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 Effects, 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

DAS Number

DAS PCTR Number

Sponsor

The Dow Chemical Company

Midland, MI 48674

BioReliance Study No. 

Dow Number: 3 

QUALITY ASSURANCE STATEMENT 
BioReliance Study No. 

Dow Number: 4 

Bacterial 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 Drive 

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:

DAS Number:

BioReliance 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 2015

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 November 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, MChem
BioReliance Study No. 

Dow Number: 5 

Analytical Laboratory (Analytical 

Chemistry Analysis):

BioReliance Corporation

9610 Medical Center Drive

Rockville, MD 20850
BioReliance 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 SUBSTANCES ..................................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: Dosing Formulation Analysis .......................................................................55

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

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

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SUMMARY

The test substance, Phosphorochloridothioic acid, O,O-diethyl ester, was tested in the bacterial 

reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535, 

TA1537 and Escherichia coli tester strain WP2 uvrA 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 preliminary mutagenicity evaluation. 

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

mutagenic potential of the test substance. 

In the initial toxicity-mutation assay, the maximum concentration 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, 1.50, 5.00, 15.0, 50.0, 

150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed. No 

precipitate was observed. 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 

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

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

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

In the confirmatory mutagenicity assay, no positive mutagenic 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, TA1537 and 

WP2 uvrA 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 uvrA 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 controls 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 Phosphorochloridothioic acid, O,O-diethyl ester was 

negative (non-mutagenic) in the bacterial reverse mutation assay.
BioReliance Study No. 

Dow Number: 8 

PURPOSE

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

Phosphorochloridothioic acid, O,O-diethyl ester, by measuring its ability to induce reverse 

mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan 

locus of Escherichia coli strain WP2 uvrA in the presence and absence of 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 Chemicals Section 4: Health Effects, No. 471 (1997). 

CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES 

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

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

Certificate of Analysis is included in Appendix III. 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 Phosphorochloridothioic acid, O,O-diethyl ester to the test system 

was acetone (OptimaTM, 99.6%, Catalog No. A929, Lot No. 156453, Exp. Date: 30 September 

2020), obtained from Fisher Scientific. Test substance dilutions were prepared immediately 

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

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

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

substances 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 assays are listed 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 -15 to -40 °C.
BioReliance Study No. 

Dow Number: 9 

Strain S9

Activation Positive Control Concentration(μg/plate)

TA98, TA1535

Rat

2-aminoanthracene

(Sigma Aldrich Chemical Co., Inc.)

Lot No. STBD3302V 

Exp. Date 31-Jul-2017

CAS No. 613-13-8 

Purity 97.5%

1.0

TA100, TA1537 2.0

WP2 uvrA 15

TA98

None

2-nitrofluorene

(Sigma Aldrich 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-aminoacridine

(Sigma Aldrich Chemical Co., Inc.)

Lot No. 09820CEV 

Exp. Date 31-Mar-2016

CAS No. 52417-22-8 

Purity 99.4%

75

WP2 uvrA 

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 levels and 

the vehicle used in the initial toxicity-mutation and confirmatory mutagenicity assays were 

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

were incubated under the same conditions as the assay.

Dose Formulation Collection and Analysis

Dose formulation samples were collected from the mutagenicity assays as follows:

Vehicle Sampling

Number of SamplesA Volume

2 0.500 mL

A One sample was used for analysis and the other served as the backup
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Solution Sampling

Dose Level Number of SamplesB Volume

High Dose 2 0.500 mL

Low Dose 2C 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 were discarded following issuance of the final analytical report. A copy of the 

analytical report is included in Appendix IV. 

MATERIALS AND METHODS 

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 uvrA as 

described by Green and Muriel (1976). Salmonella tester strains were derived from Dr. Bruce 

Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine 

Bacteria, Aberdeen, Scotland. 

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

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

reverted by mutagens that cause base-pair substitutions. Tester strain TA100 can be reverted by 

mutagens that cause both frameshift and base-pair substitution mutations. Specificity of the 

reversion mechanism in E. coli is sensitive to A:T base-pair substitution mutations, rather than 

frameshift mutations (Green and Muriel, 1976).

Metabolic Activation System

Aroclor 1254-induced rat liver S9 was used as the 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 prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, 

the S9 was stored 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 S9 Quality Control Certificate is 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 MgCl2 and 

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

containing 100 mM phosphate buffer at pH 7.4, was prepared 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.
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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 preliminary mutagenicity evaluation. Vehicle control, 

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

per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA 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 uvrA on selective minimal agar in the presence and absence of 

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 final 

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

25 mL of sterile water for each 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 levels and the vehicle used in each assay were plated on selective agar with an aliquot 

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

as the assay.

The standard treatment volumes 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) and

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 minutes at 37±2 °C. Following the preincubation, 0.65 mL of treatment 

mixture was added to2.0 mL of selective top agar for each plate and the mixture was vortexed 

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

controls, the test substance aliquot was replaced by a 0.375 mL aliquot of appropriate positive 

control. Each plate was sealed with Petri-SealTM 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.
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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 without magnification. Toxicity and degree of precipitation were scored 

relative 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 Slightly 

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 compared 

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 precipitate 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 activation 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 checked for the appropriate 

genetic markers.
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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 presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. 

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

plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA 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 uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, 

tester strain culture titers must be greater than or equal to 0.3x109 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 control. 

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-dependent drop in the 

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

code 3, 4 or 5). Historical control data are included 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 uvrA 

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 response 

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 criteria for evaluation as positive. This could be a concentration-responsive 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 equivocal.
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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 (Microsoft 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 electronic 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 DISCUSSION

Solubility Test

Acetone was selected as the solvent 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.
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Tester Strain Titer Results

Experiment

Tester Strain

TA98 TA100 TA1535 TA1537 WP2 uvrA

Titer Value (x 109 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

Initial Toxicity-Mutation Assay

The results of the initial-toxicity mutation assay are presented in Tables 1 and 2. These data 

were generated in experiment B1. 

In experiment B1 (initial toxicity-mutation assay), the maximum concentration 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, 1.50, 

5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic 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 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 

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

S9 activation) and 2000 μg per plate (with tester strains TA1537 and WP2 uvrA 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 presented in Tables 3 and 4. These data 

were generated in experiment B2. 

In experiment B2 (confirmatory mutagenicity assay), no positive mutagenic 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 uvrA 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 uvrA 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 observed previously with TA98 in the absence of 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 copy of the analytical report will be included in Appendix IV. The results of the 
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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 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. Although the 

actual concentration 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 recommended 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 vehicle 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 met as described in the protocol. The results of the bacterial 

reverse mutation assay indicate that, under the conditions of this study, was negative (nonmutagenic) both in the presence and absence of Aroclor-induced rat liver S9.

REFERENCES

Ames, B.N., J. McCann and E. Yamasaki (1975). Methods for detecting carcinogens and 

mutagens with the Salmonella/mammalian microsome mutagenicity test. 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: partial purification and some 

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. 

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DATA TABLES
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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 / 

solvent

Individual revertant

colony counts and 

background codes

TA98

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 5, 0M 5, 0M 5 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

150 μg 14 4 0.6 17A 3, 14A 3, 10A 3

50.0 μg 32 1 1.4 32A, 31A, 32A

15.0 μg 33 7 1.4 39A, 34A, 26A

5.00 μg 21 2 0.9 18A, 22A, 22A

1.50 μg 22 8 1.0 22A, 30A, 14A

0.500 μg 23 1 1.0 22A, 24A, 23A

0.150 μg 26 8 1.1 36A, 21A, 22A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube) 

23 5 18A, 24A, 27A 

TA100

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

150 μg 75 8 0.8 73A 3, 68A 3, 84A 3

50.0 μg 121 18 1.3 137A, 101A, 125A

15.0 μg 116 5 1.2 121A, 114A, 112A

5.00 μg 106 20 1.1 87A, 127A, 105A

1.50 μg 102 4 1.1 97A, 104A, 104A

0.500 μg 112 13 1.2 98A, 115A, 124A

0.150 μg 120 19 1.3 98A, 130A, 133A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

96 4 92A, 97A, 100A 

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 count
BioReliance 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

Mean 

revertants 

per plate

Standard 

Deviation

Ratio 

treated / 

solvent

Individual revertant

colony counts and 

background codes

TA1535

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

150 μg 20 5 1.1 16A 3, 26A 3, 18A 3

50.0 μg 16 2 0.8 18A, 16A, 15A

15.0 μg 21 2 1.1 18A, 22A, 22A

5.00 μg 19 3 1.0 21A, 15A, 21A

1.50 μg 18 4 0.9 22A, 16A, 15A

0.500 μg 16 6 0.8 13A, 11A, 23A

0.150 μg 16 1 0.8 17A, 15A, 15A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

19 5 22A, 21A, 13A 

TA1537

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 13 4 1.3 11A 3, 18A 3, 11A 3

150 μg 11 3 1.1 14A, 10A, 8A

50.0 μg 10 3 1.0 9A, 13A, 7A

15.0 μg 11 1 1.1 11A, 10A, 11A

5.00 μg 14 2 1.4 11A, 15A, 15A

1.50 μg 11 3 1.1 9A, 9A, 14A

0.500 μg 10 3 1.0 9A, 13A, 8A

0.150 μg 12 3 1.2 11A, 15A, 10A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

10 5 16A, 7A, 7A 

Key to Plate Postfix Codes

4 

3 

Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M: Manual count A: Automatic count
BioReliance Study No. 

Dow Number: 20

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

Mean 

revertants 

per plate

Standard 

Deviation

Ratio 

treated / 

solvent

Individual revertant

colony counts and 

background codes

WP2uvrA

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 32 7 1.1 39A 3, 26A 3, 32A 3

150 μg 25 4 0.9 29A, 23A, 22A

50.0 μg 36 5 1.2 30A, 38A, 39A

15.0 μg 33 5 1.1 38A, 30A, 30A

5.00 μg 34 7 1.2 27A, 35A, 40A

1.50 μg 36 2 1.2 35A, 35A, 38A

0.500 μg 35 11 1.2 27A, 48A, 30A

0.150 μg 29 3 1.0 31A, 30A, 25A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

29 5 34A, 27A, 25A 

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

TA100 SA 1.0 μg 451 107 4.7 519A

, 506A, 328A

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

TA1537 9AAD 75 μg 162 46 16.2 136A

, 215A, 135A

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

Key to Positive Controls Key to Plate Postfix Codes

2NF

SA

9AAD

MMS

2-nitrofluorene

sodium azide

9-Aminoacridine

methyl methanesulfonate

4 

3 

Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M: Manual count A

: Automatic count
BioReliance 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,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 20 3 0.9 17A 3, 21A 3, 23A 3

150 μg 31 7 1.3 38A, 32A, 24A

50.0 μg 26 5 1.1 29A, 29A, 21A

15.0 μg 24 3 1.0 22A, 27A, 22A

5.00 μg 22 5 1.0 17A, 24A, 26A

1.50 μg 24 8 1.0 22A, 32A, 17A

0.500 μg 27 3 1.2 29A, 29A, 23A

0.150 μg 27 5 1.2 30A, 21A, 29A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

23 6 18A, 22A, 29A 

TA100

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 5, 0M 5, 0M 5 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

150 μg 83 4 0.9 81A 3, 88A 3, 80A 3

50.0 μg 93 26 1.1 68A, 120A, 91A

15.0 μg 78 9 0.9 71A, 88A, 74A

5.00 μg 83 14 0.9 95A, 87A, 68A

1.50 μg 87 6 1.0 92A, 80A, 90A

0.500 μg 96 9 1.1 90A, 106A, 93A

0.150 μg 97 11 1.1 86A, 97A, 107A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

88 9 95A, 91A, 78A 

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 count
BioReliance Study No. 

Dow Number: 22

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 revertant

colony counts and 

background codes

TA1535

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 12 5 0.9 11A 3, 17A 3, 8A 3

150 μg 14 6 1.1 10A, 21A, 11A

50.0 μg 17 3 1.3 17A, 14A, 19A

15.0 μg 14 3 1.1 11A, 15A, 16A

5.00 μg 12 5 0.9 17A, 8A, 11A

1.50 μg 16 2 1.2 15A, 15A, 18A

0.500 μg 8 3 0.6 8A, 11A, 6A

0.150 μg 16 6 1.2 16A, 10A, 21A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

13 4 9A, 14A, 16A 

TA1537

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

150 μg 6 2 0.5 5A 3, 5A 3, 8A 3

50.0 μg 9 2 0.8 10A, 9A, 7A

15.0 μg 8 2 0.7 8A, 6A, 9A

5.00 μg 5 2 0.5 7A, 3A, 6A

1.50 μg 9 1 0.8 8A, 10A, 8A

0.500 μg 7 2 0.6 7A, 5A, 8A

0.150 μg 7 4 0.6 3A, 8A, 10A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

11 2 13A, 10A, 10A 

Key to Plate Postfix Codes

4 

3 

Extremely reduced background

Moderately reduced background

Key to Automatic & Manual Count Flags

M: Manual count A: Automatic count
BioReliance 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 revertant

colony counts and 

background codes

WP2uvrA

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

5000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

1500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

500 μg 0 0 0.0 0M 4, 0M 4, 0M 4

150 μg 26 2 1.0 26A 3, 24A 3, 27A 3

50.0 μg 26 2 1.0 27A, 27A, 24A

15.0 μg 31 7 1.1 29A, 25A, 38A

5.00 μg 24 1 0.9 25A, 24A, 24A

1.50 μg 30 6 1.1 33A, 33A, 23A

0.500 μg 29 11 1.1 21A, 25A, 42A

0.150 μg 26 4 1.0 25A, 31A, 23A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

27 4 31A, 24A, 26A 

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

TA100 2AA 2.0 μg 614 300 7.0 822A

, 270A, 751A

TA1535 2AA 1.0 μg 69 4 5.3 65A, 70A

, 73A

TA1537 2AA 2.0 μg 63 18 5.7 58A

, 83A, 49A

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

Key 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 count
BioReliance Study No. 

Dow Number: 24

Table 3

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 revertant

colony counts and 

background codes

TA98

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

600 μg 0 0 0.0 0M 5, 0M 5, 0M 5 

200 μg 11 3 0.6 13A 2, 13A 2, 8A 2

60.0 μg 17 3 0.9 19A, 17A, 14A

20.0 μg 15 1 0.8 14A, 14A, 16A

6.00 μg 9 3 0.5 7A, 7A, 13A

2.00 μg 13 3 0.7 11A, 13A, 16A

0.600 μg 12 3 0.7 14A, 14A, 8A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

18 4 15A, 16A, 23A 

TA100

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

200 μg 0 0 0.0 0M 4, 0M 4, 0M 4

60.0 μg 79 7 0.9 72A, 80A, 86A

20.0 μg 80 2 0.9 78A, 79A, 82A

6.00 μg 80 2 0.9 CPN#, 81A, 78A

2.00 μg 81 11 1.0 88A, 87A, 68A

0.600 μg 73 10 0.9 80A, 62A, 78A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

85 4 89A, 82A, 83A 

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 count
BioReliance 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

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,Odiethyl ester

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

200 μg 8 0 1.1 8A 3, 8A 3, 8A 3

60.0 μg 11 3 1.6 11A, 8A, 13A

20.0 μg 11 2 1.6 10A, 10A, 13A

6.00 μg 9 3 1.3 6A, 11A, 11A

2.00 μg 12 2 1.7 13A, 13A, 10A

0.600 μg 10 1 1.4 10A, 9A, 10A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

7 2 7A, 5A, 8A 

TA1537

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

2000 μg 0 0 0.0 0M 5, 0M 5, 0M 5 

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4

200 μg 3 1 0.6 2A 2, 3A 2, 3A 2

60.0 μg 9 0 1.8 9A, 9A, 9A

20.0 μg 5 4 1.0 9A, 5A, 2A

6.00 μg 5 1 1.0 5A, 6A, 5A

2.00 μg 5 2 1.0 5A, 6A, 3A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

5 0 5A, 5A, 5A 

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 count
BioReliance 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 revertant

colony counts and 

background codes

WP2uvrA

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

2000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4

200 μg 22 3 1.4 24A, 22A, 19A

60.0 μg 23 1 1.4 24A, 23A, 22A

20.0 μg 20 3 1.3 24A, 19A, 18A

6.00 μg 26 5 1.6 31A, 21A, 26A

2.00 μg 15 2 0.9 14A, 17A, 15A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

16 6 21A, 17A, 10A 

TA98 2NF 1.0 μg 126 50 7.0 73A

, 173A, 132A

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

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

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

WP2uvrA MMS 1000 μg 453 41 28.3 472A

, 480A, 406A

Key to Positive Controls Key to Plate Postfix Codes

2NF

SA

9AAD

MMS

2-nitrofluorene

sodium azide

9-Aminoacridine

methyl methanesulfonate

4 Extremely reduced background

Key to Automatic & Manual Count Flags

M: Manual count A

: Automatic count
BioReliance 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 revertant

colony counts and 

background codes

TA98

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

2000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4

200 μg 17 3 0.8 21A, 15A, 16A

60.0 μg 16 3 0.7 19A, 15A, 14A

20.0 μg 25 5 1.1 29A, 27A, 19A

6.00 μg 15 8 0.7 23A, 7A, 14A

2.00 μg 21 2 1.0 22A, 19A, 22A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

22 4 18A, 25A, 22A 

TA100

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

200 μg 0 0 0.0 0M 4, 0M 4, 0M 4

60.0 μg 117 22 1.1 116A, 139A, 95A

20.0 μg 102 9 1.0 99A, 95A, 112A

6.00 μg 93 4 0.9 98A, 90A, 91A

2.00 μg 87 25 0.8 63A, 84A, 113A

0.600 μg 105 15 1.0 95A, 122A, 99A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

106 20 129A, 93A, 97A 

Key to Plate Postfix Codes

4 Extremely reduced background

Key to Automatic & Manual Count Flags

M: Manual count A

: Automatic count
BioReliance 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,Odiethyl ester

2000 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4

200 μg 16 1 1.3 15A, 15A, 17A

60.0 μg 10 3 0.8 7A, 13A, 10A

20.0 μg 9 2 0.8 10A, 7A, 9A

6.00 μg 11 7 0.9 15A, 14A, 3A

2.00 μg 9 4 0.8 13A, 8A, 5A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

12 6 9A, 19A, 9A 

TA1537

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

600 μg 0 0 0.0 0M 5, 0M 5, 0M 5 

200 μg 8 2 1.3 6A 3, 10A 3, 9A 3

60.0 μg 8 3 1.3 6A, 7A, 11A

20.0 μg 9 4 1.5 9A, 13A, 6A

6.00 μg 4 2 0.7 2A, 6A, 5A

2.00 μg 10 3 1.7 7A, 13A, 10A

0.600 μg 9 3 1.5 11A, 6A, 9A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

6 1 7A, 6A, 6A 

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 count
BioReliance Study No. 

Dow Number: 29

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

WP2uvrA

Phosphorochl

oridothioic 

acid, O,Odiethyl ester

600 μg 0 0 0.0 0M 4, 0M 4, 0M 4 

200 μg 0 0 0.0 0M 4, 0M 4, 0M 4

60.0 μg 35 3 1.2 36A, 38A, 32A

20.0 μg 27 4 0.9 24A, 25A, 32A

6.00 μg 28 6 1.0 35A, 24A, 24A

2.00 μg 29 9 1.0 22A, 25A, 39A

0.600 μg 27 12 0.9 17A, 25A, 40A

Acetone

50.0 μL/ 

plate 

(0.375 

mL/tube)

29 2 27A, 30A, 30A 

TA98 2AA 1.0 μg 166 30 7.5 171A

, 133A, 193A

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

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

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

WP2uvrA 2AA 15 μg 288 37 9.9 251A

, 288A, 325A

Key to Positive Controls Key to Plate Postfix Codes

2AA 2-aminoanthracene 4 Extremely reduced background

Key to Automatic & Manual Count Flags

M: Manual count A

: Automatic count
BioReliance Study No. 

Dow Number: 30

APPENDIX I: Historical Control Data
BioReliance Study No. 

Dow Number: 31

Historical Negative and Positive Control Values

2014

Revertants 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 63 164 66-138

Pos 681 176 213 1767 681 259 186 2793

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

Pos 586 226 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 uvrA Neg 25 7 7 62 11-39 28 8 10 55 12-44

Pos 376 123 99 1026 302 102 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 control
BioReliance Study No. 

Dow Number: 32

APPENDIX II: Study Protocol and Amendments
BioReliance Study No. 

Dow Number: 33
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Dow Number: 34
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Dow Number: 42
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Dow Number: 51
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Dow Number: 52
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Dow Number: 53

 

APPENDIX III: Certificate of Analysis
BioReliance Study No. 

Dow Number: 54
BioReliance Study No. 

Dow Number: 55

APPENDIX IV: Dosing Formulation Analysis
Page 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

Phosphorochloridothioic 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. 

Dow Number: 

56
BioReliance Study No. 

Dow Number: 

57
BioReliance Study No. 3 

2. TABLE OF CONTENTS

 Page

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

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

3. ANALYTICAL CONDITIONS ................................................................................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 ........................................................10

BioReliance Study No. 

Dow Number: 

58
BioReliance Study No. 4 

3. ANALYTICAL CONDITIONS 

The analysis of the test substance formulations, Phosphorochloridothioic acid, O,O-diethyl 

ester in acetone, for study was performed by gas chromatography 

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

Number GCGTCHEM.BTL. The formulations were also analyzed in accordance 

with BioReliance SOPs “Dose Formulation Analysis” and “Dose Formulation Stability 

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

The solvent standards were prepared per Table 2. 

Due 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. 

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Table 1: Analytical Conditions

Instrument: Agilent 6890N Gas Chromatograph

Detector: Flame Ionization (FID)

Software: Agilent ChemStation with Open Lab CDS

Diluent: Acetone

Vehicle: Acetone-Fisher scientific, Optima Grade

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

Inlet Temperature: 100°C

Inlet Pressure: 11.4 psi

Split Ratio: 10:1

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

Makeup Gas: Helium

Syringe Needle Washes (A & B): Acetone

Injection Volume: 2 μL

Pre-Injection Solvent A Washes: 2

Post-Injection Solvent A Washes: 2

Pre-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 3

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Table 2: Preparation of the Solvent Standard Solutions

Standard ID SS

(mL)

Final Volume with 

Diluent (mL)

Final TS

Concentration (μg/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 4 120

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 dosing solution from each experiment was 

also analyzed to verify that it did not contain test substance. 

The dosing formulations were diluted to bring the test substance concentration to a suitable 

level within the calibration range. The concentration of Phosphorochloridothioic acid, O,Odiethyl 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 formulations analyzed met the acceptance criteria 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 Formulations 

Experiment 

No./Phase Date of Preparation Date of Analysis (Start/End)

Concentration

(mg/mL)

Homogeneity 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 N

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Table 4: Solvent Standards for the B1 Dosing Formulation and Stability Analyses 

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 TS Mean Mean Target x Final Mean S/L

ID Form. Sample Peak Peak Conc. Dilution Conc.

(mg/mL) (μg/mL) Area Area (μg/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

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

Table 6: Solvent Standards for the B2 Dosing Formulation Analysis 

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: B2 Dosing Formulation 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) (μg/mL) Area Area (μg/mL) (%) Factor (mg/mL)

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

0.0400 A 0.0400 40 24.98 25.05 29.62 74.1 1 0.0296 0.995 0.0400 B 0.0400 40 25.11

40 A 40 40 36.68 36.35 42.51 106.3 1000 42.5 0.982 40 B 40 40 36.06

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5. STABILITY OF PHOSPHOROCHLORIDOTHIOIC ACID, O,O-DIETHYL 

ESTER IN ACETONE DOSING FORMULATIONS

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

room temperature for 3 hours and reanalyzing as described 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 temperature for at least 3 hours. 

Table 8: Stability T=3 Hours Analysis 

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

ID Form.1 Sample1 Peak Peak Conc. Dilution Conc.

(mg/mL) (μg/mL) Area Area (μg/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 Analysis 

Item Value Acceptance Criterion

Slope 0.9174 NA

Intercept -1.159 NA

Correlation Coefficient 0.99994 ≥0.99

Recovery % (Range) 99.5-101.2 90-110

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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 Sample1 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 indicate that the actual mean concentrations of the analyzed 

formulation samples were between 74.1% and 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 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, were stable at room temperature for at least 5 hours. 

7. DEVIATIONS 

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

study. 

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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 TS Peak Area 

 Large TS Peak Area

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APPENDIX V: S9 Quality Control Certificate
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APPENDIX VI: Measures to address US EPA's comments on the study protocol
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