UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

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

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

## ~ S

## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals.
The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID

PATHWAYS

CHEMICAL: PENTACHLOR ONITROBENZENE (PCNB)

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYPCNB FINAL

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 Rele vant 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-Estr ogenic Pathway for PCNB ..........................................12
Table 3. Androgenic/Anti-Andr ogenic 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 Utiliz ed in the PCNB WoE Analysis.................................62PCNB FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g enerationPCNB FINAL

iv Abbreviation Terminology

F2 Second filial g eneration
Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low
MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated ConcentrationPCNB FINAL

v Abbreviation Terminology

MTD Maximum Tolerated Dose
N Ne g ative
NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant
N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)PCNB FINAL

vi Abbreviation Terminology

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating Hormone

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidencePCNB FINAL

Page 1 of 62 Executive Summary

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry 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 pestic ide chemical interacts with E, A or T hormone pathways, the number and type of effects i nduced, the magnitude and pattern of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in part icular, whether or not endocrine-rela ted 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 we ight-of-evidence (WoE) analysis of the potential interaction of PCNB with the E, A or T hormone path ways. 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 poten tial interaction of PC NB 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 in crease of estradiol production (<1.4 fold) at the 10-20 mM concentrations. Additionally, PCNB show ed 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 o ffspring survival and or weight s 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 ob served; no reproductive effects observed in the bobwhite quail. In the FSTRA, fecundity at the highest concentrati on tested (0.074 mg/L) was decreased (67%) and GSI was in creased by 35% and histological changes were noted in the ovaries, including decreased post-ov ulatory follicles and increased oocyte atresia. However, it is
noted that there were no effects on fertilization or serum vitell ogenin (VTG) levels. While there is limited evidence of redundancy across the battery (all assays negative ex cept for the Tier 1 steroidogenesis assay), there is complementarity of evidence within the FSTRA which suggests potential interaction with th e estrogen pathway in fish.PCNB FINAL

Page 2 of 62 There was no evidence of interaction of PCNB with the androgen pathway in the Tier 1 in vitro ( i.e , AR binding and steriodogenesis a ssays were negative) or Tier 1 in vivo mammalian assays ( i.e ., Hershberger and male pubertal assays were negative in the ab sence 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 re duction in number/weight of offspring were observed, these effects lack dia gnostic specificity with regards to the androgen pathway. While there was a significant increase in male GSI in the FSTRA at the mid a nd high concentrations, there was a lack of complementarity evidence as there were no othe r effects noted in the males. Therefore, the Tier 1 battery data do not support a conclusion of potentia l 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 as says, there were signi ficant decreases in T 4 levels and increases in thyroid stimulating hormone (TSH) levels, but there were no significant changes in thyroid weight or histology. A lthough there was a slight, dose-re lated trend toward decreased
colloid area and increased follicular cell height in the female pubert al study. It is noted that the thyroid effects were seen in the presence of si gnificant (p <0.05) increas es 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 indicativ e 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 PC NB levels compared to the negative control, no treatment-related effects were seen in thyr oid histopathology, asynchr onous 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 gr oup. Overall, these findings do not suggest that the delay in development or decreas ed growth is thyroid-related.

In the mammalian Part 158 studies , alterations in thyroid hormone s, thyroid weights and thyroid lesions were seen in the subchronic, chronic and two-generation reproduction studies in rats. Additionally, alterations in T 3 , T 4 and TSH levels were observed in a special non-guideline subchronic study with male rats, conducted specif ically to examine poten tial thyroid effects of PCNB. Across these studies, the obs erved effects were concurrent wi th 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 in teract with the estrogen pathway in wildlife based on the complementarity effects observed wi thin the FSTRA along with the results of the Tier 1 steroidogenesis assay. For the androge n pathway, there no convincing evidence of potential interaction of PCNB with the mammals or wildlife. Additiona lly, there is no evidencePCNB FINAL

Page 3 of 62 of potential interaction of PCNB with the thyroid pathway in amphibians. However, there is evidence of potential interacti on of PCNB with the thyroid pa thway 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 wa s 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 (P OD) of 1 mg/kg/day fo r human health risk assessment is based on hepatocellular hypertr ophy and hyperplasia seen at 150 mg/kg /day (LOAEL) in a combined chronic toxicity/carcinogenici ty study in rats. Thyroid hypertrophy and hyperplasia were seen at the next higher dose (3 57 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 endpo ints of concern are base d 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. Howe ver in the case of PCNB, alt hough no data exists for potential thyroid toxicity in the young, a comparative thyr oid assay (CTA) is not recommended since: 1) the POD is approximately 300-fold lower than th e dose that caused thyroid lesions seen at 357 mg/kg/day; 2) in subchronic st udies conducted with young rats cha nges 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 rela ted through the liver and
liver lesions are used as the t oxicity endpoint of concern for es tablishing the POD; 5) the CTA would be tested at higher doses and would be expected to yi eld a POD lower than the level currently used for risk assessm ent; and 6) the RfD and the MOEs are protective of the thyroid effects seen in the pubertal assa ys and the OSRI. Therefore, ma mmalian EDSP Tier 2 testing is not recommended for PCNB since additional testing is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

In the FSTRA, fecundity was decreased, GSI was in creased and there were histological changes in the ovaries at 74 μg a.i./L. Currently, the ch ronic fish risk assessments are based on a NOAEC of 13 μg a.i./L and a LOAEC of 32 μg a.i./L estab lished in the fish early-life cycle (ELS) toxicity study with the rainbow trout. Base d on the concentrations at wh ich responses observed in the
FSTRA occurred in at similar concentrations as the current chronic endpoint, the EDSP Tier 2 Medaka Extended One-Generation Reproduc tion Test (MEGORT) is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for PCNB. In mallard duc ks, effects on reproductive parameters were observed; no reproductive effects were observed in bobwhite quail. The type of data obtainedPCNB FINAL

Page 4 of 62 from Part 158 avian reproduction studies (O CSPP 850.2300) are considered sufficient for evaluating potential reproductive ef fects to birds from PCNB expos ure. Relative to the EDSP, additional testing is not recommended.PCNB FINAL

Page 5 of 62 I. Introduction

The Endocrine Disruptor Screening Programs (EDSP) Tier 1 assay ba ttery is designed to provide the necessary empirical data to evaluate the pote ntial of chemicals to interact with the estrogen (E), androgen (A) or thyroid (T) signaling pa thways. This interacti on includes agonism and antagonism at the estrogen and androgen recep tors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (H PG) and hypothalamic-pituitary thyroid (HPT) perturbations.
In addition to the available Ti er 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. Additi onally, the conditions under which effects occu r were considered, in particular, whether or not e ndocrine-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 we ight-of-evidence (WoE) analysis of the potential interaction of pentachlorobenzene with the E, A or T hor mone 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 wi ldlife studies (40 CFR Pa rt 158 and literature).

Pentachloronitrobenzene is a contact organochl orine 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 tende ncy to sorb to organic matter. However, on sandy soils low in organic matter, PC NB may leach to groundwater. Once in the atmosphere, PCNB is not expected to degrade by phot o-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 stud ies 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 (U SEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recommendations ar e presented in Section V.PCNB FINAL

Page 6 of 62 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 believe d to be relevant to one or more of the Tier 1 Screening assays were submitted as OSRI by the Te st Order recipient(s) and/or the public. This included studies published in the open literatu re and/or data submitted to support pesticide registration (e.g., Part 158 guideli ne studies). The Agency’s revi ew of the initial OSRI is provided in the Report of the Endocrine Disr uptor 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 info rmation. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/ studies considered but not uti lized 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 presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of eviden ce process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual 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 indivi dual 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 endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting 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 werePCNB FINAL

Page 7 of 62 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 in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach 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 speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A, or T path ways are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, 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 ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment 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 rele vant, robust and consistent evidence in terms of complementarity and redundancy, as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of biological organization? Finally, under what conditions did the responses occur including co nsideration 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 eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.PCNB FINAL

Page 8 of 62 This uncertainty in distinguishi ng whether the responses were endoc rine-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, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was ge nerally 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 include d, but were not limited to: mortal ity; 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, es pecially 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/concentra tion 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 decr eases 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 ef fects were generally cons idered as less severe forms of toxicity ( e.g., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular 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/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addres sing the three component s described above is fundamental to the WoE process and in determ ining 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 eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availa ble toxicity data into account.PCNB FINAL

Page 9 of 62 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 bi ologically 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)

ER α 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)PCNB FINAL

Page 10 of 62 B. Effects on Hypothalamic-P ituitary-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 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This table also includes HPG-relevant findings from data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but are not reporte d in the table and not considered further in the WOE assessment.

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 equivo cal 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 orde r 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,
hER α , or hER β . Additionally, PCNB was negative in a yeast based ER transactivation assay using hER α . 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 uterot rophic 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). Howeve r, 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 th e 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 wa s 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 se rum vitellogenin levels. Also at the high test concentration, GSI was also increased by 35% an d histological change s were noted in the ovaries, including decreased post-ov ulatory follicles and increased oocyte atresia. There was a dose-dependent increase in body weight in female fish that attained si gnificance at the high treatment ( ↑ 10%).

No estrogen-related effects were observe d in the Part 158 m ammalian studies.PCNB FINAL

Page 11 of 62 In the Part 158 bobwhite quail re production studies, decreased su rvival of 14-day old chicks were observed at the respective high concentr ations 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 e ggs/hatchlings) at 5000 mg/kg-diet. In two Part 158 mallard studies, there were no effects on reproduc tive parameters. In the Part 158 rainbow trout fish early life-cycle study, hatcha bility was decreased along with th e 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.PCNBFINALPage 12 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNBLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ LiteratureCitation 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) Vitellogenin Systemic toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssayER BindingRequirement satisfied by OSRI (Ashbyet al, 2005; see below)ERTARequirement satisfied by OSRI (Ashbyet al, 2005; see below)Aromatase(MRID 48916701)NSteroidogenesis(MRID 48916704)PUterotrophicRequirement satisfied by OSRI (Ashbyet al, 2005; see below)Female Pubertal Rat(MRID 49042103)NNNNNNLW:↑8, 6%KW:↑8%(L, H)NFSTRA(MRID 49042102)NE↑35%(H)P4(H)NFrt: NFcd:↓67% (H)NNOSRIER Binding,Transactivation(Ashbyet al, 2005)N NReceptorTransactivation(Kojimaet al., 2004)N NPCNBFINALPage 13 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNBLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ LiteratureCitation 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) Vitellogenin Systemic toxicity Observed 2 Overt Toxicity Observed 3ReceptorTransactivation(Vinggaardet al.,1999)NReceptorTransactivation(Zouet al., 2002)NUterotrophic assay(Ashbyet al, 2005)NNNFemale Pubertal assay(Ashbyet al., 2005)NNN NThree-generationreproduction (Rat;MRIDs 00001666 and40865903)NR NR N NRNN NTwo-generationreproduction (Rat;MRID 41918701)NRNRNNRNNNTwo-generationreproduction (Rat;MRID 43469301)NENNNRNX(H)NDevelopmentaltoxicity (Rat; MRID40588601)NRNNNDevelopmentaltoxicity (Rat; MRID41361201)NENNNPCNBFINALPage 14 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNBLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ LiteratureCitation 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) Vitellogenin Systemic toxicity Observed 2 Overt Toxicity Observed 3Developmentaltoxicity (Rabbit;MRID 41361301)NRNNX(H)Developmentaltoxicity (Rabbit;MRID 40717102)NRNNNChronic toxicity/carcinogenicity (Rat;MRID 41987301)NENNNEX(M,H)XChronic toxicity/carcinogenicity (Rat;MRID 43015801)NNNNEX(MH,H)NCarcinogenicity(Mouse; MRID00114224)NENENNRX(M,H)NCarcinogenicity(Mouse; MRID45609101)NENENNRNX(H)Chronic toxicity (Dog;MRID 00001667)NENENNEX(MH,H)NChronic toxicity (Dog;MRID 41718601)NENNNX(H)NSubchronic oraltoxicity (Rat dietary;MRID 40636701)NNNNEX(MH)NPCNBFINALPage 15 of 62Table 2: Estrogenic/Anti-Estrogenic Pathway for PCNBLines of Evidence Indicating Potential Interaction with the Estrogenic/Anti-Estrogenic Pathway for PCNB1Study Type/ LiteratureCitation 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) Vitellogenin Systemic toxicity Observed 2 Overt Toxicity Observed 3Subchronic oraltoxicity (Rat gavage;MRID 42416001)NE N N NEX(H)NSubchronic toxicity(Rat dermal; MRID42413901)NENENNEN NSubchronic toxicity(Rat dermal; MRID42416002)NENNNEN NAvian reproduction(Quail; MRID41332801)P5(H)N NAvian reproduction(Quail; MRID43980301)P6(ML,MH, H)NNAvian reproduction(Duck; MRID41332802)NN N7Avian reproduction(Duck; MRID43903301)NN N1.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. Changes in weight areabsolute unless otherwise indicated. LW: Liver weight; KW: Kidney weight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For detailssee Section IV. A4. At the high concentration (0.074 mg/L),the incidences of severe oocyte atresiawere increased and decreases in post-ovulatory follicles were reported.PCNBFINALPage 16 of 625. Reductions in 14-d offspring survival at 1200 mg/kg-diet6. Reductions in 14-d offspring survivors and body weightat 2500 mg/kg-diet; effects on embryo viability/hatchling success at 5000 mg/kg-diet.7. Decreases in male body weight at 1200 mg/kg-diet.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examineNR Not reportedPCNB FINAL

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-re levant findings from da ta evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

The in vitro Tier 1 AR binding and steroidogenesis assa ys were both negative, as was an AR transactivation assay conducted by Kojima et al . (2004). PCNB was also negative for androgenic or anti-androgeni c activity in the in vivo Hershberger and male pubert al 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 note d 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, decrea ses in offspring survival and/or hatch were observed; no effect in the bobwhite quail studies.PCNBFINALPage 18 of 62Table 3: Androgenic/Anti-Androgenic Pathway for PCNBLines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for PCNB1Study Type/ LiteratureCitation 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 Systemic Toxicity Obseved 2 Overt Toxicity Observed 3EDSP Tier 1 AssayAR Binding(MRID 48946801)NSteroidogenesis (MRID48916704)NHershberger(MRID 48916702)NENLW:↑7-8%(H)NMale Pubertal Rat(MRID 49042103)NNNNNNNNLW:↑8, 10%KW:↑8, 6%(L, H)NFSTRA(MRID 49042102)NE↑42,59%(M, H)NNNNNOSRIReceptorTransactivation(Kojimaet al., 2004)NThree-generationreproduction (Rat;MRIDs 00001666 and40865903)NRNNRNNRNRN NTwo-generationreproduction (Rat;MRID 41918701)NRNNRNNRNNNX(H)PCNBFINALPage 19 of 62Table 3: Androgenic/Anti-Androgenic Pathway for PCNBLines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for PCNB1Study Type/ LiteratureCitation 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 Systemic Toxicity Obseved 2 Overt Toxicity Observed 3Two-generationreproduction (Rat;MRID 43469301)NNNNNRNNX(H)NChronic toxicity/carcinogenicity (Rat;MRID 41987301)NNNENNENEX(M.H)X(H)Chronic toxicity/carcinogenicity (Rat;MRID 43015801)NNNENNENEX(MH,H)NCarcinogenicity(Mouse; MRID00114224)NENNENENENEX(M,H)NCarcinogenicity(Mouse; MRID45609101)NRNNENNRNRN NChronic toxicity (Dog;MRID 00001667)NNNENENENEX(MH,H)NChronic toxicity (Dog;MRID 41718601)NNNENNNEX(H)NSubchronic oraltoxicity (Rat dietary;MRID 40636701)NNNENNENEX(M.H)NSubchronic oraltoxicity (Rat gavage;MRID 42416001)NNNENENENEX(H)NSubchronic toxicity(Rat dermal; MRID42413901)NNNENNENEN NPCNBFINALPage 20 of 62Table 3: Androgenic/Anti-Androgenic Pathway for PCNBLines of Evidence Indicating Potential Interactionwith the Androgenic/Anti-Androgenic Pathway for PCNB1Study Type/ LiteratureCitation 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 Systemic Toxicity Obseved 2 Overt Toxicity Observed 3Subchronic toxicity(Rat dermal; MRID42416002)NNNENENENEN NAvian reproduction(Quail; MRID41332801)P4(H)N NAvian reproduction(Quail; MRID43980301)P5(ML,MH,H)N NAvian reproduction(Duck; MRID41332802)NN N6Avian reproduction(Duck; MRID43903301)NN N1. 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. Changes in weight areabsolute unless otherwise indicated. Abbreviations for androgensensitive tissues: Seminal vesicles (SV), Ventral prostate (VP), Dorsal prostate (DP), Prostate(PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). LW: Liver weight; KW: Kidneyweight2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in thiscolumn. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. For details seeSection IV. A4 Reductions in 14-d offspring survival at 1200 mg/kg-diet5. Reductions in 14-d offspring survivors and body weightat 2500 mg/kg-diet; effects on embryo viability/hatchling success at 5000 mg/kg-diet.6. Decreases in male body weight at 1200 mg/kg-diet.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedNR Not reportedPCNB FINAL

Page 21 of 62 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal female and male rat assays, and the AMA (frog).

The potential interactions of PCNB w ith thyroid regulation are summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. Three Tier 1 studies were evaluated for potential effects of PCNB. This table also include s 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 furt her in the WOE assessment.

In both the Tier 1 male and female pubertal as says, there were signif icant decreases in T 4 levels
and increases in TSH levels in both the lo w- and high-dose groups (500 and 1000 mg/kg/day). Although the increased TSH levels in males (39 and 25% at the lo w and high dose, respectively) were not statistically significant, the effect wa s considered biologically meaningful. There were no significant changes in thyroi d weight or histology, although th ere was a slight, dose-related
trend toward decreased colloid area and increase d 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 developm ent was noted and there was no effect on normalized hind limb length (HLL). Mild thyroi d atrophy was noted at the highest test concentration (0.016 mg/L) compared to the contro l but were not statisti cally significant; and, the histopathologist report stat ed these effects were not a result of treatment. The high
concentration also resulted in an obser ved 19% decrease in tadpole body weight.

In the Part 158 two-generation rat reproducti ve study that included analyses of thyroid histopathology, incidences of thyroid follicular cell hypertrophy/hyperplasi a 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 weight s were significantly (p <0.01) increased in both sexes at the mid and high dose groups at the terminal sacrifi ce. Absolute thyroid wei ghts 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-neoplas tic lesions in the high-dose males and females included colloidPCNB FINAL

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 ad enomas, 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 carcinogenic ity 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 s ubchronic rat studies (oral gavage and dermal), follicular cell hypertrophy and hyperplasi a were observed at the high dose (985-1000 mg/kg/day). When examined, none of the Part 158 mamma lian toxicity studies showed effects of PCNB on pituitary weight or histopathology.

To specifically examine potential thyroid eff ects of PCNB, a special, non-guideline subchronic study was also conducted with male rats at tw o dietary dose levels (e quivalent to 1 or 300 mg/kg/day) . At the high-dose, T 3 levels were decreased by 19-26%, T 4 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. H epatocellular hypertrophy was also observed in the low- and high-dose groups, w ith the severity increasing with dose.PCNB FINAL

Page 23 of 62 Table 4. Thyroid Pathway for PCNB.

Lines of Evidence Indicating Potential Int eraction with the Thyroid Pathway for PCNB 1

Study Type/ Literature CitationThyroid receptorsThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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 P 5 (M, H) NE X (H) N

Chronic toxicity/ carcinogenicity (Rat; MRID 41987301) ↑ 21- 26% (M) P 5 (M) NE X (M, H) X

Chronic toxicity/ carcinogenicity (Rat; MRID 43015801) N P 5 (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) NPCNB FINAL

Page 24 of 62 Table 4. Thyroid Pathway for PCNB.

Lines of Evidence Indicating Potential Int eraction with the Thyroid Pathway for PCNB 1

Study Type/ Literature CitationThyroid receptorsThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH levelsPituitary WeightDevelopmental stage(±or asynchronous,HLL)Growth(BW, SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

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 P 5 (H) NE X (H) N

Subchronic toxicity (Rat dermal; MRID 42416002) NE P 5 (H) NE N N

Special Subchronic toxicity (Rat; MRID 42630801) ↓ (H) P 5 (L, H) T 3 : ↓ 19-26%
T 4 : ↓ 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 re sponse. 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, 0.82 μ g/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 reportedPCNB FINAL

Page 25 of 62 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of PCNB to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing.. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tire 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l 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/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).PCNB FINAL

Page 26 of 62 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 incr eases liver (6-10%) and kidney (6-8%) weights and changes in liver enzymes. No signs of ove rt toxicity were obser ved 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 injectio n, as opposed to oral dosing in the above Tier 1

assays.

In part 158 mammalian studies, toxi city was typically characterize d by increases in liver weight, changes in liver histopathology and enzyme activ ities, and decreases in body weight and/or body weight gain.

No overt toxicity was noted in any of the mu lti-generation reproductive studies in rats, but decreases in body weight were reported at th e 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 toxic ity was observed in the two rat development studies and one of the rabbit developmental studies, bu t 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 wh ich 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 consis ted of increases in liv er weight (11-20%), decreases in body weight ( 11%) and body weight gain ( ≤ 10%), and microscopi c changes in the liver (hypertrophy). Increased liver weight was also noted in high-dose males in the other chronic rat study, along with increased incidenc es of hepatocellular hypertrophy in males at ≥ 36 mg/kg/day and females at ≥ 357 mg/kg/day. Dose-dependent incr eases 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 toxic ity 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 se veral subchronic studies, and liver weights were inc reased in all the studies at the highest doses tested (300-985 mg/kg/day).PCNB FINAL

Page 27 of 62 In the avian reproduction studies, no overt or systemic toxicity we re noted in the quail or duck studies at dietary concentrati on of PCNB up to 5500 ppm, although transient decreases in bodyweight (Week 8) were observed in one of the duck studies at 1200 ppm. In the early fish lifecycle study, overt toxicity was obs erved at the highest test con centration (52 μg/L) based on the reduction in fry survival (75%), and systemic effects were al so observed at 32 μg/L, based on reductions in body length and weight. In a fathea d minnow fish early life-cycle study, larval growth was significantly re duced (20%) at 54 μg/L.

B. Estrogen Pathway

There was no convincing evidence of potential inte raction of PCNB with the estrogen pathway.
The Tier 1 or OSRI in vitro assays were negative with the ex ception of the steroidogenesis assay where there was a slight increase of estr adiol 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 to xicity studies. In the Part 158 avian reproduction studies, decreases in o ffspring survival and or weight s 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 ob served; no reproductive effects observed in the bobwhite quail. In the FSTRA, fecundity at the highest concentrati on tested (0.074 mg/L) was decreased (67%) and GSI was in creased by 35% and histological changes were noted in the ovaries, including decreased post-ov ulatory follicles and increased oocyte atresia. However, it is
noted that there were no effects on fertilization or serum vitell ogenin (VTG) levels. While there is limited evidence of redundancy across the battery (all assays negative ex cept for the Tier 1 steroidogenesis assay), there is complementarity of evidence within the FSTRA which suggests potential interaction with th e 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 a ssays were negative) or Tier 1 in vivo mammalian assays ( i.e ., Hershberger and male pubertal assays were negative in the ab sence 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 re duction in number/weight of offspring were observed, these effects lack dia gnostic specificity with regards to the androgen pathway. While there was a significant increase in male GSI in the FSTRA at the mid a nd high concentrations, there was a lack of complementarity evidence as there were no othe r effects noted in the males. Therefore, the Tier 1 battery data do not support a conclusion of potentia l interaction with the androgen pathway.PCNB FINAL

Page 28 of 62 D. Thyroid Pathway

There is evidence of potential in teraction 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 as says, there were signi ficant decreases in T 4 levels and increases in TSH levels, but th ere were no significant changes in thyroid wei ght 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 kidn ey weights, were noted in both studies at both dose levels along with cha nges in clinical chemistry parameters ( ↑ ALP and ↑ bilirubin) that are indicativ e of altered liver function.

In the AMA, while there was a slight but significa nt delay in the median developmental stage at Day 21 at all PCNB levels compared to the negati ve control, no treatment-related effects were seen in thyroid histopathol ogy, 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 hormone s, thyroid weights and thyroid lesions were seen in the subchronic, chronic and two-generation reproduction studies in rats. Additionally, alterations in T 3 , T 4 and TSH levels were observed in a special non-guideline subchronic study with male rats, conducted specif ically to examine poten tial thyroid effects of PCNB. Across these studies, the obs erved effects were concurrent wi th 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 th e FSTRA along with the results of the Tier 1 steroidogenesis assay. For the androgen path way, there no convincing evidence of potential
interaction of PCNB with the mammals or wi ldlife. Additionally, th ere is no evidence of potential interaction of PCNB with the thyroid pathway in amphibians. However, there is evidence of potential interacti on of PCNB with the thyroid pa thway in mammals, although it is
noted that the observed thyroid effects were consistently associated with alterations in the liver.PCNB FINAL

Page 29 of 62 V. EDSP Tier 2 Testing Recommendations

In the EDSP Tier 1 assays and OSRI, there wa s 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 thyr oid hyperplasia were seen at the next higher dose (357 mg/kg/ day). This POD is used for deri ving 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 endpo ints of concern are base d 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. Howe ver in the case of PCNB, alt hough no data exists for potential thyroid toxicity in the young, a comparative thyr oid 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 st udies conducted with young rats cha nges 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 rela ted through the liver and
liver lesions are used as the t oxicity endpoint of concern for es tablishing 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 assessm ent; and 6) the RfD and the MOEs are protective of the thyroid effects seen in the pubertal assa ys and the OSRI. Therefore, ma mmalian EDSP Tier 2 testing is not recommended for PCNB since additional testing is not expected to impact EPA’s current regulatory point of departur es and endpoints for human h ealth risk assessments.

In the FSTRA, fecundity was decreased, GSI was in creased and there were histological changes in the ovaries at 74 μg a.i./L. Currently, the ch ronic fish risk assessments are based on a NOAEC of 13 μg a.i./L and a LOAEC of 32 μg a.i./L esta blished in the Fish Early Life Cycle toxicity study with the rainbow trout. Base d on the concentrations at wh ich responses observed in the
FSTRA occurred in at similar concentrations as the current chronic endpoint, the EDSP Tier 2 Medaka Extended One-Generation Reproductio n Test (MEDGORT is recommended.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard
duck are available for PCNB. In mallard duc ks, 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 (O CSPP 850.2300) are considered sufficient forPCNB FINAL

Page 30 of 62 evaluating potential reproductive ef fects to birds from PCNB expos ure. Relative to the EDSP, additional testing is not recommended.PCNB FINAL

Page 31 of 62 VI. References

Akhurst, L. C. (2012) PCNB Technical H295R Steroidogenesis Assay. Huntingdon Life Sciences Ltd., Cambridgeshire, England. Laboratory Study No. BD G0126, 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 Pube rtal 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 Internati onal, 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: Hershberg er bioassay in the castrated rat (oral administration). Huntingdon Life Sciences, Camb ridgeshire, England. Laboratory Project Study ID: BDG0121, June 19, 2012. Unpublished. (MRID 48916702)

Foster, J.R. (2012) PCNB Technical: Aromatas e (Human Recombinant) Assay. Huntingdon Life Sciences Ltd., Huntingdon, Cambridgeshire, Engl and. 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 carcinogenici ty study in rats. International Research and Development Corporation

. Study #399-072. [900-CHR-014]. (MRID 41987301)PCNB FINAL

Page 32 of 62 Goldenthal, E.I. (1992) 21-day dermal toxicity study in rats (PCNB). Laboratory Project
Number: 399-125. International Research and De velopment 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 pentachloron itrobenzene (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 toxici ty study in rats with pentachloronitrobenzene (MRD-89505). EXXON Biomedical Sciences, Inc. Project #250570A

. [900-SCH-001]. (MRID 42416001)

Keller, K. (1988a) Developmental toxicity st udy in rats: Laboratory Project ID 399-068. International Research and Developm ent Corporation. Unpublished. (MRID 40588601)

Keller, K (1988b) Developmental toxicity study in New Zealand White rabbits:
Pentachloronitroben zene (PCNB): Laboratory Project ID 399-070. International Research and Development Corporation. Unpublished. (MRID 40717102)

Larson, P

. S and Borzelleca, J.F (1968a) Three ge neration reproduction study on rats receiving
Terraclor in their diet. Medi cal College of Virginia, Dept. of Pharmacology. CDL:091391-B. Unpublished. (MRIDs 00001666 and 40865903)

Larson, P.S and Borzelleca, J.F (1968b) Toxicologi cal 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, Cambri dgeshire, UK. Laboratory Study No.: BDG0128, July 26, 2012. Unpublished. (MRID 48946801)PCNB FINAL

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) Toxico logy 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). Pentachloron itrobenzene (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 repro duction 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) T oxicity and reproducti on 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 reproductio n toxicity study in rats with pentachloronitroben zene (PCNB) (MRD-89-505). EXXON Biom edical Sciences, Inc. Project #1S0535. [900-REP-001]. (MRID 43469301)

Plutnick, R.T. (1993) 2-year Chronic t oxicity/oncogenicity study in rats with pentachloronitroben zene (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 Thyr oid Active Substances. Wildlife International, Ltd., Easton, Maryland. Labor atory project number 246A-1 11. Unpublished. (MRID 49042101)

Schneider, S.Z., K.H. Martin, T.Z. Kendall, a nd H.O. Krueger. (2013) PCNB Technical: Fish Short-Term Reproduction Assay with the Fathead Minnow ( Pimephales promelas ). Wildlife International, Ltd., Easton, Maryland. La boratory report number 246A-110A. Unpublished. (MRID 49042102)

Trimmer, G. (1992) 21-day re peated dose dermal toxic ity study in rats with pentachloronitroben zene (Final Report). Laboratory Pr oject Number: 150509. Exxon Biomedical Sciences, Inc. (MRID 42416002)PCNB FINAL

Page 34 of 62 USEPA (2009) Reregistration E ligibility 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 Disrupt or Review Team- Test Order#: EDSP056502-105. December 9, 2010. Office of Chemical Safety and Pollution Prevention. U.S. Environmental Protection Ag ency, Washington, D.C.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention Science Coordina tion and Policy. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

van der Heijden, C.A. and Til, H.P. (1974) Pe ntachloronitrobenzene (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)PCNB FINAL

Page 35 of 62 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA, OCSPP 890.1100)

The 21-day assay (MRID 49042101) of PCNB t echnical (95.6% purity) on amphibian metamorphosis of African clawed frog ( Xenopus laevis ) was conducted under flow-through conditions. Amphibian larvae at Nieuwkoop-Fa ber (NF) stage 51 ( 80 tadpoles/group, 20 tadpoles/replicate) were exposed to a negativ e 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 (calculate d 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 22.0 o C and a pH of 7.9 to 8.1.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. By test termination, mean surviv al was 98% in both the negative control and the mid treatment group, 97% in the low treatmen t group, and 100% in the high treatment group. Clinical signs ( i.e. , behavioral and other subl ethal effects) including tail curvature and small size were observed in all treatment groups, with no ap parent pattern. Tail curvature was seen in 54% (32/59) of tadpoles in the nega tive 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 reduc ed at all treated levels, (NF 56; p<0.05, Jonckheere-Terpstra) relative to the negative cont rol (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 c ontrol (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 pa thology and histopathology (follicle size) findings was visually observed to be gr eater with increasing test conc entrations. The study author analyzed the incidence and seve rity counts using the Jonckheere- Terpstra step-down test and
determined that the differences were not statistically significant (p>0.05).

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

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

Page 36 of 62 the competitive binding experiments, a satu ration binding experiment was conducted to demonstrate that the AR in the rat prostate cy tosol was present in reasonable numbers and was functioning with appropriate affinity for the radiolab eled 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 us ed 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 r un included dexamethasone as a weak positive control, and R1881 as the lig and 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 K d for [ 3 H]-R1881 was 1.6246±0.3283 nM, and the mean estimated B max was 0.1805±0.0199 nM for the prepared rat prostate cytosol.
The K d was within the guideline recommended range of 0.03 to 1.5 nM for two of the three runs,
but the K d for Run 2 (2.2810±0.2936 nM) was above the recommended range.

The protein concentration for the competitive bindi ng assay was determined to be 2.0 mg/mL as it bound 13% of the [ 3 H]-R1881 at 1 nM. Across all three valid runs, the estimated mean IC 50 was 1.95 × 10

− 9 M for R1881 and 1.01 × 10

− 4 M for the weak positive control, dexamethasone. The corresponding log IC 50 values for R1881 and dexamethasone were

− 8.72 and

− 4.00 M, respectively. The mean relative binding affi nity (RBA) for dexamethasone compared to R1881 was 0.0019%, for a log RBA of

− 4.72%. Three valid assay runs we re 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 [ 3 H]-R1881 to the AR was 64, 83 and 80% in the three valid runs, which corresponded to an equivocal cla ssification (50-75% [ 3 H]-R1881 binding) for the first valid run and non-binder (>75% [ 3 H]-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 ([1 β

3 H(N)]-androst-4-en e-3,17-dione; [ 3 H]-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 ar omatase activity.PCNB FINAL

Page 37 of 62 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. Tritia ted water was quantified using liquid scintillation counting (LSC). Th ree runs were conducted and each run included a full activity control, a bac kground activity control, a pos itive control series (10

− 10 to 10

− 5 M) using a known inhibitor (4-hydroxyandrostenedi one; 4-OH ASDN; formestane), and the test chemical series (10

− 10 to 10

− 4 M) with three repetiti ons per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.251 to 0.340 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a m ean 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 IC 50 , and %CV for replicates of each concentration within runs. The estimated mean log IC 50 for 4-OH ASDN was

− 7.39 M and the mean Hill slope was

− 0.955.

For PCNB, aromatase activity av eraged 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 fr om 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 mode l to extrapolate possible log IC 50 and Hill slope values for PCNB (

− 1.23 M and

− 0.465, respectively), these values ar e 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) Bi nding Assay (OCSPP 890.1250)

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

ER α Transcriptional Activatio n (ERTA) Assay (OCSPP 890.1300)

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

Page 38 of 62 Fish Short Term Reproduction Assay (FSTRA, OCSPP 890.1350)

The 21-day short-term reproduction assay (M RID 49042102) of PCNB w ith fathead minnow ( Pimephales promelas ) was conducted under flow-through conditi ons. Adult fish (2 males and 4 females/group; 4 groups/treatment; ~5.5 months old) were exposed to nomi nal concentrations of PCNB (95.6% purity) of 0 (negative cont rol), 0.0010, 0.010, and 0.100 mg a.i./L.; meanmeasured concentrations were <0.00 0600 (<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 mainta ined at 24.1 to 25.6°C and a pH of 7.6 to 8.3.

Unless otherwise indicated, all e ffects are reported based on comparison to the negative (clean water) control. Survival was 100% in all treatm ent groups, and all fish appeared normal and healthy throughout the exposure pe riod, with the exception of two females in the high treatment group who appeared to be eggbound. One female wa s 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 cont rols was 97.4%. PCNB significantly decreased fecundity by 67% (p<0.01) at the high trea tment concentration; fertilization success was comparable between the control and treatment gro ups. 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 gonada l 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 a nd increased oocyte atre sia. 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) screeni ng 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 includedPCNB FINAL

Page 39 of 62 a positive control group consisting of 10 castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutan eous (s.c.) injection.

To screen for potential anti-andr ogenic activity, PCNB in 1% SCMC was administered daily via oral gavage to 54-day old, castrat ed male Sprague Dawley rats (10/group) at dose levels of 100, 300, or 1000 mg/kg/day in conjunction with a da ily 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 animal s 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 weight s 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 tr eatment-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). Animal s 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 Co wper’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 a nd body weight gains for the 100, 300, and 1000 mg/kg/day dose groups were comparable to cont rols 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 an y of the accessory sex organs of PCNB-dosed animals. The unadjusted liver weights were incr eased by 8% (NS) at 100 0 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 we ights were increased by 10% (p ≤ 0.01). All %CV values were less than or equa l to the maximum recommended values.PCNB FINAL

Page 40 of 62 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 androgeni city and anti-androgenicity in the Hershberger

assay.

Female Pubertal Assay (OCSPP 890.1450)

In a Female Pubertal Assay (MRI D 49042103A), 15 Sprague Dawley [Crl:CD ® (SD) IGS BR] rats/dose group were treated daily via oral gava ge with PCNB (95.6% a.i.; Lot # 107030L052) in aqueous 1% sodium carboxymethyl cellulose at doses of 0 (v ehicle), 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, an d age and weight at day of attainment were recorded. Following sacrifice on PND 42, blood was collect ed for clinical chemistry and hormone analyses. Total serum thyroxine (T 4 ) was determined by radioimmuno-assay, but the method used for determination of thyroid stimulating horm one (TSH) levels was not reported. Adrenal, liver, pituitary, thyroid, and uroge nital organs were weighed, and microscopic examinations were performed on the ovaries, uter us, thyroid, liver, and kidneys.

There were no effects of treatmen t on mortality, clinical signs of toxicity, final body weights or body weight gains, food consumption, or gro ss 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 ch emistry parameters were observed at 500 and 1000 mg/kg/day, respectively: alkaline phosphata se (ALP) activity was increased (p<0.05) by 20 and 26%; alanine aminotransferase (ALT) ac tivity 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 bo dy 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 female s classified in metestrus by examination of the uterus (with cervix) was increased (6 treate d vs. 1 control; p<0.05); however, the biological significance of this finding is unclear.PCNB FINAL

Page 41 of 62 Serum T 4 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-re lated 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 gava ge with PCNB (95.6% a.i.; Lot # 107030L052) in aqueous 1% sodium carboxymethyl cellulose at doses of 0 (v ehicle), 500, or 1000 (limit dose) mg/kg/day from post-natal day (PND) 23 to 53. An imals were examined for preputial separation (PPS) daily beginning on PND 30, a nd age and weight at day of attainment were recorded. Following sacrifice on PND 53, blood was collect ed for clinical chemistry and hormone analyses. Total thyroxine (T 4 ) and testosterone serum le vels were determined by radioimmunoassay, but the method used for determ ination of thyroid stim ulating hormone (TSH) levels was not reported. Adrenal, liver, pituit ary, thyroid, and urogenita l organs were weighed, and microscopic examinations were performed on the epididymis, test is, thyroid, liver, and kidneys.

There were no treatment-related effects on morta lity, clinical signs of toxicity, body weights or body weight gains, food consumption, or gross pa thology. Compared to the control, absolute, adjusted, and relative (to body) liver weights were increas ed (p<0.05) by 8-10% at 500 mg/kg/day and by 10-11% at 1000 mg/kg/day, an d relative kidney weights were increased (p<0.05) by 8 and 6% at 500 a nd 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) wa s increased (p<0.05) by 15%. Minimal increased inflammatory foci with/without single cell necrosis was obser ved in livers of 5/15 males at 1000 mg/kg/day (NS) co mpared to 2/15 controls.

There were no effects of treatment on age and bo dy weight at attainment of PPS. All males achieved complete PPS, and serum te stosterone levels were unaffect ed by treatment. There were also no treatment-related changes in weights or microscopic findings fo r the male reproductive
tissues.PCNB FINAL

Page 42 of 62 Compared to the controls, serum T 4 levels were decreased (p<0.001) by ≥ 78% at 500 and 1000 mg/kg/day, with T 4 levels being below the method limit of detection (<LOD, 0.82 μ g/dL) in 6/15 males at 500 mg/kg/day and 12/15 males at 1000 mg/kg/day. Conc omitantly, serum TSH levels were increased (not significant [NS]) by 39 a nd 25% at 500 and 1000 mg/kg/day, respectively. However, no treatment-related change s were noted in thyroid histology.

The high dose (1000 mg/kg/day) was adequate ba sed on increased liver a nd kidney weights, and changes in clinical chemistry pa rameters in the study, and consid ering that 1000 mg/kg/day is the limit dose for the assay.

Steroidogenesis Assay (OCSPP 890.1550)

In a steroidogenesis assay (M RID 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 inde pendent 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 ea ch independent run of a test chem ical plate to demonstrate that the assay responded properly to pos itive control agents at two con centration levels. The positive
controls included a known inhibito r (prochloraz) and a known indu cer (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 μ M in Run 1. Therefore, the highest PCNB
concentrations evaluated were 10 μ M in Runs 1 and 2 and 20 μ M in Run 3.

For the test plates, the coefficients of variat ion (% CVs) ranged from 4.8–5.8% for absolute
testosterone concentrations a nd 1.7–3.2% for absolute estradiol c oncentrations 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 be tween-plate CV for testosterone. Although the between plate CVs fo r estradiol exceeded the guideline performance criteria, the estradiol data is considered acceptabl e in light of the low within-plate CV values.

Basal hormone production in the SC wells wa s acceptable both in te rms 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. Fo r estradiol, the fold induction resulting from forskolin couldPCNB FINAL

Page 43 of 62 not be confirmed to be above the guideline speci fied >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 induc tion 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 synthesi s 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 estr adiol. The between plate CVs for the SC
wells was 15.9% for testosterone and 45.0% for estrad iol. 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 accepta ble in light of the w ithin-plate CV values.

There were no significant differenc es in testosterone concentrati ons in the PCNB-treated cells relative to the SC, or in estradiol concentrations at PCNB levels ≤ 1 μ M. PCNB caused small, but statistically significant (p ≤ 0.05), increases in estradiol con centrations in the culture medium

at 10 μ M in all three runs (1.09- to 1.21-fold) and at 20 μ M in the third run (1.36-fold). However, all significant increases we re <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 repr oducible 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.

.PCNB FINAL

Page 44 of 62 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-e strogenic activity including in vitro competitive binding assays to
recombinant human α and β estrogen receptors (ERs; hER α and hER β ) 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 hER α and hER β were performed according to procedures that were previously described for the rat assay. Briefly,
recombinant hER α and hER β were diluted in hER buffer, and incubated for 17 h at 4°C with
DMSO or 10-fold dilutions of either 17 β -estradiol (E 2 ; 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]E 2 (~0.22 mCi, 88 Ci/mmol) at a final concentration of 5 x 10 -9 M E 2. 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 hER α gene together with expression plasmids containing an estroge n responsive element and the lac -Z reporter gene (encoding the

enzyme β -galactosidase), were incu bated in medium containing the test chemical and the chromogenic substrate, CPRG. Active ligan ds (which bind to the receptor) induce β

galactosidase expression, causing CP RG (initially yellow) to cha nge 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 concentra tion 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 (Hep es) were used as negative contro ls, 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
hER α . 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 hER β or rat uterine ER was observed. Overall, it was concluded that PNCB does not show biologically relevant binding to

ER.PCNB FINAL

Page 45 of 62 In the yeast hER assay, PCNB did not show a ny anti-estrogenic activ ity. The anti-estrogen hydroxytamoxifen gave the expected inhi bition of E2-induced activation of β -galactosidase, and the negative controls niacinamide and Hepes s howed 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 (incl uding PCNB; purity >99%) were evaluated in transactivation assays for hER α , hER β , 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 17 β -estradiol or dihydrotestost erone (DHT) for the ER or AR assays, respectively. In addition, for measurement of antagonist ac tivity, PCNB was co-administered with 17 β -estradiol (10

− 11 M for ER α ; 10

− 10 M for ER β ) 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 prolifer ation assay and in a y east estrogen screening (YES) assay. MCF-7 cells were incubate d at 0.001, 0.1, 1,0 and 10 μM with PCNB, and proliferation was determined by measuring DNA c ontent. After 6 days of exposure, PCNB did not have any effect on cell prolif eration. PCNB was negative in th e 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.PCNB FINAL

Page 46 of 62 Uterotrophic Assay

In a multi-component, published study by Ashby et al

. ( 2005, MRID 48233802 ), PCNB was tested using several assays for estrog enic/anti-estrogenic activity including in vivo weanling rat uterotrophic assays assessing both estrogenic and anti-estroge nic 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 (Wis tar derived) female rats were obtained from the AstraZeneca breeding unit (Alder ley 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 (Experime nts 2-6), PCNB was tested for estrogenic and anti-estrogenic activity. Test comp ounds were administer ed 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 abse nce of estradiol (E2) over a dose range of 1-10 μg/kg/day; Faslode x (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 presen ce of either E2 at 0.1-5 μg/kg/day (Expt. 5) or diethylstilbestrol (DES), a s ynthetic estrogen, at 0.01-0.5 μg/k g/day (Expt. 6). Animals were
killed as described above 24 h after the fina l dose. Uteri were removed and weighed. In Experiment 5, blood was collected in heparini zed tubes by cardiac puncture, and plasma was prepared by centrifugation for use in various biochemical assays which measured the following: alkaline phosphatase (ALP), alanine transamina se (ALT), aspartate transaminase (AST), and glutamate dehydrogenase (GLDH). Livers from animals in Experiment 5 were removed, weighed, and used to prepare microsomes and cy tosol; 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 an tagonize the uterotrophi c activity of E2 in Experiments 2 and 3, while Faslodex abolishe d it. Unexpectedly, PC NB 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 indicat ed that the potentiation by PCNB of the uterotrophic activity of E2 was specific to E2.PCNB FINAL

Page 47 of 62 PCNB had no effect on liver weight or the plas ma markers of hepatotoxicity ALP, AST, and GLDH. A small reduction in ALT (~75% of contro ls) was consistently ob served in all groups receiving PCNB; this effect was considered to be biologically insigni ficant. 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 aut hors concluded that PC NB is neither an estrogen nor an antiestrogen in vivo . The enhancement of estradiol uterotroph ic activity by PCNB was attributed to possible alteration of the me tabolism 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-e strogenic activity including an in vivo female pubertal rat assay for anti-estrogens. PCNB (99% pur ity) 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 AstraZene ca 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 as say; food and water were provided ad libitum

.
For the pubertal rat anti-estrogen assay (des ignated 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-est rogen; control animals received peanut oil only. Com pounds were administered once da ily, 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 di slocation. 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 no t related to treatment with PCNB. The positive control Faslodex inhibited uterine gr owth and delayed vaginal opening.

The authors concluded that PCNB is ne ither an estrogen nor an anti-estrogen in vivo

.

Three-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a three-generation re production toxicity study ( MRIDs 00001666 and 40865903 ), PCNB (98.2% purity) was administered to 25 CD Spra gue Dawley rats/sex/dos e 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 PPCNB FINAL

Page 48 of 62 generations received the test diet for 11 weeks prior to mating to produce the F 1a offspring. After
the F 1a offspring were weaned and sacrificed, the P animals were mated again to produce the F 1b offspring. The F 1 parental animals were mated when they reached 105 days of age to produce the

F 2a and F 2b litters; parents for the third generation were selected from the F2b offspring.

Significant differences in body we ights were observed for the F 2a females at weaning and the F 2b females at mating in the 5 ppm gr oup (7.7% less than control), the F 2b at mating and F 3a at weaning in the 50 ppm group (9.7 and 7.9% less than control), and the F 2b at the second mating (6.3% less than control).

No treatment-related effects were seen in the ma le 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 (F 1a , F 2a and F 3a ). No treatment-related gross pathological changes were seen in the gonads, thyroid, adrenal and pituitary glands of the F 3b litters.

The NOAEL is 500 ppm (25 mg/kg/day) for re production effects, and the LOAEL was not
determined.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction 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 F 1 generations, resp ectively); with exposure continuing in dams through gestation, lactation and weaning. The P generation rats were mated once to produce F 1 litters, and the F 1 generation rats were mated twice to produce F 2a and F 2b litters.

There were no treatment-related eff ects on mortality in either P or F 1 animals. Clinical signs included small size in high-dose F 1 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 F 2a gestation period. In the 3000 ppm group, so me small size males and one emaciated female was observed. Significant bodyweight reduc tions were observed in the mid- and highdose P generation males and females during the pr e-mating periods and in the high-dose females during the first mating (gestati on and lactation) periods. Food consumption was consistently reduced in the high-dose group with effects obs erved early during the study periods for both generations. No treatment-related effect s 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 gestati on length in either mating, mean number of live newborns, sex ratio and offspring survival. For the F 1 parental animals, noPCNB FINAL

Page 49 of 62 treatment-related effects were s een 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 F 2a litters were seen at the mid-( 6.1) and high (5.3) dose groups comp ared 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 F 2a newborn females or the litter
indices for F 2b mating.

No treatment-related histopatholog ical changes were seen in th e testes, epididymides, seminal vesicle, prostate, coagulating gl and, ovaries, uterus, vagina, cervi x, 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 LOA EL for neonatal effects are 20 and 3000 ppm, respectively, based on decreas ed number of males in F 2a litters.

Two-Generation Reproduction Toxicity (Rat, OCSPP 870.3800)

In a two-generation repr oduction toxicity study ( MRID 43469301 ), PCNB (98% purity) was administered to 35 Sprague Dawley rats/sex/dos e by gavage at dose leve ls equivalent to 0, 10, 100, or 1000 mg/kg/day in 0.1% car boxymethylcellulose/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 F 1 generation. The F 1 males and females were treated from weaning for at least 10 weeks prior to mating and through the mating period for the F 2 litters. The females were treated until the weaning of the F 2 offspring.

There were no treatment-related effects on mort ality, 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, gestati on length, mean litter size, mean number of live offspring or sex ratio in either generation.

In the F 1 generation, there were no treatment-related effect s on the absolute or relative weights of the testes, epididymides, seminal vesicle, pr ostate, or ovaries. At 1000 mg/kg/day in the F 1 generation, the absolute (0.128 g) ovarian weight was significantly (p< 0.05) increased when compared to controls (0.109 g). There was no eff ect on the mean relative ovary weight or any corroborative histopat hological 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.PCNB FINAL

Page 50 of 62 No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uter us, vagina, cervix and pituitary gl ands 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 1 000 mg/kg/day females of the P and F1 generations. In the P generation adults, the incidences of thyroi d follicular cell hypert rophy/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 female s at 1000 mg/kg/day (54% vs. 14% in controls).

The NOAEL values are 10 mg/kg/day in male s 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 developm ental toxicity study ( MRID 40588601 ) groups of 25 pregnant Sprague Dawley COBS CD rats were admini stered 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 we re euthanized on GD 20.

There were no deaths and no treatment related clinical signs were obs erved in any group. Body weights were slightly reduced ( not significant) in the high dose group during the entire gestation period. Food consumption was significantly decr eased in the high dose group on days 6-9. No other significant differences in food consump tion 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, ea rly 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.PCNB FINAL

Page 51 of 62 Developmental Toxicity (Rat, OCSPP 870.3700)

In a prenatal developm ental toxicity study ( MRID 41361201 ) groups of 25 pregnant CRL: CDTM(SD)BR Sprague Dawley rats were admini stered 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 we re 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 betw een 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 tr eatment. The maternal NOAEL is 1500 mg/kg/day,
and the LOAEL was not determined.

No treatment-related changes were observed fo r pregnancy rate, number of corpora lutea, implantation sites, post implantation loss, ea rly 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, signifi cant 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 developm ental 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 we re 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 ulcera tion and/or litter w ith six conceptuses. One high dose doe delivered prematurely on day 28 and exhibited the clinical signs of wei ght 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 NOA EL 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 fo r pregnancy rate, number of corpora lutea, implantation sites, post implantation loss, ea rly 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.PCNB FINAL

Page 52 of 62 Developmental Toxicity (Rabbit, OCSPP 870.3700)

In a prenatal developm ental 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, earl y or late resorptions/doe, number of live fetuses/litter, fetal sex ratio, or soft tissue abnormalities at any dose.

Chronic Toxicity/Carcin ogenicity (Rat, OCSPP 870.4300)

In a combined chronic toxicity/carcinogenicity study ( MRID 41987301, 1991 ), groups of 50 CD
rats/sex were fed diets containi ng 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 c linical 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, semina l vesicles, uterus, and mammary gland.

No treatment related clinical si gns or differences in mortality were observed during the study. In the high-dose male and female rats, body wei ght gains were decreased (p<0.05) up to 10% compared to control throughout the study. The oc casionally significant body weight differences
observed in the mid-dose males and females we re not considered biologically significant; however, mean body weights in high-dose male s and females were decreased by 11% by studytermination (Week 104). Food consumption, as g/animal/day, was significantly lower for highdose males during the first 10 weeks of th e study. Food consumption, as g/kg/day, was significantly higher in the hi gh-dose males and females.

No treatment-related effects were observed fo r hematological or urinalysis parameters. Significant increases in cholesterol were obs erved 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 incr eased by 20% in high-dose females. Histologic findings in the liver included increased hepatoce llular hypertrophy at the mid and high doses in
both sexes. For thyroids, the absolute and/or relative weights were significantly (p <0.01)PCNB FINAL

Page 53 of 62 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 histopat hological lesions were observe d in the testes, prostate, ovaries, uterus, and cervix, ma mmary 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/Carcin ogenicity (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 dos es 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 sacrif ice. For clinical chemistry ex aminations, additional groups of
10 rats/sex were sacrificed at the 6-, 12-, 18- and 24-month interv als. Organs that were weighed included the liver, kidneys, testes, ovaries, uter us, 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 c linical signs, mortality, body wei ght, or food consumption were observed during the study. Dose-dependent decrea ses 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 in creased by 22% (NS) compared to control males (0.0502 g), and the rela tive thyroid weight was increased by 24%. No treatment-related changes were seen in absolute or relative weights of th e testes, ovaries, uterus or adrenal glands.

At the high dose, increased incidences of non-neoplastic lesi ons of the thyroid glands were observed in both sexes. Follicular cell hypertro phy/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 hepatoce llular 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).PCNB FINAL

Page 54 of 62 No treatment-related histopa thological lesions were observed in the testes, seminal vesicles,
prostate, ! ovaries, uterus, mammary, ad renal or pituitary glands.

Based on thyroid follicular cell hyper trophy/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% pur ity) 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 reproduc tive organs were weighed. The tissues subjected to histological examination include d liver, kidneys, gonads, pituitar y, 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%. Re lative (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 histopatholog ical 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 ), B6C3F 1 mice (50/sex/dose gr oup) 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 grou p only contained 49 male mice. Organ weights
were not determined

. The tissues subjected to histological examination included the kidneys, liver, adrenal glands, pituitary, thyroid/parath yroid, ovaries, uterus, ma mmary glands testes, prostate, and seminal vesicles.

Mortality was significantly increased by study te rmination in females at 5000 ppm (22/50) compared to the control (13/50). No treatment-related effects were re ported for body weights.PCNB FINAL

Page 55 of 62 No treatment-related histopatholog ical lesions were observed in the testes, ovaries, seminal vesicle, prostate, uterus , mammary, thyroid, adrena l and pituitary glands. No treatment related nonneoplastic or neoplastic lesions were observe d 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, pituit ary, adrenals, thyroid and parathyroids. Gross and microscopic pathological examinations were con ducted on these tissues as well as the prostate, seminal vesicles, uterus, and mammary glands.

No treatment-related effects on mo rtalities, 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 si gnificantly 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, t hyroid, 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), a nd 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 pi tuitary, thyroid, adrenal, and gonads.

No treatment-related effects on mo rtalities, clinical signs, body weights, or food consumption were observed during the study. No treatment-related effects were observed for the urinalysisPCNB FINAL

Page 56 of 62 parameters. Significantly decreased hematocrit values were observed in the 30 and 180 ppm groups. In the high dose group, signi ficant 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-dos e 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, thyroi d, adrenal or pituitary glands. Cholestatic hepatosis with secondary bi le 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% purit y) 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 adrena ls, brain, heart, kidneys, liver, testis and ovary. Gross and microscopic pathological examinations were co nducted on these tissues as well as the mammary gland, pituitary, prostate, seminal vesicl e, 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 wei ght 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, ova ries, or adrenal glands. No treatment-related histopathological lesions were observed in the testes, epidi dymides, 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 male s/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 gavagePCNB FINAL

Page 57 of 62 at nominal doses of 0, 5, 10, 100, or 1000 mg/kg/da y for 13 weeks; actual dose levels were equivalent to 0, 4, 8.7, 100, or 985 mg/kg/day. Orga ns that were weighed included the liver, kidneys, testes, and ovaries. Gross and microsco pic pathological examinations were conducted on these tissues, as well as th e spleen, thymus, uterus, oviducts, mammary gland, adrenals, parathyroids, thyroid, and pituitary gland.

There were no treatment-related effects on mort ality, clinical signs, body weights, hematology, clinical chemistry, or gross pat hology parameters. The significant increases in food consumption for treatment groups (not specified) compared to the controls were not considered biologically significant. No treatment-related cha nges were observed in the absolu te or relative testicular or ovarian weights. In the high-dos e 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 histopatholog ical 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, he patocellular centrilobular hypertrophy was observed in both sexes of the high dose group.

Based on the observed thyroid fo llicular 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 PC NB (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 incl uded the liver, pituitary, and pa rathyroids/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 T 3 , T 4 and TSH levels.

No treatment-related effects were observed on mo rtality, clinical signs, or gross pathology. At 300 mg/kg/day , mean weekly body weights were signifi cantly reduced thr oughout 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, T 3 levels were decreased 19-26% (p<0.05) compared to the controls on Days 7, 14 and 90, T 4 was decreased by 48-54% (p<0.05) on Days 7, 14, 30 and 90, and TSH wasPCNB FINAL

Page 58 of 62 increased by 33-132% (p<0.05) on Days 14, 30 a nd 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 ab solute 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 d ecrease in pituitary weight observed at 300 mg/kg/day on Day 7 was not considered biol ogically significant. Th e relative liver to body weight ratios were significantly increased on Days 14, 20, and 90.

No treatment-related histopatholog ical lesions were observed in the pituitary glands. In the thyroid, follicular epithelial hype rtrophy 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; tr ace) and 300 mg/kg/day (1 5/15; mild) groups.

The LOAEL is 20 ppm (equivalent to 1 mg/kg/da y) 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/da y and 7 days/week. Organs that were weighed included the liver, kidneys, and te stes. Gross and microscopic pat hological examinations were conducted on these tissues as well as the epididymides, prostate, seminal vesicles, ovaries, uterus, pituitary, adrenal, and mammary gl ands. The thyroid gland was not examined.

No treatment-related effects were observed on mortality, clinical si gns, body weights, food consumption, dermal evaluations, hematology, clin ical chemistry, urinalysis, or gross pathology. No treatment-related changes were observed in ab solute or relative testicular weights. No treatment-related histopathological lesions were observed in th e testes, epididymides, seminal vesicle, prostate, ovaries, uterus, mamma ry 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 16PCNB FINAL

Page 59 of 62 applications over 22 days. Organs that were we ighed included the liver, kidneys, testes, and ovaries. Gross and microscopic pathological exam inations were conducted on these tissues as well as the thyroid.

No treatment-related effects were observed on mortality, clinical si gns, body weights, food consumption, clinical chemistry, dermal evalua tions, 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 ovaria n weights. No treatment-related histopathological lesions were observed in the test es or ovaries. Dilatation of the thyroid follicles (3/5) and hypertrophy of the thyroid follicular ep ithelium (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 observe d 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 wa s comprised of sixteen mating pairs, with one pair per pen. A progressive photo period was em ployed in this study: 8 hours/day through Week 7 and 17 hours/day until the end of the study. M ean temperature was maintained at 20.0±2.5°C throughout the study and mean humidity was ma intained at 48±12%. Individual adult body weights were recorded at study initiation, biw eekly through Week 8, and at study termination. Food consumption in each replicate was measured once each week throughout the study. Individual chick body weights we re recorded after hatching a nd on Day 14 after hatching; egg
shell thickness was measured weekly during egg production. A dult birds and chicks were observed routinely for mortal ity 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, si gnificant (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 nomi nal concentrations of 0, 1000, 2500, 4000, and 5500 ppm (not adjusted for pur ity) 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 progres sive photo period wasPCNB FINAL

Page 60 of 62 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 cons umption in each replicate was measured at least biweekly. Individual chick body weights were recorded afte r hatching and on Day 14 after hatching; egg shell thickness was measured weekly during egg produc tion. Adult birds and chicks were observed routinely fo r mortality and clinical signs.

No treatment-related effects were observed on ad ult 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 percen tage of hatchlings, and 14-day old survivor weights. In the

≥ 4000 ppm groups, significant reductions were obse rved for 14-day old survivors/hen, 14-day old survivors as a percentage of eggs lai d, and hatchling weights. In the 5000 ppm group, significant reductions were observe d for normal eggs as a percenta ge 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 prio r to the start of e gg 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 replic ate 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; e gg shell thickness was measured weekly during egg production. Adult birds and chicks were observe d routinely for mortalit y and clinical signs.

No treatment-related effects were observed fo r adult mortality, signs of toxicity, or food consumption. At 1200 ppm, body wei ghts were reduce for females ( ↓ 7%, p<0.05) at Week 8. No other significant differences in body weights were observed duri ng the study. No significant effects were observed for reproduc tion parameters, egg shell thic kness, 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,PCNB FINAL

Page 61 of 62 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 pr oduction). 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 th e study and mean humidity was maintained at 55%. Individual adult body weight s were recorded at study initi ation, biweekly through Week 8, and at study termination. Food consumption in each replicate was measured at least biweekly. Individual chick body weights we re recorded after hatching a nd on Day 14 after hatching; egg
shell thickness was measured weekly during egg production. A dult birds and chicks were observed routinely for mortal ity and clinical signs.

No treatment-related effects were observed for a dult mortality, signs of toxicity, body weight, or food consumption. No significant effects were obs erved for any of the reproduction parameters. The NOAEC is 5500 ppm, and the LOAEC was not determined.

Fish Early Life-Cycle Toxici ty (Rainbow trout, OCSPP 850.1400)

In a fish early life st age toxicity study ( MRID 41663401 ), groups of rainbow trout ( Oncorhynchus mykiss ) embryos (<24 hours old) were expos ed to PCNB (99% purity) at nominal concentrations of 3.8, 7.5,15, 30, or 60 μ g/L from egg stage to 62 days post-hatch in a flow-through system. A negative control group (d ilution 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 evaluate d for percent hatch, time to hatch, and time to swim-up. The fry were evaluated fo r clinical signs and survival daily. Growth was determined as standard length at 35 days post-hatch and at st udy termination (62 days post-hatch) and as wet weight at study termination.

Sublethal effects were observed with the greate st variety and frequency in the 52 μg/L group. At 52 μg/L, significant (p<0.05) reducti ons were observed in hatchabil ity (73% vs. 89% in control) and fry survival (75% vs 100% in control) at 35 days post-hatc h. 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 μ g/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 la rval 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 c oncentration was 20% less than that of the pooled
control larvae.PCNB FINAL

Page 62 of 62 APPENDIX 3: References Not Utiliz ed in the PCNB WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for PCNB but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the com pound of interest was not us ed in the study; 3) the test material was not adequately described; 4) a formulated end-use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.