FINAL REPORT

Unpublished Work

Testing Facility Study No.

Sponsor Report No.

Work Request No.

Service Code

Bacterial Reverse Mutation Assay Using Gas-phase Exposure

SPONSOR:

STUDY COMPLETED ON 03 June 2010

Page 1 of 50



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TABLE OF CONTENTS

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

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

3. CERTIFICATION STATEMENT ......................................................................................3

LIST OF TABLES .........................................................................................................................7

LIST OF FIGURES .......................................................................................................................8

LIST OF APPENDICES ...............................................................................................................9

4. STUDY INFORMATION...................................................................................................10

5. SUMMARY INFORMATION...........................................................................................11

6. INTRODUCTION ...............................................................................................................12

7. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS ..................................12

7.1. Control Atmosphere...................................................................................................12

7.2. Test Substance ............................................................................................................12

7.3. Positive Controls.........................................................................................................12

7.3.1. In the Absence of S9 Mix ............................................................................12

7.3.2. In the Presence of S9 Mix ...........................................................................13

8. EXPERIMENTAL PROCEDURES..................................................................................13

8.1. Test System .................................................................................................................13

8.2. S9 Mix..........................................................................................................................14

8.3. Sterility Controls ........................................................................................................14

8.4. Atmosphere Concentration and Stability.................................................................14

8.5. Test Method and Test Atmosphere Generation ......................................................14

9. EVALUATION AND INTERPRETATION OF RESULTS ...........................................16

10. QUALITY ASSURANCE...................................................................................................18

11. PROTOCOL DEVIATIONS..............................................................................................18

12. ARCHIVES..........................................................................................................................18

13. RESULTS.............................................................................................................................18
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13.1.Bacterial Mutation Test .............................................................................................18

14. CONCLUSION....................................................................................................................19
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LIST OF TABLES

Table 1 - Initial Test in the Absence of S9 Mix.................................................. 20

Table 2 - Initial Test in the Presence of S9 Mix ................................................. 21

Table 3 Positive Controls for the Initial Test..................................................................... 22

Table 4 - Confirmatory Test in the Absence of S9 Mix ..................................... 23

Table 5 - Confirmatory Test in the Presence of S9 Mix..................................... 24

Table 6 Positive Controls for the Confirmatory Test......................................................... 25
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LIST OF FIGURES

Figure 1 TA1535 Revertant Colony Count Historical Results............................................ 26

Figure 2 TA1537 Revertant Colony Count Historical Results............................................ 26

Figure 3 TA98 Revertant Colony Count Historical Results................................................ 27

Figure 4 TA100 Revertant Colony Count Historical Results.............................................. 27

Figure 5 WP2 uvrA Revertant Colony Count Historical Results ........................................ 28
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LIST OF APPENDICES

Appendix 1 Certificate of Analysis .......................................................................................... 29

Appendix 2 Historical Positive Control Results....................................................................... 31

Appendix 3 Protocol and Amendments.................................................................................... 33
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4. STUDY INFORMATION

Substance Tested: ï

CAS Number)

Number:

Composition: (mole %)

Purity: See composition, above

Physical Characteristics: Colorless gas

Stability: The test substance appeared to be stable under the conditions

of the study; no evidence of instability was observed.

Study Initiated/Completed: 09 March2010/(see report cover page)

Experimental Start/Termination: 12 March 2010/30 March 2010
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5. SUMMARY INFORMATION

The purpose of this study was to evaluate the genotoxicity of using the bacterial reverse

mutation test.

Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia coli strain

WP2 uvrA were treated with the gaseous test substance at a range of concentrations separated by

a half-log10 dose interval up to 100%, i.e., the maximum practical level) in the presence and

absence of a supplemented liver fraction (S9 mix) using the plate incorporation version of the

bacterial mutation test. Since no evidence of genotoxicity and only slight toxicity was seen in

this initial test, the study was repeated using a narrower (approximately 2-fold) dose interval to

confirm the results.

Bacteria were incubated with standard positive control agents, and the response of the various

bacterial strains to these agents confirmed the sensitivity of the test system and the activity of

the S9 mix.

No substantial increases in the revertant colony counts were obtained with any strain following

exposure to the test substance in either the initial or confirmatory assays in the absence or

presence of S9 mix. It is concluded that did not show any evidence of genotoxic

activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.
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6. INTRODUCTION

The bacterial mutation test detects point mutations, which are the cause of many human genetic

diseases and can play a role in tumor initiation and development.

7. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS

7.1. Control Atmosphere

Ambient laboratory air was used to treat negative/vehicle control plates and as the diluent for the

test substance.

7.2. Test Substance

Identity

CAS number

Chemical name

Molecular weight

Lot number

Expiry date 30 June 2011

Description Colorless gas under normal room conditions, boiling point 7.5°C

Storage conditions Kept in a pressurized cylinder in a ventilated area at

ambient/controlled room temperature

Supplier Sponsor

Purity/Correction Assumed to be 100%; all concentrations are expressed in terms of

material as is

The above information relating to the nature of the test substance is the responsibility of the

Sponsor (see Appendix 1 for Certificate of Analysis).

7.3. Positive Controls

7.3.1. In the Absence of S9 Mix

Identity Sodium azide (NaAz)

CAS number 26628-22-8

Vehicle Sterile water for irrigation USP

Supplier Sigma-Aldrich
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Identity 9-Aminoacridine (9AC)

CAS number 52417-22-8

Vehicle Sterile water for irrigation USP

Supplier Sigma-Aldrich

Identity 2-Nitrofluorene (2NF)

CAS number 607-57-8

Vehicle DMSO

Supplier Sigma-Aldrich

Identity 4-Nitroquinoline N-oxide (NQO)

CAS number 56-57-5

Vehicle DMSO

Supplier Sigma-Aldrich

7.3.2. In the Presence of S9 Mix

Identity 2-Aminoanthracene (2AA)

CAS number 613-13-8

Vehicle DMSO

Supplier Sigma-Aldrich

Identity Benzo[a]pyrene (BaP)

CAS number 50-32-8

Vehicle DMSO

Supplier Supelco

Full details of positive controls including expiry date, lot number, appearance, storage, copies of

certificates of analysis and formulation are retained as raw data.

8. EXPERIMENTAL PROCEDURES

8.1. Test System

The bacteria were originally supplied by Moltoxô, NC, USA. Each batch of frozen bacteria

was tested for appropriate phenotype characteristics and spontaneous reversion rates; response to

diagnostic mutagens is also routinely assessed. The following bacterial strains were employed:
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S. typhimurium TA1535 hisG46 rfa ∆uvrB

S. typhimurium TA1537 hisC3076 rfa ∆uvrB

S. typhimurium TA98 hisD3052 rfa ∆uvrB pKM101

S. typhimurium TA100 hisG46 rfa ∆uvrB pKM101

E. coli WP2 trp uvrA

Fresh bacterial cultures were prepared so that they were in the late-log phase of growth at the

time of use. The density of the cultures was confirmed to be ≥ 1000 × 106 bacteria/mL using a

bacterial counting chamber before the cultures were used in the test.

8.2. S9 Mix

The S9 mix, used as a model of intact mammalian metabolism, was prepared on the day of use

and contained 10% v/v S9 fraction (phenobarbital/5,6-benzoflavone induced male

Sprague-Dawley rat liver fraction supplied by Moltox, stored in a freezer set to maintain -80°C,

thawed before use) and the following sterile cofactors: 8 mM MgCl2, 33 mM KCl, 100 mM

sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP. The S9 mix was

stored at in a refrigerator set to maintain 4°C or on ice until required. A copy of the

manufacturerís quality control certificate(s) for the S9 fraction is retained as raw data.

8.3. Sterility Controls

Sterility of appropriate components used in the test including buffer, S9 mix, positive controls

(high dose where more than one dose is tested) was confirmed on the day of the test using

appropriate preparations without bacteria (detailed results not reported here).

The spontaneous mutation rates of the bacterial strains were assessed using concurrent control

samples in which the bacteria were exposed to the control atmosphere (air).

8.4. Atmosphere Concentration and Stability

Chemical analysis of test atmosphere for test substance content and stability was not performed

as part of this study.

The positive control formulations were not subjected to analysis for safety reasons and because

the biological response of the test system is considered to be the best measure of the

appropriateness of the formulations.

8.5. Test Method and Test Atmosphere Generation

The study employed the standard plate incorporation method. In the initial test, a 0.5 mL aliquot

of S9 mix (+S9) or phosphate buffer 0.2M pH 7.4 (0S9) was combined with 0.1 mL bacterial
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culture in a sterile container. Where appropriate, a standard volume (0.1 mL) of the test positive

control was added. Approximately 2 mL of molten top agar supplemented with 0.05 mM biotin

and minimal (0.05 mM) histidine and minimal (0.05 mM) tryptophan was added immediately

afterward. The solution was mixed and overlaid onto a minimal glucose plate (1.5% agar,

Vogel-Bonner medium E, 2% glucose). After the overlay solidified, the plates were inverted and

incubated at ca. 37°C for 48 to 72 h. The sterile containers and each plate were labeled with:

study number, a unique number that identifies the contents by reference to the experimental

design held with the raw data, and the day code. After the overlay solidified, the plates were

inverted, stacked and placed in Tedlar bags that had been slit on one side (one bag per

treatment). The bags were sealed with adhesive tape and the excess air inside the bags was

evacuated - note that this initial evacuation was omitted from the protocol in error, but was

correctly performed as per SOP and procedural records in the study raw data.

Appropriate volumes of ambient laboratory air and stock test gas were added via the

septum/valve into each bag to achieve the final concentrations indicated in the Study design.

For the highest dose of the test gas, the gas was added to the bag, then purged prior to the

addition of a second volume of gas.

The plates in the bags were transferred to incubators at ca. 37°C for 48 to 72 h. The sterility

plates for the buffer and S9 mix as well as the plates treated with the routine liquid positive

controls were not placed in Tedlar bags. They were inverted and incubated at ca. 37°C for the

same duration as the test atmosphere-treated plates. After the treatment period, the bags were

opened in a fume hood or extracted laminar flow cabinet and the plates allowed to equilibrate for

ca. 20 mins before counting colonies.

The initial test used a range of dose levels separated by a half-log10 dose interval up to the

maximum practical limit (100% Based on the results of the initial test, a confirmatory

test was performed using the same methodology and the same upper exposure level but, in this

case, a narrow (approximately 2-fold) concentration interval was employed.
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Text Table 1 Study Design for Initial and Confirmatory Tests

Dose Number of replicates

number Material Exposureconc. % v/v 0S9 +S9

Number of

strains

Initial test

0 Vehicle - 3 3 5

1 Test substance 0.032 3 3 5

2 0.100 3 3 5

3 0.316 3 3 5

4 1.000 3 3 5

5 3.16 3 3 5

6 10.0 3 3 5

7 31.6 3 3 5

8 ~100 a 3 3 5

Positive control á 3 3 5

Confirmatory test

0 Vehicle - 3 3 5

1 Test substance 3.2 3 3 5

2 6.4 3 3 5

3 12.8 3 3 5

4 25.6 3 3 5

5 51.2 3 3 5

6 ~100 3 3 5

Positive control á 3 3 5

a The maximum practical dose (nominally 100%) was achieved by removing as much air as possible from the bag

using a vacuum pump, gassing with test substance, purging the atmosphere then re-gassing.

á Depends on the test organism and positive control agent.

9. EVALUATION AND INTERPRETATION OF RESULTS

After the incubation/equilibration period, the plates were examined visually and, if necessary,

with the aid of an inverted microscope. Plates were evaluated for the quality of the background

lawn and the number of revertant colonies. Revertant colony counts were routinely collected and

saved directly into an electronic database using a ProtoCOL automated colony counter

(version 3.06 supplied by Synbiosis). The electronic raw data were transferred to CD-ROM and

held with the rest of the study data. Colony numbers were enumerated visually if precipitation or

other artifacts interfered with the colony counter or at the discretion of the Study Director.

Visual counts and the presence of visible precipitate, if any, were manually recorded in the

raw data.
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If available, the plates from at least five non-toxic dose levels of the test substance were assessed

in each experiment, i.e., the five highest levels below the toxic level. Toxic effects of the test

substance are normally indicated by the partial or complete absence of a background lawn

(colony counts, if any, are not reported) or a substantial dose-related reduction in revertant

colony counts compared with lower dose levels and concurrent vehicle controls (i.e., fold

response < 0.6) taking into account the laboratory historical control range. Where five

(relatively non-toxic) dose levels were analyzed, the remaining plates from lower dose levels

may not have been evaluated, and are not reported.

Means and standard deviations for appropriate dose levels were calculated using MicrosoftÆ

Excel. The mean number of revertant colonies for all treatment groups was compared with those

obtained for the concurrent vehicle control group. The mutagenic activity of the test substance

was assessed by applying the following criteria:

1. The results are considered positive (i.e. indicative of mutagenic potential) if both the

following criteria are met.

! The results for the test substance show a substantial increase in revertant colony

counts, i.e. response ≥ 2 times the concurrent vehicle control values for TA98,

TA1535, TA1537 and WP2 uvrA, or ≥ 1.5 times for TA100, with mean value(s)

outside the laboratory historical control range (beyond the 98% tolerance limit).

Otherwise results are considered negative.

! The above increase must be dose related and/or reproducible i.e. increases must

be obtained at more than one experimental point (more than one strain or dose

level, more than one occasion or with different methodologies).

2 If the second criterion is not met, further testing might be appropriate to clarify equivocal

results using an appropriately modified study design, e.g., a narrower dose interval with

the appropriate strain. In such cases, if no substantial increase is obtained in the

confirmatory test, the results would be considered negative.

For an assay to be considered valid, the mean revertant colony counts of the vehicle controls for

each strain should lie close to or within the current historical control range of the laboratory.

All positive control substances (with S9 mix where required) should produce increases in

revertant colony numbers to at least twice the concurrent vehicle control levels with the

appropriate bacterial strain (1.5× for strain TA100). Any values that do not comply with these

criteria are discussed in the Results section of this report.
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10. QUALITY ASSURANCE

The Quality Assurance department of performed and documented inspections and

audits of critical phases of the study performed at during its conduct and audited

the study report. Data audits were conducted according to standard procedures.

The Quality Assurance Statement is presented on page 3.

11. PROTOCOL DEVIATIONS

Occasional minor deviations from the protocol, if any, are documented in the raw data and,

where appropriate, the text of this report. No deviation had any impact on the outcome of the

study or upon the interpretation of the results.

12. ARCHIVES

The following items will be retained in the scientific archives of for a period of

approximately 1 year after dispatch of the final report:

! All raw data

! Documents generated at during this study

! The original copy of the protocol (including amendments)

! The final report

Subsequent storage details will be documented in the raw data.

13. RESULTS

13.1. Bacterial Mutation Test

Occasional reductions in the revertant colony count were obtained with some bacterial strains at

the highest concentration of indicating that the substance was somewhat toxic to the

bacteria and confirming exposure. Revertant colony counts were recorded and reported here for

at least the five highest non-toxic concentrations of the test substance; i.e. at least the five highest

non-toxic concentrations. The results are presented in Table 1, Table 2, Table 3, Table 4,

Table 5 and Table 6; footnotes are explained in Table 1. The mean and standard deviation values

quoted have been rounded to the nearest whole number.

The absence of colonies on sterility check plates confirmed absence of microbial contamination

(results not shown). The mean revertant colony counts for the negative controls were close to or

within the laboratory historical control range (see Figure 1, Figure 2, Figure 3, Figure 4 and

Figure 5 for individual colony count historical results). Appropriate positive control compounds

(with S9 mix where required) induced increases in revertant colony numbers to at least twice the

concurrent vehicle control levels with the appropriate bacterial strain (1.5× for strain TA100),
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confirming sensitivity of the test system and activity of the S9 mix (see also Appendix 2 for

positive control historical results).

No substantial increases in revertant colony numbers were obtained with any of the tester strains

following exposure to at any concentration in either the initial or confirmatory assay in

the presence or absence of S9 mix.

14. CONCLUSION

No substantial increases in the revertant colony counts were obtained with any strain following

exposure to the test substance in either the initial or confirmatory assays in the absence or

presence of S9 mix. It is concluded that did not show any evidence of genotoxic

activity in this in vitro mutagenicity assay when tested in accordance with regulatory guidelines.
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Table 1 - Initial Test in the Absence of S9 Mix

Number of revertants Plate observations * Strain Conc.

(μg/plate) S9

x1 x2 x3 mean SD x1 x2 x3

Fold

response Ü

TA1535 Air 0 16 16 19 17 2 - - - 1.0

1.00 0 25 20 17 21 4 - - - 1.2

3.16 0 25 20 22 22 3 - - - -

10.0 0 17 15 18 17 2 - - - 1.0

31.6 0 17 20 28 22 6 - - - 1.3

~100 0 19 20 16 18 2 - - - 1.1

TA1537 Air 0 12 6 8 9 3 - - - 1.0

0.316 0 8 17 7 11 6 - - - 1.2

1.00 0 12 6 11 10 3 - - - 1.1

3.16 0 11 11 13 12 1 - - - 1.3

10.0 0 10 6 13 10 4 - - - 1.1

31.6 0 7 8 4 6 2 - - - 0.7

~100 0 7 3 4 5 2 - - - 0.5 T

TA98 Air 0 42 27 19 29 12 - - - 1.0

0.316 0 20 21 15 19 3 - - - 0.6

1.00 0 33 22 34 30 7 - - - 1.0

3.16 0 38 28 24 30 7 - - - 1.0

10.0 0 28 27 25 27 2 - - - 0.9

31.6 0 31 22 20 24 6 - - - 0.8

~100 0 10 6 10 9 2 - - - 0.3 T

TA100 Air 0 108 113 117 113 5 - - - 1.0

1.00 0 116 119 128 121 6 - - - 1.1

3.16 0 118 106 135 120 15 - - - 1.1

10.0 0 96 119 120 112 14 - - - 1.0

31.6 0 120 103 118 114 9 - - - 1.0

~100 0 89 83 106 93 12 - - - 0.8

WP2 uvrA Air 0 57 58 80 65 13 - - - 1.0

1.00 0 62 63 65 63 2 - - - 1.0

3.16 0 63 70 43 59 14 - - - 0.9

10.0 0 52 67 63 61 8 - - - 0.9

31.6 0 58 56 52 55 3 - - - 0.9

~100 0 53 56 47 52 5 - - - 0.8

* Comments on the plate or background lawn, if applicable: incomplete lawn (IL), no lawn (NL), precipitate (ppt).

Ü Fold response in mean revertants compared to concurrent vehicle control.

SD Sample standard deviation (note that SDs based on two values may be unreliable).

T If noted, toxic as indicated by low revertant colony counts (fold response < 0.6), or incomplete/

no background lawn (no meaningful count results for plates with IL or NL).

A If noted, low count considered to be due to normal variation rather than toxicity since not clearly dose-related

and not outside normal limits based on historical control values.
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Table 2 - Initial Test in the Presence of S9 Mix

Number of revertants Plate observations * Strain Conc.

(μg/plate) S9

x1 x2 x3 mean SD x1 x2 x3

Fold

response Ü

TA1535 Air + 17 17 26 20 5 - - - 1.0

0.316 + 12 16 11 13 3 - - - 0.7

1.00 + 22 19 26 22 4 - - - 1.1

3.16 + 20 22 26 23 3 - - - 1.1

10.0 + 24 20 17 20 4 - - - 1.0

31.6 + 12 17 13 14 3 - - - 0.7

~100 + 16 8 7 10 5 - - - 0.5 T

TA1537 Air + 10 11 7 9 2 - - - 1.0

1.00 + 10 8 15 11 4 - - - 1.2

3.16 + 6 12 11 10 3 - - - 1.0

10.0 + 18 9 6 11 6 - - - 1.2

31.6 + 7 4 8 6 2 - - - 0.7

~100 + 9 6 4 6 3 - - - 0.7

TA98 Air + 35 62 40 46 14 - - - 1.0

0.316 + 22 24 22 23 1 - - - 0.5 A

1.00 + 42 47 40 43 4 - - - 0.9

3.16 + 49 28 37 38 11 - - - 0.8

10.0 + 44 39 35 39 5 - - - 0.9

31.6 + 44 27 36 36 9 - - - 0.8

~100 + 13 20 18 17 4 - - - 0.4 T

TA100 Air + 117 139 126 127 11 - - - 1.0

1.00 + 139 169 126 145 22 - - - 1.1

3.16 + 147 134 117 133 15 - - - 1.0

10.0 + 107 126 124 119 10 - - - 0.9

31.6 + 113 122 115 117 5 - - - 0.9

~100 + 71 85 82 79 7 - - - 0.6

WP2 uvrA Air + 91 96 78 88 9 - - - 1.0

0.316 + 57 44 36 46 11 - - - 0.5 A

1.00 + 67 65 48 60 10 - - - 0.7

3.16 + 80 65 63 69 9 - - - 0.8

10.0 + 68 80 43 64 19 - - - 0.7

31.6 + 70 62 70 67 5 - - - 0.8

~100 + 47 39 33 40 7 - - - 0.4 T

For explanatory footnotes, please see Table 1.
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Table 3 Positive Controls for the Initial Test

Conc. Number of revertants Fold Strain Treatment (μg/plate) S9

x1 x2 x3 mean SD response Ü

TA1535 NaAz 0.5 0 303 371 310 328 37 19

TA1537 9AC 50 0 384 380 308 357 43 41

TA98 2NF 1 0 193 172 171 179 12 6.1

TA100 NaAz 0.5 0 473 520 485 493 24 4.4

WP2 uvrA NQO 0.5 0 227 253 230 237 14 3.6

TA1535 2AA 5 + 522 495 479 499 22 25

TA1537 BaP 5 + 107 113 97 106 8 11

TA98 BaP 5 + 411 422 417 417 6 9.1

TA100 BaP 5 + 1139 984 1003 1042 85 8.2

WP2 uvrA 2AA 15 + 239 227 248 238 11 2.7

Ü Fold response in mean revertants compared to concurrent vehicle control.

SD Sample standard deviation (note that SDs based on two values may be unreliable).
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Table 4 - Confirmatory Test in the Absence of S9 Mix

Number of revertants Plate observations * Strain Conc.

(μg/plate) S9

x1 x2 x3 mean SD x1 x2 x3

Fold

response Ü

TA1535 Air 0 20 9 18 16 6 - - - 1.0

6.4 0 22 15 8 15 7 - - - 1.0

12.8 0 15 18 28 20 7 - - - 1.3

25.6 0 15 12 12 13 2 - - - 0.8

51.2 0 18 20 11 16 5 - - - 1.0

~ 100 0 12 15 17 15 3 - - - 0.9

TA1537 Air 0 14 5 9 9 5 - - - 1.0

6.4 0 12 11 11 11 1 - - - 1.2

12.8 0 10 9 10 10 1 - - - 1.0

25.6 0 11 6 5 7 3 - - - 0.8

51.2 0 8 9 7 8 1 - - - 0.9

~ 100 0 12 9 6 9 3 - - - 1.0

TA98 Air 0 35 22 29 29 7 - - - 1.0

6.4 0 31 25 28 28 3 - - - 1.0

12.8 0 23 26 40 30 9 - - - 1.0

25.6 0 22 24 17 21 4 - - - 0.7

51.2 0 14 20 15 16 3 - - - 0.6

~ 100 0 18 23 26 22 4 - - - 0.8

TA100 Air 0 121 124 118 121 3 - - - 1.0

6.4 0 105 117 95 106 11 - - - 0.9

12.8 0 120 106 137 121 16 - - - 1.0

25.6 0 110 123 103 112 10 - - - 0.9

51.2 0 92 91 100 94 5 - - - 0.8

~ 100 0 86 100 92 93 7 - - - 0.8

WP2 uvrA Air 0 49 54 61 55 6 - - - 1.0

6.4 0 39 64 42 48 14 - - - 0.9

12.8 0 71 38 71 60 19 - - - 1.1

25.6 0 53 46 69 56 12 - - - 1.0

51.2 0 43 63 43 50 12 - - - 0.9

~ 100 0 54 50 51 52 2 - - - 0.9

For explanatory footnotes, please see Table 1.
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Table 5 - Confirmatory Test in the Presence of S9 Mix

Number of revertants Plate observations * Strain Conc.

(μg/plate) S9

x1 x2 x3 mean SD x1 x2 x3

Fold

response Ü

TA1535 Air + 20 17 12 16 4 - - - 1.0

6.4 + 22 12 17 17 5 - - - 1.0

12.8 + 17 20 16 18 2 - - - 1.1

25.6 + 12 27 17 19 8 - - - 1.1

51.2 + 14 16 17 16 2 - - - 1.0

~ 100 + 14 16 15 15 1 - - - 0.9

TA1537 Air + 9 3 9 7 3 - - - 1.0

6.4 + 11 8 11 10 2 - - - 1.4

12.8 + 5 6 7 6 1 - - - 0.9

25.6 + 20 9 11 13 6 - - - 1.9

51.2 + 12 8 8 9 2 - - - 1.3

~ 100 + 11 5 12 9 4 - - - 1.3

TA98 Air + 45 33 38 39 6 - - - 1.0

6.4 + 53 38 32 41 11 - - - 1.1

12.8 + 50 40 37 42 7 - - - 1.1

25.6 + 42 34 42 39 5 - - - 1.0

51.2 + 36 43 40 40 4 - - - 1.0

~ 100 + 42 49 26 39 12 - - - 1.0

TA100 Air + 103 118 119 113 9 - - - 1.0

6.4 + 108 109 116 111 4 - - - 1.0

12.8 + 139 114 128 127 13 - - - 1.1

25.6 + 127 119 95 114 17 - - - 1.0

51.2 + 91 115 100 102 12 - - - 0.9

~ 100 + 97 110 105 104 7 - - - 0.9

WP2 uvrA Air + 77 66 45 63 16 - - - 1.0

6.4 + 78 60 52 63 13 - - - 1.0

12.8 + 77 83 72 77 6 - - - 1.2

25.6 + 60 64 57 60 4 - - - 1.0

51.2 + 81 55 50 62 17 - - - 1.0

~ 100 + 71 54 57 61 9 - - - 1.0

For explanatory footnotes, please see Table 1.
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Table 6 Positive Controls for the Confirmatory Test

Conc. Number of revertants Fold Strain Treatment (μg/plate) S9

x1 x2 x3 mean SD response Ü

TA1535 NaAz 0.5 0 362 360 338 353 13 23

TA1537 9AC 50 0 455 201 490 382 158 41

TA98 2NF 1 0 173 183 141 166 22 5.8

TA100 NaAz 0.5 0 590 599 590 593 5 4.9

WP2 uvrA NQO 0.5 0 243 256 270 256 14 4.7

TA1535 2AA 5 + 503 487 568 519 43 32

TA1537 BaP 5 + 89 76 105 90 15 13

TA98 BaP 5 + 334 352 379 355 23 9.2

TA100 BaP 5 + 1137 1015 1037 1063 65 9.4

WP2 uvrA 2AA 15 + 168 198 182 183 15 2.9

Ü Fold response in mean revertants compared to concurrent vehicle control.

SD Sample standard deviation (note that SDs based on two values may be unreliable).
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Figure 1 TA1535 Revertant Colony Count Historical Results

Means of triplicate plate counts from non-GLP and QA-audited GLP studies from

11 March 2005 to 21 April 2009. The grand mean is 22 and SD is 5.

2 6

17

41

74

117

94

75

54

38

8 3 3 7 11 1111

112

0

20

40

60

80

100

120

140

<5 8 12 16 20 24 28 32 36 40 44 48 52

Revertant colony count

Frequenc

y

Figure 2 TA1537 Revertant Colony Count Historical Results

Means of triplicate plate counts from non-GLP and QA-audited GLP studies from

11 March 2005 to 21 April 2009. The grand mean is 15 and SD is 4.

1 3 10

54

108

153

127

89

63

21 16

5 4 1 2 1 1

0

20

40

60

80

100

120

140

160

180

<3 6 10 14 18 22 26 30 34 38

Revertant colony count

Frequenc

y
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Figure 3 TA98 Revertant Colony Count Historical Results

Means of triplicate plate counts from non-GLP and QA-audited GLP studies from

11 March 2005 to 21 April 2009. The grand mean is 35 and SD is 9.

3 3

13

34

110

101

115

95

81

63

23

15

2 2 1 1

0

20

40

60

80

100

120

140

<9 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 >72

Revertant colony count

Frequenc

y

Figure 4 TA100 Revertant Colony Count Historical Results

Means of triplicate plate counts from non-GLP and QA-audited GLP studies from

11 March 2005 to 21 April 2009. The grand mean is 145 and SD is 20.

1 7 14

41

86

135 141

98

73

35

18

8 6 2

0

20

40

60

80

100

120

140

160

<61 80 100 120 140 160 180 200 220

Revertant colony count

Frequenc

y
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Figure 5 WP2 uvrA Revertant Colony Count Historical Results

Means of triplicate plate counts from non-GLP and QA-audited GLP studies from

11 March 2005 to 21 April 2009. The grand mean is 51 and SD is 12.

1

10 14 17 21

62

83

113

101

84

77

42

16

8

1

0

20

40

60

80

100

120

<10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 >90

Revertant colony count

Frequenc

y
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Appendix 1 Certificate of Analysis
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Appendix 2 Historical Positive Control Results
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Appendix 2 Historical Positive Control Results

Mean of the mean results of triplicate plate counts from non-GLP and QA-audited GLP studies

from 11 March 2005 to 21 April 2009.

Strain Treatment Dose μg/plate S9 Mean SD Range

TA1535 NaAz 0.5 0 308 45 150 - 653

TA1537 9AC 10 a 0 1633 1094 234 - 4678

50 b 0 328 165 28 - 1009

TA98 2NF 1 0 154 37 41 - 247

TA100 NaAz 0.5 0 524 71 246 - 1012

WP2 uvrA NQO 0.5 0 658 511 75 - 1495

TA1535 2AA 5 + 342 79 108 - 700

TA1537 BaP 5 + 114 25 34 - 190

TA98 BaP 5 + 378 72 178 - 550

TA100 BaP 5 + 1042 214 404 - 1665

WP2 uvrA 2AA 15 + 330 120 109 - 666

Where a and b indicate standard dose levels for the pre-incubation and plate incorporation

versions of the test, respectively
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Appendix 3 Protocol and Amendments
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Testing Facility Study No. 

1. STUDY SPECIFIC DETAILS 

Purpose To evaluate the genotoxicity of the test substance using the 

bacterial mutation test. 

Sponsor's Representative 

 

Testing Facility

Compliance Statement This study will be conducted in accordance with the OECD 

Principles of Good Laboratory Practice (revised 1997, published 

1998) and EPA GLP Standard 40 CFR Part 792 (TSCA) with the 

following exceptions: 

● Concentration, stability and homogeneity of test atmosphere will 

not be determined in this study. 

● Test substance characterization was not conducted in compliance 

with GLP. Characterization of the test substance was conducted 

by the Sponsor’s laboratory according to established SOPs, 

controls and approved test methodologies to ensure integrity and 

validity of the results generated. 

The procedures described in this protocol comply with the 

recommendations of the ICH S2A (Federal Register. Adopted 

1995; 61:18197-18202), the OECD guideline 471 for the testing of 

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Testing Facility Study No. 

chemicals (1997), the US EPA health effects test guidelines (1998, 

OPPTS 870.5100) and the US FDA Redbook 2000 (Short-Term 

Tests for Genetic Toxicity, July 7, 2000). 

Archiving Period Approximately 1 year at the Testing Facility. 

Test Substance 

Identity

CAS number 

Chemical name

Molecular weight 

Lot number 

Expiry date 30 June 2011 

Description Colorless gas under normal room conditions, boiling point 7.5°C 

Storage conditions Kept in a pressurized cylinder in a ventilated area at ambient room 

temperature 

Supplier Sponsor 

Purity/Correction Assumed to be 100%; all concentrations to be expressed in terms 

of material as is 

Vehicle/diluent Ambient laboratory air 

Test Atmosphere 

Preparation 

Appropriate mixture prepared in situ immediately at the start of 

exposure

Stability of Test 

Atmosphere 

Not assessed in this study. 

Formulation Analysis for 

Achieved Concentration 

Not assessed in this study. 

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Study Design 

Dose Number of replicates 

number 

Material Exposure 

conc. % v/v 0S9 +S9 

Number of 

strains 

Initial test a

0 Vehicle - 3 3 5 

1 Test substance 0.032 3 3 5 

2 0.100 3 3 5 

3 0.316 3 3 5 

4 1.000 3 3 5 

5 3.16 3 3 5 

6 10.0 3 3 5 

7 31.6 3 3 5 

8 ~100 a 3 3 5 

Positive control ‡ 3 3 5 

CE 20 3 3 5 

Confirmatory test b

0 Vehicle - 3 3 5 

1 Test substance 3.2 3 3 5 

2 6.4 3 3 5 

3 12.8 3 3 5 

4 25.6 3 3 5 

5 51.2 3 3 5 

6 ~100 3 3 5 

Positive control ‡ 3 3 5 

CE 20 3 3 5 a The maximum practical dose (nominally 100%) will be achieved by removing as much air as possible from the 

bag using a vacuum pump, gassing with test substance, purging the atmosphere then re-gassing 

 b The study design for the confirmatory test is dependent on results obtained in the initial test. In the event of 

toxicity or apparent treatment-related increases in revertant counts, the study design will be discussed with the 

Study Monitor and altered by protocol amendment, if necessary. c

 Standard positive controls dosed as solutions; concentration dependant on test organism and positive control 

Modifications and Notes Gas testing procedures as per Appendix 3 of Test Facility SOP 

 Sponsor report number: 

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2. REASON FOR CHOICE OF TEST SYSTEM 

The bacterial mutation test detects point mutations, which are the cause of many human genetic 

diseases and can play a role in tumor initiation and development. 

3. REASON FOR DOSE LEVEL SELECTION 

Typically, the test substance is dosed at a range of concentrations but is only assessed at the five 

highest levels below the toxic level or, if non-toxic, at five levels up to the standard limit or 

maximum practical concentration, as appropriate. The highest dose for the initial test is the 

maximum practical. 

4. PARTICIPATING PERSONNEL 

Study Director 

 

Participating Scientist(s) 

Assistant Scientist 

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5. SUGGESTED SCHEDULE 

Experimental start (first day of 

exposure of test system to test 

substance) 

 12 March 2010 

Results (provisional experimental 

completion) 

29 March 2010 

Draft report (non-audited) 16 April 2010 

Suggested date for client comments Within 2 weeks of receipt of draft report (see below) 

Final report May 2010, see below 

In order to ensure compliance with GLP, if no requested revisions or instructions to finalize have 

been communicated by the Sponsor six months after issuance of the draft report, the report will 

be issued as the signed final version following QA audit and review. Otherwise, report 

finalization normally occurs about 4 weeks following receipt of client comments and any 

contributing reports. 

6. COMPUTER SYSTEMS 

• Revertant colony counts are routinely collected and saved directly into an electronic 

database using a ProtoCOL automated colony counter (version 3.06 supplied by 

Synbiosis) which minimizes potential bias and transcription errors. 

• Results from ProtoCOL are saved and archived as ProtoCOL database files with backed 

up versions in Microsoft Excel 2000/2003 file format. 

• Results are tabulated, mean values and sample standard deviations calculated (if required) 

using Microsoft Excel 2000/2003. 

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7. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS 

7.1. Documentation 

Full details of the vehicle, test and positive control substances, including supplier, appearance, 

lot number, storage, expiry date, formulation methods and concentrations (if applicable), will be 

recorded in the raw data; appropriate details will be presented in the final report. 

7.2. Vehicle 

The vehicle/diluent will be ambient laboratory air. 

7.3. Test substance and Sponsor's Responsibilities 

The batch/lot number, concentration, purity, expiry date, description, handling precautions and 

supplier will be provided by the Sponsor. The appropriate details will be included in the final 

report. 

The test substance should be characterized by the Sponsor by performing analyses according to 

GLP regulations – refer to STUDY SPECIFIC DETAILS for any exceptions. All data relevant 

to such analyses will be reviewed, audited and retained by the Sponsor who will provide a test 

substance Certificate of Analysis for inclusion in the study report. 

The Sponsor’s approval of the protocol indicates that appropriate documentation of the method 

of synthesis, fabrication or derivation of the test and any non-commercial control substance(s) 

supplied by the sponsor is on file and that it is available to the appropriate regulatory agencies 

should it be requested. 

Unless specifically requested by the Sponsor, all test substance remaining at study completion 

will be returned to the Sponsor (storage conditions, contact, address, fax number and telephone 

to be provided). Alternatively, the test substance can be retained at for subsequent 

studies. The final disposition of the test substance at will be documented but not 

reported unless specifically requested by the Sponsor. 

7.4. Gaseous positive control 

The positive control will be mixed in appropriate proportions with ambient laboratory air in the 

treatment bags at dosing. 

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Identity: Chloroethane (CE) 

Vehicle: Ambient laboratory air 

Concentration: 20% v/v (positive in strains TA1535 and WP2 uvrA and weak/borderline

positive in TA100) 

7.5. Standard Positive Controls 

Sodium azide, 9-aminoacridine, 2-nitrofluorene, 4-nitroquinoline N-oxide, 2-aminoanthracene 

and benzo[a]pyrene will be used as positive control substances. Appropriate details will be 

presented in the report. The positive control formulations will not be subjected to analysis for 

safety reasons and because the biological response of the test system is considered to be the best 

measure of the appropriateness of the formulations. 

8. EXPERIMENTAL PROCEDURES 

8.1. Test system 

The bacteria were originally supplied by MoltoxTM, NC, USA. Each bacterial culture is 

identified by its name (TA1535, TA1537, TA98, TA100, WP2 uvrA) and batch number. Each 

batch of frozen bacteria is tested for appropriate phenotype characteristics, spontaneous reversion 

rate and response to diagnostic mutagens. The following bacterial strains will be employed: 

S. typhimurium TA1535 hisG46 rfa ΔuvrB 

S. typhimurium TA1537 hisC3076 rfa ΔuvrB 

S. typhimurium TA98 hisD3052 rfa ΔuvrB pKM101

S. typhimurium TA100 hisG46 rfa ΔuvrB pKM101 

E. coli WP2 trp uvrA

The batch identification for each strain used will be recorded in the raw data. Fresh bacterial 

cultures, labeled with initiation date, study and strain number, will be prepared and will be in the 

late-log phase of growth at the time of use. The density of the cultures will be confirmed to be 

≥ 1000 × 106

 bacteria/mL using a bacterial counting chamber before the cultures are used in the 

test. 

8.2. S9 Mix 

The S9 mix, used as a model of intact mammalian metabolism, will be prepared on the day of 

use and will contain 10% v/v S9 fraction (phenobarbital/5, 6-benzoflavone induced male 

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Sprague-Dawley rat liver fraction supplied by Moltox) and the following sterile cofactors: 8 mM 

MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 

4 mM NADP. The S9 mix will be stored at ca. 4°C or on ice until required. A copy of the 

manufacturer’s quality control certificate(s) for the S9 fraction will be retained as raw data. 

8.3. Sterility and vehicle controls 

The sterility of the buffer, S9 mix, and positive controls (high dose where more than one dose is 

tested) will be assessed on the day of the test using appropriate preparations without bacteria. 

The spontaneous mutation rates of the bacterial strains will be assessed using concurrent control 

samples in which the bacteria are exposed to the vehicle. Where an unusual vehicle is used, a set 

of untreated (negative) control plates may be included to assess potential effects of the vehicle – 

refer to the study design. 

8.4. Plate Incorporation Method 

A 0.5 mL aliquot of S9 mix (+S9) or phosphate buffer 0.2M pH 7.4 (0S9) will be combined with 

0.1 mL bacterial culture in a sterile container. Where appropriate, a standard volume (0.1 mL) of 

the test positive control will be added. Approximately 2 mL of molten top agar supplemented 

with 0.05 mM biotin and minimal (0.05 mM) histidine and minimal (0.05 mM) tryptophan will 

be added immediately afterward. The solution will be mixed and overlaid onto a minimal 

glucose plate (1.5% agar, Vogel-Bonner medium E, 2% glucose). After the overlay solidifies, 

the plates will be inverted and incubated at ca. 37°C for 48 to 72 h. The sterile containers and 

each plate will be labeled with: study number, a unique number that identifies the contents by 

reference to the experimental design held with the raw data, and the day code. After the overlay 

solidifies, the plates will be inverted, stacked and placed into Tedlar bags (or equivalent) that 

have been slit on one side (one bag per treatment). The bags will be sealed with adhesive tape 

and the air inside the bags (high dose only) will be evacuated. 

Inside a fumehood or laminar hood, appropriate volumes of ambient laboratory air, stock test gas 

or positive control gas will be added via the septum/valve into the bag to achieve the final 

concentrations indicated in the Study design. For the highest dose of the test gas, the gas will be 

added to the bag, then purged prior to the addition of a second volume of gas. 

The plates in the bags will be transferred to incubators at ca. 37°C for 48 to 72 h. After the 

treatment period, the bags will be opened in a flame hood or laminar hood and the plates 

equilibrated in the hood for ca. 20 mins before counting colonies. 

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The sterility plates for the buffer and S9 mix as well as the plates treated with the routine liquid 

positive controls will not be placed into Tedlar bags. They will be inverted and incubated at ca. 

37°C for the same duration as the test atmosphere-treated plates. 

9. EVALUATION AND INTERPRETATION OF RESULTS 

After the incubation period, the plates will be examined visually and, if necessary, with the aid of 

an inverted microscope. Plates will be evaluated for the quality of the background lawn and the 

number of revertant colonies. Revertant colony counts are routinely collected and saved directly 

into an electronic database using a ProtoCOL automated colony counter (version 3.06 supplied 

by Synbiosis). The electronic raw data will be transferred to CD-ROM and held with the rest of 

the study data. Colony numbers will be enumerated visually if precipitation or other artifacts 

interfere with the colony counter (if practical), or at the Study Director’s discretion. Any visual 

counts and the presence of visible precipitate are manually recorded in the raw data. 

If precipitation interferes with observation of the background lawn, this is recorded and noted in 

the report. In such cases, the background lawn is assumed to be normal if the colony counts are 

similar to the vehicle control. In the rare event that precipitation prevents counting of revertant 

colonies, this is noted in the report and the colony count is considered not readable. 

In the event of an apparent treatment-related increase in colony numbers for the test substance, 

the Study Director may authorize replica plating to confirm the phenotype of colonies to 

preclude potential artifacts. 

If available, the plates from at least five non-toxic dose levels of the test substance will be 

assessed in each experiment, i.e. the five highest levels below the toxic level. If the test 

substance is nontoxic, then the five levels up to the highest will be assessed and reported. Toxic 

effects of the test substance are normally indicated by the partial or complete absence of a 

background lawn (colony counts, if any, will not be reported) or a substantial dose-related 

reduction in revertant colony counts compared with lower dose levels and concurrent vehicle 

controls (i.e. fold response < 0.6) taking into account the laboratory historical control range. 

Means and standard deviations for appropriate dose levels will be calculated using Microsoft®

Excel 2003 or 2007 and presented in the report together with laboratory historical control 

information. The mean number of revertant colonies for all treatment groups will be compared 

with those obtained for the concurrent vehicle control group. The mutagenic activity of the test 

substance is routinely assessed by applying the following criteria: 

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1. The results will be considered positive (i.e. indicative of mutagenic potential) if both the 

following criteria are met. 

● The results for the test substance show a substantial increase in revertant colony 

counts, i.e. response ≥ 2 times the concurrent vehicle control values for TA98, 

TA1535, TA1537 and WP2 uvrA, or ≥ 1.5 times for TA100, with mean value(s) 

outside the laboratory historical control range (beyond the 98% tolerance limit). 

Otherwise results are considered negative. 

● The above increase must be dose related and/or reproducible i.e. increases must 

be obtained at more than one experimental point (more than one strain or dose 

level, more than one occasion or with different methodologies). 

2. If the second criterion is not met, further testing may be appropriate to clarify equivocal 

results using an appropriately modified study design, e.g. a narrower dose interval with 

the appropriate strain. In such cases, if no substantial increase is obtained in the 

confirmatory test, the results will be considered negative. 

For an assay to be considered valid, the mean revertant colony counts of the vehicle/negative 

controls for each strain should lie close to or within the current historical control range of the 

laboratory. All positive control substances (with S9 where required) should produce increases in 

revertant colony numbers to at least twice the concurrent vehicle control levels with the 

appropriate bacterial strain (1.5× for strain TA100). Invalid assay results will normally lead to 

an automatic repeat of the appropriate part of the assay. If that is not practical then any values 

that do not comply with these criteria will be discussed in the report. 

10. QUALITY ASSURANCE 

The Quality Assurance department of will undertake and document inspections and 

audits of critical phases of the study performed at during its conduct and audit the 

study report. Data audits will be conducted according to standard procedures. 

Authorized designates of the Sponsor may inspect their study during regular working hours for 

quality assurance purposes. 

11. ARCHIVES 

The following items will be retained in the scientific archives of for the period 

described in the STUDY SPECIFIC DETAILS section after dispatch of the final report: 

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Testing Facility Study No. 

• All raw data 

• Documents generated at during this study 

• The original copy of the protocol (including amendments, if any) 

• The final report 

Subsequent disposition will be documented at the Testing Facility. 

12. COMMUNICATION 

Untoward changes or events occurring during the study will be communicated to the Sponsor by 

telephone, FAX or email. Agreed upon changes in study conduct or design will be documented 

in protocol amendments. 

Hard and/or electronic copies of each protocol amendment will be sent to the Sponsor who will 

return formal approval of the document to 

13. REPORTS 

A complete detailed non-audited draft report will be submitted to the Sponsor electronically for 

review. Any revised draft reports will be submitted to the Sponsor electronically for further 

review. Subsequent to the inclusion of any modifications or corrections (agreed upon by the 

Sponsor and a submission ready version (linked, searchable PDF) of the final report 

will be provided electronically. If no requested revisions or instructions to finalize have been 

communicated by the Sponsor six months after issuance of the draft report, the draft report will 

be issued as a signed final report. 

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