Vo. L DIAZINON EDSP TEST ORDER

OrderNo.EDSP-057801-36

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 Diazinon

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 ;•. •.

February 12, 2010

• •••

•..• •

STUDY NO.

MA-DZN-EDSP-20100213

Makhteshim Chemical Works, Ltd.

Beer Sheva 84100, Israel

c/o Makhteshim Agan of North America, Inc.

4515 Falls of Neuse Road, Suite 300

Raleigh, NC 27609

TOTAL PAGES: 42
Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

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:

Company Representative

Makhteshim Chemical Works, Ltd.

Beer Sheva 84100, Israel

P. LeannexPryiiett

Authorized Representative - MCW

Regulatory Product Manager - MANA

Date

•••••I

: • :
Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

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.

2-/2-20Q

Submitter:

Date

**•*

:•••!,

P. Leanne

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

••••

Sponsor:

Date

P. Leanne vfu

Authorized Agent (MCW) and Regulatory Product Manager (MANA)
Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

Table of Contents

I. Introduction 5

II. Background 5

III. Discussion of DiazinonOSRI 5

A. Directly or Functionally Equivalent Diazinon OSRJ Identified in the Published

Literature 16

B. Directly or Functionally Equivalent Diazinon OSRJ Identified in Diazinon

Registration Studies 21

IV. Discussion of the EDSP Tier 1 Tests Needed for Assessment of the Endocrine

Disruption Potential of Diazinon 32

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) 34

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) ..35

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

Assay (890.1100) 35

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

(890.1450) 36

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

(890.1500) 38

V. Overall Summary 40

VI. References Cited 41
Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

I. Introduction

On November 5, 2009, the U.S. Environmental Protection Agency (EPA) issued a

Testing Order (TO) for diazinon (CAS # 333-41-5) related to testing under the EPA

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

submission of EDSP Tier 1 testing for diazinon. 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 data already available on the endocrine disruption potential of diazinon,

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

how available OSRI provides for assessment of many of the same endpoints included in

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

specific EDSP Tier 1 testing, including rationale for waivers for certain EDSP Tier 1

assays.

II. Background

EDSP Tier 1 assays were designed to provide data relevant to assessing the

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 assessed 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.
Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

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

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

diazinon, 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)
Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

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

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

support registration of diazinon. Before discussion of each diazinon 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 diazinon 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 that particular endpoint is

measured in the that 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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Study No. MA-DZN-EDSP-20100213

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 diazinon.

Before discussing the OSRI for diazinon 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 diazinon, two sources were examined: 1) the peerreviewed, published scientific literature; and 2) the diazinon pesticide registration studies.

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

assessment for diazinon, 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 diazinon OSRI included identifying a reliability indicator for each

10
Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

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 diazinon OSRI, only studies that could be given a category rating

of either 1 or 2 were used in the OSRI evaluation presented here. This means, that only

studies that were GLP-compliant1 or comparable to a 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 diazinon OSRI was evaluated by scientists with

training in toxicology and familiarity with pesticide registration and EDSP testing

protocols.

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

diazinon that is either directly equivalent or functionally equivalent to the EDSP Tier 1

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

Tier 1 endpoints are adequately assessed through use of OSRI and which endpoints may

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

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

provided a specific rationale for why certain EDSP Tier 1 assays should be waived based

on the available OSRI (Section IV of this document).

III. Discussion of Diazinon OSRI

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

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

listing of each source of OSRI for diazinon 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. Following Table 3, the various OSRI are

discussed in more detail, including a discussion of whether the OSRI are either directly

equivalent, functionally equivalent, or both.

1

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

outlined by EPA.

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Makhteshim Chemical Works, Ltd.

Study No. MA-DZN-EDSP-20100213

A. Directly or Functionally Equivalent Diazinon OSRI Identified in the

Published Literature

A search of the published scientific literature identified several sources of

diazinon OSRI that are directly equivalent to data collected in EDSP Tier 1

assays. These studies include Soto et al. (1995); Nishihara et al. (2000); Kojima

et al. (2004); Kojima et al. (2005); Manabe et al. (2006); Oh et al. (2007); and

Judson et al. (2009). The following is a discussion of the relevant OSRI from each

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

OSRI.

1. Soto et al (1995)

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

diazinon.

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

diazinon, the authors report results of testing using the E-SCREEN assay.

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 (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 17Pestradiol as well as a variety of other known estrogen receptor agonists to

validate the responsiveness of their assay. 17p-estradiol is the positive

control agent used in EDSP Tier 1 assays. In Table 5 from the paper, the

authors report findings from the E-SCREEN for dozens of pesticides, as

well as other classes of chemicals. Diazinon is identified as being "nonestrogenic" in the E-SCREEN assay. The data provided are directly

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

and are reliable diazinon OSRI.

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Study No. MA-DZN-EDSP-20100213

2. Nishihara<*a£(2000)

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

potential of diazinon.

In this in vitro study, a yeast two-hybrid assay (ERa receptor and

TIF2 co-activator, S. cerevisiae Y190 cells) was used to screen for the

ability of a group of 500 individual test chemicals to activate the estrogen

receptor. The assay used was based on the ligand-dependent interaction of

two proteins, the hormone receptor and its co-activator, with hormonal

activity identified by detection of the presence of p-galactosidase.

Diazinon was one of the 500 test chemicals screened for estrogen activity

in this assay. Of 500 test chemicals, only 64 were found to be "positive" as

having estrogenic activity. Diazinon was negative, showing no significant

estrogenic activity at doses up to the highest test dose of 100 uM (1 x 10"4

M). The authors reported a RECio > 1 x 10"4 M for diazinon, where RECio

is the concentration of test chemical showing 10% of the agonist activity

of the positive control, l?p-estradiol. The data provided in this study are

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

890.1300 assay, and are functionally equivalent to the data collected in the

890.1250 assay, and are reliable diazinon OSRI.

3. Kojimae/a/. (2004)

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

transactivation potential of diazinon.

In this study, 200 individual pesticides were screened for their

potential agonist and antagonist activity at two human estrogen receptor

subtypes (ERa and ER0), and a human androgen receptor, in sensitive

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

the pesticides tested. The assays measured firefly luciferase gene

expression. The activity of the pesticides, including diazinon, was

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Study No. MA-DZN-EDSP-20100213

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

control agents. The authors reported that diazinon was not shown to have

activity in any of the three systems, at doses tested from 10"8 to 10~5 M.

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

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

equivalent to the data collected in the 890.1250 assay and the 890.1150

assay. Thus, these data also provide reliable diazinon OSRI.

4. Kojima et al. (2005)

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

potential of diazinon.

In this study, BG1 human ovarian carcinoma cells were transfected

with luciferase reporter plasmid. This luciferase gene expression assay

was used to assess estrogenic activity of diazinon, where luciferase

expression is under estrogen-mediated control. Diazinon was weakly

estrogenic, with effects seen only at a dose of 460 uM diazinon, or 4.6 x

10"4 M diazinon. It should be noted that the potency of diazinon in this

assay was similar to the potency reported by Nishihara et al. (2000). 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 diazinon OSRI.

5. Manabee/a/. (2006)

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

of diazinon.

In this in vitro study, the authors report results for six pesticides

tested individually using a rat pituitary tumor cell line, MtT/Se, which

expresses both ERa and ERp. This is a standard assay employed in many

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Study No. MA-DZN-EDSP-20100213

laboratories and measures the comparative potency of test compounds to

induce proliferation of these cells in culture. Stimulation of these receptors

by compounds such as estradiol leads to increased cell proliferation. The

authors report use of l?p-estradiol as an agonist and 4-hydroxytamoxifen

as an antagonist to validate the responsiveness of their assay. In Table 2

from the paper, the authors report findings for the six pesticides. Diazinon

is identified as being a weak estrogenic compound. The reported RECio,

which is the dose equivalent to 10% of the maximum estradiol response

(RECio), was 9.7xlO~5 M, or 97 \iM diazinon. Again, it is noted that the

potency of diazinon in this assay was similar to the potency seen by

Nishihara et al. (2000) and Kojima et al. (2005). 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 functionally equivalent to the data collected in

the 890.1300 assay. Thus, these data also provide reliable diazinon OSRI.

6. Oh et aL (2007)

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

of diazinon.

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

pesticides tested individually using the E-SCREEN assay. This is a

standard assay employed in many laboratories and measures the

comparative potency of test compounds to induce proliferation of MCF-7

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

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

receptors by compounds such as estradiol leads to increased proliferation

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

responsiveness of their assay. In Figure 2 from the paper, the authors

report findings from the E-SCREEN for all tested compounds. Diazinon is

identified as being non-estrogenic in the E-SCREEN assay. The data

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Study No. MA-DZN-EDSP-20100213

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

Tier 1 890.1250 assay, and are reliable diazinon OSRI.

7. 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 assessing

estrogen receptor binding, androgen receptor binding, estrogen receptor

transactivation, aromatase activity, and thyroid hormone binding potential

of diazinon.

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 diazinon and diazoxon. Diazinon and its

major metabolite diazoxon, as well as the all of the other compounds, were

tested for estrogen receptor binding, androgen receptor binding, estrogen

receptor transactivation, aromatase activity, and thyroid hormone binding

potential, among a variety of other endpoints. The specific tests performed

included the following: binding to bovine estrogen receptors from uterine

membrane (bER); binding to human estrogen receptors from breast cancer

cells (hER); binding to bovine progesterone receptors from uterine

membrane (bPR); binding to human progesterone receptors from MCF-7

cells (hPR); binding to rat androgen receptors (rAR; recombinant);

binding to human androgen receptors from LnCAP cells (hAR); binding to

rat thyrotropin-releasing hormone receptor from forebrain membranes

(rTRH); binding to human thyroid hormone receptor-alpha (hTRa;

recombinant); activity to inhibit the aromatase enzyme; estrogen receptor

transactivation; androgen receptor transactivation; and thyroid hormone

receptor transactivation.

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-DZN-EDSP-20100213

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, diazinon tested at a single initial concentration of 25 uM was

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

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

diazinon did not show evidence of binding to bovine or human estrogen

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

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

hormone receptor-a. In cell-based assays, diazinon 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, diazinon did not activate the human androgen

receptor, human estrogen-related receptor-a, human estrogen-related

receptor-y, or thyroid hormone receptor-a. Diazinon showed weak activity

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

concentrations of between 19 and 38 uM. It is noted that the positive

control compound, 17p-estradiol, had activity in this same assay in the low

nanomolar range. The major diazinon metabolite, diazoxon, was tested in

all of these same systems and it was uniformly negative in all systems.

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 diazinon OSRI.

B. Directly or Functionally Equivalent Diazinon OSRI Identified in

Diazinon Registration Studies

A search of the guideline toxicology studies performed to support the

registration of diazinon identified several sources of diazinon OSRI that are

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Study No. MA-DZN-EDSP-20100213

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

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

toxicity study in rats (Singh 1988), a chronic toxicity study in rats (Kirchner et al.

1991), a chronic toxicity study in dogs (Rudzki et al. 1991), a multi-generation

reproductive toxicity study in rats (Giknis 1989), a prenatal developmental

toxicity study in rats (Infurna 1985), and a developmental neurotoxicity study in

rats (Mandella 2003). 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. Singh (1988)

Review of this GLP-compliant, guideline subchronic toxicity study

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

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

relating to estrogen, androgen and thyroid hormone system activity.

In this guideline subchronic toxicity study in rats, young adult

male and female Sprague-Dawley (CD) rats were administered 87.7%

pure diazinon in diet at doses of 0, 0.5, 5, 250 and 2500 ppm diazinon for

90 days. It should be noted that a dietary route of administration is more

relevant to the expected human exposure to diazinon than data that might

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

gavage dosing, intraperitoneal dosing, subcutaneous dosing). As in any

standard guideline rodent subchronic toxicity study, endpoints relevant to

endocrine activity were assessed (see Table 3). The endpoints measured in

the subchronic study that are directly equivalent to endpoints monitored in

EDSP Tier 1 assays (i.e., 890.1350, 890.1400, 890.1450, 890.1500,

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

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

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

prostate, seminal vesicle); organ histopathology (adrenal, thyroid,

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

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Study No. MA-DZN-EDSP-20100213

mammary gland, ovaries, uterus, testes, epididymides, prostate, seminal

vesicle). It should be noted as well that the subchronic rat study involves

repeated dosing for 90 days, a time period much longer than any time

period of exposure in an EDSP Tier 1 assay (i.e., maximum time of

exposure of less than one month).

The developmental period assessed in the subchronic study is

young adult, for both male and female animals. The most sensitive noobserved adverse effect level (NOAEL) in the study was 0.5 ppm and was

based on decreased red blood cell acetylcholinesterase levels at the 5 ppm

exposure level. As shown in Table 3, the estrogen-responsive (e.g.,

ovaries, uterus), androgen-responsive (e.g., testes, prostate) and thyroidresponsive 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, except for an increased liver weight in female rats

only. No effects on organ histopathology were reported except for

centrilobular hypertrophy in female rats at doses greater than 250 ppm

diazinon. Body weight decreased in males and females but only at the

highest test dose (2500 ppm diazinon). These data demonstrate that

diazinon was not toxic to endocrine organs systems in this study in rats.

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

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

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

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

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

diazinon OSRI.

2. KirchnerefaM1991)

Review of this GLP-compliant guideline chronic (1 and 2 year

sacrifices) toxicity study in rats indicates that it provides reliable data for

assessing a variety of EDSP Tier 1 study endpoints as shown in Table 3

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Study No. MA-DZN-EDSP-20100213

above, endpoints relating to estrogen, androgen and thyroid hormone

system activity.

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

Sprague-Dawley (CD) rats were administered 87.7% pure diazinon in diet

at doses of 0, 0.1,1.5, 125 and 250 ppm diazinon for 54 or 99 weeks (1 or

2 years). It should be noted that a dietary route of administration is more

relevant to the expected human exposure to diazinon 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, uterus, female mammary gland, testes, epididymides,

prostate, seminal vesicle); 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 1 to 2 years, a time period much longer than

any time period of exposure in EDSP Tier 1 assays, and covering a

developmental period not covered by EDSP Tier 1 studies, i.e.,

senescence.

The developmental periods assessed in the chronic study include

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

most sensitive NOAEL in the study was 1.5 ppm and was based on

decreased red blood cell acetylcholinesterase levels at the 250 ppm

exposure level. As shown in Table 3, estrogen-responsive (e.g., ovaries,

uterus), androgen-responsive (e.g., testes, prostate) and thyroid-responsive

tissues (e.g., thyroid, brain), were examined histopathologically in this

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Study No. MA-DZN-EDSP-20 1 002 1 3

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). Body weight increased in

males and females but only at the highest test dose (250 ppm diazinon).

These data demonstrate that diazinon 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, 890.1600. The

data provide information on estrogen, androgen, and thyroid hormone

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

3.

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

study in dogs indicates that it provides reliable data for assessing 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 87.7% pure diazinon in diet at doses of 0,

0.1, 0.5, 150 and 225 ppm diazinon for 1 year. It should be noted that a

dietary route of administration is more relevant to the expected human

exposure to diazinon 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 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,

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Study No. MA-DZN-EDSP-20100213

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 0.5 ppm and was based on decreased

red blood cell acetylcholinesterase levels at the 150 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

diazinon 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, 890.1600. The

data provide information on estrogen, androgen, and thyroid hormone

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

4. GiknistVa/. (1989)

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

reproductive toxicity study in rats indicates that it provides reliable data

for assessing 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 diazinon study and covers all developmental

periods covered by EDSP Tier 1 assays (in utero, lactation, post-natal,

peri-pubertal, young adult, and adult).

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Study No. MA-DZN-EDSP-20100213

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

rats were administered 94.9% pure diazinon in diet at doses of 0, 10, 100

and 500 ppm diazinon for 10 weeks pre-mating (PI), 3 weeks mating (PI),

and 11 weeks pre-mating (Fl). Pups of the Fl generation were, therefore,

exposed in tttero, during lactation, and then placed on diazinon 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 diazinon 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 diazinon 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. It is an OPP 83-4 study design, and like the most current

guideline study design it was designed to examine potential effects of

diazinon on endocrine organs, in particular the estrogen and androgen

systems that are critical to reproductive function. As a result of being a

reproductive toxicity study, many endpoints relevant to endocrine activity

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

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Study No. MA-DZN-EDSP-20100213

pups); reproductive organ weights (ovaries and testes); reproductive organ

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

testes, epididymides, prostate, seminal vesicle, coagulating gland); fertility

indices; fecundity indices; and indirect measures of estrous cyclicity.

The most sensitive NOAEL in the study was 10 ppm and was

based on a decreased Fl maternal and pup body weight and decreased pup

survival at the 100 and 500 ppm exposure levels. There were no effects on

endocrine organ weights except for a decreased testes weight at the

highest test dose only (500 ppm diazinon), a dose level that was also

associated with maternal toxicity. 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 statistically significant

effects on reproductive indices in either generation (all monitored

endpoints). There were no reproductive system abnormalities noted in

pups of either generation. There was a trend towards a prolonged gestation

in both generations at the high dose level (500 ppm), but the effect was not

statistically significant. In the Fl generation, a trend also was observed for

a decreased number of pregnancies and the number of viable newborns, as

well as adverse effects on mating and fertility, again only at the test dose

that produced signs of maternal toxicity (500 ppm diazinon). These data

demonstrate that diazinon 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

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

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Study No. MA-DZN-EDSP-20100213

endocrine toxicity potential of diazinon. 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 diazinon (see Table 1). For example, the lack of effects on

fertility and female and male endocrine organ histopathology in the multigeneration study is consistent with findings in vitro that diazinon is not a

potent estrogenic agent, is not an androgen receptor binding agent, and has

no effects on aromatase activity (e.g., Soto et al. 1995; Nishihara et al.

2000; Kojima et al. 2004; Kojima et al. 2005; Manabe et al. 2006; Judson

et al. 2009). Further, the lack of effects on thyroid organ histopathology

and ultimate effects of altering thyroid hormone activity in the multigeneration study are consistent with the lack of thyroid hormone receptor

binding by diazinon (Judson et al. 2009)

5. Infurna 1985

Review of this GLP-compliant guideline prenatal (teratology)

toxicity study in rats indicates that it provides reliable data for assessing 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 Charles River CD rats were

administered diazinon of unspecified purity by gavage at doses of 0, 10,

20, and 100 mg diazinon/ kg body weight from gestation day six (GD6)

through GDIS. It should be noted that the gavage route of administration

is not as relevant for human health risk assessment but is the most routine

route of exposure for teratology studies.

Like all guideline prenatal toxicity studies, this study was designed

to examine the teratogenic potential of diazinon. 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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Study No. MA-DZN-EDSP-20100213

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;

testes, ovary, uterus); and fertility indices (survival, pre- and postimplantation loss).

The most sensitive NOAEL in the study was 20 mg/kg/day and

was based on decreased maternal and fetal body weight at the 100 mg/kg

exposure level. There were no reported teratogenic effects in pups at any

dose level. There were no effects on endocrine organ histopathology at

any dose level tested (e.g., various reproductive organs in males and

females exposed even during development). There were no statistically

significant effects on fertility indices. These data demonstrated that

diazinon 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 diazinon in vivo, and

are reliable diazinon OSRI.

6. Mandella 2003

Review of this GLP-compliant guideline developmental

neurotoxicity study in rats indicates that it provides reliable data for

assessing 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

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.

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In this guideline study in rats, female Sprague-Dawley rats were

administered 92.9% pure diazinon in diet at doses of 0, 0.3, 30, and 300

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

evaluated at PND60. It should be noted that a dietary route of

administration is more relevant to the expected human exposure to

diazinon 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 toxicity 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 0.3 ppm diazinon and

was based on decreased maternal acetylcholinesterase levels in blood and

brain. No gross lesions of any kind were reported, including lesions in

endocrine organs. No effects on any reproductive indices were reported.

There were no observed effects on pup body weight or lactation or nursing

behavior and no effects on pup survival. Pup growth was decreased at the

highest test dose of 300 ppm diazinon at both PND7 and PND21. Vaginal

opening and preputial separation in pups were delayed by less than 2 days

but only at the highest test dose (300 ppm), a dose that was associated

with maternal toxicity as well as significant clinical signs of toxicity

(inhibition of acetylcholinesterase activity in blood and brain of pups).

These data demonstrated that diazinon was not directly toxic to endocrine

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

periods of development, in utero and during lactation.

The data provided in this prenatal toxicity study are functionally

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

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

890.1500, and 890.1600). The data on vaginal opening and preputial

separation are endpoints that had not been included in other guideline

diazinon toxicity studies and are monitored in EDSP Tier 1 assays

(890.1450 and 890.1500). This study provides reliable diazinon OSRI.

IV. Discussion of the EDSP Tier 1 Tests Needed for Assessment of the Endocrine

Disruption Potential of Diazinon

In September 2008, the Agency for Toxic Substances and Disease Registry

(ATSDR) published an update to the diazinon toxiciological profile document that

included a discussion of the potential endocrine toxicity of diazinon based a review of the

published data on diazinon. In that document, it is stated that "Results of available animal

studies indicate that the reproductive system is not particularly sensitive to diazinon

toxicity." (see page 110 of ATSDR. 2008. lexicological Profile for Diazinon.

September). The document also states "Results of available in vitro assessments of

diazinon estrogenicity indicate a potentially weak estrogenic effect at best." (see page

111 of ATSDR. 2008. Toxicological Profile for Diazinon. September). Review of the

information presented in Table 3 (above) is consistent with the statements found in the

2008 toxicological profile for diazinon.

In the case of diazinon's potential for estrogenic activity in vitro, the compound

has demonstrated only limited, weak estrogenic activity in vitro in some published

studies (e.g., Kojima et al. 2005; Manabe et al. 2006), with the majority of the diazinon

OSRI demonstrating that the compound would not act through estrogenic or antiestrogenic mechanisms to produce toxicity. The OSRI studies with diazinon provide both

directly equivalent and functionally equivalent measures of the potency of diazinon to

bind to ERs and affect the function of ERs. When the concentrations of diazinon shown

to have effects in these studies are considered (concentration in the micromolar range or

> 1 x 10"4 M), it is unlikely that ER binding assays from EDSP Tier 1 testing, if

performed, would indicate that diazinon is a potent or biologically relevant ER agonist or

antagonist. The in vivo data for diazinon relevant to estrogenic activity is consistent in

showing that diazinon does not affect female reproductive function in mammals.

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With respect to androgen activity in vitro, the recent publication of data collected

as part of the ToxCast program (Judson et al. 2009) has demonstrated that diazinon was

inactive as either an AR agonist or antagonist, and had no activity to affect aromatase

function, the enzyme that converts androgens to estrogens (anti-androgenic activity).

Moreover, there are a variety of in vivo studies available examining the effects of

diazinon on androgen systems in mammals. When considered together, the data do not

indicate that diazinon is a potent reproductive toxicant that produces androgen-specific

effects.

Review of the diazinon in vivo guideline registration studies, by themselves,

confirms the findings of the ATSDR (2008) that diazinon is not a potent reproductive or

developmental toxicant. 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 diazinon 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

diazinon exposure that might be linked to endocrine activity were limited generally to

high doses (100 ppm diazinon in diet and greater). These effects included decreased pup

survival and growth in the two-generation reproductive toxicity study (at doses of 100

ppm diazinon and greater) and delays in preputial separation and vaginal opening in the

developmental neurotoxicity study (at doses of 300 ppm diazinon).

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

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

effects in humans. Regulatory bodies rely on such studies to make decisions regarding

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

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

assessment.

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

available diazinon endocrine toxicity data that most of the Tier 1 EDSP tests need not be

conducted if the goal is to screen for potential endocrine activity. However, there are

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certain endpoints of EDSP Tier 1 testing that have not been specifically measured in

available diazinon studies. Considering the available data, MANA proposes that only the

following Tier 1 studies be conducted as part of the EDSP testing program for diazinon:

Fish Short-Term Reproduction (890.1350); Hershberger assay in rats (890.1400);

Uterotrophic assay in rats (890.1600); and Steroidogenesis (Human Cell Line H295R)

(890.1500). The rationale for MANA's position is outlined below regarding particular

studies that do not need to be performed.

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

Assays for Estrogen Activity (Estrogen Receptor Binding 890.1250 and

Estrogen Receptor Transcriptional Activation 890.1300)

EDSP Tier 1 assays 890.1250 and 890.1300 are screening assays to

identify modes of action for estrogenic activity involving binding to and

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

activity data available for determining the ability of diazinon 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 diazinon to appropriate control agents. The results have been consistent

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

orders of magnitude less than positive control agents (i.e., l?p-estradiol); activity

of diazinon in vitro is only seen at doses in the high micromolar range. The major

metabolite of diazinon, diazoxon was shown to be inactive in all ER assays in

which it has been tested (Judson et al. 2009). Therefore, MANA believes there is

no scientific basis for performing additional in vitro estrogen receptor activity

studies, specifically the Estrogen Receptor Binding assay (890.1250) and the

Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa-9903)

assay (890.1300). The available data are adequate for assessing the activity of

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Study No. MA-DZN-EDSP-20100213

diazinon at ER receptors. As a result, a waiver of both 890.1250 (Estrogen

Receptor Binding assay) and 890.1300 (Estrogen Receptor Transcriptional

Activation assay) is sought.

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

for Androgen Activity (Androgen Receptor Binding 890.1150 and Aromatase

Activity 890.1200)

EDSP Tier 1 assays 890.1150 and 890.1200 are screening assays to

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

binding to androgen receptors or alterations in conversion of androgens to

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

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

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

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

aromatase activity in vitro. Diazinon and its major metabolite, diazoxon, were

inactive in all these assays. Further, the available studies provide directly and

functionally equivalent data on essentially the same endpoints, including

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

are adequate OSRI for assessing the activity of diazinon at AR receptors and on

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

vitro androgen receptor activity studies, specifically the Tier 1 Androgen

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

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

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

Binding assay) and 890.1200 (Aromatase assay) for diazinon is sought.

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

Metamorphosis Assay (890.1100)

The Amphibian Metamorphosis assay (890.1100) is described in the

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

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

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Study No. MA-DZN-EDSP-20100213

(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 diazinon has no effects on functionally equivalent

endpoints in mammalian species. For example, the diazinon 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 diazinon 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 diazinon

were producing direct effects on either thyroid hormone synthesis or function.

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 diazinon OSRI, is not scientifically justified

when robust in vivo data in developing organisms is already available and does

not show that the thyroid hormone system is a target for diazinon 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 diazinon OSRI are considered. It should also be pointed out that in the

discussion below of the waivers being sought for the pubertal assays in rats (both

male and female), MANA is proposing to add measurement of thyroid hormone

levels to the Uterotrophic and Hershberger assays that will be performed with

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diazinon. Thus, direct effects of diazinon to affect thyroid hormone synthesis

through actions in the brain and the thyroid gland will be measured in vivo.

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

Pubertal Assay (890.1450)

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

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

endocrine system by identifying effects on pubertal development and thyroid

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

diazinon demonstrating that this chemical is not a potent estrogenic or antiestrogenic agent in vivo. The OSRI includes the robust, guideline registration

studies (Table 3). These in vivo assessments 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 subchronic and chronic toxicity

studies, all included assessments of diazinon's effects on female reproductive

tissues and organs, both in adult animals and in developing animals (including

peri-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

guideline reproductive toxicity and developmental toxicity studies provide

adequate data on female reproductive organ effects following diazinon exposure,

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

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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 diazinon

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

following diazinon 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), there does not appear to be a strong scientific basis for

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

simply to provide for measures of thyroid hormone levels in rats. Therefore,

MANA proposes that monitoring of thyroid hormone levels be added as an

endpoint for the Uterotrophic assay, where rats do not have an intact HPG axis. In

this way, the potential mode of action of diazinon to affect thyroid hormone levels

will be directly addressed. MANA proposes to conduct the Tier 1 Uterotrophic

assay in rats because the test follows uterine weight changes in ovarectomized

female rats as a screen for estrogenic activity, a model not currently covered with

diazinon OSRI, while OSRI directed to diazinon and estrogen activity are

available in peripubertal female rats (multi-generation reproductive toxicity

study). Therefore, a waiver of EDSP Tier 1 890.1450 (Pubertal Female Rat

Development) is sought.

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

Assay (890.1500)

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

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

endocrine system by identifying effects on pubertal development and thyroid

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

male reproductive function and pathology to indicate that the systems that involve

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

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

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

toxicity study, the developmental toxicity study, the developmental neurotoxicity

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Study No. MA-DZN-EDSP-20100213

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

assessments 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

diazinon toxicity studies in rats (Table 3) all included assessments of diazinon's

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

animals and in developing animals (including peri-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 diazinon guideline

toxicity studies are the levels of sex hormones (i.e., testosterone) and the level of

thyroid hormones in serum of animals following diazinon exposure. In the case of

measurement of sex hormone levels in animals (i.e., testosterone), the fact that

none of the in vivo data, including the multi-generation reproductive toxicity

study in rats, indicate toxicity induced by diazinon that could be related to

significant changes in hormone levels alone, there does not appear to be a strong

scientific basis for requiring performance of EDSP Tier 1 screening studies

simply to provide for measures of sex hormone levels in male rats. Further, given

that the ultimate effects of either increasing or decreasing thyroid hormone levels

in mammals have been monitored in the available studies (e.g., growth, metabolic

rate and function, fetal brain development), 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. As

a result, MAN A proposes that monitoring of thyroid hormone levels be added as

an endpoint for the Hershberger assay, where rats do not have an intact HPG axis.

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Study No. MA-DZN-EDSP-20100213

In this way, the potential mode of action of diazinon to affect thyroid hormone

levels will be directly addressed. MANA proposes to conduct the Hershberger

assay because the test follows changes in castrated male rats as a screen for

androgen activity effects, a model not currently covered with diazinon OSRI,

while OSRI directed to diazinon and androgen activity are available in

peripubertal male rats (multi-generation reproductive toxicity study). Therefore, a

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

V. Overall Summary

The preceding discussion provides a detailed description of available diazinon

OSRI that can be used to support waivers for certain EDSP Tier 1 screening assays. The

diazinon OSRI is rich in information including information specific to assessing the

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

of diazinon on specific endpoints (i.e., potential modes of action). MANA is seeking

waivers for performance of seven 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); 890.1450 (Pubertal Female Rat Development); and 890.1500 (Pubertal Male

Rat Development). MANA also proposes to add measurement of thyroid hormone levels

to the protocols for both the Uterotrophic assay in rats (890.1600) and the Hershberger

assay in rats (890.1400). The addition of measurement of thyroid hormone levels to these

two assays will provide data on the potential direct effects of diazinon to affect thyroid

hormone release or synthesis in the brain and pituitary gland.

MANA proposes to conduct the Steroidogenesis assay (890.1550) in order to

collect data on the potential for diazinon to affect synthesis of steroid hormones, both

androgens and estrogens. Additionally, MANA proposes to conduct the Fish Short-Term

Reproduction assay (890.1350) in order to screen for potential endocrine toxicity of

diazinon in fish.

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VI. References Cited

Giknis, M.L.A. (1989) Diazinon Technical: A Two Generation Reproductive Study in

Albino Rats. Project ID MIN 852218. Unpublished report. Ciba-Geigy

Corporation, Research Department, Pharmaceuticals Division. February 9. MRID

41158101.

Infurna, R.N. (1985) A Teratology Study (Segment II) in Albino Rats. Report No. 52-83.

Ciba-Geigy Corporation, Research Department, Pharmaceuticals Division. April

19. MRID 00153017.

Judson, R.S., Houck, K.A., Kavlock, R.J., Knudsen, T.B., Martin, M.T., Mortensen,

H.M., Reif, D.M., Rotroff, D.M., Shah, I., Richard, A.M., and Dix, D.J. (2009) In

vitro screening of environmental chemicals for targeted testing prioritization - the

ToxCast project. Environ. Health Perspect. Online December 2009.

Kirchner, F.R., McCormick, G.C., Arthur, A.T. (1991) Diazinon (MG-8): One/two Year

Oral Toxicity Study in Rats. Lab Project No. 882018. Ciba-Geigy Corporation,

Research Department, Pharmaceuticals Division. June 14. MRID 41942002.

Klimisch, H-J., Andreae, M., and Tillman, U. (1997) A systematic approach for

evaluating the quality of experimental toxicological and ecotoxicological data.

Regul. Toxicol. Pharmacol. 25:1-5.

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

for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter

gene assays using Chinese hamster ovary cells. Environ. Health Perspect.

112:524-531.

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Kojima, M., Fukunaga, K., Sasaki, M., Nakamura, M., Tsuji, M. and Nishiyama, T.

(2005) Evaluation of estrogenic activities of pesticides using an in vitro reporter

gene assay. Int JEnviron Health Res 15:271-280.

Manabe, M; Kanda, S; Fukunaga, K; et al. (2006) Evaluation of the estrogenic activities

of some pesticides and their combinations using MtT/Se cell proliferation assay.

IntJHyg Environ Health 209:413-421.

Mandella, R.C. (2003) A developmental neurotoxicity study in rats. OPPTS 870.6300.

Huntingdon Life Sciences, Study No. 01-4532. November 17. MRID 46105601.

Nishihara, T., Nishikawa, J-I. Kanayama, T., Dakeyama, F., Saito, K., Imagawa, M.,

Takatori, S., Kitagawa, Y., Hori, S., and Utsumi, H. (2000) Estrogenic activities

of 517 chemicals by yeast two-hybrid assay. J. Health Sci. 46:282-298.

Oh, Y.J., Jung, Y.J., Kang, J-W., and Yoo, Y.S. (2007) Investigation of the estrogenic

activities of pesticides from Pal-dang reservoir by in vitro assay. Sci. Total

Environ. 388:8-15.

Rudzki, M.W., McCormick, G.C., Arthur, A.T. (1991) Diazinon (MG-8): 52-Week Oral

Toxicity Study in Dogs. Lab Project No. 882014. Ciba-Geigy Corporation,

Research Department, Pharmaceuticals Division. June 14. MRID 41942001

Singh, A.R. (1988) Diazinon (MG-8): 90-Day Oral Toxicity Study in Rats. Project ID

882011. Ciba-Geigy Corporation, Research Department, Pharmaceuticals

Division. August 4. MRID 40815003.

Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., and Serrano, P.O.

(1995) The E-SCREEN assay as a tool to identify estrogens: an update on

estrogenic environmental pollutants. Environ. Health Perspect. 103:113-122.

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