### Page 1 of 73 FINAL REPORT

Study Title

Bacterial Reverse Mutation Assay with Phosphorochloridothioic acid, O,O- diethyl ester

Test Substance

Phosphorochloridothioic acid, O,O- diethyl ester

Test Guidelines

OECD Guidelines for the Testing of Chemicals Section 4: Health Ef fects, No. 471 (1997)

Authors

Valentine O. Wagner, III, M.S.

Gayathri Jayakumar, MPS

.

Study Completion Date

07 January 2016

Testing Facility

BioReliance

9630 Medical Center Drive

Rockville, MD 20850

BioReliance Study Number

Dow Number

D AS Number

DAS PCTR Number

Sponsor

The Dow Chemical Company

Midland, MI 48674BioReliance Study No.

Dow Number: 3 

### QUALITY ASSURANCE STATEMENTBioReliance Study No.

Dow Number: 4 

### Ba cterial Reverse Mutation Assay with Phosphorochloridothioic acid, O,O- diethyl ester

STUDY INFORMATION

Sponsor: The Dow Chemical Company

Midland, MI 48674

Sponsor Study Monitor : Kristy Swiecicki, B.S.

Testing Facility: BioReliance

9630 Medical Center Driv e

Rockville, MD 20850

Test Substance I.D.: Phosphorochloridothioic acid, O,O

- diethyl ester

Test Substance Lot No.: C032013081501

Test Substance Purity: 99.3% (as provided on the Certificate of

Analysis )

Dow Number :

D AS Number:

BioRelian ce Study No.:

Test Substance Description: Pale whitish yellow clear liquid

Storage Conditions: Room temperature, protected from light, with

desiccant and blanketed by inert gas (Nitrogen)

Test Substance Receipt / Login Date : 29 April 201 5

Study Initiation Date : 09 November 2015

Experimental Starting Date (first day

of data collection): 12 November 2015

Experimental Start Date (first day test

substance administered to test

system) : 13 November 2015

Experimental Completion Date: 30 Nove mber 2015

Laboratory Supervisor: Jessica Heavin, M.S.

Testing Facility Management: Rohan Kulkarni, MSc, PhD

Director, Study Management Genetic Toxicology

Contributing Scientist (Analytical

Chemistry Analysis): Philip Atkins, MChemBioReliance Study No.

Dow Number: 5 

### Analytical Laboratory ( Analytical

Chemistry Analysis): BioReliance Corporation

9610 Medical Center Drive

Rockville, MD 20850BioReliance Study No.

Dow Number: 6 

### TABLE OF CONTENTS

Page

STATEMENT OF COMPLIANCE ...........................................................................................2

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

STUDY INFORMATION .........................................................................................................4

TABLE OF CONTENTS ...........................................................................................................6

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

PURPOSE ..................................................................................................................................8

CHARACTERIZATION OF TEST AND CONTROL SUBSTANC ES ..................................8

MATERIALS AND METHODS .............................................................................................10

RESULTS AND DISCUSSION ..............................................................................................14

CONCLUSION ........................................................................................................................16

REFERENCES ........................................................................................................................16

DATA TABLES ......................................................................................................................17

APPENDIX I: Historical Control Data ....................................................................................30

APPENDIX II: Study Protocol and Amendments ...................................................................32

APPENDIX III: Certificate of Analysis ..................................................................................53

APPENDIX IV: Do sing Formulation Analysis .......................................................................55

APPENDIX V: S9 Quality Control Certificate .......................................................................66

APPENDIX VI: Measures to address US EPA's comments on the study protocol .................68BioReliance Study No.

Dow Number: 7 

### SUMMARY

The test substance , Ph osphorochloridothioic acid, O,O

- diethyl ester , was tested in the b acterial

r everse m utation a ssay using Salmonella typhimurium t ester strains TA98, TA100, TA1535

, TA1537 and Escherichia coli tester strain WP2 uvr A in the presence or absence of

Aroclor

- induced rat liver S9. The assay was performed in two phases using the preincubation

method. The first phase, the initial toxicity

- mutation assay, was used to establish the dose

- range

for the confirmatory mutagenicity assay and to provide a preli minary mutagenicity evaluation.

The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the

mutagenic potent ial of the test substance

.

In the initial toxicity

- mutation assay, the maximum concentrati on tested was 5000 μg per plate,

which is the maximum concentration recommended by test guidelines. This concentration was

achieved using a concentration of 100 mg /mL and a plating aliquot of 50.0 μL (0.375 mL/tube

to minimize headspace )

. The concentrations tested were 0.150, 0.500, 1.5 0 , 5. 0 0, 15 .0 , 50 .0

, 150, 500, 1500 and 5000 μg per plate. N o positive mut agenic responses were observed

. No

precipitate was obse rved. Toxicity was observed beginning at 150 or 500 μg per plate. Based

on the findings of the initial toxicity

- mutation assay, the maximum dose plated in the

confirmatory mutagenicity assay was 600 μg per plate (with tester strains TA98, TA100 and

TA153 5 in the absence of S9 activation and TA100, TA1537 and WP2 uvr A in the presence of

S9 activation) and 2000 μg per plate (with tester strains TA1537 and WP2 uvr A in the absence

of S9 activation and TA98 and TA1535 in the presence of S9 activation)

.

In the confirmatory mutagenicity assay, no positive mut a genic responses were observed

. The

dose levels tested were 0.600, 2.00, 6.00, 20.0, 60.0, 200 and 600 μg per plate (with tester

strains TA98, TA100 and TA1535 in the absence of S9 activation and TA100, TA15 37 and

WP2 uvr A in the presence of S9 activation) and 2.00, 6.00, 20.0, 60.0, 200, 600 and 2000 μg

per plate (with tester strains TA1537 and WP2 uvr A in the absence of S9 activation and TA98

and TA1535 in the presence of S9 activation)

. No precipitate was observed. Toxicity was

observed beginning at 200 or 600 μg per plate

.

All criteria for a valid study were met as described in the protocol. The vehicle control s and

positive controls in the initial toxicity

- mutation and confirmatory mutagenicity assays were

within the acceptable historical ranges and fulfilled the requirements for a valid assay

. Under

the conditions of this study, test substance Ph osphorochloridothioic acid, O,O

- diethyl ester was

negative (non

- mutagenic) in the bacterial reverse mutati on assay.BioReliance Study No.

Dow Number: 8 

### PURPOSE

The purpose of this study was to evaluate the mutagenic potential of the test substance

, Ph osphorochloridothioic acid, O,O

- diethyl ester

, by measuring its ability to induce reverse

mutations at selected loci of several strains of Salmone lla typhimurium and at the tryptophan

locus of Escherichia coli strain WP2 uvr A in the presence and absence o f Aroclor

- induced rat

liver S9. Copies of the study protocol and amendments are included in Appendix II

. Measures

to address US EPA's comments on the study protocol are summarized in Appendix VI

.

This study was conducted in compliance with the testing guidelines of the OECD Guidelines for

the Testing of Chemica ls Section 4: Health Effects, No. 471 ( 1997 )

.

CHARACTERIZATION OF TEST AND CONTROL SUBSTANCE S

The Sponsor has determined the identity, strength, purity and composition or other

characteristics to define the test substance and the stability of the test sub stance . A copy of the

Certificate of Analysis is included in Appendix I II

. Based on the re

- certification date provided

on the Certificate of Analysis, the test substance was considered stable through 13 January

2016

.

The vehicle used to deliver Ph osphorochloridothioic acid, O,O

- diethyl ester to the test system

was acetone ( Optima TM 

### , 99.6%, C atalog No. A929 , Lot No. 156453 , Exp. Date: 30 September

2020 ), obtained from Fisher Scientific

. Test substance dilutions were prepared imme diately

before use and delivered to the test system at room temperature under yellow light.

The negative and positive control substance s have been characterized as per the Certificates of

Analysis on file with the testing facility. The stability of the negative and positive control

substance s and their mixtures was demonstrated by acceptable results that met the criteria for a

valid test.

Positive controls plated concurrently with the initial toxicity

- mutation and confirmatory

mutagenicity assay s are list ed in the following table. All positive controls were diluted in

dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in deionized water. All

subdivided solutions of positive control were stored at -1 5 to -40 °C.BioReliance Study No.

Dow Number: 9 

### Strain S9

Activation Posi tive Control Concentration

(μg/plate)

TA98, TA1535

Rat 2

- aminoanthracene

(Sigma Aldrich Chemical Co., Inc.)

Lot No

. STB D3302V

Exp. Date 31

- Jul

- 2017

CAS No. 613

- 13 -8

Purit y 97.5% 1.0

TA100, TA1537 2.0

WP2 uvr A 15

TA98

None 2

- nitrofluorene

(Sigma Ald rich Chemical Co., Inc.)

Lot No. S43858V

Exp. Date 31

- Mar

- 2016

CAS No. 607

- 57 -8

Purity 99.4% 1.0

TA100, TA1535 sodium azide

( Sigma Aldrich Chemical Co., Inc. )

Lot No. MKBH5113V

Exp. Date 30

- Jun

- 2016

CAS No. 26628

- 22 -8

Purity 99.6% 1.0

TA1537 9

- aminoacr idine

(Sigma Aldrich Chemical Co., Inc.)

Lot No

. 09820CEV

Exp. Date 31

- Mar

- 2016

CAS No . 52417

- 22 -8

Purity 99.4% 75

WP2 uvr A methyl methanesulfonate

( Sigma Aldrich Chemical Co., Inc. )

Lot No . MKBR6050V

Exp. Date 31

- Oct

- 2017

CAS No. 66

- 27 -3

Purity 100.0% 1 ,000

To confirm the sterility of the test substance and the vehicle

, all test substance dose leve ls and

the vehicle used in the initial toxicity

- mutation and confirmatory mutagenicity assays were

plated on selective agar with an aliquot volume equal to th at used in the assay. These plates

were incubated under the same conditions as the assay.

Dose Formulation Collection and Analysis

D os e formulation samples were collected from the mutagenicity assays as follows:

Vehicle Sampling

Number of Samples A 

### Volume

2 0.5 00 mL

A 

### One sample was used for analysis and the other served as the backupBioReliance Study No.

Dow Number: 10 

### Solution Sampling

Dose Level Number of Samples B 

### Volume

High Dose 2 0.5 00 mL

Low Dose 2 C 

### 5 .00 mL

B 

### One sample was used for analysis and the other served as the backup

C 

### The lowest analyzable dose (0.0300 mg/mL (Initial Toxicity-Mutation Assay) and 0.0400

mg/mL (for Confirmatory Mutagenicity Assay)) was collected within the validated range

Samples were brimmed to minimize headspace in the vials. High dose vial contained 0. 800 mL,

low dose vial contained 6.00 mL and vehicle vial contained 0.800 mL.

All samples were submitted to the analytical chemistry laboratory at BioReliance for analysis.

Unused samples w ere discarded following issuance of the final analytical report. A copy of the

analytical report is included in Appendix IV

.

MATERIALS AND METHOD S

Test System

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100,

TA1535 and TA1537 as described by Ames et al . (1975) and Escherichia coli WP2 uvr A as

described by Green and Muriel (1976)

. Salmonella tester strains were derived from Dr. Bruce

Ames’ cultures; E

. coli tester strains were from the Na tional Collection of Industrial and Marine

Bacteria, Aberdeen, Scotland

.

Tester strains TA98 and TA1537 can be reverted from histidine dependence (auxotrophy) to

histidine independence (proto trophy) by frameshift mutagens. Tester strain TA1535 can be

reve rted by mutagens tha t cause base

- pair substitutions. Tester strain TA100 can be reverted by

mutagens that cause both frameshift and ba se

- pair substitution mutations. Specificity of the

reversion mechanism in E. coli is sensitive to A:T base

- pair substitu tion mutations, rather than

frameshift mutations (Green and Muriel, 1976).

Metabolic Activation System

Aroclor 1254

- induced rat liver S9 was used as t he metabolic activation system. The S9 was

prepared from male Sprague

- Dawley rats induced with a single intraperitoneal injection of

Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 (Lot No. 3503 , Exp. Date: 28 July

2017 ) was prepa red by and purchased from Mol t ox (Boone, NC)

. Upon arrival at BioReliance,

the S9 was s tored at

- 60 °C or colder until used. Each bulk preparation of S9 was assayed for its

ability to metabolize benzo(a)pyrene and 2

- aminoanthracene to forms mutagenic to Salmonella

typhimurium TA100. A copy S 9 Quality Control Certificate i s included in Appendix V

.

The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM

glucose -6- phosphate, 4 mM ß

- nicotinamide

- adenine dinucleotide phosphate, 8 mM MgCl 2 

### and

33 mM KCl in a 100 mM phosphate buffer at pH 7.4. Th e Sham S9 mixture (Sham mix),

containing 100 mM phosphate buffer at pH 7.4, was prepa red immediately before its use. To

confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective

agar.BioReliance Study No.

Dow Number: 11 

### Initial Toxicity

- Mutation Assay

The initial toxicity

- mutation assay was used to establish the dose

- range for the confirmatory

mutagenicity assay and to provide a preli minary mutagenicity evaluation. Vehicle control,

positive controls and a minimum of ten dose levels of the test substanc e were plated, three plates

per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvr A on

selective minimal agar in the presence and absence of Aroclor

- induced rat liver S9.

Confirmatory Mutagenicity Assay

The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential

of the test substance . A minimum of seven dose levels of test substance along with appropriate

vehicle control and positive controls were plated with overnight cultures of TA98, TA100,

TA1535, TA1537 and WP2 uvr A on selective minimal agar in the presence and absence o f

Aroclor

- induced rat liver S9. All dose levels of test substance , vehicle control and positive

controls were plated in triplicate.

Treatment of Test System

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V),

was melted and supplemented with L

- histidine, D

- biotin and L

- tryptophan solution to a fin al

concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with

25 mL of sterile water for e ach 100 mL of minimal top agar. Bottom agar was Vogel

- Bonner

minimal medium E ( Vogel and Bonner, 1956 ) containing 1.5 % (W/V) agar. Nutrient bottom

agar was Vogel

- Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented

with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel

- Bonner

salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder).

To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on

selective agar. To confirm the sterility of the test substance and the vehicle

, all test substance

dose leve ls and the vehicle used in each assay were plated on selective agar with an aliquot

volume equal to that use d in the assay. These plates were incubated under the same conditions

as the assay.

T he standard treatment volume s were increased 7.5X to minimize the headspace of the

treatment vessel, 3.75 mL S9 mix or sham mix

, 0.75 mL of tester strain (cells seeded) a nd

0.375 mL of vehicle or test substance dilution were added to 4 mL air tight sealed, capped glass

culture tubes pre

- heated to 37±2 °C . After vortexing, these mixtures were incubated with

shaking for 20±2 mi nutes at 37±2 °C. Following the preincubation, 0.65 mL of treatment

mixture was added to 2.0 mL of selective top agar for each plate and the mixture was vortexed

and overlaid onto the surface o f 25 mL of minimal bottom agar. When plating the positive

controls, the test substance aliquot was replaced b y a 0.375 m L aliquot of appropriate positive

control. Each plate was sealed with Petri

- Seal TM 

### adhesive sealing film (Sigma

- Aldrich Cat. No.

Z376922). After the overlay had solidified, the plates were inverted and incubated in an

appropriate number of ≥ 9

- liter desiccators by dose level and incubated for 48 to 72 hours at

37±2 ° C

. Plates that were not counted immediately following the incubation period were stored

at 2 -8 ° C until colony counting could be conducted.BioReliance Study No.

Dow Number: 12 

### Scoring

The condition of the bacterial background lawn was evaluated for evidence of test substance

toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period

by visual examination with out magnification. Toxicity and degree of precipitation were scored

relat ive to the vehicle control plate using the codes shown in the following table. As

appropriate, colonies were enumerated either by hand or by machine.

Code Description Characteristics

1 or no

code Normal Distinguished by a healthy microcolony lawn.

2 Sli ghtly

Reduced Distinguished by a noticeable thinning of the microcolony lawn

and possibly a slight increase in the size of the microcolonies

compared to the vehicle control plate.

3 Moderately

Reduced Distinguished by a marked thinning of the microcolony lawn

resulting in a pronounced increase in the size of the microcolonies

compared to the vehicle control plate.

4 Extremely

Reduced Distinguished by an extreme thinning of the microcolony lawn

resulting in an increase in the size of the microcolonies comp ared

to the vehicle control plate such that the microcolony lawn is

visible to the unaided eye as isolated colonies.

5 Absent Distinguished by a complete lack of any microcolony lawn over

more than or equal to 90% of the plate.

6 Obscured by

Precipitate The background bacterial lawn cannot be accurately evaluated due

to microscopic test substance precipitate.

NP Non

- Interfering

Precipitate Distinguished by precipitate on the plate that is visible to the

naked eye but any precipitate particles detected by the automated

colony counter total less than 10% of the revertant colony count

(e.g., less than 3 particles on a plate with 30 revertants).

IP Interfering

Precipitate Distinguished by precipitate on the plate that is visible to the

naked eye and any prec ipitate particles detected by the automated

colony counter exceed 10% of the revertant colony count (e.g.,

more than 3 particles on a plate with 30 revertants). These plates

are counted manually.

Revertant colonies for a given tester strain and activati on condition, except for positive controls,

were counted either entirely by automated colony counter or entirely by hand unless the plate

exhibited toxicity.

Tester Strain Verification

On the day of use in each assay, all tester strain cultures were checke d for the appropriate

genetic markers.BioReliance Study No.

Dow Number: 13 

### Criteria for a Valid Test

The following criteria must be met for the initial toxicity

- mutation and confirmatory

mutagenicity assays to be considered valid. All Salmonella tester strain cultures must

demonstrate the p resence of the deep rough mutation ( rfa ) and the deletion in the uvr B gene.

Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101

plasmid R

- factor. All WP2 uvr A cultures must demonstrate the deletion in the uvr A gene. All

cultures must demonstrate the characteristic mean number of spontaneous revertants in the

vehicle controls as follows (inclusive): TA98, 10

- 50; TA100, 80

- 240; TA1535, 5

- 45;

TA1537, 3

- 21; WP2 uvr A, 10

- 60. To ensure that appropriate numbers of ba cteria are plated,

tester strain culture titers must be greater than or equal to 0.3x10 9 

### cells/mL. The mean of each

positive control must exhibit at least a 3.0

- fold increase in the number of revertants over the

mean value of the respective vehicle contro l.

A minimum of three non

- toxic concentrations is required to evaluate assay data. A

concentration is considered toxic if one or both of the following criteria are met: (1) A >50 %

reduction in the mean number of revertants per plate as compared to the mean vehicle control

value. This reduction must be accompanied by an abrupt concentration

- depende nt drop in the

revertant count. (2) At least a moderate reduction in the background lawn (b ackground lawn

code 3, 4 or 5). Historical control data are inclu ded in Appendix I

.

Evaluation of Results

For each replicate plating, the mean and standard deviation of the number of revertants per plate

were calculated and are reported.

For the test substance to be evaluated positive, it must cause a concentration

- related increase in

the mean revertants per plate of at least one tester strain over a minimum of two increasing

concentrations of test article as specified below:

Strains TA98, TA1535, TA1537 and WP2 uvr A

Data sets were judged positive if the increase in mean revertants at the peak of the dose response

was equal to or greater than 3.0

- times the mean vehicle control value.

Strain TA100

Data sets were judged positive if the increase in mean revertants at the peak of the dose re sponse

was equal to or greater than 2.0

- times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets

the crite ria for evaluation as positive. This could be a concentration

- respon sive increase that

does not achieve the respective threshold cited above or a non

- concentration responsive increase

that is equal to or greater than the respective threshold cited. A response was evaluated as

negative if it was neither positive nor equivo cal.BioReliance Study No.

Dow Number: 14 

### Electronic Data Collection Systems

The primary computer or electronic systems used for the collection of data or analysis

included, but were not limited to , the following:

System Purpose

LIMS Labware System Test Article Tracking

Excel 2007 (Microsof t Corporation) Calculations

Sorcerer Colony Counter and Ames Study Manager

(Perceptive Instruments) Data Collection/Table

Creation

Kaye Lab Watch Monitoring system (Kaye GE) Environmental Monitoring

BRIQS Deviation and audit reporting

Records and Archives

All raw data, the protocol and all reports, generated by BioReliance, will be returned to the

Sponsor after issuance of the final report. All study materials returned to the Sponsor or

destroyed will first be copied onto electronic media and the elect ronic copy will be retained in

the BioReliance archives in accordance with BioReliance’s Standard Operating Procedures.

The raw data, reports, and other documents generated at locations other than BioReliance will

be archived at the Test Site. All unused test substance was disposed of prior to finalization of

the report.

Deviation

No known deviations from the protocol or assay

- method SOPs occurred during the conduct of

this study.

RESULTS AND DISCUSSI ON

Solubility Test

Acetone was selected as the solven t of choice based on information provided by the Sponsor,

and compatibility with the target cells. The Sponsor indicated the test substance formed clear

solutions in acetone at a concentration of approximately 500 g/L.

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test

substance dilutions or the S9 and Sham mixes.BioReliance Study No.

Dow Number: 15 

### Tester Strain Titer Results

Experiment Tester Strain

TA98 TA100 TA1535 TA1537 WP2 uvr A

Titer Value (x 10 9 

### cells per mL)

B1 2.7 1.8 3.5 2.3 3.0

B2 3.3 5.7 3.7 4.2 4.6

I nitial Toxicity

- Mutation Assay

The results of the initial

- toxicity mutation assay are presented in Tables 1 and 2

. These data

were generated in e xperiment B1

.

In e xperiment B1 ( i nitial t oxicity -m utation a ssay), the maximum concentrati on tested was

5000 μg per plate, which is the maximum concentration recommended by test guidelines. T his

concentration was achieved using a concentration of 100 mg/mL and a plati ng aliquot of 50.0

μL (0.375 mL/tube to minimize headspace). The concentrations tested were 0.150, 0.500, 1.50,

5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No positive mut agenic responses were

observed. No precipitate was observed. Toxicity was observed beginning at 150 or 500 μg per

plate. A non

- dose responsive, 1.4

- fold increase was observed with tester strain TA98 in the

absence of S9 activation with some of the counts being outside of the 95% vehicle control

limits. Based on the finding s of the initial toxicity

- mutation assay, the maximum dose plated in

the confirmatory mutagenicity assay was 600 μg per plate (with tester strains TA98, TA100 and

TA1535 in the absence of S9 activation and TA100, TA1537 and WP2 uvr A in the presence of

S9 activation) and 2000 μg per plate (with tester strains TA1537 and WP2 uvr A in the absence

of S9 activation and TA98 and TA1535 in the presence of S9 activation)

.

Confirmatory Mutagenicity Assay

The results of the confirmatory mutagenicity assay are presente d in Tables 3 and 4

. These data

were generated in e xperiment B 2.

In e xperiment B 2 (c onfirmatory m utagenicity a ssay), no positive mut agenic responses were

observed. The dose levels tested were were 0 .600, 2.00, 6.00, 20.0, 60.0, 200 and 600 μg per

plate (with tester strains TA98, TA100 and TA1535 in the absence of S9 activation and TA100,

TA1537 and WP2 uvr A in the presence of S9 activation) and 2.00, 6.00, 20.0, 60.0, 200, 600

and 2000 μg per plate ( with tester strains TA1537 and WP2 uvr A in the absence of S9

activation and TA98 and TA1535 in the presence of S9 activation). No precipitate was

observed. Toxicity was observed beginning at 200 or 600 μg per plate. The non

- dose

responsive increase obser ved previously with TA98 in the absence o f S9 activation did not

reproduce and was not indicative of mutagenic activity

.

Dosing Formulation Analysis

Dosing formulations were analyzed by the analytical chemistry laboratory at BioReliance for

analysis

. A c opy of the analytical report will be included in Appendix IV

. The results of theBioReliance Study No.

Dow Number: 16 

### analysis indicate that the actual mean concentrations of the analyzed formulation samples were

between 74.1% and 106.3% of their respective low and high concentrations, with S/L ratio s of >

0.925. The 0.0400 mg/mL formulation for Experiment B2 was found to be below the acceptable

range for concentration (85.0 to 115.0% of target) but met the S/L ratio of > 0.925. Although the

actual concent ration of the low dose formulation was lower than the protocol

- specified

acceptance criteria for a solution (85.0 to 115.0% of target concentration), the critical top dose

formulation met the acceptance criteria indicating that the test guideline recommend ed limit

dose was achieved. Therefore, the Study Director has concluded that the difference in nominal

concentration at the low dose had no adverse impact on the study results or validity of the study

conclusion. No test article was detected in the vehic le control sample. Additionally,

Phosphorochloridothioic acid, O,O

- diethyl ester in acetone, at concentrations of 0.300 and 105

mg/mL, was stable at room temperature for at least 3 hours. Also Phosphorochloridothioic acid,

O,O

- diethyl ester in acetone, at concentrations of 0.0219 and 92.6 mg/mL, was stable at room

temperature for at least 5 hours

.

CONCLUSION

All criteria for a valid study were me t as described in the protocol. The results of the b acterial

r everse m utation a ssay indicate that, unde r the co nditions of this study, was negative (non

mutagenic) both in the presence and absence of Aroclor

- induced rat liver S9.

R EFERENCES

Ames, B.N., J. McCann and E. Yamasaki (1975)

. Methods for d etecting c arcinogens and

m utagens with the Salmonella /m ammalian mi crosome m utagenicity te st. Mutation

Research 31:347

- 364.

Green, M.H.L. and W.J. Muriel (1976)

. Mutagen testing using trp+ reversion in Escherichia

coli

. Mutation Research 38:3

- 32.

OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum

to the OECD Guidelines for the Testing of Chemicals, published by OECD, Paris, February

1998.

Vogel, H.J. and D.M. Bonner (1956)

. Acetylornithinase of E. coli : p artial p urification and s ome

properties. Journal of Biological Chemistry 218:97

- 106.

Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T. and Okada, M. (1977)

. Mutagenicities of N

- nitrosamines on salmonella. Mutation Research 48:121

- 130.BioReliance Study No.

Dow Number: 17 

### DATA TABLESBioReliance Study No.

Dow Number: 18 Table 1

Initial Toxicity

- Mutation Assay without S9 activation

Study Number: Study Code:

Experiment: B1 Date Plated: 11/13/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/17/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

sol vent Individual revertant

colony counts and

background codes

TA98 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 5, 0 M 5, 0 M 5

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

150 μg 14 4 0.6 17 A 3, 14 A 3, 10 A 3

50.0 μg 32 1 1.4 32 A , 31 A , 32 A

15.0 μg 33 7 1.4 39 A , 34 A , 26 A

5.00 μg 21 2 0.9 18 A , 22 A , 22 A

1.50 μg 22 8 1.0 22 A , 30 A , 14 A

0.500 μg 23 1 1.0 22 A , 24 A , 23 A

0.150 μg 26 8 1.1 36 A , 21 A , 22 A

Acetone 50.0 μL /

plate

(0.375

mL/tube ) 23 5 18 A , 24 A , 27 A

TA100 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

150 μg 75 8 0.8 73 A 3, 68 A 3, 84 A 3

50.0 μg 121 18 1 .3 137 A , 101 A , 125 A

15.0 μg 116 5 1.2 121 A , 114 A , 112 A

5.00 μg 106 20 1.1 87 A , 127 A , 105 A

1.50 μg 102 4 1.1 97 A , 104 A , 104 A

0.500 μg 112 13 1.2 98 A , 115 A , 124 A

0.150 μg 120 19 1.3 98 A , 130 A , 133 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 96 4 92 A , 97 A , 100 A

Key to Plate Postfix Codes

5

4

3 Absent background

Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 19 Table 1 (CONT.)

Initial Toxicity

- Mutation Assay without S9 activation

Study Number: Study Code:

Experiment: B1 Date Plated: 11/13/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/17/2015

Strain Article Dose level

per plate Mea n

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual revertant

colony counts and

background codes

TA1535 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

150 μg 20 5 1.1 16 A 3, 26 A 3, 18 A 3

50.0 μg 16 2 0.8 18 A , 16 A , 15 A

15.0 μg 21 2 1.1 18 A , 22 A , 22 A

5.00 μg 19 3 1.0 21 A , 15 A , 21 A

1.50 μg 18 4 0.9 22 A , 16 A , 15 A

0.500 μg 16 6 0.8 13 A , 11 A , 23 A

0.150 μg 16 1 0.8 17 A , 15 A , 15 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 19 5 22 A , 21 A , 13 A

TA1537 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 13 4 1.3 11 A 3, 18 A 3, 11 A 3

150 μg 11 3 1.1 14 A , 10 A , 8 A

50.0 μg 10 3 1.0 9 A , 13 A , 7 A

15.0 μg 11 1 1.1 11 A , 10 A , 11 A

5.00 μg 14 2 1.4 11 A , 15 A , 15 A

1.50 μg 11 3 1.1 9 A , 9 A , 14 A

0.500 μg 10 3 1.0 9 A , 13 A , 8 A

0.150 μg 12 3 1.2 11 A , 15 A , 10 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 10 5 16 A , 7 A , 7 A

Key to Plate Postfix Codes

4

3 Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 20 Table 1 (CONT.)

Initial Toxicity

- Mutation Assay without S9 activatio n

Study Number: Study Code:

Experiment: B1 Date Plated: 11/13/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/17/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual revertant

colony counts and

background codes

WP2uvrA Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 32 7 1.1 39 A 3, 26 A 3, 32 A 3

150 μg 25 4 0.9 29 A , 23 A , 22 A

50.0 μg 36 5 1.2 30 A , 38 A , 39 A

15.0 μg 33 5 1.1 38 A , 30 A , 30 A

5.00 μg 34 7 1.2 27 A , 35 A , 40 A

1.50 μg 36 2 1.2 35 A , 35 A , 38 A

0.500 μg 35 11 1.2 27 A , 48 A , 30 A

0.150 μg 29 3 1.0 31 A , 30 A , 25 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 29 5 34 A , 27 A , 25 A

TA98 2NF 1.0 μg 269 94 11.7 226 A , 377 A , 204 A

TA100 SA 1.0 μg 451 107 4.7 519 A , 506 A , 328 A

TA1535 SA 1.0 μg 769 81 40.5 829 A , 801 A , 676 A

TA153 7 9AAD 75 μg 162 46 16.2 136 A , 215 A , 135 A

WP2uvrA MMS 1000 μg 337 63 11.6 410 A , 295 A , 306 A

Key to Positive Controls Key to Plate Postfix Codes

2NF

SA

9AAD

MMS 2

- nitrofluorene

sodium azide

9- Aminoacridine

methyl methanesulfonate 4

3 Extre mely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 21 Table 2

Initial Toxicity

- Mutation Assay with S9 activation

Study Number: Study Code:

Experiment: B1 Date Plated: 11/13/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/17/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual revertant

colony counts and

background codes

TA98 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 20 3 0.9 17 A 3, 21 A 3, 23 A 3

150 μg 31 7 1.3 38 A , 32 A , 24 A

50.0 μg 26 5 1.1 29 A , 29 A , 21 A

15.0 μg 24 3 1.0 22 A , 27 A , 22 A

5.00 μg 22 5 1.0 17 A , 24 A , 26 A

1.50 μg 24 8 1.0 22 A , 32 A , 17 A

0.500 μg 27 3 1.2 29 A , 29 A , 23 A

0.150 μg 27 5 1.2 30 A , 21 A , 29 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 23 6 18 A , 22 A , 29 A

TA100 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 5, 0 M 5, 0 M 5

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

150 μg 83 4 0.9 81 A 3, 88 A 3, 80 A 3

50.0 μg 93 26 1.1 68 A , 120 A , 91 A

15.0 μg 78 9 0.9 71 A , 88 A

, 74 A

5.00 μg 83 14 0.9 95 A , 87 A , 68 A

1.50 μg 87 6 1.0 92 A , 80 A , 90 A

0.500 μg 96 9 1.1 90 A , 106 A , 93 A

0.150 μg 97 11 1.1 86 A , 97 A , 107 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 88 9 95 A , 91 A , 78 A

Key to Plate Postfix Codes

5

4

3 Absent background

Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 22 Table 2 (CONT.)

Initial Toxicity

- Mutation Assay with S9 activation

Study Numbe r: Study Code:

Experiment: B1 Date Plated: 11/13/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/17/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent I ndividual revertant

colony counts and

background codes

TA1535 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 12 5 0.9 11 A 3, 17 A 3, 8 A 3

150 μg 14 6 1.1 10 A , 21 A

, 11 A

50.0 μg 17 3 1.3 17 A , 14 A , 19 A

15.0 μg 14 3 1.1 11 A , 15 A , 16 A

5.00 μg 12 5 0.9 17 A , 8 A , 11 A

1.50 μg 16 2 1.2 15 A , 15 A , 18 A

0.500 μg 8 3 0.6 8 A , 11 A , 6 A

0.150 μg 16 6 1.2 16 A , 10 A , 21 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 13 4 9 A

, 14 A , 16 A

TA1537 Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

150 μg 6 2 0.5 5 A 3, 5 A 3, 8 A 3

50.0 μg 9 2 0.8 10 A , 9 A , 7 A

15.0 μg 8 2 0.7 8 A , 6 A , 9 A

5.00 μg 5 2 0.5 7 A , 3 A , 6 A

1.50 μg 9 1 0.8 8 A , 10 A , 8 A

0.500 μg 7 2 0.6 7 A , 5 A , 8 A

0.150 μg 7 4 0.6 3 A , 8 A , 10 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 11 2 13 A , 10 A , 10 A

Key to Plate Postfix Codes

4

3 Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 23 Table 2 (CONT.)

Initial Toxicity

- Mutation Assay with S9 activation

Study Number: Study Code:

Experiment: B1 Date Plated: 11/13/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/17/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual reverta nt

colony counts and

background codes

WP2uvrA Phosphorochl

oridothioic

acid, O,O

diethyl ester 5000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

1500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

500 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

150 μg 26 2 1.0 26 A 3, 24 A 3, 27 A 3

50.0 μ g 26 2 1.0 27 A , 27 A , 24 A

15.0 μg 31 7 1.1 29 A , 25 A , 38 A

5.00 μg 24 1 0.9 25 A , 24 A , 24 A

1.50 μg 30 6 1.1 33 A , 33 A , 23 A

0.500 μg 29 11 1.1 21 A , 25 A , 42 A

0.150 μg 26 4 1.0 25 A , 31 A , 23 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 27 4 31 A , 24 A , 2 6 A

TA98 2AA 1.0 μg 209 24 9.1 220 A , 225 A , 182 A

TA100 2AA 2.0 μg 614 300 7.0 822 A , 270 A , 751 A

TA1535 2AA 1.0 μg 69 4 5.3 65 A , 70 A , 73 A

TA1537 2AA 2.0 μg 63 18 5.7 58 A , 83 A , 49 A

WP2uvrA 2AA 15 μg 295 39 10.9 335 A , 291 A , 258 A

K ey to Positive Controls Key to Plate Postfix Codes

2AA 2

- aminoanthracene 4

3 Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 24 Table 3

Confir matory Mutagenicity Assay without S9 activation

Study Number: Study Code:

Experiment: B2 Date Plated: 11/24/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/30/2015

Strain Article Dose level

per plate Mean

rever tants

per plate Standard

Deviation Ratio

treated /

solvent Individual revertant

colony counts and

background codes

TA98 Phosphorochl

oridothioic

acid, O,O

diethyl ester 600 μg 0 0 0.0 0 M 5, 0 M 5, 0 M 5

200 μg 11 3 0.6 13 A 2, 13 A 2, 8 A 2

60.0 μg 17 3 0.9 19 A , 17 A , 14 A

20.0 μg 15 1 0.8 14 A , 14 A , 16 A

6.00 μg 9 3 0.5 7 A , 7 A , 13 A

2.00 μg 13 3 0.7 11 A , 13 A , 16 A

0.600 μg 12 3 0.7 14 A , 14 A , 8 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 18 4 15 A , 16 A , 23 A

TA100 Phosphorochl

oridothioic

acid, O,O

diethyl ester 600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

60.0 μg 79 7 0.9 72 A , 80 A , 86 A

20.0 μg 80 2 0.9 78 A , 79 A , 82 A

6.00 μg 80 2 0.9 CPN#, 81 A , 78 A

2.00 μg 81 11 1.0 88 A , 87 A , 68 A

0.600 μg 73 10 0.9 80 A , 62 A , 78 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 85 4 89 A , 82 A , 83 A

Key to Plate Postfix Codes

5

4

2

CP

N# Absent background

Extremely reduced background

Slightly reduced background

Contaminated plate

Not counted

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 25 Table 3 (CONT.)

Confirmatory Mutagenicity Assay without S9 activation

Study Number: Study Code:

Experiment: B2 Date Plated: 11/24/2015

Exposur e Method: Pre

- incubation assay Evaluation Period: 11/30/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual revertant

colony counts and

background codes

TA1535 Phosphorochl

oridothioic

acid, O,O

diethyl ester 600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 8 0 1.1 8 A 3, 8 A 3, 8 A 3

60.0 μg 11 3 1.6 11 A , 8 A , 13 A

20.0 μg 11 2 1.6 10 A , 10 A , 13 A

6.00 μg 9 3 1.3 6 A , 11 A , 11 A

2.00 μg 12 2 1.7 13 A , 13 A , 10 A

0.600 μg 10 1 1.4 10 A , 9 A , 10 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 7 2 7 A , 5 A , 8 A

TA1537 Phosphorochl

oridothioic

acid, O,O

diethyl ester 2000 μg 0 0 0.0 0 M 5, 0 M 5, 0 M 5

600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 3 1 0.6 2 A 2, 3 A 2, 3 A 2

60.0 μg 9 0 1.8 9 A , 9 A , 9 A

20.0 μg 5 4 1.0 9 A , 5 A , 2 A

6.00 μg 5 1 1.0 5 A , 6 A , 5 A

2.00 μg 5 2 1.0 5 A , 6 A , 3 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 5 0 5 A , 5 A , 5 A

Key to Plate Postfix Codes

5

4

3

2 Absent background

Extremely reduced background

Moderately reduced background

Slightly reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 26 Table 3 (CONT.)

Confirmatory Mutagenicity Assay without S9 activation

Study Number: Study Code:

Experiment: B2 Date Plated: 11/24/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/30/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual r evertant

colony counts and

background codes

WP2uvrA Phosphorochl

oridothioic

acid, O,O

diethyl ester 2000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 22 3 1.4 24 A , 22 A , 19 A

60.0 μg 23 1 1.4 24 A , 23 A , 22 A

20.0 μg 20 3 1.3 24 A , 19 A , 18 A

6.00 μg 26 5 1.6 31 A , 21 A , 26 A

2.00 μg 15 2 0.9 14 A , 17 A , 15 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 16 6 21 A , 17 A , 10 A

TA98 2NF 1.0 μg 126 50 7.0 73 A , 173 A , 132 A

TA100 SA 1.0 μg 513 14 6.0 521 A , 497 A , 522 A

TA1535 SA 1.0 μg 149 62 21.3 78 A , 193 A , 177 A

TA1537 9AAD 75 μg 286 46 57.2 233 A , 317 A , 309 A

WP2uvrA MMS 1000 μg 453 41 28.3 472 A , 480 A , 406 A

Key to Positive Controls Key to Plate Postfix Codes

2NF

SA

9AAD

MMS 2

- nitrofluorene

sodium azi de

9- Aminoacridine

methyl methanesulfonate 4 Extremely reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 27 Table 4

Confirmatory Mutagenicity Assay with S9 activation

Study Number: Study Code:

Experiment: B2 Date Plated: 11/24/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/30/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual revert ant

colony counts and

background codes

TA98 Phosphorochl

oridothioic

acid, O,O

diethyl ester 2000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 17 3 0.8 21 A , 15 A , 16 A

60.0 μg 16 3 0.7 19 A , 15 A , 14 A

20.0 μg 25 5 1.1 29 A , 27 A , 19 A

6.00 μg 15 8 0.7 23 A , 7 A , 14 A

2.00 μg 21 2 1.0 22 A , 19 A , 22 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 22 4 18 A , 25 A , 22 A

TA100 Phosphorochl

oridothioic

acid, O,O

diethyl ester 600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

60.0 μg 117 22 1.1 116 A , 139 A , 95 A

20.0 μg 102 9 1.0 99 A , 95 A , 112 A

6.00 μg 93 4 0.9 98 A , 90 A , 91 A

2.00 μg 87 25 0.8 63 A , 84 A , 113 A

0.600 μg 105 15 1.0 95 A , 122 A , 99 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 106 20 129 A , 93 A , 97 A

Key to Plate Postfix Codes

4 Extremely reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 28 Table 4 (CONT.)

Confirmatory Mutagenicity Assay with S9 activation

Study Number: Study Code:

Experiment: B2 Date Plated: 11/24/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/30/2015

Strain Article Dose level

per plate Mean

revertants

per plate Standard

Deviation Ratio

treated /

solvent Individual revertant

colony counts and

background codes

TA1535 Phosphorochl

oridothioic

acid, O,O

diethyl ester 2000 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 16 1 1.3 15 A , 15 A , 17 A

60.0 μg 10 3 0.8 7 A

, 13 A , 10 A

20.0 μg 9 2 0.8 10 A , 7 A , 9 A

6.00 μg 11 7 0.9 15 A , 14 A , 3 A

2.00 μg 9 4 0.8 13 A , 8 A , 5 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 12 6 9 A , 19 A , 9 A

TA1537 Phosphorochl

oridothioic

acid, O,O

diethyl ester 600 μg 0 0 0.0 0 M 5, 0 M 5, 0 M 5

200 μg 8 2 1.3 6 A 3, 10 A 3, 9 A 3

60.0 μg 8 3 1.3 6 A , 7 A , 11 A

20.0 μg 9 4 1.5 9 A , 13 A , 6 A

6.00 μg 4 2 0.7 2 A , 6 A , 5 A

2.00 μg 10 3 1.7 7 A , 13 A , 10 A

0.600 μg 9 3 1.5 11 A , 6 A , 9 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 6 1 7 A , 6 A , 6 A

Key to Plate Postfix Codes

5

4

3 Absent background

Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 29 Table 4 (CONT.)

Confirmato ry Mutagenicity Assay with S9 activation

Study Number: Study Code:

Experiment: B2 Date Plated: 11/24/2015

Exposure Method: Pre

- incubation assay Evaluation Period: 11/30/2015

Strain Article Dose level

per plate Mean

revertants

p er plate Standard

Deviation Ratio

treated /

solvent Individual revertant

colony counts and

background codes

WP2uvrA Phosphorochl

oridothioic

acid, O,O

diethyl ester 600 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

200 μg 0 0 0.0 0 M 4, 0 M 4, 0 M 4

60.0 μg 35 3 1.2 36 A , 38 A , 32 A

20.0 μg 27 4 0.9 24 A , 25 A , 32 A

6.00 μg 28 6 1.0 35 A , 24 A , 24 A

2.00 μg 29 9 1.0 22 A , 25 A , 39 A

0.600 μg 27 12 0.9 17 A , 25 A , 40 A

Acetone 50.0 μL /

plate

(0.375

mL/tube) 29 2 27 A , 30 A , 30 A

TA98 2AA 1.0 μg 166 30 7.5 171 A , 133 A , 193 A

TA100 2AA 2.0 μg 426 12 4.0 431 A , 413 A , 435 A

TA1535 2AA 1.0 μg 676 62 56.3 748 A , 644 A , 637 A

TA1537 2AA 2.0 μg 82 34 13.7 51 A , 119 A , 75 A

WP2uvrA 2AA 15 μg 288 37 9.9 251 A , 288 A , 325 A

Key to Positive Controls Key to Plat e Postfix Codes

2AA 2

- aminoanthracene 4 Extremely reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic countBioReliance Study No.

Dow Number: 30 

### APPENDIX I : Historical Control DataBioReliance Study No.

Dow Number: 31 

### Historical Negative and Positive Control Values

2014

R evertants per plate

Strain Control Activation

None Rat Liver S9

Mean SD Min Max 95% CL Mean SD Min Max 95% CL

TA98 Neg 16 5 5 42 6- 26 24 7 5 53 10

- 38

Pos 232 258 57 2691 400 165 109 1382

TA100 Neg 94 14 66 152 66

- 122 102 18 63 16 4 66

- 138

Pos 68 1 176 213 1767 681 259 186 2793

TA1535 Neg 11 4 2 31 3- 19 13 5 2 36 3- 23

Pos 586 22 6 16 2509 117 99 23 1060

TA1537 Neg 7 3 1 19 1- 13 9 4 1 23 1- 17

Pos 411 355 32 2921 72 52 10 562

WP2 uvr A Neg 25 7 7 62 11

- 39 28 8 10 55 12

- 44

Pos 376 123 99 1026 30 2 102 91 687

SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than

zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and

acetone); Pos=positive controlBioReliance Study No.

Dow Number: 32 

### APPENDIX II : Study Protocol and AmendmentsBioReliance Study No.

Dow Number: 33BioReliance Study No.

Dow Number: 34BioReliance Study No.

Dow Number: 35BioReliance Study No.

Dow Number: 36BioReliance Study No.

Dow Number: 37BioReliance Study No.

Dow Number: 38BioReliance Study No.

Dow Number: 39BioReliance Study No.

Dow Number: 40BioReliance Study No.

Dow Number: 41BioReliance Study No.

Dow Number: 42BioReliance Study No.

Dow Number: 43BioReliance Study No.

Dow Number: 44BioReliance Study No.

Dow Number: 45BioReliance Study No.

Dow Number: 46BioReliance Study No.

Dow Number: 47BioReliance Study No.

Dow Number: 48BioReliance Study No.

Dow Number: 49BioReliance Study No.

Dow Number: 50BioReliance Study No.

Dow Number: 51BioReliance Study No.

Dow Number: 52BioReliance Study No.

Dow Number: 53 

### APPENDIX I II : Certificate of AnalysisBioReliance Study No.

Dow Number: 54BioReliance Study No.

Dow Number: 55 

### APPENDIX IV : Dosing Formulation An alysisPage 1 of 10 

### FINAL 

### ANALYTICAL 

### REPORT

Study Title

Bacterial Reverse Mutation Assay with Phosphorochloridothioic acid, O,O- diethyl ester

Report Title

Determination of Phosphorochloridothioic acid, O,O- diethyl ester in Acetone Dosing

Formulations

Test Substance

Phosph orochloridothioic acid, O,O

- diethyl ester

Author

Philip Atkins, MChem.

Final 

### Analytical 

### Report 

### Date

07 January 2016

Analytical 

### Laboratory

BioReliance Corporation

9610 Medical Center Drive

Rockville, MD 20850

BioReliance 

### Study 

### Number

Sponsor

The Dow Chemical Company

Midland, MI 48674

Dow Number:

DAS Number:

DAS PCTR Number:BioReliance Study No. 3 

### 2. TABLE OF CONTENTS

Page

1. COMPLIANCE STATEMENT .................................................................................2

2. TABLE OF CONTENTS ...........................................................................................3

3. ANALYTICAL CONDITION S ................................................................................4

4. DOSING FORMULATION ANALYSIS ..................................................................6

5. STABILITY OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O

- DIETHYL ESTER

IN ACETONE DOSING FORMULATIONS .....................................................................8

6. CONCLUSION ..........................................................................................................9

7. DEVIATIONS ...........................................................................................................9

8. ABBREVIATIONS AND CALCULATIONS ........................................................10BioReliance Study No. 4 

### 3. ANALYTICAL CONDITION S

The analysis of the t est substance formulations, Phosphorochloridothioic acid, O,O

- diethyl

ester in a cetone , fo r study was performed by gas chromatography

with flame ionization detection (G C /FID ) using a method validated under BioReliance Study

Number GCGTCHEM.BTL

. The formulations were also analyzed in accordance

with BioReliance SOP s “D ose Formulation Analysis” and “Dose Formulation Stability

Determination”. T he analytical conditions used in this study are summarized in Table 1

. The solvent standards were prepared per Table 2

.

Du e to potential volatility concerns of the Test Substance, all of the samples and standards

were prepared and analyzed on the same day. Additional efforts were made to the

preparation of the samples and standards to minimize headspace where appropriate and

diluted aliquots were transferred to autosampler vials in excess then capped and sealed to

eliminate as much headspace as possible.BioReliance Study No. 5 

### Table 1 : Analytical Conditions

Instrument: Agilent 6890N Gas Chromatograph

Detector: Flame Ion ization (FID)

Software: Agilent ChemStation with Open Lab CDS

Diluent: Acetone

Vehicle: 

### Acetone

- Fisher scientific, Optima Grade

(SS) Stock Solution: 200 μ g/mL Test Substance in Diluent

Inlet Temperature: 100°C

Inlet Pressure: 11.4 psi

Split Ratio: 1 0 :1

Split Flow: 8 mL/min

Total Inlet Flow: 11.4 mL/min

Inlet Gas: Helium

Column: Agilent DB

- 5 30 m x 250 μm, 0.25 μm film thickness

Column Flow Mode: Constant Flow

Flow rate: 0.8 mL/min

Detector Temperature: 300° C

Hydrogen Flow: 40 mL/min

Air Flow: 400 mL/min

Detector Flow Mode: Constant Makeup Flow

Makeup Flow: 25 mL/min

M akeup Gas: Helium

Syringe Needle Washes (A & B): Acetone

Injection Volume: 2 μL

Pre

- Injection Solvent A Washes: 2

Post

- Injection Solvent A Washes: 2

P re

- Injection Solvent B Washes: 2

Post

- Injection Solvent B Washes: 2

Injections/Sample: 1

Run Time : 20.6 minutes

Test Substance Retention Time: ~12.11 minutes

Calibration Curve: y = Ax + B (not weighted)

Oven Temperature Profile: Gradient (see below)

Rate (°C/min) Final Temperature ( °C) Hold Time (min)

0 80 4

3 110 0

25 200 3BioReliance Study No. 6 

### Table 2: Preparation of the Solvent Standard Solutions

Standard ID SS

(mL) Final Volume with

Diluent (mL) Final T S

Concentration ( μ g/mL)

S

- 0 0 4 0

S

- 1 1 

### 4 20

S

- 2 2 

### 4 40

S

- 3 3 

### 4 60

S

- 4 4 

### 4 80

S

- 5 5 

### 4 100

S

- 6 6 

### 4 1 2 0



### 4. DOSING FORMULATION ANALYSIS

Dosing formulations of Phosphorochloridothioic acid, O,O

- diethyl ester in acetone were

collected and analyzed by GC /FID on the day of preparation to assess accuracy of the

preparation (per Table 3 ). A sample of vehicle d osing solution from each experiment was

also analyzed to verify that it did not contain t est substance.

The dosing formulation s were diluted to bring the t est substance concentration to a suitable

level within the calibration range

. The concentration of Phosphorochloridothioic acid, O,O

diethyl ester was calculated by reference to the solvent standard solutions prepared (per

Table 2 ) and analyzed concurrently with the dosing formulations

. All solvent standard

curves met the acceptance criteria ( Table 4 and Table 6 ).

All dosing formulation s analyzed met the acceptance criteri a of 85.0 – 115.0% of target

concentration and the ratio of the small/large (S/L) obtained concentrations for the duplicate

dilutions of each solution was found to be > 0.925

, except for the 0.0400 mg/mL formulation

for Experiment B2 which was found to be below the acceptable range but met the S/L ratio

of > 0.925 ( Table 5 and Table 7 ). No Test Substance was detected in the vehicle control

(VC) samples.

Table 3 : Summary of the Dosing Formulation s

Experiment

No./Phase Date of Preparation Date of Analysis

(Start/End) Concentration

(mg/mL) Homogen eity Testing

(Y) Yes or (N) No

B1 13 November 2015 13 November 2015/

14 November 2015 0 N

0.0300 N

100 N

B2 24 November 2015 24 November 2015/

24 November 2015 0 N

0.0400 N

40.0 NBioReliance Study No. 7 

### Table 4: Solvent Standards for the B1 Dosing Formulation and Stability Analys es

Item Value Acceptance Criterion

Slope 0.8702 NA

Intercept

- 1.396 NA

Correlation Coefficient 0.99991 ≥ 0.99

Recovery % (Range) 98.2

- 101.8 90

- 110



### Table 5: B1 Dosing Formulation Analysis

Formulation Conc. of Conc. of T S Mean Mean Target x Final Mean S/L

ID Form. Sample Peak Peak Conc. Dilution Conc.

(mg/mL) ( μ g/mL) Area Area ( μ g/mL) (%) Factor (mg/mL)

VC (0) 0 0 ND ND NA NA 1 NA NA

100 A 100 100 90.64 90.00 105.0 105.0 1000 105 0.986 100 B 100 100 89.36

0.03 00 A 0.03 00 30 24.62 24.68 29.97 99.9 1 0.0300 0.996 0.03 00 B 0.03 00 30 24.73



### Table 6: Solvent Standards for the B2 Dosing Formulation Analysi s

Item Value Acceptance Criterion

Slope 0.8765 NA

Intercept

- 0.9113 NA

Correlation Coefficient 0.9998 ≥ 0.99

Recovery % (Range) 98.9

- 101.4 90

- 110



### Table 7 : B 2 Dosing Formulation Analysis

Formulation Conc. of Conc. of T S Mean Mean Target x Final Mean S/L

ID Form. Sample Peak Peak Conc. Dilution Conc.

(mg/mL) ( μ g/mL) Area Area ( μ g/mL) (%) Factor (mg/mL)

VC (0) 0 0 ND ND NA NA 1 NA NA

0.04 00 A 0.04 00 40 24.98 25.05 29.62 74.1 1 0.0296 0.995 0.04 00 B 0.04 00 40 25.11

40 A 40 40 36.68 36.35 42.51 106.3 1000 42.5 0.982 40 B 40 40 36.0 6BioReliance Study No. 8 

### 5. STABILITY OF PHOSPHOROCHLORIDOTHI OIC ACID, O,O

- DIETHYL

ESTER IN ACETONE D OSING FORMULATIONS

Stability of the dosing formulations was determined by storing the B1 dosing formulations at

room temperature for 3 hours and reanalyzing as describe d above. The acceptance criterion

of 90-110% of the concentration determined at T=0 was met ( Table 8 ).

Phosphorochloridothioic acid, O,O

- diethyl ester in acetone , at concentrations of 0.0300 and

105 mg/mL, was stable at room t emperature for at least 3 hours.

Table 8 : Stability T= 3 Hours Analysis

Formulation Conc. of Conc. of T S Mean Mean Target x Final Mean S/L

ID Form. 1 

### Sample 1 

### Peak Peak Conc. Dilution Conc.

(mg/mL) ( μ g/mL) Area Area ( μ g/mL) (%) Factor (mg/mL)

100 A T=3 105 105.0 86.83 86.11 100.6 95.8 1000 101 0.983 100 B T=3 105 105.0 85.38

0.03 A T=3 0.0300 29.97 24.23 24.45 29.70 99.1 1.001 0.0297 0.983 0.03 B T=3 0.0300 29.97 24.66

1 

### Concentration determined at T=0 ( Table 5 )



### Additionally , stability was performed to cover the time of dosing by preparing independent

formulations at concentration of 0.030 mg/mL and 100 mg/mL. The formulations were

analyzed immediately and stored room temperature for 5 hours and reanalyzed as described

above. The acceptance criterion of 90

- 110% of the concentration determined at T=0 was met

( Table 11 ).

Phosphorochloridothioic acid, O,O

- diethyl ester in acetone , at concentrations of 0.0219 and

92.6 mg/mL, was stable at room temperature for at least 5 hours.

Table 9: Solvent Standards for the Stability T=0 Analysi s

Item Value Acceptance Criterion

Slope 0.9174 NA

Intercept

- 1.159 NA

Correlation Coefficient 0.99994 ≥ 0.99

Recovery % (Range) 99.5

- 101.2 90

- 110BioReliance Study No. 9 

### Table 10: Stability T=0 Analysis

Formulation Conc. of Conc. of TS Mean Mean Target x Final Mean S/L

ID Form. Sample Peak Peak Conc. Dilution Conc.

(mg/mL) (  g/mL) Area Area (  g/mL) (%) Factor (mg/mL)

0.03 A 0.030 30 19.06 18.93 21.90 73.0 1 0.0219 0.986 0.03 B 0.030 30 18.80

100 A 100 100 83.04 83.82 92.63 92.6 1000 92.6 0.982 100 B 100 100 84.59



### Table 11: Stability T=5 Hours Analysis

Formulation Conc. of Conc. of TS Mean Mean Target x Final Mean S/L

ID Form. 1 

### Sample 1 

### Peak Peak Conc. Dilution Conc.

(mg/mL) (  g/mL) Area Area (  g/mL) (%) Factor (mg/mL)

0.03 A T=5hrs 0.0219 21.9 18.91 18.92 21.89 100.0 1 0.0219 0.999 0.03 B T=5hrs 0.0219 21.9 18.93

100 A T=5hrs 92.6 92.6 80.86 81.28 89.86 97.0 1000 89.9 0.990 100 B T=5hrs 92.6 92.6 81.70

1 

### Concentration determined at T=0 ( Table 10 )



### 6. CONCLUSION

The results of the analysis in dicate that the actual mean c oncentrations of the analyzed

formulation samples were between 74.1% a nd 106.3% of their respective concentrations

, with S/L ratios of > 0.925. The 0.0400 mg/mL formulation for Experiment B2 was found to

be below the acceptable range for concentration (85.0 to 115.0% of target) but met the S/L

ratio of > 0.925. This indicates that the formulations were accurately prepared, except as

indicated above. No test substance was detected in the vehicle control sample. Additionally,

Phosphorochloridothioic acid, O,O- diethyl ester in acetone, at concentrations of 0.0300 and

105 mg/mL, were stable at room temp erature for at least 3 hours. Also

Phosphorochloridothioic acid, O,O- diethyl ester in acetone, at concentrations of 0.0219 and

92.6 mg/mL, were stable at room te mperature for at least 5 hours.

7. DEVIATIONS

No deviations from the protocol or assay- method SOPs occurred during the conduct of this

study.BioReliance Study No. 10 

### 8. ABBREVIATIONS AND CALCULATIONS

Calc. = Calculated

Conc. = Concentration

Form = Formulation

HPLC = High Performance Liquid Chromatography

M = Matrix

NA = Not Applicable

ND =Not Detected

RSD = Relative Standard Deviation

S/L = Small/Large

T = Time

TS = Test Substance

UV = Ultra

- Violet

VC = Vehicle Control

The following formulas were used for the calculations:

1. Mean Concentration (μg/ mL) = (Mean Peak Area - Intercept) / Slope

Intercept and slope calculated using linear regression analysis

2. Final Mean Conc. (mg/mL) = (Mean Conc. (μg/ mL) x Dilution Factor) / 1000

3. % of Target, % Recovery = Mean Calculated Concentration 

### x 100

Concentration of Sample

4. S/L = Small T S Peak Area

Large T S Peak AreaBioReliance Study No.

Dow Number: 66 

### APPENDIX V : S9 Quality Control CertificateBioReliance Study No.

Dow Number: 67BioReliance Study No.

Dow Number: 68 

### APPENDIX V I : Measures to address US EPA's comments on the study protocolBioReliance Study No.

Dow Number: 69BioReliance Study No.

Dow Number: 70BioReliance Study No.

Dow Number: 71BioReliance Study No.

Dow Number: 72BioReliance Study No.

Dow Number: 73