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Report ID: 101563

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STUDY TITLE

Chlorpyrifos: Scientific Rationale for Citation of Other Scientifically Relevant Information (OSRI) in

Fulfillment of OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I Assays

ORDER NUMBERS: EDSP-059101-30, EDSP-059101-31, EDSP-059101-33, EDSP-059101-34

DATA REQUIREMENT

OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I Assays

AUTHOR

Daland R. Juberg

STUDY COMPLETED ON

April 12,2010

PERFORMING LABORATORY

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, IN 46268

REPORT ID

101563

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STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

Compound: Chlorpyrifos

Title: Chlorpyrifos: Scientific Rationale for Citation of Other Scientifically Relevant

Information (OSRI) in Fulfillment of OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I

Assays

No claim of confidentiality, on any basis whatsoever, is made for any information contained in

this document. I acknowledge that information not designated as within the scope of FIFRA

Section 10 (d)(l)(A)(B) or (C), and which pertains to a registered or previously registered

pesticide is not entitled to confidential treatment and may be released to the public, subject to

the provisions regarding disclosure to multinational entities under FIFRA Section 10(g).

Company: Dow AgroSciences LLC

Company Agent: Kenneth D. Racke

Title: Regulatory Manager

Signature:

Date: April 12. 20 10 / ^ A "& ^O I O

THIS DATA MAY BE CONSIDERED CONFIDENTIAL OUTSIDE THE UNITED

STATES.
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STATEMENT OF COMPLIANCE WITH GOOD LABORATORY PRACTICE

STANDARDS

Title: Chlorpyrifos: Scientific Rationale for Citation of Other Scientifically Relevant

Information (OSRI) in Fulfillment of OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I

Assays

Compound: Chlorpyrifos

This report does not meet the definition of a GLP study as it appears in:

United States Environmental Protection Agency

Title 40 Code of Federal Regulations Part 160

Federal Register, 17 August 1989

Organization for Economic Cooperation and Development

ENV/LMC/CHEM(98)17, Paris - January 26,1998

NON-GLP STUDY

Kenneth D. Racke, Ph.D.

Sponsor

Dow AgroSciences LLC

Kenneth D. Racke,

Submitter

Dow AgroSciences LLC

Daland R. Juberg, mD.

Study Director/Author

Dow AgroSciences LLC

April 12,2010

/

April 12, 2010

2-

April 12, 2010
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QUALITY ASSURANCE STATEMENT

Compound: Chlorpyrifos

Title: Chlorpyrifos: Scientific Rationale for Citation of Other Scientifically Relevant

Information (OSRI) in Fulfillment of OPPTS 890.1450 and OPPTS 890.1500 EDSP Tier I

Assays

Report Completion Date: April 12,2010

NON-GLP STUDY
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1.1 Summary

This document is being submitted to address the requirements under the Tier I Endocrine

Disruption Screening Program (EDSP) for chlorpyrifos. Studies conducted on chlorpyrifos as

well as studies published in the open literature were reviewed to determine whether sufficient

functionally equivalent information comparable to that developed in EPA's proposed Tier I

battery is available for the purpose of assessing the endocrine-modulating potential of

chlorpyrifos. Other Scientifically Relevant Information (OSRI) is cited here in addressing two

of these requirements because the critical endpoints (as defined in the OPPTS 890.1450 and

OPPTS 890.1500 guidelines) and the associated endocrine modes-of-action have either been

previously evaluated or are in the process of being evaluated in one or more of three ways: a)

the endpoint has been directly assessed in existing regulatory mammalian toxicity studies; b)

in rare cases, surrogate or relevant endpoints have been evaluated in regulatory toxicity

studies; or c) the relevant endocrine mode-of-action will be assessed in other Tier I EDSP

assays (See Tables 8 and 9).

The stated purpose of the Endocrine Disrupter Screening Program is ".. .to determine whether

certain substances may have an effect in humans that is similar to an effect produced by a

naturally occurring estrogen, or such other endocrine effect as the Administrator may

designate." The stated purpose of Tier I testing "... is to identify substances that have the

potential to interact with the estrogen, androgen, or thyroid hormone systems using a battery

of assays."

Dow AgroSciences believes that conducting the following tests from the Tier I battery will

provide complementary data and sufficient scientific redundancy to assure that any potential

endocrine-related effects of chlorpyrifos would be clearly identified:

• Estrogen receptor binding

• Estrogen receptor transcriptional activation

• Androgen receptor binding

• Steroidogenesis

• Aromatase

• Uterotrophic

• Hershberger

• Amphibian metamorphosis

• Fish short-term reproduction

However, male and female pubertal assays are not required to make these determinations since

existing data from mammalian regulatory toxicity studies cited as OSRI in combination with

the data from other Tier I studies make these assays redundant and unjustified from the
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standpoint of animal use. Consequently, consistent with EPA's mandate to avoid duplicative

testing, these studies should not be conducted.

We respectfully ask that EPA carefully examine the OSRI cited in this rationale and request

that, based on its evaluation of the OSRI and the other EDSP assays to be conducted, the

Agency consider the requirement for chlorpyrifos for conducting the female and male pubertal

assays (OPPTS 890.1450, OPPTS 890.1500) to have been met.

1.2 Background

The EPA document entitled: "EPA's Approach for Considering Other Scientifically Relevant

Information (OSRI) under the Endocrine Disruptor Screening Program" (EPA, March 2009)

provides guidance to registrants on how existing data or "...other scientifically relevant

information" (OSRI) is to be considered in full or partial fulfilment of Tier I EDSP data

requirements. OSRI is defined as".. .information that informs the determination as to whether

the substance may have an effect that is similar to an effect produced by a substance that

interacts with the estrogen, androgen, and/or thyroid hormonal systems."

According to the EPA's OSRI guidance document, the following types of information may be

useful as OSRI: studies that correspond to the assays in Tier 1 or Tier 2, metabolism studies,

and other vertebrate or invertebrate toxicity studies, including, but not limited to,

developmental toxicity tests, carcinogenicity tests, toxicogenomic data, and reproductive

toxicity tests.

Review of Peer Reviewed Literature

In addition to the 40 CFR Part 158 registration studies evaluated, a literature search was also

performed for the purpose of identifying any published chlorpyrifos studies or data that could

potentially serve as OSRI in fulfilment of EDSP Tier I data requirements. The search was

performed in a broad-based manner and yielded a number of potentially relevant studies;

however, upon closer examination the majority were determined to not be directly relevant to

the EDSP Tier I data requirements. Of the original search, a sub-set of seven studies was

identified and determined to be more directly germane to several of the Tier I screening assays

or individual end-points within these assays. These include: one study that evaluated thyroid

hormone levels in mice, several studies examining estrogen or androgen receptor binding

and/or activation by various methods, and two aromatase assays.

Following identification of the seven publications, a data quality evaluation was performed on

each. To ensure that this step adhered to a standard and well-documented process, a publiclyavailable data quality evaluation tool was utilized. This tool is called the "ToxRTool"

(Toxicological data Reliability Assessment Tool) and is available to the public on the

ECVAM (European Centre for the Validation of Alternative Methods) web-site
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(http://ecvam.jvc.ec.europa.eu/index.htm). The purpose of the tool is to provide a transparent

and harmonized method for evaluation of data quality for in vivo and in vitro toxicology

studies. The tool provides data quality criteria and guidance and in the end assigns a Klimisch

category of 1 (reliable without restrictions), 2 (reliable with restrictions), or 3 (not reliable) to a

given study (Klimisch, 1996). As shown in Table 1, evaluation of the seven chlorpyrifos

literature studies did not yield any reliable (1 or 2) results. The primary reason that a more

favorable reliability score could not be assigned was due to the lack of dose-response data

where in multiple studies only one dose-level of chlorpyrifos was tested in the relevant assay.

Since none of the chlorpyrifos literature studies identified was determined to be reliable, they

will not be utilized as OSRI in fulfilment of any data requirements and new data will be

generated for these endpoints. These publications may be useful in future weight of evidence

determinations following completion of Tier I screening.

Table 1; Data quality and reliability of chlorpyrifos literature citations

'•"•StndhK^

1

2

3

Simona, D.A., et al.

Developmental Exposure

to Chlorpyrifos Induces

Alterations in Thyroid

and Thyroid Hormone

Levels Without Other

Toxicity Signs in Cdl

Mice. (2009)

Toxicological Sciences

108:311-319.

Lemaire, G. et al.

Activation of alpha and

beta estrogen receptors by

persistent pesticides in

reporter cell lines.

(2006). Life Sciences 79:

1160-1169.

Grunfeld, H.T. et

a/.Effect of in vitro

estrogenic pesticides on

human oestrogen receptor

alpha and beta mRNA

levels. (2004).

Toxicology Letters 151:

467480.

Thyroid hormone and

thyroid morphological

alterations in dams and

Fl GDI mice

Chlorpyrifos did not

cause a statistically

significant change in

transactivation activity

of ER-alpha or ER-beta

compared to control.

A small increase in ER

mRNA levels observed

with chlopyrifos

treatment

m^'A^^^^^K^^^

3

3

3

Study utilized a

subcutaneous route

of exposure for

post-natal

exposure; not a

relevant route for

human exposure.

Number of animals

per group was

insufficient.

Insufficient doselevels tested or

presented in the

paper

Measure of mRNA

level is not

considered specific

nor relevant to

EDSP
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' •» >*•• • f ^''^IwJvrarffi

iNumDerKv'

4

5

6

7

Vinggaard, A.M., ef

al. Screening of Selected

Pesticides for Inhibition

ofCYP19Aromatase

Activity In Vitro. (2000).

Toxicology in Vitro 14:

227-234.

Andersen, H.R., et

a/.Effects of Currently

Used Pesticides in Assays

for Estrogenicity,

Androgenicity, and

Aromatase Activity in

Vitro. (2002). Toxicology

and Applied

Pharmacology. 179: 1-12.

Kojima, H. et

al. Screening for Estrogen

and Androgen Receptor

Activities in 200

Pesticides by In Vitro

Reporter Gene Assays

Using Chinese Hamster

Ovary Cells. (2004). EHP

112:524-531.

Vinggaard, A.M., et

al. Screening of Selected

Pesticides for oestrogen

receptor activation in

vitro. (1999). Food

Additives and

Contaminants 16: 533-

542.

No statistically

significant effect of

chlorpyrifos treatment

compared to control

Chlorpyrifos induced a

weak response in

estrogenicity assay(s);

No effect relative to

control in aromatase

assay

Chlorpyrifos induced a

very weak response in

ER alpha transactivation

assay

No statistically

significant effect of

chlorpyrifos treatment

compared to control

;-KUinlscl0

3

3

3

3

Insufficient doselevels tested or

presented in the

paper

Insufficient doselevels tested or

presented in the

paper; MCF-7 test

system is of limited

relevance to EDSP

Insufficient doselevels tested or

presented in the

paper

Insufficient dose- •

levels tested or

presented in the

paper; Yeast and

MCF-7 test systems

is of limited

relevance to EDSP;

significant toxicity

issues observed

In addition to the types of data noted previously as relevant OSRI information, the OSRI

guidance document gives specific mention to toxicity studies conducted in fulfilment of the

data requirements for pesticide registration (40 CFR Part 158). Evaluation of these data in

addressing Tier I data requirements is a significant priority since these data delineate a broad

range of robust toxicity studies that must be completed and critically assessed by the EPA

prior to registration of a pesticide active ingredient in the United States. Among the required

data-set are multiple repeat-dose toxicity studies of various duration, from 4-13 weeks to

exposures encompassing the majority of these organism's lifespan.
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In addition, studies are conducted under 40 CFR Part 158 to determine any potential for

treatment-related effects on reproduction as well as pre- and post-natal development. The

doses used in all of these studies appropriately challenge the test organisms and the doses

employed are much higher than anticipated human exposure levels.

Examination of this complete data-set, which is already available for all pesticide active

ingredients, allows for determination of target organ toxicity and any other adverse effects that

may be elicited by exposure. These existing studies are capable of detecting adverse

biological effects of treatment that could be elicited by one or more modes-of-action,

including the endocrine modes-of-action being addressed by the EDSP. Potential adverse

effects resulting from modulation of the endocrine system have already been intrinsically

evaluated in the exiting registration toxicity studies. Based on the no-effect levels determined

by these studies, application of the principles of risk assessment allows for appropriate

margins of exposure in the use of these active ingredients.

Both in terms of good scientific principles and particularly in light of the scientific

community's responsibility for judicious use of animal resources, it is appropriate to consider

existing data in the context of the EDSP requirements, and EPA should grant exemptions

from screening where existing data already provide adequate information. In fact, according

to Federal Food, Drug and Cosmetic Act (FFDCA section 408(p)(5)(B)), EPA is obliged to

''.. ..minimize duplicative testing of the scone substance for the scone endocrine effect." This

policy is echoed by the Office of Management and Budget's response to EPA's EDSP

Information Collection Response (ICR) which directs the Agency to ".. .promote and

encourage test order recipients to submit Other Scientifically Relevant Information (OSRI) in

lieu of performing all or some of the Tier I assays, and EPA should accept OSRI as sufficient

to satisfy the test orders to the greatest extent possible."

In summary, for pesticide active ingredients there is a wealth of existing information available

that can reduce or eliminate the need for Tier I screening. In particular, the multi-generation

reproduction study already available for pesticide active ingredients is a higher-tiered

assessment of potential endocrine-mediated toxicity, and EPA is in fact planning to use it as

the mammalian Tier n testing requirement. Logic therefore dictates that having the highertiered study completed lessens or eliminates the need for supplying lower-tiered screening

assays of a less robust nature. With higher-tier data available, the practical utility of

performing screening studies is highly questionable. In addition, EPA has already evaluated

these existing studies for pesticide active ingredients, including any potential adverse effects

resulting from modulation of the endocrine system, and the Agency has already made a

determination that these active ingredients can be used without undue risk to human health.

1.3 Chlorpyrifos: Overview of Existing Database

First registered by the USEPA in 1965, chlorpyrifos is a pest control product now registered

and used for agricultural applications in more than 100 nations. No pest control product has
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been studied more extensively and present applications rest on 4 decades of use, health

surveillance of manufacturing workers and applicators, and a scientific database of more than

3600 studies on health and the environment.

The toxicological database for chlorpyrifos is one of the more extensive for any regulated

chemical, including a full set of reproductive and developmental studies required for

registration which provides in-depth assessment of numerous potential toxicological endpoints

of interest, including endocrine function. Specific studies that meet or exceed USEPA

guidelines and which are most relevant for evaluation of potential interaction with the

endocrine system are noted in Table 2 and include subchronic and chronic studies hi several

species, 4 developmental toxicity studies in three species, 2 multigeneration reproduction

studies, and a developmental neurotoxicity study. These studies were conducted hi

accordance with internationally accepted test guidelines in place at the time, and the more

recent studies adhere to GLP standards. Many of the studies utilize rats and, therefore, are

considered the most relevant to the mammalian Tier IEDSP data requirements. Taken

together, this suite of studies involving numerous endpoints of interest, and with the additional

strength afforded through direct analysis of effects on potential endocrine target organs and

inclusion of clinical chemistry and histopathology, provides the basis for a robust

determination of any potential effect on general toxicity, development and maturation, as well

as other endpoints that could signify an interaction or effect on the endocrine system.

Chlorpyrifos, along with other organophosphorous insecticides, exert their toxicity to target

organisms through inhibition of the enzyme cholinesterase. This is the well-characterized and

accepted mode of action by regulatory authorities worldwide. The multigeneration

reproduction and developmental studies for chlorpyrifos demonstrate that even at doses

thousands of times greater than estimated human exposure levels (Barr et al., 2005),

chlorpyrifos has no effect on male or female fertility, litter size or viability, or number of

embryos implanted in the uterus. Collectively, the studies reported in Table 2 have assessed a

wide range of biological effects related to endocrine function as part of the FIFRA required

testing for registered pesticides, and no endocrine effects have been found in experimental

animals that have been exposed from conception through adulthood to old age, at levels

thousands of times higher than potential human exposure. Regulatory agencies worldwide

have evaluated these studies and have not identified endocrine effects as a toxicological

endpoint or mode of action of concern. The EPA has also evaluated chlorpyrifos under FQPA

and has not identified it as an endocrine concern. More recently (December, 2008), the

California Office of Environmental Health Hazard Assessment's (OEHHA) Developmental

and Reproductive Toxicant Identification Committee (DART) declined to list Chlorpyrifos as

a reproductive (male or female) or developmental toxicant under California Proposition 65.

The DART concluded that evidence does not exist for chlorpyrifos which is necessary to meet

the standard that a chemical must be clearly shown to cause reproductive and/or

developmental toxicity in order for its listing under Proposition 65. Finally, there are no

known or reported endocrine effects in humans exposed occupationally.
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Table 2: Existing studies (OSRI) cited in fulfilment of Tier I data requirements

Citation Code

A

B

C

D

E

Study Citation

Rubin etal., 1987.

Pyrinex: Teratogenicity

study in the rabbit.

Makhteshim-Agan

(America), Inc. Life

Science Research Israel

Ltd. Ness Ziona, Israel

Rubin et al., 1987.

Pyrinex: Teratogenicity

study in the rat.

Makhteshim-Agan

(America), Inc. Life

Science Research Israel

Ltd. Ness Ziona, Israel

Deacon etal. 1979. The

effects of orally

administered

Chlorpyrifos on

embryonal and fetal

development in mice.

Dow Chemical.

Midland, Michigan

Ouellette et al. 1983.

Chlorpyrifos: Oral

teratology study in

Fischer 344 rats. Dow

Chemical. Midland,

Michigan

Warner etal. 1980.

Results of a two-year

toxicity and

oncogenicity study of

Chlorpyrifos

administered to CD-I in

the diet. The Dow

Chemical Company.

Indianapolis, Indiana

GLP Study

Yes

Yes

No

No

No

iVIRID Number

40436408

40436407

00095268

00130400

00054352 &

00142902
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Citation Code Study Citation GLP Study VIRID Number

Young and Grandjean,

1988. Chlorpyrifos: 2-

year dietary chronic

toxicity-oncogenicity

study in Fischer 344

rats. The Dow Chemical

Company, Freeport, TX

Yes 40952802

Thompson, et al. 1971.

Three generation

reproduction and

teratology study in the

rat following prolonged

dietary exposure to

Dursban: 0,0-diethyl 0-

3, 5,6-trichloro-2-

pyridyl

phosphorothioate. The

Dow Chemical

Company, Zionsville, IN

No 00029064 &

00064934

H Crown et al. 1985.

Pyrinex Technical:

Toxicity in dietary

administration to rats for

13 weeks. MakhteshimAgan (America), Inc.

Ness Ziona, Israel

Yes 40436406

Gur. 1992. Pyrinex

Technical. Oncogenicity

study in the mouse.

Makhteshim-Agin of

North America. Ness

Ziona, Israel

Yes 42534201

Breslinetal. 1991.

Chlorpyrifos: Twogeneration dietary

reproduction study in

Sprague-Dawley rats.

The Dow Chemical

Company. Midland, MI

Yes 41930301
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Citation Code

K

M

N

Study Citation

Szabo et al. 1988.

Chlorpyrifos: 13-week

dietary toxicity study in

Fischer-344 rats. The

Dow Chemical

Company. Freeport,

TX.

Corleyetal. 1986.

Chlorpyrifos: 13-week

nose-only vapor

inhalation exposure

study in Fischer 344

rats. The Dow Chemical

Company. Midland, MI

McCollister et al., 1971.

Results of two-year

dietary feeding studies

on DOWCO 179 in rats.

The Dow Chemical

Company. Midland, MI

Hoberman. 1998.

Developmental

neurotoxicity study of

Chlorpyrifos

administered orally via

gavage to Crl:CD®BR

VAF/Plus® presumed

pregnant rats. Argus

Research Laboratories.

Horsham, PA

GLP Study

Yes

No

No

Yes

MRID Number

40952801

40013901 &

40166501

00095178

44556901
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1.4 Chlorpyrifos: Scientific Rationale for Sufficiency of OS/77 in

Fulfilment of OPPTS 890.1450 and OPPTS 890.1500

In preparing this scientific rationale, the following process was followed:

1. Evaluate appropriate existing chlorpyrifos data (OSRI), including primarily guideline

toxicity studies and other published studies.

2. Examine existing data for endpoints relevant to EDSP Tier I.

3. Determine which Tier I endpoints are adequately addressed by OSRI.

4. Determine if any EDSP Tier I assays and endpoints are sufficiently addressed by

OSRI.

For registered active ingredients like chlorpyrifos, there is an abundance of available

information that is relevant to determining if any endocrine-mediated effects occur with

treatment. Studies submitted as OSRI in fulfilment of Tier I requirements should be carefully

considered and not dismissed if only one or a few specific endpoints requested by the Tier I

screening studies were not evaluated. For example, TSH and T4 measurements are required

endpoints under OPPTS 890.1450 and OPPTS 890.1500 assays, but these measurements

provide only one insight (and one based on a snapshot in time) on potential thyroid toxicity. It

is well-established that assessment of organ weight and histopathological review of the thyroid

gland are more deterministic and informative relative to this organ being a target and potential

implicator of endocrine-related toxicity. In this specific case, these types of data are superior

to discrete measurements on thyroid hormones. Further to this point, it should be noted that in

the case of the pubertal assays, many of the endpoints are not specific to the endocrine system

and can be altered as a result of general systemic toxicity (Stoker, 2000). For example,

endpoints evaluated in the pubertal assays can be altered secondary to effects on body weight

(Carney, 2004). This is significant as the guidelines for pubertal assays define reduced body

weight as evidence for having achieved an appropriate high dose-level for testing. Given the

lack of specificity for many of the apical pubertal endpoints, it is appropriate to consider

surrogate endpoints in place of these less-specific endpoints which may not have been

examined in the existing database for a given chemical. This approach will allow for a robust

weight of evidence determination based upon the totality of the available data. While all of

these endpoints may not be of equal sensitivity, consistent results found across multiple

studies and end-points will often provide a high degree of confidence in the conclusion

without the need for conducting additional testing.

Tables 3-5 below provide examples of end-points commonly assessed in 40 CFR 158

guideline studies that can serve as surrogates for other endpoints that assess estrogenicity, antiestrogenicity, and anti-androgenicity (androgenicity not included as xenobiotic androgen

agonists are rare). These tables present a clear and predictable pattern of adverse biological

outcomes following exposure to chemicals acting through estrogenic, anti-estrogenic or antiandrogenic modes-of-action. If a pattern consistent with the effects presented in these tables

is not observed, then a test chemical can be considered negative for adverse effects elicited
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through various endocrine mode(s)-of-action. Therefore, individual endpoints that are lacking

or even isolated statistically significant findings are of limited importance in determining a

chemical's potential to interact with the endocrine system since it is the array or pattern of

effects that is most useful in making this determination.

Table 3; Adverse effects elicited by estrogenic substances

Endpoint

Estrous cyclicity

Time to mating

Uterine weight

Vaginal patency

PPS

Ovarian follicle count

Fertility

Mating index

Corpora lutea

Implantations

Gestational length

Litter size

Anogenital distance

Epidydimis sperm count

Testis Histopathology

Epidydimis histopathology

Prostate histopathology

Ovary weight

Ovary histopathology

Mammary histopathology

Expected Change

Decrease frequency;

anestrous

Increase

Increase

Accelerated

Delayed

Decrease

Decrease

Decrease

Decrease

Increase

increase

Decrease

Decrease

Decrease

Atrophy

Lesions, decreased sperm

content

Involution of ventral prostate

epithelium; stromal

proliferation

Decrease

Stromal hyperplasia;

follicular cysts

Increased

References

Terry, 2004; Abbott, 2006;

Delia Seta, 2008; Sawaki,

2003
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Table 4; Adverse effects elicited by anti-estrogenic substances

Endpoint

Estrous cyclicity

Time to mating

Testis weight

Vaginal patency

Corpora lutea

Litter size

Testis Histopathology

Epidydimis histopathology

Prostate histopathology

Seminal Vesicle

histopathology

Ovary histopathology

Expected Change

Decrease frequency;

anestrous

Increase

Decrease

Delayed

Decrease or absent

Decrease

Leydig cell atrophy

Degenerative changes

Degenerative changes

Degenerative changes

Decrease primordial follicles;

hemorrhagic follicles;

interstitial/stromal

hyperplasia

References

Vanage, 1997; Abbott, 2006;
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Table 5; Adverse effects elicited by anti-androgenic substances

Endpoint

Preputial separation

Sperm count, motility

Testis Weight

Vaginal patency

Epididymis weight

Prostate weight

Seminal Vesicles Weight

Testis histopathology

Epididymis histopathology

Prostate histopathology

Seminal Vesicle

histopathology

Anogenital distance

Uterine histopathology

Mammary gland

Estrous cyclicity

Time to mating

Fertility

Ovary histopathology

Uterine histopathology

Expected Change

Delayed

Decrease

Generally Decrease

Delayed

Decrease

Decrease

Decrease

Carcinoma; leydig cell

hypertrophy/hyperplasia;

germinal degeneration

Agenesis; spermatic

depletion

Fibrosis, cellular infiltration;

epithelial hyperplasia;

agenesis

Fibrosis, cellular infiltration;

epithelial hyperplasia

Decrease

Cystic endometrial

hyperplasia

Decrease adenomas,

adenocarcinomas

Persistent estrus

Increase

decrease

Hypertrophy

Cystic endometrial

hyperplasia

References

Gray, 2001; Shin, 2002;

Lund, 2004; Van Der School,

1992; York, 2006; Bowman,

2005; Horsley, 2005; Rider,

2008; O'Connor, 2002;

Specific endpoints defined and required under OPPTS 890.1450 and OPPTS 890.1500

guidelines are summarized in Tables 6 and 7, respectively. For endpoints where the requested

data are not available, other functionally equivalent or surrogate endpoints generated in other

studies that have been conducted for chlorpyrifos are listed (citation codes refer to studies

listed in Table 2). In addition, other OPPTS EDSP Tier I assays may be performed to provide

the missing data-points since there is significant overlap in the Tier I testing program for the

various endocrine-mediated modes-of-action.

The conclusion from this process, after careful consideration of the weight of the evidence

provided by existing toxicity studies and endpoints available for chlorpyrifos (i.e., OSRI),
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combined with the EDSP studies that will be conducted, is that the female and male pubertal

assays are redundant of existing relevant information, and are, therefore not justified from an

animal use or scientific needs perspective. In granting an exemption from performing these

studies. EPA will prevent the unnecessary use of at least 90 animals.
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Dow AgroSciences LLC

Report ID: 101563

Page 26 of 31

7.7 Summary and Conclusions

Potential effects of treatment on growth and development are evaluated in multi-generation

reproduction studies. As this is essentially the purpose of the EDSP pubertal assays, it is logical to

conclude that data from multi-generation studies should be carefully evaluated prior to conducting the

pubertal assays. In addition to the multi-generation study for chlorpyrifos, there is a robust and diverse

set of toxicological studies covering numerous other endpoints and endocrine-sensitive tissues in

multiple species and including a vertical array of biological assessments (i.e., organ weight, growth,

fertility, histopathology, clinical chemistry). For chlorpyrifos, there is no empirical evidence that is

suggestive of endocrine toxicity nor has a mode-of-action involving endocrine-mediated effects been

postulated, investigated or confirmed. When multi-generation study results for chlorpyrifos are

combined with other existing data and perhaps supplemented with EDSP Tier I screening data (to be

generated), it may be concluded that the pubertal assays would be redundant and, therefore, not

necessary to make a robust determination of potential effects on the endocrine system. A

summarization of the collective data available for chlorpyrifos pertaining to female and male

reproductive toxicity follows:

Female Reproductive Toxicitv

There is no evidence from the multigeneration reproduction study in rats or from other

toxicological studies in which female reproductive organs/tissues were evaluated that

chlorpyrifos is associated with female reproductive toxicity. There is no evidence that

chlorpyrifos is associated with effects on female reproductive structure or function including

genetic damage to the ovum, alterations in ovulation or menstruation, impaired or altered

endocrine function or complications during pregnancy. Similarly, there is no evidence that

chlorpyrifos affects female reproductive performance as evaluated in the studies for which such

effects would nominally be determined, primarily the multigeneration reproduction study in the

rat. Additionally, an evaluation of both long-term studies in rats and mice for evidence of

treatment-related effects to the reproductive organs/tissue of females revealed no adverse effects.

There were no observed treatment-related effects on reproductive organ weights nor were there

any non-neoplastic or neoplastic treatment-related findings in female reproductive organs/tissues

at either the interim sacrifice or terminal sacrifice periods.

Male Reproductive Toxicity

There is no evidence from the multigeneration reproduction study in rats or from other

toxicological studies in which male reproductive organs/tissues were evaluated that chlorpyrifos

is associated with male reproductive toxicity. There is no evidence that chlorpyrifos is associated

with effects on male reproductive structure or function including genetic damage to the

spermatozoon or its precursor, impaired sperm or seminal fluid production, impaired or altered

endocrine function, or impaired reproductive performance. An evaluation of long-term studies in

both rats and mice for evidence of treatment-related effects to the reproductive organs/tissue of
Dow AgroSciences LLC

Report ID: 101563

Page 27 of 31

males revealed no adverse effects. There were no observed treatment-related effects on

reproductive organ weights nor were there any non-neoplastic or neoplastic treatment-related

findings in male reproductive organs/tissues at either the interim sacrifice or terminal sacrifice

periods.

For chlorpyrifos, all of the critical endpoints defined in the OPPTS 890.1450 and OPPTS 890.1500

guidelines and the associated endocrine mode(s)-of-action have been evaluated in one or more of three

ways: a) the endpoint has been directly assessed in existing regulatory mammalian toxicity studies; b)

in some cases, surrogate or relevant endpoints have been evaluated in regulatory toxicity studies; or c)

the relevant endocrine mode-of-action will be assessed in other Tier IEDSP assays (See Tables 8 and

9). It is clear from these tables that no additional or critical information informing on endocrine-related

effects will be generated through the conduct of the male and female pubertal assays.

Table 8; Summary of OSRI for female pubertal assay

OPPTS 890.1450

Endpoint

Growth

Age at Vaginal

Opening

Uterine Weight

Ovarian Weight

Thyroid Weight

Liver Weight

Kidney Weight

Pituitary Weight

Adrenal Weight

Uterine

Histopathology

Ovarian

Histopathology

Thyroid

Histopathology

Kidney

Histopathology

TSHandT4

Estrous cyclicity

Clinical Chemistry

Endpoint assessed

in existing studies

X

X

X

X

X

X

X

X*

X

X

X

X

X

X

Surrogate endpoints

assessed in existing

studies

X

X

Mode-of- Action to

be assessed in other

Tier I EDSP assays

X

X

X

X

X

X

X

* histopathology data available which is a surrogate indicator of target organ toxicity
Dow AgroSciences LLC

Report ID: 101563

Page 28 of 31

Table 9: Summary of OSRI for male pubertal assay

OPPTS 890.1500

Endpoint

Growth

Age at preputial

separation

SV Weight

VP Weight

DLP Weight

Levator ani- weight

Epididymides

Weight

Testes weight

Thyroid weight

Liver Weight

Kidney Weight

Pituitary Weight

Adrenal Weight

Epididymis

Histopathology

Testes

Histopathology

Thyroid

Histopathology

Kidney

Histopathology

TSHandT4

Testosterone

Clinical Chemistry

Endpoint assessed

in existing studies

X

X

X

X

X

X

X*

X

X

X

X

X

X

Surrogate endpoints

assessed in existing

studies

X

Mode-of- Action to

be assessed in other

Tier I EDSP assays

X

X

X

X

X

X

X

X (optional)

X (optional)

X (optional)

X

X (optional)

X

* histopathology data available which is a surrogate indicator of target organ toxicity

The confidence in the existing studies cited in this rationale and ability to use them in lieu of

performing new studies is increased by examining the totality of available information and performing

a weight of evidence evaluation. The existing database for chlorpyrifos, generated over 40+ years,

does not demonstrate any pattern of effects consistent with adverse endocrine-mediated effects on

reproductive organs or processes.

The stated purpose of the Endocrine Disrupter Screening Program is "to determine whether certain

substances may have an effect in humans that is similar to an effect produced by a naturally occurring

estrogen, or such other endocrine effect as the Administrator may designate." The purpose of Tier I

screening "is to identify substances that have the potential to interact with the estrogen, androgen, or

thyroid hormone systems using a battery of assays." Based on the information presented above, it is
Dow AgroSciences LLC

Report ID: 101563

Page 29 of 31

clear that the male and female pubertal assays are not required to make this determination. Adequate

information is available based on existing data and through other Tier I studies that will be conducted

for chlorpyrifos. The pubertal assays are, therefore, redundant, not justified from an animal use

position, and should not be conducted per EPA's mandate to avoid duplicative testing. We encourage

EPA to carefully examine the OSRI cited in this rationale and request that the requirement for

conducting the female and male pubertal assays (OPPTS 890.1450, OPPTS 890.1500) be considered

complete for chlorpyrifos.
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Report ID: 101563

Page 30 of 31

References:

Abbott, DH, V Padmanabhan, and DA Dumesic. (2006). Contributions of androgen and estrogen to

fetal programming of ovarian dysfunction. Reprod Biol Endocrinol 4:17.

Barr, D. B., et al. 2005. Concentrations of selective metabolites of organophosphorous pesticides in

the United States population. Environ. Res. 99(3): 829-833.

» Bowman, CJ. (2005). Validation of the Pubertal Male Assay in Rats (WA 4-15). WIL-431005. Final

report. 13 December 2005.

Carney, E.W., C. L. Zablotny, M. S. Marty, J. W. Crissman, P. Anderson, M. Woolhiser, and M.

Holsapple The Effects of Feed Restriction during in Utero and Postnatal Development in Rats

Toxicol. Sci., Nov 2004; 82: 237 - 249.

Delia Seta, D, F Farabollini, F Dessi-Fulgheri, and L Fusani. (2008). Environmental-like exposure to

low levels of estrogen affects sexual behavior and physiology of female rats. Endocrinology

149(11):5592-5598.

EPA, 2009. EPA's Approach for Considering Other Scientifically Relevant Information (OSRI)

under the Endocrine Disrupter Screening Program.

Gray, Jr, LE, J Ostby, J Furr, CJ Wolf, C Lambright, L Parks, DN Veeramachaneni, V Wilson, M

Price, A Hotchkiss, E Orlando, and L Guillette. (2001). Effects of environmental antiandrogens on

reproductive development in experimental animals. Hum Reprod Update 7(3):248-264.

« Horsley, EMT. (2005). Assessment of Pubertal Development and Thyroid Function in Juvenile Male

Rats. Study No. 04-4276. Final report. 22 December 2005.

Klimisch, H.J., Andreae, M., Tillmann, U. A Systematic Approach for Evaluating the Quality of

Experimental Toxicological and Ecotoxicological Data. (1996). Regulatory Toxicology and

Pharmacology 25: 1-5.

Lund, TD, DJ Munson, ME Haldy, KDR Setchell, ED Lephart, and RJ Handa. (2004). Equol is a

novel anti-androgen that inhibits prostate growth and hormone feedback. Biol Reprod 70:1188-1195.

O'Connor, JC, SR Frame, and GS Ladies. (2002). Evaluation of a 15-day screening assay using intact

male rats for identifying antiandrogens. Toxicol Sci 69:92-108.

Ride:', CV, J Furr, VS Wilson, and LE Gray, Jr. (2008). A mixture of seven antiandrogens induces

reproductive malformations in rats. IntJ Androl 31:249-262.
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Sawaki, M, S Noda, T Muroi, H Mitoma, S Takakura, S Sakamoto, and K Yamasaki. (2003). In utero

through lactational exposure to ethinyl estradiol induces cleft phallus and delayed ovarian dysfunction

in the offspring. Toxicol Sci 75:402-411.

Shin, JH, HS Kim, HJ Moon, IH Kang, TS Kim, JH Seok, IY Kim, KL Park, SY Han, and SY Nam.

(2002). Effects of flutamide on puberty in male rats: An evaluation of the protocol for the assessment

of pubertal development and thyroid function. J Toxicol Environ Health, PtA 65(5-6):433-445.

Stoker, T.E., Louise G. Parks, L. Earl Gray, Ralph L. Cooper (2000). Endocrine-Disrupting

Chemicals: Prepubertal Exposures and Effects on Sexual Maturation and Thyroid Function in the

Male Rat. A Focus on the EDSTAC Recommendations. Critical Reviews in Toxicology, Jan 2000,

Vol. 30, No. 2, Pages 197-252.

Terry, KK, GD Cappon, ME Hurtt, MS Tassinari, and U Gupta. (2004). Reproductive toxicity

assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in female rats. Birth

Defects Res B Dev Reprod Toxicol 71(3): 150-60.

Vanage GR, B Dao, X-J Li, CW Bardin and SS Koide. (1997). Effects of Anordiol, an Antiestrogen,

on the Reproductive Organs of the Male Rat. Systems Biology in Reproductive Medicine Vol. 38, No.

1, Pages 13-21

Van der School, P. (1992). Disturbed testicular descent in the rat after prenatal exposure to the

antiandrogen flutamide. J Reprod Pert 96:483-496.

*York, RG. (2006). Interlaboratory Validation of the Pubertal Male Assay. Final Report. 3 January

2006.

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