg/day (≥3000 ppm). 

Chronic Toxicity/Carcinogenicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study (MRID 43015801, 1993), groups of 60 

Crl:CDBR Sprague-Dawley rats/sex were dosed with PCNB (98% purity) in 1% carboxy 

methylcellulose/0.1% Tween by gavage at doses of 0, 5, 50, 500, or 1000 mg/kg/day for 104 

weeks. The dose levels adjusted for continuous exposure are equivalent to 0, 3.6, 36, 357, or 714 

mg/kg/day, respectively. Additional rats were randomly allocated for 12-month 

(10 rats/sex/group) interim sacrifice. For clinical chemistry examinations, additional groups of 

10 rats/sex were sacrificed at the 6-, 12-, 18- and 24-month intervals. Organs that were weighed 

included the liver, kidneys, testes, ovaries, uterus, brain, adrenals, and thyroid/parathyroids. 

Gross and microscopic pathological examinations were conducted on these tissues as well as the 

prostate, seminal vesicles, and mammary gland. 

No treatment-related effects on clinical signs, mortality, body weight, or food consumption were 

observed during the study. Dose-dependent decreases in serum AST and ALT and excretion of 

triple phosphate crystals in urine were observed in both sexes of all dose groups. The mean 

absolute thyroid weight of males at the high dose (0.0612 g) was increased by 22% (NS) 

compared to control males (0.0502 g), and the relative thyroid weight was increased by 24%. No 

treatment-related changes were seen in absolute or relative weights of the testes, ovaries, uterus 

or adrenal glands. 

At the high dose, increased incidences of non-neoplastic lesions of the thyroid glands were 

observed in both sexes. Follicular cell hypertrophy/hyperplasia of thyroids was observed in 

12/60 (20%) males and in 10/60 (16%) females at the high dose compared to control males (3%) 

and females (2%), respectively. Significantly increased hepatocellular hypertrophy (minimal to 

slight) was observed in ≥36 mg/kg/day males (p<0.05) and in ≥357 mg/kg/day females (p<0.01). 

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No treatment-related histopathological lesions were observed in the testes, seminal vesicles, 

prostate,!ovaries, uterus, mammary, adrenal or pituitary glands. 

Based on thyroid follicular cell hypertrophy/hyperplasia, the NOAEL is 3.6 mg/kg/day for males 

and 36 mg/kg/day for females. The LOAEL is 36 mg/kg/day for males and 357 mg/kg/day for 

females. 

Carcinogenicity (Mouse, OCSPP 870.4200)

In a carcinogenicity study in mice (MRID 00114224), Swiss mice (100/sex/dose group) were fed 

diets containing PCNB (98.2% purity) at nominal concentrations of 0, 100, 400, or 1200 ppm (0, 

15, 60, or 180 mg/kg/day) for 80 weeks. Organs that were weighed included the liver and 

kidneys. No male or female reproductive organs were weighed. The tissues subjected to 

histological examination included liver, kidneys, gonads, pituitary, thyroid, uterus, and seminal 

vesicles. 

No treatment-related effects were observed on mortality or clinical signs. In the 1200 ppm males, 

significant decreases (≤7%) in mean body weight were observed after the Week 43. The 

hematological changes were within historical ranges and included decreased lymphocytes and 

increased neutrophils. In the 1200 ppm females, the relative (to body) kidney weight was 

significantly increased by 21%. Relative (to body) liver weights were also significantly increased 

in males and females at the mid dose (↑10-11%) and high dose (40-54%). 

No treatment-related histopathological lesions were observed in the testes, seminal vesicle, 

coagulating gland, ovaries, uterus, thyroid and pituitary glands. Lesions were observed in the 

liver and lungs. 

Based on increased liver to body weight ratios, the LOAEL is 60 mg/kg/day (400 ppm) and the 

NOAEL is 15 mg/kg/day (100 ppm).

Carcinogenicity (Mouse, OCSPP 870.4200) 

In a carcinogenicity study (MRID 45609101), B6C3F1 mice (50/sex/dose group) were fed diets 

containing PCNB (98.2% purity) at nominal concentrations of 0, 2500, or 5000 ppm (0, 375, or 

750 mg/kg/day) for 103 weeks. The control group only contained 49 male mice. Organ weights 

were not determined. The tissues subjected to histological examination included the kidneys, 

liver, adrenal glands, pituitary, thyroid/parathyroid, ovaries, uterus, mammary glands testes, 

prostate, and seminal vesicles. 

Mortality was significantly increased by study termination in females at 5000 ppm (22/50) 

compared to the control (13/50). No treatment-related effects were reported for body weights. 

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No treatment-related histopathological lesions were observed in the testes, ovaries, seminal 

vesicle, prostate, uterus, mammary, thyroid, adrenal and pituitary glands. No treatment related 

nonneoplastic or neoplastic lesions were observed during the study. PCNB is not considered 

carcinogenic in mice under the conditions of this study. 

Based on mortalities, the LOAEL is 750 mg/kg/day (5000 ppm) and the NOAEL is 375 

mg/kg/day (2500 ppm).

Chronic Toxicity (Dog, OCSPP 870.4300) 

In a chronic toxicity study (MRID 41718601), groups of 6 beagle dogs/sex were fed diets 

containing PCNB (99.4% purity) at levels equivalent to doses of 0, 0.375, 3.75, or 37.5 

mg/kg/day (0, 15, 150, or 1500 ppm) for one year. Organs that were weighed included the liver, 

spleen, kidneys, testes, ovaries, brain, pituitary, adrenals, thyroid and parathyroids. Gross and 

microscopic pathological examinations were conducted on these tissues as well as the prostate, 

seminal vesicles, uterus, and mammary glands. 

No treatment-related effects on mortalities, clinical signs, body weights, or food consumption 

were observed during the study. No treatment-related effects were observed for the 

hematological, urinalysis, or gross pathology parameters. At the high dose, significant increases 

were observed for alkaline phosphatase in males and females (p<0.05) and for cholesterol in 

males (p<0.05). Significant decreases in ALT (p<0.01) were observed in both sexes of the mid 

and high dose groups. No treatment-related changes were seen in absolute or relative weight of 

the testes, ovaries, adrenals, or thyroid. Liver weights were significantly increased in high-dose 

males (relative, ↑26%) and females (relative, ↑30%; absolute, ↑39%), and relative (to body) 

kidney weights were increased in high-dose females (↑17%). No treatment-related 

histopathological lesions were seen in the testes, ovaries, thyroid, adrenal or pituitary glands. 

Mild hepatocyte hypertrophy was observed for both sexes in the high dose group. 

The NOAEL is 3.75 mg/kg/day (150 ppm), and the LOAEL is 37.5 mg/kg/day (1500 ppm). 

Chronic Toxicity (Dog, OCSPP 870.4300) 

In a chronic toxicity study (MRID 00001667), groups of 4 beagle dogs/sex were fed diets 

containing PCNB (98.2% purity) at levels equivalent to doses of 0, 0.125, 0.75, 4.5, or 27 

mg/kg/day (0, 5, 30, 180, or 1080 ppm) for two years. Organs that were weighed included the 

heart, spleen, kidneys, liver, and testes. Gross and microscopic pathological examinations were 

conducted on these tissues as well as the pituitary, thyroid, adrenal, and gonads. 

No treatment-related effects on mortalities, clinical signs, body weights, or food consumption 

were observed during the study. No treatment-related effects were observed for the urinalysis 

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parameters. Significantly decreased hematocrit values were observed in the 30 and 180 ppm 

groups. In the high dose group, significant increases in the SGOT and SAP levels were observed. 

Absolute and relative (to body) liver weights were increased by 25 and 39%, respectively, in 

high-dose males and by 26 and 39% in high-dose females, compared to the controls. No 

treatment-related changes were seen in absolute or relative testicular weights. No treatmentrelated histopathological lesions were seen in the testes, ovaries, thyroid, adrenal or pituitary 

glands. Cholestatic hepatosis with secondary bile nephrosis was observed in all dogs in the 180 

and 1080 ppm groups. 

The NOAEL is 0.75 mg/kg/day (30 ppm), and the LOAEL is 4.5 mg/kg/day (180 ppm). 

Subchronic Toxicity (Rat, OCSPP 870.3100) 

In a subchronic toxicity study (MRID 40636701), groups of 15 CD® rats/sex were fed diets 

containing PCNB (99.5% purity) at 0, 50, 3000 or 6000 ppm (equivalent to 0/0, 3.07/3.69, 

187/223 or 381/455 mg/kg/day in males and females, respectively) for 13 weeks. Organs that 

were weighed included the adrenals, brain, heart, kidneys, liver, testis and ovary. Gross and 

microscopic pathological examinations were conducted on these tissues as well as the mammary 

gland, pituitary, prostate, seminal vesicle, thyroid/parathyroid, and uterus. 

No treatment-related effects were observed on mortality, ophthalmic examination, hematology or 

gross pathology. At 3000 ppm, body weights in females were decreased (p<0.05) Weeks 10-13 

(↓5%; except Week 11) and body weight gains in males and females were decreased by 11-14%. 

Additionally, at 6000 ppm, body weights in males were decreased (p<0.05; except Week 11) 

throughout the study (↓7-10%), and body weights in females were decreased (p<0.05) during 

Weeks 6-13 (↓5-7%; except week 8). No treatment-related changes were observed in the 

absolute or relative weights of the testes, ovaries, or adrenal glands. No treatment-related 

histopathological lesions were observed in the testes, epididymides, seminal vesicle, prostate, 

ovaries, uterus, mammary, thyroid, adrenal or pituitary glands. Increased (p<0.05) relative liver 

weights were observed in males (↑24%) and females (↑9%) at the high dose and were associated 

with an increased incidence of liver hypertrophy; trace to mild liver hypertrophy was observed in 

7/15 males and 8/15 females, both compared to 0/15 in controls. 

The LOAEL is 3000 ppm (equivalent to 187/223 mg/kg/day in males/females) based on reduced 

body weight in females and reduced body weight gains in females. The NOAEL is 50 ppm 

(equivalent to 3.07/3.69 mg/kg/day). 

Subchronic Toxicity (Rat; OCSPP 870.3100)

In a subchronic toxicity study (MRID 42416001), groups of 10 Crl:CD BR Sprague-Dawley 

rats/sex were dosed PCNB (98% purity) in 2% carboxymethylcellulose/0.2% Tween by gavage 

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at nominal doses of 0, 5, 10, 100, or 1000 mg/kg/day for 13 weeks; actual dose levels were 

equivalent to 0, 4, 8.7, 100, or 985 mg/kg/day. Organs that were weighed included the liver, 

kidneys, testes, and ovaries. Gross and microscopic pathological examinations were conducted 

on these tissues, as well as the spleen, thymus, uterus, oviducts, mammary gland, adrenals, 

parathyroids, thyroid, and pituitary gland. 

 

There were no treatment-related effects on mortality, clinical signs, body weights, hematology, 

clinical chemistry, or gross pathology parameters. The significant increases in food consumption 

for treatment groups (not specified) compared to the controls were not considered biologically 

significant. No treatment-related changes were observed in the absolute or relative testicular or 

ovarian weights. In the high-dose males, the mean absolute kidney weight was significantly 

increased. In the high-dose females, the mean relative liver weight was significantly increased. 

No treatment-related histopathological lesions were observed in the testes, ovaries, oviducts, 

uterus, mammary, adrenal or pituitary glands. Changes in the thyroid glands included an 

increased incidence and severity of hypertrophy and hyperplasia of the follicular epithelium at 

the high-dose males (6/10; 60%) and females (5/10; 50%) compared to control males (2/10; 

20%) and females (0/10; 0%). In the liver, hepatocellular centrilobular hypertrophy was observed 

in both sexes of the high dose group. 

Based on the observed thyroid follicular epithelium hypertrophy and hyperplasia, the NOAEL is 

100 mg/kg/day and the LOAEL is 985 mg/kg/day. 

Special - Subchronic Toxicity (Rat, OCSPP 870.3100) 

In a subchronic toxicity study (MRID 42630801) designed to examine potential effects of PCNB 

on the thyroid, groups of 75 male CD® rats were fed diets containing PCNB (99.5% purity) at 0, 

20 or 6000 ppm (equivalent to 0, 1 or 300 mg/kg/day) for 90 days with a 90-day recovery period. 

Organs that were weighed included the liver, pituitary, and parathyroids/thyroids. Gross and 

microscopic pathological examinations were also conducted on these tissues. Blood samples 

were collected from15 rats/group sacrificed at 7, 14, 30, 90, and 183 days, for clinical chemistry 

measurements including T3, T4 and TSH levels. 

No treatment-related effects were observed on mortality, clinical signs, or gross pathology. At 

300 mg/kg/day, mean weekly body weights were significantly reduced throughout most of the 

exposure period, and overall body weight gain was decreased by 19%. Mean food consumption 

was significantly decreased at 300 mg/kg/day during Week 1. 

At 300 mg/kg/day, T3 levels were decreased 19-26% (p<0.05) compared to the controls on Days 

7, 14 and 90, T4 was decreased by 48-54% (p<0.05) on Days 7, 14, 30 and 90, and TSH was 

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increased by 33-132% (p<0.05) on Days 14, 30 and 90. Hormone levels in the high-dose group 

returned to the control group values following the 90-day recovery period. 

No treatment-related changes were observed in absolute or relative pituitary gland weights. At 

300 mg/kg/day, mean absolute thyroid weights were decreased on Day 30 (p<0.05) and at 

termination (NS). These results differ from other PCNB studies (not specified) which resulted in 

increased thyroid weights. The significant decrease in pituitary weight observed at 300 

mg/kg/day on Day 7 was not considered biologically significant. The relative liver to body 

weight ratios were significantly increased on Days 14, 20, and 90. 

No treatment-related histopathological lesions were observed in the pituitary glands. In the 

thyroid, follicular epithelial hypertrophy was observed in all 300 mg/kg/day rats (15/15) at 14, 30 

and 90 days compared to the controls (0/15). Mild follicular epithelial hypertrophy of the thyroid 

was observed in all 1 mg/kg/day rats (15/15) at Day 90. In the liver, hepatocellular hypertrophy 

was observed in the 1 mg/kg/day (14/15; trace) and 300 mg/kg/day (15/15; mild) groups.

The LOAEL is 20 ppm (equivalent to 1 mg/kg/day) based on observed thyroid effects. The 

NOAEL was not determined. 

Subchronic Dermal Toxicity (Rat, OCSPP 870.3200) 

In a subchronic dermal toxicity study (MRID 42413901), groups of 6 CD rats/sex were dermally 

exposed to PCNB (98.72% purity) at 0, 30, 300, or 1000 mg/kg/day for 21 days. The dermal 

applications exposed the rats for 6 hours/day and 7 days/week. Organs that were weighed 

included the liver, kidneys, and testes. Gross and microscopic pathological examinations were 

conducted on these tissues as well as the epididymides, prostate, seminal vesicles, ovaries, 

uterus, pituitary, adrenal, and mammary glands. The thyroid gland was not examined. 

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

consumption, dermal evaluations, hematology, clinical chemistry, urinalysis, or gross pathology. 

No treatment-related changes were observed in absolute or relative testicular weights. No 

treatment-related histopathological lesions were observed in the testes, epididymides, seminal 

vesicle, prostate, ovaries, uterus, mammary glands, adrenal or pituitary glands.

The NOAEL is 1000 mg/kg/day, and the LOAEL was not determined. 

Subchronic Dermal Toxicity (Rat, OCSPP 870.3200) 

In a subchronic dermal toxicity study (MRID 42416002), groups of 5 Crl:CDBR rats/sex were 

dermally exposed to PCNB (99.5% purity) at 0, 100, 300, or 1000 mg/kg/day for 21 days. The 

dermal applications exposed the rats for 6 hours/day and 5 days/week for a total of 16 

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applications over 22 days. Organs that were weighed included the liver, kidneys, testes, and 

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

well as the thyroid. 

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

consumption, clinical chemistry, dermal evaluations, or gross pathology. For hematology, the 

white blood cell count was significantly less in the mid-dose females. No treatment-related 

changes were observed in absolute or relative testicular or ovarian weights. No treatment-related 

histopathological lesions were observed in the testes or ovaries. Dilatation of the thyroid follicles 

(3/5) and hypertrophy of the thyroid follicular epithelium (4/5) was observed in high-dose males. 

No histopathological lesions were seen in females at any dose level.

The LOAEL is 1000 mg/kg/day based on observed thyroid effects, and the NOAEL is 300 

mg/kg/day. 

Avian Reproduction (Quail; OCSPP 850.2300)

In an avian reproduction study (MRID 41332801), Bobwhite quail (Colinus virginianus; 24-

weeks old) were administered PCNB (99.4% purity) in the feed at nominal concentrations of 0, 

300, 600, or 1200 ppm for 20 weeks (11 weeks prior to the start of egg production and during 9 

weeks of egg production). Each treatment group was comprised of sixteen mating pairs, with one 

pair per pen. A progressive photo period was employed in this study: 8 hours/day through Week 

7 and 17 hours/day until the end of the study. Mean temperature was maintained at 20.0±2.5°C 

throughout the study and mean humidity was maintained at 48±12%. Individual adult body 

weights were recorded at study initiation, biweekly through Week 8, and at study termination. 

Food consumption in each replicate was measured once each week throughout the study. 

Individual chick body weights were recorded after hatching and on Day 14 after hatching; egg 

shell thickness was measured weekly during egg production. Adult birds and chicks were 

observed routinely for mortality and clinical signs. 

No treatment-related effects were observed for adult mortality, clinical signs of toxicity, body 

weight, or food consumption. For the reproduction parameters, significant (p<0.05) reduction in 

the number of 14-day old survivors as a percentage of hatchlings was observed at 1200 ppm. The 

LOAEC is 1200 ppm based on the reduction in 14-day old survivors, and the NOAEC is 600 

ppm. 

In another avian reproduction study (MRID 43980301), Bobwhite quail (Colinus virginianus; 

28-weeks old) were administered PCNB (97.4% purity) in the feed at nominal concentrations of 

0, 1000, 2500, 4000, and 5500 ppm (not adjusted for purity) for 22 weeks (12 weeks prior to the 

start of egg production and during 10 weeks of egg production). Each treatment group was 

comprised of sixteen mating pairs, with one pair per pen. A progressive photo period was 

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employed in this study: 7 hours/day through Week 8 and 17 hours/day until the end of the study. 

Mean temperature was maintained at 17-19°C throughout the study and mean humidity was 

maintained at 67%. Individual adult body weights were recorded at study initiation, biweekly 

through Week 8, and at study termination. Food consumption in each replicate was measured at 

least biweekly. Individual chick body weights were recorded after hatching and on Day 14 after 

hatching; egg shell thickness was measured weekly during egg production. Adult birds and 

chicks were observed routinely for mortality and clinical signs. 

No treatment-related effects were observed on adult mortality, clinical signs of toxicity, body 

weight, or food consumption. In the ≥2500 ppm groups, significant reductions were observed for 

14-day old survivors as a percentage of hatchlings, and 14-day old survivor weights. In the 

≥4000 ppm groups, significant reductions were observed for 14-day old survivors/hen, 14-day 

old survivors as a percentage of eggs laid, and hatchling weights. In the 5000 ppm group, 

significant reductions were observed for normal eggs as a percentage of eggs laid and normal 

hatchlings as a percentage of live embryos. The LOAEC is 2500 ppm based on the reductions in 

14-day old survivor weights and percentage of 14-day old survivors of normal hatchlings. The 

NOAEC is 1000 ppm. 

Avian Reproduction (Duck; OCSPP 850.2300) 

In an avian reproduction study (MRID 41332802), Mallard ducks (Anas platyrhynchos; 26-

weeks old) were administered PCNB (99.4% purity) in the feed at nominal concentrations of 0, 

300, 600, or 1200 ppm ai for 19 weeks (10 weeks prior to the start of egg production and during 

9 weeks of egg production). Each treatment group was comprised of sixteen mating pairs, with 

one pair per pen. A progressive photo period was employed in this study: 8 hours/day through 

Week 8 and 17 hours/day until the end of the study. Mean temperature was maintained at 

20.0±2.5°C throughout the study and mean humidity was maintained at 46±22%. Individual 

adult body weights were recorded at study initiation, biweekly through Week 8, and at study 

termination. Food consumption in each replicate was measured biweekly throughout the pretreatment 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 weekly during 

egg production. Adult birds and chicks were observed routinely for mortality and clinical signs. 

No treatment-related effects were observed for adult mortality, signs of toxicity, or food 

consumption. At 1200 ppm, body weights were reduce for females (↓7%, p<0.05) at Week 8. No 

other significant differences in body weights were observed during the study. No significant 

effects were observed for reproduction parameters, egg shell thickness, or offspring body weight. 

The LOAEC is 1200 ppm based on the reductions in adult body weights. The NOAEC is 600 

ppm. 

In another avian reproduction study (MRID 43903301), Mallard ducks (Anas platyrhynchos; 20-

weeks old) were administered PCNB (97.4% purity) in the feed at nominal concentrations of 0, 

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1000, 2500, 4000, and 5500 ppm (not adjusted for purity) for 20 weeks (9 weeks prior to the start 

of egg production and during 11 weeks of egg production). Each treatment group was comprised 

of sixteen 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. Mean 

temperature was maintained at 21°C throughout the study and mean humidity was maintained at 

55%. Individual adult body weights were recorded at study initiation, biweekly through Week 8, 

and at study termination. Food consumption in each replicate was measured at least biweekly. 

Individual chick body weights were recorded after hatching and on Day 14 after hatching; egg 

shell thickness was measured weekly during egg production. Adult birds and chicks were 

observed routinely for mortality and clinical signs. 

No treatment-related effects were observed for adult mortality, signs of toxicity, body weight, or 

food consumption. No significant effects were observed for any of the reproduction parameters. 

The NOAEC is 5500 ppm, and the LOAEC was not determined. 

Fish Early Life-Cycle Toxicity (Rainbow trout, OCSPP 850.1400) 

In a fish early life stage toxicity study (MRID 41663401), groups of rainbow trout 

(Oncorhynchus mykiss) embryos (<24 hours old) were exposed to PCNB (99% purity) at 

nominal concentrations of 3.8, 7.5,15, 30, or 60 μg/L from egg stage to 62 days post-hatch in a 

flow-through system. A negative control group (dilution water) and solvent control group (6.9 

μL DMF/L) were included in the study. Mean measured concentrations were 3.0, 6.5, 13, 32, and 

52 μg/L. Groups consisted of 160 embryos/treatment (40 embryos/replicate; 4 

replicates/treatment). The embryos were evaluated for percent hatch, time to hatch, and time to 

swim-up. The fry were evaluated for clinical signs and survival daily. Growth was determined as 

standard length at 35 days post-hatch and at study termination (62 days post-hatch) and as wet 

weight at study termination. 

Sublethal effects were observed with the greatest variety and frequency in the 52 μg/L group. At 

52 μg/L, significant (p<0.05) reductions were observed in hatchability (73% vs. 89% in control) 

and fry survival (75% vs 100% in control) at 35 days post-hatch. Significant (p<0.05) reductions 

in length (↓5%) and wet weight (↓17%) were observed at 32 μg/L. The overall LOAEC and 

NOEAC were 32 and 13 μg/L, respectively, based on growth effects. 

Fish Early Life-Cycle Toxicity (Fathead minnow, OCSPP 850.1400) 

A freshwater fish early life-cycle test using PCNB (98.2%) (MRID 408840-01) was conducted 

using fathead minnow (Pimephales promelas) Embryo hatchability and larval survival were not 

affected at any exposure levels. Larval growth was significantly reduced at the 54 μg/L treatment 

level. The mean larval weight at the highest concentration was 20% less than that of the pooled 

control larvae. 

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APPENDIX 3: References Not Utilized in the PCNB 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 PCNB 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. 

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