DATA TRANSMITTAL DOCUMENT

Oxamyl

Name arid Address of Submitter:

E.I. DuPont de Nemours and Company

1090 Elkton Road

P.O. Box 30

Newark; DE 19714

Phone (302) 366-5274

Regulatory Action in Support of Which this Package is Submitted:

Information Submitted in Support of continued registration of Oxamyl

Product Name: DuPontTM Oxamyl® Technical 42 Insecticide/Nematicide

EPA Registration No: 352-400

Transmittal Date: February 19, 2009

List of Submitted Studies:

ol

Data

Requirement

NA .

' Doc Number

DuPont-29827

Study Title

Oxamyl: Other Scientifically Relevant

Information Pertaining To EPA Test

Order EDSP- 103 80 1-63

MRID

Submitter:

WUUM.

Patricia p. Devine

U.S. Product Registration Manager

Company Name: E.I. du Pont de Nemours and Company

Company Contact: Patricia G. Devine

Date:

• •

L •«•••-
DuPont-29827

TRADE SECRET

Position Paper Title

OXAMYL: OTHER SCIENTIFICALLY RELEVANT INFORMATION

PERTAINING TO EPA TEST ORDER EDSP-1 03801 -63

Authors

Karin S. Bentley, Ph.D.

Kristin E. Brugger, 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

1 090 Elkton Road

Newark, DE 19711

DuPont Project Identification .

DuPont-29827 - ' •.•

.Page 1 of 39
DuPont-29827

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

Company Agent:

U.S. Product Registration Manager

• • • •

Date ' ' ••••••» • •!•

We have submitted this material to the United States Environmental I*t»^e«tion * * * *

Agency specifically under the provisions contained in FIFRA as amefidSd and, . .

hereby, consent to use and disclosure of this material by EPA according to FIFR&.***0

Notwithstanding the wording of our marking TRADE SECRET, this marking, bj*«»»,

itself, conveys no supplemental claims of confidentiality under FIFRA Sections VQ$)

or 10(b). In submitting this material to EPA according to method and format

requirements contained in PR Notice 86-5, we do not waive any protection or right

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

had not been submitted to the EPA, nor do we waive any protection or right provided

under FIFRA Section 10(g).
DuPont-29827

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

U.S. EPA Good Laboratory Practice Standards (40 CFR Part 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-29827

SIGNATURE

OXAMYL: OTHER SCIENTIFICALLY RELEVANT INFORMATION

PERTAINING TO EPA TEST ORDER EDSP-103801 -63

Position Paper by:

Karm S. Bentley, Ph.D. ff . Date (/

Senior Regulatory Toxicologist

Kristin E. Brugger, Ph.

Senior Regulatory-Ecotoxicologist

Date Completed:

February 18,2010

Applicant/Sponsor:

E. I. du Pont de Nemours and Company

Wilmington, Delaware 19898

U.S.A.
DuPont-29827

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 (OSPJ) 8

2.2 Toxicological Profile of Oxamyl 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 25

3.2.1 Summary of the Potential to Modulate Estrogen or Androgen

Receptor Function ..25

3.2.2 Summary of the Potential of Methomyl Modulate Placental

Aromatase Activity In vitro '. '.....21

3.3 Available Information from the EPA ToxCastTM Project ..27

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 32

5.1 Potential for Oxamyl to Interact with the Endocrine System 32

5.2 Functionally Equivalent Information Fulfilling the EDSP Tier IBattery 33

5.3 Overall Conclusion 35

6.0 References 36

TABLES

Table 1 Cited Oxamyl Toxicity Studies Supporting Registration 38

Table 2 Endocrine Tissues Evaluated in Mammalian Toxicology Studies

Conducted with Oxamyl ..39
DuPont-29827

OXAMYL: OTHER SCIENTIFICALLY RELEVANT INFORMATION

PERTAINING TO EPA TEST ORDER EDSP-103801 -63

1.0 SUMMARY

This document is in response to EPA Test Order EDSP-103801-63 requiring DuPont

to test the pesticide active ingredient Oxamyl (CAS# 23135-22-0) 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. Oxamyl 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 oxamyl in estrogen and androgen

transactivation assays, and oxamyl 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 oxamyl with estrogen,

androgen, or thyroid. Therefore, further assessment of oxamyl 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-29827

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 (CYP19) 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 20.09, EPA

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

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

EDSP-103801-63, issued on November12, 2009. .
DuPont-29827

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 toxiciiy 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-29827

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 doss - 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 in 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).

• Multiseneration 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. Procyrnidone-induced hypospadias and vinclozolin-induced feminization of male

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

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

chronic toxicity studies in dogs — 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 al., 1983), are often detected in these studies.

2.2 Toxicological Profile of Oxamyl

The mammalian toxicity of oxamyl 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

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

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

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

The primary symptoms are those associated with cholinesterase inhibition.

No cumulative effects on the nervous system occur with repeated exposure.
DuPont-29827

Oxamyl does not induce tumors in rats or mice and is classified by EPA as Group E

(not likely to be carcinogenic to humans). Oxamyl is not mutagenic and has

produced negative results in a battery genotoxicity studies. Increased sensitivity of

offspring was not observed 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 clinical signs of

cholinesterase inhibition, body weight reductions, and reduced pup weight. Oxamyl

is considered neither a reproductive nor developmental toxicant. Throughout the

mammalian toxicology database, there is no evidence suggestive of an adverse effect

on any component of the endocrine system.

The ecotoxicity of oxamyl is similarly well characterized based on the results of

extensive acute and chronic testing with birds, fish, aquatic and terrestrial

invertebrates as well as aquatic and terrestrial plants. The primary toxicological effect

of oxamyl in vertebrate and invertebrate animal species is the inhibition of

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

oxamyl demonstrates high acute toxieity by the oral and aquatic routes of exposure.

The principle effects in chronic exposure studies in fish were reduced embryo

hatching and larval swim-up, and in birds were reduced numbers of eggs laid, total

egg mass, and number of fertile eggs produced, all related to frank toxicity of the

carbamate. Throughout the ecotoxicology database, there is no evidence suggestive

of 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 oxamyl 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 oxamyl

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. In addition, as requested by EPA, hard copies of the

front page of each report which 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 oxamyl 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 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-29827

3.0 SUMMARY OF RELEVANT MAMMALIAN TOXICOLOGY STUDIES

3.1 Discussion of studies supporting registration

Snee, D.A. (1969); Ninety-day feeding study in rats with l-(Dimethylcarbamoyl)-N-

(Methylcarbamoyloxy)thioformimidic acid, methyl ester (IND-1410).

DuPont Report No. HLR 308-69, MRID 00066911, does not satisfy guideline but

acceptable chronic study is available.

Materials and Methods;

Groups of 16 male and 16 female weanling albino rats (Charles River-CD strain)

were orally administered oxamyl (100% purity) in pulverised basal diet (containing

1% corn oil) at concentrations of 0, 50, 100 and 500 ppm for 91-95 days. Because of

severe toxicity at the 500 ppm dose level during the first four days of the study, the

animals in the 500 ppm dose group were fed a control diet for three days and then

placed on a diet containing 150 ppm of the test substance.

All animals were observed for clinical signs of toxicity, and mortality. Body weights

and food consumption were recorded throughout the study. Haematology, clinical

chemistry (alkaline phosphatase) and urinalysis evaluations were performed on

10 male and 10 female rats from the control, mid-, and high-dose groups at months

1,2, and 3. Glutamic-pyruvic transaminase (GPT) was measured in the control and

high-dose groups monthly. All but 6 male and 6 female rats in each group were

sacrificed after 91-95 days. Histopathology was conducted on sacrificed animals

from the control and the high-dose groups (including adrenals, pituitary, testes,

prostate, ovary, uterus, and thyroid); absolute and relative organ weights (including

adrenals, pituitary, and testes) were determined for all animals at termination.

A reproductive toxicity assessment was conducted on the remaining 6/sex/group of

the test animals following 91-95 days on study. Each female was co-housed with

three males from the same group (5 days with each male). At the end of the 15-day

mating period, females were separated and examined daily for parturition. Four days

after parturition, the number of F)a pups'was reduced to a maximum of 10 per litter.

Weaning of the F]a pups was done on day 21 of lactation. The same procedure was

followed to produce an Fib litter. Mating behaviour, number of pregnancies, number

of young in each litter at birth and at 4, 12, and 21 days of age, and body weights of

the young at weaning were recorded.

Findings:

General Observations: One 500 ppm male was found dead on Day 5. One 100 ppm

:

 male was found dead on day 30. Males in the 500 ppm group started to fasciculate

after 2 days; after 4 days, all males and females exhibited fasciculations, ruffled fur,

mild diarrhoea, bulging eyes, lacrimation, excessive food spillage, and weight loss.

When animals were placed on control diet, the signs disappeared or lessened in

severity. No treatment-related clinical signs of toxicity were noted at 50, 100, or

150 ppm. Food consumption was significantly lower in 150 ppm males.; Food

consumption was significantly increased in 50 ppm males. There were ho treatment11
DuPont-29827

related effects on food efficiency. Significantly depressed body weight gain in

150 ppm males and in 100 ppm males and females was noted.

Haematology, clinical chemistry, urinalysis: No treatment-related abnormalities in

haematology parameters were detected. No treatment-related abnormalities were

detected in alkaline phosphatase and glutamic-pyruvic transaminase activities. An

increased incidence of protein and blood in urine was noted in 150 ppm males and

females.

Reproductive effects: No significant differences between control and test groups

with respect to reproduction and lactation performance were noted. Body weights of

weanlings at all dietary levels in both Fla and Fib generations were reduced.

Gross pathology, organ weights, histopathology: No significant treatment-related

gross lesions were detected. Statistically significant decreases in absolute kidney,

heart, thymus, and spleen weights in 100 and 150 ppm males were noted.

Statistically significant decreases in absolute liver weight in 150 ppm males and

females and increases in stomach weight in 150 ppm females were noted.

Statistically significant decreases in absolute kidney, liver, and lung weights in

lOQ ppm females were noted. No changes in relative organ weight to body weight

ratios were evident. No significant treatment-related microscopic lesions were

detected. There were no test substance related effects suggestive of an adverse effect

on the endocrine system.

Conclusion:

No-observed-effe.ct level for adult animals: 50 ppm (equivalent to 3.9 mg/kg bw/day

for males and 4.3 mg/kg bw/day for females) - based on depressed body weight gain

and organ weight effects at 100 ppm. There was no evidence of a direct adverse

effect on the endocrine system in this study.

Noising, G.C. (1969); 13 week oral administration - dogs - insecticide 1410.

DuPont Report No. HLO 328-69, MRID 00113398, does not satisfy guideline but

acceptable chronic study available.

Materials and Methods:

Groups of 4 male and 4 female Beagle dogs each received oxamyl, 100% purity"

(assumed), in pulverised basal diet at concentrations of 0, 50, 100, and 150 ppm for

13 weeks.

All animals were observed daily for clinical signs of toxicity and mortality; body

weights and food consumption were recorded weekly. Haematology, blood

chemistry, and urinalysis evaluations were performed prior to initiation and at weeks

4 and 13 on all animals. Gross necropsy was performed and organ weights (including

adrenals, testes with epididymis, and thyroid) were recorded for all animals at

termination. Histopathology was performed on selective tissues (including adrenals,

testes, ovary, uterus, and thyroid) of the control and 150 ppm animals.

Histopathology was performed on the liver, kidneys, and unusual lesions of the

SOppmand lOOppm animals: .

12
DuPont-29827

Findings:

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

course of this study. No test substance-related clinical signs of toxicity were noted.

No test substance-related effects on food consumption or body weights were noted.

Haematology, clinical chemistry, urinalysis: No treatment-related haematology,

clinical chemistry or urinalysis effects 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 were apparent at any dietary concentration. It was noted by the study

pathologist that the level of activity in the testes, ovaries, and thyroid were within

normal limits and comparable between the control and treated animals. There were

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

system.

Conclusion:

No-observed-effect level: >150 ppm (5.0 mg/kg bw/day for males and 4.2 mg/kg

bw/day for females) - highest dose tested. There was no evidence of a direct adverse

effect on the endocrine system in this study.

Mebus, C.A. (1990); Chronic toxicity study with oxamyl (IN D1410-196) one-year

feeding study in dogs. DuPont Report No. HLR 381-90, MRID 41697901, satisfies

Guideline 83-1b. '

Materials and Methods:

Four groups of 5 male and 5 female Beagle dogs each received oxamyl, purity 97.1%,

in the diet at concentrations of 0, 50, 150, and 250 ppm for 12 months. Body weights

and food consumption were recorded weekly. Clinical signs of chronic toxicity were

recorded weekly; clinical signs of acute toxicity were recorded daily, three hours after

the dogs were fed, for the first three months of the study. Haematology, clinical

chemistry, and urinalysis evaluations were conducted for all animals at pretest and at

months 3, 6, 9, and 12. On Days 7, 34, 88, 187, 270, and 363, blood was drawn for

analysis of cholinesterase (ChE) levels. An ophthalmoscopic examination was

conducted at pretest and on Day 358. Gross necropsy, organ weight (including testes

and thyroid) and histopathology (including adrenals, pituitary, testes, prostate,

epididymis, ovary, uterus, vagina, mammary, and thyroid) evaluations were

performed at study termination on all surviving dogs. Cholinesterase activity was

determined in sections of brains taken from the caudate nucleus, cerebellum/medulla,

and cerebrum!

Findings:

General Observations: One 250 ppm female was found dead on day 348. The

.cause of death could not be determined from the gross and histological examinations,

and there were no apparent compound-related lesions. A statistically significant

increase in the incidence of tremors and vomiting in 150 and 250 ppm males and/or

13
DuPont-29827

females was noted. Increased incidence of diarrhoea, vomiting, and mucoidal stool in

150 and 250 ppm males and/or females was not statistically significant but was

consistent with symptoms of ChE inhibition and, therefore, was considered

biologically significant. There was no statistically significant difference between

control and treatment groups with regard to clinical observations of chronic toxicity.

There was a higher incidence of vomiting in males at 150 and 250 ppm and in

females at 250 ppm, which represents a continuation of the acute toxicity beyond

three hours. Statistically significant decreases in food consumption and food

efficiency were noted in 250 ppm males throughout the study; statistiqally significant

decreases in food efficiency were noted in 150 and 250 ppm females during the first

week of the study. Statistically significant reductions in mean body weights and

mean body weight gains were noted in 250 ppm males. Decreased mean body weight

and body weight gain were noted in 150 and 250 ppm females (not statistically

significant) but were correlated with decreased plasma ChE activity and, therefore,

considered biologically significant. A slight dose-related decrease in mean body

weight and body weight gain were noted in 50 and 150 ppm males (not statistically

significant)-but were also considered biologically significant because the decreases

were correlated with decreases in plasma ChE activity. There were no test

substance-related ophthalmoscopically visible changes in the eyes of either male or

female dogs at any dietary concentration.

Haematology, clinical chemistry, urinalysis: No treatment-related haematology or

urinalysis effects were noted. Significantly decreased plasma and brain (caudate

nucleus) cholinesterase activity occurred in male dogs administered 50 ppm and

above. Plasma cholinesterase activity was also significantly lower in females fed 150

or 250 ppm diets. Biologically significant decreases in cholinesterase activity were

observed in the caudate nucleus sections of the brain for females in the 150 and

250 ppm groups.

Gross pathology, organ weights, histopathology: No treatment-related gross

pathology effects were noted. Statistically significant increases in mean relative brain

and kidney weights were noted in 250 ppm males and were attributed to the decreased

mean final body weight in these animals. Minimal to mild renal tubular epithelial

alterations were noted in three 250 ppm males, which were judged to be regenerative

and did not correlate with any clinically observable or clinical pathology findings.

Terminal aspiration of ingesta was a treatment-associated effect seen in all male

treatment groups and was attributed to the emetic properties of oxamyl. There were

no other compound-related microscopic findings observed in treated animals at

sacrifice. There were no test substance related effects suggestive of an adverse effect

on the endocrine system.

Conclusion:

No-observed-effect level: <50 ppm (<1.56 mg/kg bw/day) in males and 50 ppm

(1.46 mg/kg bw/day) in females based on decreases in plasma and brain

cholinesterase activity at >50 ppm in males and >150 ppm in females, accompanied

by decreased body weight and clinical signs indicative of cholinesterase inhibition at

150 and 250 ppm. There was no evidence of a direct adverse effect on the endocrine

system in this study.

14
DuPont-29827

Dickrell, L. (1991); 52-week dietary toxicity study with IND-1410 (oxamyl) in male

dogs. DuPont Report No. HLO 555-90, MRID 42052 701, in combination with the

above 1-year study in dogs, satisfies Guideline 83-1 b.

Materials and Methods;

Five groups of 5 male Beagle dogs each received oxamyl, purity 97.1%, in the diet at

concentrations of 0, 12.5, 20, 35, and 50 ppm for 52 weeks. This study was

conducted to establish a NOEL for cholinesterase inhibition in males. Animals were

observed for clinical signs of toxicity. Body weights and food consumption were

recorded throughout the study. An ophthalmic examination was performed at pretest

and at weeks 26, 39, and 52. Haematology and clinical chemistry evaluations were

performed at pretest and at weeks 13, 26, 39, and 53. At study termination, the

animals were necropsied and examined macroscopically, selected organs were

weighed (including testes and thyroid), and selected tissues were preserved. Tissues

were not examined microscopically as the primary purpose of this study was to define

a NOAEL for cholinesterase inhibition in male dogs.

Findings;

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

course of this study. No clinical signs of toxicity were observed. There were no test

substance-related effects on body weights, body weight gains, or food consumption.

There were no test substance-related ophthalmoscopically visible changes in the eyes

at any dietary concentration.

Haematology, clinical chemistry: No treatment-related haematology effects were

noted. No treatment-related effects on plasma, RBC, and brain ChE activity were

noted. • ,

Gross pathology and organ weights: No treatment-related gross lesions were

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

apparent at any dietary concentration. There were no test substance-related

macroscopic findings observed in treated animals at sacrifice. Based on the endpoints

evaluated, there was no test substance related findings suggestive of an adverse effect

on the endocrine system.

Conclusion:

No-observed-effect level: 50 ppm (1.36 mg/kg bw/day in males) - highest dose

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

this study.

15
DuPont-29827

Malley, LA. (1991); Combined chronic toxicity/oncogenicity study with oxamyl

(IN-D1410-196) long-term feeding study in rats (4 volumes).

DuPont Report No. HLR 278-91, MRID 41963201, satisfies Guideline 83-5

Materials and Methods:

Five groups of 62 male and 62 female Sprague Dawley rats were fed diets containing

0, 25, 50, 100, and 150 ppm oxamyl, purity 97.1%, for approximately 2 years.

Clinical signs of toxicity, body weights, and food consumption were recorded

throughout the study. Haematology, clinical chemistry, and urinalysis evaluations

were performed on all rats at approximately 3, 6, 12, 18, and 24 months. Plasma and

RBC cholinesterase activity was determined at one month and at all other clinical

chemistry sampling times. Ophthalmoscopic examinations were performed on all rats

prior to initiation and at 12 and 24 months. At 12 months, 10 rats per sex per group

were sacrificed for organ weight determination and histopathological evaluation.

At 24 months, all surviving rats were sacrificed for organ weight (including testes)

determination and histopathological (including adrenals, pituitary, testes, epididymis,

prostate, seminal vesicles, ovary, uterus, vagina, mammary, and thyroid) evaluation.

Brains were collected at the interim and final sacrifice and assessed for brain

cholinesterase activity.

Findings:

General Observations: There was no test substance-related effect on mortality.

A test substance-related trend for greater survival in 100 and 150 ppm males and in

50, 100, and 150 ppm females was noted, which correlated with lower body weight

observed in 100 and 150 ppm males and;females. Hyperreactiviry and swollen

legs/paws were noted in 100 and 150 ppm males and females (swollen legs/paws in

females at 150 ppm only). Skin lesions including sores and alopecia were noted in

100 and 150 ppm females. Decreased body weight and body weight gain were noted

in 100 and 150 ppm males and females (as much as 22% and 30%, respectively).

No treatment-related effects were noted on food consumption; however, food

efficiency was decreased in males and females at 100 to 150 ppm. A higher

incidence of retinal photoreceptor cell atrophy occurred in 150 ppm females at the

2-year sacrifice only and was considered to be a secondary effect of nutritional status

and ageing. A dose-related increase in the incidence of pale ocular fundi was noted in

150 ppm males. Microscopic evaluation of the ocular tissues in males did not reveal

compound-related effects. The higher incidences of pale, ocular fundi in 150 ppm

males may be due to anaemia, thinning of the retina, or the lower number of animals

which survived until the ophthalmic exam in the 0 and 25 ppm groups compared with

the 100 and 150 ppm groups.

Haematology, clinical chemistry, urinalysis: No treatment-related changes in

haematology or urinalysis were noted. Decreased plasma ChE activity was noted in

100 and 150 ppm .males at several sampling intervals and in 100 and 150 ppm

females at the one-month sampling interval only. No effects on RBC and brain ChE

were observed.

16
DuPont-29827

Gross pathology, organ weights, histopathology: At the one-year sacrifice,

absolute liver weights were decreased and mean relative heart and brain weights were

increased in 150 ppm males. Females in the 150 ppm group had increased relative

kidney weights, and females in the 100 or 150 ppm groups had increased relative

heart and brain weights. The changes in organ weight were secondary effects due to

the lower body weights. At the one-year sacrifice,- no test substance-related effects

were noted on tissue morphology. At the two-year sacrifice, absolute brain weight

was significantly higher in 150 ppm males and in 100 and 1'50 ppm females. In

addition, 150 ppm males had decreased absolute kidney weights. Females in the 100

and 150 ppm groups had increased relative kidney, liver, and brain weights. Females

in the 150 ppm group also had increased relative heart weights. There were no

histopathologic changes in these organs; and, therefore, these changes were

considered to be secondary to the lower body weight and/or greater survival. At the

two-year sacrifice, dose-related increases in the incidence of ulcer/erosions on the

limbs, nodules on the tail, tail deformity, periocular chromodacryorrhea, stomach

ulcer or erosion, large aorta, brain compression, and discoloration of the kidneys were

noted. The observations at the two-year sacrifice are common in geriatric or stressed

animals, and the apparent dose response is considered related to the increased

survival and decreased body weight in 100 and 150 ppm rats. There were no test

substance related findings suggestive of an adverse effect on the endocrine system.

Conclusion: No-observed-effect level: 50 ppm (1.97 mg/kg bw/day in males and

2.69 mg/kg bw/day in females) - based on lower body weight and body weight gain

and plasma cholinesterase inhibition at 100 and 150 ppm. Oxamyl was not oncogenic

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

system in this study.

Adamik, E.R. (1981); Long term feeding study in mice with oxamyl.

DuPont Report No. HLO 252-81, MRID 00076813, satisfies Guideline 83-2:

Materials and Methods:

Four groups of 80 male and 80 female weanling CD-I mice were fed diets containing

0, 25, 50, and 100 ppm oxamyl, purity 97.1%, for approximately 2 years. Because of

unexpected high mortality rates at the 50 and 100 ppm dose levels during the first few

weeks, the 100 ppm group was decreased to 75 ppm at week 6.,

Body weights, food consumption, and clinical observations were recorded for all

animals throughout the study. All mice were palpated once weekly for masses.

Haematological evaluations were performed on 10 males and 10 females from each

group after 1, 3, 6, 12, and 18 months of dosing and prior to termination of the study.

Gross and relative organ weights (including testes) were determined for all animals at

termination. Gross necropsies were performed on all surviving animals at study

termination. Histopathological evaluations were performed (including adrenals,

testes, epididymis, prostate, seminal vesicles, ovary, uterus, cervix, mammary, and

thyroid).

17
DuPont-29827

Findings:

General observations: Early deaths (through Week 4: 2 males, 9 females at 50 ppm;

14 males and 11 females at 100 ppm) were likely due to an acute exposure to the test

compound; no long-term treatment-related increases in mortality were noted. No

treatment-related clinical signs of toxicity were noted. Decreased body weights were

noted in males up to Week 81 (50 ppm) or up to Week 42 (100/75 ppm) and in

females up to week 21. Statistically significant1 decreases in food consumption were

noted for 100/75 ppm males from Week 11 through Week 84.

Haematology: Sporadic statistically significant increases and decreases in

haematology parameters were observed at 100/75 ppm were suggestive of an effect

on RBC mass, particularly at Week 4. This effect appeared sporadically during

weeks 4-52; however, it was not noted after Week 52.

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

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

weights were apparent at any dietary concentration. No treatment-related increase in

microscopic findings was noted. There were no test substance related findings

suggestive of an adverse effect on the endocrine system.

Conclusion:

No-observed-effect level: 25 ppm (equivalent to 4.2 mg/kg bw/day for males and

5.2 mg/kg bw/day for females) - based on haematology effects at 100/75 ppm and

mortality and decreased body weights at >50 ppm. Oxamyl was not oncogenic in this

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

this study.

Sherman, H. (1972); Long-term feeding study in rats and dogs with 1-

(dimethylcarbamoyl)-N-(methylcarbamoyloxy)-thioformimidic acid, methyl ester

(IND-1410). DuPont Report No. HLR 37-72, MRID 00083352.

Materials and Methods:

Groups of 36 male and 36 female Sprague-Dawley rats received oxamyl, purity

95.0%, in the diet at concentrations of 0 (two groups), 50, 100, and 150 ppm for

2 years. The animals were evaluated for clinical signs of toxicity, body weights, and

food consumption, which were recorded throughout the study. Clinical chemistry

(alkaline phosphatase and glutamic-pyruvic transaminase) and haematology (RBC,

haemoglobin, hematocrit, and leukocyte count), and urinalysis were conducted on

10 rats/sex/group at 100 and 150 ppm and on 5 rats/sex in the control group at

Months 1, 3, 6, 9, 12, 18, and 24. (The same animals were used for haematology and

urinalysis evaluations; different animals were used for clinical chemistry evaluations.)

Blood cholinesterase evaluations were conducted on 10/sex/group from the control

and high-dose groups on Days 4 and 8 and at Months 1,6, 12, and 24 (additional

cholinesterase evaluations were conducted on the 100 ppm group at months 1 and 6).

Aliesterase activity was measured on blood from these rats at Months 1> 12, and 24.

Gross pathology and histopathology (including adrenal, pituitary, testis, epididymis,

18
DuPont-29827

prostate, ovary, uterus, mammary, and thyroid) evaluations were conducted, and

organ weights (including adrenal, pituitary, and testes) were recorded for interimsacrifice animals at 12 months and for all surviving animals at 24 months. This study

also contained a reproductive assessment in rats.

After approximately 12 weeks of oxamyl administration in the diet at concentrations

of 0, 50, 100, and 150 ppm, 16 females rats/group were housed separately and mated

(for approximately 15 days, 5 days each with 3 separate males). Animals were

observed during gestation for the birth of young. The number of pups (Fia) in each

litter was determined at birth and at 4, 12, and 21 days of age. Pup body weights

were recorded at weaning (21 days). Litters were culled at day 4 postpartum.' The

F]a litters were sacrificed. The parental animals were mated a second time to produce

Fib litters. The Fib animals were fed their respective diets. When the Fib generation

was 110 days of age, they were mated to produce the p2a and F2b litters. This process

was repeated to produce F3a and Fab litters. At weaning, two males and two females

from each of five Fsb litters from the control and high-dose group were evaluated

histopathologically (including adrenal, testis, epididymis, ovary, and thyroid).

Because of decreased body weights observed consistently in the weanling rats at 100 A

and 150 ppm, a study was conducted to ascertain whether these lower body weights .

reflected toxic or .nutritional effects. Twenty male and 20 female pups of the Fab litter

were selected at weaning from 5 or more mothers in the control and 150 ppm groups

and divided into subgroups of 10 males and 10 females each. One subgroup from the

controls and one subgroup from the 150 ppm group were maintained on their

respective diets; the other subgroup from the controls was transferred to the 150 ppm

group while that from the 150 ppm group was transferred to the control group.

The animals were maintained on their diets for eight weeks.

Groups of 4:male and 4 female Beagle dogs each received diet at concentrations of

0, 50, 100, and 150 ppm for 2 years. Body weights and food consumption were

recorded weekly. Clinical signs of toxicity were recorded daily. Haematology, blood

biochemistry, and urinalysis evaluations were conducted for all animals at pretest and

at Months 1, 2, 3, 6, 9, 12, 15, 18, 21, and 24. Cholinesterase and aliesterase activity

were measured at pretest and at 1, 6, 12, and 24 months. Gross necropsy and

histopathology (including adrenal, pituitary, testis, epididymis, prostate, ovary,

uterus, fallopian tubes, mammary, and thyroid) evaluations were performed for one

male and one female dog each from the control and 150 ppm group at one year.

At study termination, all surviving dogs were given a gross necropsy. Complete

histopathology evaluations were performed on all dogs in the control and 150 ppm

dose groups. Only liver, kidney and testis were examined microscopically from the

low- and mid-dose groups. Organ weights were recorded for a}l animals at

termination.

Findings:

General observations: No treatment-related effects on mortality were noted.

No treatment-related clinical signs of toxicity were observed. Decreased body weight

in 100 and 150 ppm rats was observed throughout the study. No treatment-related

body weight effects in dogs were noted. Slightly decreased food consumption was

19
DuPont-29827

noted in 150 ppm male and female rats. No treatment-related effects on food

efficiency were noted in male or female rats. No treatment-related food consumption

effects in dogs were noted.

Haematology, clinical chemistry, urinalysis: No treatment-related haematology

effects were noted in rats or dogs. Blood ChE activity was decreased in 150 ppm

female rats at day 4 and in male rats at Day 8. Increased alkaline phosphatase and

.cholesterol levels were noted in 150 ppm male and female dogs. No treatment-related

urinalysis effects were noted in rats or dogs.

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

lesions were observed at necropsy in rats or dogs. No test substance-related changes

in mean organ weights were apparent at any dietary concentration in rats or dogs.

No treatment-related microscopic findings were noted in rats or dogs.

Reproductive effects: Litter size and viability and lactation indices were slightly

decreased in the 100 and 150 ppm groups. There were no treatment-related effects on

reproductive or lactation parameters.

Effects on pups: Slightly decreased weanling body weights were noted in the 100

and 150 ppm groups. These body weight effects appear to reflect an adverse

nutritional effect rather than a toxic one; the reversibility of this effect was seen by a

greater rate of body weight gain when pups were transferred from the test diet to the

control diet. No treatment-related histopathological effects were noted at 150 ppm in

the Fsb weanling pups.

Conclusion:

No-observed-effect level in adult rats was 50 ppm (equivalent to 2.3 mg/kg bw/day in

males and 2.8 mg/kg bw/day in females) - based on decreased body weight at > 100

ppm.

No-observed-effect level for reproduction was >150 ppm (>7.5 mg/kg bw/day1) -

highest dose tested. (Calculated based on standard conversion method of ppm x

0.05 mg/kg bw/day). The NOEL in pups was 50 ppm (2.5 mg/kg bw/day1)-based

on slight pup body weight effects at >100 ppm.

No-observed-effect level in dogs was 100 ppm (equivalent to 2.8 mg/kg bw/day in

males and 3.0 mg/kg bw/day in females) - based on increased alkaline phosphatase

and cholesterol levels at 150 ppm.

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

rats or dogs in this study.

20
DuPont-29827

Hurtt, M. E. (1990); Reproductive and fertility effects with oxamyl (IN D1410) '

multi-generation reproduction study in rats. DuPont Report No. HLR 423-90,

MRID 41660801, satisfies Guideline 83-4.

Materials and Methods:

Groups of 30 male and 30 female Sprague Dawley rats each received oxamyl, purity

97.1%, in the diet at concentrations of 0, 25, 75, and 150 ppm fofe approximately

70 days pre-mating and during mating, gestation, and lactation. Fj weanlings

(30/sex/group; 1 rat/sex/each litter where possible) were randomly selected and

continued on their respective diets. The FI animals were bred to produce ¥2 offspring

when all had been on test at least 105 days.

On Day 0 postpartum, live and dead pups in each litter were counted; live pups were

weighed collectively by sex. On Day 4 postparrum, pup counts and weights were

determined, and the litters were culled randomly to 8 (4/sex) where possible. Litter

counts and weights were also determined after culling on Day 4 and on Days 7 and 14

postparrum. On Day 21 (weaning), individual pup weights were taken. On Days 0, 4,

7, 14, and 21, offspring were individually examined for abnormal behaviour and

appearance.

Clinical signs of toxicity were evaluated in all PI and FI rats weekly. All PI and FI .

rats were weighed weekly during premating. During gestation and lactation, females

were weighed on Days 0, 7, 14, and 21 of each period; males were weighed weekly.

Food consumption was determined weekly throughout premating for PI and FI rats;

food consumption of pregnant rats was also recorded on Days 0, 7, and 14 of

gestation. All parental rats (Pi and FI) were evaluated for gross lesions. Organ

weights of selected tissues (including testes) were recorded. Tissues from parental

rats from the control and 150 ppm dose groups in both generations were examined

histologically (including pituitary, testes, epididymis, prostate, seminal vesicles,

coagulating gland, ovary, uterus, and vagina). Twenty FI (from those not continuing

to the next generation) and ¥2 weanlings per sex per group were also necropsied and

given a gross exam. Reproductive function indices were calculated for PI and FI

parental rats.

Findings:

General observations: In the 150 ppm group, three FI males and one PI female were

found dead during the study (not considered to be treatment related). Treatmentrelated decreases were observed in mean body weight and body weight gain in 75 and

150 ppm PI and FI parental rats during premating and gestation. During lactation,

decreased body weight and increased body weight gain were observed in 75 and

150 ppm PI and FI females. In 75 ppm (males) and 150 ppm (males and females)

significant reductions in food consumption and food efficiency were noted in PI and

FI rats during premating. Food efficiency was significantly decreased in 150 ppm

PI and FI females during gestation. Significant upward trend in the incidence of

hyperreactivity in FI males and females (premating) was observed (statistically

significant in males at 150 ppm). During the premating and/or gestation periods, 75

and 150 ppm FI females showed a statistically significant dose-related increase in

alopecia.

21
DuPont-29827

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

lesions were observed at necropsy in parental rats, weanlings, or pups. No test

substance-related changes in mean organ weights were apparent at any dietary

concentration. Mean relative testes weight was significantly higher in 75 and 150

ppm PI and FI males due to lower body weight. No treatment-related microscopic

findings were noted in PI or FI adult males and females.

Reproductive parameters: There were no treatment-related effects on mating,

fertility, and gestation length in the PI and FI rats.

Litter size, pup survival, and pup weight: In the 150 ppm group, statistically

significant decreases in mean number of pups (males and females combined) born per

litter, born alive per litter, and the number of pups alive on day 4 preculling in the FI

and'F2 generations were noted. A significant increase in the number of pups observed

with no milk spot was noted in the F2 offspring (attributed to low pup weight). In the

75 and 150 ppm dose groups, a statistically significant decrease in pup body weight

was observed in FI and F2 males and females throughout most of the lactation period.

There was no impact of treatment on the sex ratio of the FI and F2 pups at any dose

level.

Conclusion:

Parental NOEL: 25 ppm (equivalent to 1.43 mg/kg bw/day) - based on body weight

effects in males and females at >75 ppm.

Pup NOEL: 25 ppm -.based on body weight effects at >75 ppm and decreased pup

survival at 150 ppm.

Reproductive NOEL: >150 ppm (equivalent to 12.2 mg/kg bw/day) - highest dose

tested.

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

study.

Richard, I.E. (1988). Teratogenicity study of IN D1410-196 in the rat. DuPont

Report No. HLR 473-88 and accompanying paper DuPont-1954, MRIDs 40859201

and44737501, satisfies Guideline 83-3.

Materials and Methods:

Groups of 25 female Sprague-Dawley rats each received oxamyl, purity 97.2%, in

distilled water at concentrations of 0, 0.2, 0.5, 0.8, and 1.5 mg/kg bw/day orally by

gavage daily from Days 7 to 16 of gestation; the dosing volume was 10 mL/kg bw.

The day copulation was confirmed (by the presence of a vaginal plug) was designated

as Day 1 of gestation. The animals were observed for clinical signs oftoxicity in the

morning on Days 1-22 of gestation and at approximately 2 hours.postdosing on

Days 7-16 of gestation. Body weights were recorded on Days 1,7-17, and 22 of

gestation. Food consumption was recorded on Days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,

and 22 of gestation. Females were sacrificed on Day 22 of gestation and were

examined for gross lesions. The liver and gravid and non-gravid uteri were weighed.

22
DuPont-29827

The dams were examined for reproductive effects. Foetuses were weighed, sexed,

and examined externally, viscerally, and skeletally. Sex was confirmed upon visceral

exam.

Findings:

Maternal effects

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

course of this study. Tremors, salivation, diarrhoea, eye discharge, and wet areas of

the body were observed during the dosing period in 1.5 mg/kg bw/day rats. Tremors

were observed in 0.8 mg/kg bw rats during dosing period. Significant dose-related

decreases in body weight changes were noted at 0.8 and 1.5 mg/kg bw during the

dosing period (reduced by -21% and 30%, respectively, compared with controls).

A slight but not statistically significant decrease in body weight gain was noted in

0.5 mg/kg bw rats during the dosing period (reduced by ~9% compared to controls).

Significant dose-related effects on food consumption were noted in 0.8 and 1.5 mg/kg

bw rats during the dosing period. ,

Reproductive outcomes: No treatment-related effects on reproductive parameters ,

(sex ratio, pregnancy rate, incidence of total resorptions, incidence of early deliveries,

corpora lutea, or the number of stunted foetuses) were observed. There was no

apparent test substance related effects on endocrine function pertaining to

maintenance of pregnancy or foetal development.

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

observed at necropsy. An upward trend in increased relative liver weights was

observed (no statistical significance was noted in any group).

Foetal effects

Significant reductions in mean foetal body weights were noted at 0.8 and 1.5 mg/kg

bw/day. A minimal (reduced by <4% compared to controls), yet statistically

significant decrease in mean foetarbody weight was noted at 0.5 mg/kg bw/day;

however, subsequent analysis of the data (DuPont-1954) indicates that the mean

foetal weight of this dose group is well within the laboratory historical control range.

Therefore, it is not considered adverse. Moreover, it occurred in the presence of a

similar slight depression in maternal body weight gain during the dosing period.

No treatment-related structural malformations were noted.

Conclusion:

No-observed-effect level (maternal toxicity): 0.5 mg/kg bw/day - based on decreased

body weight and food consumption and tremors at >0.8 mg/kg bw/day.

No-observed-effect level (foetal toxicity): 0.5 mg/kg bw/day - based on adversely

decreased foetal weights at >0.8 mg/kg bw/day.

No-observed-effect level (developmental toxicity): >1.5 mg/kg bw/day - highest dose

tested.

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

study.

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DuPont-29827

Hoberman, A.M. (1980); Teratology study in rabbits - oxamyl. DuPont Report No.

HLO 0801-80, MRID 00063009, satisfies Guideline 83-3b:

Materials and Methods;

Groups of 17 female pregnant New Zealand White rabbits each received oxamyl,

purity 97.1%, in distilled water at concentrations of 0, 1, 2, and 4 nig/kg bw/day

orally by gavage daily from Days 6 to 19 of gestation; the dosing volume was

1 mL/kg bw/day.

Females were artificially inseminated; the day after artificial insemination was

designated as Day 0 of gestation. Animals were observed for clinical signs of toxicity

daily. Individual body weights were recorded on Days 6, 11, 15, 19, and 29 of

gestation; individual food consumption was recorded on Days 7 through 29 of

gestation. On Day 29 of gestation, female rabbits were sacrificed, the foetuses were

taken by^caesarean section, and the does were examined for visceral gross pathology.

The ovaries and uterus (with and without foetuses) of each rabbit were weighed and

the number of corpora lutea and implantations determined. Each foetus was

examined externally, weight and crown-rump distance determined, and a visceral

examination conducted. The heads of one-third of the foetuses were removed and

sectioned by Wilson's technique for examination. All foetuses were, evaluated for

skeletal anomalies following fixation and staining. Each pup was sexed by internal

exam.

Findings:

Maternal effects

General Observations: No treatment-related mortality was observed; one low dose

pregnant rabbit and one high dose nonpregnant rabbit died during the study (deaths

likely due to intubation error). No treatment-related clinical signs of toxicity were

noted. Sporadic incidences of eye discharge and red stains in the tray under the cage

were noted in the control and mid-dose groups. Statistically significant decreases in

body weight gain was noted in the 2 and 4 mg/kg bw/day groups during the treatment

period. No treatment-related effects in food consumption were noted.

Gross pathology, organ weight: No test substance-related gross lesions were

observed at necropsy. Slightly decreased ovarian and uterine weights with and

without foetuses were noted (not statistically significant) in the 2 and 4 mg/kg bw/day

groups presumably due to the effects of treatment on body weight.

Reproductive outcomes: No treatment-related effects were noted on pregnancy rate,

numbers of corpora lutea and implantations, implantation efficiencies, or sex ratio.

No treatment-related effects were noted on foetal body weights and lengths. In the 2

and 4 mg/kg bw/day groups, slightly increased incidence of resorption was noted (not

statistically significant: 2 high-dose does had 100% resorption; 1 dead foetus in the

mid-dose group). Foetal viability was slightly lower at 4 mg/kg bw/day but was not

statistically significant. There was no apparent test substance related effects on

endocrine function pertaining to maintenance of pregnancy or foetal development.

24
DuPont-29827

Foetal effects

No test substance-related effects were noted.

Conclusion:

No-observed-effect level (maternal toxicity): 1 mg/kg bw/day - based on statistically

significant decreased body weight gain at 2 and 4 mg/kg bw/day.

No-observed-effect level (foetal toxicity): 2 mg/kg bw/day - based on increased

percent resorptions at 4 mg/kg bw/day.

No-observed-effect level (developmental toxicity): >4 mg/kg bw/day - 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

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

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

In the report by Klotz, Arnold, and McLachlan (1997), oxamyl and methomyl 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, oxamyl and methomyl 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, oxamyl and methomyl were tested at concentrations of

100 nM, IjaM, and 10 uM 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 oxamyl and methomyl

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"4 M, and the response was compared to the activity of

17p-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

_
DuPont-29827

[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 17p-estradiol (10 nM in

MCF-7 cells) or the PR agonist Rl 881 (0.1 nM in CHO cells). In the cell

proliferation assays, methomyl was tested over a concentration range of 10"8 to 5x10"5

M in MCF-7 cells and the responses were compared to the activity of 17p-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), oxamyl and methomyl were evaluated

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

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

methomyl 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 17p-estradiol (10'" M for ERa; 10'10 M for ERP) or DHT (10'10

M) for the ER and AR trasactivation assays, respectively. Under the conditions of the

study, oxamyl and methomyl showed no significant activity in any of ER or AR

transactivation assays.

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

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

assay, oxamyl and methomyl were tested over a concentration range of 100 mg/1 to

50 ug/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, oxamyl

and methomyl 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. I

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

analogue methomyl 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 coworkers, oxamyl and

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

26
DuPont-29827

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 oxamyl or methomyl are steroid hormone receptor

agonists/antagonists, and the authors themselves state that the reported activity was

not receptor mediated. The weight of evidence suggests that neither oxamyl nor

methomyl interact with the ER or AR.

3.2.2 Summary of the Potential of Methomyl Modulate Placental Aromatase Activity

In vitro

In the report by Andersen and coworkers, the close structural analogue of oxamyl,

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 |^M) and the response was

compared to the activity of 4-androsten-4-ol-3,17-dione (1 uM). When tested at a

single concentration (50 |im) 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 oxamyl and methomyl.

3.3 Available Information from the EPA ToxCastTM 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 oxamyl, using assays

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

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

27
DuPont-29827,

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, oxamyl 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

Roberts, N.L., Phillips, C.N.K., Chanter, D.O., McAllister, A. (1982b); The effects of

dietary inclusion of oxamyl on reproduction in the bobwhite quail.

DuPorit Report No.: HLO 453-82, MRID-00116610, USEPA guideline 163.71-4

(1978).

Materials and Methods:

Groups of young adult bobwhite quail (Colinus virginianus), were offered diet

containing technical oxamyl (purity 97.1%) at concentrations of 0, 10 or

50-mg a.s./kg diet for 28 weeks (sixteen weeks prior to the start of egg production

and twelve weeks during egg production). Each treatment group comprised

14 replicates of one male and two females. Individual adult body weights were

recorded on Days 0, 14, 28, 42, 56, 70, 84, and 196; food consumption in each

replicate was measured weekly throughout the pre-treatment and treatment periods of

the adult phase. Individual chick body weights were recorded within 24 hours of

hatching and on Day 14 after hatching; egg shell thickness was measured on the first

two days of Weeks 1, 3, 5, 7, 9, and 11 of the egg production period. Adult birds and

chicks were observed daily for mortality and clinical signs; all adult birds were

subjected to post mortem examination.

Findings:

Four birds died or were sacrificed (two in the control and two in the

50-mg a.s./kg diet treatment group) during the pre-egg production period; all were

replaced with spare birds. There was no treatment-related effect on adult mortality;

clinical observations and post mortem changes were consistent with long-term

housing of birds in cages, and were not treatment-related. Adult body weight changes

over the treatment period were similar in all groups and there was no evidence of any

statistically significant treatment-related effect. Adult food consumption was

significantly lower in the two oxamyl treated groups compared to the control during

both pre-egg production and egg production periods.

Initial chick body weights were similar overall in all treatment groups; chicks that

died after hatching during the 14-day observation period were examined and no

abnormalities were detected. Of the reproductive parameters measured, few

differences were detected between the control and oxamyl treatments, with two

exceptions: the proportion of damaged eggs was lower in the 10-rng a.s./kg diet

28'
DuPont-29827

treated group compared to the control, and the proportion of chicks surviving to

14 days was significantly higher in the 10-mg a.s./kg diet treatment compared to the

control (Williams test, PO.05).

Conclusion:

The reproductive NOEC for bobwhite quail exposed to oxamyl in the diet for

28 weeks was 50-mg a.s./kg diet, based on the absence of reproductive effects at the

highest dose level tested (50-mg a.s./kg diet).

Roberts, N.L., Phillips, C.N.K., Chanter, D.O., McAllister, A. (1982a); The effects of

dietary inclusion of oxamyl on reproduction in the mallard duck.

DuPont Report No.: HLO 337-82, MRID-00116609, USEPA guideline 163.71-4

(1978).

Materials and Methods:

Groups of young adult mallard ducks (Anas platyrhynchos), were offered diet

containing technical oxamyl (purity 97.1%) at concentrations of 0, 10, or

50-mg a.s./kg diet for 22 weeks (ten weeks prior to the start of egg production and

during twelve weeks of egg production). Each treatment group comprised six

replicates of two male and five females. Individual adult body weights were recorded

on Days 0, 14, 28, 42, 56, and 154; food consumption in each replicate was measured

weekly throughout the pre-treatment and treatment periods of the adult phase.

Individual chick body weights were recorded within 24 hours of hatching and on

Day 14 after hatching; egg shell thickness was measured on the first two days of

Weeks 1, 3, 5, 7, 9, and 11 of the egg production period. Adult birds and chicks were

observed daily for mortality and clinical signs; all adult birds were subjected to post

mortem examination. '

Findings:

Seven birds died or were sacrificed during the pre-egg production period mainly as a

result of bullying (two in the 10-mg a.s./kg diet treatment and five in the

50-mg a.s./kg diet treatment), all of which were replaced with spare birds. During the.

egg production period, six birds died in the control, two birds died in the

10-mg a.s./kg diet treatment and nine birds died in the 50-mg a.s./kg diet treatment, of

which one was sacrificed. There were no treatment-related effects on adult mortality,

food consumption or body weight. Initial duckling body weights were similar in all

treatment groups. Ducklings that died during the 14-day observation period after

hatching were examined and no abnormalities were detected.

There were no statistically significant effects of treatment on the proportion of eggs*

cracked, mean egg weight, embryonic deaths, hatchability of fertile eggs, or survival

of ducklings to 14 days. In the 50-mg a.s./kg diet treatment group, statistically

significant reductions compared to the control were recorded on the numbers of eggs

laid, total egg mass, and numbers of fertile eggs produced.

29
DuPont-29827

Conclusion;

The NOEC for mallard ducks exposed to oxamyl in the diet for 22 weeks was

10-mg a.s./kg diet, based on egg production and egg viability effects observed at the

highest dose level tested (50-mg a.s./kg diet).

Hutton, D.G. (1988); Early life stage toxicity ofIND1410-196 (oxamyl) to rainbow

trout. DuPont Report No.: HLR 468-88. MRID 40901101. (MRID 41472301 for

HLR 468-88, Supplement 1, supplemental data for statistical analysis), USEPA

guideline 72-4 (1988).

Materials and Methods:

Rainbow trout (Salmo gairdnerii, equivalent to Oncorhynchus mykiss) embryos and

larvae were continuously exposed to oxamyl (purity: 97.2%) in a flow-through test

system at measured concentrations of 0.06, 0.10, 0.23, 0.40, 0.77, and 1.5-mg a.s./L

for 61 days. Observations were made on embryo hatching and larval survival and

growth.

The test vessels (aquaria made of stainless steel) were filled with 7 litres of filtered

well water (water depth: 17 cm), maintained at the test temperature (11.8-13.9°C),

with a dissolved oxygen concentration of 9.4 to 10.0 mg/L. Each aquarium was

partitioned in the middle to give two replicates. For the embryo phase, two 200 mL

screen bottomed glass cups were suspended in each aquarium replicate (water

depth: 2 to 5 cm), and twenty embryos were added to each cup (80 embryos per

concentration). The first larvae hatched on Day 20 and hatching was considered

complete by Day 29. As the larvae hatched, they were transferred from the embryo

cup to the appropriate aquarium, with up to 15 larvae per replicate; excess larvae

were counted and then discarded. Survival of the remaining larvae was assessed for

32 days, and at the end of the study all surviving larvae were weighed and measured.

Findings:

The mean measured concentrations of oxamyl in the test vessels ranged from 75% to

100% of the nominal values.' No statistically significant effects on embryo hatching,

larval survival, first day of larval swim-up, or larval growth were observed at

 i

 .

measured test concentrations of 0.06 to 0.77-mg a.s./L. At 1.5-mg a.s./L (the highest

concentration tested), statistically significant effects on embryo hatching and a delay

in the first day of larval swim-up were observed compared to the control.

Conclusion:

The NOEC for rainbow trout exposed to oxamyl was 0.77-mg a.s./L, based on

statistically significant effects on embryo hatching and larval swim-up at

1.5-mg a.s./L (mean measured concentrations).

30
DuPont-29827

Driscoll, R.R. (1982); Early life stage toxicity ofoxamyl to fathead minnow.

DuPont Report No. HLR 877-81, MRIDs 00094663, 00116607, 00126510, and

00248463, guideline "Standard Practice for Conducting Toxicity Tests with Early

Life Stages of Fishes" Committee E-47 of American Society for Testing and Materials

(DraftNo. I.February 1981).

Materials and Methods:

Duplicate groups of 50 fathead minnow (Pimephales promelas) eggs (fertilised

embryos, less than twenty four hours old at study initiation) were exposed to oxamyl

(purity 97.1 %) at nominal concentrations of 0 (control), 0.25, 0.5, 1.0, 2.0, and

4.0-mg a.s./L in a flow through system. Egg viability was assessed daily until

hatching was complete; any dead embryos or embryos covered in fungi were

removed.

To initiate the 28-day larval exposure study, 20 newly hatched fathead minnows were

randomly selected from each embryo replicate and transferred to the respective test

aquarium, with two replicates per concentration; the excess larvae were counted and

then discarded. The test larvae were fed at least once daily on live brine shrimp

.nauplii (Anemia salind). The test conditions were 16 hours light, 8 hours dark; a

temperature of 25°C; pH 7.5 to 7.7; and dissolved oxygen 7.7-7.8 mg/L. Daily

•observations were made on larval mortality, behaviour and appearance. The number

of live fry in each aquarium was recorded weekly. At test termination, surviving

larvae were measured (mean standard length) and individually weighed.

Findings: >

Analytical determinations ofoxamyl concentrations were within 25% of nominal

levels. Percent embryo hatch was reduced in one or both replicates at nominal test

concentrations of 0.25, 2.0, and 4.0-mg a.s./L; however these reductions, which were

not statistically significant, were considered to be a result of fungal growth rather than

a treatment related effect. Significant reductions in larval survival were detected at

nominal test concentrations of 1.0-mg a.s./L and above. However significant effects

on larval growth (mean standard length) occurred only at the highest concentration

tested (4.0-mg a.s./L). No treatment-related effects on egg viability, larval survival or

growth were detected at nominal concentrations of 0.5-mg a.s./L or below.

Conclusion:

The NOEC ofoxamyl to the early life-stages of fathead minnow was 0.5-mg a.s./L,

based on the reduced survival of fathead minnow larvae exposed for 28 days. The

LOEC was 1.0-mg a.s./L.

31
. DuPont-29827

4.2 Discussion of available information in the open literature

Additional information was sought in the published literature by searching the

ECOTOX data base and TOXCENTER. The search is summarized below:

TOXCENTERwas searched by CAS # (16752-77-5 OR23135-22-0) and key terms:

• hormone? or endocrine? or endocrine disruption or ?androgen? or

?estrogen? or testosterone? or receptor binding or reprod? or develop? or

tox?

• in utero? or terat? or steroid? or aromatase? or thyroid? or hershberger? or

pubert? or utero? or ovary or ovaries or teste? or sex?

The above search was narrowed down to those papers/reports relevant to

• amphibian metamorph? or fish or amphibian? or frog? or bird?

No additional relevant ecotoxicology data was identified by these searches.

5.0 DuPoNT POSITION AND CONCLUSIONS

5.1 Potential for Oxamyl to Interact with the Endocrine System

Oxamyl 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 a 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

adrenals, pituitary, thyroid, testis, epididymis, prostate, coagulating gland, seminal

vesicles, ovary, fallopian tubes, uterus, vagina, cervix, and mammary gland. In none

of these studies were there treatment-related 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 tumors of the

endocrine tissues, were induced by oxamyl.

Oxamyl 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, or feminization or

masculinization of pups (sex ratio). The only effects noted in offspring were

decreases in body weight the high dose levels of 0.8 and 1.5 mg/kg bw/day, which

occurred in the presence of similar reductions in maternal weight and clinical signs of

. cholinesterase inhibition. In the rabbit study, the observed effects at the high doses

(2 and 4 mg/kg bw/day by gavage) were a reduction in body weight gain in the dams

and a slight increase in resorptions and reduced viability in the pups.

The outcome of a multigeneration reproduction study in rats also demonstrated a lack

of an adverse effect by oxamyl on the hormonal control of sexual development,

32
DuPont-29827

, growth, and reproduction. Oxamyl 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,

and gestation length. There were also no data in these studies to indicate that sexual

differentiation (sex ratio), pubertal development, production of germ cells, and

accessory sex organs had been adversely affected. The primary effects noted were

body weight decreases and reduced food consumption in parental animals at the two

high doses of 75 and 150 ppm in diet. In addition, a reduction numbers of pups, pup

survival, and pup weight at these same doses occurred and were consistent with the

body weight effects observed in the parental animals. In a three-generation

reproduction study conducted in conjunction with a long-term chronic study, litter

size and pup viability were slightly decreased at the same dose levels at which

consistent depressions in parental body weight were observed. No treatment-related

: effects occurred on reproductive parameters. There were no histopathological

findings due to treatment with oxamyl in a number reproductive tissues examined

from 2 male and 2 female Fsb pups/litter at weaning.

Histopathology of the ovary, testes, and accompanying accessory reproductive tissues

have been evaluated in a number of studies in rats, mice, and dogs following

subchronic and chronic exposures to oxamyl. 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

oxamyl 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, or rabbits indicate that oxamyl produces no adverse effects on the

endocrine system of mammals.

Exposure to bobwhite quail resulted in no reproductive effects at the highest dose

tested. In contrast, reductions in numbers of eggs laid, total egg mass, and numbers

of fertile eggs produced were noted in the high dose group of a reproduction study in

mallard duck. Chronic exposure to fish resulted in reductions of embryo hatching and

larval swim-up. In the absence of any evidence of adverse effects suggestive of

interference of icstrogen, androgen, or thyroid hormone process in mammalian

toxicity studies, however, the changes in these apical endpoints are attributed to a

direct toxic effect of the carbamate rather than endocrine system dysfunction in these

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 oxamyl as well as information from the open literature and ToxCastTM,

DuPont proposes that the primary purpose of the Tier 1 EDSP screening battery, to

33
DuPont-29827

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, oxamyl, as well as its structural analogue methomyl, 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 oxamyl

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

oxamyl.

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 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

oxamyl that would suggest interference of steroid hormone production (e.g., adrenal

and reproductive organ weight or histopathology, fertility, gestation, sex ratio), it is

DuPont's position that no further testing of oxamyl 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 (with and without the products of conception) has been

assessed in developmental toxicity studies in rats and rabbits conducted with oxamyl.

Histopathology of a number of female reproductive tissues has been evaluated in one.

or more of the studies summarized above (ovary, fallopian tubes, uterus, vagina, and

mammary gland). Testes weights were measured in the majority of these studies as

well. One study also included epididymis 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. Seminal vesicles and coagulating

glands wre also examined in several studies. In the absence of any adverse findings,

the weight of the evidence suggests that oxamyl 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 oxamyl.

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

34
DuPont-29827

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, a three-generation

reproduction study was conducted in which there were no effects on reproductive

performance over three generations; the study also included histopathological

evaluation of a number of reproductive tissues in F^ weanlings. Therefore, under

these circumstances, it is DuPont's position that the evaluation of oxamyl 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 oxamyl

does not interact with the HPT axis. No treatment-related effects were observed on

the thyroid gland in the studies supporting registration of oxamyl 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 oxamyl in the Tier 1 AMA screen.

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. DuPont is of the position that a fish short-term reproduction

assay should not be required for oxamyl. Sufficient information is available from the

mammalian toxicology data base and the chronic ecotoxicology studies with birds

and fish to demonstrate the lack of interference with estrogen and androgeri"

dependent processes and that any effect on the normal function of the reproduction

system of vertebrates is due to frank toxicity of the carbamate, not to interference

with the reproductive endocrine system.

5.3 Overall Conclusion

Oxamyl is a carbamate insecticide which exerts its biological activity by inhibiting

acetylcholinesterase in the nervous system of insects, mammals, and other vertebrate

species. Oxamyl 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

_
DuPont-29827

suggest that oxamyl directly interacts with the function of estrogen, androgen, or

thyroid hormone processes to cause endocrine dysfunction. .A similar toxicology

database exists for methomyl, another DuPont carbamate insecticide which shares

close structural analogy to oxamyl and which also demonstrates low potential to

interact with the endocrine system (see DuPont response to Test Order

EDSP-090301-62, DuPont -29838). Oxamyl 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 oxamyl 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 oxamyl based on the availability of a large database which indicates a lack of a

direct adverse effect on the endocrine system of mammals and wildlife species.

6.0 REFERENCES

Andersen, H. R., A. Vinggaard, T. Rasmussen, I. Gjermandsen, E. BonefeldJorgensen (2002). Effects of Currently Used Pesticides in Assays for

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.

European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) (1996).

'Environmental Oestrogens' A compendium of test methods. Document No. 33,

July 1996. Brussels, Belgium.

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.

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

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DuPont-29827

TABLE 1 CITED OXAMYL TOXICITY STUDIES SUPPORTING REGISTRATION

MRID No.

00066911

00113398

41963201

00076813

41697901

42052701

00083352

41660801

40859201

44737501

00063009

00116610

00116609

40901101

41472301

00094663

00116607

00126510

00248463

REPORT No

HLR 308-69

HLO 328-69

HLR 278-91

HLO 252-81

HLR 381-90

HLO 555-90

HLR 37-72

HLR 423-90

HLR 473-88

DuPont-1954

HLO 0801-80

HLO 453-82

HLO 337-82

HLR 468-88

HLR 877-81

TITLE

Ninety-day feeding study in rats with 1-

(Dimethylcarbamoyl)-N-

(Methylcarbamoyloxy)thioformimidic acid,

methyl ester (IND-1410)

13 week oral administration - dogs -

insecticide 1410

Combined chronic toxicity/oncogenicity study

with oxamyl (IN-D1410-196) long-term feeding

study in rats

Long term feeding study in mice with oxamyl

Chronic toxicity study with oxamyl (IN D1410-

196) one-year feeding study in dogs

52-week dietary toxicity study with IND-1410

(oxamyl) in male dogs

Long-term feeding study in rats and dogs with

1 -(dimethylcarbamoyl)-N-

(methylcarbamoyloxy)-thioformimidic acid,

methyl ester (IND-1410)

Reproductive and fertility effects with oxamyl

(IN D1410) multi-generation reproduction

study in rats

Teratogenicity study of IN D1410-196 in the .

rat

DuPont's position on foetal weight changes in

rats following developmental toxicity testing

with oxamyl

Teratology study in rabbits - oxamyl

The effects of dietary inclusion of oxamyl on

reproduction in the bobwhite quail

The effects of dietary inclusion of oxamyl on

reproduction in the mallard duck

Early life stage toxicity of IN D1410-196

(oxamyl) to rainbow trout

Early life stage toxicity of oxamyl to

fathead minnow

REVIEWED

BY EPA

(Y OR N)

Y

Y

Y

Y

Y

Y

Unknown

Y

Y

Y

Y

Y

Y

Y

Y

EPA

CLASSIFICATION

Unacceptable

(waived due to

acceptable chronic

study)

Unacceptable

(waived due to

acceptable 1 year

studies)

Acceptable

Acceptable

Acceptable

Acceptable

Unknown

Acceptable

Acceptable

Acceptable

Acceptable

Supplemental

Acceptable

Acceptable

Acceptable

i

38
DuPont-29827

TABLE 2 ENDOCRINE TISSUES EVALUATED IN MAMMALIAN TOXICOLOGY

STUDIES CONDUCTED WITH OXAMYL .

TISSUE

Adrenal

Pituitary

Thyroid

Testes

Epididymis

Prostrate

Seminal

vesicles

Coagulating

gland

Ovary

Follopian

tube

Uterus

Vagina

Mammary

gland

90-D

RAT

W, H

W, H

H

W, H

H

H

H

90-D

DOG

W, H

W, H

W, H

W

H

H

1-YR

DOG

H

H

W, H

W, H

H

H

H

H

H

1-YR

DOG

w

W

2-YR

RAT

H

H

H

W, H

H

H

H

H -

H

H

H

2-YR

RAT

W, H

W, H

H

W, H

H

H

H

H

H

2-YR

MOUSE

H

H

W, H

H

H

H

H

H

H

2-YR

DOG

W, H

W, H

W, H

W, H

H

H

H

H

H

H.

2-GEN

REPRO

H

W, H

H

H

H

H

H

H

H

3-GEN

REPRO*

H

H

H

H

H

RAT

DEV

G

G, W

RABBIT

DEV

,

G

G, W

W = organ weight; H = histopathology; G.= gross pathology only

* F3b pups

39