### 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 Chemical s 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

### 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.

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### 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

### BioReliance Study No. Dow Study No. 5 TABLE OF CONTENTS Page

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

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

CHARACTERIZATION OF TE ST AND CONTROL ART ICLES.........................................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

### 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 Sal monella typhi murium test er strains TA98, TA 100, TA1535 and TA1537 a nd Escherichia coli tester strain WP2 uvr A 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 provid e a preli minary mutagenicity ev aluation. The s econd phase, the confirmatory mu tagenicity assay, was used to eval uate and confirm the mu tagenic 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 xi mum concentration tested i n the solubility test.

In the initial toxici ty-mutation assay, the maxi mum concentration tested was 5000 μg per plate, which is the ma ximum concentra tion recommended by test guidelin es. This concentr ation was achieved using a concentration of 100 mg/mL and a 50 μL plati ng 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 abse nce of S9 acti vation. Pr ecipitate was observed beginning at 50, 150, 500, 1500 or at 50 00 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 uvr A 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 a ssay was 5000 μg per plate.

In the confir matory mutagenicity assay, the c on centrations 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, TA 1535 and WP2 uvr A in the presence of S9 activation.

All criteria for a valid st udy were met as des cribed in the pr otocol. The vehicle controls and positive controls in the initial toxicity-mutation and conf irmato ry 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 stud y was to eval uate the mutagenic potenti al of the test ar ticle, by measuring its ability to induce reverse mutatio ns at selected loci of several strains of Salmonella typhimurium and at the tryp tophan locus of Escherichia coli strain WP2 uvr A in the presence and absence of Ar oc lor-induced rat liver S9. A co py of t he st udy pr otocol is included in Appendix II

.

### 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 A nnex 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 recei ved 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 descri bed 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 ficat ion 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 an alysis. The remaining se t of samples from the confirmatory mutagenicity assa y and all sample s from t he initial toxici ty-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.

### 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 uvr A 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 uvr A 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 us ed 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 uvr A as

### BioReliance Study No. Dow Study No. 9 described by Green and Muriel ( 1976)

. Salmonella tester st rains were fr om Dr. Bruc e Ames’ Master cultures, E

. coli tester strains were from the Nationa l 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 su bstitutions. Tester strain TA100 is reverted by mutagens that cause both fra meshift a nd bas epair substit u tion mutations. Specif ic ity of the reversion mechanism in E. coli is se nsitive t o ba se-pai r substitution mu tations, rather th an fram eshift mutations (Green and Muriel, 1976).

Overnight cultures were prepared by inoculating from th e appropriate master plate, a ppropriate 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 l owing inoculation, each f las k wa s place d in a resting shaker/incubator at room temper ature. 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 fo r turbidity and wa s harvested at a percent transmittance yiel ding a titer of greater than or equal to 0.3x10 9 

### cells per milliliter. The actual titers were dete rmined by viab le count assays on nu trient 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-Da wley rats induced with a singl e intraperitoneal injection o f Aroclor 1254, 500 mg/ kg, fi ve 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 m mediately be fore its use a nd contai ned 10% S9, 5 mM glucose-6-phosphate, 4 mM ß-nicotinamide-ad enine dinucleotide phosphate, 8 mM MgCl 2 

### and 33 mM KCl in a 100 mM phos ph ate buffer at pH 7.4. The Sham S9 mi xture ( Sham mix) , containing 100 mM phosphate buffe r at pH 7.4, wa s prepared imme diately before its use. To confirm the sterility of the S9 an d 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 aqu eous solvents and up to 500 mg/mL in organic solvents.

### 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-ra nge for the c onfirmatory mutagenicity assa y and t o provi de a preli minar y 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 uvr A 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 fi ve 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 uvr A on selective minimal a gar i n the pr esence and a bsence of Aroclor-induced rat li ver 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 arti cle via the pr eincubatio n methodology described by Yahagi et al. (1977).

On the da y of its use , minimal top a gar, cont aining 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. To p agar not used with S9 or Sh am 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 (d ry 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 incu bation 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

### BioReliance Study No. Dow Study No. 11 without magnification. Toxicity and degree of precip itation were scored re lative to the vehicle control plate using the codes s hown 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 m ean 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 uvr A 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 vehi cle control value. Data sets for tester strains TA100 were judged posi tive if th e increase in mean revertants at the peak of the response w as greater than or equal to 2.0-time s the mean vehicle control value.

### 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 co uld be a dos e-responsive increase that does not achieve the respective threshold cited above or a n on-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 d ered valid. Al l Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation ( rfa ) and the deletion in the uvr B gene. Cultures of tester strain s TA98 and TA100 must demonstrate the presence of t he pKM101 plasmid R-factor. All WP2 uvr A cultures must demonstrate the deletion i n t he uvr A ge ne. All cult ures must de monstrat e 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 uvr A, 10 - 60. To e nsure that a ppropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x10 9 

### 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 mini mum of three non-toxic concentra tions 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 o n 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. Copi es 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 betwee n that site and the Sponsor.

Deviation

No known deviati ons from the protoc ol or as sa y-method SOPs occ urred during the c onduct of this study.

### BioReliance Study No. Dow Study No. 13 RESULTS AND DISCUSSION

Solubility Test

DMSO) was selected as the solvent of choice ba sed 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 approx imately 100 mg/mL, the maximum conc entration 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 Sha m mixes.

Tester Strain Titer Results

Tester Strain
TA98 TA100 TA1535 TA1537 WP2 uvr A Experiment Titer Value (x 10 9 

### 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 xi mum concentration test ed was 5000 μg per plate. Thi s c oncentration was ac hieved usi ng a concentration of 100 mg/ m L 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 cipit ate 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 te ster strains TA100, TA1537 and WP2 uvr A in the presence of S9 activation. Based on th e findings of the initial toxicity -mutation assay, th e maximum dose plated in the confirmatory mutageni city 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 eith er the presence or ab sence of S9 activation.

Confirmatory Mutagenicity Assay

The results of the conf irmatory mutageni city assay are presented in Tables 3 and 4 . These data were generated in Expe riment B2. I n the c onfir matory mutagenicity assa y, the concentrations tested were 15, 50, 150, 500, 15 00 and 5000 μg per plate. Precip itate was observed beginning at 500, 1500 or at 5000 μg per plate. Toxicity wa s observed, as reductions in revertant counts, at 5000 μg per plate with t ester str ains TA100, TA1535 a nd WP2 uvr A in the presence of S9 activation.

### BioReliance Study No. Dow Study No. 14 In Experiment B2 (Confirm ator y Mutagenicity Assay), no pos itive mutagenic responses were observed with any of the tester strains in eith er the presence or ab sence of S9 activation.

Dosing Formulation Analysis

Dosing formulations were analyzed by the Sponsor. A copy of the analytic al report is i ncluded 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 wa s within 85 to 115% of target , the dosing formulations were considered stable for th e 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 pr esence and absence of Aroc lor-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. Mu riel (1976) Mutagen testing using trp+ reversion in Escherichia coli

, Mutation Research 38:3-32.

OECD Guideline 471 (Genetic Toxicology: Bact erial Reverse Mutation Test), Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, p ublished 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 Purifica tion 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-nitros amines 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 26 M 6 IP, 14 M 6 IP
1500 μg 19 1 0.5 20 M 6 NP, 18 M 6 NP 500 μg 31 1 0.9 32 M 6 NP, 30 M 6 NP 150 μg 34 2 0.9 35 M 1 NP, 32 M 1 NP 50 μg 32 10 0.9 39 M 1 NP, 25 M 1 NP 15 μg 31 4 0.9 33 M , 28 M 5.0 μg 27 8 0.8 21 M , 32 M 1.5 μg 34 2 0.9 35 M , 32 M
DMSO 50 μL 36 5 39 M , 32 M

TA100 5000 μg 85 4 1.0 88 M 6 IP, 82 M 6 IP
1500 μg 91 6 1.1 95 M 6 NP, 86 M 6 NP 500 μg 82 6 1.0 77 M 6 NP, 86 M 6 NP 150 μg 90 1 1.1 91 M 1 NP, 89 M 1 NP 50 μg 73 16 0.9 61 M , 84 M 15 μg 76 6 0.9 72 M , 80 M 5.0 μg 85 5 1.0 81 M , 88 M 1.5 μg 88 7 1.1 83 M , 93 M
DMSO 50 μL 82 3 84 M , 80 M

TA1535 5000 μg 9 0 0.5 9 M 6 IP, 9 M 6 IP
1500 μg 14 1 0.8 13 M 6 NP, 15 M 6 NP 500 μg 11 1 0.6 10 M 6 NP, 12 M 6 NP 150 μg 14 1 0.8 14 M 1 NP, 13 M 1 NP 50 μg 15 3 0.9 17 M 1 NP, 13 M 1 NP 15 μg 16 4 0.9 18 M , 13 M 5.0 μg 18 9 1.1 11 M , 24 M 1.5 μg 14 3 0.8 16 M , 12 M
DMSO 50 μL 17 7 22 M , 12 M

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

### 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 3 M 6 IP, 8 M 6 IP
1500 μg 5 3 0.6 7 M 6 IP, 3 M 6 IP 500 μg 4 1 0.5 5 M 1 NP, 3 M 1 NP 150 μg 6 0 0.8 6 M 1 NP, 6 M 1 NP 50 μg 5 0 0.6 5 M , 5 M 15 μg 5 1 0.6 4 M , 6 M 5.0 μg 5 2 0.6 6 M , 3 M 1.5 μg 6 1 0.8 6 M , 5 M
DMSO 50 μL 8 0 8 M , 8 M

WP2 uvr A 5000 μg 26 0 0.8 26 M 6 IP, 26 M 6 IP
1500 μg 28 6 0.9 23 M 6 NP, 32 M 6 NP 500 μg 22 4 0.7 19 M 1 NP, 25 M 1 NP 150 μg 24 6 0.8 19 M , 28 M 50 μg 29 4 0.9 32 M , 26 M 15 μg 31 5 1.0 27 M , 34 M 5.0 μg 29 6 0.9 33 M , 24 M 1.5 μg 31 5 1.0 27 M , 34 M
DMSO 50 μL 32 2 30 M , 33 M

TA98 2NF 1.0 μg 291 11 8.1 283 A , 298 A
TA100 SA 1.0 μg 839 52 10.2 802 A , 875 A
TA1535 SA 1.0 μg 631 66 37.1 584 A , 677 A
TA1537 9AAD 75 μg 279 20 34.9 293 A , 265 A
WP2 uvr A MMS 1000 μg 638 16 19.9 626 A , 649 A

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

### 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 21 M 6 IP, CPN# *
1500 μg 33 1 1.2 32 M 1 NP, 33 M 1 NP 500 μg 25 2 0.9 26 M , 23 M 150 μg 27 1 1.0 26 M , 28 M 50 μg 36 4 1.3 39 M , 33 M 15 μg 38 5 1.4 34 M , 41 M 5.0 μg 41 3 1.5 43 M , 39 M 1.5 μg 33 6 1.2 37 M , 29 M DMSO 50 μL 28 6 32 M , 23 M

TA100 5000 μg 43 10 0.4 50 M 6 IP, 36 M 6 IP 1500 μg 56 1 0.6 55 M 1 NP, 57 M 1 NP 500 μg 58 3 0.6 56 M , 60 M 150 μg 78 15 0.8 67 M , 88 M 50 μg 79 1 0.8 80 M , 78 M 15 μg 76 16 0.8 87 M , 64 M 5.0 μg 72 4 0.7 69 M , 75 M 1.5 μg 81 6 0.8 77 M , 85 M DMSO 50 μL 99 9 105 M , 92 M

TA1535 5000 μg 5 1 0.5 4 M 6 IP, 6 M 6 IP 1500 μg 6 1 0.6 6 M 6 NP, 5 M 6 NP 500 μg 8 3 0.8 10 M , 6 M 150 μg 9 3 0.9 7 M , 11 M 50 μg 12 1 1.2 11 M , 12 M 15 μg 10 4 1.0 7 M , 12 M 5.0 μg 8 4 0.8 11 M , 5 M 1.5 μg 11 1 1.1 11 M , 10 M DMSO 50 μL 10 1 9 M , 11 M

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 inva lidate the results since the remaining plate at this dose level is comparable to the negative control.

### 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 2 M 6 IP, 1 M 6 IP
1500 μg 5 0 0.7 5 M 1 NP, 5 M 1 NP 500 μg 3 0 0.4 3 M , 3 M 150 μg 6 1 0.9 5 M , 7 M 50 μg 8 5 1.1 4 M , 11 M 15 μg 10 3 1.4 8 M , 12 M 5.0 μg 10 1 1.4 9 M , 10 M 1.5 μg 12 4 1.7 9 M , 15 M
DMSO 50 μL 7 2 8 M , 5 M

WP2 uvr A 5000 μg 14 1 0.4 13 M 6 IP, 15 M 6 IP
1500 μg 22 7 0.7 17 M 6, 27 M 6 500 μg 30 1 0.9 31 M , 29 M 150 μg 28 4 0.9 30 M , 25 M 50 μg 27 3 0.8 25 M , 29 M 15 μg 27 1 0.8 28 M , 26 M 5.0 μg 32 9 1.0 38 M , 25 M 1.5 μg 30 1 0.9 30 M , 29 M
DMSO 50 μL 32 6 36 M , 28 M

TA98 2AA 1.0 μg 505 20 18.0 519 A , 491 A
TA100 2AA 2.0 μg 997 355 10.1 746 A , 1248 A
TA1535 2AA 1.0 μg 100 18 10.0 87 A , 112 A
TA1537 2AA 1.0 μg 55 6 7.9 51 A , 59 A
WP2 uvr A 2AA 10 μg 175 40 5.5 203 A , 147 A

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. 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 27 M 6 IP, 31 M 6 IP, 12 M 6 IP 1500 μg 34 5 0.8 37 M 6 NP, 36 M 6 NP, 28 M 6 IP
500 μg 39 4 0.9 39 M 1 NP, 43 M 1 NP, 35 M 1 NP 150 μg 44 5 1.0 45 M , 39 M , 49 M 50 μg 35 7 0.8 38 M , 39 M , 27 M 15 μg 37 11 0.9 30 M , 32 M , 50 M
DMSO 50 μL 43 8 50 M , 34 M , 46 M

TA100 5000 μg 81 13 1.0 93 M 6 IP, 67 M 6 IP, 82 M 6 IP
1500 μg 84 12 1.0 72 M 6 NP, 85 M 6 NP, 95 M 6 NP
500 μg 77 8 0.9 74 M 1 NP, 86 M 1 NP, 72 M 1 NP 150 μg 90 12 1.1 83 M , 84 M , 104 M 50 μg 89 4 1.0 88 M , 93 M , 85 M 15 μg 89 7 1.0 92 M , 81 M , 93 M
DMSO 50 μL 85 6 89 M , 87 M , 78 M

TA1535 5000 μg 11 2 0.9 8 M 6 IP, 12 M 6 IP, 12 M 6 IP
1500 μg 14 2 1.2 12 M 6 NP, 14 M 6 NP, 16 M 6 NP
500 μg 11 2 0.9 11 M 6 NP, 13 M 6 NP, 10 M 6 NP 150 μg 9 4 0.8 4 M , 12 M , 10 M 50 μg 9 3 0.8 6 M , 11 M , 9 M 15 μg 11 6 0.9 18 M , 7 M , 7 M
DMSO 50 μL 12 1 13 M , 12 M , 12 M

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

### 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 6 M 6 IP, 7 M 6 IP, 7 M
6 IP 1500 μg 3 1 0.6 4 M 6 IP, 4 M 6 IP, 2 M
6 IP
500 μg 5 2 1.0 4 M 1 NP, 3 M 1 NP, 7 M 1 NP 150 μg 3 2 0.6 5 M , 4 M , 1 M 50 μg 5 2 1.0 5 M , 7 M , 3 M 15 μg 5 2 1.0 4 M , 8 M , 4 M
DMSO 50 μL 5 1 4 M , 6 M , 6 M

WP2 uvr A 5000 μg 23 2 0.7 22 M 6 IP, 22 M 6 IP, 25 M 6 IP
1500 μg 34 6 1.0 38 M 6 NP, 28 M 6 NP, 37 M 6 NP
500 μg 31 8 0.9 24 M 1 NP, 39 M 1 NP, 29 M 1 NP 150 μg 31 8 0.9 27 M , 40 M , 26 M 50 μg 30 2 0.9 31 M , 30 M , 28 M 15 μg 29 1 0.9 30 M , 29 M , 29 M
DMSO 50 μL 34 3 36 M , 36 M , 31 M

TA98 2NF 1.0 μg 432 111 10.0 305 A , 488 A , 504 A
TA100 SA 1.0 μg 825 32 9.7 805 A , 807 A , 862 A
TA1535 SA 1.0 μg 555 70 46.3 494 A , 539 A , 631 A
TA1537 9AAD 75 μg 311 62 62.2 370 A , 316 A , 246 A
WP2 uvr A MMS 1000 μg 502 26 14.8 482 A , 531 A , 494 A

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

### 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 20 M 6 IP, 28 M 6 IP, 20 M 6 IP 1500 μg 34 6 0.8 39 M , 37 M , 27 M 500 μg 30 2 0.7 28 M , 30 M , 31 M 150 μg 46 7 1.1 38 M , 47 M , 52 M 50 μg 40 9 0.9 42 M , 48 M , 30 M 15 μg 49 4 1.1 52 M , 45 M , 50 M
DMSO 50 μL 43 11 32 M , 53 M , 45 M

TA100 5000 μg 40 6 0.4 34 M 6 IP, 43 M 6 IP, 44 M 6 IP 1500 μg 57 13 0.6 44 M , 69 M , 58 M 500 μg 63 17 0.7 72 M , 44 M , 74 M 150 μg 77 22 0.9 52 M , 96 M , 82 M 50 μg 76 13 0.9 62 M , 82 M , 85 M 15 μg 76 7 0.9 76 M , 83 M , 69 M
DMSO 50 μL 89 10 100 M , 81 M , 87 M

TA1535 5000 μg 6 2 0.5 7 M 6 IP, 7 M 6 IP, 4 M
6 IP 1500 μg 4 1 0.3 5 M 1 NP, 3 M 1 NP, 4 M 1 NP 500 μg 7 2 0.5 8 M , 9 M , 5 M 150 μg 7 1 0.5 7 M , 6 M , 8 M 50 μg 9 1 0.7 10 M , 8 M , 8 M 15 μg 13 4 1.0 17 M , 9 M , 13 M
DMSO 50 μL 13 1 12 M , 14 M , 12 M

TA1537 5000 μg 3 1 0.6 3 M 6 IP, 3 M 6 IP, 2 M
6 IP
1500 μg 2 1 0.4 3 M 1 NP, 1 M 1 NP, 2 M 1 NP 500 μg 6 2 1.2 4 M , 7 M , 7 M 150 μg 4 1 0.8 5 M , 4 M , 3 M 50 μg 4 3 0.8 8 M , 2 M , 3 M 15 μg 6 1 1.2 6 M , 5 M , 6 M
DMSO 50 μL 5 2 7 M , 4 M , 4 M 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

### 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

WP2 uvr A 5000 μg 14 4 0.5 18 M 6 IP, 11 M 6 IP, 14 M 6 IP 1500 μg 17 2 0.5 18 M 1 NP, 15 M 1 NP, 17 M 1 NP 500 μg 34 3 1.1 32 M , 37 M , 34 M 150 μg 31 5 1.0 26 M , 32 M , 36 M 50 μg 35 5 1.1 29 M , 36 M , 39 M 15 μg 36 8 1.2 40 M , 41 M , 26 M
DMSO 50 μL 31 4 35 M , 30 M , 28 M

TA98 2AA 1.0 μg 722 41 16.8 736 A , 676 A , 754 A
TA100 2AA 2.0 μg 844 45 9.5 807 A , 831 A , 895 A
TA1535 2AA 1.0 μg 91 10 7.0 79 A , 99 A , 94 A
TA1537 2AA 1.0 μg 98 17 19.6 115 A , 82 A , 98 A
WP2 uvr A 2AA 10 μg 247 54 8.0 185 A , 269 A , 287 A

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

### BioReliance Study No. Dow Study No. 25 Historical Negative and Positive Control Values
2007 – 2009

revertants per plate
Activation

None Rat Liver Strain Control

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 uvr A 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: In formation for Ja panese 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 uvr A 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 Ar oclor-induced rat liver S9.

Referential matters

The vehicle and positive contro l values indicate that all te ster 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 

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### BioReliance Study No. Dow Study No. 52 APPENDIX V: Dosing Formulation Analysis

### BioReliance Study No. Dow Study No. 53 Toxicology Inf omlation Co ll ection (TIC ) Report 111e Dow C hemical C ompany



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"Oject I Samp le Description (optional): S am p le s in OM S O ( B io R e l iallCe S t udy ID : Test nlaterial (s ): Test nlateriallot #: Report I"ccipienl ( s ) : Jill S mal ley Ana l y s is request date: 6123120 1 0 Date s ubmittor prelJrared s amp l es: 612SflO l0 Numi>eror s amples orestimate: 7

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in dim ethy l sul f oxide ( DM SO). Th e analys i s wa s pe r forme d u s i ng

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PAGE I

### BioReliance Study No. Dow Study No. 54TableI.Results ofconcentrationanalysesDoseOl,eMi,cd;06121110OverallOveralll..Ret:;n.:2010·91'c.(m&,mL):0.000.0",",cdCone:dlQ'loorTargttCone:(n):(2)Tar,,"lCone.(m&,mL):0.3000.3MeanMU5W"cdCone:0.274'loorTargetCone:9l.l%91.2%NA(n):(2)Tar,,",Cone.(m&,mL):U"'.0MeanMU5W"cdCone:0.974'loorT..,,,Cone:91.4'lo97.4%NA(n):(2)TugttCon<;.(m&,mL):3003.0MeanMea$ll"NCone:2.92'loorT.r,ttCone:97.4'lo97.4%NA(n):(2)Targe'Cone.(m&,mL):10.0'"MeanMUllI"cdCone!9.62'loorTar,,,Cone'96.2%96.2%NA(n):(2)TargttCo...:.(m&,mL):30.030MeanMe_edCone:29.0'loofT.,",,,Cone:96.1%96.1%NA(n):(2)TargelCOne.(m&,mL):100100MeanMel5W"wConc:93.7%of1llrguCone'93.1%93.7% NA(n):(2)LLQ.(mymL):u.UOJOi0.00401Nit:<lLQ.qlhan10........limitof1jIWI1IIiouonNA_NotApplicableI)AGE2

### BioReliance Study No. Dow Study No. 55 Samp l e Prepar a tion:

Dose so lution s of in dim ethyl su lf oxide (DMSO) and a DMSO
veh icl e co ntr o l were prepared a l B io R eliance o n 6125110 ( Bi o R elia n ce Study 10 : Sample s were shipped o n 6128110, r eceived o n 612911 0. and pla ced in a -2 0 {l C freezer. Sa mple s were removed fro m storage o n 6130110. allowed t o thaw t o a mbient temperature. and prepared in the following mann e r. Samples were vonex-mixe d prior 10 s amplin g . D uplicate a l iquols r a n ging from 1 0

- 100 f.lL we r e ta k en f r o m th e ce nte r of eac h sa mple. A ll sam ple s were firs t dilute d with aceton i tr il e. Samples

were then dil ut ed w i th 50150 Milli-Q water/acetonilTile 

### + 0. 1 % formic acid t o obta in final

concentrations of approximately 1 0.0

- 15.0 IJ.gl mL

. Th e final di l ution s of each s amp l e

were vo n ex-mixed and aliquo t s we r e transf e rred 1 0 glass autosamp l e r v i al s for a n alys i s. All sa mpl es were analyzed for by high perf or m ance liquid

c hromat ography with u l trav i olet detection ( HPL C/UV). The leve l s of

were quantified by using externa l s tandard ca librati o n.

Standard a nd Spike Pr ep aration:

Standards were prepar ed co vering th e concen trati o n range of 0.100

- 50.2 f.lg

per m L 50150 Milli -Q waterlacetonitrile + 0

. 1 % f orm i c acid. Spikes were p repared

a t approximate co nc en tration s o f 0. 3, 10 and 1 00 mg per rnL DMSO.

R es ults:

S t andards were found to be l inear ove r t h e range o f 0.201

- 25.1 I-tglmL with an 

### R 2 value

of 0.9999. Ave r age sp ike rec ove ry was found to be 102 % with a s t andard d ev i ati o n of

1.38 %.

PAGE 3

### BioReliance Study No. Dow Study No. 56 COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDA RDS

Compound:

Title: Salmonella

- escherichia co lil Mm nmalian-Microsome R everse Mutation As s ay Pr e in cuba ti on Meth od with a Confirmatory A ss ay with

All pha ses of this s tudy were conducted in comp li anc e wi t h the following G ood
lab orato r y Pra c ti ce Standards:

U S Depanmcnt o f H ea lth and Human S e rv i ces Food and Dru g Admi ni s tration

- GlP s; Title 21 C FR
, P art 58

- Good Lab o ratory Pra ctice Re g ulat io n s for Noncli n ical lab ora tory Studies, Fina l Rul e
J apa n ese Mini s tr y o f Econo m y, Trade, and I ndu st ry Good L aboratory Pr actices (METO

Or ganisation for Ec onom i c Co- O peration and Dev e l o pm e nt ( DECO ) DECO Series on P rinciples o f Good l a borat o r y P ractice and Compliance M on itoring, Number I

. DEC O P rinciples on G ood L aboratory Pr actice (as revised in 199 7) ENVIMC/CHEM (98) 17

Ex ce pti on : Te st Mat er ial Characterizat i o n wa s conducted conc u rrent l y with the st u dy.

PAGE4

### BioReliance Study No. Dow Study No. 57 QUA LITY ASSURANCE STATEMENT

Compound:

Title: Salmollella

- Escherichia co li/Mammalian-Microsome Reverse Mutation Assay Preincubation M ethod with a Confirmatory Assay with

Thi s study was exa min ed for confomlUnce with Good Laborat ory Practic es as pub l ished by the USF DA, METI. and DECO. The final report wa s d e t ermined to be an accurate reflection of the data obtained. The date s of Qua l ity Assurance activ i ties on this s tud y are listed below.

Study I nitiation Date : 08 JUN 2010

TY P E OF AUPIT

Study conduct

PrOt OCO l. data, and

draft report PATE OF AU DIT

30JUN2010-

06 AUG 20 10

07 AUG 2010-

09 AUG 2010 PATE REPORTEP TO PRINCIPLE INVESTI GATOR! PI MANAGEMENT

06 AUG 2010



### 10 AUG 2010 DATE REPORTED TO STU DY DIRECTOR! Sp MANAGEMENT

06 AUG 2010



### 10 AUG 2010

Final report The date of the s i gnature below i s the date of lh e final report audit.

The final report accurately r eflects the raw data of the s t udy.

1803 Bui lding Midland, Michigan 48674

PAGE 5

### BioReliance Study No. Dow Study No. 58 APPENDIX VI: S9 Quality Control Certificate

### BioReliance Study No. Dow Study No. 59