PCNB OSRI for Tier 1 Screening March 5, 2010

REPORT TITLE

Evaluation of Functional Equivalence of Other Scientifically Relevant Information (OSRI) to EPA's Tier 1

Screening Battery for Evaluating the Potential Estrogen, Androgen or Thyroid Effects of Quintozene or

Pentachloronitrobenzene (PCNB)

DATA REQUIREMENT

Order/Data Call-In Notice for Quintozene or Pentachloronitrobenzene (PCNB)

Dated December 3, 2009 (EDSP-056502-105)

AUTHORS

Karyn L Hentz, M.S., RAC, D.A.B.T,

Amy Williams, Ph.D., D.A.B.T., and

James C. Lamb, IV, Ph.D., D.A.B.T

Exponent

1800 Diagonal Road, Suite 300

Alexandria, Virginia 22314

SPONSOR

AMVAC Chemical Corporation

2110 Davie Avenue

Commerce

CA 90040

SUBMITTER

AMVAC Chemical Corporation

2110 Davie Avenue

Commerce

CA 90040

DATE

March 5, 2010

• •

••

0907311.000 BOTO 0310 AW01

Page 1 of 61
PCNB OSRI for Tier 1 Screening March 11,2010

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this document on the basis of its

falling within the scope of FIFRA § 10(d)(l){A), (B) or (C). This statement supersedes any other

statement of confidentiality that may appear in this document

Company: AMVAC Chemical Corporation

Company Agent: Ann Manley, MSc DipRCPath(Tox)

Title: Director of Toxicology

EPA Agent of AMVAC Chemical Corporation

Signature: (£X^-~ J^\~^-A Date:

This document is the property of AMVAC Chemical Corporation. As such, it is considered to be •..•.;

confidential for all purposes other than compliance with FIFRA §10. Submission of tljis djxjiment in •

compliance with FIFRA does not constitute a waiver of any right to confidentiality, wjflcj^m^y exisj.,. J .

under any other statute or in any other country.

Page 2 of 109
PCNB OSRI for Tier 1 Screening March 11, 2010

STATEMENT OF GOOD LABORATORY PRACTICE COMPLIANCE

This document provides an evaluation of functional equivalence of Other Scientifically Relevant

Information (OSRI) to EPA's Tier 1 screening battery for evaluating the potential estrogen, androgen,

and thyroid effects of quintozene or pentachloronitrobenzene (PCNB). As such, compliance with Good

Laboratory Practice standards is not applicable to this document.

Authors:

Karyn L Hentz, M.S., RAC, DABT

Managing Scientist

Exponent

Amy Williams, Ph.D., DABT

Managing Scientist

Exponent

James C. Lamb, IV, Ph.D., DABT,

Fellow ATS

Principal Scientist and Center Director

Exponent

Signature:

Date: March 11.2010

Signature: A,Ulj / f. ],•;..,.,>,

V <L,

Date: March 11. 2010

Signature:

Date: March 11.2010

Sponsor/Submitter:

Ann Manley, MSc DipRCPath(Tox)

Director of Toxicology

AMVAC Chemical Corporation

EPA Agent of AMVAC Chemical Corporation

Signature:

Date:

• • • • • •»*

• • •

•• •

•

. .

Page 3 of 109
PCNB OSRI for Tier 1 Screening March 5, 2010

Table of Contents

1. Executive Summary 7

2. Introduction 10

3. Review of PCNB Regulatory Toxicology Studies Conducted for Registration 12

3.1. Mammalian Studies 12

3.1.1. Reproductive Toxicity Studies.... 12

3.1.2. Subchronic/Short-term Toxicity Studies 14

3.1.3. Chronic Toxicity/Oncogenicity Studies 17

3.1.4. Developmental Toxicity Studies 20

3.2. Ecotoxicology Studies 22

3.2.1. Fish Studies 22

3.2.2. Avian Studies .-.-. 22

4. Review of Publically Available OSRI 23

4.1. Published Studies with In Vitro Data and ToxCast Data 23

4.1.1. Peer-reviewed published Studies 23

4.1.2. ToxCast Data 24

4.2. Published Studies with In Vivo Data 25

4.2.1. Ashbyetal (2005) Uterotrophic Assay 25

4.2.1. Ashby et al (2005) Female Pubertal Anti-Estrogen Assay 26

5. Weight-of-Evidence for Tier 1 Assay Requirements 26

5.1. Endocrine-modulated Modes of Action 27

5.1.1. Assessment of Potential for Estrogen/Anti-estrogen Modulation by PCNB 30

5.1.2. Assessment of Potential for Androgen/Anti-androgen Modulation by PCNB 31

5.1.3. Assessment of Potential for Thyroid-mediated Endocrine Effects by PCNB 32

5.2. Tier 1 In Vitro Testing Requirements 33

5.3. Tier 1 In Vivo Mammalian Testing Requirements 34

5.4. Tier 1 In Vivo Ecotoxicological Testing Requirements 35

6. Conclusions 35

Appendix I: PCNB Data Referenced as OSRI 39

Appendix II: Endocrine Disruption Screening Matrix-PCNB Mammalian Regulatory Studies 42

Appendix III: Endocrine Disruption Screening Matrix- PCNB Ecotoxicology Regulatory Studies 43

Page 4 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Appendix IV: Published In Vitro Assays and ToxCast Program Results for PCNB 44

I. Published in vitro studies 45

a. Ashby et al., 2005 '. 45

b. Zou et al., 2002 46

c. Vinggaard et al., 1999 46

d. Kojima et al., 2004 46

II. ToxCast 52

a. ToxCast HTS Test Battery and Relevance to Potential Endocrine Modulation 52

1. Cell-Free HTS Assays 53

2. Cell-Based HTS Assays 55

3. Multiplex Transcription Reporter Assays 56

b. Comparison to Tier 1 In Vitro Screening Requirements 57

c. Data Specific to PCNB 59

Appendix V: Title Pages of Regulatory Toxicology Studies Submitted as OSRI 61

Page 5 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table of Tables

Table l:Tier ITest Battery and Requested EPA Action Based on OSRI Weight-of-the-Evidence 9

Table 2: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to Endocrine

Modulation from the PCNB Reproductive Toxicity Studies 13

Table 3: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to Endocrine

Modulation from the PCNB Subchronic and Short-term Toxicity Studies 15

Table 4: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to Endocrine

Modulation from the PCNB Chronic Toxicity/Oncogenicity Studies 18

Table 5: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to Endocrine

Modulation from the PCNB Developmental Toxicity Studies 21

Table 6: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to Endocrine

Modulation from the PCNB Fish Early Life Stage Studies 22

Table 7: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to Endocrine

Modulation from the PCNB Avian Reproductive Toxicity Studies 23

Table 8: Indicators of Potential Estrogen, Androgen and Thyroid'Endbcrine Disruption from Mammalian

Studies 28

Table 9: Tier 1 Test Battery and Requested EPA Action Based on OSRI Weight-of-the-Evidence 36

Table 1-1: Submitted Regulatory Toxicological Studies Referenced as OSRI 40

Table 1-2: Literature Studies Referenced as OSRI1 41

Table IV-1: Published In Vitro ER Binding Studies of PCNB Referenced as OSRI: Comparison to EDSP Tier 1

Guidelines1 48

Table IV-2: Published In Vitro ER Transactivation Study of PCNB Referenced as OSRI: Comparison teEESP

Screening Guidelines1 50

Table IV-3: Parameter Specifics Regarding Endocrine-Related Endpoints of the Cell-Free HTS Assays ....54

Table IV-4: Parameter Specifics Regarding Endocrine-Related Endpoints of the Cell-Based HTS Assays . 56

Table IV-5: Endocrine-Related Endpoints and Reference Compounds Tested in the Multiplex

Transcription Reporter Assays 57

Page 6 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Evaluation of Other Scientifically Relevant Information (OSRI) for Functional Equivalence to

EPA's Tier 1 Screening Battery for Evaluating the Potential Estrogen, Androgen, or Thyroid

Effects of Quintozene or Pentachloronitrobenzene (PCNB)

1. Executive Summary

The 1996 Food Quality Protection Act requires the US Environmental Protection Agency (EPA) to assess

the potential endocrine-modulating effects of a variety of chemicals. EPA's Endocrine Disrupter

Screening Program (EDSP) was the result of this mandate and currently consists of two tiers of screening

tests to assess whether chemicals may modulate hormone function in humans or wildlife. In this report,

we have evaluated the available toxicity database for quintozene or pentachloronitrobenzene (PCNB) to

determine whether sufficient functionally equivalent information is available for the purpose of

assessing the endocrine-modulating potential of this chemical comparable to that developed in EPA's

proposed Tier 1 screening battery. Functionally equivalent, in this context, refers ttrdata of a suitable

nature and quality to provide the same essential predictive information on a chemical, even if different

methods and procedures may be used for obtaining the data. The objective of defining functionally

equivalent endpoints and studies is to avoid unnecessary use of testing resources, delays in the

regulatory process, and sacrifice of animals while ensuring that sufficient data are available to evaluate

both the potential for endocrine activity and safety. Such data will be used to determine further testing

needs, and if necessary, to develop protective risk assessments.

Studies conducted for regulatory purposes as well as studies published in the open literature were

identified and reviewed. An objective assessment of these studies was performed to determine

whether, as a body of data, they provide data functionally equivalent to that in EPA's Tier 1 screening

battery and include sufficient information to characterize potential endocrine-modulating effects on

estrogen, androgen or thyroid hormone pathways. Conclusions are based on a weight-of-evidence

(WOE) analysis of the data.

AMVAC is citing multiple PCNB studies as "Other Scientifically Relevant Information" or OSRI, which it

believes provide data that are functionally equivalent to, and effectively substitutes for, the information

that would be developed through many of the Tier 1 endocrine screening assays and provide the

redundancy that EPA considers critical for adequate evaluation of potential effects on endocrine target

organs. We have reviewed the available data, and believe that these data, in conjunction with selected

Tier 1 tests, provide a test battery that conserves animal usage, uses existing information appropriately,

and will adequately identify any potential endocrine modulating effects of PCNB.

AMVAC believes that conducting the following tests from the Tier 1 screening battery will provide

complementary data and sufficient scientific redundancy to assure that potential adverse endocrine

Page 7 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

effects would be identified and sufficient data are available to perform a WOE evaluation of whether

any Tier 2 testing would be necessary:

In vitro: Steroidogenesis and aromatase inhibition assays;

In vivo mammalian:, Hershberger assay; and ...... _. . ..

In vivo ecotoxicological: Frog metamorphosis and fish short-term reproduction tests.

The cited (previously submitted) regulatory toxicology studies tabulated in Appendix I, Table 1 are

considered OSRI for assessment of the potential of PCNB to affect endocrine function. Copies of the

title pages of these studies, as requested in the Test Order/Data Call-In Notice for PCNB dated

December 3, 2009 (EDSP-056502-105) (the "Test Order"), are included in Appendix V. Published

studies cited as OSRI are tabulated in Appendix I, Table 2.

AMVAC believes that sufficient data for identification of potential endocrine effects are available from

the studies cited as OSRI, including the extensive regulatory mammalian toxicology data base developed

for supporting the registration of PCNB, peer-reviewed published literature, and other publically

available data in combination with the Tier 1 studies AMVAC is committed to performing. Therefore,.

AMVAC requests the following tests be waived:

• In vitro: Estrogen and androgen receptor binding assays; estrogen receptor transactivation

assay; and

• In vivo mammalian: Uterotrophic assay, male and female pubertal assay.

The following table (Table 1) summarizes the assays that AMVAC will conduct and those assays for

which we are requesting waivers.

Page 8 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table 1: Tier 1 Test Battery and Requested EPA Action Based on OSRI Weight-of-the-Evidence

Endocrine Disrupter Screening Program Tier 1 Test

••"*' ;.A( . Guideline , ,

, " Requested Action ..:> ;

 :<^ i

In Vitro Screening Assays

OPPTS 890.1250: Estrogen Receptor Binding Assay

Using Rat Uterine Cytosol (ER-RUC)

OPPTS 890.1300: Estrogen Receptor Transcriptional

Activation (Human Cell Line (HeLa-9903))

OPPTS 890.1150: Androgen Receptor Binding

(Rat Prostate Cytosol)

OPPTS 890.1200: Aromatase

(Human Recombinant)

OPPTS 890.1550: Steroidogenesis

(Human Cell Line -H295R)

WAIVER requested based on functional

equivalence of OSRI

WAIVER requested based on functional

equivalence of OSRI

WAIVER requested based on functional

equivalence of OSRI

AMVAC willconduct (no waiver requested)

AMVAC will conduct (no waiver requested)

'•.'In Vivo Screening Assays

OPPTS 890.1600: Uterotrophic Assay

OPPTS 890.1400: Hershberger Bioassay

OPPTS 890.1450: Pubertal Development and Thyroid

Function in Intact Juvenile/ Peripubertal Female Rats

OPPTS 890.1500: Pubertal Development and Thyroid

Function in Intact Juvenile/ Peripubertal Male Rats

OPPTS 890.1100: Amphibian Metamorphosis (Frog)

OPPTS 890.1350: Fish Short-Term Reproduction

Assay

WAIVER requested based on functional

equivalence of OSRI

AMVAC will conduct (no waiver requested)

WAIVER requested based on functional

equivalence of OSRI

WAIVER requested based on functional

equivalence of OSRI

AMVAC will conduct (no waiver requested)

AMVAC will conduct (no waiver requested)

Page 9 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

2. Introduction

The 1996 Food Quality Protection Act requires the US EPA to assess the potential endocrine-modulating

effects of a variety of chemicals. EPA has established the EDSP and is utilizing validated tests to assess

whether chemicals may modulate hormone function in humans or wildlife. Screening is designed to

involve a two-tiered approach. The purpose of Tier 1 screening assays is to identify chemicals with the

potential to interact with the endocrine system. The specific Tier 1 screens include a battery of in vitro

tests (including estrogen receptor [ER] binding and transactivation assays, an androgen receptor [AR]

binding assay, and assays of aromatase activity and steroidogenesis); certain in vivo mammalian tests

(including male and female pubertal assays, the uterotrophic assay and Hershberger assay); and two

ecotoxicological assays (the fish lifecycle assay and the frog metamorphosis assay).

The Tier 1 screening assays provide potential hazard characterization and mechanistic information

regarding possible endocrine modulation; however, this information is not useful for risk assessment

purposes because it does not characterize the dose-response or establish a no observable adverse effect

level (NOAEL). Therefore, chemicals that test positive for possible endocrine modulation in the Tier 1

screening assays must be further evaluated in Tier 2 tests to determine what functionally relevant

endocrine-related changes are associated with exposure in vivo, if any, and to provide information

regarding dose-response. To date, Tier 2 tests have not been defined, but are proposed to include

apical tests of reproduction and development in mammalian as well as other species.

To meet the registration requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA),

pesticide manufacturers must conduct guideline-compliant mammalian and ecological toxicity tests that

are designed to characterize the potential hazards associated with exposure to a chemical via a variety

of different routes. These tests include apical studies of development and reproduction, neurotoxicity,

oncogenicity, subchronic and chronic toxicity. The studies conducted in fulfillment of FIFRA

requirements include measurement of a wide-range of toxicity endpoints, many of which assess

endocrine modulation either through evaluation of impacts on hormone-producing tissues or endocrinetarget organs, or by assessing secondary functional endpoints such as reproduction and fertility.

Although these regulatory studies do not measure all of the specific parameters assessed in the Tier 1

battery assays, they provide functionally equivalent information. Further, these studies are designed to

provide information that may be used to determine NOAELs and may be incorporated into a risk

assessment, which the Tier 1 screening assays are not designed to do. In particular, the two-generation

reproductive toxicity study—especially when evaluated in conjunction with the current guideline

subchronic toxicity studies—is similar in scope and breadth to the proposed mammalian Tier 2 test and

provides sufficient information to assess whether endocrine targets are, in fact, altered with in vivo

exposure. Conducting Tier 1 screening assays when Tier 2 equivalent data are available is wasteful of

animal lives as well as both industry and EPA resources. Existing studies need to be considered carefully

when deciding whether all or some of the Tier 1 screening battery is necessary. Indeed, EPA is

Page 10 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

specifically directed by statute to minimize duplicative testing under the EDSP (see Section 408 of the

Federal Food, Drug, and Cosmetic Act (FFDCA); 21 U.S.C. § 346a(p)(5)).

As noted above, the endpoints examined in guideline-compliant studies generally are not identical to

those measured in the Tier 1 screening assays; however, the information derived from these studies

may be similar enough to provide confidence that the study results adequately characterize whetherthe -

compound is likely to be an endocrine disrupter at environmentally relevant exposure levels. For

example, the male pubertal assay, which is part of the Tier 1 screening battery, measures exposurerelated changes in organ weights and histopathological changes in various estrogen, androgen or thyroid

hormone-producing or impacted tissues following exposure of an immature intact animal through the

time of sexual maturation. Many of these same tissues are assessed in the multi-generation

reproductive toxicity study conducted under the current guideline (OPPTS 870.3800), which includes

exposure to the same developmental stages assessed in the pubertal assay, as well as during the

potentially more vulnerable peri-natal period. Endpoints also receive redundant assessments across

multiple life stages and in several species, as many of the same organs are also evaluated following

subchronic and chronic exposures. Additionally, clinical, gross and histopathological evaluations

conducted as part of these guideline studies measure many related endpoints, which provide additional

confidence that potential endocrine-related effects have not been missed.

Even the EPA Guideline 83-4 reproductive toxicity study, which lacks some of the critical endocrinerelated endpoints of the newer OPPTS 870.3800 guideline study, has been demonstrated as competent

and sensitive for detecting aromatase inhibitors, anti-androgens and estrogenic compounds. It may not

provide sufficient information to assess the mechanism of toxicity, but it provides significant sensitivity

to detect the potential hazard. For example, the adverse effects of linuron, which EPA characterizes as a

weak anti-androgen (and uses as a weak positive control), were detected in an 83-4 guideline

reproductive toxicity study (EPA, 1995).

Furthermore, the data derived from guideline studies are more informative than much of the data from

the Tier 1 screening battery in that they address whether exposure is associated with an actual

functional endocrine—rather than mechanistic—change in the intact animal system, at what doses, and

how these doses compare to those shown to cause other toxic effects that may drive risk assessment.

Thus, they provide information on the potential for endocrine disruption as defined in the Weybridge

conference:

"An endocrine disrupter is an exogenous substance that causes adverse health effects in an

intact organism, or its progeny, secondary to changes in endocrine function."

- European Workshop on the Impact of Endocrine Disrupters on Human Health and Wildlife

(Weybridge, UK; 1996). European Union Report EUR17459.

The following section, Section 3, of this report provides a summary of the guideline-compliant studies

reviewed with a description of the endpoints examined in these studies that are either functionally

equivalent to the endpoints in the Tier 1 assays or provide supplementary data on endocrine

modulation. Section 4 is a summary of the publically available studies that provide additional

Page 11 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

information on PCNB and associated endocrine-modulated effects. Section 5 is a WOE review on the

collective body of data, guideline-compliant studies and relevant published literature, evaluating the

sufficiency of the existing data in context of EPA's Tier 1 screening battery requirements. We conclude

with our assessment of the functional equivalence of these OSRI data and present AMVAC's request for

EPA action regarding each of the Tier 1 screening assays.

3. Review of PCNB Regulatory Toxicology Studies Conducted for Registration

3.1. Mammalian Studies

A total of 25 regulatory mammalian toxicity studies of PCNB conducted for pesticide registration

purposes were reviewed. These studies consisted of three reproductive toxicology studies; seven

toxicity studies involving either subchronic or short-term exposures; eight chronic toxicity and/or

oncogenicity studies; and seven developmental toxicology studies. These studies are discussed below in

terms of their functional equivalence for assessment of possible endocrine modulation by PCNB.

3.1.1. Reproductive Toxicity Studies

Three rat reproductive toxicity studies of PCNB were reviewed. These include two EPA 83-4 guidelinecompliant two-generation reproductive studies (900-DAR-008 and 900-REP-001) and a three-generation

reproductive study (900-DAR-001). The first two-generation reproductive study was considered

acceptable by EPA (900-DAR-008). EPA data evaluation records were not available for the other two

studies. Although the DER is not available for the second two-generation reproductive study (900-REP001), sufficient data are provided on the methods and results to assure that it was well conducted.

Limited data are available in the rat three-generation study; in particular, statistics were not conducted

for many of the key endpoints so that determination of treatment-related effects is more difficult.

Specific information regarding each study's design, test species, number of animals per group, doses,

and route and duration of exposure can be found in Table 2, along with information regarding the

various endpoints evaluated in these studies relevant to the assessment of endocrine disruption.

Page 12 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table 2: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to

Endocrine Modulation from the PCNB Reproductive Toxicity Studies

AMVAC Study Number

MRIDtf ' 1 - - -

Study acceptability

Study design

Test material purity

i Route of exposure

Exposure duration

Species (strain)

# Animals/sex/group

Age at study initiation

Doses (ppm)

Doses (mg/kg/d)1

i

i

f

„„ J ! - j i ''

] Body weights - male

Body weights - female

Gestational body weights

Lactational body weights

' Pre-coital interval

Gestation duration

Female reproductive indices

Male reproductive indices

# Pups ,

Dystocia

Lactational indices

Fetal/pup sex ratio

Pup survival

Pup growth

Nipple retention (at necropsy)

External genitalia (pups)

; Male external genitalia (Fl adults)

Fetal/pup reproductive tract

900-DAR-008

. '. - 41918701 „';_ :

Acceptable/guideline

reproductive "-'• ?\'l '

99.4-99.94%

diet

81 d pre-lst mating of FO

to weaning of F2b

"Rat(SD)

26

FO: 55 days of age

0, 20, 3,000, 6,000

FO ci 0, 0 8-1 7, 132-2~3~7,~263-486,

2 0, 1-1 9, 155-278, 361-537

Fl i 0, 0 8-2 7, 126-409, 274-900,

'- 0, 0 9-2 7, 132 422, 364-977

, .(prematjng).

Selected Endpoints

V

X

 " "'""

X

X

X

X

X

X

X

X

X

x"

X

"x

X

X

x" ' "

X

X

900-REP-001

". 43469301

DER not avail

'. '. • ^productive

94.15-99.7%

gayage

10 wks pre-FO mating

to weaning of F2

1

 Rat(SD)

35

FO: 56"days of age

0, 10,"lOO, 1,000 ' '

i

.1"

,,

 r x , "

X

_ ^ .._

X

X

X " "

" " X

X

"" X

x~""

* X

X

X

x j ..". ".

X

X

x

900-DAR-001

__

 Jj Mf _ , *, „

DER not avail

reproductive

98.2%

diet

11 wks pre-FO mating

to weaning of F3

Rat (SD)

20

FO: 28 days of age

0, 5, 50, 500

Not determined '

, '

i<( ;, ' • j' '

. ' ''

, j

. . ,,L . . . '..'1

_- -- j ,,^ , »3

, fc &

i

X

X

X

X

X

' x ~

X

'*«' ' ' ' ' ' J

Page 13 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

AMVAC Study Number

Organ weights - ovaries (O),

'uterus (U)

Organ weights - testes (T),

900-DAR-008 900-REP-001

O

T, P+SV

900-DAR-001

prostate (P), seminal vesicles (SV)

Histopathology - adrenal , X

Histopathology-pituitary X X X

Histopathology-thyroid .„„,', ,

 x x

Histopathology - ovaries (O), 0, U, V, C 0, U, V, C

uterus (U), vagina (V), cervix (C)

; Histopathology-testes (T), " " ' T, Ef>,'Sv', CG ' T ~ T, E,'<R,SV ""' ~ "TM

epididymides (E), prostate (P), j

seminal vesicles (SV), coagulating ;

gland (CG) 1

Gross pathology X X X

For studies in which PCNB was provided in the diet, calculated doses in mg/kg/d are for listed for males/females.

3.1.2. Subchronic/Short-term Toxicity Studies

Seven subchronic and short-term toxicity studies of PCNB were reviewed. These include three 13 week

studies in the rat (900-CHR-012, 900-SCH-001, and 900-SCH-003) and one in the mouse (900-CHR-001); a

four-week study in the dog (900-TOX-005); and two three-week studies in the rat (900-TOX-010,900-

TOX001). One of the 90-day studies in rats (900-SCH-001) was considered acceptable by EPA. EPA DERs

were not available for the remaining studies; thus, no conclusions can be drawn regarding the

acceptability of these studies. Two of these studies, the 13-week mouse study and the four-week dog

study were pilot studies for subsequent chronic studies and as such have limited data. Specific

information regarding each study's designfiest species, number of animals per group, doses, and route

and duration of exposure can be found inTable 3, along with information regarding the various

endpoints evaluated in these studies relevant to assessment of endocrine disruption.

Page 14 of 61
o

4-f Ol i

n

U fll ? .2

5" •TT ^§j

.£ %

o *£

3 •• » -x

c o in <u "^ -= o 1 bf o-o J "> *n *f 2 ° t S 5 '5 "> Q. -a ~o c c re

111 . . **• U

'>>• *r- ^ C•c S "x -c ° -Q £ 5 £ mo z o 5 Ol Q.

£. u .2E c <-•

re p = Ire *

.

01 C Q .2 ^ 4-» TO (U

+•• _.

<U "O

11

Ol QJ Q- c X •- LU b

«•§

01 C

60 3 ^

.£ ,2

c ^ Ol Ol u10^HL. (UpTM£ceCOOCQUQ.

«-l OoXoo0cno

«-t aXOi-* oocn

IS) ooS

I

H- CL

OOen-CHR-001

(pilot)

oocnroOoXuLOoocn«HoOiu"?o0cnCMrH9ccXuQ0cnt_Ol oE_3_AMVAC Study h

;;v;

,

-•*' i

r^i;' C3 '

<

Sv

T— f «t ' (N^- ;

'•>*, • ': \t \ •

rH' . 0 .

<3- m

,

h~3H ' V P!

:»i''

iHSi'

frill!' F&IS ;

'!,

:

••;,>•!,*:'

'»;!;'

''irij

1

O

:

^:

i-H

:

't

I•in;

,^.1;

!:'li£'

OM;;

•;<| rij:

*-' i''

!

^

=§;

'Si!

;J2-i;

:S; -" -;! : 42416QP1>,:: •{

'-'.

;*H'<

Ol'

.r>ij

LO;:

•ro:;

;LOo«

;Sii <*$\->

p'lityl . \'

^•'.-N

f!'

ilfl

'''tjii i

cc

:

i.?; H

3>CD «ca: Q

'roS

4.1oco: LU

/•>—

ro

4-*gQ£ LU

j^s^ not avail LUQfD>2occLUQAcceptable/

guideline

—

'ro>

•4-* : 8ccLUQ

!.§•Study acceptab

-^f— ---

'!!':••

U'c - ' O' ' '' •' i_ • • JCu

f*i DVI

• • : '

_y. p i

S,;''. j:« '.:/ -D ;.- . •

5}< .j;''

"•;,>

.0' • •,' ;

g'^ :,9,!3; >

-6";yi: •>&^. ;y

•*•"" - '• X'

5';*'o

•~ " •'r'

jj%';:-;:-

^fpj-chronic

ieify;stBdyS

*;"i x ;O,-:0

•'5. :?!•

_;: , :-;;

,'• ,- -?

:' ubchronic

;.wi"

:'',' '•

. subchronic - u' '•'

".E

 :^<

•S

 :;

,-5 '

>JD

13' • '' ^;V->-:

ii'{" +!•'

•J^ i '<•'

, *feii;

}

* ,. •• -I '

 :''ij'n

;

-">fj-ir •',;' M^ '::;

'''

:

;it,

y^: #^' - *M, ! SjStudylclesigrvSs."

^oocn' SSrM

r~»

06

cn^j 5skOo^3?cn

ft.

S?

cn

cn^oocnseLOcn§L. 3Test material pi

•

ro§

1

-£•

T)• ,

j

^;*

s

1

*-^ o>

•T3;;.

•ti . ' IjSf Sll1

\.,'i'ft

s.

ja-

';; .;.>'

•<u

'.(30

'ifOi

:•>

:ro

fi£

'-^; . •:,

:'.,

•4->'

'<U.

.^li

ha-EiRoute-pfr^xposi

, p

-^ ••'"•' S" ^ -S2.. ^ j1

 , _,

_jt j 4^1

> TO <. |:cc "O ! '. ' LD :;, .

V-: . \: '•

-.-,,—

|.'' 1 i

^j§

1-1 ]

iJ

fM TO '

i

'•^:!' ' : .-.

i - : ,^

I;:!'!''!

l;aj!.

1 ao

•o IIS.;' s is;

;^no'

1:0

|.Q •, ' ^ =-J'

s;- «11:

jt;.-'fe

Sf

v, P"

!

js; ^\D

5 'S-

;

<^ PcLli?

r^ i- i/i^

!

 ;3Cf

S

S .'O* •- "sr * !

;•<£_.'

p|j|

ft

;l

fcs l

?^

!

-o i^ o t — ' TM i^^5 -

 : •

i'u-- '

=j .; •

I.,-

I;';;'' '

•« E:;:-

Sfa-

^1^

5 |%

5 £B' i

 J

^ :<'t

' ^'f

"> I:S g S2- ro !«;-

,

«-< jS;

if'

JV;. i

V'T'N

1

cO * if{

:rMej

._ eilt

Exposure durat

L;:Sp^ecii|f(st|ain);

LOIDMOtH

^V'

-

,

in

r-*orHLO

rlCL38M# Animals/sex/

|||

\&i-'s

:^"'.

QJ •"*

^Of :' ^

•^;,

'?$"• Wli } i' ''','.'•

'!."fj|;

!^'

:!T

.MI::

^c.

SO), w.

'0';

itH:;

' r:

<«.'.'•

:

'i •'

 :

;;':i

j

;.

s.'*,i

li*

,;6*

;.;ESJ

;y?'"

LO; t ,:..>-

,

&aj-

oy

tig;

3i'";a

iil

•

:

-vir;

t'-^i

fa»

!

!<

;

5

:i W

i'op."

^.•^;

;

i:-3

!

:..-.-:-•; i!

i-8ii

iiS'i

!££'• i«~*'.f

r,. •:• !' ;-.;''

Hl^H

?»•'

^:^ !!

.tu,

;

fl

r-»!

Jill

!;•' /'•

r v>'/

i ^ -- • oi.1-: m\'t9f,< fci-fl'i f^t- iij,§%

[ij*HM

1

 !

lr! 1

M02

e«

*.2-i

r**j

Is!

P>^D::i

iftts

;

jrei ;1(uH

•158

SlSil oo0 0

rsg

§ ^ o0 § S8 rH ^

.-°' " LT 8)

'"° <N

Oo0

LO" o"01ooofS ._

.

*— ' O "I 8 s * 0?CL 5:VI Oi O) OQ

.-S

£&?r*. iSiii-*

^^^;LD?IP:-.:

f

tLO . cci

0)wi

X

•x-i' x x- X I X X:^ X

IpiMil ^J

;jaj;l "

tw'l E N- fti^ i wi I w»

^il£

.SPij .w>

^«u-;j oi ^?

 5

'^'1-g-

fOs; O

CO CQ

10M—O

LT) rHO) 00OS a.
ooIN

in"

"§

re5BOC'EHI SI u10iHQ) P1.£EinOCOzuQ.

OO1X0ooen0

tHI 0Xo1-to0eninoo§

1

H 0.

8en

rl 8* i"" X ° v a 0oenooXo1ooen

tt o9xu6oenINTHoi

t£. XU60en,Ol X)

|

^^ T33552**

t

X X ' }

' \ ' ( 5

t 1 ' * I '•

;,'; .... ' i ' ' x>

~i ! ' •i, !• ', '! J'' '!

!

 ' ^ " ' *t ? t ' * ' t X X X X X { i t] 1 •1 '1

 X 'X

1 ' 1 ' '' \ ', • I x x !

x

< ' !

* ' ( s ' f i ' ' ' J i i ' , .H c j 1

 j E a ' t 1, ' ' * 1 X XX X , t

' , ' \ i I 1 J 1 } t i 1'

 h { •

*

x

 : x x x !

x

!\

\

y* > , • * I

- > * "'tn ' '

:"''' * ' • '•"' i i

VI VI , VI l/J IA | |^ft Cjf

£ £ £ hjJc

 f'£

l o o

Cto * bo bo

 r WJ < &

) "ID

 f "

o

*5

 T *

S *(U o^ 'cw •*"* -C

£

 c S *

5 m.ti mTM raO

 C O

2? -1 2? ^, S? 2 S? 5 S?. .? -S .^ [ 1 , „. x O

x

o

X

(/)

x' ^

U3

•s0100

roa.

Z3O

ioo

! Q.

•So

OJ TJ_C-oSQ

. nCDuO- JCulc ' o « »'
PCNB OSRI for Tier 1 Screening March 5, 2010

3.1.3. Chronic Toxicity/Oncogenicity Studies

Eight chronic toxicity and/or oncogenicity studies of PCNB were reviewed. These include three rat

chronic toxicity/oncogenicity studies (900-CHR-005, 900-CHR-014, 900-CHR-024), three mouse chronic

toxicity/oncogenicity studies (900-CHR-010, 900-CHR-001, 900-CHR-024), and two chronic toxicity in the

dog (900-CHR-013, 900-CHR-016). Two of the rat chronic toxicity/oncogenicity studies-(900-eHR^005/

900-CHR-014) were found by EPA to be guideline-compliant. EPA DERs were not available for the

remaining six studies. Specific information regarding each study's design, test species, number of

animals per group, doses, and route and duration of exposure can be found in Table 4, along with

information regarding the various endpoints evaluated in these studies relevant to the assessment of

endocrine disruption.

Page 17 of 61
V

o4->

S

1.2

_CJ ^

<u S

o oe v)

*"* "S *"

(M 'C 'u

in 5 =

*" a o» _^ Q £yQ

£ °- 8

5 0 = 2

 2 5.

.c >

§. 5

UJ 1-

.ti 'c

— •= i_

^^ n -C

^P ° m ^^^^r U ^p

11

"o e

s i

vt •*-

•« p

Q .2

g |

.§.«

i- C

a* -c

O. u

ul -o

<* uj

0)

do JO

c ^

E

0)

cu

u

(/)

^

.2

^~

S

V)

O

m

•

z

ai

I

oc

s

 s

en

ec

v s

o o

o

en

cc

so §en

cc.

u "*

6 S

o

en

cc

TU o 6 o

o

cc

v s 6 o

o

en

cc

o

*

 o§o

en

i

cc

6 o

o

en

cu

"1

z

TO

1

u

$5

<

" 'ro x ' « '" \ ' sn

> O . — . -C

, ro ^''S ? i i/i .Si • , , £

' 0 ''E ^ .S! >• 0 re3 •* 2

i C ; o 00 ^T3 ,vi ;Q

 !CJ j E

•1 ft •„' ' ' I 2" . i*

m '""... ,fc v j.^^u. ^ » . *, . „. T . ., l«vi ', „ ii * -.~.- _ — — —£_: - • —

''"

 S iu ^'- - 'ao5

" '^ '

*o P ij3!?i! **" ,On s .{UT) 2

c o Oi '~° ** 'Q o ' < 1C.

, OE , J= ' S ' r^

•• !g 1-5' -i "

i , , t i * J , * 1

5 £ ' ^ ^

j» ;-E s? 4, | 'is s., *„'£

g '& & f-|

 ;

C §u 'S o o '5

 0 ;"30 O "^<J3

 tj."i

-l^

,OC 1 <J , . ,.

>r^ j^C Q O,< U

1 UJ i C ' I — ' (N > ' T3

," i "" "' ••

' i ' * ' ' ;

'5 l-t " ,. ' ••'« ^! i" -g

TM ,-E S? 4,,| ,« £ *, -g 4. ^

o 'o . r^ .cu , ? roOcgQ.O'c

c

 o , ' ; "^ oo •«,«; u2"

cc u ."i^- oS o <u

uj c ii ^ rM , T3

- ° i° • ' ;•„.•" ,

^

 <:s '> rr | • . .w '

SB S'l* ^»| :«£. ', ;|'

S ,| ,£ Sg 2 ^ g 0 . g .1

S '«

 je 8' A • s s g M «•

•* uj j= c rt . — lob

',. Q .U 01 ,j • ,i' ,

— i >, ' • I I1. ,i- 5 £ ss • ^

i re ' u "^ " w cu • — ' l~5

' ^ ''c 'oo'^'- ^ !«£ 'o

 !$

•-S ll $."§ § ||':3 :*• •

-,| •§' . s i ,,:" . irj ,1 • s i •'' ' •; J :

•^^ '"--^ >» " ^

rt CU ml X •-, ,

R.l^^ig - e s •;„ '1 ' <*• ' 4-* GJ i CU ' ^^ ' CU ^. *"* C3 ftj"'1

00 CL 73" COOm^ S 4J *D >

CfiCU—ooS^'^ 1 <S >

3 - .H al^01

- : !« •• :• * ••

<• ' u o '

.,1

 l

 , ' '

1 ' . ''

l

1

^ ^ >* * , "' , " >

g.sSdi. g-^^ o .!'. ^'

SlS^iugg'raSg.ii ' 0 !.g

<-" • Q. -D- E S) S >,^-4 ^ " "3 ! SJ

O ' • CU ~ o ' O " W n> 'i "0 (N ro , S,,

(Y5 « l (J « «>3 ti ^ _ tj G 0O0 t- UOl ^v^ " 1*^1

 iOC ^ tp

•j < I o o; ' • i ' ' i

1 S , ' •!• •' " '

'

s

••I )' : , f • | j s c •

i ' ' ' D. • o

•" & , «|'r«: c : ' :''§ ^

»

 r=

 '/' 3 3 < -S : , ,60 S

s

 en __ o P , ^ x «—

I ;5 -S.fl .'B 'IS. ^ 3 *s , £j . * u (5. ''.2 3'

S-L>"S . Sioj.v ) . «. ' • E •".

2"U '-0 4^

 !^' 0 'o c '" •'

CC 5 5 To 3 Q. cu ,.? (U

^•'4J !4^ CUO' X t O '^- GO

S iri in, i— cc uj (/>' ' st <

f~ i - , --^^ 1

• 0" T3 '

oo cu

0 § 0 £

"I r-T "^ fc

0- , -g ,

o o . *r

•H Q Q TT Q ^

. . ^1" . -v ^f

o" ' ro . c-t

{f

us m "S - S^i en — 2

no 4/1 C) r1*-* JJ , , .h;

f~, £3- -M , '

-0 C> • -g .

( '

*" "* "O

LO C3 5r f1^ ^D ^> O * c

° ° b. ° 3 £ Z |

i "° ,

-D

go ^,.1

^ in S < '

O QJ

i "O

'

8 ,' «'

•* o ^ .E

0" ! TO

,,t

crT

8 «-r •„, 5 '

O o "O) O

rrT 0 ;0 ^ rH O

0" in 0~ 5J" ^ ^

^ V' ' T-* o

O ^"' ,-'1 -

' ' , V

• o"

'o ' , ;"l § -

»' r-f

in-

• • 'o • "•'

I • .,

E ' '

tH

tXO

1

a. .JE 1_

t/l j VI

cu cu

t/i in

0 O

Q Q

O

00

00

roCL
i

orHOfMin"§

re2

t>o

0) <uwu

I/)rHL. P1_OCOuQ.

CCv

*

I °

ecv s § ° Ol ecV N o ° o

cr>a: V Si o o oen<r "C i v s 6 o oOl ceu S 0o ° Cha: I -

.

v s o o oai

tc. u g 8 ° cnu. iuAMVAC Study Numbi

"';. ~ ~' IX X ' ' i ' ' ' 1

i/i c*QQ

.

T>C i * *

._- ,*»,,„.,„* - .

XXX X;X X

•' 1 '

','

'x x * '" "

, , 1 ' ' • ^ ''

x,x „• • : ;

i,,. • 1

 I ' '. 1 ' > • \

^ f ! * , '

xx' ' ,! < * • li

t

•« ,

"g ' ' <U(U ! ,«M i ! XX j ; | ' t } , | i ' i ,' 1 ' , ,

 !

 . i '

xxx x! • x

t <

•

, 1 ' j 1 * i t ^ i i :

X' X X "X 'X

i- . j , | , ,,•

i

S '„ '' ' s ' -O «

«

*t'i

 i

 {

 J}nJ

 &!

 ' • ' 5 1• ' TM ' •—

-1 El/ :^l' • §

E (U *- O

7

lHr.-'i M

M i

' ' tfi t tfl W i Vt

tA, M £ +1 j ;g , «

•£>•£. °° - no no -so

,2?. .5? '3 i 'S i '5 '53

5 3 ' »', _i _ as -.

•^.-*.5c£ ~*j S

*^ ^" re (y fo i ro *TM (u

m CQ O fo;O O 'a. O

"", ' :'"

!

 "• '

!

X ! X X ' X : O '< h-

, • ' i : 1 ' J' ' < > S ' ' ' j ' i ;' ' , • ;S , 'a_ X X , X X ID | tu"

 ! X , O •" ' • ' ! ' ! 1 ' 1 ' ' ''!5 ' '! 1 , - ' • 'cu - 'X

 !

X i X '^ ' . 1 » ' 1— i ', ' '°

 !

i ' i i ! ' l ' • , ( - ' ' 1 ' J 1 ' ! ,' ' .5 ,• /X 'X ., ,X tzf "' i^"- : 1

 1—

;,, i " 0. ; , ; ' • , ' f ;

 ' ' ' >

1

 I i t ' ' '

;

 x !x x 'ID ' D-

• • ! ' . - i iu '!>

 0 ! . i 'i i \ •"* ^ ' V * ' | J

n I ' .., 1 ' ,5 . i

~ 8

( X , X „ < X 1

^ ' j ^ IX

i i ' -O , .

 m

" . • » ' , ' H

!

~

 I

1

! ' 'I'MI > I ', t : |2 i • ,i 1 "1 S ; X ', X i X X z> ' ' ^ x '" • ; i°" 1^- 1

 ! I I; '• I-'1

 •>- !> j : . • , =5 .- j'.'l'C- • X X ,X X ~- , "^ X i ' § u "' ; d • ,r !

 '

* fB V)

.«• • : .£ — !S 3"

u> ' . v >

:

 4-2 2? ^ i ai " * " ^J

<y * u 2 > ' ' •*— ' j- i^j- c **"""*

*

J

 i i i i H3 ^ i/j i trt *? "O

.„• >• >• >» >* •=: ~"T

 :

c ^ >• "n <u ra i2 tto CUD cto tuo •* ci- * m bo •— 55 —••

^cO

 !

o ^o '^^^ ^rj

1

 J2 [ o P - ,-£ *^ *ft

w>"o "o, "o >"o— =^ ^

-

'??"o>Sr>SPo

cu' .c ^ jc ' £ O Jn t>< cl .c "° ^^ HI -- *S

5t to T=- n, ? re ^3 to ^ % £ g w 2 S w> « ^

f" ' Q. ,2 ' Q. "3 1 QL *•«• > Q. QJ <U C CL Q. TO ^ ^ ••'

*TM2 S

;

2'TM£ S'B'^-^" E j 2 ^wS wj

 ; S:

O'ir "refx *E ir 5~x o 2- 2- E.x t_- a > Sfo:

* . ., US:

'-u

i

"roE^

"roEi£"O•2.2~^oa>ra ^Ec01oT3•a

<1J

•Mrc-^_yreu

•4-*"

OJ

-C

•*-•

<LJ T3'§a

.

re1 For studies in which PCNB

O01rH(U

QJO

roo_
PCNB OSRI for Tier 1 Screening March 5, 2010

3.1.4. Developmental Toxicity Studies

Seven developmental toxicity studies of PCNB were reviewed. These include four developmental

studies in the rat (900-CHR-002, 900-DAR-004, 900-DAR-005, 900-DAR-006) and three developmental

studies in the rabbit (900-CHR-004; 900-CHR-004, pilot; 900-NEU-003). Two of the studies in rats (900-

CHR-002,900-DAR-006) and'one of the-studies in rabbits are considered by EPA to be guidelinecompliant. EPA DERs are not available for the remaining four studies; one of these four studies is a pilot

study and only provides limited data. Specific information regarding each developmental toxicity study's

design, test species, number of animals per group, doses, and route and duration of exposure can be

found in Table 5, along with information regarding the various endpoints evaluated in these studies

relevant to the assessment of endocrine disruption.

Page 20 of 61
o4-101C5Ol V

I

-5 .a

0

 K

 ~g

S "5 **

2 -43 ">

S H •5 ,? x " °- 0 « o t ^ v» (0

4-* +•»

C C 1 E •O Q. C O

>"5>

£ S U Q

i is

J *4=K Ol

Srf •«=

Ol 4-

Ol C £ Ic ^

re c J2 .2 (0 (0 O ^3 T5 ^ •M ^ C o) 01 c E .—^ | o ,2 £ inOl

00 'E * ^~ Ol u10tHk-HI £Ein0CQ

kgQro9UJZCDOen1Co0ccQOoeno9cc<QO'oen.ooQOOen^u '

a

ooen^.o0ccIuooen

rvi ooCCIV0oenu.ai JQE•3Z•o3

(NO

r--'

r-. oo

CO' ooLno ', '

i<t <f

"',

»

ift,

*--) OtHVDro

rt

>

rHO<Nro1 1 *acc

'ro0!occc1 1 1acceptable/

guideline

«^. 3

TOoCCCLUQ1

ro0cOLUJQ-—

'ro

rofyUJQ!a a. J3 C S =5 Q- T3 gu a

 1o

<cceptable/

guideline

<>Study acceptabilil

—QJ Q

.

O,

W0) T3 ' "

"

veippSiht"; 0) T3

"ro ' o>Q. _0UJo> —rocOl D. o"3Oi T5

~roc

'

CDE

•Ocu -ot

~ro

^~>5EQ

.

0OJ CU T3

"rocdl E1<31 <u "D

.2?ST3f

4-*

in

qen1OU3s; OO8; LOcoenLn0000en2-Test material puri

no

ro

roMOf

BO'

ro

roao

.')

CU

CU!

ro

ro00

'

ao

ro

roOBon

ro;

1

 ro

, 00

an

roaoQJ CO

ro

ro00O>£30a

.

xCU

**o-a; I Occ

1 ' 1 NJ

*

 :

l»^ .tr sr i Q -0

 • Cj

^ "ra"-

f

!

S a" ' 'J2

. in

§jl,"

1/1

 'o"

u> " j tn

Q ' ro (J cc . • 1 * !

'"*

LA g"

8 '5 ' •', s |

ub ±i i •*

Q JD i S. , '

°o "za \

rH — -• '

(n _D

^"^ H3

CC '

i i

t9 a:

t '

*

,'. j , ti t

Exposure duratioi

p5p'j|t| ^(strjunlp!

# Animals/group

•^'T?

fN CL

Ln r\i m

(N / — • (N

cf ^

i ^ ! O ' §8 o" ^ m r-i O | ' '

§\§"

in R

rsi , tH

, ~ 'o' 0 0 in in >

,

00Ln°~ (N

OO'

o",

( ' ^^ '

- O

o" § ',

S '''

o" ' o. m. o CD O o cn i

t-i • ' o" ' ,• .1 • O ,,' in i '• ' P^ C3 ^ O o to 'O , -, i * t-i Si | ; ,,

reT3 ^

1

 ao '•'

'JLoo

•g'oQ

.

TJCLU•ouCU01 V>

1

X | X ' X X ! X j\ * ' ' ' • ' 1 ll< ' x 1 x x ' x x 1

 ! • ' , I '•'.(

•X ' • X ! X ' X'

f

1

 \ i

IX XX X i i i •;! • ,

x._ jx , x x

'•' I'l ••' ' ' * * w , i, : x , x x ,. x x i • ' -I. ti 1

 ,

1

 ' i -J

x x x!j x • x

'' " i .'.! 'E

(.,,''

il § a

) .1

cy "* 1

33il S . f' JJ

iGestatidnailbbdy; !S=si««SBasSSzMiSS=Is

Body weights - fe

f|8|orp^ra|{Mt^5

# Implantation sil

|l|^sMiii|gi|i

}

sx * s^

1

^v *^- i X Xif1 { , X XsI'( X X X ,X

re

<U' +3

If

x a.

ai a>

W) ' k.

"re 15

4»* 4-*

0) , 01

LL. U-

VD^-O

<-H

(N0>&Dm

CL.
PCNB OSRI for Tier 1 Screening March 5, 2010

3.2. Ecotoxicology Studies

3.2.1. Fish Studies

One fish early life stage study using rainbow trout (Oncorhynchus mykiss) was reviewed. This study was

-concluded by EPA to-be acceptable and guideline-compliant. Specific information regarding the study's --

design, test species, number of animals per group, doses, and route and duration of exposure can be

found in Table 6, along with information regarding the various endpoints evaluated in this study relevant

to the assessment of endocrine disruption.

Table 6: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to

Endocrine Modulation from the PCNB Fish Early Life-stage Studies

AMVAC Study Number 900-AQU-008

MRID # 41663401

Study acceptability _ Acceptable/guideline

Study design ' early life-stage

 !

Route of exposure water

1

 Species (strain) , trout

_» _i' _ , i (Rainbow) _i _

# Animals/group 160

(200 control)^ _

Test material purity 99%

 !

Concentration (ug/L) 0, 3, 6.5,13, 32, 52 (measured)

_J Selected Endpoints '

Mortality (FO) X

Abnormal behavior , X (, _ ,

Terminal body wt & length X

JHatching success_ _ __ ___ w X _

Gross malformations X

3.2.2. Avian Studies

Four avian reproductive toxicity studies of PCNB were reviewed. These included two studies conducted

in bobwhite quails (Cotinus virginianus) and two studies conducted in mallards (Anas platyrynchos). All

of these studies were deemed acceptable by EPA and considered either guideline-compliant or core

studies. Specific information regarding each study's design, test species, number of animals per group,

doses, and route and duration of exposure can be found in Table 7, along with information regarding the

various endpoints evaluated in these studies relevant to assessment of endocrine disruption.

Page 22 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table 7: Experimental Details and Selected Toxicity Endpoints of Potential Relevance to

Endocrine Modulation from the PCNB Avian Reproductive Toxicity Studies

AMVAC Study Number

MRID#

Study acceptability

Study design , .

Route of exposure

. Species (strain) " > "

# Animals/sex/group

Test material purity

Concentration (ppm)

900-AVW-016

41332801

Acceptable/

guideline

reproductive,

diet

,' quail

..... (Bpbwhite)

16/sex

,' " 96% .":

0, 300, 600, 1,200

900-AVW-005

43980301, .

Acceptable/core

reproductive

diet

, quail

(Bqbwhite)

16/sex

"• ' '97 A%'"'-

0, 1,000, 2,500,

4,000, 5,500

900-AVW-015

41332802

Acceptable/

guideline

, - reproductive

diet

• ' duck

(Mallard) __

16/sex

", -~96%

0,300,600,1,200

900-AVW-007

•' 43903301 ., •

Acceptable/core

reproductive "

diet

• duck

(Mallard) „,

16/sex

•'• • 97.4% ,"

 :'iT •

0, 1,000, 2,500,

4,000, 5,500

Selected Endpoints

Mortality (FO)

Mortality (Flj

 ;

Feed consumption

'•Gross ,

malformations/lesions

Egg production (fecundity)

Hatching success

Growth and development

<

FO>

Growth and development

Chick weight gain

Chick survival, , i, lV

aOvary weight

> testes weight '•'

X

, '

X

, x

X

, X

X

, X „ , •,,", ,

"x

. _,,„ _

x

 . ..••>• . ,

, ' ' _ , ' '!

X( . — - ,,j.> -. T

X

X

' '

X

X

X

'

x

 • • ,„. ''

X a

c

!

.x • „„_ « .

X

x' '>:~ '•"'" •""

X

'

X "

X

X

X

X

.X '

X

x ,,

X

X

, x

1

 ! '

X

X

X

x

 ;•

, ..^

___. . .. , _ x % _ r .

. __ nr./)f ,„.,_. ^

4. Review of Publically Available OSRI

4.1. Published Studies with In Vitro Data and ToxCast Data

PCNB has been examined for activity at various endocrine-related receptors in published in vitro studies

and as part of the EPA ToxCast Program. A more detailed review of each of the published studies

presented below, a description of the ToxCast program, and the findings for PCNB are provided in

Appendix IV.

4.1.1. Peer-reviewed Published Studies

Ashby et al. (2005) investigated the ability of PCNB to bind to human recombinant estrogen receptor-ct

(hERa) and estrogen receptor-R (hERli) or rat uterine cytosolic ER. Within the range of concentrations

tested, PCNB exhibited no binding to hERR or rat uterine ER. Although PCNB resulted in approximately

Page 23 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

25% displacement of radioligand binding to hERa, this was not considered to be biologically relevant.

PCNB was also assessed for anti-estrogenic activity using a yeast strain containing DNA encoding the

human ER and a R-galactosidase reporter gene containing an estrogen-responsive promoter region. At

the range of concentrations tested, PCNB did not inhibit activity of estrogen acting at the human ER.

This ER binding assay was conducted using both rat uterine ER, as specified in the EPA guidelines, as well

as recombinant human ERa and ERR. fhus, this study fulfills the Tier 1 screening requirements for an ER

binding assay. The study additionally provides evidence for the lack of binding at the human ERa and

ERR and a lack of anti-estrogenic activity at the human ER (as assessed in the yeast transcriptional

system). Therefore, this study provides more robust information than is required in Tier 1 screening.

Two studies were identified that examined the effects of PCNB on MCF7 cells (Zou et al. 2002, Vinggaard

et al. 1999). Zou et al. reported PCNB treatment significantly inhibited foci formation of both untreated

and E2-treated cultures. The results suggest that this action was mediated via a non-endocrine related

pathway since inhibition also occurred in the absence of E2. In contrast, Vinggaard et al. did not observe

MCF-7 proliferation due to PCNB-treatment. Although evaluation of MCF7 cell proliferation (also known

as the E-SCREEN) was considered as a potential EDSP Tier 1 screening assay, ultimately it was not

selected as part of the EDSP Tier 1 battery, likely because MCF7 cell proliferation may be affected by

more than alteration of the estrogenic pathway.

Kojima et al. (2004) specifically addresses some of the data requirements of Tier I screening. Within the

concentration range tested, PCNB did not demonstrate agonist or antagonist activity at the human ERa,

human ERR, or human AR. Although this study was conducted in a different model system and using a

narrower concentration range of test chemicals than that specified in the ER transcriptional activation

guidelines (EPA 2009a), it demonstrated an absence of ERa transactivation by PCNB. Additionally, this

study went further than required in the guidelines to show a lack of antagonist activity at the ERa and

the absence of both agonist and antagonist activity at ERR. Finally, while the Tier I screening

recommends the examination of AR binding activity (EPA, 2009b), Kojima et al. (2004) examined the

ability of PCNB to transactivate the AR or antagonize the activity of DHT at the AR. Thus, this study

provides more robust data regarding activity at the AR and potential estrogenic activity than can be

expected from the results of the Tier I screening assays.

4.1.2. ToxCast Data

ToxCast is an EPA program that includes a battery of in vitro, high-throughput screening (HTS) assays to

screen chemicals for potential human toxicity. Approximately 69 chemicals have undergone HTS under

the ToxCast program, the majority of these being pesticides. A number of these chemicals, including

PCNB, have current or forthcoming requirements for Tier 1 screening. Of the assay technologies

available through ToxCast, those that provide information most directly relevant to the issue of

potential EAT endocrine modulation include the cell-free HTS assays, the cell-based HTS assays, and the

multiplex transcription reporter assays.

In the above three assay systems, PCNB demonstrated a complete lack of activity at the androgen

receptor. That is, PCNB did not bind to the rat or human androgen receptors in the cell-free assay and

Page 24 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

did not activate or antagonize transcriptional activity in the cell-based assays, and did not activate

transcription by a chimeric protein containing the ligand binding site of the human AR. PCNB also

demonstrated a complete lack of binding or activity at all thyroid-related receptor sites assessed in

these assays.

PCNB'did not bind to either the bovine or human ER in the cell-free assay and did not act as-an~agonist ~-

or antagonist at the human ER in the cell-based assay. However, in the multiplex transcription reporter

assay, PCNB activated a protein containing the binding domain of the human ER-a and activated

transcription from an estrogen responsive element. The reason for the discrepancy in potential

activation of the ER is not known. The multiplex transcription reporter assay is conducted in HepG2

cells, which possess metabolic activity. Therefore, it is possible that the differences may be due to a

metabolite of PCNB. Nevertheless, PCNB has been negative for estrogenic activity in the vast majority of

published in vitro studies, suggesting that the findings of the multiplex reporter transcription assay are

spurious.

4.2. Published Studies with In Vivo Data

Only one study, Ashby et al. (2005) was identified that provided sufficient and relevant information on

potential endocrine modulated effects following the administration of PCNB to animals. Ashby et al.

(2005) performed an uterotrophic assay and an abbreviated female pubertal assay. A description of the

studies conducted and their relevance to EPA's Tier 1 screening battery is provided below.

4.2.1. Ashby et al (2005) Uterotrophic Assay

Ashby et al. (2005) conducted a uterotrophic assay with immature females rats administered PCNB

subcutaneously for three days; the study design employed was Protocol B from the OECD validation

process (Kanno et al. 2001, 2003). Although the EDSP protocol recommends the use of the

ovariectomized animal due;to its "increased specificity over the immature model," use of either

immature animals or ovariectomized animals was found to be robust, reproducible, and transferable

across laboratories in the OECD validation process.

Multiple doses of PCNB were administered to the rats, ranging from 100 mg/kg/day to 800 mg/kg/day.

In addition, an uterotrophic assay was also conducted with PCP (a metabolite of PCNB) at doses ranging

from 1 mg/kg/day to 10 mg/kg/day. No changes in blotted uterine weights were observed at doses up

to 800 mg/kg/day PCNB or 10 mg/kg/day PCP.

Although not directly relevant to the EDSP protocol, additional experiments were conducted where

PCNB was administered in conjunction with 17p-estradiol (E2) or diethylstilbestrol (DES). In these

experiments, PCNB was reported to potentiate the effect of E2, but not DES. The authors concluded that

the potentiation of E2 by PCNB is specific to E2 and may be associated with an inhibition of E2

metabolism. While these findings suggest a potential for indirect endocrine activity, they would not be

replicated in EPA's protocol for the uterotrophic assay. It is also important to note that the lowest

concentration at which these potentiating effects were tested was 100 mg/kg/day and that these results

may be related to the effect of liver toxicity on estrogen metabolism. Liver toxicity is the basis of the noPage 25 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

observed-effect-level (NOEL, 0.75 mg/kg/day) for EPA's reference dose; furthermore, the NOEL is over

two orders of magnitude lower than dose tested by Ashby et al. (2005). Thus, liver toxicity is the

endpoint of concern for human risk assessment.

4.2.1. Ashby et al (2005) Female Pubertal Anti-Estrogen Assay

Ashby et al. (2005) also conducted a female pubertal anti-estrogen assay that examined uterine weights

as well as age and weight at vaginal opening following the administration of 800 mg/kg PCNB

subcutaneously beginning on post-natal day 25 or 26. This assay is comparable to the EDSP

recommended protocol with a few exceptions. A strain of Wistar rats was used instead of SpragueDawley rats. However, EPA notes that strain is unlikely to be a major determinant in this assay (OPPTS

890.1450). Further, Wistar rats and Sprague-Dawley rats are genetically related and both have been

demonstrated to be sensitive to estrogen-related endpoints. Additionally, the administration of PCNB

was not initiated until post-natal day 25 instead of the recommended post-natal day 22. However, this

discrepancy is not likely to have impacted the results since none of the control animals terminated at

the start of the experiment to measure uterine weight had open vaginas; therefore, it is assumed that

sexual maturation had not yet commenced. A number of secondary endpoints were not measured in

this assay including: organ weights (excluding uterine weight), histopathology, thyroid hormones,

estrous cyclicity, and serum chemistry.

PCNB did not affect uterine weights, or age and weight at vaginal opening. It was concluded that PCNB

failed to delay the onset of puberty in the female pubertal assay.

5. Weight-of-Evidence for Tier 1 Assay Requirements

This weight-of-evidence evaluation examines the functional equivalence of AMVAC's OSRI to EPA's Tier

1 screening battery for evaluating the potential estrogen, androgen, and thyroid effects of PCNB.

Endpoints in toxicology studies that provide information regarding potential estrogen or androgen

modulation include reproductive and accessory sex organ weights, reproductive organ histopathology

(including tumor incidence and types in chronic studies), pituitary and adrenal histopathology, gross

findings in reproductive organs, parameters related to reproductive function (e.g., estrous cyclicity,

mating success and fertility), parameters related to reproductive maturation (e.g., day and weight of

vaginal opening and preputial separation), and endpoints related to pregnancy and in utero

development (e.g., gestational length, number of corpora lutea and implantations, and pup sex ratio).

Characterization of these effects relative to other indications of systemic toxicity provides an indication

of whether any direct endocrine modulation is occurring at exposure levels below that producing

systemic toxicity, which is important for risk assessment. Similarly, fecundity in fish and reproductive

success in birds may also show adverse effects via estrogen-, androgen-, or thyroid-related mechanisms;

however, these endpoints may also be adversely impacted by nutrition and systemic toxicity.

The unpublished mammalian and ecological toxicology studies of PCNB conducted for pesticide

registration purposes were compared to the Tier 1 study designs and parameters for

correspondence/overlap of dosing period and endpoints. The resulting matrices, which can be found in

Page 26 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Appendices II and III, were designed as a tool for demonstrating the functional equivalence between the

existing data and the individual Tier 1 screens. The matrices include both the specific requirements of

the Tier 1 assays, as well as other endpoints which closely correspond to and/or supplement and

provide functionally equivalent data to the endpoints included in each Tier 1 assay. For example, the

Tier 1 Hershberger assay requires evaluation of ventral prostate weight. Although no regulatory

toxicology studies assessed ventral prostate weight, many assessed both prostate weight and/or

histopathology, which in combination are considered as sensitive and reliable for detecting potential

estrogenic, androgenic or anti-androgenic effects, as ventral prostate weight. Therefore, information on

ventral prostate weight for a regulatory study will not be available, but the findings based on prostate

weight and/or histopathology data from the study would be provided in the matrix.

Within the matrices, endpoints that were not assessed in a particular regulatory study are represented

by empty boxes or cells. Endpoints that were assessed, but showed no statistically significant or

biologically significant effects, are marked by a dash and highlighted with a green background.

Endpoints with effects that might have a relationship to an endocrine effect, but where the relationship

is very weak or equivocal are highlighted with a blue background and an arrow showing the direction of

any statistically significant changes. Slightly stronger, but still equivocal, endpoints are highlighted with

a yellow background. Finally, any-findings with a strong perceived relationship to an endocrine

mechanism (for example a delay of more than 2 days in balano-preputial separation) are highlighted

with a pink background.

Below we present an overview of the endocrine-modulated modes of action assessed in this weight-ofthe-evidence (WOE), followed by a summary of the WOE for PCNB's OSRI for each category of endocrine

modulation: estrogen/anti-estrogen, androgen/anti-androgen, and thyroid-mediated action.

5.1. Endocrine-modulated Modes of Action

Table 8 provides a list of some, but not all, of the indicators f rom-mammalian studies that assess

pesticide exposure-related disruption of the endocrine system. As seen in Table 8, modulation of either

estrogen or androgen function often affects many of the same endpoints or parameters. However, the

direction of the change will often depend on which hormone system is affected, whether the chemical

mimics or blocks the hormone's function, and whether male or female animals are being evaluated.

This table is not a complete list of possible endocrine-mediated effects and other endocrine-related

changes that may be observed in mammalian studies conducted for pesticide registration purposes, but

these are the most commonly observed effects in response to endocrine modulation. Consequently,

this table serves as a useful starting point for the evaluation of mammalian studies available for PCNB.

Page 27 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table 8: Indicators of Potential Estrogen, Androgen and Thyroid

Endocrine Disruption from Mammalian Studies

Mechanism Potential Effects in Males Potential Effects in Females

Estrogenicity -Delayed-preputial separation

(marked, or in absence of

significant body weight decreases)

Reduced fertility

Reduced sperm counts

Decreased male reproductive

organ weights (except prostate

weight may be increased)

Histopathological findings of the

testes (e.g., Leydig cell

proliferation and/or tumors)

Increased incidence, growth of

pituitary tumors

Precocious vaginal.opening .

Persistent estrus

Increased time to mating

Reduced gestation duration

Increased uterine weights

Histopathological findings of the

female reproductive organs (e.g.,

vaginal cornification, uterine

hypertrophy and hyperplasia)

Increased mammary tumors

(adenomas or adenocarcinomas)

Increased incidence, growth of

pituitary tumors

Ovarian, uterine and vaginal

tumors in female-offspring

Anti-estrogenicity Decreased height of epithelium in

testicular tubules

Testicular weight increases

(short term)

Testicular atrophy (long term)

Infertility and testicular atrophy

(moderate term)

Delayed vaginal opening (VO)

Delayed start of estrous cycling

and irregular or absent estrous

cyclicity

Reduced fertility

Decreased corpora lutea,

implantations

Decreased female reproductive

organ weights

Decreased mammaiytumor

incidence

Increased or decreased male

reproductive organ weights

Reduced sperm counts

Testicular atrophy

Androgenicity Increased anogenital distance

Accelerated vaginal opening

Reduced fertility

Altered differential follicle count

Histopathological findings of the

female reproductive organs (e.g.,

vaginal agenesis)

Induced male sex accessory

tissues

Page 28 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Mechanism

Anti-androgenicity

Reduced steroid

biosynthesis

Aromatase

inhibition

Thyroid hormone

modulation

Potential Effects in Males

Delayed preputial separation

Reduced anogenital distance

Ectopic testes

Hypospadias/epispadias

~Re~ducecl fertility ~ - - -

Reduced reproductive organ

weight (particularly prostate,

seminal vesicles)

Retained nipples/areolas

Histopathological findings of the

reproductive organs (e.g.,

epididymal agenesis, testicular

tumors)

Similar to anti-androgenicity

Possible increased serum

cholesterol levels

Increased Leydig cell tumors

Increased time to mating

Decreased male mounting

behavior

Decreased body weight (chronic)

Increased testis weight (chronic)

Potential Effects in Females

Altered pup sex ratios between

external and internal sexing

Similar to anti-estrogenicity/

aromatase inhibition

Increased body weight

Decreased uterine weight

Increased uterine weight

Increased ovary size

Polycystic ovaries

Stromal hyperplasia in ovary

(chronic)

Hyalinization in ovary (chronic)

Increased ureter and bladder

infection

Decreased mammary tumor

incidence (SD)

Decreased T3 and/or T4 levels, increased TSH levels

Increased thyroid weights

Decreased colloid content and thyroid follicle size

Follicularcell hypertrophy

Thyroid follicularcell hyperplasia

Thyroid follicular cell tumors

These parameters that were assessed in the PCNB studies conducted for pesticide registration purposes

are as follows:

• Reproductive and/or accessory sex organ weights were assessed in a two-generation

reproductive study, four of the seven subchronic studies, and three of the eight chronic studies;

Page 29 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

• Reproductive and/or accessory sex organ histopathology was examined in both two-generation

reproductive toxicity studies in the FO and Fl generations (with two litters per generation), four

of the seven subchronic studies, and all eight oncogenicity/chronic studies;

• Histopathology of the adrenals was conducted in the three-generation reproductive study, three

of the_seven subchronic toxicity studies, and all eight oncogenicity/chronic toxicity studies;

• Histopathology of the pituitary was examined in the two two-generation reproductive toxicity

studies, the three-generation reproductive study, four of the seven subchronic toxicity studies,

and all eight of the oncogenicity/chronic toxicity studies;

• Gross examination of the reproductive organs was conducted in all reproductive,

developmental, and chronic toxicity studies, and five of the seven subchronic studies;

• Endpoints related to reproductive function were measured in the two two-generation

reproductive toxicity studies and the three-generation reproductive study; in the fish early life

stage study and the avian reproductive toxicity studies in quail and duck; and

• Endpoints related to pregnancy and in utero development were evaluated in both twogeneration reproductive toxicity studies, the three-generation reproductive study, and the

seven developmental toxicity studies.

5.1.1. Assessment of Potential for Estrogen/Anti-estrogen Modulation by PCNB

PCNB has been found to be negative for estrogenic activity in the vast majority of publically available in

vitro studies (Ashby et al. 2005, Kojima et al. 2004). The estrogenic activity of PCNB has been evaluated

though binding, competitive binding, transactivation, agonist or antagonist activity to a range of

estrogen receptors including: human ERa, ER[5, rat uterine ER, and bovine ER. Only in the multiplex

transcription reporter assay from ToxCast were contradictory findings seen for estrogen-mediated

activity; PCNB activated a transcription protein with a binding domain for the human ER-a and

transcription at an estrogen response element. Given the body of evidence suggesting a lack of

estrogen-related activity in other in vitro and in vivo assays, these findings are considered spurious.

Additionally, PCNB was considered negative for aromatase inhibition in the ToxCast cell-free assay.

These studies provide functionally equivalent or more robust information regarding potential estrogenic

activity than can be expected from the results of the in vitro Tier I screening assays.

Findings from the in vivo studies in females were generally negative and do not suggest either

estrogenicity or anti-estrogenicity. In the guideline compliant studies ovary weights were generally not

affected, except increased absolute weight was reported at the highest dose in the second generation of

the two-generation study. No histopathological changes were reported for any of the reproductive or

accessory sex organs, including the ovaries from any of the reproductive, developmental, subchronic or

chronic studies. Additionally, no exposure-related changes in incidence of reproductive organ,

mammary, or endocrine tumors were observed in the cancer bioassays of rats or mice. Further, no

effects were seen on reproductive function or performance. The lack of estrogenic/anti-estrogenic

activity by PCNB is supported by the uterotrophic assay and female pubertal assay conducted by Ashby

et al. (2005). Body weights, uterine weights, and age and weight at vaginal opening were not affected

Page 30 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

by PCNB administration to pubertal rats. In the uterotrophic assay, PCNB did not affect uterine weights

up to 800 mg/kg/day.

In males, data on estrogen or anti-estrogen modulation are more limited, but do not suggest

estrogenicity or anti-estrogenicity. Testicular weights were not affected in any of the subchronic studies

or in two of the chronic studies;'but an increase in testicular weight (absolute and relative) was seen'in -

rats in a two year study and a decreased relative weight was seen in a two-year dog study. Therefore,

overall testicular weight changes can be considered equivocal at best; further, they were not correlated

with histopathological changes. Prostate weight was only measured in combination with seminal

vesicles in the two-generation reproductive study and were not statistically different from controls. In

addition, no treatment-related histopathological changes were observed in the prostate in the

subchronic or chronic studies. No exposure-related histopathological changes were reported for any

other reproductive or accessory sex organ. Specifically, no exposure-related testicular atrophy,

testicular tumors, or pituitary tumors were observed in the eight chronic/oncogenicity studies.

Additionally, no impact on fertility was seen in either of the two two-generation reproductive studies or

the three-generation reproductive study. None of the available studies evaluated sperm counts or

measured preputial separation

Limited data are available in the fish early life stage study and the four avian reproductive studies, such

that the findings cannot be attributed to any endocrine-mediated effect.

5.1.2. Assessment of Potential for Androgen/Anti-androgen Modulation by PCNB

PCNB has been found to be negative for androgen activity in all of the publically available in vitro studies

(Kojima et al. 2004). The androgenic activity of PCNB has been evaluated though binding,

transactivation, agonist or antagonist activity to the human or rat AR. These studies provide either

functionally equivalent or more robust information regarding potential androgenic activity than is

required by the in vitro Tier I screening battery.

Data on androgen/anti-androgen modulation in females generally are negative in the regulatory studies

conducted for PCNB. No exposure-related histopathological changes were seen in the reproductive and

accessory sex organs from any of the studies, including the two-generation reproductive studies and 2-

year chronic bioassays. No impact on fertility was seen in either of the two-generation studies or the

three-generation study. Further, no differences in sex ratio were reported and no effects on male sex

accessory organs were observed in the offspring through two and three generations of maternal

exposure. Anogenital distance and vaginal opening was not assessed in any of these studies; however,

Ashby (2005) conducted a female pubertal assay and did not observe any impact on vaginal opening

following exposure to PCNB.

Findings related to androgenicity or anti-androgenicity in males are more limited. In the guidelinecompliant studies, testicular weights were equivocal (discussed above). Prostate weights were not

affected in a two-generation reproductive study, but are limited by the collection of combined weights

with seminal vesicles. Overall, no exposure related histopathological changes were seen in the various

male reproductive organs, including no testicular atrophy or tumors in the cancer bioassays. No

Page 31 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

reductions in fertility were seen in the two-generation or three-generation reproductive studies.

Furthermore, there is no evidence of ectopic testes, hypospadias or other reproductive tract

malformations, or retained nipples in the males in these studies. However, these studies are limited by

the lack of information on sperm counts, preputial separation, and anogenital distance.

Limited data are available in the fish early life~stage study~and'the~four avian reproductive studies;^

further, none of the findings can be attributed to any endocrine-mediated effect.

5.1.3. Assessment of Potential for Thyroid-mediated Endocrine Effects by PCNB

Limited data are available on PCNB-induced thyroid-mediated effects from in vitro studies in ToxCast,

but all data are negative. PCNB demonstrated a complete lack of binding or activity at all thyroidrelated receptor sites tested.

Thyroid effects, and the mechanism of action for these effects in rats are well-characterized in PCNB in

vivo studies. A number of parameters related to thyroid function were measured in the PCNB studies

conducted for pesticide registration and include:

• Thyroid organ weight changes in two of the seven subchronic studies and three of the eight

chronic studies;

• Thyroid histopathology in two of the three reproductive studies, six of the seven subchronic

studies, and all eight chronic studies; and

• Thyroid hormones were measured in one of the rat subchronic studies.

Thyroid organ weight changes were evaluated in both rats and dogs, but changes were only observed in

rats. Histopathological changes were examined in two strains of rats, two strains of mice, and dogs. An

increased incidence of follicular cell hypertrophy/hyperplasia and thyroid follicularcell adenomas were

observed only in Sprague-Dawley rats at the highest doses administered in two studies; no

histopathological changes were observed in the other six chronic studies. The thyroid findings observed

in the Sprague-Dawley rats were accompanied by increased liver weights and liver hypertrophy,

suggesting that the thyroid effects were secondary to liver enzyme induction. Based on these

observations, additional studies were conducted to investigate the mechanism of action. In a

subchronic study (900-SCH-003), increased levels of thyroid stimulating hormone (TSH) and decreased

levels of T3 and T4 were measured and biliary excretion of thyroxine was confirmed in studies 900-CHR018 and 900-CHR-019. Collectively, these studies indicate that PCNB induces liver enzyme induction

resulting in excretion of thyroxine in the bile, which activates the thyroid-pituitary feedback mechanism

leading to TSH stimulation and ultimately thyroid follicular cell tumors. These findings clearly involve a

thyroid-mediated mode of action, but one which is secondary to liver effects. Most importantly, these

types of thyroid follicular cell tumors have been demonstrated to be a threshold effect and not are

relevant for human health risk assessment (EPA 1998).

Page 32 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

5.2. Tier 1 In Vitro Testing Requirements

The Tier 1 in vitro testing requirements include evaluation of ER binding (OPPTS 890.1250), androgen

receptor binding (OPPTS 890.1150), ER transactivation (OPPTS 890.1300), aromatase inhibition (OPPTS

890.1200)7and steroidogenesis (OPPTS'890.1-550). As^can beseen'from the matrix found in Appendix-|l,~

many of the endpoints evaluated in the mammalian studies conducted for pesticide registration

purposes address endpoints of relevance to the Tier 1 in vitro assays. Furthermore, the available data

indicate that PCNB has low potential for interaction with the estrogen or androgen receptors,

aromatase, or other enzymes and pathways related to steroidogenesis.

Additionally, other mechanistic data are available for PCNB that generally shows a lack of activity at the

estrogen and androgen receptors and aromatase enzyme. Two studies are available in the published

literature (Ashby et al. 2005, Kojima et al. 2004) that evaluated PCNB and its ability to interact with the

ER-a, ER-li, or androgen receptor. These studies are reviewed in Appendix IV, which provides the results

of the experiments and how closely the described assays correspond with those required in EDSP Tier 1

screening. A high degree of confidence is placed in these studies as they specify test material purity,

assay conditions and provide a clear discussion of test methodology. Furthermore, Kojima et al. (2004)

provides more robust information regarding potential interaction with the estrogen and androgen

receptors than would be derived from the in vitro assays required in Tier 1 screening. Thus, both of

these studies are being referenced as OSRI.

PCNB was reported generally to not bind to ortransactivate either estrogen or androgen receptors as

evaluated by several laboratories under contract to EPA for the ToxCast Program. In addition, PCNB was

considered negative for aromatase inhibition. The ToxCast report and supplementary materials, and

data files provided on the ToxCast Program website (www.epa.gov/ncct/toxcast/ index.html) are being

referenced as OSRI. Additionally, the assays used in the ToxCast program relevant to Tier 1 screening

objectives, and a comparison to Tier 1 assay guidelines are described in the review of ToxCast data for

PCNB, attached as Appendix IV. The ToxCast report provides links to the commercial suppliers of the

assays used, which provide some additional information on assay methodology; the attached review

summarizes this information and the assay results as a convenience to EPA. Please note that additional

information on the ToxCast assays is expected to be published in the near future; we plan to provide a

follow-up OSRI submission to EPA with any new information on the in vitro ToxCast assays relevant to

Tier 1 testing objectives.

Based on the availability of existing data from mammalian regulatory studies (as summarized in the

matrix of Appendix II), which show a lack of PCNB activity at the estrogen and androgen receptors and

the existence of additional mechanistic data in the published literature and from the ToxCast Program,

AMVAC is requesting a waiver for the estrogen and androgen receptor binding assays (OPPTS 890.1250

and OPPTS 890.1150, respectively), as well as the ER transactivation assay (OPPTS 890.1300). Finally,

although the available data do not show indications that PCNB affects aromatase or steroidogenesis,

specific mechanistic data are not available. ToxCast did assess aromatase inhibition, and found no

inhibition from PCNB, but documentation available for this assay is limited. Thus, AMVAC will conduct

Page 33 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

the aromatase inhibition (OPPTS 890.1200) and steroidogenesis (OPPTS 890.1550) assays according to

the EDSP Tier 1 Screening guidelines.

5.3. Tier 1 In Vivo Mammalian Testing Requirements

.-The-Tier--l-/'/7v/Vo-mammalian testing requirements include the male pubertal assay (OPPTS-890.1500),—

the female pubertal assay (OPPTS 890.1450), the Hershberger assay (OPPTS 890.1400), and the

uterotrophic assay (OPPTS 890.1600). As can be seen in Appendix II, the matrix shows a good

correlation in exposure periods and endpoints assessed in the key mammalian regulatory toxicology

studies of PCNB (identified in orange in the Appendix II matrices and referenced as OSRI in Appendix I)

with many of the data requirements of both the male and female pubertal assays. The key reproductive

toxicity studies of PCNB have many characteristics likely to be included in a Tier 2 apical assay, including

exposure during the peri-natal period and histopathological evaluation and measurement of

reproductive organs subsequent to peri-natal exposure. As two-generation studies, these studies

evaluated animals exposed as weanlings as well as animals exposed during the per-natal life stage,

which is arguably more critical for evaluation of potential endocrine effects that the post-weaning lifestage evaluated in the pubertal studies. Although detailed necropsies were conducted on adult animals

only in these two studies, endpoints such as retained nipples or urogenital malformations (which are

particularly sensitive indicators for anti-androgens and/or steroidogenesis inhibitors) would have been

detected. Unfortunately, neither of these studies included evaluation of vaginal opening or balanopreputial separation, the timing of which are quite sensitive to estrogen and androgen modulation and

are required in the pubertal assays. Sperm motility, estrous cyclicity and differential follicle counts were

also not evaluated in the guideline studies. Evaluation of secondary endpoints including thyroid, adrenal

and pituitary weights, as well as histopathology, are available from the guideline-compliant subchronic

toxicity studies of PCNB.

In addition to the fairly robust dataset for PCNB on estrogen and anti-estrogen related effects, an

uterotrophic assay and female pubertal assay is available in the published literature (Ashby et al. 2005).

Ashby et al. (2005) provides key sensitive endpoints including uterine weights and age and weight at

vaginal opening for PCNB. Although the uterotrophic assay does not include an evaluation of estrous

cyclicity, it provides a very sensitive evaluation of potential estrogenic effects. The other endpoints

required in the female pubertal assay (e.g., organ weights, histopathology, and thyroid hormones) have

been repeatedly measured in other guideline-compliant studies and with the exception of thyroid effect

have not demonstrated any endocrine-mediated changes. Thus, sufficient functionally equivalent data

are available to waive the female pubertal assay (OPPTS 890.1450) and the uterotrophic assay (OPPTS

890.1600).

Although the reproductive studies of PCNB involve exposures during a sensitive period of development,

they do not evaluate the comprehensive suite of male accessory sex tissues. Therefore, to compensate

for the absence of these potentially sensitive endpoints in the PCNB dataset, AMVAC will conduct the

Hershberger assay (OPPTS 890.1400) for PCNB according to the Tier 1 guidelines. The Hershberger assay

provides a sensitive assessment of anti-androgenicity and includes evaluation of male accessory sex

organ weights not captured in the regulatory study database, such as those of the Cowper's gland,

Page 34 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

ventral prostate and glans penis. The Hershberger assay will provide redundancy for the identification

of potential androgen and anti-androgen effects. As described above, the guideline-compliant studies

have clearly demonstrated effects on the thyroid-pituitary axis which manifest as increased thyroid

weights and histopathological changes, secondary to hepatic enzyme induction. Given the

acknowledged thyroid effects of PCNB in rats and the lack of relevance to human risk assessment based

on the mechanism of action, these endpoints do not require further assessment in the Tier 1 screen.

Therefore, AMVAC is requesting to waive the male pubertal assay (OPPTS 890.1500) based on sufficient

functionally equivalent data.

5.4. Tier 1 In Vivo Ecotoxicological Testing Requirements

The available ecotoxicology studies of PCNB are not considered adequate to address the possibility of

endocrine-modulated effects in frogs or fish. No frog studies have been conducted for pesticide

registration purposes. Additionally, although a fish early life-stage study was conducted, the endpoints

assessed related to possible endocrine modulation are few. Finally, although the available data on birds

do not provide any direct evidence of PCNB toxicity to the endocrine system, the species differences are

sufficient that these data cannot realistically substitute for that in frogs or fish. Therefore, AMVAC

agrees to conduct both a metamorphosis assay (OOPTS 890.1100) and a fish short-term reproduction

study (OPPTS 890.1350), as specified in the EDSP Tier 1 guidelines.

6. Conclusions

As a whole, the PCNB database of studies conducted for pesticide registration purposes provides a

strong foundation upon which to evaluate for potential endocrine-related effects, particularly in

mammals. Evidence to suggest that PCNB is a potential endocrine modulator is limited to thyroid

effects, which are secondary to liver toxicity and hepatic enzyme induction. The available published

data-andTjDxCast program results for PCNB, as a whole, indicate a lack of activity at the estrogen and

androgen receptors. Additionally, an uterotrophic assay and a pubertal female rat anti-estrogen assay

were conducted and no endocrine-mediated effects were reported. Nevertheless, certain sensitive

endpoints related to androgenic or anti-androgenic effects have not been evaluated. Therefore, AMVAC

believes that the following Tier 1 assays should be performed to supplement the mammalian regulatory

study database: aromatase, steroidogenesis, Hershberger, and pubertal male assay. These assays

provide redundancy to the endocrine endpoints characterized in the mammalian regulatory studies of

PCNB conducted for pesticide registration purposes, thus fulfilling the objective of the Tier 1 screen to

provide a conservative and redundant assessment of potential endocrine-mediated toxicity.

The available ecotoxicology studies of PCNB are not considered adequate to address the possibility of

endocrine modulation resulting from exposures to frogs or fish. Therefore, AMVAC will conduct the frog

metamorphosis assay and the fish short-term reproductive toxicity study.

The following table summarizes the assays that AMVAC will conduct and those assays for which the

company is requesting waivers:

Page 35 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table 9: Tier 1 Test Battery and Requested EPA Action Based on OSRI Weight-of-the-Evidence

Endocrine Disruptor Screening Program Tier 1 Test

" , :,; - , >i$ <" - - . • •- G uideline •'•• ;>,.'"' . i,:;; '

In Vitro Screening Assays

OPPTS 890.1250: Estrogen Receptor Binding Assay

Using Rat Uterine Cytosol (ER-RUC)

OPPTS 890.1300: Estrogen Receptor Transcriptional

Activation (Human Cell Line (HeLa-9903))

OPPTS 890.1150: Androgen Receptor Binding

(Rat Prostate Cytosol)

OPPTS 890.1200: Aromatase

(Human Recombinant)

PVnMvo Screening Assays

OPPTS 890.1550: Steroidogenesis

(Human Cell Line -H295R)

OPPTS 890.1600: Uterotrophic Assay

OPPTS 890.1400: Hershberger Bioassay

OPPTS 890.1450: Pubertal Development and Thyroid

Function in Intact Juvenile/ Peripubertal Female Rats

OPPTS 890.1500: Pubertal Development and Thyroid

Function in Intact Juvenile/ Peripubertal Male Rats

OPPTS 890.1100: Amphibian Metamorphosis (Frog)

OPPTS 890.1350: Fish Short-Term Reproduction

Assay

Requested Action

"

WAIVER requested based on functional

equivalence of OSRI

WAIVER requested based on functional

equivalence of OSRI

WAIVER requested based on functional

equivalence of OSRI

AMVAC will conduct (no waiver requested)

AMVAC will conduct (no waiver requested)

WAIVER requested based on functional

equivalence of OSRI

AMVAC will conduct (no waiver requested)

WAIVER requested based on functional

equivalence of OSRI

WAIVER requested based on functional

equivalence of OSRI

AMVAC will conduct (no waiver requested)

AMVAC will conduct (no waiver requested)

Page 36 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Bibliography

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 Phormocp/pgy._.20(2Q05):199-208.

Environmental Protection Agency (EPA). (1995). Reregistration Eligibility Decision (RED)

Linuron. March 1995.

Environmental Protection Agency (EPA). 1998. Assessment of thyroid follicular cell tumors. Risk

Assessment Forum. U.S. Environmental Protection Agency. Washington, DC EPA/630/R-97/002.

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

Corporation. Study #399-087. [900-CHR-013].

Goldenthal, E.I. 1991. Two year dietary toxicity and oncogenicity study in rats. International Research

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

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

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

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

of pentachloronitrobenzene(PCNB) administered orally via gavage to CRL:CDtm(SD)BR presumed

pregnant rats. Argus Research Laboratories, Inc. Protocols 310-005. MRID# 41361201. [900-CHR-002]

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. Protocol 310-006. MRID# 41361301. [900-CHR-004]

Judson, R.S., Houck, K.A., Kavlock, R.J., Knudserr, T.B., Martin, M.T., Mortensen, H.M., Reif, D.M., Rotroff,

D.M., Shah, I., Richard, A.M., and Dix, D.J. (2009). In vitro screening of environmental chemicals for

targeted testing prioritization -the ToxCast Project. Environmental Health Perspectives. Epub (doi:

10.1289/ehp.0901392; available at http://dz.doi.org/): and supplemental data.

Kanno J, Onyon L, Haseman J, Fenner-Crisp P, Ashby J, and Owens W. (2001). The OECD program to

validate the rate uterotrophic bioassay to screen compounds for in vivo estrogenic responses: phase 1.

Environmental Health Perspectives. 109(8):785-94.

Kanno J, Onyon L, Peddada S, Ashby J, Jacob E, and Owens W. 2003. The OECD program to validate the

rat uterotrophic bioassay. Phase 2: dose-response studies. Environmental Health Perspectives.

lll(12):1530-49.

Keefe, R.T. 1992. 90-Day subchronic oral toxicity study in rats with pentachloronitrobenzene (MRD-89-

505). EXXON BiomedicalSciences, Inc. Project #250570A. MRID# 42416001. [900-SCH-001]

Page 37 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

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

androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster

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

Kossor, D.C. 1996. Biliary excretion of [125l]-thyroxine and thyroid uptake of

 125I in pentachloronitrobenzene (PCNB)-treated rats. MPI Research. Study #399-179. MRID

Martin, M.T., Dix, D.J., Judson, R.S. Kavlock, R.J., Reif, D.M., Richard, A.M., Rotroff, D.M., Romanov, S.,

Medvedev, A., Poltoratskaya, N., Gambarian, M., Moeser, M., Makarov, S.S., and Houck, K.A. (2010).

Impact of environmental chemicals on key transcription regulators and correlation to toxicity endpoints

within EPA's ToxCast program. Accepted for publication.

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

International Research and Development Corporation. Study #399-071. MRIDft 40636701.

[900-CHR-008].

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

(PCNB) (MRD-89-505). EXXON BiomedicalSciences, Inc. Project #150535. MRID# 43469301.

[900-REP-001]

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

(PCNB) (MRD-89-505). EXXON Biomedical Sciences, Inc. Project #250570A. MRID# 43015801.

[900-CHR-005]

Shea, J.P. 1996. Biliary excretion of labeled thyroxine and thyroid uptake of labeled iodine in PCNB

treated rats. BIODEVELOPMENT Laboratories. Preject # 10601. [900-CHR-019]

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

Central Institute for Nutrition and Research. Rapport # 4365. [900-CHR-010]

Vinggaard, A., Breinholt, V., and Larsen, J. 1999. Screening of selected pesticides for oestrogen receptor

activation in vitro. Food Additives and Contaminants. 16(12):533-542.

Weybridge: European Workshop on the Impact of Endocrine Disrupters on Human Health and Wildlife

(Weybridge, UK; 1996). European Union Report EUR17459.

Wirth MJ and Schardien JL. 1991. Two generation reproduction study in rats. Amendment to the Final

Report issued February 1,1991. International Research and Development Corporation. Study #399-086.

MRID# 41918701. [900-DAR-008].

Zou, E., Hatakeyama, M., and Matsumura, F. 2002. Foci formation of MCF7 cells as an in vitro screening

method for estrogenic chemicals. Environmental Toxicology and Pharmacology. ll(2002):71-77.

Page 38 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Appendix I: PCNB Data Referenced as OSRI

Page 39 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table 1-1: Submitted Regulatory Toxicological Studies Referenced as OSRI

'!

 : : Study Type' i Reference " > MRID EPA

Classification

Reproductive/Developmental Toxicity

Two Generation Reproduction Study in Rats

Two Generation Reproduction Toxicity Study

in Rats with PCNB

Developmental Toxicity Study of PCNB

Administered Orally via Gavage to Rats

Developmental Toxicity Study of PCNB

Administered Orally to Rabbits

Wirth and Schardien 1991

900-DAR-008

Phillips 1994

900-REP-001

Hoberman 1989a

900-CHR-002

Hoberman 1989b

900-CHR-004

41918701

43469301

41361201

41361301

Acceptable/

guideline

DER not available

Acceptable/

guideline

Acceptable/

guideline

Short-Term Toxicity

13-Week Dietary Toxicity Study in Rats with

PCNB

90-Day Subchronic Oral Toxicity Study in Rats

with PCNB

90-Day Dietary Toxicity Study in Rats

McGee 1988

900-CHR-012

Keefe 1992

900-SCH-001

Goldenthal 1993

900-SCH-003

40636701

42416001

42630801

DER not available

Acceptable/

guideline

DER not available

Long-Term Toxicity

2-Year Chronic Toxicity/Oncogenicity Study in

Rats with PCNB

Two Year Dietary Toxicity and Oncogenicity

Study in Rats

PCNB Carcinogenicity Study in Mice

One Year Chronic Dietary Study in Dogs

Plutnick 1993

900-CHR-005

Goldenthal 1991

900-CHR-014

van der Heijden and Til 1974

900-CHR-010

Goldenthal 1990

900-CHR-013

43015801

41987301

Acceptable/

guideline

Acceptable/

guideline

DER not available

DER not available

Other Studies

Biliary excretion of [125l]-thyroxine and

thyroid uptake of

 125I in pentachloronitrobenzene (PCNB)-treated rats

Biliary Excretion of Labeled Thyroxine and

Thyroid Uptake of Labeled Iodine in PCNB

Treated Rats

Kossor 1996

900-CHR-018.

Shea 1996

900-CHR-019

DER not available

DER not available

Page 40 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table 1-2: Literature Studies Referenced as OSRI1

, Authors, ,t - ,

Ashby, J., et al.

Kojima, H., et al.

Vinggaard, A., et al.

Zou, E., et al.

'• : . - ' '" ' Title

The report in vitro anti-estrogen

pentachloTcThitrobenzehe enhances

the estrogenic activity of estradiol

in vivo in the rat.

Screening for estrogen and

androgen receptor activities in 200

pesticides by in vitro reporter gene

assays using Chinese hamster ovary

cells.

Screening of selected pesticides for

oestrogen receptor activation in

vitro.

Foci formation of MCF7 cells as an

in vitro screening method for

estrogenic chemicals.

Journal ^

Environmental

Toxicology and

Pharmacology

20:199-208.

Environmental Health

Perspectives

112:524-531.

Food Additives and

Contaminants

16:533-542.

Environmental

Toxicology and

Pharmacology

11:71-77.

Year

2005

2004

1999

2002

lOnly those published studies being referenced as OSRI are listed in the Table. Other published studies are not being

referenced as OSRI due to the lack of information regarding chemical composition and purity, as well as limited description of

methods and results.

Page 41 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Appendix II: Endocrine Disruption Screening Matrix - PCNB Mammalian Regulatory Studies

Page 42 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Appendix III: Endocrine Disruption Screening Matrix - PCNB Ecotoxicology Regulatory Studies

Page 43 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Appendix IV: Published In Vitro Assays and ToxCast Program Results for PCNB

Page 44 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

PCNB, also known as quintozene, has been examined for activity at various endocrine-related receptors

in several published in vitro studies and as part of the EPAToxCast Program. Descriptions of these

studies, their results as they relate to PCNB, and how closely the described assays correspond to those

required in EDSP Tier 1 screening can be found below.

I. Published in vitro studies

a. Ashby et al., 2005

The ability of PCNB (99% purity) to bind to human recombinant estrogen receptor-cc (hERa) and

estrogen receptor-R (hERR) or rat uterine cytosolic estrogen receptor (ER) was assessed. Duplicate

aliquots of commercially available hERa and hERR (diluted in hER buffer) were incubated with DMSO, 5 x

10~10-5 x 10"6 M 17R-estradiol (E2; 10-fold dilutions), or 5 x 10'10-5 x 10~" M PCNB (10-fold dilutions) in

the presence of 5 x 10"9 M 3H-E2 for 17 hrs at 4°C. Ligand-bound receptor complexes were precipitated

using hydroxylapatite, sedimented by centrifugation, and washed three times before being measured

for radioactivity using a scintillation counter. Binding to rat uterine cytosolic ER was conducted in a

similar manner. Uterine cytosols were prepared from immature (21-22 days old) female Alpk:ApfSD

(Wistar derived) rats and diluted in TEGM buffer with 5nM

 3H-E2 and 5 x 10"10

 - 5 x 10"4 M PCNB (10-fold

dilutions). Following 18 hours incubation at 4°C, receptor complexes were precipitated with

hydroxylapatite, washed in TEGM and measured for radio-ligand binding using a scintillation counter.

Within the range of concentrations tested, PCNB exhibited no binding to hERR or rat uterine ER;

however, PCNB treatment resulted in approximately 25% displacement of radioligand binding to hERa.

These latter results were not considered to be biologically relevant.

PCNB was also assessed for anti-estrogenic activity using a yeast strain containing DNA encoding the

human ER and a R-galactosidase reporter gene containing an estrogen-responsive promoter region.

PCNB was serially diluted in EtOH, aliquots transferred to a 96-well plate and the solvent evaporated

away. Aliquots of assay media containing the recombinant yeast and the R-galactosidase substrate

CPRG were then added to each well along with 5 nM E2. After mixing, cultures were incubated at 32°C

for 3 days before R-galactosidase activity was measured. Niacinamide and hydroxytamoxifen were used

as negative and positive control agents, respectively. Reported results were the means of triplicate

determinations. At concentrations of 5 x 10"12 -1 x 10~3 M, PCNB did not inhibit activity of estrogen

acting at the human ER.

This study specifically addresses some of the data requirements of Tier I screening. The ER binding assay

was conducted using both rat uterine ER, as specified in the EPA guidelines, as well as recombinant

human ERa and ERR. Further, the range of test chemical used was similar to that required in Tier 1

screening, although the number of replicates is slightly less than desired and the number of test runs is

not known. Thus, this study fulfills the Tier 1 screening requirements for an ER binding assay. The study

additionally provides evidence for the lack of binding at the human ERa and ERR and a lack of antiestrogenic activity at the human ER (as assessed in the yeast transcriptional system). Therefore, this

study provides more robust information than is required in Tier 1 screening.

Page 45 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

b. Zou et al., 2002

Zou et al. examined the effects of chemical treatment on the ability of MCF7 cells to form foci in vitro.

Human MCF7 cells were seeded in six-well cell culture plates at an initial density of 1 x 105 cells/well in

phenol red-free DMEM media containing 5% FBS (which had been previously heat-inactivated and

charcoal-dextran treated), 100 U/ml penicillin and 100 ug/ml streptomycin. After two days, cultures

were treated with 100 nM PCNB (98.6% purity), 1 nM 17B-estradiol (E2), or 100 nM PCNB + 1 nM E2.

Control cultures for which no treatments were initiated were also established. Medium plus treatments

were changed twice per week. Seventeen days after treatment initiation, the cell cultures were fixed in

10% formalin in PBS, stained with GIEMSA, and examined for the number of foci formed. The number of

cultures examined in each treatment group was not reported. PCNB treatment significantly inhibited

foci formation of both untreated and E2-treated cultures. The mechanism of action by which this

inhibition was mediated was not examined. However, the fact that PCNB inhibited foci formation in the

absence of E2 suggests that it occurred via a non-endocrine related pathway. Although evaluation of

MCF7 cell proliferation (also known as the E-SCREEN) was considered as a potential EDSP Tier 1

screening assay, ultimately it was not selected as part of the EDSP Tier 1 battery, likely because MCF7

cell proliferation may be affected by more than alteration of the estrogenic pathway.

c. Vinggaard et al., 1999

MCF-7 cells were seeded in 12-well cell culture plates at an initial density of 1.5 x 104 cells/well in RPMI

1640 medium containing 10 mM HEPES, 1 mM sodium pyruvate, 2 mM L-glutamine, 5 ml MEM nonessential amino acids, 100 U/ml penicillin and 100 ug/ml streptomycin, and 10% dextran-charcoaltreated FBS. After an overnight incubation, the cells were treated with media containing 10 u,M test

compounds. Culture media with test compounds were changed every third day. After six days, 0.1

mg/ml solution of resazurin in PBS was added to the cultures and plates incubated for 4 hrs at 37°C.

Reduction of the resazurin dye was measured by the emission of fluorescence. PCNB (purity of 97-99%)

"tested in two separate experiments did not activate MCF-7 cell proliferation. The results of this study

contrast with those of Zou et al. (2002); the conflicting results may be due to differences in methodology

or how MCF7 proliferation was assessed. Again, assessment of MCF7 cell proliferation is not part of the

EDSP Tier 1 battery.

d. Kojima et al., 2004

This study specifically addresses some of the data requirements of Tier I screening. Two hundred

pesticides, including PCNB (purity between 95-100%), were tested for agonist and antagonist activity at

the human ERct, human ERR, and human AR. Chinese hamster ovary cells (CHO Kl) were plated in 96-

well plates at a density of 8,400 cells per well in DMEM/F-12 media containing 5% FBS pretreated with

dextran-coated charcoal. One day later, the cells were transiently transfected with ERct, ERR, or AR.

Cells were simultaneously transfected with a luciferase reporter plasmid containing either an estrogen

responsive element (ERE) or an androgen responsive element (ARE) upstream of the firefly luciferase

gene, and a plasmid containing the Renilla luciferase gene (used as an internal control for transfection

efficiency). Three hours post-transfection, cells were treated with 10'8-10"5 M of each test compound

Page 46 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

or 0.1% DMSO (vehicle control). To evaluate antagonist activity at the ERa, ERR, and AR, test

compounds were added in the presence of either 10"11 M E2, 10"10 M E2, or 10"10 M DHT, respectively.

After a 24-hour incubation period, the cells were lysed and firefly luciferase activity measured and

normalized against expression of Renilla luciferase activity. A test compound was determined to have

agonist activity if it showed an activity level within the range of concentrations tested that was >20% of

that induced by 10"10 M E~2,l6~9 M E2~ or IfJ9 M DHT at the ERa, ERR, or AR, respectively, similarly, a

test compound was determined to have antagonist activity if it inhibited the activity of the agonist by at

least 20% within the range of concentrations tested. Mean results of three independent experiments

were reported. Within the concentration range of 10'8 -10"5 M, PCNB did not demonstrate agonist or

antagonist activity at the ERa, ERR, or AR.

Although this study was conducted in a different model system and using a narrower concentration

range of test chemicals than that specified in the ER transcriptional activation guidelines (EPA, 2009a), it

demonstrated an absence of ERa transactivation by PCNB. Additionally, this study went further than

required in the guidelines to show a lack of antagonist activity at the ERa and the absence of both

agonist and antagonist activity at ERR. Thus, this study provides more robust information regarding

potential estrogenic activity than that which would be derived from the in vitro Tier I screens. Finally,

Tier I screening requests the examination of AR binding activity (EPA, 2009b). Kojima et al. (2004) did

not look at this parameter specifically, but went further to examine the ability of the test chemicals to

transactivate the AR or antagonize the activity of DHT at the AR. Thus, this study provides more robust

data regarding activity at the AR than would be derived from the AR binding assay prescribed in Tier I

screening.

The following tables provide a comparison of the EDSPTier 1 screening guidelines. Table 1 compares

the methodology of Ashby et al. (2005) to the ER binding assay protocol guideline and Table 2 compares

the methodology of Ashby et al. (2005) and Kojima et al. (2005) to the ER transactivation assay protocol

guideline.

Page 47 of 61
O<MUk.

ra

-2LDOa>

100)

"a;

TJ0) a. LOacaa.ou

oc. inO

in-

to

-OOJuQJ<u

{£.

COzuQ.

«*•oVI OJ

4-*

COU> TJ03i.T^J x:u> !53Qoc

LT) OCO

•

LnO,0•

ra

4-»QJ XI x:^'cOJE

'Ql '501

ce. Ql _C

"33 -a'5W£' Q)1

ra

ra

li-

rain

.

<?;

| Model System

cc.' LU

4-<C

roc:

laE

.

ou

.01

tj

'ro£Xaa: LUC

rocXIIaj c

roE

*4—Q_

_Dl

4-*

roo; Qtoa:c

ro

4—' | Animal species/s !' i

f; ' i ' O . C TJ

' — ' QJ

OJ M

:

DO 'c

ra ' t

14- 0 ® > tj "° .5i LO i: 2. i

rvi

ro

"DO^85-100 days of age/ovariectomized

prior to sac

Age/condition

1' ,Uterus Uterus (number not specified) 01u

l/l

l/l hOl

!fcXI ccLU-CN.QJ !tXI cc1 1 1

x:.

LU•

-,,TEDG+PMSF (lml/0.1 mg tissue) l_itXI| Homogenization

o

1 ^Ol ^| Competitive Bin

OJ

'Dor LUx:QJ DXI DCLU

•x: 1TEGD+PMSF o3 itXI

rai/i

i/i

otoQOtoQOLOQL-Qj

roo- o

THOtoQomOLUOJ "5to

o

•z. o2iO

-z. 15"5

i/icin£

i/i QJ

1—

CLOCLOCLOC

Tl 0cou| 3H-17(J-estradiol

|

2"c0

-*:c3"oDO _c "DTo a level that results in 10-15% bin

total radiolabeled estradiol at 1 nM

Cytosol dilution

i/t Ql

l/l Ql

l/l Ql 13OXI

ro

rocQJ "5toI Solvent control i , o

•z. 0

•z. o2;.' nouOctyltriethoxysilane (log molar com

5, -6, -7, -8, -9, -10)

Negative control

0 ,

« ( • * , 1o

•z.'< o

•z.»

i ,»

LO" LO1

LO"1Norethynodrel (log molar cones: -4,

-6, -6.5, -7, -7.5, -8.5)

o

4-" CouOJ 4^

'l/lO0

.

roOJ

<D0

Tt XLOI 3bxLO

ID^0T— 1 XLO1 o

toX1 ^

to to X X LO LO »LO°Poo17R-estradiol (log molar cones: -7, - -9.5, -10, -11)

rouReference chemi

„

to*— i xLOI D

toxLO^TO

TlXLO1 o

to

TlXLOo I ^ X X LO LO o

T-)Log molar cones: -3, -4, -5, -6, -7, -8

(unless data suggest other cones)

53Test chemical coi

(SI

(M-

fMro

l/l QJ

rou

"Q.

<u1 No. tubes/cone (i

uu0uu0Ol1 Assay temperatu i_SI

*"*

x;

inxl ooDOC'xOl 0-CofSLOrH| Incubation time

OJ

•—

roQ.

rog-oTJXQJ *—

roa.

raxOk_ -ax014— >

roQ.

_roxOT3XHydroxylapatite 01OJ H—OJ

roReagent to separ

from bound

o00<u00

roa.
V

I

ai

"53

•p'5a. toOcovt

*tZna. EouEinO

i/i

ra

-aQJuai oeCOu0.

0>HI £GC

</»OCOz

COaciu•ouVI Q. (Ucou.a

re

•

lii

IB*

ill

lli|

|?fe;| itI'flSi

ii}

BS|

111

!lHt'

iOf

!O't

SI!

ic5?: ®

"ifc

1

5ifl!>

ss

i.!«i

Il|

|J1

Jill$

if

ill

PR^fc

ft

;^l$

i!i|

ill

3iW^

:'.• un*

jEl

laif

SE:

I

5

'

;3i'

'rcr; uSI

MS'i1:

,(3;- P|1

tfl ••:£*'.

tsoiji

;.;£!-

'si

^S; g

JID;,

I

||Ia'••!'>

'CS

,s*

,O'0: .

 c

i

'_^£i:

^CK

3j1diiif^i ?i il,;'l

ii;«SI

:f:sp; ;io;

JC^i

Jz:' . Ci ;T^

''

:

:f

;||i

, ^i¥-

Jl^i

si

.5":

O

>fc

\^

:i

.c: 3l

•!;i;

•'('•It'

t|l

m>-

TOCOCO

ra

i4—OCOC3I.dZ

K|

,ii;j'

;'S|

v;| 1

;^^

-

j i''8|,."

;?;1p5:

''it

•o?

'21

' ,:,%

t^i^-

'.'•A

S)|

'""ii'-

(i'.JfiS ' d^1

-

1

 • >'

s

-

;

:¥f^

i|I-:

|

ijife

'rtlf

•'*%5

i.*

*fe

T3QJ tO0

.

01

l_S

(_)to

'co

_>-

tuC

ra

ra

4-»

roQ

V) tDV)

LLI

CC

.04B^| Doto Interpret

ozo

"Z. ozwas obtained oy00 JDnj| Interactive

Ozoz0zc </

>

S 'oo

ai .£

1 si

o - _a

g.

5

 ^

1 'S B

5°3 T*3 ° — ^ QJ O

S

^ y *-

3 ro :p-

0 Q. Q,

<-" .!2 .0

a> TJ 4_, § -!2 c « g S

£^i 1

TM S 3 TO -0 00 "° *•" -E

 >s

0 «= ^ §

Z QJ .£ in

J3 J2 A

M—o

4-*EOJEquivocal to th

cones tested

COCU >_COCU >-COOJ >-

•(_! t

ro

^ <->

«/i m 1

 £

5 3 «s ? a

?

^1 5

S f .^

CD O DO

> * C _§ * "5 "•2 A K -

 5Q

Q. .-2 se

4_ra QJ l

i

* :r t/i ro C OJ There are d point on cu be fit & low Not interactive

OzoozQJo_Qet any of the criteria noted a QJ

4-1

"cCOQJ OQ| Equivocal

tf0Q

.

01

t_>- T33en01 .CE0

_OJ

e~i m

'ro>

roOcVI

ro

ro

-oQJ

.C

+-•oO) c

"QJ

T33U

<-HQJthe study does not match the Ti ndicate where o

~oi >

.

c•o01*J.cop

~JC.

tuo

Ic1^QJXoCO

10

t+-oOl

*frOJ 0003 D.
PCNB OSRI for Tier 1 Screening March 5, 2010

Table IV-2: Published In Vitro ER Transactivation Study of PCNB Referenced as OSRI: Comparison to

EDSP Screening Guidelines1

Assay Parameters

!

Model System

Cell line

Estrogen receptor

Reporter construct

Characterization

Culturing Conditions

Media

Antibiotic

FBS

Plating vessel

Plating density

C02 concentration

Temperature

Pre-incubation time

Assay Conditions

Maximum test cone.

Dilutions

No. wells per test cone.

Test limits

Cytotoxicity cut-off

Temperature

Incubation period

No. of replicates

, • Guideline '__ •

. Requirements'"

hERa-HeLa-9903

(stably transfected)

Human ERa

Luciferase gene with

vitellogenin ERE

Mycoplasma-free;

passaged <40 times

EMEM w/o phenol red

60 mg/L Kanamycine

10% dextran-coatedcharcoal-treated FBS

Plastic microplate free

of estrogenic activity

1 x 10" / 100 uL/ well

5%

37°C

3hrs

1 u.1 or mg/ml or 1 mM

Log serial dilutions

(7 cones)

3 (run in triplicate) '

Insolubility, cytotoxicity

80%

37°C

20-24 hrs

2-3

, .Kojimaetal.^OOa2,

•s ' / * ' ' s

CHO-K1 cells (transiently transfected)

t,t

Human ERa Human ER(i

ERE-containihg pGL3-tkERE reporter plasmid

Unknown

3 ' •' f

DMEM/F-12 (doesn't mention phenol red status)

.Penicillin-streptomycin (doesn't mention amount)

5% dextran-coated-charcoal-treated FBS

96-well plate

8,400 cells/well

5%

37°C

3 hrs post-transfection

(§,:.: ':-|iii' Om mM ' '

10"8 - 10"5 M

(exact cones unknown)

Unknown

Unknown

Unknown -

Unknown

24 hrs

3

' JQ.Ol'-mM

10"8 - 10"5 M

(exact cones unknown)

'Unknown

Unknown

Unknown

>•, Unknown

24 hrs

3

Ashby et al., 20053

i * ' '

• '- ^ : ,'

Yeast cells, ,

(transfected)

Human ER (form [a or

1?] not reported)

ERE-contaihing 1?-

galactpsidase reporter

N/A

Minimal media

•None •

None

'• ' t ,t

96-well plate

Unknown ,

Unknown

' ' - 32°C ,

Unknown •

ImM

5 x 10"12 - 1 x 10'3 M

3

Turbidity

Unknown

32'C

' • . ,|,3days

Unknown

Boxes highlighted in yellow indicate where the study does not match the Tier 1 Guideline or the data is not available from the

study report.

2Also tested for antagonist activity at ERa and ERI? in the presence of 10"" M and 10"10

 M (5-estradiol, respectively.

3

Tested antagonist activity at the hER in the presence of 5 x 10'9 M R-estradiol only.

Page 50 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table IV-2 (continued): Published In Vitro ER Transactivation Study of PCNB Referenced as OSRI:

Comparison to EDSP Screening Guidelines1

Assay Parameters

Data Analysis

For all chemicals

For all positive

chemicals

Data Interpretation

Positive finding

Negative finding

Reference Chemicals

Positive control

Weak estrogen

Very weak agonist

Negative control

Controls

Positive control

Vehicle control

Guideline

1

 , Requirements

Report RPCMax, PCMax

Report PC10, PC50 (if

appropriate)

RPCMax *PCio in 2/2 or

2/3 replicate runs.

RPCMax < PC10 in 2/2 or

2/3 replicate runs.

176-estradiol (10~14 -

10"8 M)

17a-estradiol(10~12-

10'6 M)

17o>

methyltestosterone

do'10 - irj4 M)

Corticosterone (10"11 -

10'5 M)

1 nM 1715-estradiol

(run in triplicate)

DMSO (preferred; H20

& EtOH acceptable)

Kojima et al., 20042

No «

No (report REC20 [PC20])

Data not reported in

this format

Yes

1

 • , No

, -

1

 No '.,•

No

1 1 1 *

0.1 nM 17B-estradiol <

DMSO

No

No (report REC20 [PC20])

Data not reported in

this format

Yes

.No

, . No

, No

1 nM 17&-estradiol

DMSO

Ashby et al., 20053

> , /• <

- -

No'

No

Data not reported in

this format

• No

No

No

, ', , No

N/A

Media only

Boxes highlighted in yellow indicate where the study does not match the Tier 1 Guideline or the data is not available from the

study report.

2Also tested for antagonist activity at ERa and ER(5 in the presence of 10"11

 M and 10'10 M (5-estradiol, respectively.

3Tested antagonist activity at the hER in the presence of 5 x 10"9

 M (5-estradiol only.

Page 51 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

II. ToxCast

ToxCast is a US EPA program that includes a battery of in vitro, high-throughput screening (HTS) assays

.to screen chemicals for potential human toxicity. Approximately.69 chemicals have undergone HTS

under the ToxCast program, the majority of these being pesticides. A number of these chemicals,

including PCNB, have current or forthcoming requirements for Tier 1 screening.

It came to our attention that there were potential redundancies between some of the ToxCast assays

and the Tier 1 screening objectives, and that ToxCast data may be useful OSRI (Other Scientifically

Relevant Information) for submittal in lieu of or to supplement Tier 1 required tests. Information

regarding the ToxCast Program results became generally available to the public through Environmental

Health Perspectives (EHP) and the EPA ToxCast Program website (www.epa.gov/ncct/toxcast/

index.html) in mid-December 2009, with the raw data for many of the assays being posted on the

ToxCast website in mid-February 2010. The data presentation in the EHP article (Judson et al., 2009)

and supplementary materials, and the ToxCast Program website is fairly opaque, with individual

chemical data tabulated and much of the detail on assay methodologies provided only through website

links.

We developed this review to evaluate the data that were the most relevant to potential endocrine

modulation and the results specific to PCNB. This review describes briefly the HTS assay technologies

incorporated in ToxCast relevant to the Tier 1 screening requirements, provides an overview of the HTS

assays most relevant to potential EAT endocrine modulation, discusses the Tier 1 screening assays that

could be potentially replaced, and provides a specific review of PCNB results based on the data available

from EPA.

a. ToxCast HTS Test Battery and Relevance to Potential Endocrine Modulation

The ToxCast HTS test battery includes nine separate in vitro assay technologies:

• Cell-free HTS assays to measure binding constants and enzyme inhibition values;

• Cell-based HTS assays to measure binding constants and enzyme inhibition values;

• High content cell-imaging assays to test for effects on a range of phenotypes in either the

human hepatoma cell line HepG2 or rat primary hepatocytes;

• Gene expression in human primary hepatocytes, focused on Phase I and II metabolic enzymes

and transporters;

• Multiplex transcription reporter assays to evaluate 48 transcription factor binding sites;

• Biologically multiplexed activity profiling (BioMAP) to characterize effects potentially relevant to

human tissue and inflammatory disease biology;

Page 52 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

• Phase I & II xenobiotic metabolizing enzyme (XME) cytotoxicity assays based on microarray

technology;

• High-throughput genotoxicity screening based on the GreenScreen high content GADD45A-6FP

HTS genotoxicity assay; and

• Real-time cell electronic sensing (RT-CES) to measure cellular changes in response to chemicals.

Of these assay technologies, those that provide information most directly relevant to the issue of

potential EAT endocrine modulation include the cell-free HTS assays, the cell-based HTS assays, and the

multiplex transcription reporter assays. Each of these assays and their data relevant to potential

endocrine modulation is described below.

1. Cell-Free HTS Assays

These assays were run under contract to EPA by Caliper Life Sciences (formally NovaScreen). Of the 239

endpoints examined, binding to the following receptor proteins relevant to the endocrine system was

evaluated:

• Bovine estrogen receptor from uterine membrane (bER);

• Human estrogen receptor from breast cancer cells (hER);

• Bovine progesterone receptor from uterine membrane (bPR);

• Human progesterone receptor from MCF-7 cells (hPR);

• Rat androgen receptor (rAR; recombinant);

• Human androgen receptor from LnCAP cells (hAR);

• Rat thyrotropin-releasing hormone receptor from forebrain membranes (rTRH); and

• Human thyroid hormone receptor-alpha (hTRa; recombinant).

All of these proteins are nuclear receptors, with the exception of the rat thyrotropin-releasing hormone

receptor (rTRH), which is a G-protein coupled receptor. Additionally, all of these, with the exception of

the hTRa, were evaluated in competitive binding assays that measured the displacement of radiolabeled

ligand from the receptor proteins. The hTRa assay, in contrast, examined receptor activation by

measuring the luminescent output from an hTRa-responsive reporter gene. Inhibition of thearomatase

enzyme (CYP19A1 or CYPC19) activity was also evaluated. Assay parameters specific to each of these

endpoints are detailed in Table 3.

Page 53 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table IV-3: Parameter Specifics Regarding Endocrine-Related Endpoints of the Cell-Free HTS Assays

Receptor/enzyme

Estrogen

receptor

Progesterone

receptor

Androgen

receptor

Thyroid

related

Aromatase

bER

HER

bPR

hPR

rAR

hAR

rTRH

hTRa"

hCYP

C19

Assay type

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Competitive

binding

Receptor

activation

Enzyme

inhibition

Reference

compound

N/Aa

17R-estradiol

promegestone

promegestone

methyltrienolone

methyltrienolone

thyroid

releasing

hormone (TRH)

Bisphenol-A

Ketoconazole

Positive

control

compound

17B-estradiol

17G-estradiol

promegestone

promegestone

methyltrienolone

methyltrienolone

TRH

N/A

N/A

Ligand

(cone., M)

3H-estradiol

(7 x 10'10)

3H-estradiol

(IxlO'10)

JHpromegestone

(7 x 10'10)

3Hpromegestone

(3 x 10'10)

3H-methyltrienolone

(IxlO'9)

3H-methyltrienolone

(5 x lO'10)

3H-3-meHis2-TRH

(2 x 1C)'9)

Triiodothyronine (T3)

(1.5 x 10'8)

Difbenzyloxymethoxy)

fluorescein0

(2.5 x 10"6)

Kd(M)

7 x 10'10

5X10'11

6 x 10'9

3xlO-10

4.6 x 10"9

3X10'10

2.3 x 10"9

3.5 x 10"9

N/A

Bmax

(fmol/mg

protein)

406

3,844

152

152

103,928

270

34

N/A

N/A

N/A = not applicable.

b

 Assay of antagonism to ligand-dependent binding of co-factor to the receptor.

c

 Aromatase substrate binding affinity = 4.4 x 10"6 M and Vmax = 0.03 pmol product/pmol CYPC19/min.

In all cases, the data were normalized to those of the solvent controls from the same plate as the tested

chemicals and results expressed as the percent of neutral control. For the parameters above, chemicals

were initially screened at a single concentration of 25 u.M (10 u.M for aromatase) in duplicate wells.

Those chemicals that were positive in the initial screen were then run using eight concentrations in the

range of 0.0229-50 u.M (0.00914-20 u,M for aromatase) to determine the AC50 (i.e., the half maximal

activity concentration).

It should be noted that the above information was derived from spreadsheets available on the EPA's

ToxCast website (http://www.epa.gov/ncct/toxcast/index.html). However, a review of the website for

Caliper Life Sciences (http://www.caliperls.com/), found a number of discrepancies between the

Page 54 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

information provided by EPA and that available from the contractor directly. These are as outlined

below.

According to Calipers Life Sciences,

• The human estrogen receptor is derived from MCF-7 cells;

• The human progesterone receptor is from T-47D cells;

• The rat androgen receptor is derived from prostate tissue;

• The ligand for the ER assays was

 125l-labeled;

• The bovine estrogen receptor ligand concentration is 1 x 10~10 M, the Kd is 5 x 10"10 M, and the

Bmax is 0.26 fmol.mg protein;

• The human estrogen receptor Kd is 1.1 x 10"10 M and the Bmax is 27 fmol/mg protein;

• The human progesterone receptor ligand concentration is 6 x 10"10 M; and

• The rat androgen receptor Kd is 4 x 10'9 M and the Bmax is 125 fmol/mg protein

It should also be noted that information regarding the human thyroid hormone receptor-alpha and

human aromatase (CYPC19) enzyme was not found in the Calipers Life Sciences website.

2. Cell-Based HTS Assays

These assays were conducted by the NIH Chemical Genomics Center (NCGC). Both agonist and

antagonist activity at various nuclear receptors, including the human androgen receptor (hAR), human

ERa (hERa), and human thyroid hormone receptor-R (hTRR), were examined using a reporter gene assay

system (misidentified as Invitrogen GeneGLAzer in EPA materials; most likely, the Invitrogen GeneBLAzer

reporter assay system). According to the Invitrogen website (http://www.invitrogen.com/), the

GeneBLAzer technology uses a beta-lactamase reporter system, but specifics about how the system

works are not provided. The nuclear receptor sources and the assay parameters related to each of the

examined endpoints, as provided by the EPA, are detailed in Table 4.

Page 55 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table IV-4: Parameter Specifics Regarding Endocrine-Related Endpoints of the Cell-Based HTS Assays

Andrpgen

related

Estrogen

related

Thyroid

related

Activity

hAR agonist

hAR antagonist

hERa agonist

hERa antagonist

hTRB agonist

hTRIS antagonist

Target source

HEK293H cells

HEK293H cells

HEK293H cells

HEK293H cells

HEK293T cells

HEK293T cells

Reference

compound

R1881

N/A

17ls-estradiol

4-hydroxytamoxifen

Triiodothyronine

(Ts)

N/A

Positive control

compound

N/Aa

N/A

N/A

4-hydroxytamoxifen

N/A

N/A

Ligand

(cone., M)

N/A

R1881

(1 x 10'8)

N/A

17(?-estradiol

(5xlO-10)

N/A

T3

(4 x 10'10)

aN/A = not applicable.

Exact concentrations of chemicals tested in this assay system are not clear. A recent publication

indicates that chemicals were tested at fifteen concentrations in the range of 0.0012-92 \iM (Judson et

al., 2009). However, the summary data file for these assays provided on the ToxCast website

(http://www.epa.gov/ncct/toxcast/index.html) indicates that chemicals were tested to a maximum

concentration of 200 u.M. In the case of PCNB, concentrations in the range of 0.0010 - 76.6 u.M were

tested. A minimum of 25% of the control efficacy was used as the cut-off for considering a chemical to

be active. The supplemental information to Judson et al. (2009) indicates that, "values where only the

highest concentration exceeded 50% activity were excluded to eliminate weak or false positives." Cell

viability was also evaluated in this assay system; however, the criteria for excluding concentrations to be

tested in the assay system based on cytotoxicity are not clear.

3. Multiplex Transcription Reporter Assays

These assays were performed byAttagene, Inc. usinga multiple reporter transcription unit (MRTU)

library consisting of 48 transcription factor binding sites transfected into HepG2 cells (Martin et al.,

2010). Cis-acting reporter genes examined the activities of transcription factors endogenous to the

HepG2 cells. Trans genes evaluated the activities of exogenous, chimeric proteins. Each chimera was

comprised of the ligand-binding domain of a nuclear receptor and a yeast Gal4 DMA-binding domain.

When activated, these chimeric proteins activated transcription of a reporter gene expressed

downstream of a 5X-UAS-TATA promoter. In all cases, the reporter gene was identical in sequence and

included a restriction enzyme cleavage site; the position of the restriction site, however, was unique to

each reporter gene. The system allowed for reporter gene products to be identified through sequencing

and separation via high resolution electrophoresis. Using this system, the following endpoints relevant

to endocrine modulation were evaluated (listed in Table 5, along with their respective reference

compounds, as provided in EPA materials).

Page 56 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Table IV-5: Endocrine-Related Endpoints and Reference Compounds Tested in the Multiplex

Transcription Reporter Assays

Androgen related

Estrogen related

Thyroid related

Endpoint

Human androgen receptor

Human ER-a

Human estrogen response element3

Human estrogen-related receptor-a

Human estrogen-related receptor-y

Human thyroid hormone receptor-a

Reference Compounds

Dioxin, FIC

1715-Estradiol, diethylstilbestrol

Estrogens

N/Ab

4-Hydroxytamoxifen

Triiodothyronine (T3)

"The human estrogen response element was tested as a cis element; all other endpoints relevant to endocrine modulation were

tested as trans factors.

bNot applicable.

Cytotoxicity was first determined using the MTT tetrazolium assay and 24-hour chemical exposure to

five test concentrations, with an upper concentration of 50 u.M and 10-fold serial dilutions. The

maximum tolerated concentration (MTC) was derived as one-third the cytotoxic IC50, or if no IC50 value

was determined, then the MTC was set at 100 u.M. Test compounds were then assayed at seven

different concentrations in the range of 0.0014-100 U.M, starting at the MTC, followed by three-fold

serial dilutions. After a 24-hour exposure, cells were harvested, reporter gene products isolated and

reverse-transcribed using fluorescent-tagged primers, and resolved by high resolution gel

electrophoresis. From the resulting data, the lowest effective concentration that activated a cis-acting

response element or transcription factor was determined (LEL; lowest concentration at which there was

a statistically significant change from the concurrent negative control).

b. Comparison to Tier 1 In Vitro Screening Requirements

The ToxCast data supplement, and in some cases, may fulfill the in vitro data requirements of the EDSP

Tier 1 ER binding assay, AR binding assay, ER transactivation assay and aromatase inhibition assay. In

addition, these data provide important OSRI that should be considered in a weight-of-the-evidence

evaluation of functionally equivalent information supporting the absence of need for many of the Tier 1

mammalian in vivo screening tests.

ER binding assay: The EDSP Tier 1 ER binding assay requires that binding be evaluated using rat ER

derived from uterine tissue taken from ovariectomized Sprague Dawley rats. In the ToxCast cell-free

HTS assays, binding to bovine and human, but not rat, ER was evaluated. In the Tier 1 screening assay,

the concentration of

 3H-17IJ-estradiol ligand used in the assay is 1 nM and the test chemicals are

evaluated at concentrations of 1 x 10"10 to 1 x 10"3 M. In the ToxCast assay, the concentration of

radiolabeled estradiol varied depending on the ER source (bER: 7 x 10"10 M; hER: 1 x 10"10 M), but was

generally similar to that recommended in the EDSP Tier 1 ER binding assay; however, compounds were

tested at an initial concentration of 2.5 x 10"5 M, and if positive for binding activity, were further

evaluated at concentrations of 2.3 x 10"8 - 5 x 10"5 M. This concentration range is narrower than the

range recommended in the Tier 1 screening assay, but still should be adequate to detect activity in a

moderate concentration range.

Page 57 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Androgen receptor binding assay: The EDSP Tier 1 screen AR binding assay requires that binding be

evaluated using rat AR derived from prostate tissue taken from castrated Sprague Dawley rats. The

concentration of

 3H-R1881 ligand used in the assay is 1 nM and the test chemicals are evaluated at

concentrations of 1 x 10~9 to 1 x 1CT4 M. In the ToxCast cell-free HTS assay, binding to both rat and

human androgen receptors was evaluated, so it provides a more comprehensive screen than does the

Tier 1 battery. Radiolabeled methyltrienolone, instead of R1881, was used as the radioligand in this

assay and concentrations in the ToxCast cell-free HTS assay varied depending on the AR source (rAR:

4.59 x 10~9 M; hAR: 3 x 10"10 M), but were similar to that recommended in the EDSP Tier 1 AR binding

assay. Finally, compounds were tested in the ToxCast cell-free HTS assay at an initial concentration of

2.5 x 10"5 M, and if positive for binding activity, further evaluated at concentrations of 2.3 x 10"8 - 5 x 10"

5

 M. This concentration range is slightly narrower that that recommended in the EDSP Tier 1 screen AR

binding assay guideline (1 x 10~9 - 1 x 10~4 M), but still should be adequate to detect activity in a

moderate concentration range.

ER transactivation assay: The EDSP Tier 1 screen ER transactivation assay requires that the assay be

conducted in hER-HELA-9902 cells, which express the human ER-ct and have been stably transfected

with a iuciferase reporter gene linked to an upstream vitellogenin estrogen-responsive element. Test

compounds are evaluated at concentrations of 1 x 10~9 to 1 x 10'3 M. Finally, a test chemical is

considered positive for ER transactivation in the Tier 1 assay if the maximum response induced is at least

10% of that induced by 1 nM 17R-estradiol.

In the case of the ToxCast cell-based HTS assays, details regarding the gene reporter system were not

available; however, activity at the human ER-a was assessed and test chemicals were evaluated at a

similar range of concentrations (1.2 x 10"9 - 0.9 x 10"4 M) as recommended in the EDSP Tier 1 screen.

The data outputs from the ToxCast cell-based HTS assays (AC50 values) are different than those required

in Tier 1 screening, and in most cases, positive control compounds were not tested. However, if a

chemical elicited a low level of activity at the highest concentration tested in the cell-based assay, it can

be assumed that it would be negative according to the EDSP Tier 1 screening criteria. It should be noted

that the ToxCast program's cell-based HTS assay provides more robust data than that required in the

EDSP Tier 1 ER transactivation assay because it also examined the test chemical's ability to act as an

antagonist at the ER-a.

The multiplex transcription reporter assay also examined transactivation of the human ER-a. In this

case, the assay is conducted in HepG2 cells (instead of HELA-9902 cells), and the output from the

reporter gene and how it is measured is different from that indicated in the EDSP Tier 1 screening assay.

HepG2 is a perpetual cell line, which was derived from the liver tissue of a 15-year old Caucasian

American male with a well differentiated hepatocellular carcinoma. The HepG2 cells and its derivatives

are often used as a model system for studies of liver metabolism and xenobiotics toxicity because of

their metabolic capabilities. The range of test chemical concentrations tested (1.4 x 10"9-1 x 10"4 M) is

similar to that recommended in the EDSP Tier 1 screen. Finally, as with the ToxCast cell-based HTS

assay, the data outputs (LEL values) are different than those required in EDSP Tier 1 screening, and

Page 58 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

positive control compounds were not tested. However, if a chemical elicited a low level of activity at the

highest concentration tested in the multiplex transcription reporter assay, it can be assumed that it

would be negative according to the EDSP Tier 1 screening criteria.

Aromatase assay: The EDSP Tier 1 screen aromatase assay requires that enzyme activity be evaluated

using human recombinant microsomes containing CYP19 plus reductase. Radio-labeled androst-4-ene3,17-dione at a concentration of 1 x 10"8

 M is used as the enzyme substrate and test compounds are

evaluated at concentrations of 1 x 10~10 M -1 x 10"3 M. In the ToxCast cell-free NTS assay, human

aromatase activity was evaluated using di(benzyloxymethoxy)fluorescein, which produces a fluorescent

product as the enzyme substrate. Compounds were initially screened at a single concentration of 1 x

10"5 M, and those chemicals that were positive in the initial screen were then run using eight

concentrations in the range of 9.14 x 10"9 M - 2 x 10~5 M. This concentration range is narrower than the

range recommended in the Tier 1 screening assay, but still adequate to detect activity in a moderate

concentration range.

It should be noted that the ToxCast HTS data set includes information that should be useful to submit as

OSRI, and in fact, goes beyond the in vitro testing required in the Tier 1 screening program to provide

additional information on the potential to interact with the EAT systems, including thyroid-related

receptors, as well as progesterone receptors. It does not, however, directly address Tier 1

steroidogenesis in vitro test requirements.

c. Data Specific to PCNB

PCNB of unknown purity was tested in all ToxCast assays.

In the cell-free HTS assays, PCNB was tested at an initial concentration of 25 U.M and possibly eight

additional concentrations -invttoe. range of 0.0229-50 u,M. At these test concentrations, PCNB was

negative for binding at all receptor proteins of relevance to potential endocrine modulation (i.e., at each

receptor site tested, PCNB exhibited less than 50% inhibition of radioligand binding). Specifically, PCNB

did not bind to the bovine and human ERs; bovine and human progesterone receptors; rat and human

ARs; rat thyrotropin-releasing hormone receptor (rTRH) or human thyroid hormone receptor-a.

Additionally, PCNB was considered negative for aromatase inhibition (i.e., it exhibited less than 50%

inhibition of human aromatase enzyme activity).

In the cell-based HTS assays, PCNB was tested at 15 concentrations in the range of 0.0010-76.6 u.M.

Under these conditions, PCNB was considered inactive for agonist activity at the human AR, human ERct, or the human thyroid hormone receptor-li. Furthermore, it did not block (or antagonize) the activity

of established ligands at these receptor sites.

In the multiplex transcription reporter assay, PCNB did not activate chimeric transcriptional proteins

containing ligand-binding domains for the human AR, human estrogen-related receptor-a, human

estrogen-related receptor-y, or human thyroid hormone receptor-a. However, PCNB activated a

Page 59 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

chimerical transcriptional protein containing the ligand-binding domain for the human ER-a with an LEL

of 52 u,M and activated transcription at a human estrogen response element with an LEL of 14 u.M.

These results are in contrast with those of the cell-free and cell-based assays, which showed no binding

of PCNB to bovine or human ERs and no agonist or antagonist activity at the human ER-a.

Overall, the ToxCast data for PCNB is mixed. PCNB demonstrated a complete lack of activity at the

androgen receptor. That is, PCNB did not bind to the rat or human ARs in the cell-free assay and did not

activate or antagonize transcription activity in the cell-based assays, and did not activate transcription

by a chimeric protein containing the ligand-binding domain of the human AR. PCNB also demonstrated

a complete lack of binding or activity at all thyroid-related receptor sites assessed in these assays.

PCNB did not bind to either the bovine or human ER in the cell-free assay and did not act as an agonist

or antagonist at the human ER in the cell-based assay. In the multiplex transcription reporter assay,

however, PCNB activated a chimeric transcriptional protein containing the ligand-binding domain of the

human ER-a and activated transcription from an estrogen responsive element. The reason for the

discrepancy in findings is not known. The multiplex transcription reporter assay is conducted in HepG2

cells, which possess metabolic activity. Therefore, it is possible that the differences may be due to a

metabolite of PCNB. Nevertheless, PCNB has been negative for estrogenic activity in the vast majority of

published in vitro studies, suggesting that the findings of the multiplex reporter transcription assay are

spurious.

Page 60 of 61
PCNB OSRI for Tier 1 Screening March 5, 2010

Appendix V: Title Pages of Regulatory Toxicology Studies Submitted as OSRI

Page 61 of 61