Makhteshim Agon of

North America, Inc.

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M ANA

Via Courier Delivery

March 11,2010

Document Processing Desk (PRD-EDSP)

Office of Pesticide Programs (7504P)

U.S. Environmental Protection Agency (South Bldg)

One Potomac Yard, Room S-4900

2777 South Crystal Drive

Arlington, VA 22202-4501

•••••• "••'•J

Attention: Ms. Melanie Biscoe

Chemical Review Manager - Endosulfan

Subject. EDSP Test Order (EDSP -079401-102)

Submission of 90-day response for Endosulfan,

Thionex Technical (EPA Reg. No. 11678-5)

Dear Ms. Biscoe:

Herewith, Makhteshim-Agan of North America, Inc. (MANA) on behalf of Makhteshim

Chemical Works Ltd. (MCW) is submitting the Initial or 90-day EDSP Test Order response for

endosulfan (Thionex Technical, EPA Reg. No. H678-5; Order/DCI #: EDSP- 079401-102).

Our response includes the following documents:

• Transmittal Document

• Executed Initial Response Form for Individual Order Recipients

• Volume 1 of 2 (3 copies): Response to the U.S. Environmental Protection Agency Testing

Order under the Endocrine Disruptor Screening Program (EDSP): Discussion of Other

Scientifically Relevant Information (OSRI) for Endosulfan

• Volume 2 of 2 (3 copies): Supplemental Data for Makhteshim Chemical Works, Ltd. -

Endosulfan Endocrine Disruption Screening Program

In the attached response (Vol. 1 of 2), we identified two sources regarding the Other Scientifically

Relevant Information (OSRI) for endosulfan: 1) the peer-reviewed, published scientific literature;

and 2) the endosulfan pesticide registration studies. In order to ensure that only reliable,

scientifically valid OSRI were included in this assessment, 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 for endosulfan OSRI included identifying a reliability indicator for

each study. As outlined by Klimisch et al. (1997), reliability can be differentiated into four

categories: Category 1: "reliable without restriction"; category 2: "reliable with restrictions";

4515 Falls of Neuse Road, Suite 300, Raleigh, NC 27609

Tel: 919-256-9305 Fax:919-256-9308
category 3: "not reliable"; and category 4: "not assignable". In our evaluation of endosulfan OSRI,

only studies that could be given a category rating of either 1 or 2 were used. This means, that only

studies that were GLP-compliant or comparable to a GLP-compliant 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 studies and publications meeting those criteria are enclosed in Volume 2 of 2 of this

submission.

The enclosed response is a detailed description of available endosulfan OSRI that is used to

support waivers for all eleven of the EDSP Tier 1 screening assays. The endosulfan OSRI is very

extensive, including information specific to assessing the potential for estrogen, androgen and

thyroid hormone system effects as well as the effects of endosulfan on specific endpoints (i.e.,

potential modes of action). MCW is seeking waivers for performance of all eleven of the 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); 890.1350 (Fish Short-Term Reproduction Assay); 890.1400

(Hershberger assay); 890.1450 (Pubertal Female Rat Development); and 890.1500 (Pubertal Male

Rat Development); 890.1550 (Steroidogenesis); and 890.1600 (Uterotrophic assay).

If you have any questions or need further information, please contact me at 610-793-3222 or send

me an e-mail at bertvolger@comcast.net.

Sincerely,

Bert Volger, Ph.D.

Consultant for MANA

cc: L. Pruett - MANA
TRANSMIT!AL DOCUMENT

EDSP TEST ORDER

ENDOSULFAN INITIAL RESPONSE

SUBMITTER

Makhteshim Chemical Works Ltd.

(EPA Company No. 11678)

c/o Makhteshim Agan of North America, Inc.

4515 Falls of Neuse Rd., Suite 300

Raleigh NC 27609

DATE

March 11, 2010

TEST ORDER NUMBER

EDSP-079401-102

DOCUMENTS

• Transmittal Document

• Executed Initial Response Form for Individual Order Recipients

• Volume 1 of 2 (3 copies): 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 Endosulfan

• Volume 2 of 2 (3 copies): Supplemental Data for Makhteshim Chemical Works, Ltd. -

Endosulfan Endocrine Disruption Screening Program
TITLE

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 Endosulfan

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 11,2010

STUDY NO.

MA-END-EDSP-20100313

SUBMITTER

Makhteshim Chemical Works, Ltd.

Beer Sheva 84100, Israel

c/o Makhteshim Agan of North America, Inc.

4515 Falls ofNeuse Road, Suite 300

Raleigh, NC 27609

VOLUME 1 OF 2

Page 1 of 64

• - • C (, •

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Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

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: Makhteshim Chemical Works, Ltd.

Beer Sheva 84100, Israel

Company Representative:

Bert Volger, Authorized Representative

Makhteshim Chemical Works, Ltd.

Date: March 11,2010
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

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:

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

Date: March 11,2010

Submitter/Sponsor:

Bert Volger

Authorized Representative

Makhteshim Chemical Works, Ltd.

Date: March 11,2010
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

Table of Contents

I. Introduction 5

II. Background 5

III.. Discussion of Endosulfan OSRI 11

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

Literature 20

B. Directly or Functionally Equivalent Endosulfan OSRI Identified in Endosulfan

Registration Studies..... 36

IV. MANA Seeks a Waiver for All of the Tier 1 EDSP Assays that Screen for Endocrine

Disruption Potential of Endosulfan 48

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) 50

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)...51

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

(890.1550) 52

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

Assay (890.1100) 52

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

(890.1450) 54

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

(890.1500) 55

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

(890.1600) 56

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

(890.1400) 57

I. MANA Seeks a Waiver for Performance of the Tier 1 Fish Short-Term Reproduction

Assay (890.1350) 58

V. Overall Summary 58

VI. References Cited 60
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

I. Introduction

On December 3, 2009, the U.S. Environmental Protection Agency (EPA) issued a

Testing Order (TO) for endosulfan (CAS # 115-29-7) related to testing under the EPA

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

submission of EDSP Tier 1 testing for endosulfan. 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 Makhteshim Chemicals Works (MCW). This response 1)

outlines the extensive data already available on the endocrine disruption potential of

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

discusses how available OSRI provides for screening for 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 all of the 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
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

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

identify compounds that are anti-androgens, compounds that are estrogenic, and

compounds that affect 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 chemicals such as

endosulfan, which are 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

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

6
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

performed to support registration of endosulfan as well as scientific studies on endosulfan

published in the open literature. Before discussion of each endosulfan 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 endosulfan 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 that particular EDSP Tier 1 assay.
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Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

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, peripubertal 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 endosulfan.

Before discussing the OSRI for endosulfan 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 multigeneration 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

evaluate the potential endocrine toxicity of chemicals.

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

studies, hi order to ensure that only reliable, scientifically valid OSRI were included in

this evaluation for endosulfan, 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 for endosulfan OSRI included identifying a reliability

10
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

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 our evaluation of endosulfan 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-compliant' 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 endosulfan 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 endosulfan 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 EDSP Tier 1 endpoints that are

adequately evaluated through use of OSRI. Following the discussion of the OSRI data,

MANA has provided a specific rationale for why certain EDSP Tier 1 assays should be

waived for endosulfan based on the available OSRI (Section IV of this document).

III. Discussion of Endosulfan OSRI

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

functionally equivalent to data collected in EDSP Tier 1 testing. Tables 3 and 4 below

provide a listing of each source of OSRI for endosulfan 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. Table 3 provides a listing of

the mammalian endosulfan OSRI organized by endpoints from the mammalian Tier I

EDSP assays. Table 4 provides a listing of non-mammalian endosulfan OSRI (i.e.,

1

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

EPA.

11
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

studies in birds, amphibians, and fish) organized by endpoints from non-mammalian Tier

1 EDSP assays. Some endosulfan published OSRI appear multiple times because they

contain multiple OSRI endpoints. The appearance of "yes" throughout the tables in any

column or row indicates that the particular endpoint is measured in the particular

endosulfan OSRI referenced, whereas no entry or a "blank box" in the table indicates that

the endpoint is not measured in the particular endosulfan OSRI studies referenced.

Following Tables 3 and 4, 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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Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

A. Directly or Functionally Equivalent Endosulfan OSRI Identified in

the Published Literature

A search of the published scientific literature identified 21 different

sources of endosulfan OSRI that are directly equivalent to data collected in EDSP

Tier 1 assays. These studies include: Soto et al. (1994); Soto et al. (1995); Wade

et al. (1997); Ramamoorthy et al. (1997); Arcaro et al. (1998);Wilson and

LeBlanc (1998); Hunter et al. (1999); Legler et al. (1999); Wong and Matsumura

(2000); Newbold et al. (2001); Anderson et al. (2002); Fang et al. (2003);

Gormley and Teather (2003); Grunfeld and Bonefeld-Jorgensen (2004); BpnefeldJorgensen et al. (2005); Lemaire et al. (2006); Laville et al. (2006); Varayoud et

al. (2008); Vinggaard et al. (2008); Judson et al. (2009); and Brunelli 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.

1. Soto et al, (1994)

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

activity of endosulfan.

In this in vitro study, six pesticides compounds, including

endosulfan, were individually tested for estrogen receptor binding activity

using the E-SCREEN assay. In addition to testing the racemic mixture of

endosulfan, both endosulfan-a and endosulfan-p were also tested. The ESCREEN is a standard assay employed in many laboratories and measures

the comparative potency of individual test compounds to induce

proliferation of MCF-7 cells (human breast cancer cells) in culture. This

cell line expresses estrogen receptors, i.e., estrogen receptor-a (ERa), and

stimulation of estrogen receptors by compounds such as estradiol leads to

increased proliferation of MCF-7 cells. The authors report use of 1?Pestradiol to validate the responsiveness of their assay. 17p-estradiol is the

positive control agent used in EDSP Tier 1 assays. In Table 1 from the

paper, the authors report findings for the tested compounds. Endosulfan is

20
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

reported to show estrogen receptor binding activity at doses of 10 and 25

uM and a relative potency as compared to 17p-estradiol of 0.0001% or

activity that is four orders of magnitude less than the positive control

compound, indicating only weak activity in this assay. The data provided

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

890.1250 assay and are reliable endosulfan OSRI.

2. Soto et al (1995)

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

activity of endosulfan.

In this in vitro study of a variety of compounds, including

endosulfan, the authors report results of testing using the E-SCREEN

assay. Again, this is a standard assay employed in many laboratories and

measures the comparative potency of individual test compounds to induce

proliferation of MCF-7 cells in culture. The authors report use of 170-

estradiol as well as a variety of other known estrogen receptor agonists to

validate the responsiveness of their assay. l?p-estradiol is the positive

control agent used in EDSP Tier 1 assays. In Table 6 from the paper, the

authors report findings from the E-SCREEN for endosulfan and each of its

isomers, separately. Endosulfan and both isomers are reported to show

estrogen receptor binding activity at doses of 10 uM that represents a

relative potency as compared to 17(3-estradiol of 0.0001%, or activity that

is four orders of magnitude less than the positive control compound,

indicating only weak activity in this assay. The data provided are directly

equivalent to the type of data collected in the EDSP Tier 1 890.1250 assay

and are reliable endosulfan OSRI.

3. Wade et al. (1997)

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

21
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

potency of endosulfan in vitro as well as providing reliable in vivo data on

uterine weight changes from an Uterotrophic assay in endosulfan-exposed

immature rats.

In the in vitro portion of the study, a rat uterine estrogen receptor

binding displacement assay was performed. The assay employed two

concentrations of rat uterine cytosol to provide for determination of

binding in the presence of a high concentration of estrogen receptors (0.48

nM) and a low concentration of estrogen receptors (0.065 nM). Data were

collected using standard competitive displacement binding techniques

with displacement of [3H]-labeled 17(3-estradiol being quantified.

Scatchard analysis was performed and the ICso values for unlabeled 17(3-

estradiol were 0.37 nM (high receptor concentration) and 0.19 nM (low

receptor concentration). In contrast, endosulfan exhibited only weak

estrogen receptor binding ability with binding inhibited only at a dose of

10 uM in the presence of high concentrations of estrogen receptors and at

2 as well as 10 uM when tested with low concentrations of estrogen

receptors. Next, endosulfan was tested in a MCF-7 cell proliferation assay.

As already discussed above, this is a standard assay for screening for

estrogen receptor binding ability of chemicals. Endosulfan increased cell

proliferation in this assay but only at the highest test dose of 50 uM. The

authors characterize endosulfan as "weak" in this assay of in vitro

estrogenic activity. The data provided in this study are directly equivalent

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

reliable endosulfan OSRI.

The in vivo portion of the study is consistent in design with the

Tier 1 EDSP Uterotrophic assay (890.1600). Immature (18 days old)

Sprague-Dawley rats were injected for three days subcutaneously with 3

mg/kg endosulfan. As a control, another group of immature rats was

injected with 0.1 mg diethylstilbesterol (DBS), a known estrogenic agent.

The positive control agent, DBS, produced an 80% increase in uterine

weight in treated rats while treatment with endosulfan was not associated

22
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

with any increased uterine weight in the immature rats. The data provided

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

EDSP Tier 1 890.1600 assay, and are functionally equivalent to the data

collected in the 890.1250 assay, and are reliable endosulfan OSRI.

4. Ramamoorthy et al. (1997)

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

potential of endosulfan.

In this study, a yeast-based estrogen receptor assay was used to

assess the transactivation potential of endosulfan. The system employed a

mouse estrogen receptor and a reporter plasmid containing a single ERE

linked to the LacZ gene. The assay measured p-galactosidase activity as an

endpoint of estrogen receptor transactivation. Endosulfan was one of four

pesticides that was tested individually. The activity of endosulfan was

reported relative to the activity of the positive control agent DES. The

authors reported that endosulfan increased gene expression only at a dose

of 2.5 x 10-5 M (25 uM) and that this level of activity was five orders of

magnitude less than DES. 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 endosulfan OSRI.

5. Arcaro et al. (1998)

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

potential of endosulfan.

The authors report results of testing in vitro using a MCF-7 foci

assay and a competitive estrogen receptor binding assay. Several

compounds were tested individually, including endosulfan. Compounds

were also tested for potential synergy in these same assays. The MCF-7

23
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

foci assay is similar to the basic cell proliferation assay used to screen for

estrogen receptor binding. In the assay, endosulfan was tested for its

ability to induce foci (estrogenic activity) and to inhibit foci induced by 1

nM 17(3-estradiol (anti-estrogenic activity). In the competitive binding

assay, human recombinant estrogen receptors (1.2 nM concentration) were

tested using [3H]-labeled 17p-estradiol with standard receptor binding

methods. Endosulfan increased foci formation in MCF-7 cells only at

concentrations greater than 10 uM and the authors reported no detectable

binding to estrogen receptors in the competitive binding assay. Therefore,

consistent with results of other studies, endosulfan exhibited only weak in

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

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

endosulfan OSRI.

6. Wilson and LeBlanc (1998)

Review of this study indicates that it provides reliable, nonguideline, in vivo data for screening for the potential for endosulfan to

affect steroid hormone homeostasis in vivo.

In this in vivo study, male and female CD-I mice that were

sexually mature were administered endosulfan in the diet for seven days

(doses = 0, 3.8, 7.5 and 15 mg/kg/day). The goal of the study was to

determine whether endosulfan affects testosterone metabolism and

elimination in male and female mice and if any effects resulted in

alterations in testosterone homeostasis (i.e., were biologically relevant).

The authors examined formation of testosterone metabolites in liver

preparations from mice at sacrifice. In one group of female mice

administered 7.5 mg/kg endosulfan for seven days, the authors also

monitored elimination of [14C]-radiolabeled testosterone that was injected

intraperitoneally. These animals were then housed in metabolism cages for

collection of feces and urine in order to monitor elimination of

testosterone.

24
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

The authors reported that there were several deaths in the two

highest dose groups in male animals only. They report also that in female

mice only there was a dose-dependent increase in the rate of testosterone

hydroxylated metabolite formation, as well as an increased rate of urinary

elimination of testosterone in females only. No changes in testosterone or

estradiol levels in serum were reported in these animals even though there

were effects on enzyme activity and rate of elimination. The authors

concluded that although there were endosulfan-induced effects on

testosterone metabolism and elimination kinetics, these changes were not

sufficient to affect hormone homeostatsis.

The data provided in this study are functionally equivalent to the

data collected in the 890.1450 and 890.1500 assays (i.e., sex hormone

levels). Thus, these data also provide reliable endosulfan OSRI.

7. Hunter et al (1999)

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

potential and the estrogen receptor transactivation potential of endosulfan.

In this study, estrogen receptor binding was directly measured as

was estrogen receptor transactivation potential. Inhibition of [3H]-labeled

estradiol binding to recombinant human ERa was measured in a Sf9

baculoviral system. The authors report that endosulfan exhibited very

weak activity in this assay as they were unable to calculate an ICso.

Competitive binding activity was only observed at a 10 uM endosulfan

concentration, a dose that was associated with less than 20% binding

inhibition. Then, in an in vitro estrogen receptor-controlled cell

proliferation assay using ELT3 cells, the ability of endosulfan to affect cell

proliferation was measured. These cells exhibit proliferative responses at

an estradiol concentration of 10 nM. When endosulfan-p was tested, it

exhibited no activity in this assay. Endosulfan-a showed limited activity at

\

a 10 uM exposure level (25% of the 10 nM estradiol response). Finally,

25
Makhteshim Chemical Works, Ltd.

Study No. MA-END-EDSP-20100313

the authors report results from a reporter gene transactivation assay (ELT3

cells transfected with firefly luciferase linked to vitellogenin ERE and a

promoter). In this assay, endosulfan-a and endosulfan-p both showed

activity at a 10 uM dose level, as compared to activity with estradiol in

this assay that exhibited an ECso of 100 pM. Data from all three of the

assays reported in this paper confirm the weak activity of endosulfan to

produce estrogenic effects in vitro. The data provided in this study are

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

890.1250 and 890.1300 assays. Thus, these data also provide reliable

endosulfan OSRI.

8. Legler et al (1999)

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

potential of endosulfan.

In this study, an ER-CALUX assay was used that employed T47D

(human breast cancer cells) Luc cells. This luciferase gene expression

assay was used to screen for estrogenic activity of endosulfan, where

luciferase expression is under estrogen-mediated control. Endosulfan was

weakly estrogenic, with an ECso of 5.9 |jM and maximal effects seen at 10

uM endosulfan. It should be noted that the potency of endosulfan in this

assay was similar to the potency reported by other papers already

discussed above. 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 endosulfan OSRI.

9. Wong and Matsumura (2000)

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

potential of endosulfan.

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The authors of this study were focused on examining the ligandindependent activity of chemicals to activate estrogen receptors. The assay

used is stated to detect estrogenic activity even in chemicals that exhibit

only weak or partial agonist activity at estrogen receptors. The assay is a

serum-free assay performed in an ovarian cell line, BG-1 cells; cell

proliferation responses are measured following exposure to chemicals.

The activity of test chemicals is compared to 17(3-estradiol. Endosulfan

was one of nine organochlorine agents tested individually and the authors

report it induced cell proliferation. Endosulfan's effects were partially

antagonized by tamoxifen, an estrogen receptor antagonist. The data

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

in the EDSP Tier 1 890.1250 assay. Thus, these data also provide reliable

endosulfan OSRI.

10. Newbold et al (2001)

Review of this study indicates that it provides reliable, nonguideline OSRI data screening for uterine weight changes from an

Uterotrophic assay in endosulfan-exposed immature mice.

This in vivo study is consistent in design with the Tier 1 EDSP

Uterotrophic assay (890.1600), however, mice are used instead of rats. In

one part of the study, immature (PND17) CD-I mice were injected for

three days subcutaneously (PND17-PND19) with various individual

compounds. The test compounds included the positive control agents 17(3-

estradiol and DES, the negative control tamoxifen, and endosulfan at

doses of from 0.1 to 1000 mg/kg/day. In a second part of the study, mature

ovarectomized CD-I mice were administered either a positive control

agent (DES; 10 ng/kg), a negative control agent (corn oil vehicle), or

endosulfan at a dose of 10 mg/kg, subcutaneously for 14 days. In each

experiment, the endpoint monitored was increased uterine weight. The

authors reported that endosulfan was not Uterotrophic in either experiment.

The data provided in this study are directly equivalent to the type of data
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Study No. MA-END-EDSP-20100313

collected in the EDSP Tier 1 890.1600 assay, and are functionally

equivalent to the data collected in the 890.1250 assay, and are reliable

endosulfan OSRI.

11. Andersen et al (2002)

Review of the data indicates that it provides reliable, non-guideline

data screening for estrogen receptor binding and transactivation, androgen

receptor transactivation, and aromatase activity that may be associated

with exposure to endosulfan.

In this study, the ability of individual compounds to affect estrogen

receptor binding, androgen receptor binding and aromatase (CYP19)

activity was examined. The study reports use of standard methods for each

type of assay. Estrogen receptor binding was examined in MCF-7 cells

using methods similar to those already discussed above. In the assay,

endosulfan exhibited a lowest-observed effect concentration (LOEC) of 1

uM and a maximum-observed effect concentration (MOEC) of 25 uM, but

was reported to be six orders of magnitude less potent that the positive

control compound 17(3-estradiol. In an estrogen receptor transactivation

assay, MCF-7BUS cells were used (measuring (3-galactosidase activity).

The authors reported that endosulfan exhibited a LOEC of 1 uM and a

MOEC of 25 uM and was five orders of magnitude less potent in the assay

as compared to the positive control 17p-estradiol. Androgen receptor

transactivation was measured in CHOK1 cells (Chinese hamster ovary

cells) transfected with the human androgen receptor expression vector

pSVARO and the MMTV-LUC reporter plasmid. The authors reported

that endosulfan exhibited a LOEC of 20 uM and a MOEC of 20 uM and

was seven orders of magnitude less potent in the assay as compared to the

positive control. Finally, the activity of endosulfan was examined in an

aromatase assay in human placental microsomes. The authors reported that

at a 50 uM dose endosulfan inhibited aromatase activity by 13% whereas

the positive control agent inhibited aromatase activity by 100% at a 1 uM

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Study No. MA-END-EDSP-20100313

dose level. Considered together, these data demonstrated the lack of

significant estrogenic and androgenic activity in vitro with endosulfan

exposure, and are consistent with other endosulfan OSRI already

discussed.

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

of data collected in the EDSP Tier 1 890.1200 assay, the 890.1250 assay,

and the 890. 1300 assay, are functionally equivalent to the data collected

in the 890.1150 assay, and are reliable endosulfan OSRI.

12. Fang et al (2003)

Review of the data indicates that it provides reliable, non-guideline

data for screening for the androgen receptor binding potential of

endosulfan.

In this in vitro study, 202 chemicals were individually screened for

androgen receptor binding activity using a system employing rat

recombinant androgen receptors expressed in E. coli. Standard techniques

for receptor binding were employed as described in the paper. Competitive

binding was assessed using the androgen receptor agonist 17R-methyl-

[

3H]methyltrienolone (also known as R1881). R1881 was employed at a

dose of 1 nM. The IC5o for endosulfan reported by the authors was 2.3 x

10"5 M. The authors describe endosulfan as a weak androgen receptor

agent. These data are also 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 endosulfan OSRI.

13. Gormley and leather (2003)

Review of the data indicates that it provides reliable, non-guideline

data for screening for the potential of endosulfan to affect development

and reproduction in fish.

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Study No. MA-END-EDSP-20100313

In this in vivo study, Japanese medaka were used and the effects of

endosulfan on behavior, development and reproduction were assessed. The

fish were exposed for 24 hours and exposure began either 4-6 hours after

fertilization or 4-6 hours after hatching. Endosulfan water concentrations

tested included 0.01, 0.1 and 1 jig endosulfan/L, levels stated by the

authors to be relevant to levels encountered in the environment. The

authors reported no effects on endpoints directly relevant to endpoints

monitored in Tier 1 EDSP assays, such as time to hatching and hatching

success. There were also no effects on survival and no consistent effects

on egg production. The authors reported decreased fry size at the two

highest exposure levels and decreased swimming behavior when the fish

were tested 2 weeks after hatching, but only when the exposure to

endosulfan was post-fertilization. The data provided in this study are

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

890.1350 assay, and are reliable endosulfan OSRI.

14. Grunfeld and Bonefeld-Jorgensen (2004)

Review of the data indicates that it provides reliable, non-guideline

data for screening for estrogen receptor transactivation potential of

endosulfan.

In this in vitro study, estrogen receptor mRNA levels were

monitored in MCF-7BUS cells. As already discussed, this is a standard

assay for examining estrogenic activity of chemicals as it relates to

transactivation of the estrogen receptor. Both ERa and ER|3 mRNA levels

were measured following exposure to endosulfan. The activity of nine

different chemicals, including endosulfan, was tested individually. The

activity was compared to the positive control agent 17(3-estradiol, present

at two different doses (low dose: 0.06 nM; high dose: 10 nM). Endosulfan

exhibited only weak activity in this assay, decreasing levels of ERa at a

dose of from 1 to 25 uM, but having no effect of levels of ER0 mRNA.

These data are also consistent with previous findings (discussed above)

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Study No. MA-END-EDSP-20100313

regarding estrogen receptor transactivation potential of endosulfan. 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 endosulfan OSRI.

15. Bonefeld-Jorgensen et al (2005)

Review of the data indicates that it provides reliable, non-guideline

data for screening for estrogen receptor transactivation potential of

endosulfan.

In this in vitro study, estrogen receptor transactivation was

examined in MCF-7BUS cells. As already discussed, this is a standard

assay for assessing estrogenic activity of chemicals as it relates to

transactivation of the estrogen receptor. The activity of four different

chemicals, including endosulfan, was tested individually. The activity was

compared to the positive control agent 17|3-estradiol. Endosulfan

exhibited only weak activity in this assay, with the authors reporting a

LOEC of 1 uM and a MOEC of 25 uM. These data are consistent with

previous findings (discussed above) regarding estrogen receptor

transactivation potential of endosulfan. 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 endosulfan OSRI.

16. Lemaire et al. (2006)

Review of the data indicates that it provides reliable, non-guideline

data for screening for estrogen receptor binding and transactivation

potential of endosulfan.

In this in vitro study, 49 different pesticides, tested individually,

were screened for estrogen receptor transactivation and binding

(competitive binding displacement) in HELN cells. In the receptor

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Study No. MA-END-EDSP-20100313

transactivation assays, HELN ERa and ER|3 responsive reporter cell lines

were used. These cells were used in a standard assay for assessing

estrogenic activity of chemicals as it related to transactivation of the

estrogen receptor. Endosulfan was one of the 49 pesticides tested at a

concentration of 10 uM. The activity of all test agents was compared to

the positive control agent 17p-estradiol at a dose of 10 nM. For

competitive binding detereminations, ERa and ER0 cells in culture were

incubated first with [3H]-labeled estradiol. Then, the cells were exposed to

pesticides, including endosulfan, at a concentration of 10 uM. Endosulfan

exhibited no appreciable activation activity in this assay, and inhibited

ER(3 binding by only 20-30% at a concentration of 10 uM. These data are

consistent with previous findings (discussed above) regarding estrogen

receptor binding and transactivation potential of endosulfan. The data

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

in the EDSP Tier 1 890.1250 assay and the 890.1300 assay, and also

provide reliable endosulfan OSRI.

17. Laville et al (2006)

Review of the data indicates that it provides reliable, non-guideline

data for screening for the potential of endosulfan to affect aromatase

activity.

In this study, the in vitro activity of aromatase in JEG-3 human

choriocarcinoma cells was examined following exposure to 30 different

pesticides, tested individually. Endosulfan was one of the compounds

tested. Additionally, the authors report measurement of CYP19 mRNA

levels in these cells. This assay has been developed to assess effects of

chemicals on steroidogenesis in embryonic and fetal tissue. The assay was

performed using both short (2 hours) and long (24 hours) exposure times

for each compound. The authors reported that endosulfan increased

aromatase activity, but only after the 24 hour exposure period and only at

the highest test dose level of 10 uM (no effects at 1 and 3 uM). There was

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Study No. MA-END-EDSP-20100313

no effect on CYP19 mRNA levels after endosulfan treatment. In

comparison, the positive control compounds produced effects to increase

aromatase activity at lower doses and after both short and long term

. exposures. These data are consistent with previous findings (discussed

above) regarding the potential of endosulfan to affect aromatase activity.

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

collected in the EDSP Tier 1 890.1200 assay, and also provide reliable

endosulfan OSRI.

18. Varayoud et al (2008)

Review of this study indicates that it provides reliable, nonguideline OSRI data for screening for uterine weight changes from an

Uterotrophic assay in endosulfan-exposed ovarectomized rats.

This in vivo study is consistent in design with the Tier 1 EDSP

Uterotrophic assay (890.1600). In the study, sexually mature Wistar rats

(90 days old) were ovarectomized. Following recovery from surgery (14

days), rats that exhibited at least 7 days of atrophic vaginal smears were

injected for three days (subcutaneously) with endosulfan (0, 0.006, 0.06,

0.6 and 6 mg/kg). The control agents tested included a known Uterotrophic

dose of estradiol (0.02 mg/kg) and a non-uterotrophic dose of estradiol

(0.0002 mg/kg). The primary endpoint monitored was increased uterine

weight. The authors reported that endosulfan was not Uterotrophic at any

test dose. The data provided in this study are directly equivalent to the

type of data collected in the EDSP Tier 1 890.1600 assay, and are

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

reliable endosulfan OSRI.

19. Vinggaard et al. (2008)

Review of the data indicates that it provides reliable, non-guideline

data for screening for androgen receptor binding potential of endosulfan.

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Study No. MA-END-EDSP-20100313

In this in vitro study, androgen receptor transactivation 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 endosulfan, were tested at doses of 1, 3, 10, and 30 uM. The

IC25 for endosulfan reported by the authors was between 3 and 10 uM,

indicating only very 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

endosulfan OSRI.

20. 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

transactivation, aromatase activity, and thyroid hormone binding potential

of endosulfan.

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 endosulfan. Endosulfan, 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

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.

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Study No. MA-END-EDSP-20100313

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 were as follows. In the cell-free screening

assays, endosulfan tested at a single initial concentration of 25 \iM was

inactive for binding to all endocrine receptor proteins. Because it was

inactive at this dose, no other doses were tested. More specifically,

endosulfan did not show evidence of binding to bovine estrogen receptors,

bovine or human progesterone receptors, rat or human androgen receptors,

rat thyrotropin-releasing hormone receptor, or human thyroid hormone

receptor-a. Endosulfan did exhibit a weak level of binding to human

estrogen receptors (11.41 ^M dose) in one assay only. In cell-based

assays, endosulfan did not activate human androgen receptors, human

ERa, or human thyroid hormone receptor-(3, and also did not block activity

of established ligands at these receptor sites (i.e., no antagonist activity

demonstrated). In the hormone receptor transactivation assays, endosulfan

did not activate the human androgen receptor, human estrogen-related

receptor-a, human estrogen-related receptor-y, or thyroid hormone

receptor-a. Endosulfan showed weak activity in a human estrogen

receptor-a transactivation assay, with activity seen at a concentration of

2.1 uM. It is noted that the positive control compound, 17p-estradiol, had

activity in this same assay in the low nanomolar range.

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Study No. MA-END-EDSP-20100313

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 endosulfan

OSRI.

21. Brunelli et al (2009)

Review of the data indicates that it provides reliable, non-guideline

data for screening for in vivo reproductive effects of endosulfan in

tadpoles, a non-mammalian species that is used in a Tier 1 EDSP assay

(890.1100).

In this study, tadpoles were exposed to endosulfan in water at

levels of 0.01, 0.05 and 0.1 ug/L for 43-52 days (from hatching

throughout metamorphosis). The larvae were observed for mortality, larval

growth, development, metamorphosis stage, and behavior (swimming ,

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

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

functionally equivalent to data collected in EDSP Tier 1 assay 890.1350.

These data are reliable endosulfan OSRI.

8. Directly or Functionally Equivalent Endosulfan OSRI Identified in

Endosulfan Registration Studies

A search of the guideline toxicology studies performed to support the

registration of endosulfan identified several sources of endosulfan OSRI that are

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

assays. These studies include the following (see Tables 3 and 4 above): a chronic

toxicity study in rats (Ruckman 1989), a chronic toxicity study in dogs (Brunk

1989), a multi-generation reproductive toxicity study in rats (Edwards 1983), a

prenatal developmental toxicity study in rats (MacKensie 1980), a developmental

neurotoxicity study in rats (Gilmore 2006), a one-generation reproduction study in

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Bobwhite quail (Beavers 1987), a one-generation reproduction study in Mallard

ducks (Beavers 1987), and a full life cycle study in fish (Dionne 2002). 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.

1. Ruckman (1989)

Review of this GLP-compliant guideline chronic toxicity study in

rats indicates that it provides reliable data relating to a variety of EDSP

Tier 1 study endpoints as shown in Table 3 above, endpoints relating to

estrogen, androgen and thyroid hormone system activity.

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

Sprague-Dawley rats were administered 97.1% pure endosulfan in diet at

doses of 0, 3, 7.5, 15 and 75 ppm endosulfan for 104 weeks. It should be

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

expected human exposure to endosulfan 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). 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); reproductive organ weights (ovaries, testes);

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

reproductive organ histopathology (female mammary gland, ovaries,

uterus, vagina, testes, epididymides, prostate, seminal vesicle). It should

be noted as well that the chronic rat study involves repeated dosing for 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.

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The developmental periods assessed in the chronic study include

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

most sensitive NOAEL in the study was 15 ppm and was based on kidney

toxicity at the 75 ppm exposure level. As shown in Table 3, estrogenresponsive (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).

These data demonstrate that endosulfan 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 endosulfan. These data are reliable endosulfan OSRI.

2. Brunk (1989)

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

study in dogs indicates that it provides reliable data relating to 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 96.5% pure endosulfan in diet at doses of

0, 3, 10, 30 and 60 ppm endosulfan for 1 year. In the high dose group, the

animals were first administered 30 ppm in diet and the dose was increased

to 60 ppm during the study. It should be noted that a dietary route of

administration is more relevant to the expected human exposure to

endosulfan than data that might be collected using other routes of

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

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subcutaneous dosing). 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); reproductive organ weights (ovaries, uterus, vagina, testes,

epididymides, prostate); organ histopathology (adrenal, thyroid, pituitary,

kidney, liver); and reproductive organ histopathology (female mammary

gland, ovaries, uterus, vagina, 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 assessed in the chronic study include

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

sensitive NOAEL in the study was 10 ppm and was based on decreased

body weight and tonic spasms observed at the 30 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

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

in dogs.

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 endosulfan. These data are reliable endosulfan OSRI.

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3. Edwards (1983)

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

reproductive toxicity study in rats indicates that it provides reliable data

relating to 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 endosulfan 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 Sprague-Dawley

rats were administered endosulfan in diet at doses of 0, 3, 15 and 75 ppm

endosulfan from 6 weeks of age (pre-mating in PI), during mating (PI),

and through PND21 in the F2b generation. Pups of the Fl generation

were, therefore, exposed in utero, during lactation, and then placed on

endosulfan 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 endosulfan 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 endosulfan study was not

performed according to the most recent EPA guideline for multigeneration reproductive toxicity studies (OPPTS 870.3300), it was

performed according the guidelines in place at the time the study was

performed. Like the most current guideline study design, the study was

designed to examine potential effects of endosulfan on endocrine organs,

in particular the estrogen and androgen systems that are critical to

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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 (uterus,

prostate, and epididymides); reproductive organ histopathology (female

mammary gland, ovaries, uterus, vagina, cervix, testes, epididymides,

prostate, seminal vesicle); fertility indices; fecundity indices; and estrous

cyclicity.

The most sensitive NOAEL in the study was 3 ppm and was based

on a decreased maternal and pup body weight and increased liver and

kidney weights at the higher exposure levels (15 and 75 ppm endosulfan).

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

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

pituitary, and various reproductive organs in males and females exposed

even during sensitive developmental periods). There were no effects on

reproductive indices in either generation (all monitored endpoints). There

were no reproductive system abnormalities noted in pups of either

generation. These data demonstrate that endosulfan was not toxic to

endocrine organ systems in this sensitive, in vivo test for reproductive

toxicity during critical periods of development.

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

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toxicity of endosulfan in vivo and are more than adequate to assess the

endocrine toxicity potential of endosulfan. 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 endosulfan (see Table 1). For example, the lack of

effects on fertility and female and male endocrine organ histopathology in

the multi-generation study is consistent with findings in vitro that

endosulfan is not a potent estrogenic agent, is not a potent androgen

receptor binding agent, and has no effects on aromatase activity. Further,

the lack of effects on thyroid organ histopathology and ultimate effects of

altering thyroid hormone activity in the multi-generation study are

consistent with the lack of thyroid hormone receptor binding by

endosulfan (Judson et al. 2009).

4. MacKenzie (1980)

Review of this GLP-compliant guideline prenatal (teratology)

toxicity study in rats indicates that it provides reliable data relating to 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 Sprague-Dawley rats were

administered endosulfan (97.3% pure) by gavage at doses of 0, 0.66, 2,

and 6 mg endosulfan/ kg body weight from gestation day six (GD6)

through GDI 9. 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 endosulfan. As a result, certain

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

endpoints measured in the prenatal toxicity study that are directly

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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, dystocia,

etc.).

The most sensitive NOAEL in the study was 0.66 mg/kg/day and

was based on decreased maternal and fetal body weight, clinical signs in

dams, and misaligned steraabrae in fetuses at the 2 mg/kg exposure level.

There were some developmental abnormalities reported in the high dose

group where maternal toxicity was also evident. 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, except when maternal toxicity was evident. These data

demonstrated that endosulfan 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 endosulfan in vivo, and

are reliable endosulfan OSRI.

5. Gilmore et al (2006)

Review of this GLP-compliant guideline developmental

neurotoxicity study in rats indicates that it provides reliable data relating

to 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, as well as

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postnatal development in rats. This study includes two sensitive measures

of development in animals, time to preputital separation in males and time

to vaginal opening in females.

In this guideline study in rats, female Wistar rats were

administered 99.1% pure endosulfan in diet at doses of 0, 50, 150, and 400

ppm endosulfan from GD6 through PND21 (weaning), with Fl pups

evaluated at about PND60, and sacrificed by PND75. It should be noted

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

human exposure to endosulfan than data that might be collected using

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

intraperitoneal dosing, subcutaneous dosing).

As a result of being a developmental neurotoxiciry study, certain

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

endpoints measured in the developmental neurotoxicity 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); and developmental measures

(fetotoxicity, preputial separation, vaginal opening).

The most sensitive NOAEL in the study was 50 ppm endosulfan

and was based on decreased maternal body weight and decreased fetal

body weight at endosulfan doses of 150 ppm and greater, and a delay in

preputital separation but only at the mid dose level (150 ppm endosulfan).

There were no accompanying changes in sperm parameters measured at

termination in this group. No gross lesions of any kind were reported,

including lesions in endocrine organs. No effects on any reproductive

indices were reported. The only finding noted that could be related to

endocrine effects was the small delay in preputial separation in males but

only at the mid test dose level (10.8 mg/kg/day). The lack of a doseresponse in the finding noted argues against endosulfan being an

endocrine toxicant in this study. These data demonstrated that endosulfan

was not directly toxic to endocrine organs systems in this sensitive, in vivo

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test for toxicity during critical periods of development, in utero and during

lactation.

The data provided in this developmental neurotoxicity study are

functionally equivalent to data collected in EDSP Tier 1 assays that

examine aspects of mammalian endocrine function (i.e., 890.1400,

890.1450, 890.1500, and 890.1600). This study provides reliable

endosulfan OSRI.

6. Beavers et al (1987)

Review of this GLP-compliant guideline one-generation

reproduction study in Bobwhite quail indicates that it provides reliable

data relating to EDSP Tier 1 study endpoints as shown in Table 4 above.

In this study, young adult Bobwhite quail were exposed to

endosulfan (96% purity) at doses in diet of 0, 15, 30, 60, and 120 ppm

endosulfan for 23 weeks from pre-laying through production of eggs in

one generation. Sixteen male and 16 female birds were paired and housed

individually. The endpoints monitored included clinical signs of toxicity in

parents, adult body weight and food consumption, gross pathological

effects at sacrifice, and a variety of reproductive measures (number of

eggs laid, number of eggs cracked, number of eggs set, viable embryos,

live 3-week embryos, number of hatchlings, hatchling body weight,

survival at 14 days, egg shell thickness). There were no effects on any

reproductive parameters in the study at any endosulfan dose. At 120 ppm

endosulfan in the diet, there was a decreased food consumption reported

for adult birds. The NOAEL in the study was 60 ppm. These data

demonstrated that endosulfan was not directly toxic to endocrine organ

systems in this sensitive, in vivo test for toxicity during critical periods of

non-mammalian development.

The data provided in this avian reproduction study are functionally

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

both non-mammalian and mammalian endocrine function (i.e., 890.1100;

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890.1350; 890; 890.1400, 890.1450, 890.1500, and 890.1600). This study

provides reliable endosulfan OSRI.

7. Beavers et al (1987)

Review of this GLP-compliant guideline one-generation

reproduction study in Mallard ducks indicates that it provides reliable data

relating to EDSP Tier 1 study endpoints as shown in Table 4 above.

In this study, young adult Mallard ducks were exposed to

endosulfan (96% purity) at doses of 0, 7.5, 15, 30, and 60 ppm endosulfan

for 18 weeks from pre-laying through production of eggs in one

generation. Sixteen male and 16 female birds were paired and housed

individually. The endpoints monitored included clinical signs of toxicity in

parents, adult body weight and food consumption, gross pathological

effects at sacrifice, and a variety of reproductive measures (number of

eggs laid, number of eggs cracked, number of eggs set, viable embryos,

live 3-week embryos, number of hatchlings, hatchling body weight,

survival at 14 days, egg shell thickness). At 60 ppm endosulfan in the diet,

there was a decreased food consumption and body weight reported in adult

birds, as well as a decrease in egg hatchability and the number of eggs

laid. The NOAEL in the study was 30 ppm. These data demonstrated that

endosulfan was not directly toxic to endocrine organ systems in this in

vivo test for toxicity during critical periods of non-mammalian

development.

The data provided in this avian reproduction study are functionally

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

both non-mammalian and mammalian endocrine function (i.e., 890.1100;

890.1350; 890; 890.1400, 890.1450, 890.1500, and 890.1600). This study

provides reliable endosulfan OSRI.

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8. Dionne (2002)

Review of this GLP-compliant guideline full life cycle study in

Fathead minnows indicates that it provides reliable data relating to EDSP

Tier 1 study endpoints as shown in Table 4 above.

In this study, 100 Fathead minnow embryos per dose level were

exposed to endosulfan (98.7% purity) continuously for one complete life

cycle (260 days). The water concentrations of endosulfan employed were

0.038, 0.075, 0.15, 0.30, and 0.60 ng endosulfan per liter. The endpoints

monitored included measures of fertility, fecundity, development of fish,

survival, body weight and length of offspring, length at maturation (males

and females), and deformities in hatchlings. The author reports a nonmonotonic and suppressed dose-response effect on reproductive endpoints

at the three highest exposure doses of endosulfan (0.15 |u.g/L and higher).

These three highest dose levels were identified as having reproductive

effects in Fathead minnows. These data are directly equivalent to data

collected in the EDSP Tier 1 890.1350 assay (Fish Short-term

reproduction). Although the study did not include measures of vitellogenin

levels or gonad size or histopathology, the data provide adequate data for

screening for reproductive hazards to fish. This study provides reliable

endosulfan OSRI.

9. Heusel (1998)

Review of this GLP-compliant study evaluating the possible

endocrine effects of endosufan in Fathead minnows indicates that it

provides reliable data relating to EDSP Tier 1 study endpoints as shown in

Table 4 above.

In this study, juvenile Fathead minnows were exposed to

endosulfan (technical substance) for 21 days under flow-through

conditions. The water concentrations of endosulfan employed were 0,

0.05, 0.1, 0.2, 0.40, and 0.80 ug endosulfan per liter. The endpoints

monitored included survival, body weight, body length, and vitellogenin

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concentrations. The positive control compound tested for validation of the

assay was 17a-ethinylestradiol (10 ng/ml). The author reported that there

were no statistically significant effects on body weight, body length, or

vitellogenin concentrations. Survival decreased significantly but only at

dose (0.8 u.g/L; actual concentration determined to be 0.62 >g/L). The

NOEC was determined to be 0.28 ug/L (corresponding to the target dose

of 0.4 ug/L). These data indicated that there was no indication of

estrogenic effects in the fish. These data are directly equivalent to some of

the data collected in the EDSP Tier 1 890.1350 assay (Fish Short-term

reproduction). This study provides reliable endosulfan OSRJ.

IV. MANA Seeks a Waiver for AH of the Tier 1 EDSP Assays that Screen for

Endocrine Disruption Potential of Endosulfan

There is a large and robust body of endosulfan OSRI data for screening for the

potential endocrine activity of endosulfan in both mammalian organisms and in nonmammalian systems (birds, amphibians, and fish). The available data include in vitro

evaluations of estrogen, androgen and thyroid hormone binding and/or function, as well

as the effects of endosulfan on enzymes that synthesize and metabolize estrogen and

androgen hormones in mammals (Soto et al. (1994); Soto et al. (1995); Wade et al.

(1997); Ramamoorthy et al. (1997); Arcaro et al. (1998); Wilson and LeBlanc (1998);

Hunter et al. (1999); Legler et al. (1999); Wong and Matsumura (2000); Newbold et al.

(2001); Anderson et al. (2002); Fang et al. (2003); Grunfeld and Bonefeld-Jorgensen

(2004); Bonefeld-Jorgensen et al. (2005); Lemaire et al. (2006); Laville et al. (2006);

Varayoud et al. (2008); Vinggaard et al. (2008); Judson et al. (2009); Brunelli et al.

(2009)). Thus, in terms of estrogenicity, androgenicity and thyroid hormone activity, the

available data discussed above provide more than adequate screening for potential modes

of action for endosulfan on these endocrine systems. Moreover, there are reliable, robust

in vivo studies that directly monitor, or are functionally equivalent to, all of the endpoints

that would be monitored in Tier 1 EDSP assays. Based on the fact that none of the

studies demonstrate that endosulfan is a direct toxicant to endocrine systems in organisms

(discussed above), there is no scientifically defensible reason to perform any of the Tier 1

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EDSP studies, particularly given the already extensive and numerous studies performed

to date. As a result, MANA is asking for a waiver of all eleven of the Tier 1 studies.

The in vitro studies screening for estrogen, androgen and thyroid hormone activity

indicated that endosulfan, in some instances, affected 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 endosulfan are useful for identifying the need for collection of in vivo data

that should be evaluated for potential interactions with estrogen-related and androgenrelated functions (e.g., reproductive function). In the case of endosulfan, this in vivo data

was indeed collected (the multi-generation study in rats, etc.). It must also be

remembered that the potency of endosulfan in these in vitro studies was very low, with

potencies in the range of 105 to 106 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

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 endosulfan 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 thyroid gland histopathology in all of the

available studies. Any effects reported with endosulfan exposure that might be linked to

endocrine activity were limited generally to high doses.

The endosulfan 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

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performance of EDSP Tier 1 assays less relevant as screening assays for hazard

assessment.

It should be clear based on the discussion already provided relating to the

available endosulfan endocrine toxicity data that none of the Tier 1 EDSP tests need to be

conducted if the goal is to screen for potential endocrine activity. Thus, considering the

available data, MANA seeks a waiver for all of the Tier 1 EDSP studies. 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 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 thirteen separate studies that providing

in vitro estrogen receptor activity data available for determining the abprovide

adequate OSRI for determining the ability of endosulfan to bind to and activate

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 assessing 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 endosulfan to appropriate control agents. The results have been

consistent in showing that endosulfan is, at best, a weak estrogenic agent, with

potency several orders of magnitude less than positive control, agents (i.e., 17pestradiol); activity of endosulfan in vitro is only seen at doses in the high

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

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available data are adequate for assessing the activity of endosulfan 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 five separate studies providing in vitro androgen receptor

activity data that provide adequate OSRI for determining the ability of endosulfan

to bind to androgen receptors. The studies by Andersen et al. (2002), Fang et al.

(2003), Laville et al. (2006), Vinggaard et al. (2008), and Judson et al. (2009)

provide data on both androgen receptor binding potency, agonist and antagonist

activity, as well as the potential for endosulfan to inhibit aromatase activity in

vitro. The available studies provide directly and functionally equivalent data on

essentially the same endpoints as found in the Tier 1 EDSP assays, including

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

are adequate OSRI for screening for potential activity of endosulfan at AR

receptors and on aromatase function, and there is no scientific basis for

performing additional in vitro androgen receptor activity studies, specifically the

Tier 1 Androgen Receptor Binding assay (890.1150), and additional in vitro

aromatase activity studies, specifically the Tier 1 Aromatase assay (890.1200). A

waiver of both the 890.1150 (Androgen Receptor Binding assay) study and the

890.1200 (Aromatase assay) study with endosulfan is sought.

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

Assay (890.1550)

The Steroidogenesis assay (890.1550) is a screening assay whose stated

intent is to identify compounds that affect the steroidogenic pathway beginning

with reactions that occur after the gonadotropin receptors through the production

of testosterone and estradiol. This does not include effects of compounds on the

hypothalamus or pituitary glands. In this assay, the levels of estradiol and

testosterone that are produced in cells in culture both before and after exposure to

the test chemical are measured. Since, by design, such an in vitro assay is less

robust and less relevant to screening for potential effects in intact organisms, any

available in vivo data would be preferred when screening for endocrine disruption

potential of a chemical. Thus, in vivo data on sex steroid hormone levels and their

metabolism and pharmacokinetics in vivo would be preferred over such studies in

cells, data that is available for endosulfan (Wilson and LeBlanc 1998). The study

by Wilson and LeBlanc (1998) provides adequate endosulfan OSRI for screening

for effects of endosulfan on Steroidogenesis, since the study provides data on

testosterone metabolism and pharmacokinetics in male and female animals. There

are also functionally equivalent OSRI on Steroidogenesis activity in the

endosulfan registration studies (e.g., apical effects on reproductive organ weights

and histopathology as well as reproductive capacity). The endosulfan OSRI even

includes a multi-generation reproductive toxicity study where organisms were

exposed to endosulfan at all critical stages of development. Thus, there are

adequate OSRI for assessing the activity of endosulfan on Steroidogenesis, and

there is no scientific basis for performing additional in vitro Steroidogenesis

studies when in vivo data on the same endpoints is available. As a result, a waiver

of the 890.1550 (Steroidogenesis assay) study with endosulfan is sought.

D. 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

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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 vivo data collected in

guideline toxicology studies that cover all stages of mammalian development, and

are consistent in showing that endosulfan has no effects on functionally

equivalent endpoints in mammalian species. For example, the endosulfan OSRI

include data from a multi-generation reproductive toxicity study in rats, a prenatal

toxicity study in rats, and a developmental neurotoxicity study in rats. All of these

studies demonstrated that endosulfan 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 endosulfan were producing direct effects on either thyroid hormone synthesis or

function. Additionally, there is endosulfan OSRI collected in developing

amphibians (Brunelli et al. 2009) where effects of endosulfan on metamorphosis

are provided. Performing the amphibian assay merely to monitor changes in

thyroid hormone levels or sex hormone levels in developing amphibians,

endpoints that have not been quantified in currently available endosulfan OSRI, is

not scientifically justified when robust in vivo data in developing organisms,

including tadpoles, is already available and those data do not show that the

thyroid hormone system is a target for endosulfan effects. 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 endosulfan

OSRI are considered.

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E. 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

endosulfan demonstrating that this chemical is not a potent estrogenic or antiestrogenic agent in vivo, and has no identifiable effects on thyroid function. The

OSRI includes the robust, guideline registration studies (Table 3). These in vivo

evaluations of female reproductive function and developmental 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 developmental neurotoxicity study, and the chronic

toxicity studies, all included assessments of endosulfan's effects on female

reproductive tissues and organs, both in adult animals and in developing animals

(including peri-pubertal animals), including evaluations of effects across

generations. These studies also included histopathological assessments 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 endosulfan

exposure, as well as fertility, fecundity, vaginal opening, and estrous cycling.

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 endosulfan

guideline toxicity studies is the level of thyroid hormones in serum of animals

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following endosulfan exposure. Given that the ultimate apical 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 no such apical effects were observed, there does not

appear to be an adequate 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 rats. Therefore, a waiver of EDSP Tier 1 890.1450

(Pubertal Female Rat Development) is sought.

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

Assay (890.1500)

The purpose of the 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 peripubertal male rats. There are ample in vivo endosulfan OSRI on male

reproductive function and pathology to indicate that the systems that involve

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

uniquely sensitive to the effects of endosulfan. The endosulfan OSRI studies

include the robust guideline studies in rats such as the two-generation

reproductive toxicity study, the developmental toxicity study, the developmental

neurotoxicity study, 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 endosulfan toxicity studies in rats (Table 3) all included

assessments of endosulfan effects on androgen-sensitive male reproductive tissues

and organs, both in adult animals and in developing animals (including peri55
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pubertal animals), including assessments of effects across generations. These

studies also included histopathological assessments of thyroid gland tissue in

animals exposed both during gestation and development, and as adults.

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

have not been directly measured in the currently available endosulfan guideline

toxicity studies is the level of thyroid hormones in serum of animals following

endosulfan exposure. The Tier 1 assay also measures levels of the sex hormone

testosterone. Functionally equivalent in vivo endosulfan OSRI data on

testosterone levels can be found in the study by Wilson and LeBlanc (1998).

Moreover, with respect to 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, indicate toxicity induced by endosulfan 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 additional measures of sex hormone levels in peri-pubertal

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 there is no evidence of such apical effects, 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 rats. Therefore, a waiver of EDSP Tier 1 890.1500 (Pubertal

Male Rat Development) is sought.

G. MANA a 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 endosulfan, there are three endosulfan

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OSRI studies that are actual Uterotrophic assays (Wade et al. 1997; Newbold et

al. 2001; Varayoud et al. 2008). Additionally, there are adequate endosulfan

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 2 years (Ruckman et al. 1989), as well as dosing

throughout all stages of development (Edwards 1983; MacKenzie 1980). As a

result, there is no scientific basis for conducting another Uterotrophic assay with

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

sought.

H. MANA a 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 endpoint monitored is changes in

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

endosulfan 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 2 years (Ruckman et al. 1989), as well as dosing throughout all

stages of development (Edwards 1983; MacKenzie 1980). The multi-generation

reproduction study even included sperm evaluation. As a result, there is no

scientific basis for conducting another in vivo assay with endosulfan 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.

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Study No. MA-END-EDSP-20100313

I. MANA a Seeks a Waiver for Performance of the Tier 1 Fish ShortTerm Reproduction Assay (890.1350)

The purpose of the assay (890.1350) is stated in the guideline to be a

screening test for reproductive fitness in fathead minnow as a measure of toxicant

effects. The test measures a variety of histological and biochemical endpoints that

relate to disturbances in the activity of the hypothalamus-pituitary-gonadal (HPG)

axis. In the assay, male and female fish (mature) are exposed for 21 days and

survival, reproductive behavior, and effects on offspring are monitored. There are

adequate in vivo endosulfan OSRI on fish reproductive function and effects on

offspring (Dionne 2002; Gormley and Teather 2003; Table 4), as well as two

reproduction studies in birds (Beavers 1987; Beavers et al. 1987), and a study

evaluating the potential endocrine toxicity of endosulfan in fish (Heusel 1999).

The study of Dionne (2002) is a GLP-compliant fish full life-cycle study and by

design is more robust in terms of exposure duration than the short-term

reproduction study that is part of Tier 1 in the EDSP. These in vivo evaluations of

fish reproduction have provided data on most of the same endpoints as generated

in the EDSP Tier 1 study (890.1350). Vitellogenin levels were measured in the

1999 study by Heusel. The only endpoints from the Tier 1 in vivo assay that are

not directly measured in the currently available endosulfan guideline toxicity

studies are gonad size and histology following endosulfan exposure. However,

functionally equivalent in vivo endosulfan OSRI on these endpoints is available in

the studies by Gormley and Teather (2003) and Dionne et al. (2002). For example,

measures of fertility and reproduction as well as morphological changes. Thus,

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

EDSP Tier 1 screening study simply to provide for a few additional measures in

fish exposed for a shorter length of time. Therefore, a waiver of EDSP Tier 1

890.1350 (Fish Short-Term Reproduction) is sought.

V. Overall Summary

The preceding discussion provides a detailed description of available endosulfan

OSRI that is used to support waivers for all eleven of the EDSP Tier 1 screening assays.

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Study No. MA-END-EDSP-20100313

The OSRI discussed in this TO response include 21 published studies and nine guideline

registration studies. Clearly the endosulfan OSRI is extensive and rich, including

information specific to screening for the potential for estrogen, androgen and thyroid

hormone system effects as well as the effects of endosulfan on specific endpoints (i.e.,

potential modes of action). As a result, there is no scientifically defensible basis for

performing additional EDSP screening studies with endosulfan. Thus, MANA is seeking

waivers for performance of all eleven of the 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); 890.1350 (Fish Short-Term Reproduction Assay); 890.1400 (Hershberger

assay); 890.1450 (Pubertal Female Rat Development); and 890.1500 (Pubertal Male Rat

Development); 890.1550 (Steroidogenesis); and 890.1600 (Uterotrophic assay).

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Study No. MA-END-EDSP-20100313

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64