DICOFOL EDSP TEST ORDER: EDSP-010501-101

Response to the U.S. Environmental Protection Agency Testing

Order under the Endocrine Disrupter Screening Program (EDSP):

Discussion of Other Scientifically Relevant Information (OSRI) for Dicofol

DATA REQUIREMENTS

N/A

STUDY AUTHORS

Laura M. Plunkett, Ph.D., DABT

Integrative Biostrategies, LLC

Houston, Texas

Pramila Singh, Ph.D, DABT

Corporate Toxicologist, MANA

Raleigh, NC

STUDY COMPLETION DATE

March 12, 2010

STUDY NO.

2010-DIC-OL-0311

Agan Chemical Manufacturers Ltd.

c/o Makhteshim Agan of North America, Inc ••••••

4515 Falls of Neuse Road, Suite 300 •ct'

Raleigh, NC 27609 .....

TOTAL PAGES: 51
Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

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

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

This statement overrides any confidentiality markings within this study.

Company: Agan Chemical Manufacturers Ltd.

c/o Makhteshim Agan of North America, Inc.

4515 Falls of Neuse Road, Suite 300

Raleigh, NC 27609

Company Representative ,

Authorized Representative - AGAN

Regulatory Product Manager - MANA

Date

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Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

The material presented in this report is descriptive in nature and, as such, is not subject to

the Good laboratory Practice regulations as set forth in 40 CFR 160.

Author:

Date

3

/l£-f 3-o ID

Laura M. Plunkett, Ph. D., DABT.

Submitter:

Date 0 *>

Authorized Agent (MCW) and Regulatory Product Manager (MANA)

Sponsor:

Date 6 $ t2 / - ^9 10

Authorized Agent (Agan) and Regulatory Product Manager (MANA)
Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

Table of Contents

I. Introduction 5

II. Background 5

III. Discussion of Dicofol OSRI 11

A. Directly or Functionally Equivalent Dicofol OSRI Identified in the Published

Literature 18

B. Directly or Functionally Equivalent Dicofol OSRI Identified in Dicofol Registration

Studies 27

IV. MANA Seeks a Waiver for Performance of Nine of Eleven EDSP Tier 1 Tests Based

on Available Dicofol OSRI 38

A. MANA Seeks a Waiver for Performance of Both Tier 1 In Vitro Assays for

Estrogen Activity (Estrogen Receptor Binding 890.1250 and Estrogen Receptor

Transcriptional Activation 890.1300) 40

B. MANA Seeks a Waiver for Performance of the Tier 1 In Vitro Assays for Androgen

Activity (Androgen Receptor Binding 890.1150 and Aromatase Activity 890.1200) ..41

C. MANA Seeks a Waiver for Performance of the Tier 1 Amphibian Metamorphosis

Assay (890.1100) 42

D. MANA Seeks a Waiver for Performance of the Tier 1 Female Pubertal Assay

(890.1450) 43

E. MANA Seeks a Waiver for Performance of the Tier 1 Male Pubertal Assay

(890.1500) 45

F. MANA Seeks a Waiver for Performance of the Tier 1 Hershberger Assay

(890.1400) 46

G. MANA Seeks a Waiver for Performance of the Tier 1 Uterotrophic Assay

(890.1600) 47

V. Overall Summary 47

VI. References Cited 49
Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

I. Introduction

On November 13, 2009, the U.S. Environmental Protection Agency (EPA) issued

a Testing Order (TO) for dicofol (CAS # 115-32-2) related to testing under the EPA

Endocrine Disrupter Screening Program (EDSP). In the TO, EPA has required

submission of EDSP Tier 1 testing for dicofol. The Tier 1 tests listed for submission

include: 1) Amphibian Metamorphosis assay (890.1100); 2) Androgen Receptor Binding

assay (rat prostate) (890.1150); 3) Aromatase assay (human recombinant) (890.1200); 4)

Estrogen Receptor Binding assay (890.1250); 5) Estrogen Receptor Transcriptional

Activation assay (human cell line HeLa-9903)(890.1300); 6) Fish Short-term

Reproduction assay (890.1350); 7) Hershberger assay (890.1400); 8) Female Pubertal

assay (rat) (890.1450); 9) Male Pubertal assay (rat) (890.1500); 10) Steroidogenesis assay

(human cell line H295R) (890.1550); and 11) Uterotrophic assay (890.1600). This

document is a response to the TO, and has been prepared by Makhteshim-Agan of North

America (MANA) on behalf of Agan Chemical Manufacturers Ltd (Agan). This

response 1) outlines the data already available on the endocrine disruption potential of

dicofol, referred to by EPA as "other scientifically relevant information" or OSRI, 2) .

discusses how available OSRI provides for screening of endpoints included in EDSP Tier

1 assays, and 3) provides MANA's response regarding the performance of specific EDSP

Tier 1 testing, including rationale for waivers for certain dicofol EDSP Tier 1 assays.

II. Background

EDSP Tier 1 assays were designed to provide data relevant to screening for

endocrine disruption potential of chemicals by identifying potential modes of action of

chemicals, specifically the effects of chemicals on estrogen, androgen and thyroid

hormone linked systems. The potential modes of action screened for in Tier 1 assays

include, in vitro estrogen and androgen hormone receptor binding, in vitro effects on

enzymes involved in hormone synthesis (e.g., assays directed towards Steroidogenesis

and aromatase activity), as well as in vivo tests of mammalian and fish reproduction

involving both male and female organisms.

Before discussing details on the EDSP Tier 1 studies, it is useful to review what

has already been established about chemically-induced endocrine toxicity. There are
Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

well-known biological effects that can be monitored in vivo in animal testing and used to

identify compounds that affect androgen, estrogen, and/or thyroid hormone activity. The

biological endpoints are listed below in Table 1. It is important to note that the various

endpoints listed in Table 1 are apical endpoints that are already routinely monitored in

standard toxicology studies that are part of the pesticide registration process. Therefore,

in the case of a chemical such as dicofol, which is currently registered for use in the

United States, there is a wealth of toxicology data available on endpoints that address

endocrine toxicity.

Table 1

Known Chemically-Induced Effects on Hormone-Related Apical Biological Endpoints

Anti-androgen Agents

Altered testis weight and

histopathology

Altered epididymis weight and

histopathology

Altered prostate weight and

histopathology

Altered prostate weight and

histopathology

Altered seminal vesicle weight

and histopathology

Decreased fertility and fecundity

Increased time to mating

Altered uterine histopathology

Altered ovarian histopathology

Estrogenic Agents

Increased uterine weight and

altered histopathology

Decreased testes weight and

altered histopathology

Decreased epididymis weight and

altered histopathology

Decreased ovary weight

Altered histopathology of vagina,

testis, and mammary gland

Decreased litter size

Decreased fertility and mating

index

Increased implantation loss

Increased gestation time and time

to mating

Decreased fecundity

Thyroid Hormone Active

Agents

Enlarged thyroid, increased

thyroid weight

Altered hormone levels (T3/T4

and TSH)

Altered thyroid histopathology

Increased liver weight

Increased body weight

Altered brain morphology

(developmental study)

With the information in Table 1 in mind, it is clear that any discussion of dicofol

OSRI will include a discussion of the guideline toxicology studies that were performed to

6
Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

support registration of dicofol, as well as scientific studies on dicofol published in the

open literature. Before discussion of each dicofol OSRI study in some detail, it is

important to understand what specific endpoints of endocrine toxicity are being

monitored in EDSP Tier 1 studies. With this approach, the OSRI discussion can be

clearly focused on the issue of the adequacy of existing dicofol OSRI to support a waiver

of certain EDSP Tier 1 studies. As a result, Table 2 below has been constructed to

provide an organizational aid that lists the Tier 1 EDSP assays and each type of endpoint

measured in the combined assays.

The endpoints listed in Table 2 were found by review of each of the EPA test

guidelines (890.1100 through 890.1600). In some cases, the endpoint listed in Table 2 has

been generalized to include more than one specific endpoint. For example, the endpoint

listed as "developmental measures" includes a variety of specific findings in in vivo

studies including objective measures such as time to preputial separation, time to vaginal

patency or vaginal opening, time to eye opening, and more subjective measures such as

behavioral changes in lactation or nursing behavior, or neurobehavioral test observations.

The endpoint listed as "reproductive organ" weight or histopathology includes a variety

of organs, which may be measured in any species and is meant to include measures

related to the mammary gland, uterus, ovaries, cervix and vagina in female animals and

the testes, epididymides, prostate, seminal vesicle, glans penis, levator anibulbocavernous muscle, and Cowper's glands in male animals. The endpoint listed as

"hormone synthesis" is meant to encompass endpoints such as enzyme activity measures.

The endpoint listed as "sex hormones" includes measurement of actual levels of

vitellogenin, estradiol, testosterone, serum luteinizing hormone, and follicular stimulating

hormone. Finally, the endpoint listed as "thyroid hormones' includes measurement of

levels of thyroxine (T4) and thyroid-stimulating hormone. Finally, the appearance of

"yes" throughout the table in any column or row indicates that the particular endpoint is

measured in the particular EDSP Tier 1 assay, whereas no entry or a "blank box" in the

table indicates that the endpoint is not measured in the particular EDSP Tier 1 assay.
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Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

Inspection of the endpoints measured in various EDSP Tier 1 assays (Table 2)

reveals there is a wide variety of data collected in rats (mammals), and one study each in

fish and amphibians. The amphibian metamorphosis assay, however, is designed to be a

screening assay for potential effects on thyroid hormone systems in mammals, including

humans (see OPPTS 890.1100). The various assays cover different stages of development

(e.g., early development, peri-pubertal period, adult). The assays screen for effects on

estrogen, androgen and thyroid hormone systems. The endpoints listed in Table 2 will be

specifically identified in OSRI available for dicofol.

Before discussing the OSRI for dicofol specifically, it is important to differentiate

between "direct" equivalence data versus "functional" equivalence data. In its EDSP TO

documents, EPA has indicated that companies can submit OSRI data that is either

"directly equivalent" to data gathered in EDSP Tier 1 tests or data that is "functionally

equivalent" to EDSP Tier 1 data. Therefore, directly equivalent data would be data

collected on the exact same endpoints in the same assays as specified under EDSP Tier 1,

although the assay involved might differ slightly in terms of the protocol used or the

conditions of the assay, i.e., data gathered in an estrogen receptor binding assay in a

tissue other than rat uterine cytosol (OPPTS 890.1250). Also it follows that functionally

equivalent refers to quality, reliable data that provides the same essential endpoint

information on a chemical, even if methods and procedures other than EDSP Tier 1

assays were used to obtain the data (i.e., fertility data gathered in a multi-generation rat

reproductive toxicity study). Use of functionally equivalent endpoints and studies is

allowed under the concept of OSRI and avoids unnecessary additional use of resources

and sacrifice of animals, while ensuring that sufficient data are available to screen for the

potential endocrine toxicity of chemicals.

In order to identify OSRI for dicofol, two sources were examined: 1) the peerreviewed, published scientific literature; and 2) the dicofol pesticide registration studies.

In order to ensure that only reliable, scientifically valid OSRI were included in this

evaluation of dicofol, each study was analyzed using the scientific method set forth by

Klimisch et al. (1997), which is consistent with the process outlined by EPA in 2003 in

its document entitled EPA Assessment Factors Guidance (2003). Thus, the process of

selecting studies that can be used as dicofol OSRI included identifying a reliability

10
Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

indicator for each study. As outlined by Klimisch et al. (1997), reliability can be

differentiated into four categories as follows, "reliable without restriction" or category 1;

"reliable with restrictions" or category 2; "not reliable" or category 3; and "not

assignable" or category 4. In this evaluation of dicofol OSRI, only studies that could be

given a category rating of either 1 or 2 were used in the OSRI evaluation presented here.

This means, that only studies that were GLP-compliant1 or comparable to a GLPcompliant study protocol were given a rating of category 1 and studies given a category 2

rating were ones that, although not performed according to GLP, were considered to be

well-documented and scientifically acceptable studies. The dicofol OSRI was evaluated

by scientists with training in toxicology and familiarity with pesticide registration and

EDSP testing protocols.

It is the objective of the OSRI identification process to determine whether

equivalent OSRI exist that support the waiver of an EDSP Tier 1 study. Interpretation of

the results gathered in specific OSRI studies is beyond the scope of this process. The

following (Section III of this document) is a discussion of the OSRI for dicofol that is

either directly equivalent or functionally equivalent to the EDSP Tier 1 assay endpoints

listed in Table 2. The discussion is focused on describing which EDSP Tier 1 endpoints

are adequately evaluated through use of OSRI and which endpoints may need to be

examined further through performance of EDSP Tier 1 assays, assays that will be

specifically identified. Following the discussion of the OSRI data, MANA has provided a

specific rationale for why certain EDSP Tier 1 assays should be waived based on the

available dicofol OSRI (Section IV of this document).

III. Discussion of Dicofol OSRI

There are OSRI for dicofol that can be identified as being either directly or

functionally equivalent to data collected in EDSP Tier 1 testing. Table 3 below provides a

listing of each source of dicofol OSRI that has been identified, and organizes the OSRI

from that source based on the EDSP Tier 1 endpoints, similar to the presentation of the

EDSP Tier 1 endpoints in Table 2 above. Some published dicofol OSRI appear multiple

' "GLP-compliant" means that a study was performed under Good laboratory Practice (GLP) as outlined by

EPA.

11
Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

times in Table 3 because they contain multiple OSRI endpoints. The appearance of "yes"

throughout the table in any column or row indicates that the particular endpoint is

measured in the particular dicofol OSRI, whereas no entry or a "blank box" in the table

indicates that the endpoint is not measured in the particular dicofol OSRI studies

referenced. Following Table 3, the various OSRI are discussed in more detail, including a

discussion of whether the OSRI are either directly equivalent, functionally equivalent, or

both.

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Agan Chemical Manufacturers Ltd.

Study No. 2010-DIC-OL-0311

A. Directly or Functionally Equivalent Dicofol OSRI Identified in the

Published Literature

A search of the published scientific literature identified 12 sources of

dicofol OSRI that are directly equivalent to data collected in EDSP Tier 1 assays.

These studies include: van den Berg et al. (1991); Vinggaard et al. (1999);

Vinggaard et al. (2000); Du and Xu (2001); Ishihara et al. (2003); Kojima et al.

(2004); Okubo et al (2004); Shimada and Yamamuchi (2004); Kojima et al.

(2005); Sugiyama et al. (2005); Vinggaard et al. (2008); and Judson et al (2009).

The following is a discussion of the relevant OSRI from each study and a

statement as to whether the data are directly or functionally equivalent OSRI. The

objective of the current OSRI identification process is to determine whether

equivalent OSRI exist that support the waiver of an EDSP Tier 1 study.

Interpretation of the results gathered in specific OSRI studies is beyond the scope

of this process.

1. van den Berg et al. (1991)

Review of this study indicates that it provides reliable, nonguideline OSRI data for screening for the potential of dicofol to affect

thyroid hormone receptors and activity.

In this in vitro study, the authors examined the ability of different

chemicals tested individually to interfere with the T4 binding site of

transthyretin in a competitive binding assay. Transthyretins are

mammalian transport proteins that take thyroid hormone into cells where

they interact with thyroid hormone receptors. Purified human serum

(containing transthyretins) was mixed with radiolabeled T4 and the ability

of test chemicals added to the serum to displace radiolabeled T4 was

quantified. Dicofol (100 uM) was one of the chemicals tested. The authors

reported that dicofol displaced T4 binding from transthyretin by 71-100%.

These in vitro data are not directly predictive of in vivo activity, and are in

fact not supported by effects seen in vivo in other dicofol OSRI (discussed

below). The data provided in this study are functionally equivalent to the

18
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Study No. 2010-DIC-OL-0311

type of data collected in the EDSP Tier 1 890.1100 assay, and are reliable

dicofol OSRI.

2. Vinggaard et al. (1999)

Review of this study indicates that it provides reliable, nonguideline study data for screening for the estrogen receptor binding and

transactivation potential of dicofol.

In this in vitro study, the authors report results for a variety of

pesticides tested individually. The authors measured the comparative

potency of test compounds to induce proliferation of MCF-7 breast cancer

cells in culture. This human breast cancer cell line expresses estrogen

receptors, i.e., estrogen receptor-a (ERa), and stimulation of these

receptors by compounds such as estradiol leads to increased proliferation

of MCF-7 cells. The authors report use of 17(3-estradiol to validate the

responsiveness of their assay. Dicofol was one of 20 pesticides tested

individually. The authors reported that when tested at concentrations from

0.001 to 10 uM, dicofol had no significant effects on cell proliferation.

Then, dicofol was tested for its ability to transactivate estrogen receptors.

£ cerevisae were transfected with human ERa and a reporter plasmid

encoding p-galactosidase. Dicofol exhibited only weak activity in this

assay with cytotoxicity seen at 10 uM dicofol concentrations. Activity on

estrogen receptor transactivation was seen only at dicofol levels of 1 to 5

uM, as compared to estradiol which showed activity at low nM levels. The

data provided are directly equivalent to the type of data collected in the

EDSP Tier 1 890.1250 and 890.1300 assays, and are reliable dicofol

OSRI.

3. Vinggaard et at. (2000)

Review of this study indicates that it provides reliable, nonguideline study data for screening for the potential for dicofol to alter

aromatase activity.

19
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Study No. 2010-DIC-OL-0311

In this in vitro study, 22 different compounds were tested

individually for their ability to affect aromatase activity in human

placental microsomes. Human placental microsomes contain the CYP19

protein (aromatase) and thus use of these cells is a standard screening

system for aromatase activity in vitro. The authors reported that dicofol

was a weak inhibitor of aromatase activity, exhibiting an ICso of greater

than 50 ^M. The data provided are directly equivalent to the type of data

collected in the EDSP Tier 1 890.1200 assay, and are reliable dicofol

OSRI.

4. Du and Xu (2001)

Review of this study indicates that it provides reliable, nonguideline study data for screening for the estrogen receptor binding

potential of dicofol.

In this in vitro study, the authors measured the comparative

potency of dicofol to induce proliferation of MCF-7 breast cancer cells in

culture. As already discussed, this human breast cancer cell line expresses

estrogen receptors, i.e., ERa, and stimulation of these receptors by

compounds such as estradiol leads to increased proliferation of MCF-7

cells. The authors report use of 1 ?p-estradiol to validate the

responsiveness of their assay. The authors reported that dicofol was tested

at concentrations from 8 x 10"12 M to 4 x 10"5 M, and that estradiol was

tested at a concentration of 8.5 x 10"12 M. Dicofol was reported to induce

cell proliferation at doses of from 4 x 10"11 to 4 x 10"6 M, being cytotoxic

at a dose of 4 x 10"5 M. These in vitro data are not directly predictive of in

vivo activity, and are in fact not supported by effects seen in vivo in other

dicofol OSRI. However, the data provided are directly equivalent to the

type of data collected in the EDSP Tier 1 890.1250 assay, and are reliable

dicofol OSRI.

20
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Study No. 2010-DIC-OL-0311

5. Ishihara et al. (2003)

Review of this study indicates that it provides reliable, nonguideline OSRI data for screening for the potential of dicofol to interact

with thyroid hormone receptors and activity.

In this in vitro study, the ability of a variety of chemicals to

interfere with thyroid hormone binding to both thyroid hormone receptors

and transthyretins was examined. The authors reported use of both

chicken and bullfrog transthyretins. Thyroid hormone receptor binding

was tested in human, chicken, tadpole and salmon plasma systems.

Radiolabeled T3 binding was used to detect binding to transthyretins and

thyroid hormone receptors. Seventeen different chemicals, including

dicofol, were tested individually. In the thyroid hormone receptor binding

assay, T3 or T4 concentrations of 8 uM produced 100% displacement of

radiolabeled T3 binding, thus all other compounds were tested at this same

concentration of 8 uM. The authors reported that 8 uM dicofol produced

80 to 90% inhibition of thyroid hormone receptor binding in human,

chicken, tadpole and salmon plasma. In the chicken transthyretin assay,

dicofol exhibited weak activity as compared to T3, with an ICso of 5 uM.

Similar results were seen in the bullfrog transthyretin assay, where dicofol

exhibited weak activity as compared to T3, with an ICso of 40 uM. These

in vitro data are not directly predictive of in vivo activity, and are in fact

not supported by effects seen in vivo in other dicofol OSRI. However, the

data provided in this study are functionally equivalent to the data collected

in the 890.1100 assay, and are reliable dicofol OSRI.

6. Okubo et al. (2004)

Review of this study indicates that it provides reliable, nonguideline study data for screening for the estrogen receptor binding

potential of dicofol, as well as the androgen receptor binding potential of

dicofol.

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Study No. 2010-DIC-OL-0311

In one part of this in vitro study, the authors measured the

comparative potency of chemicals to induce or inhibit proliferation of

MCF-7 breast cancer cells in culture. As already discussed, this human

breast cancer cell line expresses estrogen receptors, i.e., ERa, and

stimulation of these receptors by compounds such as estradiol leads to

increased proliferation of MCF-7 cells. The authors report use of 17Pestradiol to validate the responsiveness of their assay. Dicofol was one of

20 chemicals tested individually. The authors reported that dicofol was

tested at concentrations from 1 to 100 uM. Dicofol was reported to induce

cell proliferation only at doses greater than 10 uM, making it a weak

estrogen receptor binding agent. Its relative potency as compared to

estradiol was 5-6 orders of magnitude less. In another part of the study, the

authors examined the ability of the test compounds, including dicofol, to

inhibit ER and AR receptor binding using commercial ligand screening

assays. The ERa ICso of dicofol was reported to be 19.8 ^M and the AR

ICso was reported to be 42 uM, confirming that dicofol has only very

weak activity at estrogen and androgen receptors. The data provided are

directly equivalent to the type of data collected in the EDSP Tier 1

890.1150 assay and the 890.1250 assay, and are reliable dicofol OSRI.

7. Kojima et al. (2004)

Review of this study indicates that it provides reliable, nonguideline study data for screening for the estrogen and androgen receptor

transactivation potential of dicofol.

In this study, 200 individual pesticides were screened for their

potential agonist and antagonist activity at two human estrogen receptor

subtypes (ERa and ERp), and a human androgen receptor, in sensitive

transactivation assays in Chinese hamster ovary cells. Dicofol was one of

the pesticides tested. The assays measured firefly luciferase gene

expression. The activity of the pesticides, including dicofol, was measured

in all three assays and reported relative to the activity of positive control

22
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Study No. 2010-DIC-OL-0311

agents. The authors reported that dicofol exhibited weak estrogen receptor

transactivation potential and weak androgen receptor transactivation

potential. The relative effective concentrations (RECs) that produced 20%

of the response as compared to estradiol were 1.8 x 10"6 M (1.8 uM)

dicofol in the ERa transactivation assay, and 1.8 x 10~6 M (1.8 uM)

dicofol in the ERP transactivation assay. In the androgen receptor

transactivation assay, dicofol's relative inhibitory concentration (RIC) that

produced 20% of the response as compared to testosterone was 1.6 x 10"6

M (1.6 uM dicofol). The data provided in this study are directly equivalent

to the type of data collected in the EDSP Tier 1 890.1300 assay, and are

functionally equivalent to the data collected in the 890.1250 assay and the

890.1150 assay. Thus, these data also provide reliable dicofol OSRI.

8. Shimada and Yamamuchi (2004)

Review of this study indicates that it provides reliable, nonguideline OSRI data for screening for the potential of dicofol to interact

with thyroid hormone receptors and activity.

In this in vitro study, the ability of chemicals to affect T3 binding

in tadpole red blood cells was examined. Dicofol was one of the 25

chemicals tested individually. The authors reported radioabeled T3 uptake

in the presence of various compounds. The T3 ICso in the study was

0.14uM, while dicofol was reported to have an ICso of 0.4 uM. The

authors also stated that the dicofol mode of action was not altering uptake

of T3 or T3 binding in cells. Again, these in vitro data are not directly

predictive of in vivo activity, and are in fact not supported by effects seen

in vivo in other dicofol OSRI. However, the data provided in this study are

functionally equivalent to the data collected in the 890.1100 assay, and are

reliable dicofol OSRI.

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Study No. 2010-DIC-OL-0311

9. Kojima et al (2005)

Review of this study indicates that it provides reliable, nonguideline study data for screening for the estrogen receptor transactivation

potential of dicofol.

In this study, BG1 human ovarian carcinoma cells were transfected

with luciferase reporter plasmid. This luciferase gene expression assay

was used to assess estrogenic activity of dicofol, where luciferase

expression is under estrogen-mediated control. Dicofol was one of 32

compounds tested individually. Dicofol exhibited no activity in the assay.

The data provided in this study are directly equivalent to the type of data

collected in the EDSP Tier 1 890.1300 assay, and are functionally

equivalent to the data collected in the 890.1250 assay. Thus, these data

also provide reliable dicofol OSRI.

10. Sugiyama et al. (2005)

Review of this study indicates that it provides reliable, nonguideline OSRI data for screening for the potential of dicofol to interact

with thyroid hormone receptors and activity.

In this in vitro and in vivo study, the thyroid system-disrupting

activity of chemicals was examined in screening assays in Xenopus laevis.

In the in vitro assays, the Xenopus laevis cell line was transduced with a

self-inactivating lentivirus vector containing a thyroid hormone-responsive

element SV40 promoter and the luciferase gene. The control compounds

T3 and T4 were used, where T3 was active at concentrations of from 2 to

20 nM. By comparison, dicofol decreased binding of T3 and T4 in the

assay but only at the highest test dose of 0.8 uM, indicating weak activity

in the assay. The authors also reported in vitro activity of compounds on

T3 activation of the TR0 gene. Dicofol activity was only seen at a

concentration of 10 uM, as compared to T3 which showed activity at a

dose of 2 nM. The data are consistent in demonstrating that dicofol had

only weak effects on T3 binding. In the in vivo assay, the effects of

24
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Study No. 2010-DIC-OL-0311

compounds on Tr(3 gene activation in tadpoles undergoing metamorphosis

were examined. Dicofol exhibited no activity in vivo, supporting the

conclusion that the in vitro data were not directly predictive of in vivo

activity. The data provided in this study are functionally equivalent to the

data collected in the 890.1100 assay, and are reliable dicofol OSRI.

11. Vinggaard et al (2008)

Review of this study indicates that it provides reliable, nonguideline study data for screening for the androgen receptor binding

potential of dicofol.

In this in vitro study, androgen receptor transact!vation was

assessed for 397 chemicals, individually, in a luciferase reporter assay

using CHOK1 cells transfected with human androgen receptor. Standard

techniques for were employed as described in the paper. R1881 was

employed as a positive control agent at a dose of 0.1 nM. Chemicals,

including dicofol, were tested at doses of 1, 3, 10, and 30 uM. The 1625 for

dicofol reported by the authors was between 1 and 3 uM, indicating only

weak activity based on the authors own ranking system. These data are

consistent with data reported by others as discussed above. The data

provided in this study are directly equivalent to the type of data collected

in the EDSP Tier 1 890.1150 assay, and are reliable dicofol OSRI.

12. Judson et al. (2009)

The citation referenced here refers to the published paper and the

accompanying files found on the EPA website for the ToxCast program2.

Review of the data indicates that it provides reliable data for screening for

estrogen receptor binding, androgen receptor binding, estrogen receptor

2

 The ToxCast program is a program within EPA that is currently validating methods for testing chemicals

using a variety of in vitro assays as a way to develop testing paradigms for replacing in vivo toxicity testing

or to predict results of in vivo toxicity testing. Included in the program are in vitro assays examining the

ability of chemicals to affect estrogen, androgen, and thyroid hormone systems. Details of the program and

the data can be found at www.epa.gov/ncct/toxcast.

25
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Study No. 2010-DIC-OL-0311

transactivation, aromatase activity, and thyroid hormone binding potential

of dicofol.

In a paper published online in December 2009 (Judson et al. 2009),

the authors report on the results of extensive in vitro testing of over 300

individual chemicals, including dicofol. Dicofol, as well as the all of the

other compounds, was tested for estrogen receptor binding, androgen

receptor binding, estrogen receptor transactivation, aromatase activity, and

thyroid hormone binding potential, among a variety of other endpoints.

The specific tests performed included the following: binding to bovine

estrogen receptors from uterine membrane (bER); binding to human

estrogen receptors from breast cancer cells (hER); binding to bovine

progesterone receptors from uterine membrane (bPR); binding to human

progesterone receptors from MCF-7 cells (hPR); binding to rat androgen

receptors (rAR; recombinant); binding to human androgen receptors from

LnCAP cells (hAR); binding to rat thyrotropin-releasing hormone receptor

from forebrain membranes (rTRH); binding to human thyroid hormone

receptor-alpha (hTRa; recombinant); activity to inhibit the aromatase

enzyme; estrogen receptor transactivation; androgen receptor

transactivation; and thyroid hormone receptor transactivation.

The data from these specific tests was examined since the

endpoints are directly or functionally equivalent to endpoints of EDSP

Tier 1 assays. The results for dicofol were as follows. In the cell-free

screening assays, dicofol, when tested at a single initial concentration of

25 uM, was inactive for binding to all endocrine receptor proteins.

Because it was inactive at this dose, no other doses were tested. More

specifically, dicofol did not show evidence of binding to bovine or human

estrogen receptors, bovine or human progesterone receptors, rat or human

androgen receptors, rat thyrotropin-releasing hormone receptor, or human

thyroid hormone receptor-a. In cell-based assays, dicofol did not activate

human androgen receptors, human ERa, or human thyroid hormone

receptor-p, and also did not block activity of established ligands at these

26
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Study No. 2010-DIC-OL-0311

receptor sites (i.e., no antagonist activity demonstrated). In the hormone

receptor transactivation assays, Dicofol did not activate the human

androgen receptor, human estrogen-related receptor-a, human estrogenrelated receptor-y, or thyroid hormone receptor-a. Dicofol showed weak

activity in a human estrogen receptor-a transactivation assay, with activity

seen at a concentration of 2.7 uM. It is noted that the positive control

compound, l?p-estradiol, had activity in this same assay in the low

nanomolar range.

The data provided in this study are directly equivalent to the type

of data collected in the following EDSP Tier 1 assays: 890.1150,

890.1200, 890.1250, and 890.1300. The data on thyroid hormone binding

are functionally equivalent to an endpoint of EDSP Tier 1 assays

890.1100, 890.1450, and 890.1500. These data are reliable dicofol OSRI.

B. Directly or Functionally Equivalent Dicofol OSRI Identified in Dicofol

Registration Studies

A search of the guideline toxicology studies performed to support the

registration of dicofol identified several sources of dicofol OSRI that are directly

or functionally equivalent to endpoints/data collected in EDSP Tier 1 assays.

These studies include the following (see Table 3 above): a subchronic toxicity

study in rats (Goldman et al. 1986), a chronic toxicity study in rats (Hazelton and

Harris 1989), a chronic toxicity study in dogs (Tegeris 1988), a multi-generation

reproductive toxicity study in rats (Solomon and Kulwich 1991), a prenatal

developmental toxicity study in rats (Hoberman and Christian 1986), and a

reproduction and endocrine effects study in rats (Hoberman 1997; Hoberman

1998; Lomax 1998). The following is a discussion of the relevant dicofol OSRI

from each study and a statement as to whether the data are directly or functionally

equivalent OSRI. The objective of the current OSRI identification process is to

determine whether equivalent OSRI exist that support the waiver of an EDSP Tier

1 study. Interpretation of the results gathered in specific OSRI studies is beyond

the scope of this process.

27
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Study No. 2010-DIC-OL-0311

1. Goldman et al. (1986)

Review of this GLP-compliant, guideline subchronic toxicity study

in rats indicates that it provides reliable data for screening for a variety of

EDSP Tier 1 study endpoints as shown in Table 3 above, endpoints

relating to estrogen, androgen and thyroid hormone system activity.

In this guideline subchronic toxicity study in rats, young adult

male and female Sprague-Dawley (Crl-CD) rats were administered 95.6%

pure dicofol (containing less than 0.1% DDT, DDE and DDD residues) in

diet at doses of 0, 1, 10, 100, 500 and 1500 ppm dicofol for 90 days. It

should be noted that a dietary route of administration is more relevant to

the expected human exposure to dicofol than data that might be collected

using other routes of administration in animals (e.g., oral gavage dosing,

intraperitoneal dosing, subcutaneous dosing). As in any standard guideline

rodent subchronic toxicity study, endpoints relevant to endocrine activity

were assessed (see Table 3). The endpoints measured in the subchronic

study that are directly equivalent to endpoints monitored in EDSP Tier 1

assays (i.e., 890.1350, 890.1400, 890.1450, 890.1500, 890.1600) include:

survival, body weight, clinical chemistry, organ weights (adrenal, thyroid,

pituitary, kidney, liver); reproductive organ weights (ovaries and testes);

organ histopathology (adrenal, thyroid, pituitary, kidney, liver); and

reproductive organ histopathology (female mammary gland, ovaries,

uterus, vagina, testes, prostate). This subchronic study also included

several special evaluations including measurement of serum and urine

corticosterone levels in rats at week 11, liver mixed function oxidase

activity at necropsy, and levels of DDT, DDE and dicofol residues at

necropsy in fat, gonads and liver. It should be noted as well that the

subchronic rat study involves repeated dosing for 90 days, a time period

much longer than any time period of exposure in an EDSP Tier 1 assay

(i.e., maximum time of exposure of less than one month).

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The developmental period assessed in the subchronic study is

young adult, for both male and female animals. The most sensitive noobserved effect level (NOEL) in the study was 1 ppm and was based on

follicular epithelial cell hypertrophy of thyroid tissue in some male rats

exposed to 10 ppm dicofol, and in some females exposed to 500 ppm

dicofol; the authors reported that these effects were equivocal. As shown

in Table 3, the estrogen-responsive (e.g., ovaries, uterus), and androgenresponsive (e.g., testes, prostate) were examined histopathologically in this

study. There were no effects on weights of reproductive organs that can be

related to androgen or estrogen activity disturbances. It should be noted

that the chronic toxicity study in rats (discussed below; Hazelton and

Harris 1989) included special measures to examine potential thyroid

hormone effects.

The data provided in this study are directly and/or functionally

equivalent to specific data collected in the following EDSP Tier 1 assays:

890.1350, 890.1400, 890.1450, 890.1500, and 890.1600. The data provide

information on in vivo estrogen, androgen, and thyroid hormone system

toxicity of dicofol in young adult animals. These data are reliable dicofol

OSRI.

2. Hazelton and Harris (1989)

Review of this GLP-compliant guideline chronic (2 year) toxicity

study in rats indicates that it provides reliable data for screening for a

variety of EDSP Tier 1 study endpoints as shown in Table 3 above,

endpoints relating to estrogen, androgen and thyroid hormone system

activity.

In this guideline study in rats, young adult male and female

CRL:CD BR rats were administered 93.3% pure dicofol (less than 0.1%

DDT-related residues confirmed) in diet at doses of 0, 5, 50 and 250 ppm

dicofol for up to 104 weeks (2 years). This study included a number of

satellite groups for special evaluations as follows: 1) urine corticosterone

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levels assessed in 10 rats/sex/group at 2, 5, 11, 17, and 23 months; 2)

clinical chemistry interim measures in 10 rats/sex/group at 2, 5, and 11

months for low and high dose groups and at 17 and 23 months for all

groups; 3) determinations of T3, T4 and TSH levels in serum at 3 months

to test for thyroid function; 4) dosing for 3 months with a 2 month

recovery period and then sacrifice in 10 rats/sex/dose group; 5) interim

sacrifices again at 12 and 18 months for clinical chemistry evaluations;

and 6) examination of liver mixed function oxidase (MFO) activity at 3

and 12 months in 5 rats/sex/group. It should be noted that a dietary route

of administration is more relevant to the expected human exposure to

dicofol than data that might be collected using other routes of

administration in animals (e.g., oral gavage dosing, intraperitoneal dosing,

subcutaneous dosing). As in any standard guideline rodent chronic toxicity

study, endpoints relevant to endocrine activity were assessed (see Table

3), with the added assessment specifically of thyroid function (thyroid

hormone levels). The endpoints measured in the chronic study that are

directly equivalent to endpoints monitored in EDSP Tier 1 assays (i.e.,

890.1350, 890.1400, 890.1450, 890.1500, and 890.1600) include: survival,

body weight, clinical chemistry, thyroid hormone levels, organ weights

(adrenal, thyroid, pituitary, kidney, liver); organ histopathology (adrenal,

thyroid, pituitary, kidney, liver); and reproductive organ histopathology

(female mammary gland, ovaries, uterus, vagina, testes, epididymides,

prostate, seminal vesicle, coagulating gland). It should be noted as well

that the chronic rat study involves repeated dosing for up to 2 years, a time

period much longer than any time period of exposure in EDSP Tier 1

assays, and covering a developmental period not covered by EDSP Tier 1

studies, i.e., senescence.

The developmental periods assessed in the chronic study include

young adult, adult and senescence, in both male and female animals. The

most sensitive NOEL in the study was 5 ppm and was based on increased

activity of MFO enzymes in liver of animals exposed at both the 50 and

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250 ppm dose levels, accompanied by increased liver weights and altered

liver histopathology at these same dose levels. As shown in Table 3,

estrogen-responsive (e.g., ovaries, uterus), androgen-responsive (e.g.,

testes, prostate) and thyroid-responsive tissues (e.g., thyroid, brain), were

examined histopathologically in this study. There were no effects on organ

weights of any organ relevant to endocrine activity, and no effects on

organ histopathology were reported, including endocrine organs (i.e.,

thyroid, pituitary, and various reproductive organs in males and females).

Of particular importance is the fact that there were no changes in serum

thyroid hormone levels or serum corticosterone levels at any dose or time

point in the study. Body weight increased in males and females but only at

the highest test dose (250 ppm dicofol). These data demonstrate that

dicofol was not toxic to endocrine organ systems in this study in rats.

The data provided in this chronic study are directly and/or

functionally equivalent to specific data collected in the following EDSP

Tier 1 assays: 890.1350, 890.1400, 890.1450, 890.1500, and 890.1600.

The data provide information on estrogen, androgen, and thyroid hormone

system toxicity of dicofol. These data are reliable dicofol OSRI.

3. Tegeris (1988)

Review of this GLP-compliant guideline chronic (1 year) toxicity

study in dogs indicates that it provides reliable data for screening for a

variety of EDSP Tier 1 study endpoints as shown in Table 3 above,

endpoints relating to estrogen, androgen and thyroid hormone system

activity.

In this guideline study in dogs, young adult male and female

Beagle dogs were administered 93.3% pure dicofol (less than 0.1% DDTrelated residues) in diet at doses of 0, 5, 30 and 180 ppm dicofol for 1

year. Blood samples were drawn from dogs at weeks 13, 26, 39, and 52 for

interim hematology and clinical chemistry assessments. It should be noted

that a dietary route of administration is more relevant to the expected

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human exposure to dicofol than data that might be collected using other

routes of administration in animals (e.g., oral gavage dosing,

intraperitoneal dosing, subcutaneous dosing). Special evaluations in the

study included: 1) blood cortisol levels after injection of ACTH just prior

to study initiation and at weeks 12, 25 and 51; 2) EKG and heart rate

measurements at weeks 1,4, 13, 26, and 52; 3) rectal temperature

measurements just prior to study initiation and at weeks 14, 26, and 52;

and 4) interim sacrifices at 14 weeks in high dose recovery group animals.

As in any standard guideline dog chronic toxicity study, endpoints

relevant to endocrine activity were assessed (see Table 3). The endpoints

measured in the chronic study that are directly equivalent to endpoints

monitored in EDSP Tier 1 assays (i.e., 890.1350, 890.1400, 890.1450,

890.1500, 890.1600) include: survival, body weight, clinical chemistry,

organ weights (adrenal, thyroid, pituitary, kidney, liver); organ

histopathology (adrenal, thyroid, pituitary, kidney, liver); and reproductive

organ histopathology (female mammary gland, ovaries, uterus, vagina,

cervix, testes, epididymides, prostate). It should be noted as well that the

chronic dog study involves repeated dosing for 1 year, a time period much

longer than any time period of exposure in EDSP Tier 1 assays.

The developmental periods examined in the chronic study include

young adult and adult, in both male and female animals. The most

sensitive NOEL in the study was 30 ppm and was based on increased liver

weight, increased serum alkaline phosphatase, and decreased serum

cortisol responses to ACTH injections at week 13 in the 180 ppm exposure

level. There were no effects on organ weights of any organ relevant to

endocrine activity, and no effects on organ histopathology were reported,

including the endocrine organs studied (e.g., thyroid, pituitary, and various

reproductive organs in males and females). These data demonstrate that

dicofol was not toxic to endocrine organ systems in this chronic study in

dogs.

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The data provided in this chronic study are directly and/or

functionally equivalent to specific data collected in the following EDSP

Tier 1 assays: 890.1350, 890.1400, 890.1450, 890.1500, and 890.1600.

The data provide information on estrogen, androgen, and thyroid hormone

system toxicity of dicofol. These data are reliable dicofol OSRI.

4. Solomon and Kulwich (1991)

Review of this GLP-compliant guideline multi-generation (two)

reproductive toxicity study in rats indicates that it provides reliable data

for screening for a variety of EDSP Tier 1 study endpoints as shown in

Table 3 above, endpoints relating to estrogen, androgen and thyroid

hormone system activity. This study has the most extensive coverage of

EDSP Tier 1 endpoints of any single dicofol study and covers all

developmental periods covered by EDSP Tier 1 assays (in utero, lactation,

post-natal, peri-pubertal, young adult, and adult).

In this guideline study in rats, male and female Charles River CD

rats were administered 93.3% pure dicofol (less than 0.1% DDT-related

residues) in diet at doses of 0, 5, 25, 125 and 250 ppm dicofol, starting

with pre-mating in the PI generation. Pups of the Fl generation were

exposed in utero, during lactation, and then placed on dicofol in diet

immediately at weaning through the post-natal period and through mating

and subsequent parturition, delivery of the F2 generation. Thus, this study

included exposure of Fl animals of both sexes during peri-puberty, an

important consideration for later discussion of the pubertal rat EDSP Tier

1 assays. As with the other guideline studies already discussed, the dietary

route of administration is more relevant to the expected human exposure

to dicofol than data that might be collected using other routes of

administration in animals (e.g., oral gavage dosing, intraperitoneal dosing,

subcutaneous dosing).

The multi-generation reproductive toxicity is expected to be a

critical EDSP Tier 2 study. Although the dicofol study was not performed

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according to the most recent EPA guideline for multi-generation

reproductive toxicity studies (OPPTS 870.3300), it was performed

according the guidelines in place at the time the study was performed. It is

an OPP 83-4 study design, and like the most current guideline study

design it was designed to examine potential effects of dicofol on endocrine

organs, in particular the estrogen and androgen systems that are critical to

reproductive function. As a result of being a reproductive toxicity study,

many endpoints relevant to endocrine activity were assessed (see Table 3).

The endpoints measured in the multi-generation reproductive toxicity

study that are directly equivalent to endpoints monitored in EDSP Tier 1

assays (i.e., 890.1350, 890.1400, 890.1450, 890.1500, 890.1600) include:

survival; body weight (both adults and pups); developmental measures

(mating index in both sexes, gestation index, gestation length, sex ratio of

two successive generations, malformations in both generations, postnatal

development measures in Fl pups); reproductive organ weights (ovaries

and testes); reproductive organ histopathology (female mammary gland,

ovaries, uterus, vagina, cervix, testes, epididymides, prostate, seminal

vesicle, coagulating gland); fertility indices; fecundity indices; and

indirect measures of estrous cyclicity.

The most sensitive NOEL in the study was 5 ppm and was based

on liver histopathology in parental animals and increased vacuolation of

ovaries in the P2 generation at the 25 ppm and higher exposure levels.

This dicofol-related effect on ovarian tissue was further examined in

guideline studies initiated in 1997 and 1998 (i.e., Hoberman 1997,

Hoberman 1998, Lomax 1998), as discussed below. There were no effects

on endocrine organ weights. There were no effects on histopathology of

any other endocrine organs at any dose level tested (e.g., thyroid, pituitary,

uterus, vagina, cervix, and any male reproductive organ in animals

exposed even during sensitive developmental periods). There were no

reproductive system abnormalities noted in pups of either generation.

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There were effects on offspring viability and the number of litters at the

125 and 250 ppm exposure levels.

The data provided in this multi-generation reproductive toxicity

study are directly and/or functionally equivalent to data collected in all of

the EDSP Tier 1 assays that examine aspects of mammalian endocrine

function (i.e., 890.1100, 890.1150, 890.1200, 890.1250, 890.1300,

890.1400, 890.1450, 890.1500, 890.1550, and 890.1600). The data

provide information on estrogen, androgen, and thyroid hormone system

toxicity of dicofol in vivo and are more than adequate to screen for the

endocrine toxicity potential of dicofol. Moreover, in conjunction with the

other available published toxicity data, the multi-generation reproductive

study data are also directly applicable to identifying actual modes of

action for dicofol (see Table 1). These data are reliable dicofol OSRI.

5. Hoberman and Christian (1986)

Review of this GLP-compliant guideline prenatal (teratology)

toxicity study in rats indicates that it provides reliable data for screening

for a variety of EDSP Tier 1 study endpoints as shown in Table 3 above.

This study covers a specific developmental period not examined in

mammals in the EDSP Tier 1 assays, in utero exposure. In utero exposure

in EDSP Tier 1 studies is limited to assays conducted with amphibians and

fish.

In this guideline study in rats, female Crl:COBSCD(SD)BR rats

were administered dicofol (95.6% purity with less than 0.1% DDT-related

impurities) by gavage at doses of 0, 0.25, 2.5 and 25 mg dicofol/ kg body

weight from gestation day six (GD6) through GDIS. It should be noted

that the gavage route of administration is not as relevant for human health

risk assessment but is the most routine route of exposure for teratology

studies.

Like all guideline prenatal toxicity studies, this study was designed

to examine the teratogenic potential of dicofol. As a result, certain

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endpoints relevant to endocrine activity were assessed (see Table 3). The

endpoints measured in the prenatal toxicity study that are directly

equivalent to endpoints monitored in EDSP Tier 1 assays (i.e., 890.1350,

890.1400, 890.1450, 890.1500, 890.1600) include: survival; body weight

(both adults and pups); developmental measures (fetotoxicity; skeletal and

soft tissue malformations); reproductive organ weights (gravid uterus);

and fertility indices (survival, pre- and post-implantation loss).

The most sensitive NOEL in the study was 0.25 mg/kg/day and

was based on clinical signs in the dams only (increased salivation at the

2.5 and 25 mg/kg exposure levels). The NOEL for developmental toxicity

was reported to be greater than 25 mg/kg/day. There were no reported

teratogenic effects in pups at any dose level. There were no effects on

endocrine organ histopathology at any dose level tested (e.g., various

reproductive organs in males and females exposed even during

development). There were no statistically significant effects on fertility

indices. These data demonstrated that dicofol was not toxic to endocrine

organ systems in this sensitive, in vivo test for toxicity during critical

periods of development in utero.

The data provided in this prenatal toxicity study are functionally

equivalent to data collected in EDSP Tier 1 assays that examine aspects of

mammalian endocrine function (i.e., 890.1350, 890.1400, 890.1450,

890.1500, and 890.1600). The data provide information on estrogen,

androgen, and thyroid hormone system toxicity of dicofol in vivo, and are

reliable dicofol OSRI.

6. Hoberman (1997), Hoberman (1998), and Lomax (1998)

Review of these GLP-compliant studies of reproduction and

postnatal endocrine effects in rats indicates that it provides reliable data

for screening for a variety of EDSP Tier 1 study endpoints as shown in

Table 3 above, endpoints relating to estrogen, androgen and thyroid

hormone system activity. Like the multi-generation reproductive toxicity

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study already discussed above, this study covers all developmental periods

covered by EDSP Tier 1 assays (in utero, lactation, post-natal, peripubertal, young adult, and adult).

In this study, male and female Crl:CDBR VAR/Plus (SD) rats

were administered 96.4% pure dicofol (less than 0.1% DDT-related

residues) in diet at doses of 0, 5, 25, and 125 ppm dicofol, starting with

pre-mating in the PI generation. Pups of the Fl generation were exposed

in utero, during lactation, and then placed on dicofol in diet immediately

at weaning through the post-natal period and through mating and

subsequent parturition. Thus, this study included exposure of Fl animals

of both sexes during peri-puberty, an important consideration for later

discussion of the pubertal rat EDSP Tier 1 assays. As with the other

guideline studies already discussed, the dietary route of administration is

more relevant to the expected human exposure to dicofol than data that

might be collected using other routes of administration in animals (e.g.,

oral gavage dosing, intraperitoneal dosing, subcutaneous dosing).

This study was specifically designed to assess potential endocrine

effects of dicofol and as a result had some specific design elements that

were unique. For example, a satellite group of animals was used for

determination of dicofol residues in blood, milk and tissues. This was

done in order to confirm that the fetus was exposed to dicofol throughout

development. The study also included evaluation of both estrous cyclicity

and ovarian follicle numbers. As a result of being a reproduction and

postnatal toxicity study, certain endpoints relevant to endocrine activity

were assessed (see Table 3). The endpoints measured that are directly

equivalent to endpoints monitored in EDSP Tier 1 assays (i.e., 890.1350,

890.1400, 890.1450, 890.1500, 890.1600) include: survival; body weight

(both adults and pups); developmental measures (fetotoxicity, preputial

separation, vaginal opening); estrous cyclicity; fertility; fecundity; and

weights and histopathological changes of endocrine-related organs and

reproductive organs.

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The NOEL for general toxicity in the study was 25 ppm dicofol

and was based on decreases in body weight and liver weight and

histopathological changes. There was an equivocal effect on ovarian

weight in Fl animals at the highest test dose (125 ppm). The NOEL for

reproductive effects was 125 ppm. The authors reported that dicofol

residues were detected in milk, blood and tissues of parental animals and

pups, confirming that exposure had occurred throughout development.

There were no adverse effects on reproduction or endocrine status of

parents or offspring. The study report dealing with evaluation of estrous

cyclicity (Hoberman 1998) revealed that there were no treatment-related

effects on estrous cyclicity in dicofol-treated rats. Moreover, the study

report on ovarian follicle counts (Lomax 1998) confirmed that the ovarian

weight changes seen in the primary study were not due to ovarian toxicity.

There were no dicofol-related effects on primordial plus growing follicle

counts in either the PI or the Fl generation at 125 ppm dicofol in diet.

These data demonstrated that dicofol was not directly toxic to endocrine

organs systems in this sensitive, in vivo test for toxicity during critical

periods of development, in utero and during lactation.

The data in this reproduction and endocrine effects study are

directly and functionally equivalent to data collected in EDSP Tier 1

assays that examine aspects of mammalian endocrine function (i.e.,

890.1350, 890.1400, 890.1450, 890.1500, and 890.1600). This study

provides reliable dicofol OSRI.

IV. MANA Seeks a Waiver for Performance of Nine of Eleven EDSP Tier 1 Tests

Based on Available Dicofol OSRI

There is a large and robust body of dicofol OSRI data for identifying the potential

endocrine activity of dicofol in mammalian organisms. The available data include in vitro

assessment of estrogen, androgen and thyroid hormone binding and/or function, and

effects of dicofol on enzymes that synthesize and metabolize estrogen and androgen

hormones in mammals (van den Berg et al. 1991; Vinggaard et al. 1999; Vinggaard et al.

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2000; Du and Xu 2001; Ishihara et al. 2003; Kojima et al. 2004; Okubo et al. 2004;

Shimada and Yamamuchi 2004; Kojima et al. 2005; Sugiyama et al. 2005; Vinggaard et

al. 2008; and Judson et al. 2009). Thus, in terms of estrogenicity, androgenicity and

thyroid hormone activity, the available data discussed above provide a wealth of

information on potential modes of action for dicofol on these endocrine systems.

Moreover, there are reliable, robust in vivo guideline toxicity studies that either directly

monitor, or provide functionally equivalent data for, all of the endpoints that would be

measured in Tier 1 EDSP assays. Based on the fact that none of the dicofol OSRI studies

demonstrate that dicofol is an overt toxicant to endocrine systems in test animals

(discussed above) and that there is a wealth of endocrine related endpoints already

measured and evaluated in available OSRI, there is no scientifically defensible reason to

perform most of the Tier 1 EDSP studies. As a result, MANA is asking for a waiver of

nine Tier 1 EDSP studies (discussed in detail below in Section IV and V).

The in vitro studies examining estrogen, androgen and thyroid hormone activity

indicated that dicofol interacted to a limited extent with estrogen and androgen functions

and/or receptors. In vitro studies, however, are only relevant as indicators of the potential

binding of a chemical to hormone receptors or indicators of the potential to affect

hormone metabolism. This is because the conditions of in vitro assays do not allow for

the known relationship of pharmacokinetics and metabolism to the pharmacodynamics of

chemical responses and chemical toxicity. Further, in the case of endocrine system

function, such isolated assays have limited predictive value for the conditions of living

organisms where endocrine regulation is complex and interrelated. Therefore, the in vitro

studies with dicofol are useful for identifying the need for collection of in vivo data that

should be evaluated for potential estrogen-related and androgen-related function (e.g.,

reproductive function). In the case of dicofol, this in vivo data was indeed collected (e.g.,

the multi-generation study in rats, and a special reproduction and endocrine effects study

in rats). The potency of dicofol in these in vitro studies was very low, with potencies in

the range of 10 to 10 times less than the naturally occurring hormones.

When evaluating the in vivo toxicological data, the types of studies and the dose

levels administered must both be considered. Such in vivo studies are more relevant

measures of endocrine toxicity potential as metabolic systems and the complex

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interactions of various hormonal systems are intact, unlike the conditions of most EDSP

Tier 1 tests (8 of 11 are either in vitro assays or involve models with non-intact hormonal

systems). The dicofol studies show that the compound does not appear to target endocrine

organs when toxicity is produced, as evidenced by a lack of effects on reproductive

organs (male and female) and function. A special study on endocrine and reproductive

effects demonstrated the lack of biologically relevant dicofol-related reproductive organ

toxicity. Although effects on thyroid gland histopathology were seen in one study

(subchronic study in rats), all of the other available guideline studies failed to report such

effects, and evaluations of thyroid hormone levels in the two year study in rats failed to

identify any significant effects on hormone levels. Any effects reported with dicofol

exposure that might be linked to endocrine activity were limited generally to high doses.

The dicofol guideline toxicology studies, like all such guideline in vivo toxicology

studies, were designed purposely to be valid, realistic evaluations of possible effects in

humans. Regulatory bodies rely on such studies to make decisions regarding safe levels

for human exposure. As a result, the availability of such studies makes the performance

of EDSP Tier 1 assays less relevant as screening assays for hazard assessment.

Based on the discussion already provided relating to the available dicofol

endocrine toxicity data, most of the Tier 1 EDSP tests need not be conducted if the goal is

to screen for potential endocrine activity. Considering the available data, MANA

proposes that only the following Tier 1 studies be conducted as part of the EDSP testing

program for dicofol: Fish Short-Term Reproduction (890.1350); and Steroidogenesis

(Human Cell Line H295R) (890.1550). The rationale for MANA's position is outlined

below regarding particular studies that do not need to be performed.

A. MANA Seeks a Waiver for Performance of Both Tier 1 In Vitro

Assays for Estrogen Activity (Estrogen Receptor Binding 890.1250 and

Estrogen Receptor Transcriptional Activation 890.1300)

EDSP Tier 1 assays 890.1250 and 890.1300 are screening assays to

identify modes of action for estrogenic activity involving binding to and

activation of estrogen receptors. There are adequate in vitro estrogen receptor

activity data available for determining the ability of dicofol to bind to and activate

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estrogen receptors in vitro. These studies have been published in the peerreviewed scientific literature (Table 3) using a variety of standard in vitro assays

for screening for ER binding potency and ER activation. Although none of the

studies is technically identical in design to the EDSP Tier 1 ER studies (890.1250;

890.1300), the available studies are both directly and functionally equivalent and

provide data on essentially the same endpoints, including comparison of the

activity of dicofol to appropriate control agents. The results have been consistent

in showing that dicofol is, at best, a weak estrogenic agent, with potency several

orders of magnitude less than positive control agents (i.e., 17(3-estradiol); activity

of dicofol in vitro is only seen at doses in the micromolar range. Therefore,

MANA believes there is no scientific basis for performing additional in vitro

estrogen receptor activity studies, specifically the Estrogen Receptor Binding

assay (890.1250) and the Estrogen Receptor Transcriptional Activation (Human

Cell Line HeLa-9903) assay (890.1300). The available data are adequate for

screening for the activity of dicofol at ER receptors. As a result, a waiver of both

890.1250 (Estrogen Receptor Binding assay) and 890.1300 (Estrogen Receptor

Transcriptional Activation assay) is sought.

B. MANA Seeks a Waiver for Performance of the Tier 1 In Vitro Assays

for Androgen Activity (Androgen Receptor Binding 890.1150 and Aromatase

Activity 890.1200)

EDSP Tier 1 assays 890.1150 and 890.1200 are screening assays to

identify modes of action for androgenic and anti-androgenic activity involving

binding to androgen receptors or alterations in conversion of androgens to

estrogens. There are adequate in vitro androgen receptor activity data available for

determining the ability of dicofol to bind to androgen receptors. The study by

Judson et al. (2009) provide data on both androgen receptor binding potency,

agonist and antagonist activity, as well as the potential for dicofol to inhibit

aromatase activity in vitro. Further, other available studies (Vinggaard et al. 2000;

Kojima et al. 2004; Vinggaard et al. 2008; Judson et al. 2009) provide directly

and functionally equivalent data on essentially the same endpoints, including

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comparison of the activity of dicofol to appropriate control agents. Thus, there are

adequate OSRI for screening for the activity of dicofol at AR receptors and on

aromatase function. Thus, there is no scientific basis for performing additional in

vitro androgen receptor activity studies, specifically the Tier 1 Androgen

Receptor Binding assay (890.1150), as well as no scientific basis for performing

additional in vitro aromatase activity studies, specifically the Tier 1 Aromatase

assay (890.1200). As a result, a waiver of both 890.1150 (Androgen Receptor

Binding assay) and 890.1200 (Aromatase assay) for dicofol are sought.

C. MANA Seeks a Waiver for Performance of the Tier 1 Amphibian

Metamorphosis Assay (890.1100)

The Amphibian Metamorphosis assay (890.1100) is described in the

guideline as a "screening assay intended to empirically identify substances which

may interfere with the normal function of the hypothalamic-pituitary-thyroid

(HPT) axis". It is intended to be a generalized vertebrate model that is based on

the conserved structures and functions of the HPT axis of various species. It is

also pointed out that amphibian metamorphosis provides "a well-studied, thyroiddependent process which responds to substances active within the HPT axis, and

it is the only existing assay that detects thyroid activity in an animal undergoing

morphological development". MANA is seeking to waive the requirement for

performance of this study because there are adequate in vitro data on thyroid

hormone receptor binding and activation (van den Berg et al. 1991; Ishihara et al.

2003; Shimada and Yamamuchi 2004; Sugiyama et al. 2005) to identify the

potential effects of dicofol on thyroid hormone activity. More importantly, there

are in vivo data collected in guideline toxicology studies that cover all stages of

mammalian development, and are consistent in showing that dicofol has no

biologically significant effects on functionally equivalent endpoints in

mammalian species (i.e., thyroid histopathology, thyroid hormone levels in rats).

For example, the dicofol OSRI include data from a multi-generation reproductive

toxicity study in rats, a prenatal toxicity study in rats, and a special reproduction

and endocrine effects study in rats. All of these studies demonstrated that dicofol

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does not induce developmental and morphological effects, including none of the

possible apical effects that would result from either increasing or decreasing

thyroid hormone levels in mammals (e.g., growth, metabolic rate and function,

fetal brain development, and Table 1 above). Further, results from the chronic

studies in rats and dogs demonstrated no effects on thyroid gland histopathology,

effects that would be expected to appear if dicofol were producing direct effects

in vivo on either thyroid hormone synthesis or function. It is also pointed out that

the guideline chronic toxicity study with dicofol in rats measured levels of T3, T4

and TSH, in order to address some equivocal effects seen in the subchronic study

in rats. Finally, Sugiyama et al. (2005) reported that dicofol lacked in vivo

activity to affect thyroid hormone activity in tadpoles undergoing metamorphosis.

Performing the amphibian assay merely to monitor changes in thyroid hormone

levels, or sex hormone levels, in developing amphibians is not scientifically

justified when robust in vivo data in developing organisms is already available

and does not indicate that dicofol is producing toxicity due to disruption of the

thyroid hormone system. Therefore, a waiver of 890.1100 (Amphibian

Metamorphosis assay) is sought because there is no scientific basis for performing

an additional study when the available dicofol OSRI are considered.

D. MANA Seeks a Waiver for Performance of the Tier 1 Female

Pubertal Assay (890.1450)

The purpose of the pubertal female rat assay (890.1450) is stated in the

guideline to be identifying chemicals that have a potential to interact with the

endocrine system by identifying effects on pubertal development and thyroid

function in intact peri-pubertal rats. There are adequate in vivo OSRI available for

dicofol demonstrating that this chemical is not a potent estrogenic or antiestrogenic agent in vivo. There are also adequate in vivo OSRI available for

dicofol, including measurements of thyroid hormone levels in vivo, demonstrating

that this chemical does not have biologically relevant effects on thyroid hormone

function. The OSRI includes the robust, guideline registration studies (Table 3).

The in vivo evaluations of female reproductive function and developmental

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toxicity in rats have provided data on exposure of animals at all stages of

development (in utero, during lactation, throughout post-natal development,

throughout peri-pubertal development, during adulthood, and during senescence)

as well as providing data on many of the same endpoints found in EDSP Tier 1

studies (Table 3). For example, in the Tier 1 Female Pubertal assay (rat),

important endocrine endpoints monitored include identification of abnormalities

associated with sex organs and puberty markers (see Table 2 above), as well as

thyroid tissue. The guideline toxicity studies in rats such as the two-generation

reproductive toxicity study, the prenatal toxicity study, the reproduction and

endocrine effects study, and the subchronic and chronic toxicity studies, all

included determination of dicofol's effects on female reproductive tissues and

organs, both in adult animals and in developing animals (including peri-pubertal

animals), including examination of effects across generations. These studies also

included histopathological examination of thyroid gland tissue in animals exposed

both during gestation and development and as adults. The guideline reproductive

toxicity and developmental toxicity studies provide adequate data on female

reproductive organ effects following dicofol exposure, as well as fertility,

fecundity, vaginal opening, and estrous cycling. Finally, in vivo measures of

thyroid hormone levels in rats were provided with the chronic rat toxicity study.

Thus, the only endpoint from the EDSP Tier 1 in vivo assay in pubertal

female rats that has not been directly measured in the currently available dicofol

guideline toxicity studies is the level of thyroid hormone in serum of peri-pubertal

following dicofol exposure. As already discussed levels of thyroid hormones were

measured in the chronic rat study. Given that the ultimate apical effects of either

increasing or decreasing thyroid hormone levels in mammals have been

monitored during all stages of development in the available studies (i.e., mutigeneration rat study, prenatal toxicity study, special endocrine and reproduction

effects study), there does not appear to be a strong scientific basis for requiring

performance of the Tier 1 pubertal female screening study (890.1450) simply to

provide for measures of thyroid hormone levels in peri-pubertal rats. Therefore, a

waiver of EDSP Tier 1 890.1450 (Pubertal Female Rat Development) is sought.

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E. MANA Seeks a Waiver for Performance of the Tier 1 Male Pubertal

Assay (890.1500)

The purpose of the pubertal male rat assay (890.1500) is stated in the

guideline to be identifying chemicals that have a potential to interact with the

endocrine system by identifying effects on pubertal development and thyroid

function in intact peri-pubertal rats. There are ample in vivo dicofol OSRI on male

reproductive function and pathology to indicate that the systems that involve

androgen activity are not uniquely targeted by dicofol and that they are not

uniquely sensitive to the effects of dicofol. The dicofol OSRI studies include the

robust guideline studies in rats such as the two-generation reproductive toxicity

study, the developmental toxicity study, the reproduction and endocrine effects

study in rats, and the subchronic and chronic toxicity studies (Table 3). These in

vivo evaluations of male reproductive function, developmental toxicity, as well as

general toxicity in rats have provided data on the same endpoints as generated in

assays performed as part of Tier 1 in the EDSP, including the male pubertal rat

study (890.1500). For example, in the Tier 1 Male Pubertal assay (rat), the test is

used to screen for androgen, anti-androgen, and thyroid activity in male animals

during sexual maturation by examining abnormalities associated with sex organs

and puberty markers (see Table 2 above), as well as thyroid tissue. The guideline

dicofol toxicity studies in rats (Table 3) all included determination of dicofol's

effects on androgen-sensitive male reproductive tissues and organs, both in adult

animals and in developing animals (including peri-pubertal animals), including

evaluation of effects across generations. These studies also included

histopathological exaination of thyroid gland tissue in animals exposed both

during gestation and development, and as adults. One study (the chronic toxicity

study in rats) even measured levels of T3, T4 and TSH in animals following

dicofol exposure for up to two years.

The only endpoints from the Tier 1 in vivo assay in pubertal male rats that

has not been directly measured in the currently available dicofol guideline toxicity

studies is the level of sex hormones (i.e., testosterone) in serum of animals

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Study No. 2010-DIC-OL-0311

following dicofol exposure, and the level of thyroid hormone in the serum of peripubertal rats following dicofol exposure. With respect to measurement of sex

hormone levels in animals (i.e., testosterone), the fact that none of the in vivo

data, including the multi-generation reproductive toxicity study in rats and the

reproduction and endocrine effects study in rats, indicated toxicity induced by

dicofol that could be related to significant changes in hormone levels alone, there

does not appear to be a strong scientific basis for requiring performance of EDSP

Tier 1 screening studies simply to provide for measures of sex hormone levels in

male rats. Further, given that the ultimate effects of either increasing or

decreasing thyroid hormone levels in mammals have been monitored in the

available studies (e.g., growth, metabolic rate and function, fetal brain

development), and that thyroid hormone levels were measured in the chronic rat

study, there does not appear to be a strong scientific basis for requiring

performance of a study, such as the male pubertal assay in rats, simply to provide

for measures of thyroid hormone levels in peri-pubertal rats. Therefore, a waiver

of EDSP Tier 1 890.1500 (Pubertal Male Rat Development) is sought.

F. MANA Seeks a Waiver for Performance of the Tier 1 Hershberger

Assay (890.1400)

The purpose of the Hershberger assay (890.1400) is stated to be an in vivo

screening assay providing data about a single endocrine mechanism,

androgenicity. In the assay, castrated rats are exposed for ten days (subcutaneous

injection) to the test chemical. The major endpoints monitored are changes in

androgen-dependent tissue and organ weights. Clearly, there are adequate dicofol

OSRI that are directly equivalent, monitoring weight changes in a variety of male

reproductive organs (androgen-dependent organs) in animals of all stages of

development. The OSRI include several guideline studies that involved dosing for

up to two years (Hazelton and Harris 1989), as well as dosing throughout all

stages of development (Solomon and Kulwich 1991; Hoberman and Christian

1986; Hoberman 1997). The fact that two reproduction studies were performed

with dicofol, one directed to endocrine effects, provides even greater assurance

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that the potential androgen toxicity of dicofol has been adequately evaluated. As a

result, there is no scientific basis for conducting another in vivo assay with dicofol

where androgen-sensitive organ weight changes are monitored as in the

Hershberger assay. Therefore, a waiver of EDSP Tier 1 890.1600 (Hershberger

assay) is sought.

G. MANA Seeks a Waiver for Performance of the Tier 1 Uterotrophic

Assay (890.1600)

The purpose of the Uterotrophic assay (890.1600) is stated to be an in vivo

screening assay providing data about a single endocrine mechanism,

estrogenicity. In the assay, ovarectomized rats are exposed for three days

(subcutaneous injection) to the test chemical. The major endpoint monitored is

uterine weight changes. In the case of dicofol, there are adequate OSRI that are

directly equivalent, monitoring uterine weight changes in animals of all stages of

development. The OSRI include several guideline studies that involved dosing for

up to two years (Hazelton and Harris 1989), as well as dosing throughout all

stages of development (Solomon and Kulwich 1991; Hoberman and Christian

1986; Hoberman 1997). The fact that two reproduction studies were performed

with dicofol, one directed to endocrine effects, provides even greater assurance

that the potential estrogenicity of dicofol has been adequately assessed. As a

result, there is no scientific basis for conducting a Uterotrophic assay with dicofol.

Therefore, a waiver of EDSP Tier 1 890.1600 (Uterotrophic assay) is sought.

V. Overall Summary

The preceding discussion provides a detailed description of available dicofol

OSRI that are used to support waivers for most of the EDSP Tier 1 screening assays. As

already discussed, the objective of the OSRI identification process was to determine

whether equivalent OSRI existed to support the waiver of an EDSP Tier 1 study.

Interpretation of the results gathered in specific OSRI studies was beyond the scope of

this process. The dicofol OSRI is rich in information including information specific to

screening for the potential for estrogen, androgen and thyroid hormone system effects as

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well as the effects of dicofol on specific endpoints (i.e., potential modes of action).

MANA is seeking waivers for performance of nine EDSP Tier 1 studies: 890.1100

(Amphibian Metamorphosis); 890.1150 (Androgen Receptor Binding); 890.1200

(Aromatase); 890.1250 (Estrogen Receptor Binding); 890.1300 (Estrogen Receptor

Transcriptional Activation); 890.1400 (Hershberger assay); 890.1450 (Pubertal Female

Rat Development); 890.1500 (Pubertal Male Rat Development); and 890.1600

(Uterotrophic assay).

MANA proposes to conduct the Steroidogenesis assay (890.1550) in order to

collect data on the potential mode of action for dicofol to directly affect synthesis of

steroid hormones, both androgens and estrogens. Additionally, MANA proposes to

conduct the Fish Short-Term Reproduction assay (890.1350) in order to screen for

potential endocrine toxicity of dicofol in fish, even though adequate dicofol OSRI are

available on the potential reproductive toxicity of dicofol in mammals.

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VI. References Cited

Du, K, Xu, X. (2001) Dicofol stimulation of cell proliferation. Bull. Environ. Contam.

Toxicol. 67:795-799.

Goldman, P.R. et al. (1986) Kelthane: three month dietary toxicity study in rats. Report

No. 85R-093. Rohm and Haas Company. MRID No. 00157324.

Hazelton, G.A. and Harris, J.C. (1989) Dicofol (Kelthane Technical Miticide): 24 month

dietary chronic/oncogenic study in rats. Report No. 86R-190. Rohm and Haas

Company. MRID No. 41150001.

Hoberman, A.M. (1997) Reproduction and postnatal study of potential endocrine effects

of Dicofol administered orally (diet) to rats. Report No. 95RC-119. Rohm and

Haas Company. MRID No. 44253801.

Hoberman, A.M. (1998) Reproduction and postnatal study of potential endocrine effects

of Dicofol administered orally (diet) to rats. Report Supplement (April 2). Rohm

and Haas Company. MRID No. 44559901.

Hoberman, A.M. and Christian, M.S. (1986) Dicofol (Kelthane Technical Miticide):

Developmental toxicity study of Dicofol administered via gavage to

Crl:COBSCD(SD)BR presumed pregnant rats. Rohm and Haas Company. MRID

No. 40042046.

Ishihara, A., Sawatsubashi, S., Yamamuchi, K. (2003) Endocrine disrupting chemicals:

interference of thyroid hormone binding to transthyretins and to thyroid hormone

receptors. Mol. Cell. Endocrinol. 199:105-117.

Judson, R.S., Houck, K.A., Kavlock, R.J., Knudsen, T.B., Martin, M.T., Mortensen,

H.M., Reif, D.M., Rotroff, D.M., Shah, I., Richard, A.M., and Dix, D.J. (2009) In

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Study No. 2010-DIC-OL-0311

vitro screening of environmental chemicals for targeted testing prioritization - the

ToxCast project. Environ. Health Perspect. Online December 2009.

Klimisch, H-J., Andreae, M., and Tillman, U. (1997) A systematic approach for

evaluating the quality of experimental toxicological and ecotoxicological data.

Regul. Toxicol. Pharmacol. 25:1-5.

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

for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter

gene assays using Chinese hamster ovary cells. Environ. Health Perspect.

112:524-531.

Kojima, M., Fukunaga, K., Sasaki, M., Nakamura, M., Tsuji, M. and Nishiyama, T.

(2005) Evaluation of estrogenic activities of pesticides using an in vitro reporter

gene assay. Int J Environ Health Res 15:271-280.

Lomax, L. (1998) Ovary follicle counts (supplemental to reproduction and postnatal

study of potential endocrine effects of Dicofol administered orally (diet) to rats).

Supplemental report July 20. Rohm and Haas Company. MRID No. 44624301.

Okubo, T., Yokoyama, Y., Karo, K., Soya, Y., Kario, I. (2004) Estimation of estrogenic

and anti-estrogenic activities of selected pesticides by MCF-7 cell proliferation

assay. Arch. Environ. Contain. Toxicol. 46:445-453.

Shimada, N., and Yamamuchi, K. (2004) Characteristics of 3,5,3'-triiodothyronine (T3)-

uptake system of tadpole red blood cells: effect of endocrine disrupting chemicals

on cellular T3 response. J. Endocrinol. 183:627-637.

Solomon, H.M. and Kulwich, B.A. (1991) Dicofol: 2-generation reproduction study in

rats. Report No. 89R-028. Rohm and Haas Company. MRID No. 41806601.

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Study No. 2010-DIC-OL-0311

Sugiyama, S.-I., Shimada, N., Miyoshi, H., Yamamuchi, K. (2005) Detection of thyroid

system-disrupting chemicals using in vitro and in vivo screening assays in

Xenopus laevis. Toxicol. Sci. 88:367-374.

Tegeris, A.S. (1988) Dicofol (Kelthane Technical Miticide): One year dietary tosicity

study in Beagle dogs. Report No. 86014. Rohm and Haas Company. MRID No.

40997101.

Van den Berg, K.J., Raaij, J.A.G.M., Bragt, P.C., Notten, W.R.F. (1991) Interactions of

halogenated industrial chemicals with transthyretin and effects on thyroid

hormone levels in vivo. Arch. Toxicol. 65:15-19.

Vinggaard, A.M., Breinholt, V., Larsen, J.C. (1999) Screening of selected pesticides for

oestrogenic receptor activity in vitro. FoodAddit. Contain. 16:533-542.

Vinggaard, A.M., Hnida, C., Breinholt, V., Larsen, J.C. (2000) Screening of selected

pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol. In Vitro

14:227-234.

Vinggaard, A.M., Niemela, J., Wedebye, E.B., Jensen, G.E. (2008) Screening of 397

chemicals and development of a quantitative structure-activity model for

androgen receptor antagonism. Chem. Res. Toxicol. 21:813-823.

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