### Page 1 of 69 SECOND AMENDED FINAL REPORT

Study Title

Bacterial Reverse Mutation Assay Using Gas-Phase Exposure

Test Article

Authors

Valentine O. Wagner, III, M.S. Renee M. Hines, B.S. Meena Jois, B.S.

Study Completion Date

Original Date: 04 September 2008 Amended Final Report 1 Date: 11 January 2011

Amended Final Report 2 Date: 27 April 2011

Testing Facility

BioReliance

9630 Medical Center Drive Rockville, MD 20850

BioReliance Study Number

AC12HE.502700.BTL

Sponsor

### BioReliance Study No. AC12HE.502700.BTL 6 Bacterial Reverse Mutation Assay Using Gas-Phase Exposure

STUDY INFORMATION

Sponsor:

Study Representative/Study Monitor:

Testing Fac ility: BioReliance

9630 Medical Center Drive Rockville, Maryland 20850

Test Article I.D.:

Synonyms:

Test Article CAS No.:

BioReliance Study No.: AC12HE.502700.BTL

Test Article Purity: 99.978 area%

Test Article Lot No.: 40883-50 (S ample 0001) Batch#1-Cyl#1 (Sample 0002)

Test Article CAS No.:

Test Article Description: Clear, liquefied gas

Storage Conditions: Room temperature, upright in the dark without

desiccant

Test Article Receipt and Login: 22 February 2008 (Sample 0001) 15 July 2010 (Sample 0002)

Study Initiation: 04 April 2008

Experimental Start Date: 09 April 2008

Experimental Completion Date: 06 October 2010

Laboratory Managers: Melissa R. VanDyke, B.S. (prior to January 2009) Emily W. Dakoulas, B.S. (effective January 2009)

### BioReliance Study No. AC12HE.502700.BTL 7 TABLE OF CONTENTS Page

SUMMARY .............................................................................................................................. 8

PURPOSE ............................................................................................................................... 10

CHARACTERIZATION OF TEST AND CONTROL ARTICLES ...................................... 10

MATERIALS AND METHODS ............................................................................................ 12

RESULTS AND DISCUSSION ............................................................................................. 18

CONCLUSION ....................................................................................................................... 19

REFERENCES ....................................................................................................................... 19

DATA TABLES ..................................................................................................................... 21

APPENDIX I: Historical Control Data................................................................................... 42

APPENDIX II: Study Protocol ............................................................................................... 45

APPENDIX III: Information for Japanese Regulatory Agencies ........................................... 61

APPENDIX IV: Test Article Characterization ....................................................................... 67

### BioReliance Study No. AC12HE.502700.BTL 8 SUMMARY

The test article, was tested in the Bacteria l Reverse Mutation Assay Using Gas-Phase Exposure using Salmonella typhimurium tester strains tester strains TA98, TA100,

TA1535 and TA1537 and Escherichia coli tester strain WP2 uvr A in the presence and absence

of Aroclor-induced rat liver S9 . The assay was performed in th ree phases, using the desiccator method, a modification of the pl ate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose -range for the mutagenic ity assay. The second phase, the mutagenicity assay was used to evalua te the mutagenic potentia l of the test article. The third phase, the confirmato ry mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article.

Ambient air was used as the negative control.

In the preliminary toxicity assa y, the maximum dose tested was 37 mmoles/L. The dose levels tested were 1, 3, 6, 8, 11, 21 and 37 mmoles/L (24,500; 73,400; 147, 000; 196,00 0, 269,000; 513,000 and 905,0 00 ppm at 25°C, respectively). The test system was exposed to the test article in the desiccators for 24 hours. No precipitate was observed. No background lawn toxicity was

observed however a reduction in revertant counts was observed beginning at 11, 21 or at

37 mmoles/L with some test cond itions. Based on the findings of th e preliminary toxicity assay, the maximum dose plated in the confirmato ry mutagenicity assay was 37 mmoles/L.

In the mutagenicity assay, no po sitive mutagenic response was obs erved. The dose levels tested were 3, 6, 8, 11, 21 and 37 mm oles/L (73,400; 147,000; 19 6,000, 269,000; 513,000 and 905,000 ppm at 25°C, respectively). The test sy stem was exposed to th e test article in the desiccators for 48 hours. No pr ecipitate was observed. Toxicity was observed beginning at 11, 21 or at 37 mmoles/L. A confirmatory mutage nicity assay was conducted at the Sponsor’s request to meet the requirements fo r Japanese regulatory submission.

The confirmatory mutagenicity assay was conducted at dose levels of 3, 6, 8, 11, 21 and 37 mmoles/L (73,400; 147,0 00; 196,000, 269,000 ; 513,000 and 905,000 ppm at 25°C, respectively). No positive mutagenic response wa s observed with tester strains TA100, TA1535

and WP2 uvr A in the absence of S9 activation and with tester strains TA100, TA1535 and TA1537 in the presence of S9 activation. No precipitate wa s observed. Toxicity was observed beginning at 21 or 37 mmoles/L. Tester strain TA98 in the presence and absence of S9 was not

evaluated due to confluen t growth but was reteste d. Due to excessive toxi city, as a result of a drop in the revertant counts beginning at 8 mmoles /L, tester strain TA 1537 in the absence of S9

was retested. Due to an una cceptable vehicle control va lue, tester strain WP2 uvr A in the

absence of S9 activation was not evaluated but was retested.

In the retest of the confirmatory mutagenicity assa y, the dose levels tested were 0.7, 1, 3, 6, 8, 11, 21 and 37 mmoles/L (17,200; 24,500; 73,400; 147,000; 196,000, 269,00 0; 513,000 and 905,000 ppm at 25°C, respectivel y). No positive respon ses were observed with tester strains

TA98 and TA1537 in the absen ce of S9 activation and with tester strains TA98 and WP2 uvr A

### BioReliance Study No. AC12HE.502700.BTL 9 in the presence of S9 activation. No precipitate was observed. To xicity was observed beginning

at 21 or 37 mmoles/L.

The results of the Bacterial Re verse Mutation Test Using Gas-Phase Exposure indicate that,

under the conditions of this st udy, test article did not cause a positive mutagenic response in either the presence or ab sence of Aroclor-induced rat liver S9.

### BioReliance Study No. AC12HE.502700.BTL 10 PURPOSE

The purpose of this study was to evaluate th e mutagenic potential of the test article gas by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvr A in the presence

and absence of Aroclor-in duced rat liver S9. A copy of th e Historical Nega tive and Positive

Control Values is included in Appendix I . Copies of the study protocol and amendments are

included in Appendix II

.

This study was conducted in co mpliance with the te sting guideline of the OECD (1998) and Japanese guidelines Official notice of J MHLW, METI and ME (21 November 2003):

YAKUSHOKUHATSU No.1121002 SEIKYOKU No.2 KANPOKIH ATSU No. 031121002.

CHARACTERIZATION OF TEST AND CONTROL ARTICLES

The test article, was receive d by BioReliance on 22 Fe bruary 2008 and was assigned the code number AC12HE. Sample 0002 of the test ar ticle was received on 15 July

2010. The test article was characterized on the Material Safety Data Sh eet as a clear volatile liquid. Per the protoc ol, the test article should be stored at ambien t conditions in its shipping cylinder. Upon arrival at BioReliance, the test ar ticle was stored upright at room temperature in

the dark without desiccant.

The Sponsor has determined the identity, strengt h, purity, composition or other characteristics to

define lot 40883-50 and batch#1 cylinder#1 of the test article. Copies of the Test Article

Characterization for each lo t/batch are in cluded in Appendix IV

.

The test article was supplied in a gas cylinder. All dosing was at room temperature under yellow light.

The positive control articles have been characterized as per the Certificates of Analysis on file with the testing facility. The st ability of the negativ e and positive control articles and their mixtures was demonstrated by acceptable results that met the criteria for a valid test.

Positive controls plated concurre ntly with the mutagenicity a ssay are listed in the following table. All positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide,

which was diluted in water. All subdivided solutions of positive control were stored at -5 to

-30°C.

### BioReliance Study No. AC12HE.502700.BTL 11 Strain S9

Activation Positive Control Concentration (μg/plate)

All Salmonella Strains 1.0 (B1)

WP2 uvr A 2-aminoanthracene

CAS No. 613-13-8

(Aldrich Chemical Co., Inc.)

Lot No. 12317CE Exp. Date 01-Feb-2009 Purity 99.9%

(Sigma Aldrich Chemical Co.)

Lot No. 03403ED Exp. Date 22-Jan-2012 Purity 99.8% 10

TA100 Rat

(Sigma Aldrich Chemical Co.)

Lot No. 03403ED Exp. Date 22-Jan-2012 Purity 99.8% 2.0 (B2)

TA98 2-nitrofluorene

CAS No. 607-57-8

(Aldrich Chemical Co., Inc.)

Lot No. 03926DC Exp. Date 18-Aug-2010 Purity 98.1%

(Sigma Aldrich Chemical Co.)

Lot No. 03319JD Exp. Date 28-Feb-2011 Purity 98.1% 1.0

TA100, TA1535 None

Sodium azide

CAS No. 26628-22-8

(Sigma Chemical Co.)

Lot No. 073K0119 Exp. Date 31-Jul-2008 Purity 99.9%

(Sigma Aldrich Chemical Co.)

Lot No.71980 Exp. Date 28-Dec-2010 Purity 99.8% 1.0

### BioReliance Study No. AC12HE.502700.BTL 12 Strain S9

Activation Positive Control Concentration (μg/plate)

TA1537 9-aminoacridine (Sigma Chemical Co.)

Lot No. 106F06682 Exp. Date 08-Nov-2009 or

30-Oct-2010

CAS No. 90-45-9 Purity >97% 75

WP2 uvr A Methyl methanesulfonate

CAS No. 66-27-3

(Aldrich Chemical Co., Inc.)

Lot No. 126K3721 Exp. Date 07-Jan-2011 Purity >99.9%

(Sigma Aldrich Chemical Co.)

Lot No.76296KJ Exp. Date 02-Jun-2012 Purity 99.8% 1,000

MATERIALS AND METHODS

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

.

Test System

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al . (1975) and Escherichia coli WP2 uvr A as described by Green and Muriel (1976)

. Salmonella tester strains were received from Dr. Bruce Ames’ designated distributor, Discovery Part ners International, San Diego, California. E

. coli

tester strains were received fr om the National Collection of In dustrial and Marine Bacteria, Aberdeen, Scotland. Th e tester strains were also obtain ed from Molecular Toxicology Inc. (MolTox) using cultures derived from the above sources.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Test er strain TA100 is reverted by mutagens that cause both frameshift and basepair substitut ion mutations. Specific ity of the reversion

mechanism in E. coli is sensitive to basepa ir substitution mu tations, rather than frameshift

mutations (Green and Muriel, 1976)

.

### BioReliance Study No. AC12HE.502700.BTL 13 Each tester strain culture was in oculated from the appropriate mast er plate, from the appropriate frozen stock or with the appropriate lyophilized pell et(s). To ensure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. At the end of the working day, each inoculated flask was placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm and incubating at 37 ±2°C for approximately 12 to 14 hours before the anticipated time of harvest. Each cu lture was monitored spectrophotometrically for turbidity and was harvested at a percen t transmittance yielding a titer of approximately 10 9 

### cells per milliliter. The actual titers were determ ined 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 was prepared from male Sprague-Da wley rats induced with a singl e intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacr ifice. The lots of S9 were prepared by and purchased from MolTox (Boo ne, NC) and stored at -60 ° C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize at least two promut agens to forms mutagenic to Salmonella typhimurium TA100. The S9 homogenate was used within 6 months after preparation.

The S9 mix was prepared im mediately before its use and contained 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 phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffe r at pH 7.4, was prepared immediately 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.

Preliminary Toxicity Assay

The preliminary toxicity assay wa s used to establish the dose-range over which the test article would be assayed. Negative (air) control and seven dose levels of the test article were plated, one plate per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvr A

on selective minima l agar in the presence and absenc e of Aroclor-induced rat liver S9.

Mutagenicity Assay

The mutagenicity assay was used to evaluate the mutagenic potential of the test article. Six dose levels of test article along wi th appropriate negative (air) control and positive controls were plated with overnight culture s of TA98, TA100, TA1535, TA 1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-i nduced rat liver S9. A ll dose levels of test article, vehicle control and positive co ntrols were plated in triplicate.

### BioReliance Study No. AC12HE.502700.BTL 14 Confirmatory Mutagenicity Assay

The mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six or eight dose levels of test article along with appropriate negative (air) control and positive controls were plated with overnight cultures of TA98, TA10 0, TA1535, TA1537 and

WP2 uvr A on selective minimal agar in the presence and absence 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 test article via the desiccat or methodology, a modification of the plate incorporation methodol ogy originally described by Ames et al. (1975) and updated by Maron and Ames (1983) . This test system has been shown to detect a wi de range of classes of chemical mutagens (McCann et al., 1975; McCann and Ames, 1976) . The desiccator methodology has been shown to be an effective method for detecting the genotoxic activity of volatile and gaseous test articles (Wagner et al ., 1992)

.

On the day of its use, minimal top agar, cont aining 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 μM each. To p agar not used with S9 or Sham mix was supplemented with

25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E cont aining 1.5 % (W/V) agar and supplemented 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 that identified the test article, test phase, dose level, tester strain and activation, as described in detail in BioR eliance's Standard Operating

Procedures.

For the positive controls, one-half (0.5) milliliter of S9 or Sham mix, 100 μL of tester strain (cells seeded) and 50 μL aliquo t of appropriate positive control were added to 2.0 mL of molten selective top agar at 45±2°C. When plating negative (air) control, the addition of positive

control was omitted. After vortexi ng, the mixture was overlaid onto the surf ace of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2 ° C. The negative control plates were placed inside of a desiccator for the duration of the incubation peri od. Plates that were not counted immediately following the incu bation period were stored at 2-8 ° C until colony counting could be conducted.

For test article, one-half (0.5) milliliter of S9 or Sham mix and 100 μL of tester strain (cells seeded) were added to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the

mixture was overlaid onto the surf ace of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inve rted and placed uncovered in th e appropriate number of 9-liter

desiccators. An a ppropriate quantity of the test article wa s introduced into ea ch desiccator by

### BioReliance Study No. AC12HE.502700.BTL 15 withdrawing an appropriate amount of air and repl acing it with the test article. The desiccators were incubated, with stirring, for approxima tely 24 hours (Experiment A1) or 48 hours (Experiments B1, B2 and B3) at 37±2°C. Follow ing the 24-hour incubation in Experiment A1, the plates were remove d from the desiccators and incubated with the lids replaced at 37±2°C for

an additional 24 hours. When plating the untreated controls, one-half (0.5 ) milliliter of S9 or Sham mix and 100 μL of tester strain were a dded to 2.0 mL of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay ha d solidified, the plates were inverted and incubated under the same conditions as the assay plat es. When plating the positive c ontrols, one-half (0.5) milliliter of S9 or Sham mix, 10 0 μL of tester strain and a 50 μL al iquot of appropriate positive control were added to 2.0 mL of molten selective top ag ar at 45±2°C. After vortexing, the mixture was

overlaid onto the surface of 25 mL of minimal bot tom agar. After the overlay had solidified, the plates were inverted an d incubated under the same conditions as the assay plates . Plates that were not counted immediately following th e incubation period were stored at 2-8 ° C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was ev aluated by visual examination 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 lawn and possibly a slight increase in the size of the microcolonies compared to the vehicle control plate.

3 Moderately

Reduced Distinguished by a marked thi nning of the microcolony lawn resulting in a pronounced increase in the size of the microcolonies compared to the vehicle control plate.

4 Extremely

Reduced Distinguished by an extreme thinning of the microcolony lawn resulting in an increase in the size of the microcolonies compared to the vehicle control plate such that the microcolony lawn is visible to the unaided eye as isolated colonies.

5 Absent Distinguished by a complete lack of any microcolony lawn over greater than or equal to 90% of the plate.

6 Obscured by

Particulate The background bacterial lawn cannot be accurately evaluated due to microscopic test article particulate.

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 or equal to 10% of the revertant colony count (e.g., less than or equal to 3 particles on a plate with 30 revertants).

### BioReliance Study No. AC12HE.502700.BTL 16 Code Description Characteristics

IP Interfering Precipitate Distinguished by precipitate on the plate that is visible to the naked eye and any precipitate particles detected b y the automated colony counter exceed 10% of the revertant colony count (e.g., greater than 3 particles on a plate with 30 revertants). These plates are counted manually.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

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.

Data sets for tester strains TA 1535 and TA1537 were judged positive if th e increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean

vehicle control value. Data sets fo r tester strains TA 98, TA100 and WP2 uvr A were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the me an vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This co uld be a dose-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 respectiv e threshold cited. A resp onse will be evaluated as negative, if it is neither positive nor equivocal.

Criteria for a Valid Test

The following criteria must be met for the initial toxicity-m utation and the confirmatory mutagenicity assays to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation ( rfa ) and the deletion in the uvr B gene.

Cultures of tester strains TA 98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvr A cultures must demonstrate the deletion in the uvr A gene. All

cultures must demonstrate the ch aracteristic mean number of sp ontaneous revertants in the vehicle controls as follows (inclusive): TA 98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvr A, 10 - 60. To ensure that appropriate numbers of bacteria are plated,

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

### cells/mL. The mean of each positive control must exhi bit at least a 3.0-fold increase in the number of revertants over the mean value of the respective ve hicle control. A minimum of th ree non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % redu ction in the mean number of re vertants per plate as compared

to the mean vehicle control value. This reduction must be accom panied by an abrupt

### BioReliance Study No. AC12HE.502700.BTL 17 dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Automated Data Collection Systems

The primary computer applications used for the collection of data include:

Minicount Colony Counter (Imagi ng Products International), Sorcerer Colony Counter and Ames Study Manager (Perceptive Instruments) , LIMS System (BioReliance), Excel 2003 (Microsoft Corporation) and Kaye Lab Watch Monitoring System (Kaye GE).

Archives

All raw data, the protocol and all reports for procedures performed at BioReliance will be maintained according to Sta ndard Operating Procedure OPQP3040 by the BioReliance RAQA unit headquartered at: Bi oReliance, 14920 Brosch art Road, Rockville, MD 20850. Per this SOP, paper records will be retained for at least th ree years after which ti me the Sponsor will be

contacted for a decision as to the final dispositio n of the materials. All study materials returned to the Sponsor or destroye d will first be copied and the copy will be reta ined in the BioReliance archives for a minimum of 10 y ears. Raw data, the pr otocol and reports generated at facilities

other than BioReliance will be archived per the contractual a rrangements between the facility and the Sponsor.

Deviations

The following deviation occurred du ring the conduct of this study:

In deviation from the study protocol, the assay pl ates for Experiment B1 were incubated in the desiccators for approximately 48 hours as opposed to 24 hours as stated in the protocol and as requested by the Sponsor (Event No. 4732). The Sponsor requested the 24 hour exposure to be

consistent with work on fluor ocarbons conducted over the pa st 20 years. Since the longer exposure time demonstrated defini tive toxicity, this demonstrates exposure of the test system to the test article and provides gr eater confidence of the negative result. Therefore, the Study

Director has concluded that this deviation did no t adversely impact the integrity of the data or the validity of the study conclusion.

### BioReliance Study No. AC12HE.502700.BTL 18 RESULTS AND DISCUSSION

Sterility Results

No contaminant col onies were observed on the sterility plates for the S9 and Sham mixes.

Preliminary Toxicity Assay

The results of the preliminary toxicity assay are presented in Table 1 through Table 5 . In the preliminary toxicity assay, the maximum dose te sted was 37 mmoles/L. The dose levels tested were 1, 3, 6, 8, 11, 21 and 37 mm oles/L (24,500; 73,400; 147, 000; 196,000, 269, 000; 513,000 and 905,000 ppm at 25°C, respectively). The test system was exposed to the te st article in the desiccators for 24 hours. No background lawn toxicity was observed however a reduction in

revertant counts was observed be ginning at 1 1, 21 or at 37 mmoles/L with some test conditions. Based on the findings of the pr eliminary toxicity assay, th e maximum dose plated in the confirmatory mutagenicity assay was 37 mmoles/L.

Tester Strain Titer Results

Tester Strain

TA98 TA100 TA1535 TA1537 WP2 uvr A Experiment

Titer Value (x 10 9 

### cells per mL)

B1 0.8 0.7 1.0 0.7 1.7

B2

-- 0.3 0.7 1.0 5.6

B3 1.1

-- -- 0.9 3.0

Mutagenicity Assay

The results of the mutagenicity assay are presented in Table 6 through Table 15 and

summarized in Table 16 . These data were generated in Experiment B1.

The dose levels tested were 3, 6, 8, 11, 21 and 37 mmoles/L (73, 400; 147,000; 196,000, 269,000; 513,000 and 905,000 ppm at 25°C , respectively). The test sy stem was exposed to the

test article in the desiccators for 48 hours. No positive mutagenic respon ses were observed with any of the tester strains in the presence or absence of S9 activatio n. No precipitate was observed. Toxicity was observed beginn ing at 11, 21 or at 37 mmoles/L.

Confirmatory Mutagenicity Assay

The results of the conf irmatory mutagenicity assay are presented in Table 17 through Table 20

. These data were generated in Experiment s B2 and B3.

### BioReliance Study No. AC12HE.502700.BTL 19 The dose levels tested were 3, 6, 8, 11, 21 and 37 mmoles/L (73, 400; 147,000; 196,000, 269,000; 513,000 and 905,0 00 ppm at 25°C, respectively). No positive mutagenic response was

observed with tester stra ins TA100, TA1535 and WP2 uvr A in the absence of S9 activation and with tester strains TA100, TA15 35 and TA1537 in the presence of S9 activation. No precipitate was observed. Toxicity wa s observed beginning at 21 or 37 mmoles/L. Tester strain TA98 in the presence and absence of S9 was not evaluate d due to confluent grow th but was retested. Due to excessive toxicity, as a result of a drop in the revertant counts beginning at 8 mmoles/L,

tester strain TA 1537 in the ab sence of S9 was retest ed. Due to an unacceptable vehicle control value, tester strain WP2 uvr A in the absence of S9 activation was not evaluated but was retested.

In the retest of the confirmatory mutagenicity assa y, the dose levels tested were 0.7, 1, 3, 6, 8, 11, 21 and 37 mmoles/L (17,200; 24,500; 73,400; 147,000; 196,000, 269,00 0; 513,000 and 905,000 ppm at 25°C, respectivel y). No positive respon ses were observed with tester strains

TA98 and TA1537 in the absen ce of S9 activation and with tester strains TA98 and WP2 uvr A in the presence of S9 act ivation. No precipitate was observe d. Toxicity was observed beginning

at 21 or 37 mmoles/L.

CONCLUSION

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay Using Gas-Phase Exposure indicate that, under the conditions of this study, did not cause a positive mutagenic respon se in either the presence or absence

of Aroclor-induced rat liver S9.

Stability data were not provided for either lot/batch of the test artic le. In the absen ce of stability data, the Study Director has conclu ded that the test article was ne gative with the caveat that the maximum require d concentration may not have been ach ieved due to instab ility or impurities.

At the request of the S ponsor, dosing formulatio n analyses for concentr ation and stability were

not conducted. Due to the lack of dose formulat ion analysis, the interpretation of the study data

were based on the nominal dose levels as document ed in the study records and not on the actual formulated test article concentrations as confirmed by analytical analysis. Nevertheless, toxicity in the assay demonstrated that the test system was dosed up to the regulatory required level.

REFERENCES

Ames, B.N., J. McCann and E. Yamasaki (1 975) Methods for Dete cting Carcinogens and Mutagens with the Salmonella /Mammalian Microsome Mutageni city Test, Muta tion Research,

31:347-364.

Green, M.H.L. and W.J. Mu riel (1976) Mutagen testing using trp+ reversion in Escherichia coli

, Mutation Research 38:3-32.

### BioReliance Study No. AC12HE.502700.BTL 20 Maron, D.M. and B.N. Ames (1 983) Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113:173-215.

McCann, J. and Ames, B.N. (1976). Detection of carcino gens as mutagens in the

Salmonella /microsome test: assay of 300 chemicals; discussion. Proc. Natl. Acad. Sci. USA

73:950-954.

McCann, J., Choi, E., Yamasaki , E. and Ames, B.N. (1975). Detection of carcinogens as mutagens in the Salmonella /microsome test: assay of 300 che micals. Proc. Natl. Acad. Sci. USA

72:5135-5139.

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

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

1998.

Wagner, III, V.O., Sa n, R.H.C. and Zeiger , E. (1992) Desiccato r Methodology for Salmonella Mutagenicity Assay of Vapor- Phase and Gas-Phase Test Ma terials. Environmental and Molecular Mutagenesis 19:68.

Wilcox, P., Naidoo, A., Wed d, D.J. and Gatehouse, D.G. (1990). Comparison of Salmonella typhimurium (TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5:285-291.

Vogel, H.J. and D.M. Bonner (1956) Acetylornithinase of E. coli : Partial Purifica tion and Some Properties, J. Biol. Chem., 218:97-106.

### BioReliance Study No. AC12HE.502700.BTL 21 Bacterial Mutation Assay

Table 1

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA98 Experiment No : A1

Vehicle : Air Control Date Plated : 9-Apr-08

Concentration

mmoles/L Without S9 Revertants per plate Background

Code With S9 Revertants per plate Background

Code

Vehicle 10 1 10 1

1 10 1 11 1

3 9 1 12 1

6 10 1 11 1

8 11 1 17 1

11 11 1 13 1

21 11 1 12 1

37 5 2 3 2

Background Lawn Code 1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent
6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 22 Bacterial Mutation Assay

Table 2

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA100 Experiment No : A1

Vehicle : Air Control Date Plated : 9-Apr-08

Concentration

mmoles/L Without S9 Revertants per plate Background

Code With S9 Revertants per plate Background

Code

Vehicle 202 1 219 1

1 206 1 207 1

3 238 1 197 1

6 241 1 180 1

8 244 1 197 1

11 265 1 229 1

21 133 1 198 1

37 263 1 228 2

Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent
6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 23 Bacterial Mutation Assay

Table 3

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA1535 Experiment No : A1

Vehicle : Air Control Date Plated : 9-Apr-08

Concentration

mmoles/L Without S9 Revertants per plate Background

Code With S9 Revertants per plate Background

Code

Vehicle 7 1 13 1

1 12 1 8 1

3 13 1 12 1

6 11 1 11 1

8 11 1 9 1

11 13 1 12 1

21 13 1 6 1

37 8 1 5 1

Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent
6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 24 Bacterial Mutation Assay

Table 4

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA1537 Experiment No : A1

Vehicle : Air Control Date Plated : 9-Apr-08

Concentration

mmoles/L Without S9 Revertants per plate Background

Code With S9 Revertants per plate Background

Code

Vehicle 8 1 11 1

1 7 1 8 1

3 8 1 10 1

6 9 1 5 1

8 6 1 8 1

11 7 1 3 1

21 7 1 4 1

37 6 2 4 2

Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent
6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 25 Bacterial Mutation Assay

Table 5

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : WP2 uvrA Experiment No : A1

Vehicle : Air Control Date Plated : 9-Apr-08

Concentration

mmoles/L Without S9 Revertants per plate Background

Code With S9 Revertants per plate Background

Code

Vehicle 17 1 18 1

1 19 1 16 1

3 17 1 17 1

6 C NA 19 1

8 17 1 14 1

11 15 1 15 1

21 12 1 16 1

37 3 1 12 1

Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent
6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

C=Contaminated
NA= Not Applicable

### BioReliance Study No. AC12HE.502700.BTL 26 Bacterial Mutation Assay

Table 6

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA98 Experiment No : B1

Activation Condition : None Cells Seeded : 0.8 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 26 1

2 45 1

3 35 1 35 10

3 1 20 1

2 25 1

3 35 1 27 8

6 1 35 1

2 20 1

3 30 1 28 8

8 1 25 1

2 25 1

3 15 1 22 6

11 1 22 1

2 20 1

3 16 1 19 3

21 1 4 1

2 4 1

3 3 1 4 1

37 1 0 4

2 0 4

3 0 4 0 0

Positive Control 2-nitrofluorene 1.0 μg per plate

1 162 1

2 158 1

3 67 1 129 54
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 27 Bacterial Mutation Assay

Table 7

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA98 Experiment No : B1

Activation Condition : Rat Liver S9 Cells Seeded : 0.8 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 32 1

2 21 1

3 20 1 24 7

3 1 17 1

2 14 1

3 14 1 15 2

6 1 25 1

2 13 1

3 38 1 25 13

8 1 17 1

2 15 1

3 21 1 18 3

11 1 14 1

2 9 1

3 15 1 13 3

21 1 2 1

2 6 1

3 2 1 3 2

37 1 0 5

2 0 5

3 0 5 0 0

Positive Control 2-aminoanthracene 1.0 μg per plate

1 635 1

2 329 1

3 363 1 442 168
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 28 Bacterial Mutation Assay

Table 8

Test Article Id : Study Number : AC12HE.502700.BTL

Strain : TA100 Experiment No : B1

Activation Condition : None Cells Seeded : 0.7 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 190 1

2 153 1

3 195 1 179 23

3 1 200 1

2 205 1

3 237 1 214 20

6 1 192 1

2 151 1

3 194 1 179 24

8 1 202 1

2 167 1

3 183 1 184 18

11 1 225 1

2 216 1

3 207 1 216 9

21 1 219 1

2 185 1

3 212 1 205 18

37 1 0 5

2 0 5

3 0 5 0 0

Positive Control sodium azide 1.0 μg per plate

1 650 1

2 630 1

3 567 1 616 43
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 29 Bacterial Mutation Assay

Table 9

Test Article Id : Study Number : AC12HE.502700.BTL

Strain : TA100 Experiment No : B1

Activation Condition : Rat Liver S9 Cells Seeded : 0.7 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 206 1

2 227 1

3 195 1 209 16

3 1 220 1

2 215 1

3 213 1 216 4

6 1 241 1

2 176 1

3 191 1 203 34

8 1 235 1

2 272 1

3 201 1 236 36

11 1 227 1

2 242 1

3 174 1 214 36

21 1 291 1

2 263 1

3 247 1 267 22

37 1 0 5

2 0 5

3 0 5 0 0

Positive Control 2-aminoanthracene 1.0 μg per plate

1 577 1

2 550 1

3 1040 1 722 275
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 30 Bacterial Mutation Assay

Table 10

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA1535 Experiment No : B1

Activation Condition : None Cells Seeded : 1.0 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 17 1

2 8 1

3 6 1 10 6

3 1 8 1

2 10 1

3 11 1 10 2

6 1 11 1

2 8 1

3 9 1 9 2

8 1 9 1

2 13 1

3 10 1 11 2

11 1 7 1

2 11 1

3 7 1 8 2

21 1 7 1

2 5 1

3 5 1 6 1

37 1 0 5

2 0 5

3 0 5 0 0

Positive Control sodium azide 1.0 μg per plate

1 283 1

2 310 1

3 286 1 293 15
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 31 Bacterial Mutation Assay

Table 11

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA1535 Experiment No : B1

Activation Condition : Rat Liver S9 Cells Seeded : 1.0 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 11 1

2 7 1

3 3 1 7 4

3 1 14 1

2 6 1

3 2 1 7 6

6 1 8 1

2 8 1

3 3 1 6 3

8 1 6 1

2 15 1

3 8 1 10 5

11 1 12 1

2 6 1

3 6 1 8 3

21 1 6 1

2 3 1

3 5 1 5 2

37 1 0 5

2 0 5

3 0 5 0 0

Positive Control 2-aminoanthracene 1.0 μg per plate

1 94 1

2 39 1

3 86 1 73 30
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 32 Bacterial Mutation Assay

Table 12

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA1537 Experiment No : B1

Activation Condition : None Cells Seeded : 0.7 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 7 1

2 8 1

3 7 1 7 1

3 1 2 1

2 5 1

3 3 1 3 2

6 1 5 1

2 1 1

3 2 1 3 2

8 1 5 1

2 2 1

3 8 1 5 3

11 1 1 1

2 0 1

3 0 1 0 1

21 1 1 1

2 1 1

3 1 1 1 0

37 1 1 3

2 0 3

3 0 3 0 1

Positive Control 9-aminoacridine 75 μg per plate

1 919 1

2 1161 1

3 896 1 992 147
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 33 Bacterial Mutation Assay

Table 13

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : TA1537 Experiment No : B1

Activation Condition : Rat Liver S9 Cells Seeded : 0.7 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 7 1

2 5 1

3 9 1 7 2

3 1 2 1

2 5 1

3 6 1 4 2

6 1 4 1

2 3 1

3 1 1 3 2

8 1 5 1

2 3 1

3 3 1 4 1

11 1 9 1

2 7 1

3 4 1 7 3

21 1 0 1

2 0 1

3 0 1 0 0

37 1 0 3

2 1 1

3 0 3 0 1

Positive Control 2-aminoanthracene 1.0 μg per plate

1 40 1

2 37 1

3 31 1 36 5
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

### BioReliance Study No. AC12HE.502700.BTL 34 Bacterial Mutation Assay

Table 14

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : WP2 uvrA Experiment No : B1

Activation Condition : None Cells Seeded : 1.7 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 11 1

2 17 1

3 14 1 14 3

3 1 14 1

2 12 1

3 12 1 13 1

6 1 C

2 13 1

3 11 1 12 1

8 1 10 1

2 15 1

3 9 1 11 3

11 1 1 1

2 5 1

3 6 1 4 3

21 1 0 1

2 1 1

3 5 1 2 3

37 1 0 4

2 0 4

3 0 4 0 0

Positive Control methyl methanesulfonate 1000 μg per plate

1 146 1

2 150 1

3 209 1 168 35
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate

C=Contamination

### BioReliance Study No. AC12HE.502700.BTL 35 Bacterial Mutation Assay

Table 15

Test Article Id :
Stu dy Number : AC12HE.502700.BTL

Strain : WP2 uvrA Experiment No : B1

Activation Condition : Rat Liver S9 Cells Seeded : 1.7 x 10 8

Vehicle : Air Control Date Plated : 22-Apr-08

Concentration

mmoles/L Plate Number Revertants per plate Background

Code Average

Revertants Standard

Deviation

Vehicle 1 20 1

2 12 1

3 24 1 19 6

3 1 31 1

2 21 1

3 17 1 23 7

6 1 12 1

2 19 1

3 18 1 16 4

8 1 6 1

2 13 1

3 20 1 13 7

11 1 7 1

2 NC

3 5 1 6 1

21 1 3 1

2 5 1

3 6 1 5 2

37 1 0 4

2 0 4

3 0 4 0 0

Positive Control 2-aminoanthracene 10 μg per plate

1 202 1

2 126 1

3 215 1 181 48
Background Lawn Code
1=Normal; 2=Slightly reduced; 3=Modera tely reduced; 4=Extremely reduced; 5=Absent 6=Obscured by particulate; NP=Non-Interfering precipitate; IP=Interfering precipitate
NC=No count due to procedural erro r in which the plate did not receive an aliquot of tester strain

### BioReliance Study No. AC12HE.502700.BTL 36 Bacterial Mutation Assay Summary of Results – Initial Mutagenicity Assay

Table 16

Test Article Id :
Stu dy Number : AC12HE.502700.BTL Experiment No : B1
Average Revertants Per Plate ± Standard Deviation

Activation Condition : None
Dose (mmoles/L) TA98 TA100 TA1535 TA1537 WP2 uvr A

Vehicle 35 ± 10 179 ± 23 10 ± 6 7 ± 1 14 ± 3

3 27 ± 8 214 ± 20 10 ± 2 3 ± 2 13 ± 1

6 28 ± 8 179 ± 24 9 ± 2 3 ± 2 12 ± 1

8 22 ± 6 184 ± 18 11 ± 2 5 ± 3 11 ± 3

11 19 ± 3 216 ± 9 8 ± 2 0 ± 1 4 ± 3

21 4 ± 1 205 ± 18 6 ± 1 1 ± 0 2 ± 3

37 0 ± 0 0 ± 0 0 ± 0 0 ± 1 0 ± 0

Positive 129 ± 54 616 ± 43 293 ± 15 992 ± 147 168 ± 35

Activation Condition : Rat Liver S9
Dose (mmoles/L) TA98 TA100 TA1535 TA1537 WP2 uvr A

Vehicle 24 ± 7 209 ± 16 7 ± 4 7 ± 2 19 ± 6

3 15 ± 2 216 ± 4 7 ± 6 4 ± 2 23 ± 7

6 25 ± 13 203 ± 34 6 ± 3 3 ± 2 16 ± 4

8 18 ± 3 236 ± 36 10 ± 5 4 ± 1 13 ± 7

11 13 ± 3 214 ± 36 8 ± 3 7 ± 3 6 ± 1

21 3 ± 2 267 ± 22 5 ± 2 0 ± 0 5 ± 2

37 0 ± 0 0 ± 0 0 ± 0 0 ± 1 0 ± 0

Positive 442 ± 168 722 ± 275 73 ± 30 36 ± 5 181 ± 48

Vehicle = Vehicle Control
Positive = Positive Control (50 μ L plating aliquot)

### BioReliance Study No. AC12HE.502700.BTL 37 Table 17 Confirmatory Mutagenicity Assay without S9 activation

Study Number: AC12HE.502700.BTL Study Code: AC12HE Experiment: B2 Date Plated: 9/14/2010 Exposure Method: Plate incorporation assay Evaluation Period: 9/20/2010 to 9/22/2010

Strain Article Dose level per plate Mean

revertants per plate Standard

Deviation Ratio

treated /

solvent Individual revertant colony counts and background codes

TA100 37mmoles/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21mmoles/L 40 13 0.4 54 A 3, 37 A 3, 28 A 3

11mmoles/L 88 77 0.9 45 A , 42 A , 176 A

8 mmoles/L 130 25 1.4 121 A , 158 A , 110 A

6 mmoles/L 113 18 1.2 107 A , 98 A , 133 A

3 mmoles/L 108 14 1.1 101 A , 125 A , 99 A

Untreated Air 96 12 110 A , 86 A , 92 A

TA1535 37mmoles/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21mmoles/L 11 3 1.0 11 A 3, 13 A 3, 8 A 3

11mmoles/L 7 3 0.6 9 A , 4 A , 9 A

8 mmoles/L 11 4 1.0 8 A , 15 A , 11 A

6 mmoles/L 9 2 0.8 11 A , 8 A , 7 A

3 mmoles/L 8 1 0.7 8 A , 7 A , 9 A

Untreated Air 11 4 11 A , 15 A , 8 A

Key to Plate Postfix Codes

4

3 Extremely reduced background Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic count

### BioReliance Study No. AC12HE.502700.BTL 38 Table 17 cont. Confirmatory Mutagenicity Assay without S9 activation

Study Number: AC12HE.502700.BTL Study Code: AC12HE Experiment: B2 Date Plated: 9/14/2010 Exposure Method: Plate incorporation assay Evaluation Period: 9/20/2010 to 9/22/2010

Strain Article Dose level per plate Mean

revertants per plate Standard

Deviation Ratio

treated /

solvent Individual revertant colony counts and background codes

TA1537 37mmoles/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21mmoles/L 1 2 0.2 3 A , 0 A , 0 A

11mmoles/L 2 1 0.4 3 A , 3 A , 1 A

8 mmoles/L 2 3 0.4 1 A , 5 A , 0 A

6 mmoles/L 8 2 1.6 9 A , 9 A , 5 A

3 mmoles/L 4 1 0.8 4 A , 4 A , 3 A

Untreated Air 5 3 8 A , 3 A , 5 A

WP2uvrA 37mmoles/L 8 4 0.2 7 A , 4 A , 12 A

21mmoles/L 15 3 0.3 13 A , 19 A , 13 A

11mmoles/L 33 6 0.7 40 A , 28 A , 31 A

8 mmoles/L 39 6 0.9 45 A , 33 A , 40 A

6 mmoles/L 55 7 1.2 48 A , 61 A , 57 A

3 mmoles/L 56 3 1.2 57 A , 53 A , 58 A

Untreated Air 45 9 37 A , 54 A , 45 A

TA100 SA 1.0 μg 772 20 8.0 749 A , 784 A , 784 A

TA1535 SA 1.0 μg 666 14 60.5 668 A , 651 A , 678 A

TA1537 9AAD 75 μg 515 62 103.0 576 A , 517 A , 452 A

WP2uvrA MMS 1000 μg 372 35 8.3 338 A , 369 A , 408 A

Key to Positive Controls Key to Plate Postfix Codes

2NF

SA

9AAD

MMS 2-nitrofluorene

sodium azide

9-Aminoacridine methyl methanesulfonate 4

3 Extremely reduced background Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic count

### BioReliance Study No. AC12HE.502700.BTL 39 Table 18 Confirmatory Mutagenicity Assay with S9 activation

Study Number: AC12HE.502700.BTL Study Code: AC12HE Experiment: B2 Date Plated: 9/14/2010 Exposure Method: Plate incorporation assay Evaluation Period: 9/20/2010 to 9/22/2010

Strain Article Dose level per plate Mean

revertants per plate Standard

Deviation Ratio

treated /

solvent Individual revertant colony counts and background codes

TA100 37mmoles/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21mmoles/L 40 4 0.4 44 A 2, 41 A 2, 36 A 2

11mmoles/L 63 6 0.6 58 A , 62 A , 70 A

8 mmoles/L 72 10 0.7 73 A , 82 A , 62 A

6 mmoles/L 83 10 0.8 72 A , 90 A , 88 A

3 mmoles/L 88 5 0.8 86 A , 94 A , 84 A

Untreated Air 106 6 113 A , 103 A , 103 A

TA1535 37mmoles/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21mmoles/L 7 2 0.8 8 A 3, 8 A 3, 4 A 3

11mmoles/L 7 4 0.8 4 A , 11 A , 7 A

8 mmoles/L 12 1 1.3 13 A , 13 A , 11 A

6 mmoles/L 5 4 0.6 5 A , 8 A , 1 A

3 mmoles/L 10 6 1.1 16 A , 5 A , 8 A

Untreated Air 9 3 7 A , 12 A , 8 A

TA1537 37mmoles/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21mmoles/L 5 4 0.7 9 A , 5 A , 1 A

11mmoles/L 5 2 0.7 3 A , 7 A , 4 A

8 mmoles/L 4 4 0.6 1 A , 8 A , 3 A

6 mmoles/L 6 2 0.9 8 A , 7 A , 4 A

3 mmoles/L 6 2 0.9 4 A , 7 A , 8 A

Untreated Air 7 2 8 A , 5 A , 8 A

TA100 2AA 2.0 μg 407 68 3.8 340 A , 405 A , 476 A

TA1535 2AA 1.0 μg 140 15 15.6 NCN#, 150 A , 129 A

TA1537 2AA 1.0 μg 473 117 67.6 536 A , 338 A , 546 A

Key to Positive Controls Key to Plate Postfix Codes

2AA 2-aminoanthracene 4

2

3

NC

N# Extremely reduced background Slightly reduced background Moderately reduced background

No cells

Not counted

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic count

### BioReliance Study No. AC12HE.502700.BTL 40 Table 19 Retest of Confirmatory Mutagenicity Assay without S9 activation

Study Number: AC12HE.502700.BTL Study Code: AC12HE Experiment: B3 Date Plated: 9/28/2010 Exposure Method: Plate incorporation assay Evaluation Period: 10/6/2010

Strain Article Dose level per plate Mean

revertants per plate Standard

Deviation Ratio

treated /

solvent Individual revertant colony counts and background codes

TA98 37 mmol/L 0 0 0.0 0 M 5, 0 M 5, 0 M 5

21 mmol/L 15 3 0.5 19 M , 13 M , 14 M

11 mmol/L 22 3 0.7 19 M , 23 M , 25 M

8 mmol/L 27 6 0.9 23 M , 33 M , 24 M

6 mmol/L 24 3 0.8 23 M , 27 M , 22 M

3 mmol/L 29 8 1.0 31 M , 36 M , 21 M

1 mmol/L 34 4 1.1 36 M , 37 M , 29 M

0.7 mmol/L 31 3 1.0 28 M , 33 M , 33 M

Untreated Air 30 10 25 M , 41 M , 24 M

TA1537 37 mmol/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21 mmol/L 4 1 0.4 4 A , 5 A , 4 A

11 mmol/L 6 5 0.6 3 A , 4 A , 12 A

8 mmol/L 7 5 0.7 12 A , 3 A , 7 A

6 mmol/L 11 3 1.1 13 A , 8 A , 11 A

3 mmol/L 10 2 1.0 8 A , 9 A , 12 A

1 mmol/L 6 4 0.6 1 A , 7 A , 9 A

0.7 mmol/L 6 3 0.6 3 A , 8 A , 7 A

Untreated Air 10 2 11 A , CPN#, 8 A

TA98 2NF 1.0 μg 351 69 11.7 282 A , 350 A , 420 A

TA1537 9AAD 75 μg 633 142 63.3 721 A , 469 A , 710 A

Key to Positive Controls Key to Plate Postfix Codes

2NF

9AAD 2-nitrofluorene

9-Aminoacridine 4

CP

N#

5 Extremely reduced background Contaminated plate

Not counted Absent background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic count

### BioReliance Study No. AC12HE.502700.BTL 41 Table 20 Retest of Confirmatory Mutagenicity Assay with S9 activation

Study Number: AC12HE.502700.BTL Study Code: AC12HE Experiment: B3 Date Plated: 9/28/2010 Exposure Method: Plate incorporation assay Evaluation Period: 10/6/2010

Strain Article Dose level per plate Mean

revertants per plate Standard

Deviation Ratio

treated /

solvent Individual revertant colony counts and background codes

TA98 37 mmol/L 0 0 0.0 0 M 4, 0 M 4, 0 M 4

21 mmol/L 10 1 0.8 11 M , 10 M , 10 M

11 mmol/L 10 1 0.8 9 M , 10 M , 10 M

8 mmol/L 11 4 0.9 15 M , 10 M , 7 M

6 mmol/L 9 3 0.8 9 M , 11 M , 6 M

3 mmol/L 10 4 0.8 5 M , 11 M , 13 M

1 mmol/L 9 3 0.8 8 M , 13 M , 7 M

0.7 mmol/L 9 0 0.8 9 M , 9 M , 9 M

Untreated Air 12 1 13 M , 11 M , 11 M

WP2uvrA 37 mmol/L 4 1 0.1 3 A 3, 5 A 3, 5 A 3

21 mmol/L 34 13 0.8 49 A , 28 A , 24 A

11 mmol/L 32 4 0.8 32 A , 29 A , 36 A

8 mmol/L 35 6 0.8 41 A , 29 A , 34 A

6 mmol/L 28 5 0.7 27 A , 24 A , 34 A

3 mmol/L 43 6 1.0 37 A , 44 A , 48 A

1 mmol/L 42 5 1.0 48 A , 38 A , 41 A

0.7 mmol/L 39 15 0.9 53 A , 42 A , 23 A

Untreated Air 42 2 40 A , 42 A , 44 A

TA98 2AA 1.0 μg 408 78 34.0 318 A , 459 A , 448 A

WP2uvrA 2AA 10 μg 198 11 4.7 186 A , 206 A , 202 A

Key to Positive Controls Key to Plate Postfix Codes

2AA 2-aminoanthracene 4

3 Extremely reduced background Moderately reduced background

Key to Automatic & Manual Count Flags

M : Manual count A : Automatic count

### BioReliance Study No. AC12HE.502700.BTL 42 APPENDIX I: Historical Control Data

### BioReliance Study No. AC12HE.502700.BTL 43 Historical Negative and Positive Control Values

2005 – 2007

revertants per plate

Activation

None Rat Liver Strain Control

Mean SD Min Max Mean SD Min Max

Neg 17 6 5 56 25 8 6 60 TA98 Pos 189 112 30 1812 526 343 73 2669

Neg 133 30 52 250 143 32 67 263 TA100 Pos 548 148 112 4349 698 343 232 2652

Neg 20 7 4 53 16 5 4 45 TA1535 Pos 420 146 54 1019 104 59 18 985

Neg 7 3 1 20 8 3 1 29 TA1537 Pos 740 356 14 2514 88 87 13 1297

Neg 21 9 5 69 23 10 4 65 WP2 uvr A Pos 214 137 28 1086 268 174 29 1178

SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

### BioReliance Study No. AC12HE.502700.BTL 44 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=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

### BioReliance Study No. AC12HE.502700.BTL 45 APPENDIX II: Study Protocol

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### BioReliance Study No. AC12HE.502700.BTL 61 APPENDIX III: Information for Japanese Regulatory Agencies

### BioReliance Study No. AC12HE.502700.BTL 62 Report of Results of Reverse- Mutation Assay in Bacteria

1. Tester Strains

(1) Procurement

Strain Obtained from Date obtained Date inspected

the strain lot in storage

TA98

TA100

TA1535

TA1537

TA1538

TA97

TA102 Dr. Bruce Ames’ designated distributor,

Discovery Partners International, San Diego,

California 13 November 2002; 16 January 2004

(TA100 only)

WP2 uvr A

WP2 uvr A (pkM101) 1 July 1987

WP2 (pKM101) National Collection of

Industrial and Marine

Bacteria Aberdeen, Scotland 19 February 1993 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. AC12HE.502700.BTL 63 2. S9 Mix

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

Prepared on Used in Experiment No.

29 January 2008 (Lot 2242) A1 and B1 Purchased from

MolTox
29 June 2010 (Lot 2639) B2 and B3

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 (Lots 2242 and 2639)

Weight Unknown (Lots 2242 and 2639) Administration period andamount (g/kgweight) single dose at 0.5 gm/kg body weight, 5 days prior to sacrifice

3. Preparation of Test Article Solution

Solvent used

Name Manufacturer Lot No. Grade and/or Purity (%)

Not Applicable Not Applicable Not Applicable Not Applicable

Stability of test article in the

solvent Unknown

Method of suspension when

test article is difficult to

dissolve Not applicable

Stability, storage time and temperature of formulation from preparation until use No change in appearance was noted in the formulations during the period of use (2 hours at room temperature)

### BioReliance Study No. AC12HE.502700.BTL 64 4. Conditions of Pre-culture

Nutrient broth Name Manufacturer Lot Nos.

Oxoid Nutrient

Broth No. 2 Oxoid Ltd. 544599, 847654

and 876774

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. AC12HE.502700.BTL 65 5. Agar Plate Medium

(1) Top agar

Name BBL Select

Manufacturer Becton Dickinson
Agar Lot No. 7078979

7253113

9265193

(2) Minimum Glucose Agar

Name BBL Select

Manufacturer Becton Dickinson

Lot No. 7253113

Batch

No. Preparation Date Used in Experiment No.

16933 A1 Made in-house Agar

16934 07 April 2008
B1

Name BBL Select

Manufacturer Becton Dickinson

Lot Nos. 7078979

9265193

0223574

Batch

No. Preparation Date Used in Experiment No.

32323 18 March 2008 B1

36101 12 August 2010 B2 Agar

36238 08 September 2010 B3 Purchased from Moltox

Volume of agar plate medium 25 mL

### BioReliance Study No. AC12HE.502700.BTL 66 6. Test Results - Judgement of the results

Judgement 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.
Stability data were not provided for either lot/batch of the test article. In the absence of stability data, the Study Direct or has concluded that the test article was negative with the caveat that the maximum required concentrat ion may not have been achieved due to instability or impurities.
At the request of the Sponsor, dosing formulation analyses fo r concentration and stability

were not conducted. Due to the lack of dose fo rmulation analysis, the interpretation of the study data were based on the nominal dose leve ls as documented in the study records and

not on the actual formulated test article concen trations as confirmed by analytical analysis. Nevertheless, toxicity in the assay demonstrat ed that the test system was dosed up to the regulatory required level.

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. AC12HE.502700.BTL 67 APPENDIX IV: Test Article Characterization

### BioReliance Study No. AC12HE.502700.BTL 68

### BioReliance Study No. AC12HE.502700.BTL 69