Page 1 of 59 

FINAL REPORT

Study Title

Salmonella – Escherichia coli/Mammalian-Microsome Reverse Mutation Assay 

Preincubation Method with a Confirmatory Assay with 

Test Article

Test Guidelines

EC Commission Directive 2008/32/EC Annex B.13/14 No. L142 

US EPA Health Effects Test Guideline OPPTS 870.5100 (1998) 

OECD Guidelines for the Testing of Chemicals Section 4: Health Effects, No. 471 (1998) 

Authors

Emily W. Dakoulas, B.S. 

Meena Jois, B.S. 

Study Completion Date

21 September 2010 

Testing Facility

BioReliance 

9630 Medical Center Drive 

Rockville, MD 20850 

BioReliance Study Number

Dow Study Number

Sponsor

Midland, MI 48674

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STATEMENT OF COMPLIANCE 

Study No. was conducted in compliance with the U.S. FDA GLPs Title 

21, CFR Part 58, the Japanese Ministry of Economy, Trade and Industry (METI) GLP 

Standards and the OECD Principles of Good Laboratory Practice ENV IMC/CHEM(98) 17 in all 

material aspects. 

GLP characterization of the test substance was conducted by the Sponsor in compliance with the 

US. EPA Good Laboratory Practice Standard, 40 CFR Part 792. 

BioReliance 

Study No. 

Dow Study No. 2 

2l'SPPI.;t010 

Date 

:;t I Sq..p;ZOlO 

Date 
BioReliance 

Study No. 

Dow Study No. 3 

- 

.. BioReliance" 

Quality Assurance Statt'llleut 

Study IlIfol'lnntioll 

i'iumber: 

Complianc(' 

Procedlm"S, documentation, e-quipmelll and other n"<:ords wert' examined in order to assnre this study was 

perfonned in accordanct' with the Tt'gulation(s) listed below and COIlductt'd according to the protocol and 

",Ie-vanl Standard Operatin g Procedures Verification oflhe- study protocol was perfOffiled and documented by 

Quality Assurance 

US FDA Good Laboratory Practices 21CFR 58 

Japanese Ministry of Economy, Trade and Industry Good Laboratory Practices 

OEeD Principles of Good LabomlOf)' Practices (C(97) 186IFinal) 

IllSp{'ctiollS 

Quality Assurance perfonned the ins~tiolls(s) below for this study 

Insp. D~te s (From/To) Phase Illsperred To Stud\' Dirl'ctor To :'.Ianagement 

25-Jun-2010 25-Jun-20 10 Adminis\Ialioo 01 Test Article To Test System 3O-Jun-20 10 3O-Jun-2010 

3O-Jul-2010 3O-Ju1-2010 Data and Dran Repof1ing 3O-Jul-2010 3O-Jut-2010 

20-Sep-2010 20-Sep-2:010 Final Reporting 20-Sep-2:010 20-Sep-2010 

The- Fina! Re-port for this study de-scribes The- me-thods and proce-dures used in the- study and the- re-p<>rte-d re-sults 

accurate-Iy re- flec t the- raw data of the- shidy 

E-siglllltllrl' 

Quality Assuranrl': 

~ason roc signallJ : QA Approval 

Pliot"" y: ~"ira Ualldari 

Pliot"" on:H -S.p-IO 

Jessica Mandaci 21-Sep-2010 357 pm GMT 
BioReliance 

Study No. 

Dow Study No. 4 

Salmonella – Escherichia coli/Mammalian-Microsome Reverse Mutation Assay 

Preincubation Method with a Confirmatory Assay with 

STUDY INFORMATION 

Sponsor: 

Midland, MI 48674 

Authorized Representative: Jill M. Smalley, B.S. 

Testing Facility: BioReliance 

9630 Medical Center Drive 

Rockville, MD 20850 

Test Article I.D.: 

Chemical Name: 

Test Article Lot No.: 

Test Article CAS No.: 

Test Article Purity: ± (per GLP Certificate of 

Analysis) 

Dow Study No.: 

BioReliance Study No.: 

Test Article Description: 

Storage Conditions: 

Test Article Receipt/Login Date: 18 May 2010 

Study Initiation Date: 08 June 2010 

Experimental Start Date: 11 June 2010 

Experimental Completion Date: 02 July 2010 

Study Director / Laboratory Manager: Emily W. Dakoulas, B.S. 

Principal Investigator: Lisa Douglass, A.S. 

Analytical Laboratory: 

1803 Building, Door E, Lab 40 

Midland, MI 48674 

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BioReliance 

Study No. 

Dow Study No. 5 

TABLE OF CONTENTS 

Page 

SUMMARY...............................................................................................................................6

PURPOSE..................................................................................................................................6

CHARACTERIZATION OF TEST AND CONTROL ARTICLES.........................................7

MATERIALS AND METHODS...............................................................................................8

RESULTS AND DISCUSSION..............................................................................................13

CONCLUSION........................................................................................................................14

REFERENCES ........................................................................................................................14

DATA TABLES ......................................................................................................................15

APPENDIX I: Historical Control Data....................................................................................24

APPENDIX II: Study Protocol ................................................................................................26

APPENDIX III: Information for Japanese Regulatory Agencies............................................45

APPENDIX IV: Certificate of Analysis ..................................................................................50

APPENDIX V: Dosing Formulation Analysis ........................................................................52

APPENDIX VI: S9 Quality Control Certificate ......................................................................58

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BioReliance 

Study No. 

Dow Study No. 6 

SUMMARY 

The t est arti cle, was t ested in t he Ba cterial Reverse Mutation Assay 

using Salmonella typhi murium test er strains TA98, TA 100, TA1535 and TA1537 a nd 

Escherichia coli tester strain WP2 uvrA in the presence or absence of Aroclor-induced rat liver 

S9. The a ssay was per formed in t wo phases using the pre incubation method. The fi rst phase, 

the initial toxicity-mutation assa y, was us ed to e stablish the dos e-range for the c onfirmatory 

mutagenicity assay and t o provide a preli minary mutagenicity ev aluation. The s econd phase, 

the confirmatory mutagenicity assay, was used to eval uate and confirm the mutagenic potential 

of the test article. 

Dimethyl sulfoxi de ( DMSO) was sel ected as the solvent of choi ce based on t he i nformation 

provided by the Sponsor, solubility of the test article, and compatibility with the target cells . 

After sonication for 60 minute s at 36° C, the test article f ormed a solubl e and clear solution in 

DMSO at a concentr ation of a pproximately 100 mg/mL, the ma ximum concentration tested i n 

the solubility test. 

In the initial toxicity-mutation assay, the maximum concentration tested was 5000 μg per plate, 

which is the ma ximum concentration recommended by test guidelin es. This concentr ation was 

achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The concentrations 

tested wer e 1.5, 5.0, 15, 50, 150, 500, 1500 an d 5000 μg pe r plate. No positi ve mutage nic 

responses were observed in the pre sence or absence of S9 acti vation. Precipitate was observed 

beginning at 50, 150, 500, 1500 or at 5000 μg per plate. Toxicity was observed, as a r eduction 

in revertant counts, at 5000 μg per pl ate with tester strains TA100, TA1537 and W P2 uvrA in 

the presence of S9 activ ation. Bas ed on t he findings of the initial toxicity-mutation assay, the 

maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. 

In the confir matory mutagenicity assay, the c oncentrations tested were 15, 50, 150, 500, 1500 

and 5000 μg per plate. No posit ive muta genic responses we re observed in t he presence or 

absence of S9 activation. Preci pitate was obser ved beginning at 500, 1500 or at 5000 μg per 

plate. Toxicity was observed, as a reduction in revertant counts, at 5000 μg per plate with tester 

strains TA100, TA1535 and WP2 uvrA in the presence of S9 activation. 

All criteria for a valid st udy were met as des cribed in the protocol. The vehicle controls and 

positive controls in the initial toxicity-mutation and conf irmatory mutagenicity assays were 

within the acceptable historical ranges and ful filled the requirements for a vali d assay. Under 

the conditions of this st udy, test article was concluded to be negati ve in 

the bacterial reverse mutation assay. 

PURPOSE 

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

by measuring its ability to induce reverse mutatio ns 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 Ar oclor-induced rat liver S9. A co py of t he st udy pr otocol is 

included in Appendix II. 

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BioReliance 

Study No. 

Dow Study No. 7 

This study wa s conduct ed in compl iance wit h t he te sting guidelines of the EC Commi ssion 

Directive 2008/ 32/EC Annex B.13/ 14 No. L142 , the US E PA Healt h E ffects Test Guideli ne 

OPPTS 870.5100 (1998) and the OECD Guideli nes for t he Test ing of Chemical s Section 4: 

Health Effects, No. 471 (1998). 

CHARACTERIZATION OF TEST AND CONTROL ARTICLES 

The test arti cle, was received by BioReliance on 18 May 2010 and was 

assigned t he code numb er Per prot ocol, t he test article shoul d be st ored at room 

temperature in the dark. Upon r eceipt, the test article was described as an and 

was stored at room temperature in the dark without desiccant. 

The Spons or has det ermined the identit y, s trength, pur ity and c omposition or other 

characteristics to define t he test article. A copy t he Certificate of Analysis for the test article is 

included in Appendix IV. A recertif ication date of 06 March 2012 was assigne d to t his lot of 

 Based on the recerti fication date in t he Certificate of Analysi s, the test 

article is considered stable for the purpose of this study. 

The vehi cle used to del iver to the test system wa s DM SO (C AS No. 

67-68-5, Lot No. 49288950, Exp. Da te: March 2013), obtai ned from EM D Che micals. Test 

article dilutions were prepared immediately before use and delivered t o the test system at room 

temperature under yellow light . To achieve a soluti on, the most c oncentrated diluti on wa s 

sonicated at 35°C for approximately 10 minutes in the B1 assay and approximately 15 minutes 

in the B2 assay. 

Dosing for mulation samples (1 x 3.0 mL fr om each do se level and the vehi cle i n t he initi al 

toxicity-mutation assay; 2 x 3.0 mL fr om each dose level and the vehicle in t he confirmatory 

mutagenicity assay) were collected. One set of sam ples from the confirmatory mutagenicity 

assay was shipped on dry ice to the Sponsor for analysis. The remaining set of samples from the 

confirmatory mutagenicity assay and all sample s from t he initial toxicity-mutation assay were 

retained at BioReliance at -15 to -40°C as a backup and were discarded following issue of th e 

final analytical report. A copy of the analytical report is included in Appendix V. 

The negati ve and pos itive c ontrol a rticles ha ve be en charact erized as per the Certi ficates of 

Analysis on f ile with the test ing f acility. The stability of th e negative and positive control 

articles and their mi xtures was demonstr ated by acceptable result s that met the crit eria for a 

valid test. 

Positive controls plated concurre ntly with the mutagenicity a ssay are listed in the following 

table. All positive controls we re diluted in di methyl sul foxide ( DMSO) exc ept for s odium 

azide, which wa s diluted in wat er. All subdi vided solutions of positive c ontrol were stored at 

-15 to -40°C. 

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BioReliance 

Study No. 

Dow Study No. 8 

Strain S9 

Activation Positive Control Concentration(μg/plate) 

TA98, TA1535 

and TA1537 1.0 

TA100 2.0 

WP2 uvrA 

Rat 

2-aminoanthracene 

(Sigma Aldrich Chemical Co., Inc.) 

Lot No. 03403ED 

Exp. Date 22-Jan-2012 

CAS No. 613-13-8 

Purity 99.8% 

10 

TA98 

2-nitrofluorene 

(Sigma Aldrich Chemical Co., Inc.) 

Lot No. 03319JD 

Exp. Date 28-Feb-2011 

CAS No. 607-57-8 

Purity 98.1% 

1.0 

TA100, TA1535 

sodium azide 

(Sigma Aldrich Chemical Co., Inc.)) 

Lot No. 71980 

Exp. Date 28-Dec-2010 

CAS No. 26628-22-8 

Purity 99.8% 

1.0 

TA1537 

9-aminoacridine 

(Sigma Aldrich Chemical Co., Inc.)) 

Lot No. 106F06682 

Exp. Date 30-Oct-2010 

CAS No. 90-45-9 

Purity >97% 

75 

WP2 uvrA 

None 

methyl methanesulfonate 

(Sigma Aldrich Chemical Co., Inc.) 

Lot No. 76296KJ 

Exp. Date 02-Jun-2012 

CAS No. 66-27-3 

Purity 99.8% 

1,000 

To confirm sterility, the highest te st article concentration used in the mutagenicity assay was 

plated on sel ective agar wit h an aliquot volume equal to that used in the assay. These plat es 

were incubated under the same conditions as the assay. 

MATERIALS AND METHODS 

For submission to Japanese re gulatory agencies, additional information is included in 

Appendix III.

Test System 

The tester st rains used were t he Salmonella typhimurium histidine auxotrophs TA98, TA100, 

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

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BioReliance 

Study No. 

Dow Study No. 9 

described by Green and Muriel ( 1976). Salmonella tester st rains were fr om Dr. Bruce Ames’ 

Master cultures, E. coli tester strains were from the National Collection of Industrial and Marine 

Bacteria, Aberdeen, Scot land an d both s pecies of t ester str ain were di stributed by Mol Tox 

(Boone, NC). 

Tester strai ns TA98 and TA1537 ar e revert ed from hist idine dependenc e (auxotr ophy) t o 

histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted 

by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that 

cause both fra meshift a nd bas epair substit ution mutations. Specif icity of the reversion 

mechanism in E. coli is se nsitive t o ba se-pair substitution mu tations, rather th an fram eshift 

mutations (Green and Muriel, 1976).

Overnight cultures were prepared by inoculating from the appropriate master plate, appropriate 

frozen permanent stock or with a lyophilized pell et into a vessel contai ning ~50 mL of culture 

medium. To assure that cultures were harvested in late log phase, the length of incubation was 

controlled a nd monit ored. Fol lowing inoculation, each f lask wa s place d in a resting 

shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking 

at approxi mately 125 rpm at 37±2 C approximately 12 hours befo re the anticipated time o f 

harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a 

percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The 

actual titers were determined by viable count assays on nutrient agar plates. 

Metabolic Activation System 

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 w as 

prepared fr om male Spr ague-Dawley rats induced with a singl e intraperitoneal injection o f 

Aroclor 1254, 500 mg/ kg, five days prior t o sacrifice. The S9 was prepared by and purchased 

from MolTox. 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 at least two promutagens 

to f orms mutagenic to Salmonella typhimurium TA 100. A copy of the S 9 Qu ality C ontrol 

Certificate is included in Appendix VI. 

The S9 mi x was prepared i mmediately be fore its use a nd contai ned 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 phos phate buffer at pH 7.4. The Sham S9 mi xture ( Sham mix) , 

containing 100 mM phosphate buffer at pH 7.4, wa s 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. 

Solubility Test 

A solubility test was conducted using water and DMSO. The vehicle of choice was the solvent, 

selected in order of pref erence that permitted preparat ion of the highest so luble or workable 

concentration, up to 50 mg/mL in aqueous solvents and up to 500 mg/mL in organic solvents. 

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BioReliance 

Study No. 

Dow Study No. 10 

Initial Toxicity-Mutation Assay 

The initial toxicity-mutation as say was used to e stablish t he dos e-range for the c onfirmatory 

mutagenicity assa y and t o provi de a preli minary m utagenicity eval uation. Ve hicle c ontrol, 

positive controls and a mini mum of e ight dose le vels of the tes t article wer e plated, two plates 

per dose , with overni ght cultur es of TA98, TA 100, TA1535 , TA1537 and WP2 uvrA o n 

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 an d confirm the mutagenic potential 

of the test article. A mi nimum of five dose levels of test article along with appropriate vehicle 

control a nd posit ive c ontrols wer e plated wit h overni ght cult ures of TA98, TA100, TA1535, 

TA1537 and W P2 uvrA on selective minimal a gar i n the pr esence and a bsence of 

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

were plated in triplicate. 

Plating and Scoring Procedures 

The test system was exposed to the t est article via the preincubation methodology described by 

Yahagi et al. (1977).

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

was melted and s upplemented wit h L-hi stidine, D-biotin and L-tryptophan solution to a f inal 

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

25 mL of water for each 100 mL of m inimal t op aga r. For t he pre paration of media and 

reagents, all references to water i mply deio nized water. Bottom ag ar was Vogel- Bonner 

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

agar wa s Vogel- Bonner m inimal medium E cont aining 1.5 % (W/V) a gar and s upplemented 

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). 

Each plate was labeled with a code system t hat identi fied the test article, test phase, 

concentration, t ester str ain and acti vation, as described in detail in BioReliance's Sta ndard 

Operating Procedures. 

One-half (0.5) milliliter of S9 or sham mix, 100 μL of tester strain (cells seeded) and 50 μL of 

vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 

37±2°C. Afte r vort exing, these mi xtures wer e incubated with shaking for 20±2 minutes at 

37±2°C. Following the preincubation, 2.0 mL of selective top agar was a dded to each tube and 

the m ixture w as vortexed and ov erlaid ont o t he sur face of 25 mL of minimal bottom agar. 

When plating the positive controls, the test arti cle aliquot was replaced by a 50 μL aliquot of 

appropriate positive control. After the ove rlay had solidi fied, the plat es were i nverted and 

incubated for appr oximately 52±6 hours at 37± 2C. Plat es that were not counted imm ediately 

following the incubation period were stored at 2-8C until colony counting could be conducted. 

The c ondition of t he bacterial ba ckground l awn was evaluated for evidence of t est arti cle 

toxicity by using a di ssecting microscope. Precipitate was ev aluated by visua l exa mination 

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BioReliance 

Study No. 

Dow Study No. 11 

without magnification. Toxicity and degree of precipitation were scored re lative to the vehicle 

control plate using the codes shown in the following table. 

Code Description Characteristics 

1 or no 

code Normal Distinguished by a healthy microcolony lawn. 

2 Slightly 

Reduced 

Distinguished by a noticeable th inning of the microcolony l awn 

and possibl y a slight increase in t he size of the m icrocolonies 

compared to the vehicle control plate. 

3 Moderately 

Reduced 

Distinguished by a marked thi nning of the microcolony law n 

resulting in a pronounced increase in the size of the m icrocolonies 

compared to the vehicle control plate. 

4 Extremely 

Reduced 

Distinguished by an ext reme thinning of the microcolony lawn 

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

to the vehicle control plate such t hat the micr ocolony la wn is 

visible to the unaided eye as isolated colonies. 

5 Absent Distinguished by a complete lack of any m icrocolony la wn 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 article 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 preci pitate particles detected by the automated 

colony counter exceed 10% of t he r evertant col ony c ount ( e.g., 

more than 3 particles on a plate w ith 30 revertants). These pl ates 

are counted manually. 

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

were counted eit her entirely by automat ed colony counter or entirely by hand unl ess the ass ay 

was the preliminary toxicity assay or the plate exhibited toxicity. 

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 article to be eval uated positive, it must cause a re producible, concentration-related 

increase in t he mean revertant s per plate of at least one tester strain over a m inimum of two 

increasing concentrations of test article. Data sets for tester strains TA98, TA1535, TA1537 and 

WP2 uvrA were judged positive if the increase in mean revertants at the pe ak of t he response 

was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains 

TA100 were judged posi tive if the increase in mean revertants at the peak of the response w as 

greater than or equal to 2.0-times the mean vehicle control value. 

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BioReliance 

Study No. 

Dow Study No. 12 

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

the criteria for evaluati on as positi ve. Thi s could be a dos e-responsive increase that does not 

achieve the respective threshold cited above or a non-dose responsive increase that is equal to or 

greater than the respective thresh old cited. A re sponse will be evaluated as negative, if it is 

neither positive nor equivocal. 

Criteria for a Valid Test 

The following criteria must be me t for t he mutage nicity as say to be consi dered valid. Al l 

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 t he pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the 

deletion i n t he uvrA ge ne. All cult ures must de monstrate t he characteri stic mean number of 

spontaneous revert ants i n the ve hicle control s as f ollows (inclusive): TA98, 10 - 50; TA100, 

80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60. To e nsure that a ppropriate 

numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 

0.3x109

 cells/mL. The mean of at least one positive control per strain and activation conditio n 

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 c oncentration is considered toxic if one or both of the fo llowing criteria are met: 

(1) A >50 % reducti on in the me an number of revert ants per pl ate as compar ed t o the mean 

vehicle control val ue. Thi s reducti on must be acc ompanied by a n a brupt 

concentration-dependent drop i n the revert ant count. (2) At least a mode rate reduction in the 

background lawn (bac kground la wn code 3, 4 or 5). A copy of the His torical Ne gative and 

Positive Control Values is included in Appendix I. 

Archives 

All ra w data , the prot ocol and all r eports, gene rated by BioReli ance, wi ll be ret urned to t he 

Sponsor upon issuance of the final report. Copies of these records may be maintained according 

to Standard Operatin g Procedure OPQP3040 by the BioR eliance Quality Assurance unit 

headquartered at: BioReliance, 14920 Broschart Ro ad, Rockville, MD 2 0850. Per this SOP, 

paper records will be retained for at least th ree years af ter which ti me the Sponsor will be 

contacted for a decision as to the final disposition of the materials. All study materials returned 

to the Sponsor or destroyed w ill first be copied ont o electronic media a nd the electronic copy 

will be retained in the BioReliance archives for a m inimum of 10 years. The raw data, reports 

and other documents generated at locations ot her than BioReliance w ill be archived per th e 

contractual arrangements between that site and the Sponsor. 

Deviation 

No known deviati ons from the protoc ol or as say-method SOPs occ urred during the c onduct of 

this study. 

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BioReliance 

Study No. 

Dow Study No. 13 

RESULTS AND DISCUSSION 

Solubility Test 

DMSO) was selected as the solvent of choice based on the information provided by the Sponsor, 

solubility of the test article, and compatibility with the targ et cells. Af ter sonication for 

60 minutes at 36°C, the test article formed a soluble and clear so lution in DMSO at a 

concentration of approximately 100 mg/mL, the maximum concentration tested in the solubility 

test. 

Sterility Results 

No contaminant colonies were ob served on the sterility plates for the vehicle cont rol, the test 

article dilutions and the S9 and Sham mixes. 

Tester Strain Titer Results 

Tester Strain 

Experiment TA98 TA100 TA1535 TA1537 WP2 uvrA 

Titer Value (x 109

 cells per mL) 

B1 0.9 0.7 0.9 0.4 3.2 

B2 1.7 0.7 1.2 0.6 3.2 

Initial Toxicity-Mutation Assay 

The results of the initial-toxicity mutation assay ar e presented in Tables 1 and 2. These dat a 

were genera ted in Expe riment B1. In the initial toxicity-mutati on as say, the ma ximum 

concentration test ed was 5000 μg per plate. Thi s c oncentration was ac hieved usi ng a 

concentration of 100 mg/ mL and a 50 μL plating aliquot. Th e concentrations tested were 1.5, 

5.0, 15, 50, 150, 500, 1500 and 5000 μg per pl ate. Pre cipitate was obser ved beginning at 50, 

150, 500, 1500 or a t 5000 μg per plate . Toxi city was observed, as r eductions i n revert ant 

counts, at 5000 μg per plate with tester strains TA100, TA1537 and WP2 uvrA in the presence 

of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum dose 

plated in the confirmatory mutagenicity assay was 5000 μg per plate. 

In Experiment B1 (Initial T oxicity-Mutation Assa y), no positive mutagenic resp onses were 

observed with any of the tester strains in either the presence or absence 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 Expe riment B2. I n the c onfirmatory mutagenicity assa y, the concentrations 

tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 

500, 1500 or at 5000 μg per plate. Toxicity was observed, as reductions in revertant counts, at 

5000 μg per plate with t ester str ains TA100, TA1535 a nd WP2 uvrA in the presence of S9 

activation. 

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BioReliance 

Study No. 

Dow Study No. 14 

In Experiment B2 (Confirm atory Mutagenicity Assay), no pos itive mutagenic responses were 

observed with any of the tester strains in either the presence or absence of S9 activation. 

Dosing Formulation Analysis 

Dosing formulations were analyzed by the Sponsor. A copy of the analytic al report is included 

in Appendix V . Concentration analysis indicates that the actual mean concentrations of the 

analyzed dose levels were bet ween 91.2 a nd 97. 4% of t arget. This indic ates t hat th e 

regulatory-required top dose level was achieved and the results support the validity of the study 

conclusion. No t est art icle was det ected i n the vehi cle c ontrol s ample. Si nce t he m ost 

concentrated dosing formulation was within 85 to 115% of target, the dosing formulations were 

considered stable for the purpose of this study. 

CONCLUSION 

All criteria for a valid study were met as described in the protocol. The results of t he Bacterial 

Reverse Mutation Assay indicate th at, under the conditions of this study, 

was negative both in the presence and absence of Aroclor-induced rat liver S9. 

REFERENCES 

Ames, B.N., J. Mc Cann and E. Ya masaki (1 975) M ethods for Dete cting Carcinogens a nd 

Mutagens with the Salmonella/Mammalian Microsome Muta genicity Test, Mutatio n 

Research, 31:347-364. 

EC: B.13/14, Mutagenicity – Reverse Mutation T est Using Bacteria. Official Journal of the 

European Communities, L142, 31/05/2008. 

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. 

U.S. EPA: Health Effects Test Guidelin es, OPPTS 870.5100, Bacterial Reverse Mutation 

Test, August 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., Na gao, M., Seino, Y., Mats ushima, T., Sugi mura, T. a nd Okada, M . (1 977) 

Mutagenicities of N-nitrosamines on Salmonella, Mutation Research, 48:121-130. 

- 
BioReliance 

Study No. 

Dow Study No. 15 

DATA TABLES 

- 
BioReliance 

Study No. 

Dow Study No. 16 

Table 1 

Initial Toxicity-Mutation Assay without S9 activation 

Study Number: Study Code: 

Experiment: B1 Date Plated: 6/11/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 6/15/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA98 5000 μg 20 8 0.6 26M 6 IP, 14M 6 IP 

1500 μg 19 1 0.5 20M 6 NP, 18M 6 NP 

500 μg 31 1 0.9 32M 6 NP, 30M 6 NP 

150 μg 34 2 0.9 35M 1 NP, 32M 1 NP 

50 μg 32 10 0.9 39M 1 NP, 25M 1 NP 

15 μg 31 4 0.9 33M, 28M 

5.0 μg 27 8 0.8 21M, 32M 

1.5 μg 34 2 0.9 35M, 32M 

DMSO 50 μL 36 5 39M, 32M 

TA100 5000 μg 85 4 1.0 88M 6 IP, 82M 6 IP 

1500 μg 91 6 1.1 95M 6 NP, 86M 6 NP 

500 μg 82 6 1.0 77M 6 NP, 86M 6 NP 

150 μg 90 1 1.1 91M 1 NP, 89M 1 NP 

50 μg 73 16 0.9 61M, 84M 

15 μg 76 6 0.9 72M, 80M 

5.0 μg 85 5 1.0 81M, 88M 

1.5 μg 88 7 1.1 83M, 93M 

DMSO 50 μL 82 3 84M, 80M 

TA1535 5000 μg 9 0 0.5 9M 6 IP, 9M 6 IP 

1500 μg 14 1 0.8 13M 6 NP, 15M 6 NP 

500 μg 11 1 0.6 10M 6 NP, 12M 6 NP 

150 μg 14 1 0.8 14M 1 NP, 13M 1 NP 

50 μg 15 3 0.9 17M 1 NP, 13M 1 NP 

15 μg 16 4 0.9 18M, 13M 

5.0 μg 18 9 1.1 11M, 24M 

1.5 μg 14 3 0.8 16M, 12M 

DMSO 50 μL 17 7 22M, 12M 

Key to Plate Postfix Codes 

6 

IP 

NP 

1 

Background obscured by particulate 

Interfering particulate 

Non-interfering particulate 

Normal background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 

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BioReliance 

Study No. 

Dow Study No. 17 

Table 1 cont. 

Initial Toxicity-Mutation Assay without S9 activation 

Study Number: Study Code: 

Experiment: B1 Date Plated: 6/11/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 6/15/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA1537 5000 μg 6 4 0.8 3M 6 IP, 8M 6 IP 

1500 μg 5 3 0.6 7M 6 IP, 3M 6 IP 

500 μg 4 1 0.5 5M 1 NP, 3M 1 NP 

150 μg 6 0 0.8 6M 1 NP, 6M 1 NP 

50 μg 5 0 0.6 5M, 5M 

15 μg 5 1 0.6 4M, 6M 

5.0 μg 5 2 0.6 6M, 3M 

1.5 μg 6 1 0.8 6M, 5M 

DMSO 50 μL 8 0 8M, 8M 

WP2uvrA 5000 μg 26 0 0.8 26M 6 IP, 26M 6 IP 

1500 μg 28 6 0.9 23M 6 NP, 32M 6 NP 

500 μg 22 4 0.7 19M 1 NP, 25M 1 NP 

150 μg 24 6 0.8 19M, 28M 

50 μg 29 4 0.9 32M, 26M 

15 μg 31 5 1.0 27M, 34M 

5.0 μg 29 6 0.9 33M, 24M 

1.5 μg 31 5 1.0 27M, 34M 

DMSO 50 μL 32 2 30M, 33M 

TA98 2NF 1.0 μg 291 11 8.1 283A, 298A 

TA100 SA 1.0 μg 839 52 10.2 802A, 875A 

TA1535 SA 1.0 μg 631 66 37.1 584A, 677A 

TA1537 9AAD 75 μg 279 20 34.9 293A, 265A 

WP2uvrA MMS 1000 μg 638 16 19.9 626A, 649A 

Key to Positive Controls Key to Plate Postfix Codes 

2NF 

SA 

9AAD 

MMS 

2-nitrofluorene 

sodium azide 

9-Aminoacridine 

methyl methanesulfonate 

6 

IP 

NP 

1 

Background obscured by particulate 

Interfering particulate 

Non-interfering particulate 

Normal background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 

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BioReliance 

Study No. 

Dow Study No. 18 

Table 2 

Initial Toxicity-Mutation Assay with S9 activation 

Study Number: Study Code: 

Experiment: B1 Date Plated: 6/11/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 6/15/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA98 5000 μg 21 0.0 0.8 21M 6 IP, CPN#*

1500 μg 33 1 1.2 32M 1 NP, 33M 1 NP 

500 μg 25 2 0.9 26M, 23M 

150 μg 27 1 1.0 26M, 28M 

50 μg 36 4 1.3 39M, 33M 

15 μg 38 5 1.4 34M, 41M 

5.0 μg 41 3 1.5 43M, 39M 

1.5 μg 33 6 1.2 37M, 29M 

DMSO 50 μL 28 6 32M, 23M 

TA100 5000 μg 43 10 0.4 50M 6 IP, 36M 6 IP 

1500 μg 56 1 0.6 55M 1 NP, 57M 1 NP 

500 μg 58 3 0.6 56M, 60M 

150 μg 78 15 0.8 67M, 88M 

50 μg 79 1 0.8 80M, 78M 

15 μg 76 16 0.8 87M, 64M 

5.0 μg 72 4 0.7 69M, 75M 

1.5 μg 81 6 0.8 77M, 85M 

DMSO 50 μL 99 9 105M, 92M 

TA1535 5000 μg 5 1 0.5 4M 6 IP, 6M 6 IP 

1500 μg 6 1 0.6 6M 6 NP, 5M 6 NP 

500 μg 8 3 0.8 10M, 6M 

150 μg 9 3 0.9 7M, 11M 

50 μg 12 1 1.2 11M, 12M 

15 μg 10 4 1.0 7M, 12M 

5.0 μg 8 4 0.8 11M, 5M 

1.5 μg 11 1 1.1 11M, 10M 

DMSO 50 μL 10 1 9M, 11M 

Key to Plate Postfix Codes Key to Plate Postfix Codes 

6 

IP 

Background obscured by particulate 

Interfering particulate 

1 

NP 

CP 

N# 

Normal background 

Non-interfering particulate 

Contaminated plate 

Not counted 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count *

 The loss of this test article-treated plate does not invalidate the results since the remaining plate at this dose 

level is comparable to the negative control. 

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BioReliance 

Study No. 

Dow Study No. 19 

Table 2 cont. 

Initial Toxicity-Mutation Assay with S9 activation 

Study Number: Study Code: 

Experiment: B1 Date Plated: 6/11/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 6/15/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA1537 5000 μg 2 1 0.3 2M 6 IP, 1M 6 IP 

1500 μg 5 0 0.7 5M 1 NP, 5M 1 NP 

500 μg 3 0 0.4 3M, 3M 

150 μg 6 1 0.9 5M, 7M 

50 μg 8 5 1.1 4M, 11M 

15 μg 10 3 1.4 8M, 12M 

5.0 μg 10 1 1.4 9M, 10M 

1.5 μg 12 4 1.7 9M, 15M 

DMSO 50 μL 7 2 8M, 5M 

WP2uvrA 5000 μg 14 1 0.4 13M 6 IP, 15M 6 IP 

1500 μg 22 7 0.7 17M 6, 27M 6 

500 μg 30 1 0.9 31M, 29M 

150 μg 28 4 0.9 30M, 25M 

50 μg 27 3 0.8 25M, 29M 

15 μg 27 1 0.8 28M, 26M 

5.0 μg 32 9 1.0 38M, 25M 

1.5 μg 30 1 0.9 30M, 29M 

DMSO 50 μL 32 6 36M, 28M 

TA98 2AA 1.0 μg 505 20 18.0 519A, 491A 

TA100 2AA 2.0 μg 997 355 10.1 746A, 1248A 

TA1535 2AA 1.0 μg 100 18 10.0 87A, 112A 

TA1537 2AA 1.0 μg 55 6 7.9 51A, 59A 

WP2uvrA 2AA 10 μg 175 40 5.5 203A, 147A 

Key to Positive Controls Key to Plate Postfix Codes 

2AA 2-aminoanthracene 6 

IP 

1 

NP 

Background obscured by particulate 

Interfering particulate 

Normal background 

Non-interfering particulate 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 

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BioReliance 

Study No. 

Dow Study No. 20 

Table 3 

Confirmatory Mutagenicity Assay without S9 activation 

Study Number: Study Code: 

Experiment: B2 Date Plated: 6/25/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 7/2/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA98 5000 μg 23 10 0.5 27M 6 IP, 31M 6 IP, 

12M 6 IP 

1500 μg 34 5 0.8 37M 6 NP, 36M 6 NP, 

28M 6 IP 

500 μg 39 4 0.9 39M 1 NP, 43M 1 NP, 

35M 1 NP 

150 μg 44 5 1.0 45M, 39M, 49M 

50 μg 35 7 0.8 38M, 39M, 27M 

15 μg 37 11 0.9 30M, 32M, 50M 

DMSO 50 μL 43 8 50M, 34M, 46M 

TA100 5000 μg 81 13 1.0 93M 6 IP, 67M 6 IP, 

82M 6 IP 

1500 μg 84 12 1.0 72M 6 NP, 85M 6 NP, 

95M 6 NP 

500 μg 77 8 0.9 74M 1 NP, 86M 1 NP, 

72M 1 NP 

150 μg 90 12 1.1 83M, 84M, 104M 

50 μg 89 4 1.0 88M, 93M, 85M 

15 μg 89 7 1.0 92M, 81M, 93M 

DMSO 50 μL 85 6 89M, 87M, 78M 

TA1535 5000 μg 11 2 0.9 8M 6 IP, 12M 6 IP, 

12M 6 IP 

1500 μg 14 2 1.2 12M 6 NP, 14M 6 NP, 

16M 6 NP 

500 μg 11 2 0.9 11M 6 NP, 13M 6 NP, 

10M 6 NP 

150 μg 9 4 0.8 4M, 12M, 10M 

50 μg 9 3 0.8 6M, 11M, 9M 

15 μg 11 6 0.9 18M, 7M, 7M 

DMSO 50 μL 12 1 13M, 12M, 12M 

Key to Plate Postfix Codes 

6 

IP 

NP 

1 

Background obscured by particulate 

Interfering particulate 

Non-interfering particulate 

Normal background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 

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BioReliance 

Study No. 

Dow Study No. 21 

Table 3 cont. 

Confirmatory Mutagenicity Assay without S9 activation 

Study Number: Study Code: 

Experiment: B2 Date Plated: 6/25/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 7/2/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA1537 5000 μg 7 1 1.4 6M 6 IP, 7M 6 IP, 7M

6 IP 

1500 μg 3 1 0.6 4M 6 IP, 4M 6 IP, 2M

6 IP 

500 μg 5 2 1.0 4M 1 NP, 3M 1 NP, 

7M 1 NP 

150 μg 3 2 0.6 5M, 4M, 1M 

50 μg 5 2 1.0 5M, 7M, 3M 

15 μg 5 2 1.0 4M, 8M, 4M 

DMSO 50 μL 5 1 4M, 6M, 6M 

WP2uvrA 5000 μg 23 2 0.7 22M 6 IP, 22M 6 IP, 

25M 6 IP 

1500 μg 34 6 1.0 38M 6 NP, 28M 6 NP, 

37M 6 NP 

500 μg 31 8 0.9 24M 1 NP, 39M 1 NP, 

29M 1 NP 

150 μg 31 8 0.9 27M, 40M, 26M 

50 μg 30 2 0.9 31M, 30M, 28M 

15 μg 29 1 0.9 30M, 29M, 29M 

DMSO 50 μL 34 3 36M, 36M, 31M 

TA98 2NF 1.0 μg 432 111 10.0 305A, 488A, 504A 

TA100 SA 1.0 μg 825 32 9.7 805A, 807A, 862A 

TA1535 SA 1.0 μg 555 70 46.3 494A, 539A, 631A 

TA1537 9AAD 75 μg 311 62 62.2 370A, 316A, 246A 

WP2uvrA MMS 1000 μg 502 26 14.8 482A, 531A, 494A 

Key to Positive Controls Key to Plate Postfix Codes 

2NF 

SA 

9AAD 

MMS 

2-nitrofluorene 

sodium azide 

9-Aminoacridine 

methyl methanesulfonate 

6 

IP 

NP 

1 

Background obscured by particulate 

Interfering particulate 

Non-interfering particulate 

Normal background 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 

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BioReliance 

Study No. 

Dow Study No. 22 

Table 4 

Confirmatory Mutagenicity Assay with S9 activation 

Study Number: Study Code: 

Experiment: B2 Date Plated: 6/25/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 7/2/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

TA98 5000 μg 23 5 0.5 20M 6 IP, 28M 6 IP, 

20M 6 IP 

1500 μg 34 6 0.8 39M, 37M, 27M 

500 μg 30 2 0.7 28M, 30M, 31M 

150 μg 46 7 1.1 38M, 47M, 52M 

50 μg 40 9 0.9 42M, 48M, 30M 

15 μg 49 4 1.1 52M, 45M, 50M 

DMSO 50 μL 43 11 32M, 53M, 45M 

TA100 5000 μg 40 6 0.4 34M 6 IP, 43M 6 IP, 

44M 6 IP 

1500 μg 57 13 0.6 44M, 69M, 58M 

500 μg 63 17 0.7 72M, 44M, 74M 

150 μg 77 22 0.9 52M, 96M, 82M 

50 μg 76 13 0.9 62M, 82M, 85M 

15 μg 76 7 0.9 76M, 83M, 69M 

DMSO 50 μL 89 10 100M, 81M, 87M 

TA1535 5000 μg 6 2 0.5 7M 6 IP, 7M 6 IP, 4M

6 IP 

1500 μg 4 1 0.3 5M 1 NP, 3M 1 NP, 

4M 1 NP 

500 μg 7 2 0.5 8M, 9M, 5M 

150 μg 7 1 0.5 7M, 6M, 8M 

50 μg 9 1 0.7 10M, 8M, 8M 

15 μg 13 4 1.0 17M, 9M, 13M 

DMSO 50 μL 13 1 12M, 14M, 12M 

TA1537 5000 μg 3 1 0.6 3M 6 IP, 3M 6 IP, 2M

6 IP 

1500 μg 2 1 0.4 3M 1 NP, 1M 1 NP, 

2M 1 NP 

500 μg 6 2 1.2 4M, 7M, 7M 

150 μg 4 1 0.8 5M, 4M, 3M 

50 μg 4 3 0.8 8M, 2M, 3M 

15 μg 6 1 1.2 6M, 5M, 6M 

DMSO 50 μL 5 2 7M, 4M, 4M 

Key to Plate Postfix Codes Key to Plate Postfix Codes 

6 

IP 

Background obscured by particulate 

Interfering particulate 

1 

NP 

Normal background 

Non-interfering particulate 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 

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BioReliance 

Study No. 

Dow Study No. 23 

Table 4 cont. 

Confirmatory Mutagenicity Assay with S9 activation 

Study Number: Study Code: 

Experiment: B2 Date Plated: 6/25/2010 

Exposure Method: Pre-incubation assay Evaluation Period: 7/2/2010 

Strain Article Dose level 

per plate 

Mean 

revertants 

per plate 

Standard 

Deviation 

Ratio 

treated / 

solvent 

Individual revertant 

colony counts and 

background codes 

WP2uvrA 5000 μg 14 4 0.5 18M 6 IP, 11M 6 IP, 

14M 6 IP 

1500 μg 17 2 0.5 18M 1 NP, 15M 1 NP, 

17M 1 NP 

500 μg 34 3 1.1 32M, 37M, 34M 

150 μg 31 5 1.0 26M, 32M, 36M 

50 μg 35 5 1.1 29M, 36M, 39M 

15 μg 36 8 1.2 40M, 41M, 26M 

DMSO 50 μL 31 4 35M, 30M, 28M 

TA98 2AA 1.0 μg 722 41 16.8 736A, 676A, 754A 

TA100 2AA 2.0 μg 844 45 9.5 807A, 831A, 895A 

TA1535 2AA 1.0 μg 91 10 7.0 79A, 99A, 94A 

TA1537 2AA 1.0 μg 98 17 19.6 115A, 82A, 98A 

WP2uvrA 2AA 10 μg 247 54 8.0 185A, 269A, 287A 

Key to Positive Controls Key to Plate Postfix Codes 

2AA 2-aminoanthracene 6 

IP 

1 

NP 

Background obscured by particulate 

Interfering particulate 

Normal background 

Non-interfering particulate 

Key to Automatic & Manual Count Flags 

M: Manual count A: Automatic count 

- 
BioReliance 

Study No. 

Dow Study No. 24 

APPENDIX I: Historical Control Data 

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BioReliance 

Study No. 

Dow Study No. 25 

Historical Negative and Positive Control Values 

2007 – 2009 

revertants per plate 

Activation 

Strain Control None Rat Liver 

Mean SD Min Max Mean SD Min Max 

Neg 18 7 4 57 25 8 6 69 TA98 

Pos 221 131 34 1299 526 245 49 2342

Neg 132 32 51 255 141 35 56 268

TA100 

Pos 613 153 226 1837 776 380 224 3206

Neg 17 7 3 58 15 5 1 49

TA1535 

Pos 456 159 33 1601 120 85 18 1216

Neg 8 4 0 28 8 4 1 41

TA1537 

Pos 1040 576 24 4814 102 156 13 2360

Neg 28 11 6 72 31 12 5 77

WP2 uvrA 

Pos 344 163 44 1178 274 131 32 1656

SD=standard deviation; Min= minimum value; Max=maximum value; Neg=ne gative control 

(including but not li mited to deionized water, di methyl sulfoxi de, ethanol and ac etone); 

Pos=positive control 

- 
BioReliance 

Study No. 

Dow Study No. 26 

APPENDIX II: Study Protocol 

- 
BioReliance 

Study No. 

Dow Study No. 27 

- 
BioReliance 

Study No. 

Dow Study No. 28 

- 
BioReliance 

Study No. 

Dow Study No. 29 

- 
BioReliance 

Study No. 

Dow Study No. 30 

- 
BioReliance 

Study No. 

Dow Study No. 31 

- 
BioReliance 

Study No. 

Dow Study No. 32 

- 
BioReliance 

Study No. 

Dow Study No. 33 

- 
BioReliance 

Study No. 

Dow Study No. 34 

- 
BioReliance 

Study No. 

Dow Study No. 35 

- 
BioReliance 

Study No. 

Dow Study No. 36 

- 
BioReliance 

Study No. 

Dow Study No. 37 

- 
BioReliance 

Study No. 

Dow Study No. 38 

- 
BioReliance 

Study No. 

Dow Study No. 39 

- 
BioReliance 

Study No. 

Dow Study No. 40 

- 
BioReliance 

Study No. 

Dow Study No. 41 

- 
BioReliance 

Study No. 

Dow Study No. 42 

- 
BioReliance 

Study No. 

Dow Study No. 43 

- 
BioReliance 

Study No. 

Dow Study No. 44 

- 
BioReliance 

Study No. 

Dow Study No. 45 

APPENDIX III: Information for Japanese Regulatory Agencies 

- 
BioReliance 

Study No. 

Dow Study No. 46 

Report of Results for Salmonella – Escherichia coli/Mammalian-Microsome Reverse 

Mutation Assay Preincubation Method with a Confirmatory Assay with 

1. Tester Strains 

(1) Procurement 

Strain Obtained from Date obtained 

 Date inspected 

the strain lot 

in storage 

TA98 

TA100 

TA1535 

TA1537 

WP2 uvrA 

Salmonella tester strains 

were from Dr. Bruce 

Ames’ Master cultures, E. 

coli tester strains were 

from the National 

Collection of Industrial 

and Marine Bacteria, 

Aberdeen, Scotland and 

both species of tester strain 

were distributed by 

MolTox (Boone, NC). 

Single use 

lyophilized pellets 

used for each 

inoculation (QC 

Statement 

maintained in 

BioReliance files) 

The genetic 

markers for each 

culture are 

confirmed on the 

day of use 

(2) Storage 

Freezing method Large quantity 

Storage temperature -60C or colder 

Bacterial suspension 1.0 mL 

Composition DMSO 0.09 mL 

- 
BioReliance 

Study No. 

Dow Study No. 47 

2. S9 Mix 

(1) Source, Storage Temperature, etc. of S9 

Prepared on Used in Experiment No. Purchased from 

Moltox 07 April 2010 (Lot 2581) B1 and B2 

Storage temperature -60C or colder 

Name and 

model of 

storage 

apparatus 

So-Low, Model 

PR120-9 

(2) Preparation of S9 

Animal used Inducing substance 

Species, Strain Rattus norvegicus, Sprague 

Dawley Name Aroclor 1254 

Sex Male Administration 

method intraperitoneal 

Age (in weeks) Unknown (Lot 2581) 

Weight Unknown (Lot 2581) 

Administration 

period and 

amount 

(g/kg-weight) 

single dose at 

0.5 gm/kg body 

weight, 5 days 

prior to sacrifice 

3. Preparation of Test Substance Solution 

Solvent used 

Name Manufacturer Lot No. Grade and/or 

Purity (%) 

DMSO 

(CAS No. 67-68-5) 

EMD Chemicals 

Incorporated 49288950  99.9% 

 Stability of test substance in 

the solvent (See Appendix V). 

 Method of suspension when 

test substance is difficult to 

dissolve 

To achi eve a sol ution, t he mo st concentrated dilutio n 

was sonicated at 35° C for approximately 10 mi nutes in 

the B1 ass ay and a pproximately 15 m inutes i n the B2 

assay. 

- 
BioReliance 

Study No. 

Dow Study No. 48 

4. Conditions of Pre-culture 

Nutrient broth Name Manufacturer Lot No. 

Oxoid 

Nutrient 

Broth No. 2 

Oxoid Ltd. 823402 

 Period of pre-culture 12±2 hours 

 Storage time and temp. from 

inoculation to beginning of 

shaking culture 

<5 hours at ambient temperature 

 Storage time and temp. from 

end of culture to use for test <12 hours at 2-8°C 

 Model and manufacturer of 

shaker New Brunswick Scientific, model G-24 

 Method of shaking 

 (shaking type, speed, etc.) Rotary (125 rev/min.) 

 Culture vessel (shape, 

capacity) shape: cylinder, 200 mL 

 Culture volume 50 mL 

 Volume of inoculum 1 colony or 1 to 2 pellets 

- 
BioReliance 

Study No. 

Dow Study No. 49 

5. Agar Plate Medium 

(1) Top agar 

Name BBL Select 

Manufacturer Becton Dickinson 

Lot No. 9265193 

Name BD Bacto 

Manufacturer Becton Dickinson 

Agar 

Lot No. 9341026 

(2) Minimum Glucose Agar 

Name BBL Select 

Manufacturer Becton Dickinson 

Lot No. 9265193 

Batch 

No. Preparation Date Used in Experiment No. 

35711 02 June 2010 B1 and B2 

Agar 

35699 01 June 2010 B2 

Purchased from MolTox 

Volume of agar plate medium 25 mL 

6. Test Results - Judgement of the results 

 Judgem ent Negative 

Reason for judgement and referential matters: 

No positive mutagenic response was observed with any of the tester strains in either the 

presence or absence of Aroclor-induced rat liver S9. 

Referential matters 

The vehicle and positive control values indicate that all tester strains were functioning 

correctly and were capable of detecting a mutagen. 

- 
BioReliance 

Study No. 

Dow Study No. 50 

APPENDIX IV: Certificate of Analysis 

- 
BioReliance 

Study No. 

Dow Study No. 51 

- 

--

IA Study

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CERTIFICATE Qf ANA LY SIS FOR TESTIREfEKW(ElCUNTROL SUBSTANCES 

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{:o "'pl .. i<on I)"I~ : 

5 n ;DY DIRECTOR M'O TESTI."G FACIUTI' -\IlUIU:.s.\;: 

Imf"l'-"1 ,\ ""))10.:.1 . 19111 'II' S I A,..Jrew5 Rd MI<lI. nd . • \1 1 J~MO 
BioReliance 

Study No. 

Dow Study No. 52 

APPENDIX V: Dosing Formulation Analysis

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BioReliance 

Study No. 

Dow Study No. 53 

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Toxicology Infomlation Collection (TIC) Report 

111e Dow Chemical Company 

L',;;-A. (nk23r.s) 1:;;..0 ID 

~ ¥, ; 1 0 

IRalph R. Abec -6 - 11 - >' 

~ '.::wI D 1 5 

cl 

IN. America ITERC In/. 

l:study number: 

Salrnonclla·Eschcrichia Co il an n:l li irc roso rrc Reverse Mutation A ss ay 

Study Title: Preincubation Mcthod with;} Confim13tory A ssay wilh

File number: Projec t #: TaskJrep: 45 1- 1 

PI"Oject I Sample Description (optional): 

Samp les in OMSO (BioRe liallCe Study ID:

Test nlaterial(s): 

Test nlateriallot #: 

Report I"ccipienl(s): Jill Smalley 

Analysis request date: 6123120 10 

Date s ubmittor prelJrared s amples: 612SflO l0 

Numi>erors amples orestimate: 7 

Date samples prepared roranalysis: 6/301201 0 

An .. 'llysis date : 6/301201 0 

Nwnber or samples analyzed: 7 

Analysis me thod: HPLCIUV 

Method rerere nce: TERCSummary: 

The dose solutions prepared at BioReliance were analyzed to confirm the concentrat ions of 

in dimethyl sulfoxide (DMSO). The analysis was performed using 

high performance liquid chromatography with ultnlviolet detection (HPLC/UV). The levels 

of were quantified by using external standard calibration. 

PAGE I 
Dow Study No. 

Study No. 

BioReliance 

54 

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Table I. Results of co

ncentration anal yses 

Dose 

Ol,e Mi,cd; 06121110 Overa ll Overall 

l.. Ret:;n.: 20 10·91 

'c. (m&,mL): 0.00 

0.0 ",",cd Cone: d l Q 

'lo or Targt t Cone: 

(n): (2) 

Tar,,"l Cone. (m&,mL): 0.300 

0.3 Mean 

MU 5W"cd Cone: 0.274 

'lo or Target Cone: 9l.l% 91.2% NA 

(n): (2) 

Tar,,", Cone. (m&,

mL): U" 

' .0 Mean 

MU5W"cdCone: 0.974 

'loorT .. , ,, Cone: 91.4'lo 97.4% 

NA 

(n): (2) 

Tugtt Con<;. (m&,mL): 300 

3.0 

Mean Mea$ll"N Cone: 2.92 

'loor T.r,t t Cone: 97.4 'lo 97.4 % NA 

(n): (2) 

Targe' Cone. (m&,mL): 10.0 

'" 

Mean MUllI"cd Cone! 9.62 

'lo or Tar, ,, Cone' 96.2 % 96.2 % NA 

(n): (2) 

Targtt Co...:. (m&,mL): 30.0 

30 Mean 

Me_edCone: 29.0 

'lo ofT.,",,, Cone: 96.1% 96.1% 

NA 

(n): (2) 

Targel COne. (m&,mL): 100 

100 

Mean 

Mel5W"wConc: 93.7 

% of1llrgu Cone' 93.1% 93.7% NA 

(n): (2) 

LLQ . (mymL): 

u.UOJOi 0.00401 Nit: 

<lLQ . q lhan 10 ........ limit of 1jIWI1IIiou on 

NA_ Not Applicable 
BioReliance 

Study No. 

Dow Study No. 55 

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Sample Preparation: 

Dose solutions of in dimethyl su lfoxide (DMSO) and a DMSO 

vehicle control were prepared al BioReliance on 6125110 (BioReliance Study 10: 

Samples were shipped on 6128110, received on 612911 0. and 

placed in a -20 {lC freezer. Samples were removed from storage on 6130110. allowed to 

thaw to ambient temperature. and prepared in the following manner. Samples were 

vonex-mixed prior 10 sampling. Duplicate aliquols ranging from 10 - 100 f.lL were taken 

from the center of each sample. All samples were first diluted with aceton itrile. Samples 

were then dil uted with 50150 Milli-Q water/acetonilTile + 0. 1 % formic acid to obtain final 

concentrations of approximately 10.0 - 15.0 IJ.glmL. The final di lutions of each sample 

were vonex-mixed and aliquots were transferred 10 glass autosampler vials for analysis. 

All samples were analyzed for by high performance liquid 

chromatography with ultraviolet detection (HPLC/UV). The levels of

were quantified by using external standard calibration. 

Standard and Spike Preparation: 

Standards were prepared covering the concentration range of 0.100 - 50.2 f.lg 

per mL 50150 Milli-Q waterlacetonitrile + 0. 1% formic acid. Spikes were prepared 

at approximate concentrations of 0.3, 10 and 100 mg per rnL DMSO. 

Results: 

Standards were found to be linear over the range of 0.201 - 25.1 I-tglmL with an R2 value 

of 0.9999. Average spike recovery was found to be 102% with a standard deviation of 

1.38 %. 

PAGE 3 
BioReliance 

Study No. 

Dow Study No. 56 

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COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDA RDS 

Compound: 

Title: Salmonella - escherichia colilMmnmalian-Microsome Reverse Mutation 

Assay Preincubation Method with a Confirmatory Assay with 

All phases of this study were conducted in compliance with the following Good 

laboratory Practice Standards: 

US Depanmcnt of Health and Human Services Food and Drug Administration -

GlPs; Title 21 CFR, Part 58 - Good Laboratory Practice Regulations for Noncli nical 

laboratory Studies, Final Rule 

Japanese Ministry of Economy, Trade, and Industry Good Laboratory Practices 

(METO 

Organisation for Economic Co-Operation and Development (DECO) 

DECO Series on Principles of Good l aboratory Practice and Compliance Monitoring, 

Number I. DECO Principles on Good Laboratory Practice (as revised in 1997) 

ENVIMC/CHEM(98) 17 

Exception: Test Material Characterization was conducted concurrently with the study. 

PAGE4 
BioReliance 

Study No. 

Dow Study No. 57 

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QUALITY ASSURANCE STATEMENT 

Compound: 

Title: Salmollella - Escherichia coli/Mammalian-Microsome Reverse Mutation 

Assay Preincubation Method with a Confirmatory Assay with 

This study was examined for confomlUnce with Good Laboratory Practices as published 

by the USFDA, METI. and DECO. The final report was determined to be an accurate 

reflection of the data obtained. The dates of Quality Assurance activities on this study are 

listed below. 

Study Initiation Date: 08 JUN 2010 

TYPE OF AUPIT 

Study conduct 

PrOtOCOl. data, and 

draft report 

PATE OF AUDIT 

30JUN2010-

06 AUG 20 10 

07 AUG 2010-

09 AUG 2010 

PATE REPORTEP 

TO PRINCIPLE 

INVESTIGATOR! 

PI MANAGEMENT 

06 AUG 2010 

10 AUG 2010 

DATE REPORTED TO 

STUDY DIRECTOR! 

Sp MANAGEMENT 

06 AUG 2010 

10 AUG 2010 

Final report The date of the signature below is the date of lhe final report audit. 

The final report accurately reflects the raw data of the study. 

1803 Building 

Midland, Michigan 48674 

PAGE 5 
BioReliance 

Study No. 

Dow Study No. 58 

APPENDIX VI: S9 Quality Control Certificate 

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BioReliance 

Study No. 

Dow Study No. 59 

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