DuPont-29838

TRADE SECRET

Position Paper Title

METHOMYL: OTHER SCIENTIFICALLY RELEVANT INFORMATION

PERTAINING TO EPA TEST ORDER EDSP-090301-62

Authors

Karin S. Bentley, Ph.D.

Carla Thomas, Ph.D.

Test Guideline

Not Applicable

Date Completed

February 18, 2010

Applicant/Sponsor

E. I. du Pont de Nemours and Company

Wilmington, Delaware 19898

U.S.A.

Authors Location

DuPont Crop Protection

Stine Haskell Research Center

1090 Elkton Road

Newark, DE 19711

DuPont Project Identification

DuPont-29838

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Page 1 of 41
DuPont-29838

STATEMENTS OF CONFIDENTIALITY

The Following Statement Applies To Countries Other Than The United States of

America:

This report is the property of E. I. du Pont de Nemours and Company and contains

confidential and trade secret information. Except as required by law, this report

should not be partially or fully (i) photocopied or released in any form to an outside

party without the prior written consent of E. I. du Pont de Nemours and Company or

its affiliates, or (ii) used by a registration authority to support the registration of any

other product without the prior written consent of E. I. du Pont de Nemours and

Company or its affiliates.

The Following Statement Applies To The United States of America:

STATEMENT OF No DATA CONFIDENTIALITY

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)(l)(A), (B), or (C).

Company

E. I. du Pont de Nemours and Company

i

Company Agent:

U.S. Product Registration Manager

'10

Date

We have submitted this material to the United States Environmental Protection

Agency specifically under the provisions contained hi FIFRA as amended and, .. ,

hereby, consent to use and disclosure of this material by EPA accordifig*lt>*f?IFR/?. *•• *

Notwithstanding the wording of our marking TRADE SECRET, this marldfig, by

itself, conveys no supplemental claims of confidentiality under FIFRA'Jftfiljons

or 10(b). In submitting this material to EPA according to method and fora&t ^ ..

requirements contained in PR Notice 86-5, we do not waive any prot&ftflto^or rigfaj . . •

involving this material that would have been claimed by the Company titffis material

had not been submitted to the EPA, nor do we waive any protection or right pro'SklsA,..

under FIFRA Section 10(g). *....

•..".".*•
DuPont-29838

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

U.S. EPA Good Laboratory Practice Standards (40 CFR Pail 160) do not apply to the

current document.

Applicant/Sponsor

E. I. du Pont de Nemours and Company

Wilmington, Delaware 19898

U.S.A.

Author

Karin S. Bentley, Ph.D. (/

Senior Regulatory Toxicologist

Date

Applicant/Sponsor

DuPont Representative

Date
-DuPont-29838

SIGNATURE

METHOMYL: OTHER SCIENTIFICALLY RELEVANT INFORMATION

PERTAINING TO EPA TEST ORDER EDSP-090301 -62

Position Paper by:

Kariri S. Bentley, Ph.D. ~jj Date (/

Senior Regulatory Toxicologist

\\

Carla N. Thomas, Ph.D. Date

Regulatory Ecotoxicologist

Date Completed:

February 18,2010

Applicant/Sponsor:

E. I. du Pont de Nemours and Company

Wilmington, Delaware 19898

U.S.A.
DuPont-29838

TABLE OF CONTENTS

Title Page 1

Statements of Confidentiality 2

Good Laboratory Practice Compliance Statement '. 3

Signature ; 4

Table of Contents .5

1.0 Summary 6

2.0 Introduction and Background 6

2.1 Other Scientifically Relevant Information (OSRI) .' 8

2.2 Toxicological Profile of Methomyl 9

3.0 Summary of Relevant Mammalian Toxicology Studies 11

3.1 Discussion of studies supporting registration 11

3.2 Discussion of available information in the open literature 23

3.2.1 Summary of the Potential to Modulate Estrogen or Androgen

Receptor Function 23

3.2.2 Summary of the Potential to Modulate Placental Aromatase

Activity In Vitro 25

3.2.3 Summary of In Vivo Studies: ..........26

3.3 Available Information from the EPA ToxCastTM Project 28

4.0 Summary of Relevant Ecotoxicology Studies : 28

4.1 Discussion of studies supporting registration 28

4.2 Discussion of available information in the open literature 32

5.0 DuPont Position and Conclusions 33

5.1 Potential for Methomyl to Interact with the Endocrine System 33

5.2 Functionally Equivalent Information Fulfilling the EDSP Tier IBattery 35

5.3 Overall Conclusion 37

6.0 References 37

TABLES

Table 1 Cited Methomyl Toxicity Studies Supporting Registration 40

Table 2 Endocrine Tissues Evaluated in Mammalian Toxicology Studies

Conducted with Methomyl ; .• 41
DuPont-29838

METHOMYL: OTHER SCIENTIFICALLY RELEVANT INFORMATION

PERTAINING TO EPA TEST ORDER EDSP-090301 -62

1.0 SUMMARY

This document is in response to EPA Test Order EDSP-090301-62 requiring DuPont

to test the pesticide active ingredient Methomyl (CAS# 16752-77-5) in the Tier 1

screening battery of the Endocrine Disruption Screening Program (EDSP). DuPont is

supplying scientifically relevant information which fulfills the purpose of the Tier 1

EDSP battery - to assess the potential to interact with estrogen, androgen and thyroid

> hormone processes. Methomyl has been evaluated in a large number of mammalian

' toxicology and ecotoxicology studies supporting the registration of this product which

have assessed potential subchronic, chronic, reproductive or developmental toxicity

in rats, mice, dogs, rabbits, fish, or birds. Several studies from the open literature

have reported results for the testing of methomyl in estrogen and androgen

transactivation assays, and methomyl has been included for testing in the many

endocrine-related in vitro tests in the EPA ToxCastTM project. The results

overwhelmingly demonstrate the lack of interaction by methomyl with estrogen,

androgen, or thyroid. Therefore, further assessment of methomyl in the Tier 1 EDSP

screening assays is not warranted.

2.0 INTRODUCTION AND BACKGROUND

In August 1996, Congress passed the Food Quality Protection Act that required the

US Environmental Protection Agency (EPA) "to develop a screening program using

appropriate validated test systems and other scientifically relevant information 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 effects as [EPA]

may designate". EPA subsequently initiated its Endocrine Disrupter Screening

Program in 1998 based on the recommendations of the Agency's Endocrine

Disruption Screening and Testing Advisory Committee (EDSTAC), which was

composed of stakeholders representing industry, governmental agencies, research

scientists, and environmental and public health groups. A two-tiered approach that

addresses potential human and ecological effects and examined effects on estrogen-,

androgen-, and thyroid hormone-related processes was recommended. The purpose

of Tier 1 "screening" is to identify substances that have the "potential" to interact

with estrogen, androgen, or thyroid hormone systems using a battery of assays. The

purpose of Tier 2 "tests" is to identify and establish "dose-response relationships" for

any adverse effects that might result from the interactions identified in the Tier 1

assays. Over the next years the Agency moved forward to develop and validate a

number of Tier I assays. In October 2009, EPA announced the availability of the

final Tier 1 screening battery and the related test guidelines. The final Tier I

endocrine screening battery is composed of the following assays:
DuPont-29838

In vitro assays: •

• estrogen receptor binding - an in vitro model which assesses the ability of a

substance to interact with the estrogen receptors in rat uterine cytosol

• androgen receptor binding - an in vitro model which assesses the ability of a

substance to interact with the androgen receptors in rat prostate cytosol

• estrogen transcriptional activation (HeLa9903 cells) - an assay that assesses the

binding of a chemical to the estrogen receptor and the subsequent downstream

transcription of a reporter gene product

• aromatase - an in vitro model to identify substances that may inhibit the catalytic

activity of the enzyme (C YP19) responsible for the conversion of androgens to

estrogens

• steroidogenesis (H295R cells) - an in vitro model intended to identify substances

that interfere with steroid hormone synthesis resulting in testosterone and

estradiol/estrone production

In vivo mammalian assays:

. • rat uterotrophic - an in vivo model to assess the ability of a substance to elicit

uterine weight changes consistent with agonists or antagonists of natural estrogen.

• rat Hershberger - an in vivo model to assess the ability of a substance to elicit

male reproductive organ weight changes consistent with androgen agonists and

antagonists and 5a-reductase inhibitors

• female rat pubertal - an assay to identify substances that interact with the

endocrine system by assessing endpoints associated with pubertal development

and thyroid function in female animals

• male rat pubertal - an assay to identify substances that interact with the endocrine

system by assessing endpoints associated with pubertal development and thyroid

function in male animals • .

In vivo wildlife assays:

• Amphibian metamorphosis - a generalized vertebrate model to identify

substances which interfere with the normal function of the hypothalamicpituitary-thyroid (HPT) axis

• Fish short-term reproduction in fathead minnow - a short-term assay measuring

reproductive fitness and biochemical endpoints that reflect effects associated with

the disturbance of the hypothalamus-pituitary-gonadal (HPG) axis

In April 2009, EPA published a final list of substances to be initially tested in the

Tier 1 screening battery (Federal Register 74; 17579). Beginning October 2009, EPA

has issued test orders to registrants of the listed substances which mandate testing

under the EDSP. A test order for Methomyl has been received by DuPont, Order/DCI

• EDSP-090301-62, issued on November 12, 2009.
DuPont-29838

2.1 Other Scientifically Relevant Information (OSRI)

EPA has provided registrants an opportunity to either submit or cite existing data that

may fulfill in part or completely the requirements of the Tier 1 EDSP screening

battery (EPA's Approach for Considering OSRI under the EDSP, March 26, 2009).

EPA has provided the following understanding of what constitutes OSRI:

"Other scientifically relevant information " is 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 (e.g., information that identifies substances as

having the potential to interact with estrogen, androgen, and/or thyroid

system(s); information demonstrating whether substances have an effect on the

functioning of the endocrine system). OSRI may either be functionally equivalent

to information obtained from the Tier 1 assays — that is, data from assays that

perform the same function as EDSP Tier 1 assays — or may include data that

provide information on a potential consequence or effect that could be due to

effects on the estrogen, androgen, or thyroid systems.

EPA further states that the following types of studies may contain information that

the Agency would likely regard as scientifically relevant information: studies that

correspond to assays in Tier 1 and 2; metabolism studies; or other vertebrate or

invertebrate toxicity studies, including, but not limited.to, developmental toxicity

tests, carcinogenicity tests, toxicogenomic data, and reproductive toxicity tests. EPA

goes on to indicate that other types of studies may also be considered scientifically

relevant. For instance, testing conducted under 40 CFR Part 158 data requirements to

support pesticide registration may be considered. Judgments by EPA about whether

the OSRI satisfies all or part of the Tier 1 Test Order will based on the "weight of the

evidence" from all relevant information available taking into account the dataset as a

whole for the subject chemical. , ;

On October 2, 2009 the White House Office of Management and Budget (OMB)

approved the pending Information Collection Request (ICR) required for the

implementation of the initial Tier 1 screening under the EDSP. In approving the

initiation of this program, OMB urged EPA to "promote and encourage" test order

recipients to submit "functionally equivalent" data in lieu of "performing all or some

of the Tier 1 assays" and to accept existing FIFRA test guideline data "to the greatest

extent possible".

As part of the 40 CFR Part 158 data requirements for registration under FIFRA

Section 3, pesticide active ingredients are required to be evaluated in a broad range of

mammalian toxicology studies including tests for acute, subchronic, and chronic

toxicity, mutagenicity, acute and subchronic neurotoxicity, reproductive and

developmental toxicity, and carcinogenicity and in a variety of wildlife studies

including tests for toxicity in fish and effects on avian reproduction. Many of these

studies provide a means for assessing the potential of a substance to alter endocrine
DuPont-29838

function (Stevens et al, 1997; ECETOC, 1996). Of particular value in the

assessment of adverse endocrine-mediated effects in the intact organism are the

following types of regulatory studies:

• Subchronic toxicity studies in rats, mice, and does - These studies are capable of

detecting hormonally mediated macroscopic and microscopic changes in morphology

and/or size of the pituitary, thyroid, adrenals, uterus, ovary, mammary gland, testes,

epididymis, and other sex accessory organs as a result of repeated treatment. Both

DBS- and DDT-induced increases in uterine weight were observed hi this type of

study (Burin et al, 1993).

• Prenatal developmental toxicity studies in rats and rabbits - These studies

examine hormonally induced effects such as abortion, resorption, or premature

delivery, abnormalities in reproductive development, and masculinization or

feminization of female and male fetuses, respectively. Substances such as the

phytoestrogen coumesterol and the anti-androgen cyproterone acetate were detected

in this type of study (van Ravenzwaay, 1992).

• Multigeneration reproduction study in rats - The multigeneration reproduction

study is recommended by EDSTAC (1998) as the definitive Tier 2 study for the

assessment of adverse effects of hormonally active substances in the intact animal.

Therefore, this study plays a pivotal role in the determination of adverse endocrine

effects relative to reproduction, growth, and development. These studies are capable

of detecting hormonally induced effects covering every phase of development and

reproduction including behavior, maintenance of pregnancy, gestation length,

difficult or prolonged parturition, fertility, sex ratio of offspring, feminization or

masculinization of offspring, number of pups, still births, and gross and microscopic

pathology of the vagina, uterus, ovaries, testes, epididymis, seminal vesicles, prostate,

etc. Procymidone-induced hypospadias and vinclozolin-induced feminization of male

pups were identified in a study of this type (van Ravenzwaay, 1992).

• Combined lifetime chronic toxicity/oncogenicity studies in rats and mice and

chronic toxicity studies in doss - These studies include extensive histopathology of

primary endocrine tissues and secondary accessory organs including pituitary,

thyroid, adrenals, ovary, uterus, vagina, mammary gland, testes, epididymis, prostate,

and seminal vesicles. Weak hormonally mediated effects which result in tumors of

endocrine tissues, such as testicular tumors (Hseuh et al., 1977) or mammary gland

tumors (Gruggs et a/., 1983), are often detected in these studies.

2.2 Toxicological Profile of Methomyl

The mammalian toxicity of methomyl is well characterized based on the results of an

extensive'battery of toxicology studies conducted in rats, mice, dogs, rabbits and

Guinea pigs and well as the results of a number of in vitro assays. The primary

tbxicological effect of this substance in mammalian species is the inhibition of

acetylcholinesterse in the nervous system, which is rapidly reversible. As a result

methomyl demonstrates high acute toxicity by the oral, ocular, and inhalation routes.

The primary symptoms are those associated with choliriesterase inhibition: No

cumulative effects on the nervous system occur with repeated exposure.
DuPont-29838

Hematology changes occurred only after chronic exposure and were generally mild

and regenerative. Methomyl does not induce tumors in rats or mice and is classified

by EPA as Group E (not likely to be carcinogenic to humans). Methomyl is not

mutagenic and has produced negative results in a battery genotoxicity studies. No

increased sensitivity of offspring was noted in multigeneration reproduction and

developmental toxicity studies as offspring NOAELs are equivalent to or greater than

those for parental animals. The principle effects in these studies were mortality,

clinical signs of cholinesterase inhibition, body weight reductions, and reduced

numbers of pups due to systemic toxicity. Methomyl is considered neither a

reproductive nor developmental toxicant. Throughout the mammalian toxicology

database, there has been no evidence suggestive of an adverse effect on any

component of the endocrine system.

Methomyl has been extensively tested in acute and chronic studies with a number of

species of ecological importance (birds, fish, aquatic and terrestrial invertebrates,

non-target plants). The ecotoxicity of methomyl is therefore well characterized. The

primary toxicological effect of methomyl in vertebrate and invertebrate animal

species is the inhibition of acetylcholinesterase in the nervous system. This effect is

rapidly reversible. As a result, methomyl demonstrates high acute toxicity. The main

effects in chronic exposure were reduced survival and growth in fish, and reduced

body weight in birds. These effects are indicative of the inherent toxicity of the

carbamate insecticide. The existing data available for methomyl from toxicology and

ecotoxicology databases provide compelling evidence that methomyl is not expected

to have an adverse effect on any component of the endocrine system.

Summaries of relevant existing 40 CFR Part 158 toxicology and ecotoxicology

studies that support the current FIFRA Section 3 registration of methomyl and

provide information on the potential of this product to interact with the endocrine

system are provided in Sections 3 and 4 of this document. These studies include

subchronic and chronic toxicity studies in rats, mice, and dogs and reproduction and

developmental studies in rats and rabbits. Also provided are summaries of studies

which have been conducted to evaluate any potential development and reproductive

effects of methomyl in birds and fish species. Table 1 lists for each study the MRID

numbers, report numbers, titles, whether the study was reviewed by EPA, and the

Agency's classification as to acceptability of each study. In addition, as requested by

EPA, hard copies of the front page of each report that has been previously submitted

to EPA are being supplied under separate cover. Finally, information available from

the open literature that is relevant to the potential interaction of methomyl with the

endocrine system is also summarized. Hard copies of these papers are being provided

under separate cover as a courtesy to EPA for the Agency's internal use with the

understanding that DuPont does not have authority to grant EPA permission to

distribute or post these third party studies on the EPA website.

10
DuPont-29838

3.0 SUMMARY OF RELEVANT MAMMALIAN TOXICOLOGY STUDIES

3.1 Discussion of studies supporting registration

Paynter, O.E. (1966); Three month dietary administration - rats insecticide 1179.

DuPont Report No. MRO-848, Hazleton Report No. 201-151, MRID 00007190,

satisfies Guideline 82-1.

Materials and Methods:

In a 13-week feeding study, Methomyl (purity 100%) was administered to male and

female Crl:CD®BR rats (10. rats/sex/group) at concentrations of 0, 10, 50, and

250 ppm. A fifth group of male and female rats was fed 125 ppm for 6 weeks and

500 ppm for the remainder of the study. In this group only, 5 animals/sex were

sacrificed at 2 months and the remainder were sacrificed at study termination.

Parameters evaluated included body weight,'body weight gain, food consumption,

clinical signs, haematology, urinalysis, plasma and red blood cell cholinesterase

activity, organ weights (including adrenals, pituitary, and testes), and gross and

microscopic pathology (including adrenals, testes, epididymis, ovary, uterus,

pituitary, and thyroid).

Findings:

General observations: No test substance-related clinical signs of toxicity were

noted at any dose level, with the exception of transient tremors observed in one

female rat from the 250 ppm group at 13 weeks. Test substance-related mortality did

not occur during the course of this study. Growth was suppressed in the 250 ppm

males and during the seventh through the thirteenth weeks in the 500 ppm males and

females (significantly different from control values in the 500 ppm males only). Food

consumption for the 500 ppm males was lower but not significantly different from'the

controls. Food consumption for the 250 ppm males and 500 ppm males and females

was slightly lower compared with controls.

Haematology, clinical chemistry, cholinesterase activity, urinalysis: Minor

haematological changes were noted. Compared with controls, significantly lower ,

haemoglobin values were observed in the 250 ppm males at 2 months. At 3 months,

red blood cell counts for the 250 ppm females were significantly lower but still within

normal limits. No significant differences were found in cholinesterase activity or

urinalysis Values.

Gross pathology, organ weights, histopathology: No test substance-related gross

morphological changes were observed at necropsy. Significantly decreased liver

weights and increased brain/body weight ratios were observed for the 125/500 ppm

and 250 ppm males sacrificed after 13-weeks. These differences from control values

were related to lower body weights of these animals when compared to controls.

Microscopic examination of tissues from the 250 ppm rats revealed moderate

erythroid hyperplasia in the bone marrow of all male rats in this group. There were

no test substance related effects suggestive of an adverse effect on the endocrine

system

11
DuPont-29838

Conclusion: The NOAEL in the 90-day feeding study in rats was 50 ppm

(approximately 3.6 mg/kg bw/day) for males based on decreased body weight/food

consumption at concentrations > 250 ppm and an increased incidence of erythroid

hyperplasia in the bone marrow of males at 250 ppm. The NOAEL for females was

50 ppm (approximately 4.1 mg/kg bw/day) based on the lower RBC and haemoglobin

at 250 ppm and body weight/food consumption effects at 500 ppm. There was no

evidence of a direct adverse effect on the endocrine system in this study.

Cox, R.H. (1979a); A subacute (90 day) evaluation ofNudrin in the Fischer-344 rat

for Shell Oil Company. DuPont Report No. 52B 101-630-78, MRID none.

Materials and Methods:

In a 90-day feeding study, Methomyl, dissolved in an acetone/com oil mixture

(2% w/w), was administered to male Fischer rats (20 rats/sex/group) at concentrations

of 0, 100, 200, 400 and 600 ppm. Additionally, one group of 20 males was fed

20 ppm from weeks 1-9 of the study followed by 1000 ppm from weeks 10-13 of the

study and another group of 20 males was fed 50 ppm from weeks 1-4 followed by

800 ppm from weeks 5-13. Female Fischer rats (20 rats/sex/group) were

administered concentrations of 0, 135, 270, 540, and 810 ppm. Additionally, one

group of 20 females was fed 27 ppm from weeks 1-9 followed by 1500 ppm from

weeks 10-13 and another group of 20 females was fed 68 ppm from week 1-4

followed by 1080 ppm'from weeks 5-13. Parameters evaluated included body

weight, food consumption, clinical signs, haematology, clinical chemistry, urinalysis,

plasma and RBC (red blood cell) cholinesterase activity, organ weights (including

adrenals, testes and epididymis, ovary and fallopian tubes, uterus), and gross and

microscopic pathology (including adrenals pituitary, testes, epididymis, ovary,

fallopian tube, uterus, cervix, and thyroid).

Findings:

General observations: Test substance-related mortality did not occur during the

course of this study. Test substance-related transient body weight differences were

noted throughout the study in males and females administered 1000 ppm and higher;

however, there were no differences noted by the end of the study. Transient

significant decreases in food consumption were also noted throughout the study.

Haematology, clinical chemistry, cholinesterase activity, urinalysis: Clinical

pathology changes indicative of a systemic effect included decreases in haemoglobin

at doses of 400 ppm and 50/800 for males and 540 ppm or greater in females. RBC

counts were decreased in 600 ppm males and 810 ppm females. Reticulocytes were

significantly increased in 600, 50/800, and 20/1000 ppm males and in females at

200 ppm and greater. Decreased glucose levels were noted for male rats given 400,

600, and 50/800 ppm for 6 weeks but not after 13 weeks. Glucose was also decreased

in females rats receiving 540 ppm or greater. Urea was increased in 27/1500 ppm

females at week 13. No distinct or apparent test substance-related differences were

observed in the results of the urinalysis conducted during week 7 or at termination.

Decreases in plasma cholinesterase activity were noted for females receiving

12
DuPont-29838

810 ppm or greater but similar changes were not observed in the male rats. In

contrast, there was an apparent treatment related decrease in RBC cholinesterase

activity from the 50/800 ppm males at termination, but similar changes were not

observed in the female rats.

Gross pathology, organ weights, histopathology: Absolute and/or relative spleen

weights were increased in males receiving 600 ppm or greater and in females

receiving 540 ppm or greater. Relative weights of the livers and kidneys were

increased for females receiving at and above 540 ppm and 810 ppm, respectively.

No.distinct or consistent test substance-related microscopic changes were noted in

tissue sections evaluated. There were no test substance related effects suggestive of

an adverse effect on the endocrine system.

Conclusion: The NOAEL in males was 200 ppm (13.6 mg/kg bw/day) based on

haematology changes at 400 ppm and greater. The NOAEL in females was 135 ppm

(10.0 mg/kg bw/day) based on haematology changes at 270 ppm and greater. There

was no evidence of a direct adverse effect on the endocrine system in this study.

Cox, R.H. (1979b); A subacute (90 day) evaluation ofNudrin in the B6C3F1 mouse

for Shell Oil Company. DuPont Report No. 52E 004-630-79. MRID none.

Materials and Methods:

In a 90-day feeding study, Methomyl was administered to male and female

Crl:CD®BR mice at dietary concentrations of 0, 75, 150, 300, 480, 600, and 960 ppm

(20/sex/group for controls and 10/sex/group for test substance groups except the

600 ppm group which had 11 males and 9 females). Parameters evaluated included

body weight, food consumption, clinical signs, clinical pathology, organ weights

(including adrenals, testes, ovaries with fallopian tubes, and uterus), and gross and

microscopic pathology (including adrenals, testes, prostate, ovaries, fallopian tube,

and uterus).

Findings:

General observations: Test substance-related mortality did not occur during the

course of this study. There were no consistent test substance-related clinical signs

noted or effects on body weights or food consumption.

Haematology: Haematologic differences indicative of a systemic effect included

decreased levels of haemoglobin in males at and above 300 ppm with apparent

compensation at 600 and 960 ppm, as indicated by increases in erythrocyte. counts

and haematocrit accompanied by decreased reticulocyte counts. Females at 150 ppm

and higher had decreased erythrocyte counts with increases in reticulocyte counts at"

and above 480 ppm indicative of the initiation of compensation. Haematocrit was

also significantly decreased for the 150 ppm and 600 ppm females. There were no

test substance related effects on clinical chemistry or urinalysis parameters.

Gross pathology, organ weights, histopathology: Male mice receiving 480 ppm

and above had increased absolute and relative liver weights. Other organ weight

differences were considered due to minor differences in final body weights and not

test substance-related. There were no consistent test substance-related gross or

13
DuPont-29838

microscopic pathology findings noted. There were no test substance related effects

suggestive of an adverse effect on the endocrine system.

Conclusion: The NOAEL in male mice was 150 ppm (approximately 26.6 nag/kg

bw/day) based on haematology changes at 300 ppm and above. The NOAEL in

-female mice was 75 ppm (approximately 15.6 mg/kg bw/day) based on haematology

changes at 150 ppm and above. There was no evidence of a direct adverse effect on

the endocrine system in this study.

Sherman, H. (1967); Three-month feeding study on dogs with S-methyl

N-[(methylcarbamoyl)oxy] thioacetimidate [Lannate Methomyl insecticide;

INX-1179]. DuPont Report No. HLR 168-67, MRID 00009010, does not satisfy

Guideline 82-1, but sufficient data from a chronic study is available.

Materials and Methods:

In a 90-day feeding study, Methomyl (purity 97.5%) was administered to male and

female beagle dogs (4 dogs/sex/group) at concentrations of 0, 50, 100, and 400 ppm

in the diet. Parameters evaluated included body weight, food consumption, clinical

signs, haematology, clinical chemistry, organ weights (adrenal, pituitary, testes,

prostate, and thyroid),, and gross and microscopic pathology (including adrenal,

pituitary, testes, epididymis, prostate, ovary, uterus, fallopian tube, mammary gland,

and thyroid).

Findings:

General observations: No test substance-related clinical signs were noted during the

course of the study. Test substance-related mortality did not occur. There were no

test substance-related effects on body weights or food consumption.

Haematology, clinical chemistry, urinalysis: No test substance-related effects on

haematology, urinalysis, or clinical chemistry parameters were noted.

Gross pathology, organ weights, histopathology: No test substance-related gross

lesions were observed at necropsy. No test substance-related changes in mean organ

weights or organ weights relative to final body weight were apparent at any dietary,

concentration. No gross or microscopic lesions were noted that could be attributed to

administration of the test substance. There were no test substance related effects

suggestive of an adverse effect on the endocrine system.

Conclusion: The NOAEL in the 90-day feeding study in dogs was 400 ppm for

males and females (14.7 and 12.5 mg/kg bw/day, respectively), the highest dose

tested. There was no evidence of a direct adverse effect on the endocrine system in

this study.

14
DuPont-29838

Kaplan, A.M. (1981); Long-term feeding study in rats with S-methyl

N-((methylcarbamoyl)oxy) thioacetimidate) (Methomyl; INX-1179).

DuPont Report No. HLR 235-81, MR1D 00078361, satisfies Guideline 83-5, 83-1 a,

83-2a. • •\

Materials and Methods;

In a 2-year chronic toxicity and carcinogenicity feeding study, Methomyl (purity

99%) was administered to male and female Chr:CD® rats (80 rats/sex/group plus an

additional 20 rats/sex/group designated for erythrocyte and brain cholinesterase

determinations) at concentrations of 0, 50, 100, and 400 ppm. Ten rats per group

were sacrificed after approximately 1 year on study, and all surviving rats were

sacrificed after approximately 2 years on study. Parameters evaluated included body

weight, body weight gain, food consumption, food efficiency, clinical signs, clinical

pathology, organ weights (including adrenal, pituitary, and testes), gross and

microscopic pathology (including adrenal, pituitary, testes, epididymis, prostate,

ovary, uterus, mammary, and thyroid), and erythrocyte and brain cholinesterase

activity. .

Findings:

General observations: No test substance-related clinical signs of toxicity were

noted during the study. There was no test substance-related effect on mortality. Male

and female rats fed 400 ppm had significantly lower body weight gain during the first

year of the study. Males in the 400 ppm group had a similar reduction in rate of

weight gain for test days 0-560. There were no test substance related effects on food

consumption or food efficiency.

Haematology, clinical chemistry, urinalysis: Females in the 400 ppm group had

significantly lower erythrocyte counts, haemoglobin values, and haematocrit when

compared to controls. A similar trend occurred in erythrocyte counts and

haemoglobin values in male rats fed 400 ppm; however, the changes were not

statistically significant. There were no test substance-related clinical chemistry or

urinalysis findings noted.

\

Erythrocyte cholinesterase activity: Erythrocyte cholinesterase activity was

slightly lower (not statistically significant) for the 50, 100, and 400 ppm males during

the 0.25, 0.5, and 1-month evaluations; however, a dose response-relationship was not

evident and there were no effects in females. There were no effects on brain

cholinesterase activity at either 1 or 2 years.

Gross pathology, organ weights, histopathology: No test substance-related gross

lesions were observed at necropsy. There were no histopathology findings noted.

There were no test substance-related absolute organ weight changes in either sex at

any concentration nor were there any relative organ weight findings in male rats.

Female rats at and above 100 ppm had significantly increased relative liver weights;

however, this did not correlate with any histopathological changes. Females fed

400 ppm also had increased relative spleen weights which may have been correlated'

with rear footpad dermatitis, a common naturally occurring lesion in wire cage-reared

ageing rats. There was no increase in neoplastic lesions in either males or females at

15
DuPont-29838

any dietary concentration. There were no test substance related effects suggestive of

an adverse effect on the endocrine system.

Conclusion: The NOAEL in the 2-year feeding study in rats was 100 ppm for males

and females (4.8 mg/kg bw/day for males and 6.3 mg/kg bw/day for females). This

NOAEL was based on decreased weight gain and haematology changes at 400 ppm.

Under the conditions of this study, Methomyl was not a carcinogen. There was no

evidence of a direct adverse effect on the endocrine system in this study.

Snyder, F. G. (1981); 104-week chronic toxicity and carcinogenicity study in mice

Methomyl; H-l 1,135. DuPont Report No. HLO 253-81, Hazelton Report No. 201-

510, MRID 00078423, satisfies Guideline 83-2b. '

Materials and Methods:

In a 104-week carcinogenicity feeding study, Methomyl (purity 99%) was

administered to male and female CD®-1 mice (approximately 80 mice/sex/group) at

concentrations of 0 ppm, 50 ppm, 100 ppm (reduced to 75 at week 39) and 800 ppm

(reduced to 400 at week 28 and further reduced to 200 at week 39). Parameters

evaluated included body weight, body weight gain, food consumption, food

efficiency, clinical signs, haematology, organ weights (including adrenal, pituitary,

and testis with epididymis), and gross and microscopic pathology (including adrenal,

pituitary, testes, epididymis, prostate, ovary, uterus, mammary, and thyroid).

Findings:

General observations: No test substance-related clinical signs were noted during the

study. The incidence of gross or palpable masses was similar for all groups.

Decreased survival occurred in 100 ppm females and 800 ppm males and females.

Due to this result, the high dose group was reduced to 400 ppm at week 28 and

subsequently reduced to 200 ppm at week 39. The 100 ppm group was reduced to

75 ppm also at week 39. Survival continued to be lower in 75 and 200 ppm males

and females and was lower in all treated males at week 104. There were no test

substance-related effects on body weights or body weight gains. There were no test

substance-related effects on food consumption or food efficiency.

Haematology: Significantly lower haematocrit was noted in 100 and 800 ppm males

at week 13. Significantly lower haemoglobin level and red cell count were noted in

800 ppm males at week 26. Decreases in haemoglobin levels were noted in the 100

and 800 ppm females at weeks 13 and 26 (significantly lower in 100 ppm females at

week 13). Decreases in red cell counts were also noted in 100 and 800 ppm females

at week 26: These changes are indicative of a test substance-related effect on red cell

mass. This trend was not noted after week 26:

Gross pathology, organ weights, histopathology: There were no test substancerelated gross or histopathological findings noted. The incidence of neoplastic lesions

was similar among all groups. The organ weight data was unremarkable except for a

significantly higher absolute and slightly elevated relative adrenal weight in 800 ppm

males. However, there was no corresponding change in the relative adrenal weight.

16
DuPont-29838

Moreover, the increased absolute adrenal weight was most likely the result of an

unusually high adrenal weight in a single animal (0.014 gm). The remaining adrenal

weights in this dose group ranged from 0.005 to 0.010 gm, which was similar to the

range of adrenal weights observed in the control .group (0.004 to O.'Olgm). Therefore,

the increased absolute adrenal weight in the 400 ppm male mice was not considered

to be test substance-related. There were no test substance related effects suggestive

of an adverse effect on the endocrine system.

Conclusion: The NOAEL in the 104-week feeding study in mice was 50 ppm for

both males and females (8.7 mg/kg bw/day for males and 10.6 mg/kg bw/day for

females). This NOAEL was based on increased mortality in 75 ppm and greater

males and females. Under the conditions of this study, Methomyl was not a

carcinogen. There was no evidence of a direct adverse effect on the endocrine system

in this study.

Busey, W.M. (1968); Two-year dietary administration —Dogs: Lannate Methomyl

insecticide (S-methyl-N-((methylcarbamoyl)oxy) thioacetimidate).

DuPont Report No. Hazleton 201-165 MRID 00007091, satisfies Guideline 83-1 b.

Materials and Methods:

In a 2-year feeding study, Methomyl (purity 90-100%) was administered to male and

female beagle dogs (4 dogs/sex/group) at concentrations of 0, 50, 100, 400, and

1000 ppm. Parameters evaluated included body weight, food consumption, clinical

signs, haematology, clinical chemistry, urinalysis, organ weights (including adrenals,

testes, and thyroid), and gross and microscopic pathology (including adrenals,

pituitary, testes, ovaries, uterus and thyroid).

Findings:

General observations: There were no test substance-related clinical signs noted at

or below 400 ppm in either sex. Two 1000 ppm males exhibited test substancerelated effects including tremors, salivation, incoordination, and circling movements.

There was also an increased incidence of diarrhoea in 1 male administered 1000 ppm

when compared to controls. One 1000 ppm female was found dead during week 9.

This dog was replaced on study and the replacement dog also died after 18 days on

test. Death of the replacement dog was preceded by convulsive seizures and coma.

The deaths of these 2 females were considered test substance-related. There were no

test substance-related effects on body weights or food consumption.

Haematology, clinical chemistry, urinalysis: Haematological studies revealed

slight to moderate anaemia in four 1000 ppm dogs at the 3-month interval and

persistent test substance-dependent anaemia in one of the 1000 ppm males.

Temporary test substance withdrawal during 10 consecutive weeks resulted in

improvement of the condition. There were no test substance-related clinical

chemistry or urinalysis findings noted. Plasma cholinesterase activity was slightly

lower than the control value in one male dog and one female dog administered

1000 ppm.

17
DuPont-29838

Gross pathology, organ weights, histopathology: One 1000 ppm male exhibited an

enlarged spleen and liver. The enlarged liver contributed to a higher mean absolute

arid relative liver for the 1000 ppm males. The spleen weight was so large it was

excluded from the calculation of the mean. Kidney/body weight ratios for three

1000 ppm males and one 400 ppm male were slightly to moderately increased

compared to controls. Test substance related histopathological alterations were seen

in the spleens and kidneys of the 400 and 1000 ppm male arid female groups and in

the livers and bone marrow of the 1000 ppm male and female groups. The changes

seen in the kidneys were characterised by an increase in pigmentation and slight

swelling of the epithelial cells of the proximal convoluted tubules. Pigment

deposition was seen in the 400 and lOOO ppm males but only at 1000 ppm in females.

Increased pigment and extramedullary hematopoiesis were observed in the spleens of

1000 ppm animals, and pigment was observed in the spleens of 400 ppm animals.

Minimal to slight bile duct proliferation in the liver and slightly increased erythroid

and myeloid cells in the bone marrow were observed in 1000 ppm animals. There

were no test substance related effects suggestive of an adverse effect on the endocrine

system. .

Conclusion: The NOAEL in the 2-year feeding study in dogs was 100 ppm for

males and females (3.0 mg/kg bw/day). This NOAEL was based on histopathology

changes in the kidney and spleen at 400 ppm and above. There was no evidence of a

direct adverse effect on the endocrine system in this study.

Lu, C.C. (1983); Nudrin 2 generation reproduction study in rats.

DuPont Report No. WRCRIR-275 (HLO 519-95), MRID 43250701 and 43769401,,

satisfies Guideline 83-3a.

Materials and Methods:

In a two-generation reproduction study, Methomyl was administered in the diet to

male and female CD® rats at concentrations of 0, 75, 600, and 1200 ppm. Each dose

group consisted of 13 male and 26 female rats for the FO generation. Due to low

conception rates for FO rats, additional rats were selected for the FI generation at week

12 to increase each group to 20 males and 40 females. The 1200 ppm dose group had

38 females instead of 40 due to a shortage of animals at the time of selection. The

FO rats were bred within their treatment groups to produce FI litters after 100 days on

test. The FI rats were bred within their respective treatment groups to produce

F2 litters after 120 days on test. Parameters evaluated included body weight, food

consumption, clinical signs, clinical pathology, blood cholinesterase activity,

reproductive indices, sperm counts, litter and pup parameters, organ weights

(including ovary, uterus, and testes in FO and FI adults and in FI and F2 pups; adrenals

in FI adults and FI and F2 pups), and gross and microscopic pathology (including

adrenal, uterus, and testes in FO adults; adrenals, pituitary, testes, prostate, ovary,

uterus, mammary, and thyroid in FI adults and FI and F2 pups).

18
DuPont-29838

Findings:

Fn and Fi Parental Rats

General observations: With the exception of alopecia observed at and above

600 ppm, there were no test substance-related clinical signs observed in either

generation throughout the duration of the study. Test substance-related mortality did

not occur during the course of this study. Body weights were reduced in parental

FO and FI males and females in the 600 and 1200 ppm groups. Body weights in the

FI parental females in the 75 ppm group were slightly lower (3-9% lower compared

to controls). Although the body weights of 75 ppm females were significantly lower

compared to control during premating and gestation, the average difference from

control during these periods was 6.5%. This slight difference was not considered

lexicologically significant. Body weights in the 75 ppm F] parental males were

reduced at sporadic intervals. These body weight reductions in the 75 ppm

FI parental males and females reflect the reduced starting weights in these groups,

since their rate of growth was comparable to the control group. Food consumption

for rats in the 75 ppm FO parentals was sporadically increased. Food consumption in

the 600 and 1200 ppm FO parentals tended to be reduced with sporadic statistically

significant differences from controls. • Sporadic reductions in food consumption in the

75 ppm FI parentals also occurred. Food consumption in'the FI 600 and 1200 ppm

parental animals was significantly reduced.

Reproductive parameters: No test substance-related effects on reproduction were

detected in analyses of mean sperm counts, gestational length, lactation, and fertility

indices in any of the dose groups in both generations. There were no apparent test

substance related effects on endocrine system function relative to reproductive

performance, foetal development, sexual maturation, and general growth and

development.

Gross pathology, organ weights, histopathology, in parental animals and pups:

No test substance-related anatomical changes were noted grossly or microscopically

in tissues of parental animals and selected pups in any dose group from either

generation. In the FO parents, the following organ weights were significantly different

from control due to body weight reduction in the 600 and 1200 ppm groups: absolute

uterus, spleen, and brain weights; relative spleen, brain, and testes weights. In the

FI parents, the following organ weights were significantly different from control due

to reduction in body weight of 600 and 1200 ppm rats: absolute brain, heart, liver,

adrenal, kidney, testes, ovary, uterus; relative spleen, brain, heart, kidney, testes.

In the FI and Fa pups, the following organ weights were significantly different from '

control due to reduction in body weight of 600 and 1200 ppm pups: absolute heart,

spleen, adrenal, kidney, testes; relative testes, liver, kidney, spleen, adrenal weights.

There were no histopathological changes in any of these tissues. Occasional spurious

organ weight differences were also noted in 75 ppm and FI and Fa rats for which there

were no histopathological changes. There were no test substance related effects on

organ weight or histopathological changes suggestive of an adverse effect on the

endocrine system.

. 19
DuPont-29838

Blood cholinesterase activity, clinical pathology in parental animals and pups:

No test substance-related effect on blood cholinesterase for the FO rats was observed

in any group. Significant reductions in mean values of red blood cell count,

haemoglobin, and haematocrit were observed only in the FO females;in the 600 and

1200 ppm groups when compared with controls. Other blood parameters were not

affected.

Fi andFi litter and pup data

\ General observations: During lactation, pup weights in the 600 and 1200 ppm

groups were significantly reduced in both generations. In the FI generation only,

body weights for the 75 ppm group were approximately 9% lower than controls at

birth and remained lower by the same proportion throughout the lactation period.

This effect became statistically significant by day 4. However, the lower weight of

the 75 ppniF] pups is not considered test substance-related for the following reasons:

1) There was no effect on body weight at 75 ppm in the F^ pups at the same

concentration whose parents were exposed for a longer period of time. 2) The litter .

size of the 75 ppm FI pups was the largest in either generation. Based on greater

nutritional and physiological demands, pup size and weight are often reduced and can

remain reduced throughout lactation. To illustrate this point, the body weight of the

75 ppm FI pups are compared to those of the F2,controls, which are closer in litter

size than the FI controls. When the 75 ppm FI mean pup weights are examined

relative to the FI controls, it is apparent that there are no test substance-related body

weight effects in the 75 ppm FI pups, when compared with a control litter of similar

size.

Litter size and pup survival: A test substance-related decrease in the litter size and

the number of live pups,was observed for 1200 ppm FI pups and 600 and 1200 ppm

p2 pups. A spurious decrease in litter size and live pups in the 75 ppm F2 generation

was observed due to one female that delivered a single dead pup. A larger p2 control

litter (larger than the FI generation) contributed to the statistical significance of the

75 ppm F2 litter size on day .1. Because the smaller litter size was primarily due to an

event in a single litter, it is considered to be neither biologically significant or

treatment related. Pup survival was reduced for both FI and ¥2 generations in the

1200 ppm group only. Reduced pup survival occurred predominantly during the first

four days of lactation. There were no test substance-related changes on the

proportion of male and female pups in either the FI or F2 generation.

Conclusion: There was no evidence of a direct adverse effect on the endocrine

system in this study. The NOAELs in this study are the following:

Parental toxicity: 75 ppm (4.6 mg/kg bw/day for FO males, 4.8 mg/kg

bw/day for FO females, 6.7 mg/kg bw/day for FI males,

6.3 mg/kg bw/day for FI females), based on decreased

body weight in FO / FI males and females at 600 ppm

and above.

Reproduction and 1200 ppm based on no effects in the FO / F] generation

fertility: (the highest concentration tested).

20
DuPont-29838

Pup growth and 75 ppm based on decreased pup weight in F] / ?2 pups

development: during lactation at 600 ppm and above.

Culik, R., Rogers, A.S. (1978); Oral teratogenic study in rats with Lannate

(INX-1179). DuPont Report No. HLR 498- 78, MRID 00008621, satisfies Guideline

8 3-3 a.

Materials and Methods;

In a developmental toxicity study, Methomyl (purity 99%) was administered in diet to

mated Chr-CD female rats (25/dose group) on gestation days 6-15. Doses were 0, 50,

100, or 400 ppm. This was equivalent to 0, 4.9, 9.4, and 33.9 mg/kgbw/day,

respectively. Parameters evaluated in dams were body weight, food consumption,

survival, clinical signs, reproductive outcomes (including corpora lutea in each ovary,

implantation sites in each horn, number and location of foetuses), and gross

pathology. Parameters evaluated in foetuses were body weight, incidences of dead

foetuses and/or foetal resorptions, crown-rump length of each live foetus, and

incidences of external, visceral, and skeletal malformations and variations.

Findings:

Maternal effects

General Observations: No test substance-related clinical signs of toxicity or

mortality were observed in any of the test groups during the study. Body weights

were significantly lower than controls in the 400 ppm dose group. Rats administered

400 ppm had lower food consumption compared to controls.

Reproductive outcomes: Parameters measuring the outcome of pregnancy and

foetal development, including the number of litters with partial and total resorptions,

and the body measurements of the foetuses were not different from those of the

concurrent controls. There were no apparent test-substance related effects on

endocrine function pertaining to maintenance of pregnancy or foetal development.

Gross pathology: Test substance-related and adverse gross morphological changes

were not observed. The uterus and ovaries were given a gross pathology

examination. ,

Foetal effects

Small subcutaneous haematomas and petechial haemorrhages on various parts of the

body were found in foetuses from litters of all groups, including controls. One runt

was found in each of the three test groups. Most anomalies and malformations found

in the soft-tissue sectioning were isolated occurrences with two exceptions: apparent

hydroriephrosis (found in all groups at approximately the same rate) and peliosis

(observed in all but 400 ppm foetuses at a rate similar to controls) of the liver.

No test substance-related malformation or abnormalities of the skeletal system were

noted.

21
DuPont-29838

Conclusion: The NOAEL is 100 ppm (9.4 mg/kg bw/day) for maternal effects. The

maternal NOAEL is based on body weight reduction at 400 ppm. The foetal NOAEL

is 400 ppm based on no effects at the highest dose tested. There was no evidence of a

direct adverse effect on the endocrine system in this study.

Feussner, E.L., Hoberman, A.M., Christian, M.S. (1983);,Embryo-fetal toxicity and

teratogenicity study ofMethomyl in the rabbit. DuPont Report No. HLO 331-83,

MRID 00131257, satisfies Guideline 83-3b.

Materials and Methods:

In a developmental toxicity study, Methomyl (purity 98.7%) was administered by oral

gavage to time-mated DLLNZW female rabbits (20/dose group) on gestation days

7-19. Gavage doses in reverse osmosis processed deionised water were 0, 2,' 6, and

16 mg/kg bw/day. The dose volume was 5 mL/kg bw. Parameters evaluated in dams

were body weight, body weight gain, food consumption, survival, clinical signs,

reproductive outcomes (including corpora lutea, number and placement of

implantations), and gross pathology. Uterine weight was measured with and without

the products of conception. Parameters evaluated in foetuses were body weight,

incidences of dead foetuses and/or foetal resorptions, and incidences of external,

visceral (including-internal confirmation of sex), and skeletal, malformations and

variations. .

 ;

Findings:

Maternal effects

General Observations: Test substance related central nervous system stimulation

was observed in the 16 mg/kg bw/day rabbits. Clinical signs of toxicity included

tremors, hyperactiviry, body jerks, excess salivation, aggressive behaviour,

hyperpnea, convulsions, ataxia, impaired or lost righting reflex, skin lesions, dyspnea,

hypersensitivity, pin-point pupils, and vocalisation. The incidence of test substancerelated anorexia was significantly decreased for rabbits fed 16 mg/kg bw/day. This

observation was considered to be a rebound in food consumption after the test

substance was no longer administered. Seven of the rabbits in this dose group died.

Mortality occurred in one rabbit fed 6 mg/kg bw/day which was not considered to be

test substance-related since the death occurred 8 days following the cessation of

dosing. A small test substance-related increase in maternal body weight gain

occurred between days 0 and 29 of gestation; however only the gain of the

16 mg/kg bw/day dose group differed significantly from controls. This difference

was due to the decreased incidence of anorexia of the dams in the 16 mg/kg bw/day

group during the latter portion of gestation.

Reproductive outcomes: A test substance-related increase in average foetal body

weight occurred; the difference from the control value was statistically significant

only'for the 6 and 16 mg/kg bw/day.dose groups. This increase in foetal body weight

was not considered to be an adverse effect since it was not observed to be due to the

presence of oedema. Methomyl did not adversely affect the incidence of pregnancy

or the average number of corpora lutea, implantations, resorptions, and live foetuses

22
DuPont-29838

per dam. There were no apparent test-substance related effects on endocrine function

pertaining to maintenance of pregnancy or foetal development.

Foetal effects

Administration of 2, 6, and 16 mg/kg bw/day did not result in adverse effects on

embryo/foetal viability or foetal sex ratio. Similarly, no gross external, soft tissue or

skeletal malformations, developmental variations or variations due to retarded

development observed for foetuses were attributed to test substance administration.

Conclusion: NOAEL: 6 mg/kg bw/day for maternal effects based on decreased body

weight, mortality, and clinical signs of cholinesterase inhibition at 16 mg/kg bw/day.

The foetal NOAEL was 16 mg/kg bw/day based no effects at the highest dose tested.

There was no evidence of a direct adverse effect on the endocrine system in this

study.

3.2 Discussion of available information in the open literature

3.2.1 Summary of the Potential to Modulate Estrogen or Androgen Receptor Function

In a series of publications, researchers used in vitro assays to evaluate the ability of

methomyl, and a close structural analogue oxamyl, to modulate activity of the

estrogen (ER), androgen (AR), or progesterone (PR) receptors.

In the report by Klotz, Arnold and McLachlan (1997), methomyl and oxamyl were

evaluated for their ability to modulate ER and PR activity using transactivation assays

(MCF-7 human breast cancer cells, Ishikawa human endometrial cancer cells, and

T47D human breast cancer cells) and whole-cell competitive binding assays. In the

transactivation assays, methomyl and oxamyl were tested at a concentration of

100 nM and the responses compared to 17p-estradiol (500 pM in MCF-7 cells; 1 nM

in Ishikawa cells) or progesterone (1 nM in T47D cells). In the whole-cell

competitive binding assays, methomyl and oxamyl were tested at concentrations of.

100 nM, 1 uM, and 10 jj,M and the ability to inhibit binding of 17p-estradiol (500 pM

in MCF-7 cells for ER binding assay) or the PR agonist R5020 (1 nM in T47D cells

for PR binding assay) was measured. They report that both methomyl and oxamyl

weakly activated ER- and PR-responsive reporter genes and decreased estradiol- or

progesterone-induced reporter gene activity in breast and endometrial cancer cells.

The authors also report that as a class, the carbamates tested demonstrated limited

capacity to displace ER or PR receptor ligands from the ER or PR in whole cell

assays. They suggest that the carbamates evaluated act as partial steroid hormone

receptor agonists/antagonists in binding assays through a mechanism separate from

ligand binding.

Nishihara and coworkers (2000) evaluated more than 500 chemicals, including

methomyl, in a yeast two-hybrid transactivation assay system with the estrogen

receptor ERa and the coactivator TIF2. Methomyl was tested up to a maximum

concentration of 2x10~4M, and the response was compared to the activity of

17(3-estradiol. Methomyl showed no activity under the conditions of the study.

In the report by Andersen and coworkers (2002), methomyl was evaluated for its

ability to modulate ER and PR receptor activity using transactivation assays

23
DuPont-29838

[MCF 7 cells, and Chinese Hamster Ovary (CHO) Cells], and its ability to induce cell

proliferation in MCF-7 human breast cancer cells, a marker of estrogenic activity.

In the transactivation assays, methomyl was tested over a concentration range of 10"8

to 5xlO"5 M and the responses were compared to the activity of 17(3-estradiol (10 nM

in MCF-7 cells) or the PR agonist R1881 (0.1 nM in CHO cells). In the cell

proliferation assays, methomyl was tested over a concentration range of 10"8 to 5xlO"5

M in MCF-7 cells and the responses were compared to the activity of 17(3-estradiol

(concentration range of 10~12 to 12"8 M). For the transactivation assays and the cell

proliferation assay, methomyl was also tested after co-administration with

17p-estradiol (0.01 nM in the cell proliferation assay in MCF-7 cells; 10 nM for the

ER transactivation assay in MCF-7 cells) or R1881 (0.1 nM for the transactivation

assay in CHO cells). Under the conditions of the study, methomyl showed no

significant activity in any of the ER or AR transactivation assays or the cell

proliferation assay.

In the report by Kojima and coworkers (2004), methomyl and oxamyl were evaluated

for its ability to modulate receptor activity using transactivation assays for human

ERa, human ERp, or human AR in CHO cells. In all of the assays, methomyl and

.oxamyl were tested over a concentration range up to 10"5 M and the responses were

compared to the activity of 17p-estradiol or dihydrotestosterone (DHT) for the ER or

AR assays, respectively. Relative luciferase activity was calculated'relative to the

positive controls (17p-estradiol for ERa and ERP; DHT for AR). In addition, for

measurement of antagonist activity, assays were evaluated with the test material

co-administered with np-estradiol (10'" M for ERa; 10"10 M for ERp) or DHT

(10"10 M) for the ER and AR transactivation assays, respectively. Under the

conditions of the study, methomyl and oxamyl showed no significant activity in any

of ER or AR transactivation assays.

In the report by Nendza and Wenzel (2006), methomyl and oxamyl were evaluated

for their ability to modulate ER activity using a Yeast Estrogen Screen (YES). In the

assay, methomyl and oxamyl were tested over a concentration range of 100 mg/L to

50 (ig/mL, and the responses were compared to the activity of 17p-estradiol

(concentration 3,000 ng/ml to 1.5 ng/ml). Under the conditions of the study,

methomyl and oxamyl showed no significant activity in the YES assay.

In the report by Jiao and coworkers (2008), a transgenic yeast screening system using

fish ER was established and used to evaluate a number of xenobiotics including

methomyl. Methomyl was tested at concentrations of 10"9, 10"7, and 10"5 M and.the

responses were compared to the activity of 17p-estradiol or dihydrotestosterone

(DHT) for the ER or AR assays, respectively. In addition, for measurement of

antagonist activity, assays were evaluated with the test material co-administered with

17p-estradiol (concentration not stated). Under the conditions of the study, methomyl

showed no activity to the fish ER in any of the transactivation assays that were

performed.

Together, the data from these reports suggest that methomyl, and its close structural

analogue oxamyl, do not interact with the ER or AR. The data from five of the six

reports were all negative. In the report by Klotz and coworkers1, the authors claim

methomyl and oxamyl activated the ER and PR. However, they also report that 'all

24
DuPont-29838

of the carbamates tested weakly activated ER- or PR-responsive reporter genes and

decreased estradiol- or progesterone-induced reporter gene activity in breast and

endometrial cancer cells. The authors also report that the carbamates tested

demonstrated limited capacity to displace ER or PR receptor ligands from the ER or

PR in whole cell assays. They suggest that the carbamates act as partial steroid

hormone receptor agonists/antagonists in binding assays through a mechanism

separate from ligand binding. However, several factors in these studies suggest that

the observed effects were non-specific and unrelated to true receptor-mediated

effects. These responses did not occur in a dose-dependent manner, a serine protease

inhibitor (phenylmethyl sulfonyl fluoride) demonstrated very similar activity, and

finally, the compounds appeared to affect both the ER and PR systems to an equal

extent. These are important points in that while there is "cross-talk" between receptor

systems it is rare that a compound, endogenous or exogenous, will equally affect

more than one receptor system. In fact, many compounds evaluated in this study had

similar potencies, suggesting that there are non-specific factors involved. This study

provides no solid evidence that methomyl or oxamyl are steroid hormone receptor

agonist/antagonist, and the authors themselves state that the reported activity was not

receptor mediated. The weight of evidence suggests that neither methomyl nor

oxamyl interact with the ER or AR.

Two recent papers describe efforts by Roncaglionre? al., (2008) and Celik et al,

(2008) to develop structural activity models for interaction with the human estrogen

receptor. Roncaglion et al., correlated the results of a 900-chemical database for

human ERa receptor binding and reporter gene assays with variety of parameters that

characterized receptor binding. The source of activity data in the ER binding assays

was the, Japanese METI database (OECD EDTA, 2002), which is noted by the

authors to be one of the largest collections of data for ER. Methomyl is indicated as

being inactive for ER activity according to this database. In the paper by Celik et al,

a comparative docking study of well known human ERa ligands and a number of

selected organic compounds was conducted to assess the potential of these substances

to interact with the ligand binding domain of the receptor. Methomyl was among .

three pesticides examined that gave low affinity scores suggesting that it may bind

very poorly or not at all to the human ERa.

3.2.2 Summary of the Potential to Modulate Placenta! Aromatase Activity In Vitro

In the report by Andersen and coworkers (2002), methomyl was evaluated for its

ability to modulate CYP19 aromatase activity in human placental microsomes using

the classic tritiated water release method. Methomyl was tested at a single

concentration (50 uM) and the response was compared to the activity of 4-androsten4-ol-3,17-dione (1 jaM). When tested at a single concentration (50 urn) under the

conditions of the assay, methomyl induced aromatase activity to approximately 120%

of control. While these data suggest that methomyl may have the potential to

modulate aromatase activity, the significance of the finding is unclear given the

results were not repeated in this study, only a single test concentration of methomyl

was evaluated, and no findings suggestive of an adverse effect on aromatase has been

observed in the many animal studies conducted with this product.

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3.2:3 Summary of In Vivo Studies:

The purpose of the research conducted by Porter and coworkers (1993) was to

determine the effects of aldicarb, methomyl, and/or metribuzin, alone or in

combination, on plasma hormone (thyroxine and somatotropin) concentrations in

Sprague-Dawley rats. Thyroxine (T4) is a thyroid hormone that participates in the

regulation of metabolism and food intake, as well as differentiation and maturation of

numerous cell types. Somatotropin, also known as growth hormone, is secreted by

the pituitary gland and regulates soffrand bony tissue growth, as well as other

metabolic processes. These hormones were selected as indicators of normal body

function. In this study, 6 Sprague-Dawley rats/sex/group were treated with aldicarb

(1 or 10 ppb), metribuzin (1000 or 10,000 ppb), and/or methomyl (100 or 1000 ppb)

in the drinking water for 6 or 16 weeks. Blood was drawn from female rats after 2

and 6 weeks of exposure and from male rates after 7, 13, and 16 weeks of exposure.

Plasma was analyzed for T4, Tj, uptake, and somatotropin.

After 2 weeks of exposure female rats had plasma T4 concentrations that were

significantly increased by either metribuzin or the combination of the three chemicals

at the high dose levels. T4 concentrations were significantly increased by metribuzin

after 6 weeks of exposure. Methomyl alone had no effect on T4 plasma

concentrations. There were no significant effects on somatotropin at 2 or 6 weeks of

exposure by compounds individually or in combination. Male rats had plasma T4

concentrations that were significantly increased by metribuzin after 7, 13, and

16 weeks of exposure. The combination of the three chemicals resulted in a slight,

though statistically significant, increase in somatotropin levels after 13 weeks of

exposure. Although not statistically significant, there were slight decreases in

somatotropin levels after 7 weeks of exposure to aldicarb as well as following

exposure to a combination of the 3 chemicals for 16 weeks.

The authors concluded that these substances, either individually or as a mixture, are

responsible for learning impairment and other neurological effects, changes in

immune function, and endocrine changes. The authors also suggest that the findings

support the concept of the interconnectedness of the nervous, endocrine, and immune

systems and raise the likelihood of impacts on all three systems if one is shown to be

affected.

However, we disagree with the conclusions of Porter et al, (1993) and believe that

there are several serious flaws in both the experimental design and the conclusions

that have been drawn from this study. These include the following:

Individual animal hormone levels are known to vary greatly. Not only were sample

sizes of 6 rats/sex/group too small to detect a reproducible effect but actual hormone

levels were not reported. Therefore, it is difficult to evaluate the biological

significance of the reported results.

Rats were anesthetized during the collection of blood, and the anesthesia may have

confounded any true compound-related effect. :

The time of day for blood collection was not specified nor was it stated as being done

consistently (i.e., the paper did not state that all blood was collected at a specific time

of day, such as between 7-8 a.m.). Since hormone levels fluctuate throughout the day

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DuPont-29838

and are also affected by food intake, a carefully controlled .blood collection is

essential to accurately assess alterations in hormone levels.

Measurement of thyroxine alone is not adequate to determine an adverse effect on the

thyroid gland since thyroid hormones in normal, healthy animals fluctuate to maintain

hormonal homeostasis.

Actual intake of each compound was not provided.

Pathology, body weights, and clinical signs were not shown so as to corroborate the

hormonal measurements. The authors depended heavily on statistical analysis to infer

biological significance, without scientific proof that the suggested biological effects

actually occurred.

No evidence was provided that either the nervous, endocrine, or immune systems

were affected in the study.

Although it was suggested that the combination of compounds may have had a weak

effect on somatotropin concentrations, the biological significance of the reported

changes are questionable since there was an inconsistency in the stated responses in

male rats (levels were slightly increased at 13.weeks and slightly decreased at

16 weeks).

As discussed in the previous section of this paper, traditional toxicology studies

conducted with methomyl have given no indication that the thyroid is a target organ

for methomyl toxicity or that the level of somatotropin is affected. Therefore, the

study by Porter and coworkers (1993) provides no evidence that methomyl is an

endocrine disrupter; in fact, it suggests the opposite since the reported thyroid effects.

appear to be related to the treatment with metribuzin..

In a study conducted by El-Medany and coworkers (1993), the authors reported a

decrease in serum testosterone and an elevation of FSH, LH, and prolactin levels in

male rats orally dosed with 17 mg methomyl/kg body weight/day for 2 months.

The authors reported that pathological evaluation of the testes revealed degenerative

changes in the seminiferous epithelium with sloughed cells in the tubule lumen,

containing nuclei that were pyknotic and lacking in nuclear and cytoplasmic

membranes. Additionally, histochemical analysis revealed increases in various

testicular enzymes (alkaline phosphatase, acid phosphatase, and alpha esterase).

While this data would appear to indicate that the testes had been affected, the LD5o

 :

for methomyl is 32 mg/kg body weight. This fact alone leads one to question the

study by El-Madany and coworkers. In their paper, they state that a dosage of 17 -

mg/kg body weight is 1/30 the LDso and cite Hayes (1975). However, Hayes (1975)

is a book entitled "Toxicology of Pesticides" and, while methomyl does appear in an

appendix with numerous other compounds, methomyl nor its LDso are found within

the text. El-Medany and coworkers state in their discussion that Salem and

coworkers (1988) reported that chronic exposure to methomyl resulted in decreased

libido, ejaculate volume, sperm viability, and increased abnormal sperm in male rats.

However, the cited work is actually a study of the effects of dimethoate (an

organophosphate) and deltamethrin (a pyrethroid) on semen characteristics in male

rabbits. Much of this study appears to have been subsequently republished in a paper

27
DuPont-29838

by Mahgoub and El-Medany (2001) as the study design, dose level, and the above

data appear to be the same as that reported earlier. This more recent publication

includes a recovery group in which treatment was withdrawn for one month. The

authors report that the hormonal and testicular changes were still present following,

this recovery period. The discrepancies in the cited literature, the presumed use of the

LDso dose level for a 2 month study, and the absence of similar effects on the testes

and sperm number in standard mammalian toxicology studies (summarized above)

raise questions regarding the validity of the results reported by El-Medany et al.

3.3 Available Information from the EPA ToxCasfTM Project

EPA has initiated a program to screen a large number of environmental chemicals in a

battery of in vitro, high-throughput screens. In the first phase of this project,

309 substances have been selected for testing, most of which are food-use pesticides

for which extensive animal toxicology testing results are available (Judson et al.,

2009). These substances are being evaluated with nine in vitro assays technologies

covering 467 cell-free or cell based assays. The total number of endpoints measured

for each chemical is 624. ToxCast has profiled many of the EDSP chemicals selected

for initial testing in the Tier I endocrine battery, including methomyl, using assays

which characterize receptor binding, activation, inhibition, and target gene regulation

that are relevant to estrogen, androgen, and thyroid and other endocrine-related

processes. Included are six assays related to estrogen, four assays related to androgen,

four assays related to thyroid, and 1 assay related to aromatase as well as 70 other

nuclear receptors and xenobiotic metabolizing enzymes that have potential for

endocrine signaling (Kavlock et al., 2009). The results for the 309 chemicals*in

Phase I have been presented by Reif et al., (2009); based on the pictorial presentation

of the results, methomyl appears to have produced no "hits" for any of the endocrinerelated in vitro endpoints.

4.0 SUMMARY OF RELEVANT ECOTOXICOLOGY STUDIES

4.1 Discussion of studies supporting registration

Beavers, J.B., Hawrot, R., Lynn, S.P., Jaber, M. (199la). A one-generation

reproduction study -with the mallard (Anas platyrhynchos);H-l 7, 940:

DuPont Report No.: HLO 336-91, MRID 41898601, U.S. EPA 71-4(1988).

Materials and Methods:

Four treatment groups were randomly distributed with 16 pairs of birds with one male

and one female mallard (Anas platyrhynchos} per pen. Each treatment group received

methomyl (98.4% purity) in feed at concentrations of 0, 50, 150, or 500 ppm for

18 weeks. Birds in the 50 ppm treatment group received approximately 150-ppm

methomyl for the first four weeks of the study, due to an error in premix preparation,

but it was determined that this had no effect on the study. Reproductive parameters

were measured beginning at the onset of egg laying. Adult birds were observed for

abnormal behaviour, mortality, signs of toxicity, and changes in body weight and

food intake.

28
DuPont-29838

Findings:

General observations: There were no mortalities, overt signs of toxicity or

treatment related effects observed upon food consumption. The 500-ppm methomyl

test rate showed apparent treatment related decreases in body weight in hens and a

statistically insignificant decrease in percent of viable embryos. The weight

difference in drakes was slight, and except for the first two weeks of the study, the

change in body weight was comparable to the control group. Thus, the statistical

difference is not deemed biologically relevant. It was not determined if the slight

decrease in embryo viability was biologically related or treatment related. No test

substance-related gross lesions were observed at necropsy.

Reproductive parameters: There were no statistically significant or apparent

treatment related effects upon reproductive parameters at test concentrations 50, 150,

and 500-ppm methomyl when compared to control values

Conclusion: The NOEC for mallards exposed to methomyl in the diet for 18 weeks

was 150 ppm, based on effects upon body weight of hens at 500 ppm.

Beavers, J.B., Hawrot, R., Jaber, M., Lynn, S.P. (199Ib). A one-generation

reproduction study with the northern bobwhite (Colinus viginianus); H-17, 940:

DuPont Report No.: HLO 337-91, MRID 41898602, U.S. EPA 71-4 (1988) .

Materials and Methods:

Sixteen pairs (male/female) of young adult northern bobwhite (Colinus virginianus)

received methomyl (98.4% purity) in the feed at concentrations of 0, 50, 150, and

500 ppm for 20 weeks. Reproductive parameters were measured beginning at the

onset of egg laying. Adult birds were observed for abnormal behaviour, mortality,

signs of toxicity, and changes in body weight and food intake. All birds that died

were necropsied after death. Those birds that survived until study termination were

also necropsied.

Findings:

General observations: No treatment-related mortalities, overt signs of toxicity or

treatment related effects were observed. At the 500-ppm methomyl test group, there

was a slight reduction in body weight of the cocks. However, this difference was

influenced by one bird, which was noted as feather pecked and penworn. There were

no treatment related body weight effects for the hens. No test substance-related-gross

lesions were observed at necropsy. x

Reproductive parameters: There were no apparent treatment related effects on

reproductive parameters for 50 or 150 ppm-methomyl groups. At the

500 ppm-methomyl concentration group, there was a reduction in the total number of

eggs laid per hen that was determined not to be statistically significant.

Subsequently, there was a reduction in the number of offspring. No other test

substance-related effects were1 observed on reproductive parameters.

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DuPont-29838

Conclusion: The NOEC for northern bobwhite quail exposed to methomyl in the

diet for 20 weeks was 150 ppm, based on the effect upon egg production (total

number of eggs laid per hen) at 500 ppm (the highest level tested).

Driscoll, R.R., Muska,C.F. (1982). Early Life Stage Toxicity of Methomyl to Fathead

Minnow. DuPont Report No.: HLR-528-82, MRID 00131255.

Materials and Methods:

The effects of methomyl (>99% purity) on embryos and larvae of fathead minnows

(Pimephales promelas) were evaluated in a 28-day post-hatch continuous exposure

study. Treatments consisted?of a dilution water control and nominal concentrations of

31, 62, 125, 250, and 500-jj.g/L methomyl. Fifty embryos were impartially

distributed to each of 12 embryo cups. Dead embryos and fungal-infected embryos

were counted and removed. To initiate the 28-day larval exposure, 20 larvae were

impartially selected from each embryo cup and transferred to the respective test

aquaria. Observations of larval mortality, behaviour, and appearance were made

daily. The number of live fry in each aquarium was estimated and recorded once per

week. At the end of the post-hatch exposure period, the number of surviving larvae

was recorded. Standard body length and wet body weight of each surviving fish was

measured.

Findings:

Mean measured concentrations of methomyl were 27, 57, 117, 243, and 491-ug as/L

methomyl. Percent embryo hatch at all the concentrations tested was not significantly

different from the controls. The 28-day larval survival was the most sensitive

indicator of methomyl toxicity in this study. The lowest test concentration resulting

in a significant reduction in survival of fathead minnow was 117 u.g/L. At the lower

test concentrations, fish survival was not statistically different from control responses.

Larval growth, as measured by standard length and wet body weight, was

significantly reduced at 243 jag/L.

Conclusion: On the basis of reduced percent survival of larvae, the MATC for the

fathead minnow (Pimephales promelas) was estimated to be greater than 57 jig/L but

less than 117-jag methomyl/L.

Rhodes, J.E. (1991). Early life-stage toxicity of IN-X1179-394 to the fathead minnow

(Pimephales promelas) underflow-through conditions. DuPont Report No. HLO

702-91. MRID 46015305, US EPA, 1986. Hazard Evaluation Division, Standard

Evaluation Procedure. Fish Early Life Stage Test.

Materials and Methods;

The effects of methomyl (98.4% purity) to eggs and fry of the fathead minnow,

Pimephales promelas, was determined in a flow-through, early life-stage 35-day

toxicity test. Treatments consisted of a dilution water control and nominal

30
DuPont-29838

concentrations of 19, 38, 75, 150, and 300-^g/L methomyl. Two replicates

containing 25 eggs each were exposed to each treatment concentration and control in

incubation cups. All incubation cups were observed daily. Dead eggs and fungalinfected eggs were removed. The 29-day post-hatch growth period began on study

Day 6, when 95% hatch was attained. Observations of abnormal behaviour, physical

changes, and mortality were recorded daily. At the end of the study, standard length

and blotted wet weight were measured.

Findings:

Mean measured concentrations of methomyl were 18.0, 39.3, 73.0, 145, and

261-fig as/L methomyl and ranged from 87 to 103% of nominal concentrations. Egg

hatchability and fry survival were not significantly reduced at any test concentration.

The no observed effect concentration (NOEC) was 73.0-ug methomyl/L. The lowest

observed effect concentration (LOEC) was 145-ug methomyl/L, and the maximum

acceptable toxicant concentration (MATC) was 104-jag methomyl/L. All chemical

and physical parameters for the 35-day study were within acceptable ranges.

The following endpoints were determined for the methomyl test concentrations and

control (in parenthesis): Mean percent survival ranged from 86 to 94% (92% for

water control); Mean % egg hatched ranged from 88 to 96% (94% for water control);

mean standard length ranged from 18.56 to 21.17 mm (21.15 mm for water control);

mean wet weight ranged from 0.112 to 0.148 g (0.146 g for water control).

Conclusion: The 35-day NOEC for the fathead minnow (Pimephalespromelas} was

73.0-jig methomyl/L, the LOEC was 145-jig methomyl/L, based on mean, measured

concentrations of methomyl. The MATC was 104-ug methomyl/L.

Strawn, T., Rhodes, J.E., Leak, T. (1993). Full life-cycle toxicity of DPX-X1179-394

(methomyl) to the fathead minnow (Pimephales promelas) underflow-through

conditions. DuPont Report No. HLO 47-93, MRID 43072101, U.S. EPA 72-5

(1988).

Materials and Methods; .

The toxicity of methomyl (98.4% purity) to the fathead minnow

(Pimephalespromelas}, was determined in a flow-through full life-cycle toxicity

study. Newly fertilised eggs were used for the study with exposure continuing for

193 days. Treatments consisted of a dilution water control, and nominal

concentrations of 19.0, 38.0, 75.0, 150, and 300-ug methomyl/L. Four replicates,

with 50 eggs each, were randomly set-up for the control and the five test

concentrations. Eggs were placed in incubation cups and allowed to hatch. At Day 7,

the fry were thinned to 25 per replicate and released into their respective chambers.

Measurements for length and blotted wet weights were recorded. Observations of

abnormal behaviour, .abnormal physical change, and mortality were recorded daily for

both the parental group and first generation offspring exposures.

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DuPont-29838

Findings:

Mean measured exposure concentrations of methomyl were 18.0, 40.7, 76.0, 142, and

280-ug methofnyl/L. These values ranged from 93.3 to 107% of the target nominal

test concentrations. Parental generation egg hatchability and survival were

significantly reduced at the 442.and 280-u£ methomyl/L. At 28, 56, and 84 days

post-hatch, parental generation growth, in terms of length, was significantly reduced

after exposure to 142 and 280-ug methomyl/L. Parental generation growth, in terms

of wet weight, showed a significant reduction after 84 days of exposure to

280-^g methomyl/L. Reproductive activity, measured by days to first spawn, number

of spawning days, number of spawns, and number of eggs per spawn, was not

significantly affected at any test concentration. FI generation egg hatchability and

survival after 56 days of growth were not significantly reduced at any test

concentration. FI generation growth measured by length and by blotted wet weight at

56 days post-hatch resulted in significant reductions after exposure to 142 and

280-u.g methomyl/L, respectively. - .

Treatment related physical abnormalities included distended abdomens and a bloated

appearance in the 280-ug/L methomyl test concentration.

Based on the growth endpoints evaluated in this fathead minnow

(Pimephales promelets), full life-cycle toxicity study, the MATC for methomyl is

104|ug/L. The no observed effect concentration is 76.0 jug/L, and the lowest

observed effect concentration is 142-(j,g/L methomyl.

Conclusion: The no-observed effect concentration is 76.0-ug methomyl/L based on

the growth endpoint evaluated in this fathead minnow (Pimephales promelas) full

life-cycle toxicity study. The MATC is 104-u.g methomyl/L and the lowest observed

effect concentration (LOEC) is 142-ug methomyl/L based on mean measured

concentrations.

4.2 Discussion of available information in the open literature

In 1997, the US EPA Risk Assessment Forum produced a Special Report on

Environmental Endocrine Disruption (Crisp et al, 1997) in response to a request from

the US EPA's Science Policy Council to provide an overview of. the current state of

the science. The US EPA report, which was published in the open literature in 1998

(Crisp et al., 1998), was intended as an interim assessment and analysis of the

environmental endocrine hypothesis, to be subsequently superseded by the National

Academy of Sciences critical review (National Research Council, 1999).

Methomyl was listed in the US EPA Special Report as impairing avian reproduction

based on information contained in the Office of Pesticide Program files; however, no

endocrine mode of action was cited for the observed effect in this document. A slight

reduction in egg production was observed in a standard regulatory avian reproduction

.study with northern bobwhite quail (HLO 337-91, 1991). The observation'was not

"statistically significant and was observed only at the highest test level (500 ppm).

However, reduced egg production is a common effect in avian toxicity studies; and

based on a summary of the US EPA avian reproduction database, approximately 50%

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DuPont-29838

of studies with northern bobwhite quail elicit a reduction in egg production

(Montague, 1997). The frequency of occurrence of this effect indicates that the

endpoint'is a sensitive parameter of toxicity. There is no evidence from reproduction

studies to support an endocrine mode of action for methomyl. Rather, a strong case .

can be made that the observed effects in the bobwhite quail study are a result of

methomyl's mode of action since similar effects on egg production have been

reported with other acetylcholinestrse inhibitors (Grue et al, 1991).

Hoffman and Albers (1984) reported the results of embryotoxicity tests conducted

with a commercial formulation of methomyl (Lannate L) which was applied,

externally to mallard eggs. The study employed two methods of application based on

the vehicle (water or oil) and estimated volume of field application, Fertile mallard

eggs were incubated in a temperature and humidity-controlled incubator. On the third

day of incubation, eggs were reported to have been immersed for 30 seconds in 3-6

geometrically spaced concentrations of Lannate L either mixed in with distilled water

(aqueous emulsion) or prepared in an inert oil (aliphatic hydrocarbon) vehicle.

Surviving embryos were removed from eggs on day 18 of the incubation period and

examined for external malformations.

From the test conducted with the aqueous preparation of Lannate L, the authors

reported an LCso that was greater than the concentration of actual emulsifiable

concentrate (>180 Ib/A at 100 gal/A) tested. It is important to note that this reported

estimate of the LCso occurred at greater than 200-times the maximum permissible

field level of application for methomyl at the time of the report. Malformations in

survivors of eggs treated with the aqueous emulsion of Lannate L were reported as

blister, edema, neck, and stunted. However, the number of abnormal survivors or the

treatment concentrations at which these malformations occurred have not been

reported in the article.

The authors reported an LCso of 10.3 Ib/A at 11 gal/A when mallard eggs were

exposed to the oil preparation of Lannate L. This LCso was reported to be

103.8-times the maximum field level of application at the time of the report. -

Malformations reported in the survivors from this test were blister, brain, gastroshisis,

joints, skeletal (axial), stunted. The observed greater toxicity of Lannate L when

mixed with the oil vehicle was postulated to have been due to greater penetration of

the shell and membranes conferred by the oil.

5.0 DuPoNT POSITION AND CONCLUSIONS

5.1 Potential for Methomyl to Interact with the Endocrine System

Methomyl has not been specifically evaluated in the 11 assays comprising the Tier 1

EDSP battery. However, existing toxicology studies supporting the registration of

this product indicate that exposure to mammals or wildlife species results in no

effects suggestive of interference with estrogen, androgen, or thyroid hormone

function. A fairly comprehensive evaluation of the structure and function of

endocrine-related tissues has been done in the combination of subchronic and chronic

studies in rats, mice, and dogs and in multigeneration reproduction and

developmental toxicity studies in rats and rabbits (Table 2). This has included

33
DuPont-29838

adrenals, pituitary, thyroid, testis, epididymis, prostate, ovary, fallopian tubes, uterus,

vagina, cervix, and mammary gland. In none of these studies were there treatmentrelated effects on either organ weight or microscopic pathology that suggested

endocrine system dysfunction. In long-term chronic/oncogenicity studies, no tumors

of any kind, including no rumors of the endocrine tissues, were induced by methomyl.

Methomyl has been evaluated for effects on prenatal development or reproduction in

three studies. In the prenatal developmental toxicity studies, no effects were observed

on the maintenance of pregnancy, number of pups/litter, fetal size (crown-rump

length), or feminization or masculinization of pups (sex ratio). The only effects noted

were decreases in body weight and food consumption at the high dose (400 ppm in

feed, equivalent to 33.9 mg/kg bw/day) in the rat. In the rabbit study, the observed

effects at the high dose (16 mg/kg bw/day by gavage) included clinical signs and

mortality consistent with cholinesterase inhibition as well as an increase in body

weight gain resulting from increased food consumption after the test substance was

no longer administered.

The outcome of a multigeneration reproduction study in rats also demonstrated a lack

of an adverse effect by methomyl on the hormonal control of sexual development,

growth, and reproduction. Methomyl produced no adverse effects that suggested the

endocrine systems association with reproduction and development were negatively

impacted. There were no effects on reproductive parameters such as mating, fertility,

gestation length, lactation, and weaning. There were also no data in the study to

indicate that sexual differentiation (sex ratio), pubertal development, production of

germ cells, and accessory sex organs had been adversely affected. Spernrcounts in

the two-generation study for all methomyl-treated groups were similar to control

values. The primary effects noted in these studies were body weight decreases and

reduced food consumption, which were consistent with effects observed at high doses

of methomyl. In addition, a reduction in pup survival and pup weight at the mid and

high doses (600 and 1200 ppm) occurred in the two-generation study and were

consistent with the severe body weight effects observed at the same doses in the

parental animals.

Histopathology of the ovary, testes, and accompanying accessory reproductive issues

have been evaluated in a number of studies in rats, mice, and dogs following

subchronic and chronic exposures to methomyl. There were no histological

abnormalities in any study. Gonadal activity was evident by the presence of follicles

and corpora lutea in the ovary and the presence of sperm in the testes and

epididymides. In all of these studies, there has been no indication'that methomyl

adversely affects estrogen or androgen dependent processes in three species.

Therefore, the overwhelming evidence from subchronic/chronic toxicity,

multigeneration reproduction, and developmental toxicity studies conducted in rats,

mice, dogs, and rabbits indicates that methomyl produces no adverse effects on the

endocrine system of mammals.

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DuPont-29838

As in mammalian toxicity studies, no reproductive effects were observed in mallard

ducks, rather the NOEC was based on parental effects of decreased hen and drake

weights. For bobwhite quail, a non-statistically significant reduction in the number of

eggs laid at the highest level tested (500 ppm) was observed. No other test substancerelated effects were observed on reproductive parameters. Thus the potential effects

of chronic exposure to methomyl in birds are not likely to have reproductive impact.

The results of the fish reproduction studies showed that reproductive activity of the

parental generation was not significantly affected at any test concentration. The FI

generation egg hatchability and survival were also not significantly reduced at any

test concentration. The traditional guideline toxicology studies (subchronic/chronic

toxicity, multigeneration reproduction, and developmental toxicity studies conducted

in rats, mice, dogs, and rabbits) conducted with methomyl also provide compelling

evidence that methomyl is not expected to produce adverse effects on the endocrine

. system of wildlife species.

5.2 Functionally Equivalent Information Fulfilling the EDSP Tier IBattery

EPA outlined in its March 26, 2009 approach for considering OSRI under the EDSP

that it will make decisions as to whether existing information satisfies all or part of

the Tier 1 Test Order based on the weight of the evidence. Based on the available

information from mammalian toxicology and ecotoxicology studies supporting the

registration of methomyl as well as information from the open literature and

ToxCastTM, DuPont proposes that the primary purpose of the Tier 1 EDSP screening

battery, to assess the potential to interact with estrogen androgen, or thyroid hormone

systems, has been fulfilled.

The EPA's Tier 1 estrogen and androgen receptor binding assays and estrogen

transcription activation assay are designed to identify substances that either bind to

the estrogen and androgen receptor or bind to the estrogen receptor and active the

subsequent downstream transcription of a reporter gene product. As noted in

Section 3.2.1 above, methomyl, as well as its structural analogue oxamyl, has been

tested in a number of estrogen or androgen transactivation assays in which the

majority of the results suggest a lack of agonist and antagonist activity. EPA

ToxCastTM program also includes a number of estrogen and androgen binding

assessments'in which the reported results suggest a lack of activity (Reif, 2009) of

both products. Further, the in vivo mammalian toxicology database for methomyl

provides considerable evidence that estrogen and androgen dependent process in rats,

mice, dogs, and rabbits are not adversely affected. Therefore, it is DuPont's position

that it is unnecessary to conduct further estrogen or androgen binding studies with

methomyl.

The purpose of the Tier 1 steroidogenesis and aromatase assays are to identify

substances that either interfere with steroid hormone synthesis resulting in

. testosterone and estradiol production or which inhibit the catalytic activity of the

enzyme responsible for the conversion of androgens to estrogens. In the absence of

any adverse effects in the in vivo mammalian toxicology studies conducted to date

'. with methomyl that would suggest interference of steroid hormone production

(e.g., adrenal and reproductive organ weight or histopathology, fertility, gestation,

35
DuPont-29838

sex ratio), it is DuPont's position that no further testing of methomyl in the

steroidogenesis and aromatase assays is necessary.

The uterotrophic and Hershberger assays are intended to determine the potential of

substances to induce uterine or male reproductive organ weight changes consistent

with agonists or antagonists of natural estrogen or androgen/5a-reductase inhibitors,

respectively. Uterine weight has been assessed in 90-day studies in rats and mice and

in the multigeneration reproduction study conducted with methomyl. Histopathology

of a number of female reproductive tissues has been evaluated in one or more of the

the studies summarized above (ovary, fallopian tubes, uterus, vagina, cervix and

mammary gland). Testes weights were measured in these studies as well. Some of -

these studies also included epididymis and/or prostate weights. Histopathology of the

testes, epididymis, and prostate were included in most of the subchronic and chronic

'studies as well as the multigeneration reproduction study. In the absence of any

adverse findings, the weight of the evidence suggests that methomyl does not produce

any effects consistent with estrogen or androgen agonists or antagonists. Therefore, it

is DuPont's position that there is no need to conduct either the uterotrophic or

Hershberger assay with methomyl.

The intent of the male and female pubertal assays is to identify substances that

interact with the endocrine system by assessing endpoints associated with pubertal

development and thyroid function in male and female animals. As noted in the above

discussion regarding the uterotrophic and Hershberger assays, organ weights and/or -

histopathology of a fairly large number of reproductive tissues have been assessed in

many of the existing mammalian toxicology<studies conducted to date. Adrenal

weights and pituitary weights and microscopic pathology have been included in the

majority of these studies as well. The multigeneration and developmental toxicity

studies have revealed no concerns for an adverse effect on pubertal development.

These studies have assessed such endpoints as mating behavior, fertility, maintenance

of pregnancy, gestation length, offspring sex ratio. In addition, sperm, counts were

evaluated in the FO and F] generations in the reproduction study and organ weights

and histopathology of a number of reproductive tissues were evaluated in the FI and

p2pups at weaning. Therefore, under these circumstances, it is DuPont's position that

the evaluation of methomyl in the female and male pubertal assays is not necessary.

The amphibian metamorphosis assay (AMA) is designed to empirically identify

substances which may interfere with the normal function of the HPT axis. Thyroid

hormone, through the HPT axis, regulates many processes in an organism including

somatic growth, development, metabolism, and reproduction. It is DuPont's position

that the existing mammalian toxicology studies sufficiently demonstrate that

methomyl does not interact with the HPT axis. No treatment-related effects were

observed on the thyroid gland in the many studies supporting registration of

methomyl and summarized above, which included an evaluation of organ weight

and/or histopathology in rats, mice and dogs. Many of the important features of the

thyroid system and the HPT axis are conserved across all taxa (OECD, Detailed

Review Paper on Thyroid Hormone Disruption Assays, April 2006). Therefore, given

the degree to which the thyroid has been evaluated in three mammalian species; there

is no need for further testing of methomyl in the Tier 1 AMA screen.

36
DuPont-29'838

The fish short-term reproduction assay is designed to measure the reproductive

performance of fathead minnows as a primary indicator of potential interaction of

xenobiotics with the endocrine system. The endpoints examined in this assay include

survival, fecundity, fertilization success, changes in morphology, and biochemical

endpoints which might be indicative of disturbances in estrogen and androgen

dependent processes. In comparison, the parameters examined in the fish full life

cycle study with methomyl include growth, changes in morphology, and reproductive

activity such as time to first spawning, number of spawns, and number of eggs

produced. It is DuPont's opinion that the existing fish full-life cycle study together .

with the early life stage study with methomyl provide sufficient information to

address the intended purpose of the fish short-term reproduction assay.

5.3 Overall Conclusion

Methomyl is a carbamate insecticide which exerts its biological activity by inhibiting

acetylcholinesterase in the nervous system of insects, mammals, and other vertebrate

. . species. Methomyl has. high acute toxicity by the oral, inhalation and ocular routes of

exposure. The primary toxicological effects observed in toxicology studies are those

associated with cholinesterase inhibition. This substance is not a carcinogen or

genotoxin, nor is it a reproductive or developmental toxicant. As summarized above,

there is no evidence in the available mammalian and ecological toxicology studies to

suggest that methomyl directly interacts with the function of estrogen, androgen, or

thyroid hormone processes to cause endocrine dysfunction. A similar toxicology

database exists for oxamyl, another DuPont carbamate insecticide which shares close

structural analogy to methomyl and which also demonstrates low potential to interact

with the endocrine system (see DuPont response to Test Order EDSP-103801-63,

DuPont -29827). Methomyl is currently being regulated by EPA using an extremely

conservative mammalian toxicology oral endpoint based on subtle changes in blood

and brain acetylcholine esterase activity at very low dose levels, substantially lower

than those evaluated in the registration studies cited above. Likewise, the aquatic risk

assessment for methomyl is based on toxicity observed in aquatic invertebrates

(Daphnia magna), being the most sensitive species. Therefore, DuPont considers that

" intent of the Tier 1 EDSP screening battery has been fulfilled for methomyl based on

the availability of a large database which indicates a lack of a direct adverse effect by

this substance on the endocrine system of mammals and wildlife species.

6.0 REFERENCES

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Estrogenicity, Androgenicity, and Aromatase Activity In Vitro. Toxicol. Appl.

Pharmacol. 179:1-12.

Burin, G., L. Chitlik, S.L. Makris, and J.N. Rowe, (1993). Reproductive toxicity

studies. Standard evaluation procedure. Health Effects Division: Draft Office of

Pesticide Programs. US Environmental Protection Agency, Washington, D:C.,

October 1993.

37
DuPont-29838

Celik, L., J. Lund, B. Schiott (2008),Exploring interactions of endocrine-disrupting

compounds with different confirmations of the human estrogen receptor a ligand

binding domain: A molecular docking study. Chem. Res. Toxicol. 21:2195-2206.

Crisp, T.M. et hi, (1997). Special Report on Environmental Endocrine Disruption:

An Effects Assessment and Analysis. Report Number EPA/63O/R-96/012

. February 1997. http://www.epa.gov/ORDAVebPubs/endocrine/endocrine.pdf

Grisp, T.M. et al, (1998). Environmental Endocrine Disruption: An Effects

Assessment and Analysis. Environ.l Health Perspectives 106, Supplement 1:11-

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European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) (1996).

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July 1996. Brussels, Belgium.

El-Medany, A,, G. El-Medany, and A.E. Rahmy (1993). Evaluation of the effect of

v

 methomyl insecticide on the testicular function in normal albino rats (hormonal,

histological and histochemical study.). J. Med. Res. Inst. 14: 165-i87!

Gruggs, C.J., J.C. Perkham, and K.D. Cato (1983). Mammary carcinogenesis in rats

in relation to the age at time of N-nitroso-N-methylurea administration. J. Natl.

Cancer Institute. 70:209-212.

Grue, C.E., A.D.M. Hart, and P. Mineau (1991). Biological consequences of

depressed brain cholinesterase activity in wildlife in: Cholinesterase-inhibiting

insecticides: Their impact on wildlife and the environment, ed. P. Mineau. •

Elsevier Science Publishers B.V., Amsterdam, pp. 151-209.

Hayes, W.J. Jr. (1975). Toxicology of Pesticides. Baltimore, The Williams and

WilkinsCo.

Hoffman, D.J and Albers, P.H. (1984). Evaluation of Potential Embryotoxicity and

Teratogenicity of 42 Herbicides, Insecticides, and Petroleum Contaminants to

Mallard Eggs. Arch. Environ. Contam. Toxicol. 13:, 15-27.

Hseuh, A.J.W., M.L. Dufau, and K.J. Catt (1977). Gonadotrophin induced hormone

receptors and desensitization of testicular 3',5'-cyclic AMP and testosterone

responses. Proc: Natl. Acad. Sci. USA 74:592-595.

Jiao, B. et al., (2008). Establishment of a Transgenic Yeast Screening System for

Estrogenicity and Identification of the Anti-Estrogenic Activity of Malachite

Green. J. Cell. Biochem. 105:1399-1409.

Judson, R., et al, (2009). In vitro screening of environmental chemicals for targeted

testing prioritization - The ToxCast Project. Environ. Health Perspect. Online,

December 14, 2009. Available at http://dx/doi/org.

Judson, R., et al, (2009). In vitro screening of environmental chemicals for targeted

testing prioritization - The ToxCast Project. Environ. Health Perspect. Online,

December 14, 2009. Available at http://dx/doi/org.

38
DuPont-29838

Kavlock, R., et al., (2009). Biological profiling of endocrine related effects of

chemicals in ToxCastTM. Poster presentation available at

http://www.epa.gov/NCCT/toxcast/files/summit/40P%20Kavlock%20TDAS.pdf.

Klotz, D. M. S. F. Arnold, and J.A. McLachlan., (1997). Inhibition of 17 BetaEstradiol and Progesterone Activity in Human Breast Cancer and Endometrial

Cancer Cells by Carbamate Insecticides. Life Sci. 60:1467-1475.

Kojima, H. et al., (2004). Screening for Estrogen and Androgen Receptor Activities

in 200 Pesticides by In Vitro Reporter Gene Assays Using Chinese Hamster Ovary

Cells. Environ. Health. Perspec. 112:524-531.

Mahgoub, A. A. and A.H. El-Medany (2001). Evaluation of chronic exposure of the

male rat reproductive system to the insecticide methomyl. Pharmacological

Research 44, 73-79.

Montague, B. (1997). Brian.dbf database, US Environmental Protection Agency,

Office of Pesticide Programs, August 13, 1997.

National Research Council (1999). Hormonally Active Agents in the Environment.

National Academy Press. Washington DC. 430 pages.

http://www.nap.edu/books/0309064198/html/

Nishihara, T. et al., (2000). Estrogenic activities of 517 chemicals by yeast twohybrid assay. J. of Health Sci. 46: 282-298

Nendza, M. and Wenzel, A. (2006). Discriminating Toxicant Classes by Mode of

Action, 1) (Eco)toxicity Profiles). Environ. Sci. &Pollut. Res. 13:192-203.

Porter, W.P., S .M. Green, N .L. Debbink, and I. Carlson (1993). Groundwater

pesticides: interactive effects of low concentration of carbamates aldicarb and

methomyl and the triazine metribuzin on thyroxine and somatotropin levels in

white rats. J. Toxicol. Environ. Health 40:15-34.

Reif, D., et al., (2009). Endocrine profiling and prioritization using ToxCast Assays.

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_id=1267&SIType=PR&TIMSType=Presentation&showCriteria=0&address=com

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Corporation, 425813-02.

39
DuPont-29838

TABLE 1 CITED METHOMYL TQXICITY STUDIES SUPPORTING

REGISTRATION

MRID NO.

00007190

Unknown,

report

supplied

Unknown,

report

supplied

00009010

00078361

00070241

00078423

00007091

43250701

43769401

00008621

00131257

41898601

41898602

00131255

46015305

43072101

REPORT No

MRO-848

Hazleton 201-1 51

52B 101-630-78

52E 004-630-79

HLR 168-67

HLR 235-81

HLO 253-81

Hazelton 201-510

Hazleton 201 -165

HLO 51 9-95

HLR 498-78

HLO 331-83

HLO 336-91 •

HLO 337-91

HLR 528-82

HLO 702-91

HLO 47-93

TITLE

Three month dietary administration - rats

insecticide 1179

A subacute (90 day) evaluation of Nudrin in the

Fischer-344 rat for Shell Oil Company

A subacute (90 day) evaluation of Nudrin in the

B6C3F1 mouse for Shell Oil Company

Three-month feeding study on dogs with

S-methyl N-[(methylcarbamoyl)oxy]

thioacetimidate [Lannate Methomyl insecticide;

INX-1179]

Long-term feeding study in rats with S-methyl

N-((methylcarbamoyl)oxy) thioacetimidate)

(methomyl; INX-1179)

104-week chronic toxicity and carcinogenicity

study in mice methomyl; H-1 1 , 1 35

Two-year dietary administration - Dogs: -

Lannate methomyl insecticide (S-methyi-N-

((methylcarbamoyl)oxy) thioacetimidate)

Nudrin 2 generation reproduction study in rats.

Oral teratogenic study in rats with Lannate

(INX-1179)

Embryo-fetal toxicity and teratogenicity study of

methomyl in the rabbit.

A one-generation reproduction study with the

mallard (Anas platyrhynchos)

A one-generation reproduction study with the

northern bobwhite (Colinus viginianus)

Early Life Stage Toxicity of methomyl to

fathead "minnow.

Flow-through, 21 -day toxicity of

DPX-X1 179-423 (Lannate 20L) to

rainbow trout (Oncorhynchus mykiss)

Full life-cycle toxicity of DPX-X1 179-394

(methomyl) to the fathead minnow (Pimephales

promelas) under flow-through conditions

REVIEWED

BY EPA

(Y OR N)

• Y

Unknown

Unknown

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Unknown

Y

EPA

CLASSIFICATION

Acceptable

Unknown

Unknown

Unacceptable

(waived due to

acceptable chronic

study)

Acceptable

Acceptable

Acceptable

Acceptable

Acceptable

Acceptable

Acceptable

Acceptable

Acceptable

Unknown

Acceptable

40
DuPont-29838

TABLE 2 ENDOCRINE TISSUES EVALUATED IN MAMMALIAN TOXICOLOGY

STUDIES CONDUCTED WITH METHOMYL

TISSUE

Adrenal

. Pituitary

Thyroid

Testes

Epididymis

Prostrate

Ovary.

Fallopian tube

Uterus

Vagina

Cervix

Mammary

gland

90-D

RAT

W, H

W, H

H

W, H

H

H

H

90-D

RAT

W, H

H

H

W, H

W, H

H

W, H

W, H

W,H

H

90-D

MOUSE

W, H

H

W, H

H

W, H

W, H

90-D

DOG

W, H

W,H

W, H

W, H

.H

W, H

H

H

H

H

2-YR

RAT

W, H

W, H

H

W, H

H

H

H

H

H

2-YR

MOUSE

W, H

W, H

H

W, H

H

H

H

H

H '

2-YR

DOG

W, H

H

W, H

W, H

H

H

2-GEN

REPRO

W, H

H

'H

W, H

H

W, H

W,H

H

H

H

RAT

, DEV

G

G

RABBIT

DEV

G

G, W

W = organ weight; H = histopathology; G = gross pathology only

41