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STUDY TITLE

EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER

(DEPCT) IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST

Test Guidelines

USEPA OCSPP 870.5395 (1998)

OECD, Guideline 474 (1997)

EC, B.12 (2008)

Author(s)

M. R. Schisler, B.S.

L. G. McFadden, M.S.

Study Completion Date

04 December 2014

Sponsor

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, Indiana 46268

Performing Laboratory

Toxicology and Environmental Research and Consulting

The Dow Chemical Company

Midland, Michigan 48674

Laboratory Project Study ID

DAS STUDY ID

PCTR Number
 

 

 

 


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TABLE OF CONTENTS

Page

STUDY TITLE ....................................................................................................................1

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS..................2

QUALITY ASSURANCE STATEMENT ..........................................................................3

SIGNATURE PAGE ...........................................................................................................4

SUMMARY.........................................................................................................................7

INTRODUCTION ...............................................................................................................8

Principles of the Micronucleus Test................................................................................8

Purpose............................................................................................................................8

Test Guidelines................................................................................................................8

Quality Assurance ...........................................................................................................8

Archiving ........................................................................................................................9

Safety...............................................................................................................................9

TEST MATERIAL INFORMATION .................................................................................9

Test Material Name.........................................................................................................9

Chemical Name...............................................................................................................9

Synonyms........................................................................................................................9

Supplier, City, State (Lot, Reference Number)...............................................................9

Purity/Characterization (Method of Analysis and Reference)........................................9

Characteristics.................................................................................................................9

Molecular Formula .......................................................................................................9

Molecular Weight.........................................................................................................9

Chemical Structure .....................................................................................................10

CAS Number ..............................................................................................................10

Positive Control Chemical ............................................................................................10

Chemical Name ..........................................................................................................10

CAS Number ..............................................................................................................10

Source.........................................................................................................................10

STUDY DESIGN...............................................................................................................10

Dose Levels...................................................................................................................10

Animal Dosing ..............................................................................................................10

Dose Range Finding Test (RFT)...................................................................................11

Micronucleus Test.........................................................................................................11

TEST SPECIES AND HUSBANDRY..............................................................................11

Species and Sex.............................................................................................................11

Strain .............................................................................................................................12

Criteria for Selecting the Strain.....................................................................................12

Supplier and Location ...................................................................................................12

Age at Study Start .........................................................................................................12

Physical Acclimation ....................................................................................................12

Housing .........................................................................................................................12
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TABLE OF CONTENTS

Page

Randomization and Identification.................................................................................13

Text Table 1. Animal Identification Numbers..................................................13

Feed and Water .............................................................................................................13

Animal Welfare.............................................................................................................13

STUDY SPECIFIC PARAMETERS.................................................................................14

Animal Observations and Body Weight Determination ...............................................14

Body Temperature Data Collection ..............................................................................14

Peripheral Blood............................................................................................................14

Details of Slide Preparation ..........................................................................................15

Cells Examined .............................................................................................................15

Evaluation Criteria ........................................................................................................15

STATISTICS AND CALCULATIONS............................................................................16

RESULTS AND DISCUSSION........................................................................................16

Dose Range-Finding Test (RFT)...................................................................................16

Micronucleus Assay (MNT) .........................................................................................20

CONCLUSIONS................................................................................................................21

ACKNOWLEDGEMENTS...............................................................................................21

REFERENCES ..................................................................................................................22

Table 1. Body Weights (g) – Males (Range Finding Test)..........................................23

Table 2. Body Weights (g) – Females (Range Finding Test) ......................................25

Table 3. Summary In-Life Observations – Males/Females (Range Finding Test) ......26

Table 4. Individual Animal Observations – Males (Range Finding Test) ...................28

Table 5. Individual Animal Observations – Females (Range Finding Test)................30

Table 6. Animal Body Temperature Data – Males (Range Finding Test)...................32

Table 7. Animal Body Temperature Data – Females (Range Finding Test) ...............34

Table 8. Body Weights (g) – Males (Micronucleus Test)............................................35

Table 9. Summary In-Life Observations – Males (Micronucleus Test) ......................37

Table 10. Individual Animal Observations – Males (Micronucleus Test)...................38

Table 11. Animal Body Temperature Data – Males (Micronucleus Test)...................42

Table 12. Summary Micronucleated Reticulocytes (MN-RET) Frequencies and

Percent Reticulocytes (% RET) – Males...........................................................44

Table 13. Individual Micronucleated Reticulocytes (MN-RET) and Percent

Reticulocytes (%RET) – Males (Micronucleus Test) .......................................45

Table 14. Recent Historical Control Data....................................................................46

Appendix A. Dose Selection Criteria...........................................................................47

Appendix B. Analytical Report....................................................................................48
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SUMMARY

The in vivo genotoxic potential of phosphorochloridothioic acid, O,O-diethyl ester

(DEPCT) was evaluated by examining the incidence of micronucleated reticulocytes

(MN-RET) in the peripheral blood. The test material was administered to male

Crl:CD1(ICR) mice by single oral gavage on two consecutive days at dose levels of 0

(negative control), 75, 150, and 300 mg/kg body weight (bw). The highest dose level

was based upon the results of a range-finding test where at higher doses, treatmentrelated deaths were observed in male and female mice. The analytically determined

concentrations of the test material in the dosing solutions used for the first day of dosing

in the micronucleus ranged from 90.7% to 92.4% of the targeted concentrations. All

animals were observed for clinical signs prior to dosing and at 2, 5, and 24 hours

following each dosing. Groups of animals were euthanized 48 hours after the second

treatment for the collection of peripheral blood and evaluation of RET (approximately

5,000/animal) for MN by flow cytometry. The proportion of RET was also determined

based upon 5,000 RET per animal and the results expressed as a percentage. Mice

treated with 40 mg/kg bw cyclophosphamide monohydrate by a single gavage dose and

euthanized 48 hours later served as positive controls.

There were no treatment-related deaths in the definitive micronucleus test of the study.

Treatment-related clinical signs including perineal soiling, decreased feces, and soft feces

were noted in the 300 mg/kg bw/day treatment groups. There were no statistically

significant increases in the frequencies of MN-RET or statistically significant effects on

the percent RET in groups treated with the test material as compared to the negative

controls. There was a significant increase in the frequency of MN-RET and a decrease in

the percentage of RET in the positive control chemical group as compared to the negative

control group. Based upon the results of the study reported herein,

phosphorochloridothioic acid, O,O-diethyl ester is considered negative in this test system

under the experimental conditions used.
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INTRODUCTION

Principles of the Micronucleus Test

The micronucleus test (MNT) is a short-term in vivo cytogenetic assay for detecting

agents that induce chromosomal breakage and spindle malfunction (Mavournin et al.,

1990). Micronuclei arise when acentric chromosomal fragments and/or whole

chromosomes are excluded from the main nucleus at the telophase stage of the cell

division (Schmid 1976; Mavournin et al., 1990).

The MNT has several advantages over a cytogenetic assay involving direct examination

of metaphase chromosomes. The MNT provides information on chromosome loss due to

spindle impairment whereas such information cannot be readily obtained with metaphase

chromosome analysis. The end point scored in the MNT is less subjective than in

metaphase analysis. The significance of micronuclei is comparatively clear versus some

parameters measured in metaphase analysis, e.g. gaps.

Purpose

The purpose of the study was to determine the cytogenetic effects of

phosphorochloridothioic acid, O,O-diethyl ester (DEPCT) in the mouse by examining the

incidence of micronuclei in peripheral blood reticulocytes. Phosphorochloridothioic acid,

O,O-diethyl ester will here after be referred to as DEPCT,

Test Guidelines

OECD Guidelines for Testing of Chemicals, #474: Genetic Toxicology -

Micronucleus Test, OECD Publication Service, 2 rue Andre-Pascal,

75775 Paris Cedex 16, France (1997).

USEPA Office of Prevention, Pesticides and Toxic Substances, OPPTS

870.5395. Mammalian Erythrocyte Micronucleus Test (1998).

EC B.12, Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus

Test. Official Journal of the European Communities, L142,

31/05/2008.

Quality Assurance

The study conduct, data, protocol, protocol deviation(s)/amendment(s) if applicable, and

final report were inspected by the Quality Assurance Unit, Toxicology and

Environmental Research and Consulting, The Dow Chemical Company, Midland,

Michigan.
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Archiving

The data, protocol, protocol deviation(s)/amendment(s) if applicable, and final report

were archived by the Toxicology and Environmental Research and Consulting archivist

and stored at The Dow Chemical Company, Midland, Michigan.

Safety

All personnel involved in the study were advised of the safety precautions to follow when

handling the test and positive control materials. Chemical safety information was made

available.

TEST MATERIAL INFORMATION

Test Material Name

Phosphorochloridothioic acid, O,O-diethyl ester

Chemical Name

O,O-Diethyl ester phosphorochloridothioic acid

Synonyms

Diethylchlorothiophosphate, DEPCT

Supplier, City, State (Lot, Reference Number)

The Dow Chemical Company, Midland, Michigan (Lot # C032013081501)

Purity/Characterization (Method of Analysis and Reference)

The purity of the test material was determined to be 99.3% area by gas chromatography

with identification by nuclear magnetic resonance and gas chromatography mass

spectrometry (Megregian, 2014).

Characteristics

Molecular Formula

C4H10ClO2PS

Molecular Weight

188.6
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Chemical Structure

CAS Number

2524-04-1

Positive Control Chemical

Chemical Name

Cyclophosphamide monohydrate (CP)

CAS Number

6055-19-2

Source

Sigma, St. Louis, Missouri

STUDY DESIGN

Dose Levels

The dosing solutions of the test material were prepared and used fresh on each of the two

consecutive days of administration. A frozen stock solution of CP dissolved in distilled

water (thawed and brought to room temperature prior to use) served as the positive

control. The vehicle used to mix the test material (i.e. corn oil; CAS number 8001-30-7),

served as the negative control. The concentrations of the test material in the dosing

solutions used for the first day of dosing were verified using gas chromatography using

electron capture (GC/ECD) and external standard quantitation.

Animal Dosing

The test material, the vehicle (negative control), and CP (positive control) were

administered by oral gavage. Oral gavage is one of the generally accepted routes of

administering the test material in this assay system. CP when administered by oral

gavage has been shown to induce micronuclei in polychromatic erythrocytes of

O P O

CH3

3CH S

Cl
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Crl:CD1(ICR) mice (De Boeck et al., 2005; Gollapudi et al., 1986). Dosing solutions

were administered in aliquots of 10 ml/kg bw.

Dose Range Finding Test (RFT)

This assay was conducted to aid in the selection of dose levels for the micronucleus test.

The number of animals included in each dose group was based upon the use of the dose

selection criteria (Appendix A). In the initial phase of the RFT, mice (one/sex/dose) were

dosed once with 1000 or 2000 mg/kg bw/day of the test material on July 29, 2014. Due

to mortality in these mice a second phase of the RFT was conducted with lower dose

levels of 250 and 500 mg/kg bw/day (one/sex/dose) on July 30 and July 31, 2014.

Mortality was observed in the female mouse dosed at 500 mg/kg bw/day; however, the

male mouse survived. Based on these results, mice were dosed with 250 mg/kg bw/day

(2/sex), 375 mg/kg bw/day (1/sex), and 500 mg/kg bw/day (2 males) on August 5 and

August 6, 2014 and observed for any signs of toxicity for at least 72 hours after dosing.

The remaining mice (2/sex/dose) were dosed with 375 mg/kg bw/day on August 12 and

August 13, 2014 and observed for any signs of toxicity for at least 72 hours after dosing.

All mortalities and/or moribund animals were necropsied to ascertain whether the

condition was related to a dosing mishap.

Micronucleus Test

Groups of male mice were administered 0, 75, 150, or 300 mg/kg bw/day of the test

material on two consecutive days. Animals were dosed on September 23, 2014 and

September 24, 2014. CP was administered only once at a dose level of 40 mg/kg bw.

The dose levels were based on the results of the range finding test. There were six

mice/dose except in the 300 mg/kg bw/day group where an additional group of two mice

were dosed as possible replacements in the event of deaths occurring among the treated

animals of this group. The mice were observed daily and signs of toxicity or deaths

occurring during the observation period were recorded. Approximately 48 hours after the

last dosing, peripheral blood samples were collected from all animals.

TEST SPECIES AND HUSBANDRY

Species and Sex

Male and female mice were used for the RFT. Only males were used for the MNT due to

the results of the RFT which showed no apparent difference in the toxicity between the

sexes.
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Strain

Outbred Crl:CD1(ICR) mice

Criteria for Selecting the Strain

1. Availability of historical negative control data

2. Suitability for utilization in the MNT

3. General suitability for toxicity testing

Supplier and Location

Charles River (Kingston, New York)

Age at Study Start

Approximately 8-10 weeks

Physical Acclimation

During the acclimation period each animal was evaluated by a laboratory veterinarian, or

a trained animal/toxicology technician under the direct supervision of a laboratory

veterinarian, to determine the general health status and acceptability for study purposes.

The Toxicology and Environmental Research and Consulting Laboratory was fully

accredited by the Association for Assessment and Accreditation of Laboratory Animal

Care International (AAALAC International). The animals were housed one per cage in

stainless steel cages, in rooms designed to maintain adequate conditions (temperature,

humidity, and photocycle), and acclimated to the laboratory for at least one week prior to

the start of the study.

Housing

After assignment, animals were housed one per cage in stainless steel cages. Cages had

wire mesh floors and were suspended above absorbent paper. Non-woven gauze was

placed in the cages to provide a cushion from the flooring for the rodents' feet. Cages

contained a hanging feeder and a pressure activated lixit valve-type watering system.

The following environmental conditions were maintained in the animal room.

Temperature: 22°C with a tolerance of ± 1°C (and a maximum

permissible excursion of ± 3C)

Humidity: 40-70%
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Air Changes: 12-15 times/hour (average)

Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Randomization and Identification

Before administration of test material began, animals were stratified by body weight and

then randomly assigned to treatment groups using a computer program designed to

increase the probability of uniform group mean weights and standard deviations at the

start of the study. Animals placed on study were uniquely identified via subcutaneously

implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were

correlated to unique alphanumeric identification numbers.

Text Table 1. Animal Identification Numbers

Dose Level

mg/kg bw/day Males

0 14A4624-4629

75 14A4360-4635

150 14A4636-4641

300 14A4642-4649

40 CP 14A4650-4655

Feed and Water

Feed and municipal water were provided ad libitum. Animals were provided LabDiet

Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted

form. Analyses of the feed were performed by PMI Nutrition International to confirm the

diet provides adequate nutrition and to quantify the levels of selected contaminants.

Drinking water obtained from the municipal water source was periodically analyzed for

chemical parameters and biological contaminants by the municipal water department. In

addition, specific analyses for chemical contaminants were conducted at periodic

intervals by an independent testing facility. Copies of these analyses are maintained in

the study file.

Animal Welfare

In accordance with the U.S. Department of Agriculture Animal Welfare Regulations,

9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of

this study were reviewed and approved by the Institutional Animal Care and Use

Committee (IACUC). The IACUC has determined that the proposed Activities were in
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full accordance with these Final Rules. The IACUC-approved Animal Care and Use

Activities to be used for this study were Genetic Tox 01, Blood Collection 01, Humane

Endpoints 01, and Animal ID 01.

STUDY SPECIFIC PARAMETERS

Animal Observations and Body Weight Determination

The mice were observed for positive signs of toxicity at least once on the day of dosing

and subsequent days. The mice were weighed prior to dosing and on the day of their

scheduled sacrifice in order to assess the extent of stress experienced by the animals

following treatment (Gollapudi et al., 1986).

Body Temperature Data Collection

The relative body temperatures of the treated animals were monitored during the RFT

using programmable transponders (BioMedic Data Systems, Seaford, Delaware). The

temperatures were generally collected by scanning the microchip prior to each dosing,

and approximately 2, 5, and 24 hours after dosing as well as prior to sacrifice.

Temperatures were monitored during the main study since a decrease of at least 3C for

more than 5 hours was observed during the RFT. Body temperature changes outside this

range have been previously reported to cause an increase in micronucleus formation

(Asanami and Shimono, 1997; Asanami et al., 1998).

Peripheral Blood

Micronucleus formation in peripheral blood reticulocytes was determined by flow

cytometry with traditional blood smears prepared as a backup. Samples were prepared

and analyzed per instructions in the Mouse MicroFlow Micronucleus Analysis Kit

Manual (Litron Laboratories, Rochester, New York). At the end of the specified interval

following treatment, a peripheral blood sample was collected from the orbital sinus of all

surviving animals into anticoagulant solution following anesthesia with isoflurane.

Briefly, the blood samples were fixed in ultracold (-70 to -80°C) methanol within five

hours of collection. All fixed blood samples were stored at -80°C. Fixed blood samples

of the first six mice of each group were washed with a cold, buffered salt solution and

isolated by centrifugation. The resulting cell pellets were stored at approximately 4°C

until staining. Blood samples were ultimately incubated with RNase to degrade the high

levels of RNA present in the reticulocytes (RET) and a fluorescently labeled antibody to

the transferrin receptor (anti-CD71-FITC) to specifically identify the RET. A propidium
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iodide solution was added to each sample immediately before flow cytometry (FCM,

Beckman Coulter Gallios flow cytometer) analysis to stain the DNA, including that of

micronuclei. Blood samples were analyzed by high-speed FCM. In this system, the

sample was moved at a high velocity past a laser set to provide 488 nm excitation. The

fluorescent wavelengths emitted by each cell were collected by photomultiplier tubes.

Using the previously described staining procedure, the propidium iodide-stained DNA of

the micronuclei emitted a red fluorescence and the anti-CD71-FITC antibody emitted a

high green fluorescent signal permitting differentiation between cells with and without

micronuclei. In addition to obtaining fluorescent profiles, FCM simultaneously provided

cell size information by determining the light scatter properties of each cell or

combination of cells.

Details of Slide Preparation

Duplicate cell smears were prepared and stored to serve as backups in the event that the

flow cytometric analysis was not possible. Blood was collected into a microtainer tube

coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears

were prepared, fixed in methanol, and stored at room temperature.

Cells Examined

Approximately 5,000 RETs were analyzed per blood sample. The number of

normochromatic erythrocytes (NCE), MN-NCE, RET, and MN-RET were recorded for

each sample and the frequency of MN-RET was determined to provide an indication of

genotoxic potential. The frequency of RETs relative to total erythrocytes was determined

to provide an indication of perturbations in hematopoietic activity indicative of cell

toxicity. For each of the treatment groups, a mean and standard deviation was calculated

to describe the frequency of RET, MN-NCE, and MN-RET observed. The analyses were

conducted utilizing a flow cytometer (Beckman Coulter Gallios flow cytometer).

Evaluation Criteria

A test was considered valid if all of the following conditions are met:

 The range of MN-RET values in the negative controls were within reasonable

limits of the recent laboratory background range.

 There was a significant increase in the incidence of MN-RET in the positive

control treatment as compared to the concurrent negative controls.
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 The mean for percent RET value in one or more of the test material treated

groups was  20% of the control value indicating no undue effect on

erythropoiesis (toxicity).

A test material was considered positive in this assay if the following criterion was met:

 Statistically significant increase in MN-RET frequency at one or more dose

levels accompanied by a dose response.

A test material was considered negative in this assay if the following criterion was met:

 No statistically significant dose-related increase in MN-RET when compared to

the negative control.

A test result not meeting the criteria for either the positive or the negative response was

considered to be equivocal.

STATISTICS AND CALCULATIONS

MN-RET and percent RET were tested for equality of variance using Bartlett's test (alpha

= 0.01; Winer, 1971). The MN-RET data was log transformed in the comparison of the

positive control to the negative control.

Based on the similarity of the response between sexes in the RFT, the MN-RET data and

the data on percent RET were analyzed by a one-way analysis of variance (only males

were used) (Winer, 1971). The alpha level at which all tests were conducted was 0.05.

The MN-NCE was not analyzed statistically and was only used as an adjunct end point to

evaluate the biological significance of the MN-RET results.

The final interpretation of biological significance of the responses was based on both

statistical outcome and scientific judgment.

RESULTS AND DISCUSSION

Dose Range-Finding Test (RFT)

Phase I

Targeted dose levels of 1000 or 2000 mg/kg bw/day DEPCT were used in the initial phase

of the RFT using male and female mice (one/sex/dose). The male mouse (animal number

14A3668) dosed with 1000 mg/kg bw/day displayed fecal perineal soiling and soft feces,

and had a body temperature decrease of 6.8 oC two hours after receiving the initial dose

(Summary data, Table 3; Individual data, Table 4 and Table 6). This mouse had
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spontaneously died prior to the five hour observation. The female mouse (animal number

14A3680) dosed with 1000 mg/kg bw/day displayed fecal perineal soiling, incoordinated

gait, soft feces, decreased activity, and was cold to the touch with a body temperature

decrease of 9.1 oC five hours after the initial dose at which point, the animal was declared

moribund and euthanized (Summary data, Table 3; Individual data, Tables 5 and 7).

The male mouse (animal number 14A3671) dosed at 2000 mg/kg bw/day displayed clear

perioral soiling, fecal perineal soiling, labored respiration with mouth breathing, no

reactivity, no activity, and was cold to the touch with a body temperature decrease of

14.3 oC two hours after receiving the initial dose (Summary data, Table 3; Individual data,

Table 4 and Table 6). This animal spontaneously died prior to the five hour observation.

The female mouse (animal number 14A3683) dosed with 2000 mg/kg bw/day displayed

clear perioral soiling, fecal perineal soiling, bluish skin and mucous membranes, soft feces,

no reactivity, no activity, and was cold to the touch with a body temperature decrease of

12.0oC two hours after receiving the initial dose (Summary data, Table 3; Individual data,

Tables 5 and 7). This animal spontaneously died prior to the five hour observation.

Examination of these mice by a pathologist revealed watery contents in the intestines and

hemolyzed blood in the stomach with no evidence of a dosing error.

Phase II

Based on the mortality observed at 1000 and 2000 mg/kg bw/day in male and female mice,

lower dose levels of 250 and 500 mg/kg bw/day were given to additional mice

(one/sex/dose). In the male mouse (animal number 14A3674) and female mouse

(14A3686) dosed with 250 mg/kg bw/day no remarkable observations or changes in body

temperature were observed (Summary data, Table 3; Individual data, Table 4, Table 5,

Table 6 and Table 7).

The male mouse (animal number 14A3677) dosed with 500 mg/kg bw/day displayed fecal

perineal soiling and watery feces on day 1 of dosing and by day 2 after receiving a second

dose, this mouse continued with these observations and had continuous general muscle

tremors and a body temperature decrease of 6.5 oC at the five hour observation time

(Summary data, Table 3; Individual data, Tables 4 and 6). This mouse had mostly

recovered by the third day of observation with perineal fecal soiling and soft feces

remaining and survived until the end of the study. The female mouse (animal number

14A3689) dosed with 500 mg/kg bw/day displayed watery feces and fecal perineal soiling

after receiving the initial dose. On the second day of dosing, this mouse displayed urine
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and fecal perineal soiling, general muscle tremors associated with movement, decreased

feces, soft feces, decreased activity, and was cold to the touch with a body temperature

decrease of 9.7 oC five hours after the second dose (Summary data, Table 3; Individual

data, Tables 5 and 7). The animal was declared moribund, was euthanized and examined

by a pathologist and determined that no gavage error had occurred.

Based on these results, two additional mice/sex were dosed at 250 mg/kg bw/day, one

additional mouse/sex/dose at 375 mg/kg bw/day, and two additional male mice at

500 mg/kg bw/day. The two male mice dose at 250 mg/kg bw/day (animal numbers

14A3675 and 14A3676) displayed no clinical observations, no significant changes in body

weight, or body temperature during the in-life portion of the RFT (Summary data, Table 3;

Individual data, Tables 4 and 6). On the initial day of dosing, one female mouse dosed at

250 mg/kg bw/day (animal number 14A36887), displayed perineal fecal soiling and watery

feces. By the second day of dosing this mouse largely recovered with no further

observations including significant changes in body temperature or body weight (Summary

data, Table 3; Individual data, Tables 5 and 7). The remaining female mouse dosed at 250

mg/kg bw/day (animal number 14A3688) displayed no remarkable observations including

changes in body weight or body temperature during the in-life phase of the RFT (Summary

data, Table 3; Individual data, Table 1, Table 5 and Table 7).

The male mouse dosed at 375 mg/kg bw/day (animal number 14A3669), displayed perineal

fecal soiling and watery feces on the initial day of dosing. On the second day, these

observations largely recovered; however, this mouse displayed soft feces and by the third

day of observation the only observation observed was decreased feces (Summary data,

Table 3; Individual data, Tables 4 and 6). This mouse did not have any significant changes

in body temperature or body weight during the in-life. The female mouse dosed at 375

mg/kg bw/day (animal number 14A3681) displayed perineal fecal soiling and watery feces

after the initial day of dosing. By the second day of dosing, these observations remained

and this animal also displayed muscle tremors associated with stimuli, decreased activity,

and was cold to touch with a body temperature decrease 9.1 oC five hours after the second

dose (Summary data, Table 3; Individual data, Tables 5 and 7). By the third day these

observations including body temperature decreases largely recovered; however, the animal

had decreased feces for the remaining day of observation (Summary data, Table 3;

Individual data, Table 5 and Table 7).
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A male mouse (animal number 14A3678) dosed with 500 mg/kg bw/day displayed fecal

perineal soiling and watery feces after the initial dose. On the second day of dosing

observations including muscle tremors associated with stimuli, decreased activity, partially

closed eyelid (bilateral), and a body temperature decrease of 9.9 oC five hours after dosing

were observed (Summary data, Table 3; Individual data, Tables 4 and 6). This mouse was

declared moribund and evaluated by a pathologist who determined that no gavage error had

occurred. The remaining male mouse dosed at 500 mg/kg bw/day (animal 14A3679)

displayed perineal fecal soiling and watery feces on the initial day of dosing (Summary

data, Table 3; Individual data, Tables 4 and 6). By the second day of dosing, these

observations remained and the animal displayed muscle tremors associated with stimuli,

decreased activity, partially closed eyelids (bilateral), and was cold to touch with a body

temperature decrease of 9.4 oC five hours after dosing (Summary data, Table 3; Individual

data, Tables 4 and 6). This mouse was declared moribund and evaluated by a pathologist,

where small ulcers in the glandular mucosa and hemolyzed blood in the lumen of the

stomach with no evidence of a gavage error.

Based on these results, two additional mice/sex were dosed at 375 mg/kg bw/day. The first

male mouse (animal number 14A3670) displayed perineal soiling and watery and soft feces

on day 1, which were mostly resolved by day 2 (Summary data, Table 3; Individual data,

Tables 4 and 6). On day 3 of dosing, decreased feces were observed which had resolved by

day 4. The second male mouse (animal number 14A3672) dosed with

375 mg/kg bw/day displayed soft feces on day one, and after receiving a second dose slight

muscle tremors were observed, both of these observations had resolved by day 3. On day 3

decreased feces were observed, which were resolved by day 4. A female mouse (animal

number 14A3682) dosed with 375 mg/kg bw/day, displayed fecal perineal soiling and

watery feces on day 1, by day 2 muscle tremors associated with movement were noted

along with a body temperature decrease of 9.3 oC five hours after receiving the second dose

(Summary data, Table 3; Individual data, Tables 5 and 7). This mouse was evaluated by a

pathologist, who determined that no gavage error had occurred. Another female mouse

(animal number 14A3684) dosed with 375 mg/kg bw/day, displayed decreased feces, soft

feces, fecal perineal soiling and watery feces on day 1, by day 2 muscle tremors associated

with movement were noted along with a body temperature decrease of 10.1 oC five hours

after receiving a second dose (Summary data, Table 3; Individual data, Tables 5 and 7). By

day 3 the mouse had mostly recovered, with only a clinical observation of absent feces.
THE DOW CHEMICAL COMPANY

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Micronucleus Assay (MNT)

Based upon the results of the range-finding study where no appreciable differences were

noted between sexes, only males were evaluated in the main study. The highest

concentration tested was a dose of 300 mg/kg bw/day. The middle- and low-doses were

150 mg/kg bw/day and 75 mg/kg bw/day, respectively. All dosing solutions were

confirmed to be homogenous in the vehicle (Appendix B). The analytically determined

concentrations of the test material in the dosing solutions used for the micronucleus test

ranged from 92.6% to 93.0% of the targeted values (Appendix B).

The treatments did not have a remarkable effect on the body weight or body temperature

of the animals (Tables 8 and 11). All animals dosed at 0 (negative control) or 75 mg/kg

bw/day had no indications of toxicity upon daily observations during the in-life portion of

the MNT (Summary data, Table 9; Individual data, Tables 10 and 11) with one exception

in the control group. That animal (14A4628) displayed urine perineal soiling and

decreased feces on day 3, but both were resolved by day 4 (Summary data, Table 9;

Individual data, Tables 10 and 11).

Five out of the six mice (14A4636-4640) dosed with 150 mg/kg bw/day had no

remarkable observations during the study (Summary data, Table 9; Individual data,

Tables 10 and 11). One mouse (animal number 14A4641) dosed with 150 mg/kg bw/day

had soft feces on the first day of dosing, but had recovered by day 2 (Summary data,

Table 9; Individual data, Tables 10 and 11).

Two out of the 8 mice (14A4646 and 14A4649) dosed with 300 mg/kg bw/day, had no

remarkable observations during the study (Summary data, Table 9; Individual data,

Tables 10 and 11). Four out of the eight mice (14A4642, 4643, 4647, and 4648) dosed

with 300 mg/kg bw/day displayed soft feces on first day of dosing, but had largely

recovered by day 2, with no further clinical observations or significant changes in body

temperature (Summary data, Table 9; Individual data, Tables 10 and 11). One mouse

(animal number 14A4644) dosed with 300 mg/kg bw/day displayed urine perineal soiling

and soft feces on day 1, but had recovered by day 2, on day 3 decreased feces were

observed which persisted through the end of the study (Summary data, Table 9;

Individual data, Tables 10 and 11). One mouse (animal number 14A4645) displayed

fecal perineal soiling and soft feces on day 1 but had fully recovered by day 2, with no

further clinical signs (Summary data, Table 9; Individual data, Tables 10 and 11).
THE DOW CHEMICAL COMPANY

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A summary of the data on the frequencies of MN-RET and percent RET observed in

various treatment groups of male mice is presented in Table 12. The individual animal

data presented in Table 13 and the laboratory historical control data are shown in Table

14. There were no significant differences in MN-RET frequencies between the groups

treated with the test material and the negative controls. The adequacy of the

experimental conditions for the detection of induced micronuclei was ascertained from

the observation of a significant increase in the frequencies of micronucleated RET in the

positive control group (Table 12).

The percent RET values observed in the test material-treated animals were not

significantly different from the negative control values (Table 12). The percent RET

values of the positive control animals were found to be significantly lower than those of

the negative control animals.

CONCLUSIONS

Treatment-related toxicity including, decreases feces, soft feces and perineal soiling were

observed in male mice administered two consecutive daily doses of DEPCT up to the

300 mg/kg bw/day indicating systemic availability of the test material. Administration of

higher doses of DEPCT (≥500 mg/kg bw/day) resulted in lethality and severe systemic 

toxicity in the treated mice, hence 300 mg/kg bw/day represented the maximum tolerated

dose (MTD). Data from the RFT clearly indicated systemic availability of the test

material when administered by oral gavage. Based upon the results of the study reported

herein, it was concluded that the phosphorochloridothioic acid, O,O-diethyl ester, did not

induce a significant increase in the frequency of micronucleated reticulocytes in the

peripheral blood when given as a single oral dose on two consecutive days to male

Crl:CD1(ICR) mice. Hence, Phosphorochloridothioic acid, O,O-diethyl ester is

considered negative in this test system under the experimental conditions used.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the contributions of the following individuals to the

conduct of the study and report preparation:

L. K. Sosinski Cellular and Molecular Technologist
THE DOW CHEMICAL COMPANY

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REFERENCES

Asanami, S. and Shimono, K. (1997). High body temperature induces micronuclei in

mouse bone marrow. Mutat. Res. 390, 79-83.

Asanami, S., Shimono, K., and Kaneda, S. (1998). Transient hypothermia induces

micronuclei in mice. Mutat. Res. 413, 7-14.

De Boeck, M., Van der Leede, B., Van Goethem, F., De Smedt, A., Steemans, M.,

Lampo, A., and Vanparys, P. (2005). Flow cytometric analysis of micronucleated

reticulocytes: Time- and dose-dependent response of know mutagens in mice using

multiple blood sampling. Environmental and Molecular Mutagenesis 46, 30-42.

Gollapudi, B. B., Bruce, R. J., Linscombe, V. A., Watanabe, P. G., and Sinha, A. K.

(1986). Effect of food and water deprivation on the parameters of the mouse bone

marrow micronucleus test. Mutat. Res. 173, 121-125.

Mavournin, K. H., Blakey, D. H., Cimino, M. C., Salamone, M. F., and Heddle, J. A.

(1990). The in vivo micronucleus assay in mammalian bone marrow and peripheral

blood. A report of the U. S. Environmental Protection Agency Gene-Tox Program.

Mutat. Res. 239, 29-80.

Megregian, J. N. (2014). Characterization of Phosphorochloridothioic acid, O,O-diethyl

ester (DEPCT), Lot C032013081501, For Use as a Test Substance. ML AL-2013-

017027. The Dow Chemical Company, Midland, Michigan.

Schmid, W. (1976). The micronucleus test for cytogenetic analysis. In: Hollaender (ed),

"Chemical Mutagens: Principles and Methods for Their Detection." Vol. 4, Plenum,

New York, pp 31-53.

Winer, B. J. (1971). Statistical Principles in Experimental Design (2nd Edition).

McGraw-Hill, New York.
THE DOW CHEMICAL COMPANY

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 1. Body Weights (g) – Males (Range Finding Test)

DAYS ON TEST

DOSE ANIMAL ------------------------

MKD NUMBER 1 4 GAIN

===========================================

250 3674 30.3 30.9 0.6

3675 31.3 31.5 0.2

3676 34.5 34.1 -0.4

MEAN 32.0 32.2 0.1

S.D. 2.2 1.7 0.5

N= 3 3 3

===========================================

375 3669 36.5 34.7 -1.8

3670 33.5 32.0 -1.5

3672 33.6 31.5 -2.1

MEAN 34.5 32.7 -1.8

S.D. 1.7 1.7 0.3

N= 3 3 3

===========================================

500 3677 32.0 28.5 -3.5

3678 34.4 *** ***

3679 32.7 *** ***

MEAN 33.0 28.5 -3.5

S.D. 1.2 . .

N= 3 1 1

===========================================

1000 3668 32.8 *** ***

MEAN 32.8 === ===

S.D. . === ===

N= 1 0 0

===========================================

*** DEAD ANIMAL

=== NO DATA AVAILABLE FOR MEAN AND S.D.
THE DOW CHEMICAL COMPANY

STUDY ID:

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 1. Body Weights (g) – Males (Range Finding Test) (continued)

DAYS ON TEST

DOSE ANIMAL ------------------------

MKD NUMBER 1 4 GAIN

===========================================

2000 3671 29.1 *** ***

MEAN 29.1 === ===

S.D. . === ===

N= 1 0 0

===========================================

*** DEAD ANIMAL

=== NO DATA AVAILABLE FOR MEAN AND S.D.
THE DOW CHEMICAL COMPANY

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 2. Body Weights (g) – Females (Range Finding Test)

DAYS ON TEST

DOSE ANIMAL ------------------------

MKD NUMBER 1 4 GAIN

===========================================

250 3686 25.4 25.2 -0.2

3687 24.7 25.5 0.8

3688 30.2 29.7 -0.5

MEAN 26.8 26.8 0.0

S.D. 3.0 2.5 0.7

N= 3 3 3

===========================================

375 3681 29.5 27.5 -2.0

3682 26.1 *** ***

3684 29.7 27.0 -2.7

MEAN 28.4 27.3 -2.3

S.D. 2.0 0.4 0.5

N= 3 2 2

===========================================

500 3689 22.9 *** ***

MEAN 22.9 === ===

S.D. . === ===

N= 1 0 0

===========================================

1000 3680 24.3 *** ***

MEAN 24.3 === ===

S.D. . === ===

N= 1 0 0

===========================================

2000 3683 24.7 *** ***

MEAN 24.7 === ===

S.D. . === ===

N= 1 0 0

===========================================

*** DEAD ANIMAL

=== NO DATA AVAILABLE FOR MEAN AND S.D.
THE DOW CHEMICAL COMPANY

STUDY ID:

PAGE 26

EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 3. Summary In-Life Observations – Males/Females (Range Finding Test)

SEX: MALES FEMALES

DOSE (MKD): 1000 2000 250 500 375 1000 2000 250 500 375

NUMBER OF ANIMALS OBSERVED: 1 1 3 3 3 1 1 3 1 3

-------------------------- ----------------------------------------------------------------------

All Categories

Within Normal Limits 0 0 3 0 0 0 0 2 0 0

Behavior

Reactivity, Absent 0 1 0 0 0 0 1 0 0 0

Activity, Absent 0 1 0 0 0 0 1 0 0 0

Activity, Decreased 0 0 0 2 0 1 0 0 1 1

Eyes

Eyelid(s) Partially Closed 0 0 0 2 0 0 0 0 0 0

Feces

Abnormal Quantity, Absent 0 0 0 0 0 0 0 0 0 1

Abnormal Quantity, Decreased 0 0 0 0 3 0 0 0 1 2

Abnormal Consistency, Soft 1 1 0 1 3 1 1 0 1 1

Abnormal Consistency, Watery 0 0 0 3 2 0 0 1 1 3

Gait

Incoordinated 0 0 0 0 0 1 0 0 0 0

Miscellaneous

Misc, Cold to Touch 0 1 0 1 0 1 1 0 1 1

Muscle

Tremor, Continuous 0 0 0 1 0 0 0 0 0 0

Tremor, Slight 0 0 0 0 1 0 0 0 0 0

Tremor, Associated with Movement 0 0 0 0 0 0 0 0 1 2
THE DOW CHEMICAL COMPANY

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 3. Summary In-Life Observations – Males/Females (Range Finding Test) (continued)

SEX: MALES FEMALES

DOSE (MKD): 1000 2000 250 500 375 1000 2000 250 500 375

NUMBER OF ANIMALS OBSERVED: 1 1 3 3 3 1 1 3 1 3

-------------------------- ----------------------------------------------------------------------

Muscle

Tremor, Associated with Stimuli 0 0 0 2 0 0 0 0 0 1

Respiration

Labored, With Mouth-Breathing 0 1 0 0 0 0 0 0 0 0

Skin/Fur/Mucous Membranes

Skin/Mucous Membranes Bluish 0 0 0 0 0 0 1 0 0 0

Soiling

Perioral, Clear 0 1 0 0 0 0 1 0 0 0

Perineal, Fecal 1 1 0 3 2 1 1 1 1 3

Perineal, Urine and Fecal 0 0 0 0 0 0 0 0 1 0

Disposition

Scheduled Euthanasia 0 0 3 1 3 0 0 3 0 2

Moribund 0 0 0 2 0 1 0 0 1 0

Spontaneous Death 1 1 0 0 0 0 1 0 0 1
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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 4. Individual Animal Observations – Males (Range Finding Test)

Animal Day Observed

Dose Number First Last Observation/Comment

-----------------------------------------------------------------------------------------------------------------------

1000 mkd

3668 1 1 Soiling, Perineal, Fecal

1 1 Feces, Abnormal Consistency, Soft

1 1 Disposition, Spontaneous Death

-----------------------------------------------------------------------------------------------------------------------

2000 mkd

3671 1 1 Soiling, Perioral, Clear

1 1 Soiling, Perineal, Fecal

1 1 Respiration, Labored, With Mouth-Breathing

1 1 Feces, Abnormal Consistency, Soft

1 1 Behavior, Reactivity, Absent

1 1 Behavior, Activity, Absent

1 1 Misc, Cold to Touch

1 1 Disposition, Spontaneous Death

-----------------------------------------------------------------------------------------------------------------------

250 mkd

3674 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

3675 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

3676 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

-----------------------------------------------------------------------------------------------------------------------

500 mkd

3677 1 2 Feces, Abnormal Consistency, Watery

1 3 Soiling, Perineal, Fecal

2 2 Muscle, Tremor, Continuous, General

3 3 Feces, Abnormal Consistency, Soft

4 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

3678 1 1 Soiling, Perineal, Fecal

1 1 Feces, Abnormal Consistency, Watery
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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 4. Individual Animal Observations – Males (Range Finding Test) (continued)

Animal Day Observed

Dose Number First Last Observation/Comment

-----------------------------------------------------------------------------------------------------------------------

500 mkd

3678 2 2 Muscle, Tremor, Associated with Stimuli, General

2 2 Behavior, Activity, Decreased

2 2 Eye, Lid(s) Partially Closed, Bilateral

2 2 Disposition, Moribund

3679 1 2 Soiling, Perineal, Fecal

1 2 Feces, Abnormal Consistency, Watery

2 2 Muscle, Tremor, Associated with Stimuli, General

2 2 Behavior, Activity, Decreased

2 2 Eye, Lid(s) Partially Closed, Bilateral

2 2 Misc, Cold to Touch

2 2 Disposition, Moribund

-----------------------------------------------------------------------------------------------------------------------

375 mkd

3669 1 1 Soiling, Perineal, Fecal

1 1 Feces, Abnormal Consistency, Watery

2 2 Feces, Abnormal Consistency, Soft

3 4 Feces, Abnormal Quantity, Decreased

4 4 Disposition, Scheduled Euthanasia

3670 1 1 Soiling, Perineal, Fecal

1 1 Feces, Abnormal Consistency, Watery

1 2 Feces, Abnormal Consistency, Soft

3 3 Feces, Abnormal Quantity, Decreased

4 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

3672 1 2 Feces, Abnormal Consistency, Soft

2 2 Muscle, Tremor, Slight, General

3 3 Feces, Abnormal Quantity, Decreased

4 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia
THE DOW CHEMICAL COMPANY

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 5. Individual Animal Observations – Females (Range Finding Test)

Animal Day Observed

Dose Number First Last Observation/Comment

-----------------------------------------------------------------------------------------------------------------------

1000 mkd

3680 1 1 Soiling, Perineal, Fecal

1 1 Gait, Incoordinated

1 1 Feces, Abnormal Consistency, Soft

1 1 Behavior, Activity, Decreased

1 1 Misc, Cold to Touch

1 1 Disposition, Moribund

-----------------------------------------------------------------------------------------------------------------------

2000 mkd

3683 1 1 Soiling, Perioral, Clear

1 1 Soiling, Perineal, Fecal

1 1 Skin/Mucous Membranes Bluish

1 1 Feces, Abnormal Consistency, Soft

1 1 Behavior, Reactivity, Absent

1 1 Behavior, Activity, Absent

1 1 Misc, Cold to Touch

1 1 Disposition, Spontaneous Death

-----------------------------------------------------------------------------------------------------------------------

250 mkd

3686 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

3687 1 1 Soiling, Perineal, Fecal

1 1 Feces, Abnormal Consistency, Watery

2 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

3688 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia

-----------------------------------------------------------------------------------------------------------------------

500 mkd

3689 1 1 Feces, Abnormal Consistency, Watery

1 2 Soiling, Perineal, Fecal

2 2 Soiling, Perineal, Urine and Fecal

2 2 Muscle, Tremor, Associated with Movement, General
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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 5. Individual Animal Observations – Females (Range Finding Test) (continued)

Animal Day Observed

Dose Number First Last Observation/Comment

-----------------------------------------------------------------------------------------------------------------------

500 mkd

3689 2 2 Feces, Abnormal Quantity, Decreased

2 2 Feces, Abnormal Consistency, Soft

2 2 Behavior, Activity, Decreased

2 2 Misc, Cold to Touch

2 2 Disposition, Moribund

-----------------------------------------------------------------------------------------------------------------------

375 mkd

3681 1 2 Soiling, Perineal, Fecal

1 2 Feces, Abnormal Consistency, Watery

2 2 Muscle, Tremor, Associated with Stimuli, General

2 2 Behavior, Activity, Decreased

2 2 Misc, Cold to Touch

3 4 Feces, Abnormal Quantity, Decreased

4 4 Disposition, Scheduled Euthanasia

3682 1 2 Soiling, Perineal, Fecal

1 2 Feces, Abnormal Consistency, Watery

2 2 Muscle, Tremor, Associated with Movement, General

3 3 Disposition, Spontaneous Death

3684 1 1 Feces, Abnormal Quantity, Decreased

1 1 Feces, Abnormal Consistency, Soft

1 2 Soiling, Perineal, Fecal

1 2 Feces, Abnormal Consistency, Watery

2 2 Muscle, Tremor, Associated with Movement, General

3 3 Feces, Abnormal Quantity, Absent

4 4 No Remarkable Observations

4 4 Disposition, Scheduled Euthanasia
THE DOW CHEMICAL COMPANY

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 6. Animal Body Temperature Data – Males (Range Finding Test)

0 HR 2 HR 5 HR 24 HR 26 HR 29 HR 48 HR 72 HR

DOSE ANIMAL CENT CENT CENT CENT CENT CENT CENT CENT

MKD NUMBER

===================================================================================

250 3674 36.9 37.5 38.1 36.4 37.0 36.5 36.0 36.7

3675 36.2 36.7 37.1 36.6 36.1 35.9 37.6 37.0

3676 35.6 36.7 37.0 36.7 37.4 36.1 36.7 36.1

MEAN 36.2 37.0 37.4 36.6 36.8 36.2 36.8 36.6

S.D. 0.7 0.5 0.6 0.2 0.7 0.3 0.8 0.5

N= 3 3 3 3 3 3 3 3

===================================================================================

375 3669 36.8 37.1 35.5 37.8 37.2 35.8 37.0 37.7

3670 36.5 35.6 37.6 36.9 36.5 35.0 37.4 36.8

3672 36.7 37.0 36.4 36.7 33.4 29.4 37.2 36.3

MEAN 36.7 36.6 36.5 37.1 35.7 33.4 37.2 36.9

S.D. 0.2 0.8 1.1 0.6 2.0 3.5 0.2 0.7

N= 3 3 3 3 3 3 3 3

===================================================================================

500 3677 37.4 35.9 35.2 37.1 35.3 30.6 34.4 36.6

3678 36.6 36.3 35.4 38.5 34.5 28.6 --- ---

3679 37.3 34.9 34.8 37.3 33.4 27.9 --- ---

MEAN 37.1 35.7 35.1 37.6 34.4 29.0 34.4 36.6

S.D. 0.4 0.7 0.3 0.8 1.0 1.4 . .

N= 3 3 3 3 3 3 1 1

===================================================================================

1000 3668 36.2 29.4 --- --- --- --- --- ---

MEAN 36.2 29.4 === === === === === ===

S.D. . . === === === === === ===

N= 1 1 0 0 0 0 0 0

===================================================================================

--- NO DATA

=== NO DATA AVAILABLE FOR MEAN AND S.D.
THE DOW CHEMICAL COMPANY

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 6. Animal Body Temperature Data – Males (Range Finding Test) (continued)

0 HR 2 HR 5 HR 24 HR 26 HR 29 HR 48 HR 72 HR

DOSE ANIMAL CENT CENT CENT CENT CENT CENT CENT CENT

MKD NUMBER

===================================================================================

2000 3671 37.8 23.5 --- --- --- --- --- ---

MEAN 37.8 23.5 === === === === === ===

S.D. . . === === === === === ===

N= 1 1 0 0 0 0 0 0

===================================================================================

--- NO DATA

=== NO DATA AVAILABLE FOR MEAN AND S.D.
THE DOW CHEMICAL COMPANY

STUDY ID:

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 7. Animal Body Temperature Data – Females (Range Finding Test)

0 HR 2 HR 5 HR 24 HR 26 HR 29 HR 48 HR 72 HR

DOSE ANIMAL CENT CENT CENT CENT CENT CENT CENT CENT

MKD NUMBER

===================================================================================

250 3686 37.4 37.2 37.4 36.9 36.6 35.9 36.6 36.7

3687 36.8 35.4 36.3 36.7 36.4 35.6 36.9 36.6

3688 36.8 36.3 37.4 37.3 35.9 36.8 37.6 37.4

MEAN 37.0 36.3 37.0 37.0 36.3 36.1 37.0 36.9

S.D. 0.3 0.9 0.6 0.3 0.4 0.6 0.5 0.4

N= 3 3 3 3 3 3 3 3

===================================================================================

375 3681 36.9 34.2 34.3 37.4 34.1 28.3 36.9 37.7

3682 37.3 36.9 35.8 37.2 32.1 27.9 --- ---

3684 37.5 36.3 36.5 37.5 34.4 27.4 36.9 37.3

MEAN 37.2 35.8 35.5 37.4 33.5 27.9 36.9 37.5

S.D. 0.3 1.4 1.1 0.2 1.3 0.5 0.0 0.3

N= 3 3 3 3 3 3 2 2

===================================================================================

500 3689 37.0 34.0 31.7 34.7 27.3 25.0 --- ---

MEAN 37.0 34.0 31.7 34.7 27.3 25.0 === ===

S.D. . . . . . . === ===

N= 1 1 1 1 1 1 0 0

===================================================================================

1000 3680 36.5 32.7 27.4 --- --- --- --- ---

MEAN 36.5 32.7 27.4 === === === === ===

S.D. . . . === === === === ===

N= 1 1 1 0 0 0 0 0

===================================================================================

2000 3683 37.0 25.0 --- --- --- --- --- ---

MEAN 37.0 25.0 === === === === === ===

S.D. . . === === === === === ===

N= 1 1 0 0 0 0 0 0

===================================================================================

--- NO DATA

=== NO DATA AVAILABLE FOR MEAN AND S.D.
THE DOW CHEMICAL COMPANY

STUDY ID:

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 8. Body Weights (g) – Males (Micronucleus Test)

DAYS ON TEST

DOSE ANIMAL ------------------------

MKD NUMBER 1 2 4

===========================================

0 4624 35.4 --- 35.1

4625 34.1 --- 34.4

4626 34.6 --- 35.1

4627 31.9 --- 31.4

4628 33.0 --- 28.7

4629 35.1 --- 35.4

MEAN 34.0 === 33.4

S.D. 1.3 === 2.7

N= 6 0 6

===========================================

75 4630 34.7 --- 34.7

4631 34.4 --- 35.0

4632 31.6 --- 31.9

4633 33.5 --- 34.9

4634 33.5 --- 34.1

4635 35.2 --- 36.1

MEAN 33.8 === 34.5

S.D. 1.3 === 1.4

N= 6 0 6

===========================================

150 4636 34.1 --- 34.4

4637 35.3 --- 36.5

4638 34.6 --- 33.9

4639 34.3 --- 35.0

4640 31.6 --- 32.8

4641 33.1 --- 33.3

MEAN 33.8 === 34.3

S.D. 1.3 === 1.3

N= 6 0 6

===========================================

--- NO DATA

=== NO DATA AVAILABLE FOR MEAN AND S.D.
THE DOW CHEMICAL COMPANY

STUDY ID:

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EVALUATION OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL ESTER IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Table 8. Body Weights (g) – Males (Micronucleus Test) (continued)

DAYS ON TEST

DOSE ANIMAL ------------------------

MKD NUMBER 1 2 4

===========================================

300 4642 33.6 --- 36.3

4643 34.4 --- 35.6

4644 32.4 --- 32.2

4645 32.6 --- 32.5

4646 35.1 --- 34.7

4647 33.2 --- 34.6

4648 35.5 --- 35.1

4649 33.8 --- 34.5

MEAN 33.8 === 34.4

S.D. 1.1 === 1.4

N= 8 0 8

===========================================

CP 40 4650 --- 35.1 35.6

4651 --- 35.0 35.3

4652 --- 32.9 33.1

4653 --- 35.8 36.3

4654 --- 33.8 34.3

4655 --- 33.1 33.4

MEAN === 34.3 34.7

S.D. === 1.2 1.3

N= 0 6 6

===========================================

--- NO DATA

=== NO DATA AVAILABLE FOR MEAN AND S.D.
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Table 9. Summary In-Life Observations – Males (Micronucleus Test)

SEX: MALES

DOSE (MKD): 0 75 150 300 CYP

NUMBER OF ANIMALS OBSERVED: 6 6 6 8 6

-------------------------- -----------------------------------

All Categories

Within Normal Limits 5 6 5 2 6

Feces

Abnormal Quantity, Decreased 1 0 0 1 0

Abnormal Consistency, Soft 0 0 1 6 0

Soiling

Perineal, Urine 1 0 0 1 0

Perineal, Fecal 0 0 0 1 0

Disposition

Scheduled Necropsy 6 6 6 8 6
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Table 10. Individual Animal Observations – Males (Micronucleus Test)

Animal Day Observed

Dose Number First Last Observation/Comment

-----------------------------------------------------------------------------------------------------------------------

0 MKD

4624 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4625 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4626 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4627 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4628 1 2 No Remarkable Observations

3 3 Soiling, Perineal, Urine

3 4 Feces, Abnormal Quantity, Decreased

4 4 Disposition, Scheduled Necropsy

4629 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

-----------------------------------------------------------------------------------------------------------------------

75 MKD

4630 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4631 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4632 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4633 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4634 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy
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Table 10. Individual Animal Observations – Males (Micronucleus Test) (continued)

Animal Day Observed

Dose Number First Last Observation/Comment

-----------------------------------------------------------------------------------------------------------------------

75 MKD

4635 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

-----------------------------------------------------------------------------------------------------------------------

150 MKD

4636 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4637 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4638 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4639 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4640 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4641 1 1 Feces, Abnormal Consistency, Soft

2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

-----------------------------------------------------------------------------------------------------------------------

300 MKD

4642 1 2 Feces, Abnormal Consistency, Soft

3 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4643 1 2 Feces, Abnormal Consistency, Soft

3 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4644 1 1 Soiling, Perineal, Urine

1 1 Feces, Abnormal Consistency, Soft

2 2 No Remarkable Observations

Animal Day Observed
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Table 10. Individual Animal Observations – Males (Micronucleus Test) (continued)

Dose Number First Last Observation/Comment

-----------------------------------------------------------------------------------------------------------------------

300 MKD

4644 3 4 Feces, Abnormal Quantity, Decreased

4 4 Disposition, Scheduled Necropsy

4645 1 1 Soiling, Perineal, Fecal

1 1 Feces, Abnormal Consistency, Soft

2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4646 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4647 1 1 Feces, Abnormal Consistency, Soft

2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4648 1 1 Feces, Abnormal Consistency, Soft

2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4649 1 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

-----------------------------------------------------------------------------------------------------------------------

CYP 40

4650 2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4651 2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4652 2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4653 2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necropsy

4654 2 4 No Remarkable Observations

Animal Day Observed

Dose Number First Last Observation/Comment
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Table 10. Individual Animal Observations – Males (Micronucleus Test) (continued)

-----------------------------------------------------------------------------------------------------------------------

CYP 40

4654 4 4 Disposition, Scheduled Necropsy

4655 2 4 No Remarkable Observations

4 4 Disposition, Scheduled Necrops
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Table 11. Animal Body Temperature Data – Males (Micronucleus Test)

0 HR 2 HR 5 HR 24 HR 26 HR 29 HR 48 HR 72 HR

DOSE ANIMAL CENT CENT CENT CENT CENT CENT CENT CENT

MKD NUMBER

===================================================================================

0 4624 36.5 37.0 35.6 37.2 36.6 35.1 36.6 36.3

4625 36.1 36.3 36.1 36.4 36.7 35.9 36.2 36.4

4626 36.4 36.8 36.2 37.0 36.9 36.3 36.3 36.3

4627 37.3 37.0 36.4 37.2 36.8 36.3 36.5 37.2

4628 37.0 34.9 35.2 36.6 36.1 36.0 36.1 37.1

4629 37.4 36.9 36.6 37.1 37.2 36.1 37.2 36.7

MEAN 36.8 36.5 36.0 36.9 36.7 36.0 36.5 36.7

S.D. 0.5 0.8 0.5 0.3 0.4 0.4 0.4 0.4

N= 6 6 6 6 6 6 6 6

===================================================================================

75 4630 36.4 36.5 36.4 36.7 36.3 35.8 36.4 36.1

4631 36.4 37.5 36.6 37.8 37.1 35.8 37.0 36.7

4632 36.8 37.2 36.4 36.4 37.2 36.6 37.4 37.2

4633 37.7 37.6 36.8 36.8 37.8 36.9 37.6 38.0

4634 36.1 37.3 37.2 37.7 37.2 36.6 37.1 37.3

4635 36.0 36.9 36.3 36.1 37.4 35.9 36.4 36.6

MEAN 36.6 37.2 36.6 36.9 37.2 36.3 37.0 37.0

S.D. 0.6 0.4 0.3 0.7 0.5 0.5 0.5 0.7

N= 6 6 6 6 6 6 6 6

===================================================================================

150 4636 36.7 37.2 36.9 36.7 37.6 37.0 37.4 37.9

4637 37.4 37.9 37.2 37.7 38.3 36.5 37.4 37.2

4638 36.5 37.4 37.0 36.7 37.6 37.1 37.4 37.5

4639 36.3 36.9 36.6 36.9 37.1 36.9 36.9 37.1

4640 35.7 36.9 36.2 36.1 36.8 36.0 36.1 35.8

4641 35.5 36.8 37.0 36.0 37.0 36.0 36.5 36.6

MEAN 36.3 37.2 36.8 36.7 37.4 36.6 37.0 37.0

S.D. 0.7 0.4 0.4 0.6 0.5 0.5 0.6 0.7

N= 6 6 6 6 6 6 6 6

===================================================================================

--- NO DATA

=== NO DATA AVAILABLE FOR MEAN AND S.D.
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Table 11. Animal Body Temperature Data – Males (Micronucleus Test) (continued)

0 HR 2 HR 5 HR 24 HR 26 HR 29 HR 48 HR 72 HR

DOSE ANIMAL CENT CENT CENT CENT CENT CENT CENT CENT

MKD NUMBER

===================================================================================

300 4642 35.9 36.9 37.4 36.7 36.5 36.1 36.8 36.6

4643 35.6 37.7 37.3 36.1 36.8 37.1 37.5 36.8

4644 35.9 37.2 37.4 36.9 36.5 34.8 36.7 37.8

4645 35.3 36.7 37.3 35.4 37.2 36.7 35.6 36.2

4646 35.4 36.0 36.2 35.6 35.3 36.1 37.3 37.0

4647 35.6 35.8 36.4 35.8 35.9 35.9 36.6 36.2

4648 35.8 36.3 37.3 36.7 37.1 37.0 36.8 37.5

4649 35.3 35.9 36.8 37.7 37.6 36.4 37.2 37.9

MEAN 35.6 36.6 37.0 36.4 36.6 36.3 36.8 37.0

S.D. 0.3 0.7 0.5 0.8 0.7 0.7 0.6 0.7

N= 8 8 8 8 8 8 8 8

===================================================================================

40 CP 4650 --- --- --- 36.3 36.6 37.0 37.1 36.8

4651 --- --- --- 36.1 36.7 36.5 37.1 36.9

4652 --- --- --- 36.3 37.4 37.0 37.4 37.7

4653 --- --- --- 36.0 36.9 37.2 37.5 37.5

4654 --- --- --- 36.7 37.0 36.6 37.3 37.2

4655 --- --- --- 35.6 37.2 36.4 37.3 37.3

MEAN === === === 36.2 37.0 36.8 37.3 37.2

S.D. === === === 0.4 0.3 0.3 0.2 0.3

N= 0 0 0 6 6 6 6 6

===================================================================================

=== NO DATA AVAILABLE FOR MEAN AND S.D.
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Table 12. Summary Micronucleated Reticulocytes (MN-RET) Frequencies and Percent Reticulocytes (%

RET) – Males

TEST MATERIAL: Phosphorochloridothioic Acid, O,O-Diethyl Ester (DEPCT)

Peripheral Blood-Males

Exposure (mg/kg bw/day) Na ‰ MN-RET % RET

0

b

6 1.87 ± 0.47 1.71 ± 0.23

75 6 1.97 ± 0.91 1.65 ± 0.28

150 6 2.00 ± 0.70 1.34 ± 0.31

300 6 1.30 ± 1.03 1.74 ± 0.40

Positive Control (40 mg/kg CPc) 6 9.40 ± 1.96d0.40 ± 0.13d

a N is the number of animals per dose group at the time of scheduled sacrifice. 5000 RET were

examined/animal for MN incidence, and expressed as MN/1000 RET (%MN-RET).

b Mice were dosed with the vehicle (corn oil).

c CP = Cyclophosphamide monohydrate (positive control).

d The values are significantly different from the negative control (alpha=0.05).
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ASSAY

Table 13. Individual Micronucleated Reticulocytes (MN-RET) and Percent Reticulocytes (%RET) – Males (Micronucleus Test)
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Table 14. Recent Historical Control Data

%RET

Year Sex N a Mean Max Min Mean Max Min

2009b Male 6 1.17 2 39 0.60 2.08 2.69 1.43

2009cFemale 12 1.48 3 19 0.60 1.76 2.43 1 16

2009c Male 18 1.19 2 39 0.20 1.67 2.52 1.02

2009d Male 6 1.30 1.80 0.80 2.09 3.04 1 30

2009f Male 6 1.83 2 99 0.80 1.97 2.20 1.71

2010b Male 30 1.35 2 59 0.60 1.86 2.64 1 25

2010d Male 12 1.58 2 20 0.60 1.95 3.91 1 31

2011b Male 12 1.50 3 19 0.60 1.92 2.48 1.42

2011c Male 24 1.68 3 98 0.60 1.61 2.20 1.02

2011e Male 12 1.58 2.61 0.80 1.83 2.79 1 20

2012b Male 6 1.55 2 52 0.80 2.02 2.58 1.46

2012d Male 12 1.58 3 20 0.80 1.94 3.37 1.02

2013b Male 6 1.77 2.40 0.80 1.94 2.10 1.83

2013d Male 6 1.73 2.40 1.00 1.87 2.93 1 16

2014d Male 12 2.22 4.80 0.80 2.07 3.54 1 14

%RET

Year Sex N a Mean Max Min Mean Max Min

2009gFemale 12 6.44 8 13 4.78 0.73 1.10 0.40

2009g Male 34 10 19 27.99 4.18 0.62 3.75 0 15

2010g Male 47 10 32 20.38 2.39 0.57 1.10 0.05

2011g Male 48 9.28 20.75 4.38 0.50 1.22 0 12

2012g Male 18 11.01 15.60 7.20 0.47 0.87 0 20

2013g Male 12 11.88 25.80 6.80 0.58 1.21 0 17

2014g Male 11 7.96 12.80 3.60 0.72 1.00 0 32

a

Number of animals

b

0.5 % aqueous METHOCELTM; oral gavage

c Water; oral gavage

d

Corn oil; oral gavage

e

Propylene Glycol; oral gavage

f

PEG-400; oral gavage

g

Positive control = 40 mg/kg cyclophosphamide; single oral gavage

Negative Control

MN-RET/1000 RET

MN-RET/1000 RET

Positive Control
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PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Appendix A. Dose Selection Criteria

1. If acute oral toxicity data is available for the rat or mouse;

a. The existing data indicates no toxicity and the calculated oral LD50 is > 2000

mg/kg

- The initial dose levels evaluated in the RFT will be 2000 and 1000 mg/kg

bw/day

b. The existing data indicates the calculated oral LD50 is < 2000 mg/kg

- The initial dose levels evaluated in the RFT will be LD50 and 1⁄2 LD50

2. If acute oral toxicity data is not available.

a. Dose 1 animal/sex at 2000 and 1000 mg/kg and observe animals for 24 hrs after

the second dosing

- If all animals survive, dose an additional 2 animals/sex at dose levels of 2000

and 1000 mg/kg.

- If the animal dosed with 2000 mg/kg dies and clinical signs are present in the

animal receiving 1000 mg/kg, dose an additional 2 animals/sex at 1000 and 3

animals/sex at 500 mg/kg.

- If animal dosed with 2000 mg/kg dies and NO clinical signs are present at

1000 mg/kg, discuss with study director/lead scientist regarding the addition

of a dose level between 2000 and 1000 mg/kg.
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PERIPHERAL BLOOD MICRONUCLEUS ASSAY

Appendix B. Analytical Report
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Summary

Three dose solutions containing Phosphorochloridothioic acid, O,O-diethyl ester in corn oil

were prepared by the TERC pharmacy for use in a Genetics study. Analysis of the dose

solutions and a corn oil vehicle control was performed using gas chromatography with

electron capture detection (GC/ECD). The levels of Phosphorochloridothioic acid, O,Odiethyl ester were quantified by using external standard calibration.

Results are presented in Table 1 on page 6.

Chemical Information

Test Material/Reference Substance

Test Material Name: Phosphorochloridothioic acid, O,O-diethyl ester

Chemical Name: O,O-Diethyl ester phosphorochloridothioic acid

Synonyms: Diethylchlorothiophosphate, DEPCT

Lot #: C032013081501

CAS Number: 2524-04-1

Purity: 99.3% (Megregian, 2014)

Detailed description of sampling process

The dose solutions were sampled using a 1-mL Eppendorf pipet with a wide-orifice tip while

solutions were still stirring. Aliquots of approximately 1 mL were transferred to tared 40-mL

glass vials, and the weights of the aliquots were recorded. The high and low dose solutions

were sampled from the top, middle, and bottom of the dose containers to determine

homogeneity. Duplicate aliquots were taken from the control and the middle dose solution from

random locations. Using a bottletop dispenser, 24 mL toluene was added to each vial. Vials

were capped and vortex-mixed. The samples were then further diluted in toluene using a

Hamilton Microlab 600 Diluter to concentrations of approximately 0.04 μg/mL. The final

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dilutions of each sample were vortex-mixed and 1-mL aliquots were transferred to glass

autosampler vials for analysis.

Standard preparation

A stock solution was prepared at approximately 120,000 μg Phosphorochloridothioic

acid, O,O-diethyl ester per mL toluene. The stock was diluted with toluene to prepare

standards at concentrations ranging between 0.0119 and 0.119 μg/mL.

Spike preparation

Triplicate corn oil spike samples fortified with Phosphorochloridothioic acid, O,Odiethyl ester were prepared at concentrations of approximately 7.50, 15.0, and 30.0 mg

Phosphorochloridothioic acid, O,O-diethyl ester per mL corn oil. The spikes were

diluted with toluene to the same approximate final concentrations as the dose solution

samples.

Representative Analysis Conditions

Gas Chromatograph: Agilent 6890N Series, Model G1530N

Autosampler: Agilent 7683 Series, Model G2614A

Injector: Agilent 7683B Series, Model G2913A

Inlet: Injection Port Liner: 4-mm ID single gooseneck w/ glass wool

Injection Volume: 2.0 μL

Mode: Splitless

Initial temperature: 80 °C

Pressure: 10.6 psi

Purge Flow: 10.0 mL/min

Purge Time: 0.20 min

Total flow: 13.4 mL/min

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Gas type: Nitrogen

Capillary Column: ZB-5MS w/ Guardian; 30.0 m x 0.25 mm x 0.25 μm

Mode: Constant flow

Initial flow: 0.8 mL/min

Oven: Initial temp: 80 °C – hold 4.00 min;

Ramp @ 3 °C/min to 110 °C;

Ramp @ 25 °C/min to 200 °C

Run time: 17.60 min

Detector: Electron Capture Detector (μECD)

Temperature: 250 °C

Mode: Constant makeup flow

Makeup flow: 45.0 mL/min

Makeup gas: Nitrogen

Data System: Agilent OpenLAB C.01.05

Translator: Agilent LC/SQ ChemStation to MassHunter Translator B.04.00

Processor: Agilent MassHunter Quantitative Analysis B.06.00

Results

The results obtained from the analysis of the dose solution samples are presented in Table 1

on page 6.

The standard calibration set was found to be linear with an R2value of 0.9841. A typical

standard calibration curve is presented in Figure 1 on page 9. The supporting standard data

used to generate the curve are presented in Table 2 on page 7.

Average spike sample recovery was found to be 96.3% with a standard deviation of

7.66%. Representative spike sample recovery is shown in Table 3 on page 8.

Typical GC/ECD chromatograms from the analysis of a standard, sample, and control are

shown in Figures 2, 3, and 4, respectively, on pages 10, 11, and 12.

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Reference

Megregian, J. N. (2014). Characterization of Phosphorochloridothioic acid, O,O-diethyl

ester (DEPCT), Lot C032013081501, For Use as a Test Substance. ML AL-2013-

017027. The Dow Chemical Company, Midland, Michigan.

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Table 1. Results of Phosphorochloridothioic acid, O,O-diethyl ester in corn oil concentration analysis

Dose Level Date Mixed: 09/23/14

mg/mL ACL Report #:

Target Conc. (mg/mL): 0.00

Control Mean Measured Conc: <LLQ

% of Target Conc:

(n): (2)

Target Conc. (mg/mL): 7.50

7.50 Mean Measured Conc: 6.80

% of Target Conc: 90.7%

Homogeneity % R.S.D. (n): 1.8% (3)

Target Conc. (mg/mL): 15.0

15.00 Mean Measured Conc: 13.7

% of Target Conc: 91.5%

(n): (2)

Target Conc. (mg/mL): 30.0

30.00 Mean Measured Conc: 27.7

% of Target Conc: 92.4%

Homogeneity % R.S.D. (n): 4.8% (3)

LLQ . (mg/mL): 0.0301

<LLQ = less than lower limit of quantitation

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Table 2. Representative standards/refit table of Phosphorochloridothioic acid, O,O-diethyl ester in toluene

Analyte Analyte Calculated

Concentration Peak Area Concentration Accuracy

Sample Name Sample ID (μg/mL) (counts) (μg/mL) (%)

Standard_A 2014_0923003.D 0.0119 280432 0.0133 111

Standard_B 2014_0923004.D 0.0238 475276 0.0235 98.6

Standard_C 2014_0923005.D 0.0298 528022 0.0262 88.0

Standard_D 2014_0923006.D 0.0397 895284 0.0455 114

Standard_E 2014_0923007.D 0.0477 889294 0.0451 94.6

Standard_F 2014_0923008.D 0.0596 1131270 0.0578 97.0

Standard_G 2014_0923009.D 0.0795 1376967 0.0707 88.9

Standard H 2014 0923010.D 0.119 2284670 0.118 99.3

Standard_A 2014_0923023.D 0.0119 300126 0.0143 120

Standard_B 2014_0923024.D 0.0238 486241 0.0240 101

Standard_C 2014_0923025.D 0.0298 567215 0.0283 94.9

Standard_D 2014_0923026.D 0.0397 949299 0.0483 122

Standard_E 2014_0923027.D 0.0477 943394 0.0480 101

Standard_F 2014_0923028.D 0.0596 1146494 0.0586 98.3

Standard_G 2014_0923029.D 0.0795 1432930 0.0736 92.6

Standard_H 2014_0923030.D 0.119 2461553 0.127 107

Standard_A 2014_0923046.D 0.0119 299297 0.0142 120

Standard_B 2014_0923047.D 0.0238 494961 0.0245 103

Standard_C 2014_0923048.D 0.0298 593626 0.0297 99.5

Standard_D 2014_0923049.D 0.0397 757047 0.0382 96.3

Standard_E 2014_0923050.D 0.0477 959940 0.0488 102

Standard_F 2014_0923051.D 0.0596 1089951 0.0556 93.4

Standard_G 2014_0923052.D 0.0795 1451694 0.0746 93.8

Standard H 2014 0923053.D 0.119 2405150 0.125 105

All calculations performed using Agilent MassHunter Quantitative Analysis B.06.00 (Full Precision) and reported to three significant figures

Calculated Concentration = ((Peak Area - b) / m)

Regression equation is y = mx + b, (See Figure 1 for actual values of m and b)

Accuracy (%) = Calculated Concentration / Analyte Concentration x 100

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Table 3. Representative recovery of Phosphorochloridothioic acid, O,O-diethyl ester from corn oil matrix

Target

Concentration Accuracy

Sample Name Sample ID (mg/mL) (%)

Low Spike_1 2014_0923018.D 7.50 89.7

Low Spike_2 2014_0923019.D 7.50 89.0

Low Spike_3 2014_0923020.D 7.50 94.1

Mid Spike_1 2014_0923035.D 15.00 91.7

Mid Spike_2 2014_0923036.D 15.00 92.2

Mid Spike_3 2014_0923037.D 15.00 96.7

High Spike_1 2014_0923041.D 30.00 97.3

High Spike_2 2014_0923042.D 30.00 102

High Spike_3 2014_0923043.D 30.00 114

96.3

7.66

7.96

Mean

Standard Deviation (n=9)

Relative Standard Deviation (n=9)

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Figure 1. Typical calibration curve of Phosphorochloridothioic acid, O,O-diethyl ester in toluene

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Figure 2. Example GC/ECD chromatogram of a standard solution of 0.0298 μg

Phosphorochloridothioic acid, O,O-diethyl ester per mL toluene

Phosphorochloridothioic

acid, O,O-diethyl ester

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Figure 3. Example GC/ECD chromatogram of the 30.00 mg Phosphorochloridothioic

acid, O,O-diethyl ester per mL corn oil dose solution diluted to a target concentration of

approximately 0.04 μg Phosphorochloridothioic acid, O,O-diethyl ester per mL toluene

Phosphorochloridothioic

acid, O,O-diethyl ester

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Figure 4. Example GC/ECD chromatogram of the corn oil vehicle control diluted

approximately 2500-fold in toluene

No peak present at

Phosphorochloridothioic acid,

O,O-diethyl ester retention time

of approximately 12.2 minutes

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