STUDY TITLE

Other Scientific Relevant Information (ORSI):

EPIC EDSP Test Order

DATA REQUIREMENT

Test Order/DCI #: EDSP-041401-64

AUTHOR

Gail T. Arce, PhD

Arce Consulting

COMPLETION DATE

Febl9,2010

SPONSOR

Gowan Company

P.O. Box 5569

Yuma,AZ 85366

PERFORMING LABORATORY

Not applicable

PROJECT IDENTIFICATION '• ' • ...

••:••:

MEC2010-2 ••;-. .

• ••

Page 1 of 64
STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality is made for any information contained in this report on the

basis of its falling within the scope of FIFRA Section 10 (d) (1) (A), (B), or (C).

Company: Go wan Company

Company Agent: Elizabeth Codrea

Director of Regulatory Science

Date: ' * """" ^-olb

Page 2 of64

• • • •

• •••

• •
GOOD LABORATORY PRACTICE STATEMENT

This submission is not a "study" within the meaning of 40 CFR Part 158, thus the Good

Laboratory Practices standards are not applicable.

Submitter: Gowan Company

Company Agent: Elizabeth Codrea

Director of Regulatory Science

Date:

• • •

• • •

•••••• • **

•• • • • • • .

.. . •••••• • • •

• • • ••

, .

Page 3 of 64
ORGANIZATION OF SUBMISSION

I. Executive Summary

II. Introduction

III. Endocrine-Mediated Effects of the HPG Axis

IV. Endocrine Mediated Effects of the HGT Axis

V. EDSP Tier I Battery vs. EPTC Database

VI. Summary and Conclusions

VII. References

VIII. EPTC Studies - Supplemental Detail

A. Reproduction Studies

a. 2-Generation Reproduction 1985

b. 2-Generation Reproduction 1982

B. Developmental Studies

a. Rabbit Developmental 1987

b. Rabbit Developmental 1985

c. Rat Developmental 1983

d. Rat Developmental 1985

e. Rat Developmental Neurotoxicity

C. Intermediate-Term Studies

a. 90-Day Rat Oral Toxicity

b. 90-Day Rat Inhalation Toxicity

c. 90-Day Rat Neurotoxicity

d. 90-Day Dog Toxicity

D. Long-term Studies

a. 1-Year Dog 1986

b. 1-Year Dog 1987

c. 1-Year Rat

d. 2-Year Rat Chronic/Oncogenicity 1983

e. 2-Year Rat Chronic/Oncogenicity 1987

f. 24-Month Mouse Oncogenicity 1978

g. 18-Month Mouse Oncogenicity 1986

E. Wildlife Studies

a. Mallard Duck Reproduction Study

b. Bobwhite Quail Reproduction Study

IX. OSRI Title Pages

Page 4 of 64
I. EXECUTIVE SUMMARY

This submission discusses the Other Significant Relevant Information (OSRI) that should

be considered in conjunction with Gowan Company's response to the November 12,

2009 Endocrine Disrupter Screening Program (EDSP) Data Call-In Notice for the active

ingredient carbamothioic acid, dipropyl-, S-ethyl ester (EPTC).

The purpose of the EDSP Tier 1 battery is to provide a preliminary assessment of

endpoints that may be indicative of estrogen, androgen or thyroid activity in a series of in

vitro or short-term in vivo tests. The assays in the Tier 1 battery include intentional

redundancy with respect to monitored endpoints. As a screen, the Tier 1 tests are not

definitive. The definitive tests for endocrine disruption are the longer-term in-life studies

that evaluate endocrine toxicity directly. If hormonal levels or endocrine organs are

disrupted, significant biologically adverse impacts on health status will be apparent in the

longer-term studies. The regulatory in vivo studies allow for dose-response assessment

and subsequent human risk assessment not possible with the Tier 1 tests and thus,

represent a higher tier in the EDSP testing program.

The EDSP Tier 1 in vivo studies primarily evaluate effects such as weight changes and/or

histopathology findings in sexual organs and tissues, or changes in developmental

milestones as indicators of possible endocrine disruption. EPTC has been evaluated in

'numerous acceptable regulatory studies that directly address most of such endpoints

included the in vivo Tier 1 screening battery. The few endpoints not directly evaluated are

associated with multiple adverse effects; these other effects would have been evident in

the regulatory studies from the other monitored parameters.

The relevant regulatory studies include reproduction studies in rats and birds, multiple

developmental studies hi rats and rabbits, and long-term studies in rats, mice and dogs.

The diversity of tests and tested species reflects the redundancy sought in the EDSP

assays. Altogether, thousands of animals in multiple species have been evaluated for

effects on relevant organ weights, gross pathology and histopathology. No effects were

observed in either sex to sexual organs or to any endocrine gland (adrenal, pituitary,

thyroid). No effects were found that would indicate endocrine-mediated impairment of

reproduction function or alteration in developmental progress. The absence of any effect

on serum cholesterol in the whole-animal regulatory studies confirms that EPTC does not

cause perturbation of steroidogenesis.

The Agency developed the ToxCast TM program to predict endocrine active agents and

profiled over 300 chemicals including EPTC. ToxCast results indicated EPTC has a very

low potential for endocrine disruption.

EPTC shows no endocrine-related toxicity, either clinical or biochemical at maximally

tolerated doses of EPTC. Histopathological effects in nerve and muscle, primarily heart,

defines the critical regulatory NOAELs at levels significantly lower than any other

adverse effects. Gowan Company believes that collectively the available data fulfill the

Page 5 of 64
requirements of the Endocrine Disruptor Screening Program Data Call-In and that the

overwhelming weight-of-the-evidence shows that EPTC is not anticipated to produce any

effect in humans similar to an effect produced by a naturally occurring estrogen.

Therefore, EPTC meets the standard for an exemption under FFDCA section 408(p)(4).

Page 6 of 64
II. INTRODUCTION

In this document, the EPTC database is discussed in relationship to anticipated

endocrine-related toxic effects on the hypothalamus-pituitary-gonadal (HPG) axis and

hypothalamus-pituitary-thyroid (HPT) axis. The EPTC studies will then be assessed

against specific Tier 1 tests for functional equivalency of data. More detailed individual

study information pertaining to the HPG or HPT axis has been provided as supplemental

detail at the end of the document.

III. ENDOCRINE-MEDIATED EFFECTS OF THE HPG AXIS

Multiplicity of Estrogen and Androgen Mediated Effects of the HPG Axis

Endocrine active chemicals disrupt a wide range of biological endpoints measured in the

reproductive process. For example, estrogen deficiency will be manifested by altered

ovarian development and function such as chronic anovulation, cystic and hemorrhagic

follicles, absent corpora lutea, interstitial/stromal hyperplasia (Abbott et. al., 2006).

Octylphenol, a known estrogen antagonist, induced early vaginal opening and persistent

estrus after maturation but also produced increased proliferation in the endometrial

epithelium in animals dosed from postnatal day (PND) 1 to 15 and increased uterine

weight and increased proliferation of the endometrial epithelium in adult females dosed

for 28 days (Hasegawa et. al., 2000). Androgen deficiency (induced by anabolic steroids)

that altered testosterone levels displayed histopathological findings such as arrest of

advanced spermatids, severe depletion of Leydig cells with altered weights of not just the

testes but prostate glands and seminal vesicles (Grokett et. al., 1992).

The effects are intertwined as antiandrogenic chemicals can also be estrogenic (Moore et.

al. 1991; Gray et. al. 1988). Multiple effects in males and females for estrogenicity have

been shown (Hasegawa et. al., 2000; Atanassove et. al., 2000: Hess et. al., 2000; Abbott

et. al., 2006; Sawaki et. al., 2003; Oliviera et. al., 2003; Terry et. al., 2004; Garcia-Florez

et. al., 2005). Antiestrogenic activity also displays multiple effects (Vanage et. al., 1997;

Cho et. al., 2003; Abbott et. al., 2006) as do androgenic and antiandrogenic chemicals

(Gray et. al. 2001; Grokett et. al., 1992; and Abbott et. al., 2006).

The mechanism of action of endocrine disruptors is varied, as are the biological effects

they produce. Some bind to receptors some do not. Some block or disrupt metabolic

pathways needed for synthesis of the hormones. Some show dual modes of action.

Regardless of the mechanism or mode of action, all manifest biological effects in the

reproductive systems in both males and females.

Biochemical parameters relevant to estrogen and androgen biosynthesis and receptor

binding are sentinel events, not necessarily adverse events, of HPG disruption. The most

direct and biologically relevant measure of HPG axis function is reproductive

performance hi whole animal studies. Organ weights and histopathology of both

reproductive and endocrine organs are critically important in assessing damage and are

inextricably linked to chemicals resulting in endocrine disruption.

Page 7 of 64
In all the literature reviewed, no short-term indicator of endocrine disruption was

observed without significant histopathology or reproductive repercussion. The responses

are multiple and concomitant. The assessment of these multiple effects form the basis of

a weight-of-the-evidence approach the Agency used to establish and prioritize the

conduct of the Tier 2 tests.

Lack of EPTC Estrogen and Androgen Mediated Effects of the HPG Axis

Key indicators collated from the literature and the Endocrine Disruptor Screening

Program: Statement of Policy: Notice (FR December 28,1998 pp 71542 - 71568) can be

used to assess the endocrine disrupting potential of EPTC. The EPTC studies collectively

evaluate nearly all relevant biological endpoints of estrogen or androgen-mediation of the

HPG axis. Multiple studies address most of the endpoints of concern. The few endpoints

not directly evaluated are associated with multiple adverse effects; these other effects

would have been evident in the regulatory studies from the other monitored parameters.

No indication of EPTC induced endocrine-related effects was observed in any study.

Potential hormonal effects can be detected through behavioral changes, mating, fertility

and gestational indices, duration of gestation, signs of difficult or prolonged parturition,

implantation sites (corpora lutea) and implantation loss, number of pups, fetal

development and growth, stillbirths, litter sizes, pup survival, sex ratio of the offspring,

postnatal growth, feminization or masculinization of offspring, gross pathology of pups

and FI adults, organ weights (ovaries, uterus, testes, epididymides, seminal vesicles,

prostate) and histopathology of the reproductive organs (ovaries, uterus, vagina, testes,

epididymides, seminal vesicles, prostate) of mating adults.

These endpoints are evaluated in the traditional regulatory developmental and

reproduction studies. The Agency has concluded that no developmental or reproductive

effects are found at doses lower than maternally toxic treatment levels (US EPA, 1990)

and no FQPA safety factor was applied based on these results. Endocrine-related organ

weight and detailed histopathological examination of long-term studies revealed no

evidence of endocrine disruption. Histopathological findings of myocardial degeneration

were used to establish the chronic regulatory NOEL of 2.5 mg/kg from the 2-generation

reproduction study (reproductive NOEL was 40 mg/kg).

Reproductive and Developmental Parameters

Reproductive performance is an indication of the functionality of the male and female

endocrine system. Results of the 2-generation rat reproduction studies demonstrate no

effect on mating, fertility, gestational indices, parturition or mating behavior at 40 mg/kg

(MacKenzie, 1985) or 50 mg/kg (Minor et. al. 1982). Severe maternal toxicity was

manifest as body weight decrements in PO and FI males and females in the high dose

groups of both reproduction studies and mortality in the rabbit developmental study

(Nemec 1983). Other indicators of reproductive function such as the number of corpora

lutea, post-implantation loss, resorption of fetuses, as measured in developmental studies

were unaffected as determined in two rat developmental studies (Nemec, 1983, James

Page 8 of 64
1985) and two rabbit (James, 1985, Gilles, 1987) and in a developmental neurotoxicity

study (Lees, 2004). .

Offspring were unaffected at doses less than maternally toxic doses in the developmental

and reproduction studies. Evaluated parameters included litter size, live births, pup

survival, pup sex ratios and developmental markers such as pre- or post implantation loss,

fetal loss or resorptions. There were no external, visceral or skeletal anomalies present in

the absence of maternal toxicity.

In the developmental neurotoxicity study, FI females were examined daily from day 29 to

determine the day of age of vaginal opening. FI males were examined from day 41 to

determine the age of preputial separation. Male and females in the FI generation were

evaluated for anal genital distance (AGD). AGD, vaginal opening and preputial

separation were normal at all treatment levels.

Organ Weight and Pathology

Whether estrogenic or androgenic, and agonist or antagonist, organ weight and

histopathology of endocrine-related organs will be affected.

Estrogenic and androgenic active chemicals result in a cascade and intertwined set of

effects described in Section III. Endocrine Mediated Effects of the HPG Axis. For

- example, decreased testicular weights associated with estrogenic or androgenic activity

show concomitant decreases in sperm count and abnormal histopathology of the Ley dig

cell, epididymal, and prostate. Estrogenic compounds that lead to testes weight decreases

also result in increased uterine weight and decreased ovarian weight. Uterine

histopathology will be present as cystic and endometrial hyperplasia and the ovaries will

be polycystic or hyperplastic. Estrogenic chemicals will also demonstrate reproductive

and developmental effects with decreases hi the number of corpora lutea. Similarly,

antiandrogenic chemicals will also lead to endometrial hyperplasia of the ovary and

uterus.

None of these indicators of endocrine disruption were evident following either short or

long term exposure to EPTC. There were no polycystic or hyperplastic ovaries, cystic

uteruses or endometrial hyperplasia, no effect on mammary carcinoma, and no adenoma

of the vagina. In the males, no degenerative or hyperplasic events were noted in the

prostate, epididymides, Leydig cells, or degeneration in the seminal vesicles. Organs

weight and histopathological evaluations in the two rat 2-generation reproduction studies

(MacKenzie, 1985; Minor et. al., 1982), rabbit developmental study (Gilles, 1987), 90-

day oral and inhalation rat toxicity studies (Tisdel 1983; Scott, 1985, 90-day dog toxicity

study (Daly 1985), two one-year dog chronic studies (Dickie 1986; Sprague and Taylor,

1987), one-year rat chronic study (Trutter, 1978) two 2-year rat chronic oncogenicity

studies (Warner 1983, Dickie 1987), an 18-month mouse oncogenicity study (Tisdel,

1986) and two-year mouse oncogenicity study (Goldenthal 1978), collectively were all

normal.

Page 9 of 64
The data represent a very large number of observations for all organs of interest in a wide

range of studies. The organ weight findings are summarized and collated in Table 1 and

histopathological findings are presented in Table 2.

- Table 1. Body and Organ Weights

Study

2-Gen Repro 1982

Rabbit Dev 1987

90 Day Rat Oral

90 Day Rat Inhal

90 Day Dog 1985

1 yr Dog 1986

1 yr Dog 1987

1 yr Rat 1978

2-yr Rat 1983

2-yr Rat 1987

2 yr Mouse 1978

Body

weight

100(4*

V.

18/3mg4-

V

V

V

V

20mg4'

25mg4'

9mgi

V

Liver

V

V

. V

V

V

V

60 mg tar

V

V

V

V

Kidney

V

V

V

V

V

V

V

V

V

V

Pituitary

ISOO^ar (f)

V

V

v .

Adrenal

V

V -

V

V

V

.V

Thyroid

V

V

V

V

V

Testes

Ovary

V(i)

V

V

V

V

18004>ar(m)

. V

V

V

V

V

Prostate

Uterus

V

(1) Testes only

V = Normal -I - decrease t = increase a

(m) = male (f) = female

= absolute r = relative # = Dose in ppm unless where indicated

Findings in liver, pituitary and testes were only seen in one dog study in one sex but were

not confirmed by the second dog study or found in any other study. These findings are

discussed below.

Liver: Alteration in liver weight was observed in only one dog study and only in

females. Weights were increased for females at the highest dose (60 mg/kg) in the oneyear dog chronic study (Sprague and Taylor, 1987). In the 60 mg/kg males, absolute

liver weights were normal but relative liver weights were increased. There were no

concomitant changes in liver related clinical chemistry. While apparently treatmentrelated, it was a relatively mild effect. All other organ weights were comparable to

control animals. Liver weights were unaffected at 54.7 mg/kg in the other one-year dog

chronic study (Dickie, 1986). Histopathological examination of the liver revealed no

abnormalities in any of the studies

Pituitary: Pituitary weights were lower in only one dog study and only in females.

Females treated with 1800 ppm (54.7 mg/kg equivalent) showed lower absolute and

relative pituitary weights when compared to control animals. In the absence of

microscopic changes and without additional supportive evidence to substantiate a direct

Page 10 of 64
relation to treatment, these weight changes were considered biologically unimportant.

No effect was seen in the other one-year dog study evaluated at doses up to 60 mg/kg.

(Dickie, 1986). Histopathological findings in the pituitary were normal in all studies (see

Table 2 for list of studies where testes histopathology was evaluated).

Testes: In one dog study (Dickie, 1986), absolute testes weights in the 1800 ppm (48.5

mg/kg equivalent) group males were lower than controls. In the absence of microscopic

changes and without additional supportive evidence to substantiate a direct relation to

treatment, these weight changes were considered biologically unimportant. Testes

weights were normal in the Sprague and Taylor (1987) study that evaluated EPTC up to

60 mg/kg and in all other studies in rats or mice. Histopathological findings in the testes

were normal in all studies (see Table 2 for list of studies where testes histopathology was

evaluated).

Ovary: No changes in ovary weights were observed in any study. These included the rat

reproduction studies, 90 day rat and dog studies, and the long term rat, mouse and dog

studies. Likewise, all ovaries examined had normal histomorphology.

Sex Accessory: All uterine and prostate weights were normal in all studies in which they

were evaluated. Histopathology was normal in the prostate,, epididymides, seminal

vesicles, spermatic cords, ovary, cervix, uterus, vagina and mammary glands in all

studies where evaluated.

Endocrine organs: No organ weight effects were observed in the thyroid or adrenal

glands. Histopathology was normal in the thyroid, parathyroid, pituitary, and adrenal

glands in all studies evaluated.

Page 11 of 64
Table 2. Mammalian Organ Histopathology Evaluation

Histopathology

Organs evaluated

Liver

Kidney

Thyroid

Parathyroid

Pituitary

Adrenal

Testes

Prostate

Bulbo-urethral gland

Coaggl gland

Epididymides

Seminal vesicles

Spermatic cords

Urethra

Ovary

Oviduct

Cervix

Uterus

Vagina

Mammary gland

STUDY

2-Generation

Reproduction

FO/F,

1982

V

V

^

VV

V

V'

V

V

V

.V

L_ ^

VTJ

VV

F,

1985

V

V -

V

V

V

V

V

V

90 Day

Rat

Oral

V

V

V

V

V

VV

V

V

V

V

VV

V

V

90 Day

Dog

" V

V

V

V

V

VV

V

V

V

VV

VV

lYr

Dog

1986

V

V

V

VV

VV

V

V

V

V

VV

lYr

Dog

1987

V

V •

V.

V

V

V

V

V

V

V

V

V

V

V

1 Yr

Rat

1978

V

V .

V

V

V

VV

V

V

V

V

V

V

V

2Yr

Rat

1983

V

V

V

V

V

V

• V

V

V

V

V

V •

V

V

V

2Yr

Rat

1987

V

V

V

V

V

V

V

V

V

V

V

V

V

V

V

2Yr

Mouse

1978

V

V

V

V

V

V

V

V

V

V

V

VV

V

V •

18 Mo

Mouse

1986

V

V

V

V

V

V

V

V

V

V

v.

V

V

V

v .

= Evaluated with normal pathology

Biochemical

Serum cholesterol is of particular importance as cholesterol is a molecular precursor of

all steroid hormones in humans, including estrogens and androgens. Early disruption of

steroidogenesis can lead to altered levels of cholesterol and disruptions in cholesterol

synthesis can affect steroidogenesis.

Cholesterol levels were unaffected in all studies in which it was measured including both

male and female rats (90 day oral and inhalation studies and two year studies), and dogs

(90 day and one-year studies).

ToxCastTM PrioritizationofEPTC

The Agency has been developing the ToxCast program designed to test the hypothesis

that multi-dimensional evaluation of chemical properties and effects across a broad

spectrum of information domains (e.g., molecular, cellular, and organ responses) will

provide data that will be predictive of toxicity. ToxCast profiled the responses of over

300 chemicals (mainly pesticides and other select chemicals), chosen because of the

lexicological understanding from existing traditional animal test data and their intended

Page 12 of 64
activities as biological agents.

In a presentation by Reif (2009) the results of the prioritization of these chemicals was

provided as it related to the EDSP. EPIC was one of the chemicals modeled for

endocrine activity. The model incorporated multi-assay profiles in combination with

information of relevant chemical properties, toxicity pathways, and in vivo study results.

Rankings were then developed for the chemicals.

EPIC had a score of 0.13 (Tropsha, 2009) which places EPIC in the low prioritity range

as depicted by Reif (2009).

HPG Endpoints vs. EPTC Database

There was no evidence of any HPG endocrine-disrupting effect in the rat reproduction"

studies, the rat or rabbit developmental studies, the rat or dog subchronic studies, and the

long-term chronic or oncogenicity studies in the rat, mouse or dog. No relevant changes

in organ weights or histopathology were identified in any of these studies. Likewise,

cholesterol levels were not affected by treatment with EPTC.

The range of available mammalian studies and the indicators for HPG endocrine

disruption are presented in Table 3 (female) and Table 4 (male). The selected endpoints

were collated from the literature and from the Agency's Statement of Policy from the

1998 Federal Register (US EPA 1998c). The EPTC study results are provided adjacent

to the endpoint of interest, together with the study reference.

Page 13 of 64
Table 3. Female Endpoints Relevant to Endocrine Disruption - HPG

Estrous cycliciry

Vaginal Opening

Ovarian follicle count

Fertility Index

Mating index

Corpora lutea

Implantation; pre and post

implantation loss

Resorptions

Parturition

Gestational length

Litter size of pups (live

births)

Sex ratios of pups

Survival of pups

Uterine weight

Study Type

2-G Repro

2-G Repro

DNT:

Not evaluated

2-G Repro

2-G Repro

2-G Repro

2-G Repro

RatDev

RatDev

Rabbit Dev

Rabbit Dev

RatDev

Rat Dev

Rabbit Dev

Rabbit Dev

RatDev

RatDev

Rabbit Dev

Rabbit Dev

2-G Repro

2-G Repro

Rat DNT

2-G Repro

2-G Repro

DNT

2-G Repro

2-G Repro

Rat Dev:

Rat Dev:

Rabbit Dev

Rabbit Dev

Rat DNT

2-G Repro

3-G Repro

Rat Dev

Rat Dev:

Rabbit Dev

Rabbit Dev

Rat DNT

2-G Repro

2-G Repro

Rat DNT

Rat Dev

Rabbit Dev

Observation

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal*

Normal

Normal

Normal

Normal*

Normal*

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal*

Normal*

Normal*

Normal

Normal

Normal*

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal*

Normal

Normal*

Normal

Normal

Study Reference

MacKenzie 1985

Minor 1982

Lees 2004

MacKenzie 1985

Minorl982

MacKenzie 1985

Minor 1982

Nemec 1983

James 1985

Gilles 1987

James 1985

Nemec 1983

James 1985

Gilles 1987

James 1985

Nemec 1983

James 1985

Gilles 1987

James 1985

MacKenzie 1985

Minor 1982

Lees 2004

MacKenzie 1985

Minor etal. 1982

Lees 2004

MacKenzie 1985

Minor 1982

Nemec 1983

James 1985 •

Gilles 1987

James 1985

Lees 2004

MacKenzie 1985

Minor 1982 .

Nemec 1983

James 1985

Gilles 1987

James 1985

Lees 2004

MacKenzie 1985

Minor 1982

Lees 2004

Nemec 1983

Gilles 1987

Normal* = Normal in the absence of maternal toxicity or systemic'toxicity

Page 14 of 64
Table 3 (con t.) Endpoints Relevant to Endocrine Disruption - HPG

FEMALES

Ovary weight

Adrenal weight

Uterine histopathology

Ovary histopathology

Adrenal histopathology

Study Type

Rabbit Dev

Rat 90 Day Oral

Rat 90 Day Inh

Dog 90 Day

Dogl yr

Dog lyr:

Ratlyr

Rat2yr

Rat2yr

Mouse 2 yr

Rat 90 Day Inh

Dog 90 Day

Dog 1 yr

Dog lyr:

Rat2yr

Mouse 2 yr

2-G Repro

2-G Repro

Rat 90 Day Oral

Dog 90 Day

Dog 1 yr

Dog lyr:

Rat 1 yr

Rat2yr

Rat2yr

Mouse 1 8 Mo

Mouse 2 yr

2-G Repro

2-G Repro

Rat 90 Day Oral

Dog 90 Day

Dog 1 yr

Dog lyr:

Rat 1 yr

Rat2yr

Rat2yr

Mouse 18 Mo

Mouse 2 yr

Rat 90 Day Oral

Dog 90 Day

Dog 1 yr

Dog lyr:

Rat 1 yr

Rat 2 yr

Rat2yr

Mouse 18 Mo

Mouse 2 yr

Observation

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Study Reference

Gilles 1987

Tisdel 1984

Scott 1985

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Goldenthal 1978

Scott 1985

Daly 1985

Dickie 1986

Sprague 1987

Dickie 1987

Goldenthal 1978

MacKenzie 1985

Minor 1982

Tisdel 1984

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1986

Goldenthal 1978

MacKenzie 1985

Minor etal. 1982

Tisdel 1984

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1986

Goldenthal 1978

Tisdel 1984

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1986

Goldenthal 1978

Page 15 of 64
Table 3 (cont.) Endpoints Relevant to Endocrine Disruption - HPG

FEMALES

Vaginal histopathology

Mammary histopathology

Serum cholesterol

AR binding

ER binding

Study Type

2-G Repro

2-GRepro

Rat 90 Day Oral

Dog 90 Day

Dog 1 yr

Dog lyr:

Ratl yr

Rat2yr

Rat2yr

Mouse 18 Mo

Mouse 2 yr

2-G Repro

Rat 90 Day Oral

Dog 90 Day

Dog 1 yr

Dog lyr:

Rat lyr

Rat2yr

Rat2yr

Mouse 18 Mo

Mouse 2 yr

Rat 90 Day Oral

Dog 90 Day

Dog 1 yr

Dog lyr:

Ratl yr

Rat2yr

Rat2yr

Not evaluated

Not evaluated

Observation

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Study Reference

MacKenzie 1985

Minor 1982

Tisdel 1984

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1986

Goldenthal 1978

Minor 1982

Tisdel 1984

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1986

Goldenthal 1978

Tisdel 1984

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Page 16 of 64
Table 4. Male Endpoints Relevant to Endocrine Disruption - HGP

Sexual behavior

Epididymal sperm count

Fertility Index

Mating index

Sex ratios of pups

Anogenital distance, AGO

Balano-preputial separation

Testes weight

Epididymis weight

Prostate weight

Seminal vesicle weight

Adrenal weight

Testes histopathology

Study Type

2-G Repro

2-GRepro

Observation

Normal

Normal

Not evaluated

2-G Repro

2-G Repro

2-G Repro

2-G Repro

2-G Repro

3-G Repro

Rat Dev

Rat Dev:

Rabbit Dev

Rabbit Dev

Rat DNT

DNT

DNT

2-GRepro

Rabbit Dev

Rat 90 Day Oral

Rat 90 Day Inh

Dog 90 Day

Dogl yr

Dog lyr:

Rat lyr

Rat2yr

Rat2yr

Mouse 2 yr

2-G Repro

Dog 90 Day

Rat lyr

Not evaluated

Not evaluated

Rat 90 Day Inh

Dog 90 Day

Dog lyr

Dog 1 yr

Rat2yr

Mouse 2 yr

2-G Repro

2-G Repro:

Rat 90 Day Oral

Dog 90 Day

Dog 1 Yr

Dog 1 Yr

Rat 1 Yr

Rat2Yr

Rat2Yr

Mouse 1 8 Mo

Mouse 2 Yr

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal .

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Study Reference

MacKenzie 1985

Minor 1982

MacKenzie 1985

Minor etal. 1982

MacKenzie 1985

Minor. 1982

MacKenzie 1985

Minor 1982

Nemec 1983

James 1985

Gillesl987.

James 1985

Lees 2004

Lees 2004

Lees 2004

Minor 1982

Gilles 1987

Tisdel 1984

Scott 1985

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Goldenthal 1978

Minor 1982

Daly 1985

Trutter 1978

Scott 1985

Daly 1985

Dickie 1986

Sprague 1987

Dickie 1987

Goldenthal 1978

MacKenzie 1985

Minor 1982

Tisdel 1984

Daly 1985

Dickie 1985

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1985

Goldenthal 1978

Page 17 of 64
Table 4 (cont.) Male Endpoints Relevant to Endocrine Disruption - HPG

MALES

Epididymal histopathology

Prostate histopathology

Seminal vesicle

Adrenal histopathology

Serum cholesterol

AR binding

ER binding

Study Type

2-G Repro

Rat 90 Day Oral

Dog 90 Day

Dog 1 Yr

Rat 1 Yr

Rat 2 Yr

Rat2Yr

Mouse 1 8 Mo

Mouse 2 Yr

2-G Repro

2-G Repro

Rat 90 Day Oral

Dog 90 Day

Dog 1 Yr

Dog 1 Yr

Rat 1 Yr

Rat 2 Yr

Rat 2 Yr

Mouse 18 Mo

Mouse 2 Yr

2-G Repro

2-G Repro

Rat 90 Day Oral

Dog 1 Yr

Rat 1 Yr

Rat2Yr

Rat 2 Yr

Mouse 1 8 Mo

Moused Yr

Rat 90 Day Oral

Dog 90 Day

Dog 1 Yr

Dog 1 Yr

Rat 1 Yr

Rat 2 Yr

Rat 2 Yr

Mouse 1 8 Mo

Mouse 2 Yr

Rat 90 Day Oral

Rat 90 Day Inn

Dog 90 Day

Dog 1 Yr

Dog 1 Yr

Rat2Yr

Rat2Yr

Observation

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Normal

Not evaluated

Not Evaluated

Study Reference

Minor 1982

Tisdel 1984

Daly 1985

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1986

Goldenthal 1978

MacKenzie 1985

Minor 1982

Tisdel 1984

Daly 1985

Dickie 1985

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1985

Goldenthal 1978

MacKenzie 1985

Minor 1982

Tisdel 1984

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1986

Goldenthal 1978

Tisdel 1984

Daly 1985

Dickie 1985

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987

Tisdel 1985

Goldenthal 1978

Tisdel 1984

Daly 1985

Dickie 1986

Sprague 1987

Trutter 1978

Warner 1983

Dickie 1987 '

Page 18 of 64
IV. ENDOCRINE-MEDIATED EFFECTS OF THE HGT AXIS

Thyroid Mediated Effects

Thyroid hormone, T3 and T4, synthesis and secretion in the thyroid gland is regulated by

a sensitive feedback mechanism that responds to the changes in the circulating levels of

the thyroid hormones. Thyroid Stimulating Hormone (TSH) is secreted by the anterior

pituitary gland and causes the thyroid to initiate new thyroid hormone synthesis.

Increases in iodine uptake, the iodination of thyroglobulin, and endocytosis and

proteolysis of colloid are all observed in response to TSH stimulation. If TSH is secreted

and thyroid function is increased, this will be evident in the thyroid histopathology.

Since thyroid hormones are cleared by the liver, increases in liver weight and altered

histopathology can sometimes be observed (Hill et. al, 1998).

Long-term disturbances in thyroid-pituitary hormonal feedback resulting in elevated TSH

leads to follicular thyroid hyperplasia, and subsequent thyroid follicular cell tumors

(adenomas, carcinomas). According to EPA (Hill et. al, 1998), "treatments of rodents

that cause thyroid-pituitary disruption result in chronic reduction in circulating thyroid

hormone levels, increase in TSH levels and the development of increased cell division,

increased size and numbers of thyroid cells, increased thyroid gland weight and, finally,

tumors of the thyroid. In some cases, there is also an increase in tumors of the pituitary

cells that produce TSH." This has also been recognized by OECD (2005, document

prepared under contract for EPA). It reads, 'Thyroid weight provides a relative measure

of its stimulation by TSH; thus, thyroid hormone levels are altered subtly, thyroid weight

may reflect a change. Thyroid histopathology may provide a more sensitive indicator of

this process." Rats are particularly sensitive to this process (Hill et. al., 1998, OECD,

2005).

OECD (2005) implies that if the biological change does not take place, the alteration of

T3, T4 or TSH has little biological relevance. As noted above, T3 and T4 levels are

delicately regulated and the small persistent changes that result in disruption of thyroid

hormone homeostasis leads to alterations in colloid area and follicular cell height. These

will be readily evident in any histopathological analysis.

The classic thyroid inhibitors, ETU, PTU and chlorate, have been extensively studied. The

effects on thyroid morphology are immediate and effects resulting from perinatal and adult

exposure are similar.

In the case of sodium chlorate (NTP 2005), exposed animals showed incidences of minimal

to mild thyroid follicular cell hypertrophy in as few as three weeks. Thyroid gland follicular

cell carcinoma in male rats and thyroid gland follicular cell adenoma or carcinoma

(combined) was observed in males and females after two years. Evaluation hi the pubertal

development assay also implies that thyroid weight and adverse histopathology will be

measurable after 20 days of exposure.

Page 19 of 64
It is unlikely that pups would be sensitive but adults refractory to thyroid disruption.

When PTU is given to dams of two rat strains from gestation day (GD) 6 through

postnatal day (PD) 10, a clear dose response relationship for thyroid hormones with

decreased T3 and T4 and increased TSH levels for dams (GD 20, PD 21), fetuses (GD

20) and pups (PD 4 & 21) is observed (Schneider et. al., 2009). At these time points,

dams, fetuses arid pups of both rat strains demonstrated comparable T3 and T4 inhibition

and TSH stimulation as well as comparable signs of diffuse hypertrophy and hyperplasia

of the thyroid gland follicular cells of dose-dependent severity.

This comparability is also borne out in longer-term studies. NTP (1992) evaluated the

role of perinatal ETU exposure by comparing the effects of three different dosage

regimens: perinatal only, combined perinatal and adult, and adult only in long-term

studies in rats and mice. It is clear from the data that there is no increased sensitivity to

thyroid hormone disruption of young rats or mice to early ETU exposure. Again, thyroid

disruption in the pups is also manifest in the adults.

Thus, histopathological changes as the result of pituitary-thyroid homeostasis disruption

appear early and progress in severity with long-term hormone disruption. Because of the

high degree of conservation of the thyroid system both across vertebrates and age of

organisms (OECD 2005), it is unlikely that a careful evaluation of the adult animals for

either gross pathology (including weights) or histopathology will miss any potential

thyroid inhibitor in either adult, juvenile or neonatal animals (mammals, birds, fish or

amphibians).

Lack of EPTC Thyroid Mediated Effects

As discussed above, minor alterations in thyroid function are sensitively manifested in

histopathology of the thyroid follicular cells. This effect was not observed in any

EPTC study. Likewise, no changes hi thyroid weights were present in the rat, dog or

mouse studies of any duration. The studies evaluating thyroid weight and

histopathology were shown in Table 1 and Table 2.

In some cases concomitant increases in liver weights are induced by chemicals that

alter thyroid function. EPTC treatment did not affect liver weights in any of the studies

performed. Histopathological evaluation of the liver tissues revealed no

histopathological effect. Similarly, the pituitary, part of the HPT axis, is not affected

by EPTC exposure.

Since the thyroid controls body temperature regulation, body temperature measures are

relevant. Temperature measurements (recorded at pre-test, 3, 6, 9 and 12 months)xin the

1-year dog study also revealed no changes following EPTC exposures to up to 60

mg/kg (Sprague and Taylor, 1987).

Page 20 of 64
The lack of thyroid weight changes, histopathology, liver weights or body temperature in.

the rat and dog intermediate- and long-term studies is a clear indication that EPTC does

not interact with the thyroid.

V. EDSP TIER I BATTERY VS EPTC DATABASE

Table 5 delineates the Guideline studies required in the Tier 1 testing battery along with

the major endocrine endpoints that are measured. For each of the Guideline tests in the

Tier 1 battery, the direct or functional equivalency of the EPTC data exists to inform the

Agency on the endpoints being evaluated.

Page 21 of 64
Table 5. Comparison of Available Data to EDSP Tier I Endpoints

Guideline

890.1100

890.1350

890.1150

890.1200

890.1550

890.1250

890.1300

Study

Amphibian

Metamorphosis (Frog)

Fish Short-Term

Reproduction

Androgen Receptor

Binding (Rat Prostate

Cytosol)

Aromatase (Human

Recombinant)

Steroidogenesis

(Human Cell Line -

H295R)

Estrogen Receptor

Binding

Estrogen Receptor

Transcriptional

Activation (Human

Cell Line HeLa-9903)

Route

in vivo

in vivo

in vitro

in vitro

in vitro

in vitro

in vitro

Major endocrine endpoints

Hind limb length, snout-vent length,

developmental stage, thyroid histology

Gonadal-somatic index, gonadal histo,

male secondary sex characteristics,

plasma VTG, plasma sex steroids

Potential to compete with

 3H ligand for

binding rat ventral prostate tissue

homogenate

Conversion of androgen to estrogen in

microsomes

Compares ability to elicit production of

testosterone or estradiol to positive

controls (forskolin and prochloraz)

Ability to bind with estrogen receptors

isolated from rat uterus

Ability to induce reporter gene product

following binding to a specific receptor

and subsequent transcriptional

activation

Available EPTC data

informing endpoint

No thyroid weight effects

No thyroid histopathology effects

No other thyroid related effects

No developmental effects

No reproductive effects in

bird, rat or rabbit studies

No effect on sex organ weight or

histopathology

No thyroid effects

No effect on sex organ weight or

histopathology

No developmental effects

No reproductive effects

No effect on AGD or PPS

No changes in cholesterol,

No imbalance of estrogen or

androgen per organ

weight or histopathology

No effect on cholesterol

No imbalance of estrogen or

androgen per organ

weight/histopathology

No effect on reproductive organs

No effect on sex organ weight or

histopathology

No effect on AGD, VO, PPS or

estrous cyclicity

No effect on sex organ weight or

histopathology

No developmental effects

No reproductive effects

No effect on AGD, VO, PPS or

estrous cyclicity

Page 22 of 64
Table 5 cont. Comparison of Available Data to EDSP Tier I Endpoints

Guideline

890.1400

890.1450

890.1500

890.1600

Study

Hershberger (Rat)

Female Pubertal (Rat)

Male Pubertal (Rat)

Uterotrophic

Route

in vivo

in vivo

in vivo

in vivo

Major endocrine endpoints

Weight changes in 5 androgendependent tissues in castrateperipubertal male: ventral prostate,

seminal -vesicle, LABC muscle,

Cowper's gland and glans penis

Growth, age/wt at vaginal opening,

organ wts, histology, serum thyroxine

and TSH, estrogen cyclicity, standard

blood panel

Growth, age/wt at preputial separation,

organ wts, histology, serum thyroxine,

TSH and testosterone, standard blood

panel

Uterine weight in ovarectomized rats

and mice

Available EPTC data

informing endpoint

No effect on sex organ weight or

histopathology

No developmental effects

No reproductive effects

No effect in F] males

No AGO or PPS effect

No effect on sex organ weight or

histopathology

No reproductive effects

No effect in F, females

No developmental effects

No effect on thyroid

No AGO or VO effect

No estrous cyclicity effect

No effect on sex organ weight or

histopathology

No reproductive effects

No effect in FI males

No developmental effects

No effect on thyroid

No AGO or PPS effect

No effect on sex organ weight or

histopathology

No developmental effects

No reproductive effects

No effect in FI females

No AGD or VO effect

No estrous cyclicity effect

EPTC clearly has no endocrine disrupting potential as evidenced by the reproduction,

developmental, intermediate-term and long-term studies. The most powerful indication

of endocrine disruption is the histopathology of endocrine-related organs and EPTC is

without effect.

Screening Endpoints Not Available in EPTC Database

Amphibian studies have not been performed. Since no thyroid histopathological effects

were observed, and since this is a very sensitive indicator of T3, T4 or TSH disruption, it

is not necessary to measure thyroid hormone levels or frog malformations caused by

Page 23 of 64
hormone alteration. Fish studies to predict reproductive outcomes in mammals, when the

mammalian reproductive studies have been performed, are not necessary.

Biochemical measures of estrogen and androgen binding have not been directly

performed. Again, in the absence of any biological indication of estrogen or androgen

disruption, the measure of these biochemical endpoints will be of little value. The

definitive in vivo studies have been performed. In a novel study, Fox (2004) evaluated a

number of pesticides, including EPTC, in an assay using nodulation D (NodD) protein

receptors from Rhizobium. Recent data have shown that many of the same synthetic and

natural environmental chemicals that disrupt endocrine signaling in vertebrates also

disrupt phytoestrogen NodD receptor signaling in soil bacteria. EPTC showed no activity

on nod expression.

The Hershberger, uterotropic, pubertals have not been performed using EPTC. However,

for these in vivo studies, clear functional equivalency exists. The data from the longer

exposure studies, i.e., 2-generation reproduction studies, provides a much richer database

than the short-term Tier 1 studies. Evaluation of FI males and females dosed immediately

post weaning in the regulatory reproduction studies matches the dosing regimen of the

Hershberger, uterotropic, male and female pubertals. Since these animals are dosed for a

longer period (up to 230 days in one 2-generation reproduction study), any effects will be

magnified. The histopathology of the sex organs and sex accessory organs, as well has

other endocrine-related organs, showed normal histomorphology. This measure of

endocrine damage is superior to that of sex organ weights after only a few weeks of

exposure and pup growth. Measures of AGD, VO, PPS and estrous cycling showed no

alterations in FI animals (Lees, 2004).

Gowan Company believes that functional equivalency exists for all the assays in the Tier

1 battery.

VI. SUMMARY AND CONCLUSIONS

The EPTC database represents a full assessment of any potential endocrine adverse effect

that might be identified in the EDSP Tier I battery. These studies allow for dose-response

assessment and subsequent human risk assessment not possible with the Tier I screen and

thus, represent a higher tier in the EDSP testing program. '

The relevant regulatory studies include reproduction studies in rats and birds, multiple

developmental studies in rats, rabbits and long-term studies in rats, mice and dogs. The

diversity of tests and tested species reflects the redundancy sought in the EDSP assays.

Altogether, thousands of animals in multiple species have been evaluated for effects on

relevant organ weights, gross pathology and histopathology. No effects were observed in

either sex to sexual organs or to any endocrine gland (adrenal, pituitary, thyroid). No

effects were found that would indicate endocrine-mediated impairment of reproduction

function or alteration in developmental progress. The absence of any effect on serum

cholesterol in the whole-animal regulatory studies confirms that EPTC does not cause

perturbation of steroidogenesis.

Page 24 of 64
No evidence exists to show EPTC has any hypothalamic-pituitary-gonadal (HPG) and

hypothalamic-pituitary-thyroid (HPT) activity. The EPTC database is comprehensive and

sufficient for risk assessment purposes.

Histopathological effects in nerve and muscle, primarily heart, defines the critical

regulatory NOAELs at levels significantly lower than any other adverse effects. No

biologically relevant effects related to endocrine disruption have been identified

following maximally tolerated doses of EPTC. Additional screening for potential

endocrine disruption would not result in either lower NOELs or a different risk

assessment analysis. Therefore, additional testing for potential endocrine effects will

have no impact on the risk assessment analysis for EPTC.

Go wan Company believes that collectively, the available data fulfill the requirements of

the Endocrine Disruptor Screening Program Data Call-in and that the overwhelming

weight-of-the-evidence shows that EPTC is not anticipated to produce any effect in

humans similar to an effect produced by a naturally occurring estrogen or androgen.

Therefore, EPTC meets the standard for an exemption under FFDCA section 408(p)(4).

Page 25 of 64
VII. REFERENCES

Literature Cited '

Abbott, D.H., Padmanabhan, V., and Dumesic, D. (2006) Contributions of androgen and

estrogen to fetal programming of ovarian dysfunction. Repro. Biol. Endocrinal. 4:17.

Published online. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459165/

Atanassove, N., McKinnell, C., Turner, K.J., Walker, M., Fisher, J.S., Morley, M.,

Millar, M.R., Groome, N.P., and Sharpe, R.M. (2000) Comparative effects of

neonatal exposure of male rats to potent and weak (environmental) estrogens on

spermatogenesis at puberty and the relationship to adult testis size and fertility.

Evidence for stimulatory effects of low estrogen levels. Endocrinology 14: 3898-

3907.

Cho, H.W., Nie, R., Carnes, K., Zhou, Q., Sharief, N.A.Q., and Hess, R.A. (2003) The

antiestrogen ICI 182,780 induces early effects on the adult male mouse reproductive

tract and long-term decreased fertility without testicular atrophy. Reprod Biol

Endocrinol 1:57.

Dussaudt, J H., and Rujel. J.I.,(1987). Thyroid hormones and brain development. Ann.

Rev. Physiol. 49:321-334.

Fox, J. (2004) Chemical Communication Threatened by Endocrine-Disrupting

Chemicals. Environ Heal Persp 112:648-653.

Garcia-Florez, Oliveira, C.A., and Carvalho, H.F. (2005) Early effects of estrogen on the

rat ventral prostate. Brazilian J Medical and Biological Research 38: 487-497.

Gray, Jr, L.E., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., Goldman, J.,

Slott, V., and Laskey, J. (1988) A Dose-Response Analysis of Methoxychlor-Induced

Alterations of Reproductive Development and Function in the Rat Fundam. Appl.

Toxicol. 12, 92-108.

Grokett, B.H., Ahmad, and Warren, D.W. (1992) The effects of an anabolic steroid

(oxandrolone) on reproductive development in the male rat. Acta Endocrinologica,

126: 173-178.

Hasegawa, R., Koizumi, M., Hirose, A., and Mecana, A. (2000) Estrogenic activity and

reproductive organ toxicity of alkylphenols. J Food Chem.

Hess, R.A., Lee, K.H., Bahr, J., Tayor, J.A., Korach, K.S., and Lubahn, D.B. (2001)

Estrogens and epididymal function. Reprod Fertil Dev 13: 273-283.

Hill, R.N., Hurley, P.M., Crisp, T., Rosenthal, S.L., Singh, D.V., Hard, G., and Wood,

W.P., Assessment of Thyroid Follicular Cell Tumors. (1998) EPA/630/R-97/002.

Page 26 of 64
Moore, R.W., Rudy, T.A., Lin, T.M., Ko, K., and Peterson, R.E. (2001) Abnormalities of

sexual development in male rats with in utero and lactational exposure to the

antiandrogenic plasticizer Di(2-ethylhexyl) phthalate. Environ Health Perspect.

March; 109(3): 229-237.

National Toxicology Program (NTP). (\992)NTP Technical Report on the Perinatal

Toxicity and Carcinogenicity Studies ofEthylene thiourea in F/344 Rats and B6C3F]

Mice. NTP Technical Report 388. U.S. Department of Health and Human Services,

Public Health Services, National Institutes of Health, Bethesda, MD.

National Toxicology Program (NTP). (2005) NTP Technical Report on the Toxicology

and Carcinogenesis Studies of Sodium Chlorate in F/344 Rats andB6C3Fi Mice

(drinking water studies). NTP Technical Report 517. U.S. Department of Health and

Human Services, Public Health Services, National Institutes of Health, Bethesda,

MD.

OECD (2005) Draft Detailed Review Paper On Thyroid Hormone Disruption Assays.

http://www.epa.gov/endo/pubs/edmvac/drp_thyroid 03 _31 _05.pdf

Oliveira, C.A., Nie, R., Games, K., Franca, L.R., Prins, G.S., Saunders, P.T.K., and Hess,

R.A. (2003) The antiestrogen ICI 182,780 decreases the expression of estrogen

receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in

rat efferent ductules. Repro Bio Endocrin 1:75.

Reif, D., Martin, M., Tan, S., Houck, K., Judson, R., Richard, A., Knudson, T., Dix, D.,

and Kavlock, R. (2009) Endocrine Profiling and Prioritization of Environmental

Chemical Using ToxCast TM CPCP Dec 17,2009 .

http://www.epa.gov/NCCT/bosc review/2009/files/CompTox BOSC 09.pdf

Sawaki, M., Noda, S., Muroi, T. Mitoma, H., Takakura, S., Sakamoto, S., and Yamasaki,

K. (2003) In utero through lactational exposure to ethinyl estradiol induced cleft

phallus and delayed ovarian dysfunction in the offspring. Toxicol Sci 75: 4042-411.

Schneider, S.S., Buessen, R., Kaufmann, W., Strauss, V., van Ravenzway, B.,

Hastings, C, and Hess, F. (2009) Comparative Developmental Thyroid Studies with 6-

Propyl-2-thiouracil (PTU) Using Two Rat Strains (Wistar versus Sprague-Dawley) -

Results of Treatment-related Effects in Thyroid Hormones and Thyroid Morphology.

The Toxicologist 108: Abstract #164.

Terry, K.K., Cappon, G.D., Hurtt, M.E., Tassinari, M.S., and Gupta, U. (2004)

Reproductive toxicity assessment of lasofoxifene, a selective estrogen receptor

modulator (SERM) in female rats. Birth Defects Res B Dev Reprod Toxicol 71: 150-

160.

Tropsha, A. (2009) Prediction of animal toxicity endpoints ToxCast Phase I compounds

Page 27 of 64
using a combination of chemical and biological in vitro descriptors.

http://www.eDa.gov/NCCT/toxcast/files/summit/6%20Tropsha%20ToxCast%20TDAS.pdf

U.S. EPA (1999) Reregistration Eligibility Decision (RED) EPIC (S-Ethyl

dipropylthiocarbamate) Case #604) EPA 738-R-99-006. December 1999.

US EPA (1998c) Endocrine Disruptor Screening Program: Statement of Policy: Notice

(FR December 28, 1998 pp 71542 - 71568)

Vanage, G.R., Dao, B., Li, X-J, Bardin, C.W., and Koide, S.S. (1997) Effects of

Anordiol, an antiestrogen, on the reproductive organs of the male rat. Systems Biol

inReproMed38: 13-21.

Studies Cited

Daly, I. (1985) A Three Month Subchronic Oral Dietary Toxicity Study of EPIC in

Beagle Dogs. Bio/Dynamics Report No. 83-2781. MRID 00150327.

Dickie, B.C. (1986) One-Year Oral Feeding Study of the Chronic Toxicity of EPTC in

Dogs. Hazleton Laboratories America, Inc. Report No. 6100-109 MRID 00161595:

Dickie, B.C. (1987) Two-Year Oral Feeding Study of the Oncogenicity and Chronic

Toxicity of EPTC in Rats. Hazelton Laboratories America, Inc. Report No. HLA

6100-106. MRID 40215001.

Gilles, P.A., (1987) Teratology Study in Rabbits with Eptam Technical. Stauffer

Chemical Company Report No. T-12982. MRID 40442302.

Goldenthal, E. (1978) Lifetime Oral Study in Mice. International Research and

Development Corporation. Report No. 153-011. MRID: 00022100

James, P., Smith, J., and John, D. (1985) Effect of EPTC on Pregnancy of the Rat.

Huntingdon Research Centre, Ltd. Report No. PPG 19/851002. MRID 00161598.

James, P., Smith, J., Masters, R., and Offer, J. (1985) Effect of EPTC on Pregnancy of

the Rabbit. Huntingdon Research Centre, Ltd. Report No. PPG 14 & 18/85601.

MRID 00161599.

Lees, D. (2004) Developmental Neurotoxicity Study in Rats. Central Toxicology

Laboratory Report No.: CTL/RR0926/Regulatory/Report. MRIDs 46319101.

MacKenzie, K., Two-Generation Reproduction Study with EPTC in Rats. (1985)

Hazelton Laboratories, Inc. Report No. 6100-108. MRID 00161597.

Minor, J.R., Downs, J., Zwicker, G. (1982) A Two-Generation Rat Reproduction Study

with Eptam Technical. Stauffer Chemical Company Report No. T-10123. MRID

00121284 (40420408 supplement).

Page 28 of 64
Nemec, M. (1983) A Teratology Study in Rats with EPTAM. Wil Research Laboratories,

Inc. Report No. WIL-27013. MRID 00138919.

Scott, J. (1985) Subchronic Inhalation Toxicity of Eptam in Rats. Stauffer Chemical

Company Report No. T-10422 MRID 00154784.

Sprague, G.L. and Taylor D.O.N (1987) One-Year Oral Toxicity Study with Eptam

Technical in Dogs. Stauffer Chemical Company Report No. T-12723. MRID

40442301.

Temple, D., Martin K., Beavers, K. and Jaber, M. (2005) EPTC: A Reproduction Study

with the Mallard. Wildlife International, Ltd. Project Number: 334/112. MRID

46554301.

Temple, D., Martin K., Beavers, K. and Jaber, M. (2005) EPTC: A Reproduction Study

with the Northern Bobwhite. Wildlife International, Ltd. Project Number: 334/111.

MRID: 46554302.

Tinston, D.J, (1995) Subchronic Neurotoxicity Study in Rats. Zeneca Central Toxicology

Laboratory. Report No. PRO929. MRIDs 43230901 (supplements 43964401,

4398301).

Tisdel, M. (1984) Thirteen-Week Subchronic Study in Rats with EPTC. Hazelton

Laboratories America, Inc. Report No. 6100-105. MRID 00144651.

Tisdel, (1986) Oncogenicity Study in Mice with EPTC. Hazelton Laboratories America,

Inc. Report No. 6100-104. MRID 00161596.

Trutter, J. (1978) 54-Week Feeding Study in Rats: Eptam Technical. Hazleton

Laboratories America, Inc. Report No.: 132-131. MRID 00022101.

Warner, M. and Goldenthal (1983) Two Year Oral Toxicity/Oncogenicity Study in Rats.

International Research and Development Corporation. Report No.: 153-100. MRIDs

00145004, addendum 00146311.

Page 29 of 64
VIII. EPTC STUDIES - SUPPLEMENTAL STUDY DETAIL

The EPTC registration studies consist of two studies for most registration requirements

since there were two registrants, Stauffer and PPG, at the time of initial data

development. All relevant studies to endocrine disruption are discussed. This is not

intended to be a full study review but only those facets of the study that impact an

assessment of EPTC on the endocrine system.

REPRODUCTION AND DEVELOPMENTAL STUDIES

Two rat reproduction studies and four developmental (rat and rabbit) studies have been

performed with EPTC that have direct relevance on the in vivo effect of endocrine

disruption in mammals and represent a higher tier test than the EDSP Tier I screening

tests.

Two-generation Reproduction Study - Mackenzie 1985 - MRID 00161597

EPTC was administered to 30 male and female immature rats at dietary concentrations of

0, 50, 200 or 800 ppm. FI animals were administered EPTC for 10 weeks post weaning.

At week 11, selected FI animals (5/sex/group) were sacrificed and necropsied in addition

to those sacrificed at the termination of the study. The breeding program was repeated

, on the remaining animals (25/sex/group) to produce an Faa litter.

Systemic Toxicity: For PO and FI adult males and PO and FI adult females, body weights

were significantly reduced at 800 ppm and in FO males and PO females and in FI males at

200ppm. • '

Body weight was decreased approximately 10% (17% body weight gain) in the PO males

at 800 ppm and 9% body weight gain decrements at 200 ppm. Mean weights were

reduced 12% for the 800 ppm PO females with significant body weight gain loss at

200ppm. Similarly, the F; males and females displayed mean body weight loss at 800

ppm during the growth phase and in the reproductive phase (females). FI males showed

significant weight gain decrements at 200 ppm.

Reproductive parameters

Mating indices: Mating indices were unaffected for either males or females in the PO for

FI generation.

Estrous cvclicity: The stage of estrous was determined for all dams in the PO an FI

generations. Time to mating in all groups for all matings were comparable to control

mating pairs.

Fertility indices: Fertility indices were unaffected for either males or females in the PO

for FI generation in either the first or second mating.

Gestation indices: Unaffected in any dam.

Page 3 Oof 64
Gestational length: Comparable in all groups for all pregnant dams.

Parturition: No observations of difficult deliveries or abnormal behavior were noted.

Behavioral: No behavioral abnormalities were noted in any group.

Offspring

Litter size: Mean litter size was not significantly affected by ETPC ingestion.

Pup Weights: Mean pup weight significantly reduced at 800 ppm.

Live birth: No effect on number of pups born alive. Number of pups found dead, though

not statistically significant, were 2-fold higher than controls at 800 ppm in the FI litter.

Viability Index: No effect

Survival index: No effect: (weaning index)

Sex ratio: No effects

Parents

Organ Weight: No organ weights were taken.

Macroscopic: All macroscopic observations for the FO, Fja weanlings and Fja adults

were similar to controls.

Histopathology FI adults: Histopathology evaluated the heart, liver and kidney and sex

organs of both males and females consisting of testes, seminal vesicle, prostate, ovaries,

uterus, and vagina. Histopathology was not performed on the PO animals.

The only histopathological finding was degenerative cardiomyopathy observed in both

males and females at 800 ppm.

NOEL: Parental 50 ppm (2.5 mg/kg)

NOEL: Reproductive 800 ppm (40 mg/kg)

NOEL: Developmental 200 ppm (10 mg/kg)

2-Generation Reproduction - Minor, Downs and Zwicker 1982 - MRID 00121284,

supplement 40420408

EPTC was administered, to Sprague Dawley 15 male and 30 female rats at concentrations

of 0, 40, 200 or 1000 ppm from 40 days of age with matings occurring at day 130 and

Page 31 of 64
again at day 186, This study is a two litter per generation design (evaluates two litters

from the PO parents and two litters from the Fib parents).

Pups from the second litter (Fib) were administered EPTC from weaning and continued

through termination at 230 days with matings at 100 and 155 days of age.

Systemic toxicitv: For PO and FI adult males and PO and FI adult females, body weights

were significantly reduced at 1000 ppm. Body weight was decreased approximately 10%

in the PO males and between 14 - 17% in the FI males. Mean weights for the 1000 ppm

females at time of first mating were reduced 13% for both PO and FI females. No effects

on any group were noted at doses of 40 or 200 ppm.

Reproductive parameters

Mating indices: Mating indices were unaffected for either males or females in the PO for

FI generation in either the first or second mating.

Estrous cyclicity: The stage of estrous was determined for all dams. Time to mating in

all groups for all matings were comparable to control mating pairs.

Fertility indices: Fertility indices were unaffected for either males or females in the PO

for FI generation in either the first or second mating.

Gestation indices: Unaffected in any dam.

Gestational length: Comparable in all groups for all pregnant dams.

Parturition: The mean delivery length was comparable between all groups. No

observations of difficult deliveries or abnormal behavior were noted.

Retrieval time: Mean retrieval times of the dams following delivery were normal.

Offspring

Litter size: Mean litter size was not significantly affected by ETPC ingestion. At 1000

ppm the litter sizes were consistently, but not significantly, lower than control. This

effect was secondary to the reduced body weights of the dams at the high dose.

Litter weights: Mean litter weights were affected at the high dose but were the result of

maternal toxicity.

Live birth: No effects were observed in any litter.

Viability Index: No effect

Lactation index: No effect

Page 32 of 64
Survival index: No effect:

Sex ratio: No effects

Developmental landmarks: Milk in stomach, detached pinna, inscisor eruption and eye

opening were unaffected in any litter.

Parents

Organ Weight: Liver, kidney, heart, brain and testes with epididymides were weighed in

the PO and Fl adults. There were decreases in absolute weight of the liver, kidney, heart

or brain (male only) in one or both generations in both males and females however,

relative weights were normal indicating the findings were either incidental or

consequential to the reduction in body weights at this dose. Testes weights

were not affected in either generation. Female gonads were not weighed.

Histopathology: Histopathology evaluated the heart, liver and kidney and nearly all sex

organs of both males and females. These consisted of the testes, epididymides, spermatic

cord, seminal vesicle, coagulating gland, prostate, urethra, bulbo-urethral gland, ovaries

and oviduct, uterine horns, cervix, and vagina.

All reproductive organs were normal in both PO, and FI males and females. The only

histopathological finding was increased severity of myocardial degeneration in all dose

groups.

Although the protocol and methods indicated that testes with epididymides and ovaries

from weanlings were examined histopathologically, we could find no data in the report or

addendum.

Gross Necropsy: No growth-related morphological changes were observed in any of the

weanlings from any litter. The DER, however, cites an increase in anomalous pups at

1000 ppm after a recalculation of pups with multiple anomalies was corrected from the

report.

NOEL: Parental systemic effects was <40 ppm (2 mg/kg) - cardiomyopathy

NOEL: Reproductive effects was >1000 ppm (50 mg/kg).

NOEL: Developmental 200 ppm (10 mg/kg) - pup weight PND14-21 decrease

Rabbit Developmental — Gilles 1987 - MRID 40492302

Gravid female rabbits were gavage administered 0, 5, 40 and 300 mg/kg/day in corn oil.

Animals were treated for 13 consecutive days from gravid day 7 to 19 (Gilles 1987).

Systemic Toxicitv: Maternal toxicity was observed at 300 mg/kg consisting of body

weight loss, reduced food consumption and signs of cholinesterase inhibition (RBC

inhibited -70%).

Page 33 of 64
Fertility of the dams was unaffected. Total corpora lutea and corpora lutea/dam were

unaffected as were total implantations and implantations/dam. There were no effects on

either early mid or late intrauterine resorptions. Pre and post-implantation loss were

similar levels in all groups. The number of live fetuses was similar in all groups. There

was no effect on sex ratio.

Embryofetal toxicity secondary to maternal toxicity occurred at 300 mg/kg and consisted

of reduced mean fetal body weight but was not biologically or statistically significant.

Fetuses showed no external, soft-tissue or skeletal anomalies at any treatment level. The

sex ratio was unaffected.

Organ Weight: Liver, ovaries, placentas, reproductive tract, uterus were not affected

NOELs: Maternal cholinesterase NOEL <5 mg/kg (decreased serum cholinesterase)

Maternal Systemic NOEL 40 mg/kg

Developmental NOEL >3 00 mg/kg

Rabbit Developmental - James, Smith, Masters, and Offer 1985 - MRID 00161599

EPTC was administered by gavage to rabbits at daily doses of 0, 30,100, and 300 mg/kg

from day 6 go 18 of gestation (James et .al. 1985).

Systemic Toxicity: There was an increase in transitory non-specific signs of toxicity.

Body weight gain was increased in dams given 300 mg/kg EPTC along with increased

food consumption.

Reproduction/Litter Parameters: EPTC has no effect on pregnancy rate either in the

rangefinder (high dose 360) or the definitive at 300 mg/kg. There were no effects of litter

size, pre- or post-implantation loss or resorptions. The mean number of corpora lutea

was similar in all dose groups.

Offspring: Mean litter and fetal weight showed no differences between treatment groups.

Sex ratios were unaffected.

There was no incidence of either visceral anomalies or skeletal variations.

Rat Developmental ~ Nemec 1983 - MRID 00138919

Gravid CD rats were gavaged administered 0, 30, 100, and 300 mg/kg/day from gestation

days 6 through 15 (Nemec, 1983).

Systemic Toxicity: Mortality (60%) was observed at the high dose of 300 mg/kg. Mean

body weights losses and decreased body weight gains occurred throughout the gestation

period in the 300 mg/kg dosage group only. Gravid uterine weights at 100 and 30 mg/kg

were comparable to controls.

Page 34 of 64
Reproduction Parameters: Fertility of the dams was unaffected at any dose. Total corpora

lutea and corpora lutea/dam were unaffected as were implantations/dam at 30 or 100

mg/kg. Post-implantation loss was increased in the 300 mg/kg group but was unaffected

at 100 mg/kg or 30 mg/kg EPTC. As a result the mean number of viable fetuses was

decreased. Fetal sex ratios were similar in all groups.

Offspring: Embryotoxicity was present at 300 mg/kg as evidenced by increase in early

litter resorption and a decrease in the number of viable fetuses. Mean fetal body weight

was also reduced. Fetal evaluations of the high dose group were unsuitable for evaluation

of teratogenicity. Fetuses showed no external, soft-tissue or skeletal anomalies at any

treatment level. The sex ratio was unaffected.

NOELs: The NOEL for systemic toxicity was 100 mg/kg. The NOEL for

embryotoxicity was 100 mg/kg.

Rat Developmental - James, Smith, and John 1985 - MRIDs 00161598

Gravid CD rats were gavaged administered 0, 30, 100, and 300 mg/kg/day from gestation

days 6 through 15 (James et. al 1985). A preliminary study was also reported at dosage

levels of 0, 160, 240, and 360 mg/kg/day.

Systemic Toxicitv: In the preliminary study, a retardation of weight gain was observed

throughout the treatment period. In the definitive study, body weight gain was decreased

at 300 mg/kg/day.

Reproductive parameters: The number of corpora lutea were determined by dissection as

well as both placental and embryonic remnants visible at termination. No effects were

observed in either the preliminary study or in the definitive in pregnancy rates or preimplantation loss.

In the preliminary study, mean values for litter size, mean litter and fetal weight were

similar in all groups. In the definitive, mean embryonic death was marginally higher at

360 mg/kg. Results were similar in the definitive study, litter size was marginally

reduced at 300 mg/kg. Mean fetal weight was reduced at 100 and 300 mg/kg/day but was

not statistically significant. Sex ratios were unaffected by treatment.

There was no incidence of either visceral anomalies or skeletal variations at doses below

300 mg/kg.

Rat Developmental Neurotoxicity -Lees2004-- MRIDs 46319101,

EPTC was evaluated in pregnant Wistar rats at 0,100, 300 or 1000 ppm in the diet from

day 7 of gestation through lactation day 23 postpartum. Pups were weaned on day 29 and

allowed to grow to adulthood. Neurotoxicity evaluations were performed.

Page 35 of 64
Systemic toxicity: At 1000 ppm EPTC there were lower bodyweights at days 14 and 22

of gestation and lover food consumption throughout gestation dosing and post partum.

Pup body weight was lower in males and females at day 1 postpartum at 1000 ppm with

no effects at lower dose levels.

Reproduction Parameters: There were no treatment-related effects on gestation length.

The proportion of live born pups was lower at 1000 ppm EPTC compared to control due

to two litters that suffered litter losses. The proportion of pups born live at 1000 ppm was

96.6% but was unaffected when the 2 late litter losses were excluded from the

assessment. There were no treatment-related effects in animals in the 100 or 300 ppm

treatment groups.

No effects were observed for gestation length or parturition.

Litter sizes were not affected at birth but were reduced at 1000 ppm from Day 11 to 5.

Pup survival was also not affected at birth but was decreases from Day 1 to'5. Pup body

weight was decreased at 1000 but total litter weight was not affected. Sex ratios were

unaffected.

Developmental Landmarks: Fl females were examined daily from day 29 to determine

the day of age of vaginal opening. F2 males were examined from day 41 to determine the

age of preputial separation. Both vaginal opening and preputial separation were normal.

Organ Weights: Only the brain weights were measured.

Histopathology: The focus'of this study was on brain and neural tissues. No other

organs were evaluated.

INTERMEDIATE-TERM STUDIES

90-Day Oral Rat ~ Tisdel, M., 1983 - MRID 00144651

Dose levels of 0, 18, 36, 72, and 120 mg/kg phosmet were administered in the diet to CD

rats for the first 6 weeks of the study and the mid and low doses were reduced to 3 and 15

mg/kg for the remained of the study.

Clinical: There was a dose-related decrease in body weights and/or weight gains in all

treatment groups but were much less at the 18/3 mg/kg dose (not statistically significant).

Mean body weight was 525 g in control males compared to 428 g in the high dose group)

Cholesterol: Cholesterol was measured pre-study and at termination. No effects were

observed that were related to treatment.

Organ weights: Endocrine relevant organ weights for liver, kidney, testes (right and left)

and ovary (right and left) were taken. Absolute weights of all organs, with the exception

Page 36 of 64
of the heart, were all similar to control animals. Any increases in the relative weights

were not considered to be biologically significant due to the large body weight losses.

The US EPA DER found only relative heart weight to be biologically significant.

Histopathology: Adrenal pituitary thyroid, parathyroid, prostate, seminal vesicle, testes,

epididymides, ovary, uterus, vagina, cervix, and mammary gland were all examine

macroscopically and microscopically. All tissues were normal.

The finding of cardiomyopathy (myocardial degeneration) in both sexes was observed in

all dose groups but showed dose-related severity.

NOEL: The NOEL was 3 mg/kg based on body weight loss and findings of

cardiomyopathy at 32/15 mg/kg.

90 Day Inhalation - Scott, 1985 - MRID 00154784

In this 90 day inhalation study, rats were exposed to 0, 8.3, 58 and 290 mg/m3 EPTC for

13 weeks. There were no changes in body weight.

Systemic Toxicity: Body weights were unaffected but food consumption was reduced in

the 58 and 290 mg/ m3 exposure groups. Some clinical chemistry parameters were also

affected at 58 mg/ m3

Brain cholinesterase was significantly inhibited at 290 mg/m3 with minor reductions at

the 8.3 mg/m3 dose level.

Cholesterol: Total cholesterol was measured and found to be within normal ranges for all

groups.

Organ Weights: Of interest, organs weighed were liver, kidney, adrenals and gonads

(testes, ovary). No weights were affected.

Histopathologv: Examination was limited to nasal passages, liver, kidney and target

tissue. The major finding was myocardial degeneration at 290 mg/m3.

NOEL: The NOEL was 8.3 mg/ m3 defined clinical signs and decreased food

consumption.

90 Day Neurotoxicity - Tinston 1995 - MRIDs 42921901 (supplements 43964401,

4398301)

Rat were fed diets containing 0, 100, 500 or 2500 ppm EPTC for 13 weeks and evaluated

for neurotoxicity. (Tinston 1995)

Systemic Toxiciry: Reductions in body weight gain were seen in males and females fed

500 or 2500 ppm EPTC.

Page 37 of 64
Organ weight: Organ weight measures were taken for the brain only. Reductions in

brain weight were seen in males and females fed 2500 ppm and in females fed 500 ppm

EPTC. In females reductions in brain weight were accompanied by reductions in brain

length and width.

Histopathology: Histopathological examinations were made in the brain and nerves only.

Minimal neuronal cell necrosis in the dentate gyrus and/or pyriform cortex was seen in

one female fed 2500 ppm. No functional impairment as the result of morphological

changes were observed.

NOEL. The NOEL for this study was 100 ppm EPTC based on bodyweight changes and

brain weight in the 500 ppm females.

90 Day dog -- Daly, 1985 - MRID 00150327

EPTC was administered in the diet at 0,200, 600 and 1800 ppm of Beagle dogs for a

period of three months.

Systemic Toxicity: The mean body weights of the 1800 ppm male group were lower than

control animals throughout the study. Bodyweights for the females were comparable in

all dose groups. Cholinesterase (plasma) was slightly reduced in high dose males at week

7 and termination.

Cholesterol: Total cholesterol was measured pre-test, at week 7 and at termination.

Levels in both males and females of all treated groups were normal.

Organ Weights: On endocrine interest, liver, kidney, adrenals, thyroid and parathyroids,

testes with epididymides and ovaries were weighed.

Histopathology: Liver, kidney, thyroid and parathyroids, pituitary, adrenals, testes with

epididymides, prostate, ovary,'uterus and cervix, and mammary glands.

NOEL: 600 ppm (15 mg/kg) based on reduced plasma cholinesterase at 180 ppm (45

mg/kg)

LONG-TERM STUDIES

1 Year Dog -Dickie 1986- MRID 00161595

EPTC was administered in the diet to six beagle dogs per group at dose levels of 0, 200,

600, and 1800 ppm for one year.

Systemic Toxicirv: Body weights and body weight gains were significantly lower in

males in the 600 ppm group. No effects were observed in males fed 200 or 1800 ppm

EPTC. No effects on body weight were observed hi females in any treated group.

Page 38 of 64
Cholesterol: No treatment-related changes were observed in total cholesterol levels.

Organ Weights: Endocrine related organ weights were taken for liver, kidney, pituitary,

adrenals, thyroids (with parathyroid), testes and ovaries.

Absolute testes weights in the 1800 ppm males were lower than controls. In the absence

of microscopic changes and without additional supportive evidence to substantiate a

direct relation to treatment, these weight changes were considered biologically

unimportant.

Females treated with 1800 ppm showed lower absolute and relative pituitary weights

when compared to control animals. In the absence of microscopic changes and without

additional supportive evidence to substantiate a direct relation to treatment, these weight

changes were considered biologically unimportant.

All other weights were comparable to control animals.

Histopathology: Endocrine related organs evaluated were liver, kidney, thyroid,

parathyroid, pituitary, adrenal, testes, prostate, ovary, uterus, cervix, vagina and

mammary glands. There were no microscopic findings related to treatment in any group.

1 Year Dog - Sprague and Taylor, 1987 -- MRID 40442301

EPTC was administered to beagle dogs by capsule at dose levels of 0,1, 8, and 60 mg/kg

for one year.

Systemic Toxicity: One high dose male dog showed progressive weight loss and was

terminated on day 84 because of severe toxicity. Reduced body weight gain in the 60

mg/kg males was observed but no effect was noted in the lower dosage groups or in any

of the female dose groups. Cholinesterase inhibition of plasma was observed at 60 mg/kg

in males and females.

Cholesterol: Total cholesterol was measured at pretest, 3, 6, and 12 months. Although

the female level was elevated in the 12 month sampling, it was not statistically

significant. No changes in cholesterol were observed in any other treated groups.

Organ weights: Of endocrine interest, liver, kidney, adrenals, pituitary thyroids

(including parathyroids), testes (without epididymides) and ovaries.

Liver weights were increased for females at the highest dose (60 mg/kg). In the 60

mg/kg males, absolute liver weights were normal but relative liver weights were

increased. There were no concomitant changes in liver related clinical chemistry. While

apparently treatment-related, this effect was relatively mild. All other organ weights

were comparable to control animals.

Page 3 9 of 64
Histopatholgy: Of endocrine interest, liver, kidney, thyroid, parathyroid, pituitary,

adrenal, testes, epididymides, prostate, ovary, uterus (horns and body), cervix, vagina and

mammary glands.

No treatment-related effects were observed in microscopic analysis of these organs. The

primary finding was in the nerves and muscle.

NOEL: The NOEL for this study was 1 mg/kg/day based on muscle degeneration

secondary to nerve degeneration at 8 mg/kg/day.

1 Year Rat - Trutter, 1978 - MRID 00022101

Eptam was evaluated for toxicological effects in albino rats (60/sex/group) following

dietary administration of 0, 5,20, and 80 mg/kg (adjusted weekly based on body weights)

for 54 weeks. The dietary levels often rats of each sex was increased from 80 to 160

mg/kg beginning at week 16.

Systemic Toxiciry: Slight to moderate growth retardation was observed in the low-dose

(5 mg/kg) males and mid-dose (20 mg/kg) males and females. Clinical chemistry assays

showed mean SGOT (serum glutamic oxaloacetic transaminase) to be higher than control

in the 80 and 160 mg/kg groups, indicative of heart tissue damage.

Organ Weight: Heart, liver, brain, spleen, kidney and testes with epididymides and

ovaries were weighed. There were no findings attributable to treatment. As a

consequence of reduced terminal body weights, some relative organ weights appeared

higher but were not considered biologically relevant.

Histopathology: Of endocrine interest, liver, kidney, thyroid, parathyroid, pituitary,

adrenal, testes, epididymides, seminal vesicles, prostate, ovary, uterus, vagina and

mammary glands.

The major finding in this study was an increased incidence of chronic myocarditis in the

80 and 160 mg/kg dose groups of both sexes.

2 Year Rat - Warner 1983 - MRID 00146311, addendum 00146311 (1985)

In a two-year chronic and oncogenicity study, levels of 5,25, or 125 mg/kg (adjusted for

body weight) were fed to 60 male and female CD rats.

Systemic Toxicity: Mortality was higher for the male rats treated with 125 mg/kg EPTC.

Body weight depressions were noted at 25 and 125 mg/kg in the males and females.

Cholesterol: No changes in total cholesterol were observed in any treatment group.

Page 40 of 64
Organ Weight: On endocrine relevance, liver, kidney, pituitary, adrenals, thyroid

(including parathyroids), testes, and ovaries were weighed at 12 months (interim sacrifice

of 10 animals/sex/group) and at 24 months. No organ weight changes were observed.

Histopathology: Of endocrine relevance, histopathological examination was made on

liver, kidney, thyroid, parathyroid, pituitary, adrenal, testes, with epididymides, seminal

vesicles, prostate, ovary, uterus, cervix, vagina, bone and mammary glands. No

macroscopic lesions were noted in either the interim or terminal sacrifice. No test article

related microscopic changes were present in the 12 month interim sacrifice. No findings

in endocrine-related organs were observed in the terminal sacrifice. Findings of nerve

and muscle degeneration were found in the 25 and 125 mg/kg treatment groups in males

and females.

NOEL: 5 mg/kg

2 Year Rat - Dickie 1987 - MRID 00145004/40215001

In this chronic and oncogenicity study in CD rats, EPTC was administered in .the feed at

0, 9,18, 36 or 72 mg/kg (adjusted for body weight) for two years. In this study 90

rats/sex/group were tested with interim sacrifices at 6, 12, and 18 months.

Systemic Toxicity: Early decreases in body weight were evident in all treatment groups

but by week 104, the body weights of males in the 9 and 18 mg/kg treatment groups and

in females in the 9 mg/kg group were comparable to control animals.

Cholesterol: Bloods were evaluated pre-test, and at 6, 12,18 and 24 months. No changes

in total cholesterol were observed in any treatment group.

Organ weight: Of endocrine relevance, liver, kidney, testes and ovaries were weighed at

6,12,18 and 24 months. No changes in organ weights were observed.

Histopathology: Of endocrine relevance, histopathological examination was made on

liver, kidney, thyroid, parathyroid, pituitary, adrenal, testes, with epididymides, seminal

vesicles, prostate, ovary, uterus, cervix, vagina, bone and'mammary glands. Macroscopic

findings were limited to the muscle and heart. No findings in endocrine related organs

was evident in any treatment groups. Findings of sciatic nerve demyelinization, muscle

degeneration and cardiomyopathy were present down to and including 9 mg/kg/day

EPTC.

NOEL: <9mg/kg

2 Year Mouse - Goldenthal 1978 - MRID 00022100

ETPC was administered in the diet to CD-I mice at dosage levels of 5,20, and 80 mg/kg

(adjusted for bodyweight) for 24 months.

Page 41 of 64
Systemic Toxicity: No signs of systemic toxicity were observed.

Organ weights: Of endocrine relevance, liver, kidney, pituitary, adrenals, thyroid, testes

and ovary were weighed after 52 weeks and at termination at 104 weeks. Although there

were numerous weight variations observed at both 12 and 24 months, they were sporadic

and non-dose responsive. In the absence of compound-related morphologic changes,

they were considered to be of undetermined biological significance.

Histopathology: Of endocrine relevance, histopathological examination was made on

liver, kidney, thyroid, parathyroid, pituitary, adrenal, testes, with epididymides, seminal

vesicles, prostate, ovary, uterus, cervix, vagina, bone and mammary glands. The

histopathology of treated animals was similar to control animals.

18 Month Mouse - Tisdel 1986 - MRID 00161596

ETPC was administered in the diet to CD-I mice at dosage levels of Q, 200, 600 or 1800

ppm for 18 months.

Systemic Toxicity: Body weight reduction was evident in females dosed 600 ppm

(slight) and in males and females receiving 1800 ppm.

Organ Weights: No organ weights were measured.

Histopathologv: Of endocrine relevance, histopathological examination was made on

liver, kidney, thyroid, parathyroid, pituitary, adrenal, testes with epididymides, seminal

vesicles, prostate, ovary, uterus, cervix, vagina, bone and mammary glands. The

histopathology of treated animals was similar to control animals. All tissues showed

normal histopathology.

NOEC 600 ppm (90 mg/kg)

NOEC Onco: > 1800 ppm (270 mg/kg)

WILDLIFE

Mallard Reproduction ~ Temple, Martin, Beavers, and Jaber 2005 — MRID

46554301

A toxicity and reproduction study was conducted with groups of mallard ducks (males

and females) fed to EPTC at 240,600, and 1500 ppm in the diet for 20 weeks beginning

10 weeks prior to egg laying.

There were no effects on mortality or overt signs of toxicity at 240 or 600 ppm. There

were no effects on reproductive parameters at 240 or 600 ppm. At the 1500 test

concentration there were minor clinical signs of toxicity, transient effects upon adult

body weight and feed consumption and treatment-related effects upon multiple

reproductive parameters, including egg production and the percentage of viable embryos

Page 42 of 64
(as a percentage of eggs set). There were no effects on egg shell thickness. Offspring

body weights were not affected.

Quail Reproduction ~ Temple, Martin, Beavers, and Jaber 2005 — MRID

46554302

A toxicity and reproduction study was conducted with groups of quail (males and

females) fed to EPIC at 240, 600, and 1500 ppm in the diet for 26 weeks beginning 13

weeks prior to egg laying.

The authors concluded that there were no treatment-related mortalities, overt signs of

toxicity or biologically meaningful treatment-related effects upon body weight or feed

consumption at any of the concentrations tested. Additionally, there were no treatment -

related effects upon any of the reproduction parameters measured at the 240, 600 or 1500

ppm a.i. test concentrations.

Page 43 of 64
IX. Title pages for OSRIEPTC submission

Page 44 of 64
Study Title: Two-Generation Reproduction Study with EPTC in Rats

Test Substance: Phosmet technical, 98.4% PPG

Data Requirement: US EPA Guideline 83.4

Authors: Karen MacKenzie

Completion Date: March 1,1985

Laboratory: Hazelton Laboratories America, Inc

3301 Kinsman Boulevard

Madison, WI53704

Laboratory Report No.: 6100-108

MRID: 00161597

Classification: Acceptable

Page 45 of 64
Study Title: A Two-Generation Rat Reproduction Study with Eptam Technical

Test Substance: Phosmet technical, 98.6% (Lot # EHC-0048-01)

Data Requirement: US EPA Guideline

Authors: J. R. Minor

Completion Date: October 8,1982

Laboratory: Stauffer Chemical Company

Environmental Health Center

Farmington, CT 06032

Laboratory Report No.: T-10123

MRID: 00121284, (pathology supplement 40420408)

Classification: Acceptable

*

.Page 46 of 64
Study Title: Teratology Study in Rabbits with Eptam Technical

Test Substance: EPTC technical,

Data Requirement: US EPA Guideline 83-3B

Authors: P.A. Gilles

Completion Date: August 12,1987

Laboratory: Stauffer Chemical Company

Environmental Health Center

Farmington, CT 06032

Laboratory Report No.: T-12982

MRID: 40442302

Classification: Acceptable

Page 47 of 64
Study Title: Effect of EPTC on Pregnancy of the Rat

Test Substance: EPTC technical, (Batch No. 518996 PPG)

Data Requirement: US EPA Guideline 83-3A

Authors: P. James, J. Smith, and D John

Completion Date: November 6, 1985

Laboratory: Huntingdon Research Centre, Ltd.,

Huntingdon,

Cambridgeshire PE18 6ES

Laboratory Report No.: PPG 19/851002

MRID: 00161598

Classification: Acceptable

Page 48 of 64
Study Title: A Teratology Study in Rats with EPTAM

Test Substance: EPTC technical, (Lot # CHK 0601)

Data Requirement: US EPA Guideline 83-3A

Authors: M.Nemec

Completion Date: Novembers, 1983

Laboratory: Wil Research Laboratories, Inc

1407 Montgomery Township Road

Ashland, OH 44805-9281

Laboratory Report No.: WIL-27013

MRID: 00138919

Classification: Acceptable

Page 49 of 64
Study Title: Effect of EPIC on Pregnancy of the Rabbit

Test Substance: EPTC technical, (Batch No. 518996 PPG)

Data Requirement: US EPA Guideline 83-3B

Authors: P. James, J. Smith, R. Masters, and J Offer

Completion Date: October 10, 1985

Laboratory: Huntingdon Research Centre, Ltd.,

Huntingdon, Cambridgeshire PE18 6ES

Laboratory Report No.: PPG 14 & 18/85601

MRID: 00161599

Classification: Acceptable

Page 50 of 64
Study Title: EPTC: Developmental Neurotoxicity Study in Rats

Test Substance: EPTC technical, (Lot # CHK 0601)

Data Requirement: US EPA Guideline OPPTS 870.6300

Authors: D. Lees

Completion Date: July 2,2004

Laboratory: Central Toxicology Laboratory

Alderley Park, Macclesfield

Cheshire, UK SKI 04TJ

Laboratory Report No.: CTL/RR0926/Regulatory/Report

MRID: 46319101,43039701 and 43964401

Classification: Acceptable

Page 51 of 64
Study Title: Thirteen-Week Subchronic Study in Rats with EPTC

Test Substance: EPTC technical, (PPG Lot No. BR 83-64)

Data Requirement: US EPA Guideline 82-1A

Authors: M. Tisdel

Completion Date: September 14, 1984

Laboratory: Hazelton Laboratories America, Inc.

2201 Kinsman Blvd

Madison, WI53707

Laboratory Report No.: 6100-105

MRID: 00144651

Classification: Acceptable

Page 52 of 64
Study Title: Subchronic Inhalation Toxicity of Eptam in Rats

Test Substance: EPTC technical,

Data Requirement: US EPA Guideline

Authors: J.B. Scott

Completion Date: March 5, 1982

Laboratory: Stauffer Chemical Company

Environmental Health Center

400 Farmington Avenue

Farmington, CT 06032

Laboratory Report No.: T-10422

MRID: 00154784

Classification: Acceptable

Page 53 of 64
Study Title: First Revision to EPTC: Subchronic Neurotoxicity Study in Rats

Test Substance: EPTC technical, 98.4% (P4/D7534/10)

Data Requirement: US EPA Guideline 82-7

Authors: D. J. Tinston

Completion Date: November 20,1995

Laboratory: Zeneca Central Toxicology Laboratory

Alderley Park,

Macclesfield, Cheshire, UK

Laboratory Report No.: PRO929

MRID: 432330901 (supplements 43964401, 4398301)

Classification: Acceptable

Page 54 of 64
Study Title: A Three Month Subchronic Oral Dietary Toxicity Study of EPTC in Beagle

Dogs

Test Substance: EPTC technical, 98.4% BR 84-1 1-9-84

Data Requirement: US EPA Guideline 82-IB

Authors: I. Daly

Completion Date: February 15,1985

Laboratory: Bio/Dynamics

PO Box 43

East Millstone, NJ 08873

Laboratory Report No.: 83-2781

MRID: 00150327

Classification: Acceptable

Page 55 of 64
Study Title: One-Year Oral Feeding Study of the Chronic Toxicity of EPTC in Dogs

Test Substance: EPTC technical, 98.4% (Lot No: 518-996)

Data Requirement: US EPA Guideline 83-IB

Authors: B.C.Dickie

Completion Date: June 10, 1986

Laboratory: Hazleton Laboratories America, Inc

3301 Kinsman Blvd

Madison WI, 53707

Laboratory Report No.: 6100-109

MRID: 00161595

Classification: Unacceptable, no NOEL. Is supplemental for assessment of other effects

Page 56 of 64
Study Title: One-Year Oral Toxicity Study with Eptam Technical in Dogs.

Test Substance: EPTC technical, 97.6% (WCR No: 4921 -4-25)

Data Requirement: US EPA Guideline 83-1B

Authors: G.L. Sprague and D.O.N. Taylor

Completion Date: September 8, 1987

Laboratory: Stauffer Chemical Company

400 Farmington Avenue

Farmington, CT 06032

Laboratory Report No.: T-12723

MRID: 40442301

Classification: Acceptable

Page 5 7 of 64
Study Title: 54-Week Feeding Study in Rats: Eptam Technical

Test Substance: EPTC technical, 98.0% (Lot No: 3905-35 Stauffer)

Data Requirement: US EPA Guideline 83-1A

Authors: J. Trutter

Completion Date: September 18,1978

Laboratory: Hazleton Laboratories America, Inc

3301 Kinsman Blvd

Madison WI, 53707

Laboratory Report No.: 132-131

MRID: 00022101

Classification: Unknown

Page 58 of 64
Study Title: Two Year Oral Toxicity/Oncogenicity Study in Rats

Test Substance: EPTC technical,

i

Data Requirement: US EPA Guideline 83-1A

Authors: M. Warner (main report)

E. Goldenthal (addendum)

Completion Date: September 30, 1983 main report, May 1985 addendum

Laboratory: International Research and Development Corporation (IRDC)

Mattawan, MI 49071

Laboratory Report No.: 153-100

MRID: 00145004,00146311 addendum

Classification: Acceptable with histopathology addendum (T-1001) April 23,1985

Page 59 of 64
Study Title: Two-Year Oral Feeding Study of the Oncogenicity and Chronic Toxicity of

EPTC in Rats

Test Substance: EPTC technical, 98.4% (Lot No. 518-996 PPG)

Data Requirement: US EPA Guideline 83-1A

Authors: B.C.Dickie

Completion Date: March 20,1987

Laboratory: Hazelton Laboratories America, Inc

3301 Kinsman Blvd

Madison, WI 53707

Laboratory Report No.: HLA6100-106

MRID: 40215001

Classification: Acceptable for oncogenicity. For the chronic portion of the study, no

NOEL was established and was upgraded to acceptable with another toxicity study that

established a NOEL.

Page 60 of 64
Study Title: Lifetime Oral Study in Mice

Test Substance: EPTC technical, (Lot No. 3905-35 Stauffer)

Data Requirement: US EPA Guideline: Pre 1982 (83-2B)

Authors: E. Goldenthal

Completion Date: December 22, 1978

Laboratory: International Research and Development Corporation (IRDC)

Mattawan, MI 49071

Laboratory Report No.: 153-011

MRID: 00022100

Classification: Unknown

Page 61 of 64
Study Title: Oncogenicity Study in Mice with EPTC

Test Substance: EPTC "technical, 98.4% (Lot No. 518-996 PPG)

Data Requirement: US EPA Guideline 83-2B

Authors: M. Tisdel

Completion Date: May 15, 1986

Laboratory: Hazelton Laboratories America, Inc

3301 Kinsman Blvd

Madison, WI53707

Laboratory Report No.: 6100-104

MRID: 00161596

Classification: Acceptable

Page 62 of 64
Study Title: EPIC: A Reproduction Study with the Mallard

Test Substance: EPTC technical, 98.1% (Batch FL-021317 Gowan)

Data Requirement: US EPA Guideline OPPTS 850.2300

Authors: Temple, D.; Martin, K.; Beavers, J, Jaber, M.

Completion Date: May 19, 2005

Laboratory: Wildlife International, Ltd.

8598 Commerce Drive

Easton,MD21601

Laboratory Report No.: Project Number: 334/112

MRID: 46554301

Classification: Acceptable

Page 63 of 64
Study Title: EPIC: A Reproduction Study with the Northern Bobwhite

Test Substance: EPTC technical, 98.1% (Batch FL-021317 Gowan)

Data Requirement: US EPA Guideline OPPTS 850.2300

Authors: Temple, D., Martin, K., Beavers, J. and Jaber, M.

Completion Date: May 19, 2005

Laboratory: Wildlife International, Ltd.

8598 Commerce Drive

Easton,MD21601

Laboratory Report No.: Project Number: 334/111

MRID: 46554302

Classification: Acceptable

Page 64 of 64