FINAL REPORT

Unpublished Work

Testing Facility Study No.

Sponsor Report No.

Work Request No.

Service Code

In Vitro Mammalian Chromosome Aberration Test Using Gas-phase

Exposure

TESTING FACILITY:

SPONSOR:

STUDY COMPLETED ON 04 June 2010

Page 1 of 48

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2. PARTICIPATING PERSONNEL


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TABLE OF CONTENTS

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

2. PARTICIPATING PERSONNEL .......................................................................................3

3. QUALITY ASSURANCE STATEMENT...........................................................................4

4. CERTIFICATION STATEMENT ......................................................................................5

LIST OF TABLES .........................................................................................................................8

LIST OF FIGURES .......................................................................................................................9

LIST OF APPENDICES .............................................................................................................10

5. STUDY INFORMATION...................................................................................................11

6. SUMMARY INFORMATION...........................................................................................12

7. INTRODUCTION ...............................................................................................................13

8. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS ..................................13

8.1. Control Atmosphere...................................................................................................13

8.2. Test Substance ............................................................................................................13

8.3. Positive Controls.........................................................................................................14

8.3.1. In the Absence of S9 Mix ............................................................................14

8.3.2. In the Presence of S9 Mix ...........................................................................14

8.4. Gaseous Positive Control ...........................................................................................14

9. EXPERIMENTAL PROCEDURES..................................................................................14

9.1. Test System .................................................................................................................14

9.1.1. Blood Sampling............................................................................................14

9.1.2. Culture Medium ..........................................................................................14

9.1.3. Lymphocyte Culture ...................................................................................15

9.2. S9 Mix..........................................................................................................................15

9.3. Stability and Chemical Analysis of Atmosphere Concentration............................15

9.4. Treatment and Test Atmosphere Generation..........................................................15

9.5. Medium Change (4-hour Treatment Only)..............................................................17

9.6. Harvesting and Metaphase Preparation ..................................................................17

9.7. Staining........................................................................................................................17
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9.8. Slide Examination.......................................................................................................17

9.8.1. Mitotic Index (MI).......................................................................................17

9.8.2. Selection of Slides for Detailed Examination............................................18

9.8.3. Detailed Examination for Chromosome Aberrations..............................18

10. EVALUATION AND INTERPRETATION OF RESULTS ...........................................19

11. QUALITY ASSURANCE ...................................................................................................19

12. PROTOCOL DEVIATIONS..............................................................................................20

13. ARCHIVES..........................................................................................................................20

14. RESULTS.............................................................................................................................20

14.1. Dose Selection .............................................................................................................20

14.2. Chromosome Aberrations .........................................................................................20

14.3.Incidental Observations.............................................................................................21

15. CONCLUSION....................................................................................................................21
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LIST OF TABLES

Table 1 Results and Statistical Analysis ............................................................................ 22
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LIST OF FIGURES

Figure 1 Historical Control Values ..................................................................................... 23
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LIST OF APPENDICES

Appendix 1 Certificate of Analysis .......................................................................................... 24

Appendix 2 Results for Individual Cultures ............................................................................. 26

Appendix 3 Protocol and Amendments.................................................................................... 28
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5. STUDY INFORMATION

Substance Tested: ï

ï (CAS Number)

Number: 2

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: 08 March 2010/(see report cover page)

Experimental Start/Termination: 11 March 2010/09 April 2010
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6. SUMMARY INFORMATION

The purpose of this study was to evaluate the genotoxicity of using an in vitro

chromosome aberration test.

Human peripheral blood lymphocytes were stimulated into division in culture then treated with

the gaseous test substance at a range of concentrations separated by a 2-fold dose interval up to

50% v/v, (i.e., the maximum practical level not expected to result in anoxia). Cultures were

treated for 4 hours in the absence and presence of rat S9 mix and for 21 hours in the absence;

appropriate concurrent vehicle and positive controls were included for each treatment regime.

Metaphases from cultures treated with the three highest dose levels of test substance not

producing excessive toxicity (together with appropriate vehicle and selected positive control

cultures) were subjected to detailed examination for the presence of chromosomal aberrations

using light microscopy.

Cultures treated with at levels up to 50% v/v did not show any statistically significant

increases in the incidence of aberrant metaphases in either the absence or presence of S9 mix.

The positive control agents caused highly significant increases in the proportion of cells with

chromosome damage, confirming the sensitivity of the system and the effectiveness of the

S9 mix.

It is concluded that did not show any evidence of genotoxicity in this in vitro test for

induction of chromosome damage when tested in accordance with regulatory guidelines
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7. INTRODUCTION

The purpose of this study was to evaluate the genotoxicity of using the in vitro

chromosome aberration test. Primary cultures of human peripheral lymphocytes are preferred

for this type of study because of their stable chromosome number and structure (karyotype)

compared with cell lines, and their low and stable background rate of aberrations. In addition,

human cells are generally the most relevant for risk assessment.

The present study was initiated on 08 March 2010 (date of approval of the protocol by the

Study Director) and completed on the date of the Study Director approval of this report (refer to

the appropriate signature page). The experimental phase of the study was performed between

11 March 2010 (date of first treatment of cultures with the test substance) and 09 April 2010

(last date of collection of results directly from the study).

8. VEHICLE, TEST SUBSTANCE AND POSITIVE CONTROLS

8.1. Control Atmosphere

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

test substance.

8.2. Test Substance

Identity

CAS number

Chemical name

Molecular weight

Action (intended use) Industrial chemical

Lot number

Expiry 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 throughout this report 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).
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8.3. Positive Controls

8.3.1. In the Absence of S9 Mix

Identity Mitomycin C (MMC)

CAS number 50-07-7

Vehicle Purified water (sterile water for irrigation USP)

Supplier Sigma

8.3.2. In the Presence of S9 Mix

Identity Cyclophosphamide monohydrate (cyclophosphamide, CP)

CAS number 6055-19-2

Vehicle Purified water (sterile water for irrigation USP)

Supplier Sigma

8.4. Gaseous Positive Control

Identity Ethylene oxide (ETOX)

CAS number 75-21-8

Vehicle Ambient laboratory air

Supplier Sigma

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

certificates of analysis (where available) and formulation are retained as raw data.

9. EXPERIMENTAL PROCEDURES

9.1. Test System

9.1.1. Blood Sampling

A peripheral blood sample was taken from a healthy, non-smoking, male donor by venipuncture.

The blood sample was taken directly into blood collecting tubes containing sodium heparin then

held at room temperature for less than 2 hours prior to culture initiation.

9.1.2. Culture Medium

Complete RPMI 1640 medium was prepared by supplementing RPMI 1640 with the following

filter-sterilized components: 10% (v/v) fetal calf serum, 50 μg gentamycin per mL and 4 units

heparin per mL.
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9.1.3. Lymphocyte Culture

Whole blood was mixed with medium (0.4 mL blood per 4.6 mL medium) and

phytohemagglutinin (1 mL per 49 mL diluted blood) was added to stimulate lymphocyte

division. Aliquots of 5 mL of cell suspension were dispensed into flat-sided culture tubes then

incubated at approximately 37°C in a humidified atmosphere containing 5% CO2.

9.2. 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 Moltoxô) 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 kept 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.

9.3. Stability and Chemical Analysis of Atmosphere Concentration

Test atmosphere stability and achieved concentration were not assessed 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.

9.4. Treatment and Test Atmosphere Generation

Treatments were performed approximately 48 hours after culture initiation. The cell suspensions

to be treated with gaseous test substances were transferred to gas-tight tubes (24 mL glass

anaerobic culture tubes supplied by Bellco Glass) and each tube was sealed with a gas

impermeable, butyl rubber septum (injectable) stopper and an aluminum seal. Using a gas-tight

syringe and needle, the appropriate volume of air was removed from each treatment tube, then

the appropriate volume of treatment gas was added. For the cell suspensions to be treated with

the routine positive controls, 10 μL positive control per mL of culture were used throughout and

added to the cultures in the original 5 mL flat-sided tubes. All the cultures were incubated at

ca. 37°C in a humidified atmosphere containing 5% v/v CO2.

was tested over a wide range of dose levels (0.1 to 50.0% v/v) using all treatment

regimes (4 hour treatment period in the absence and presence of S9 mix and a 21 hour treatment

period in the absence of S9 mix) so that analyzable cells would be available for at least

three dose levels for each regime. Duplicate cultures were treated at each experimental point.
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Cultures tested in the absence of S9 mix were treated as indicated in the study design then

returned to the incubator for either 4 or 21 hours. For cultures tested in the presence of S9 mix,

1 mL culture medium was removed and replaced with 1 mL of S9 mix; each culture was treated

as indicated in the study design then returned to the incubator for 4 hours.

Text Table 1 Study Design

Number of cultures Dose

number Material Exposure conc. %v/v 4 Hours

(0S9)

4 Hours

(+S9)

21 Hours

(0S9)

0 Vehicle control - 2 2 2

1 Test substance 0.1 2 2 2

2 0.2 2 2 2

3 0.4 2 2 2

4 0.8 2 2 2

5 1.6 2 2 2

6 3.2 2 2 2

7 6.4 2 2 2

8 12.8 2 2 2

9 25.6 2 2 2

10 50.0Ü 2 2 2

1 ETOX 0.05 2 2 2

2 1.0 2 2 2

3 2.0 2 2 2

1 MMCa 0.05 2 2

2 0.10 2 2

3 0.20 2 2

1 CP a 8.0 2

2 12 2

3 16 2

Ü The highest dose tested was the maximum practical level without resulting in anoxia

a Standard positive controls dosed as solutions; in units of μg/mL

Note that the 21 hour incubation (ëconfirmatory phaseí) is required by regulatory authorities

where the initial phase does not show any indication of genotoxicity for the test substance;

however, it is routinely performed at the same time as the initial phase to minimize potential

delays in performance of the study.
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9.5. Medium Change (4-hour Treatment Only)

After the 4-hour treatment, the appropriate gas-treated cultures were opened in a laminar flow

hood to allow the gas to dissipate, and the cells were transferred to flat-sided culture tubes

(Nunc). All 4-hour treatment cultures were then centrifuged at 1000 rpm for 5 minutes.

The supernatant was discarded and replaced with fresh complete medium. The cultures were

returned to the incubator for another 17 hours of incubation before metaphase harvesting.

For 21-hour treated cultures (i.e., cultures in the confirmatory phase), no replacement of medium

was performed.

9.6. Harvesting and Metaphase Preparation

After colcemid addition at a final concentration of 0.1 μg per mL, all cultures were incubated for

approximately two hours prior to harvesting. The addition of colcemid into gas-tight tubes for

the 21 h 0S9 regime was done through a needle and syringe. After 2 h, the gas-treated cultures

from the 21 h 0S9 regime were opened in a laminar flow hood to allow the gas to dissipate and

the cells were transferred to flat-sided culture tubes (Nunc). Cultures were harvested by

centrifugation at 1000 rpm for 5 min. The supernatant was discarded and the cell pellet was

resuspended in 0.075 M KCl (hypotonic solution) and incubated for approximately 12 min at

ca. 37°C. Fixative (3 vol methanol:1 vol acetic acid) was mixed with the cells suspended in

hypotonic solution to reach a final concentration of approximately 20% v/v. Following another

centrifugation, the supernatant was discarded and the cells were treated with three changes of

fixative. After the third change of fixative, the cell pellet was collected by centrifugation and

resuspended in fixative at an appropriate density for slide preparation.

The fixed cells were dropped onto clean slides and air-dried before staining. At least two slides

were prepared from each culture. Fixed cells not used for slide preparation were discarded after

completion of the experimental phase of study.

9.7. Staining

At least two slides per culture were washed with 3 changes of purified water (in-house), at

approximately 1 minute per change. The washed slides were stained with 10% v/v Giemsa stain

for 15 minutes, washed in one change of purified water (prepared in-house) for approximately

1 minute, washed in running tap water for up to 5 minutes, air-dried then mounted with

coverslips using synthetic mountant.

9.8. Slide Examination

9.8.1. Mitotic Index (MI)

Lymphocyte toxicity is primarily indicated by a decreased MI compared to the concurrent

control group. The MI was determined by examination of at least 500 cells per culture for
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selected treatment groups, i.e., relevant dose levels not showing extreme toxic effects.

The relative mitotic index (RMI) for each treatment group was calculated as a percentage ratio

compared with the appropriate concurrent vehicle control group.

9.8.2. Selection of Slides for Detailed Examination

Justification for selection of dose levels for examination is presented in the results section of this

report. Routinely, slides for examination of aberrations are selected primarily on the basis of the

RMI results. For each treatment regime and phase, the highest level selected for examination of

aberrations is the highest dose tested in the case of test substances showing relatively low

toxicity. Otherwise the highest level examined is the lowest concentration which causes a

reduction in the RMI to below 50%.

On occasion the treatment procedure may have a clear effect on the quality of metaphases or

cause an extreme reduction in the absolute number of metaphases, in which case a different

maximal dose for examination may be justified. At least two additional lower dose levels of the

test substance are assessed. Usually these are the two adjacent lower dose levels, but this range

may be extended at the Study Directorís discretion if necessary to include at least one level

showing little or no toxicity.

In addition, one relatively non-toxic dose level of positive control agent was selected for

examination from each of the three treatment regimes/phases.

9.8.3. Detailed Examination for Chromosome Aberrations

Slides selected for examination were randomized using MicrosoftÆ Excel 2000/2003 then

encoded to minimize potential operator bias. They were examined by light microscopy, and

(where practical) a total of 200 readable metaphases per experimental point were examined for

the presence of chromosome aberrations using oil-immersion optics. Usually, only one slide was

examined per culture, the remaining slide(s) being held temporarily in reserve in case of

technical problems with the first slide, e.g., insufficient metaphases available to complete

analysis. Unexamined slides and those not needed for archiving were discarded after completion

of the experimental phase of the study.

Readable metaphases are identified by the following criteria:

! Chromosome number between 44 and 48 in a single stage of condensation

! Well-spread with minimal overlap of chromosomes and chromosome arms

! Chromatids separate with centromere intact

! Structure of chromosomes clear and well-defined
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The International System for Chromosome Aberration Nomenclature (1995) was followed to

designate the observed aberrations. Since the nature of chromosomal and chromatid gaps is

uncertain (they may or may not represent true breaks in chromatid structure), these two types of

aberration were recorded but were not included in statistical analysis of aberrations.

The incidences of numerical aberrations, such as polyploidy and endoreduplication,

were recorded. As required by OECD guideline 473, obvious treatment-related increases in the

incidence of polyploidy (if present) are described in this report, although the biological relevance

of such effects in an in vitro system is unknown.

The location (Vernier reading) of observed aberrant metaphases was recorded for potential

peer review.

10. EVALUATION AND INTERPRETATION OF RESULTS

Results from replicate cultures were combined to facilitate interpretation and maximize the

power of statistical analysis. The results obtained for each treatment group were compared with

the results obtained for the concurrent vehicle control group from the same treatment regime

using Fisherís Exact Test (SAS version 8.1).

A positive response is normally indicated by a statistically significant (dose-related, if

applicable) increase in the incidence of aberrant cells for the treatment group compared with the

concurrent control group (p ≤ 0.01); individual and/or group mean values should exceed the

laboratory historical control range (99% limit). A negative result is indicated where group mean

incidences of aberrant metaphase cells for the group treated with the test article are not

significantly greater than incidences for the concurrent control group (p > 0.01) and where these

values fall within or close to the historical control range. An equivocal response is obtained

when the results do not meet the criteria specified for a positive or negative response.

For an assay to be considered valid, the vehicle/negative control results should lie within or close

to the historical negative control range, while the positive control should produce a significant

increase in the incidence of aberrant cells compared with the concurrent control.

11. 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 4.
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12. PROTOCOL DEVIATIONS

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

appropriate, the text of this report. The incorrect CAS number was used in the protocol and

corrected in this report. No deviation had any impact on the outcome of the study or upon the

interpretation of the results.

13. 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

! Permanent specimens

! Documents generated at during this study

! The original copy of the protocol (including amendments)

! The final report

Subsequent disposition will be documented at the Testing Facility.

14. RESULTS

14.1. Dose Selection

In the absence of overt toxicity, the highest dose level of the test article selected for detailed

analysis was the highest dose level tested. In addition, the next two lower dose levels were also

subjected to examination.

14.2. Chromosome Aberrations

Cultures treated with did not show any statistically significant increases in the

proportion of aberrant metaphases at any experimental point (see Table 1 for summary and

Appendix 2 for detailed results). In addition, the proportion of aberrant metaphases for all

vehicle and test substance groups was within the laboratory negative historical control range

(see Figure 1 for negative and positive historical control results).

The gaseous and conventional positive control agents caused substantial and highly significant

increases in the proportion of aberrant metaphases in each phase of the study confirming the

sensitivity of the test system and the effectiveness of the S9 mix.
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14.3. Incidental Observations

No substantial increases in the incidence of chromatid or chromosome gaps or polyploidy were

observed at any experimental point.

15. CONCLUSION

It is concluded that did not show any evidence of genotoxicity in this in vitro test for

induction of chromosome damage when tested in accordance with regulatory guidelines.
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Table 1 Results and Statistical Analysis

Number of

aberrations

Incidental

Treatment observations Ü Conc.

(% v/v) MI RMI

(%)

Number

cells

examined

% Aberrant

b e B E other (g G P C)

4 hours treatment in the absence of S9 (0S9)

Vehicle - 9.1 100 200 3.5 4 0 4 0 0 2 0 0 0

Test substance 12.8 9.2 101 200 0.5 1 0 0 0 0 0 0 0 0

25.6 8.2 90 200 1.5 1 0 2 0 0 3 0 0 0

50.0 8.6 95 200 3.5 4 0 3 0 0 0 0 1 0

ETOX 1.0 6.7 73 200 12.0** 18 3 5 0 0 0 0 0 0

MMC 0.10 a 8.8 97 200 16.0** 23 8 7 0 0 1 0 0 0

4 hours treatment in the presence of S9 (+S9)

Vehicle - 7.2 100 200 0.0 0 0 0 0 0 4 0 1 1

Test substance 12.8 8.4 116 200 1.5 2 0 1 0 0 2 0 0 0

25.6 8.1 112 200 2.0 4 0 1 0 0 0 0 1 0

50.0 8.7 121 200 0.5 1 0 0 0 0 3 0 0 0

ETOX 0.5 6.5 91 200 8.5** 12 2 6 0 0 0 1 1 0

CP 8.0 a 4.1 57 200 27.5** 39 4 33 0 0 9 0 0 0

21 hours treatment in the absence of S9 (0S9)

Vehicle - 7.9 100 200 0.5 1 0 0 0 0 3 0 1 0

Test substance 12.8 6.7 84 200 1.5 0 0 3 0 0 1 0 0 0

25.6 5.7 73 200 3.0 2 0 5 0 0 2 0 0 1

50.0 6.7 85 200 2.0 4 0 0 0 0 3 1 0 0

ETOX 0.5 4.4 56 200 9.5** 16 2 5 0 0 2 0 0 1

MMC 0.05 a 7.5 94 200 14.5** 12 5 16 0 0 0 1 0 0

MI, RMI Mitotic Index, Relative Mitotic Index (vehicle = 100%)

a Standard positive controls dosed as solutions; in units of μg/mL

b, e, g Chromatid break, exchange, gap

B, E, G Chromosome break, exchange, gap

other Includes pulverized chromosomes and cells with > 8 aberrations

P Polyploidy and endoreduplication

C Centromeric disruption

Ü g, G, P and C are excluded from the calculation of % aberrant cells

Results of statistical analysis using one-tailed Fisherís exact test

* p ≤ 0.01 (significant)

** p ≤ 0.001 (highly significant)

Otherwise, p > 0.01 (not significant)
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Figure 1 Historical Control Values

305

132

64

26 18 8 4 1 000

0

50

100

150

200

250

300

350

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 >5

Mean % aberrant cells

Frequency

The laboratory historical mean incidence of aberrant metaphase cells for negative/vehicle control

cultures for the human lymphocyte chromosome aberration test is 0.43% (SD 0.63)

for 558 treatments. These QA audited results were collected from GLP compliant studies

performed from 05 February 2003 to 19 February 2009.

The historical positive control values (for QA-audited and GLP compliant studies) are listed

below:

Mitomycin C (4 hour 0S9): mean 10.3%, SD 5.0, 144 treatments

Mitomycin C (21 hour 0S9): mean 11.8%, SD 4.7, 149 treatments

Cyclophosphamide (4 hour +S9): mean 21.2%, SD 7.7, 148 treatments
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Appendix 1 Certificate of Analysis
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Appendix 2 Results for Individual Cultures
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Appendix 2 Results for Individual Cultures

Number of aberrations Incidental

Treatment observations Conc.

(% v/v) MI

Number

cells

examined

Number

aberrant cells b e B E other (g G P C)

4 hours treatment in the absence of S9 (0S9)

Vehicle - 7.7 100 5 3 0 3 0 0 1 0 0 0

10.5 100 2 1 0 1 0 0 1 0 0 0

Test substance 12.8 7.9 100 0 0 0 0 0 0 0 0 0 0

10.6 100 1 1 0 0 0 0 0 0 0 0

25.6 8.9 100 3 1 0 2 0 0 1 0 0 0

7.5 100 0 0 0 0 0 0 2 0 0 0

50.0 7.8 100 5 3 0 2 0 0 0 0 1 0

9.4 100 2 1 0 1 0 0 0 0 0 0

ETOX 1.0 5.4 100 13 9 3 3 0 0 0 0 0 0

8.0 100 11 9 0 2 0 0 0 0 0 0

MMC 0.10 a 10.9 100 19 12 6 2 0 0 0 0 0 0

6.7 100 13 11 2 5 0 0 1 0 0 0

4 hours treatment in the presence of S9 (+S9)

Vehicle - 6.2 100 0 0 0 0 0 0 2 0 1 0

8.2 100 0 0 0 0 0 0 2 0 0 1

Test substance 12.8 7.7 100 1 0 0 1 0 0 0 0 0 0

9.0 100 2 2 0 0 0 0 2 0 0 0

25.6 7.5 100 3 3 0 1 0 0 0 0 0 0

8.7 100 1 1 0 0 0 0 0 0 1 0

50.0 10.2 100 0 0 0 0 0 0 1 0 0 0

7.1 100 1 1 0 0 0 0 2 0 0 0

ETOX 0.5 7.3 100 9 8 2 2 0 0 0 1 0 0

5.7 100 8 4 0 4 0 0 0 0 1 0

CP 8.0a 4.2 100 26 16 3 18 0 0 2 0 0 0

4.1 100 29 23 1 15 0 0 7 0 0 0

21 hours treatment in the absence of S9 (0S9)

Vehicle - 7.4 100 1 1 0 0 0 0 2 0 0 0

8.4 100 0 0 0 0 0 0 1 0 1 0

Test substance 12.8 7.7 100 2 0 0 2 0 0 0 0 0 0

5.6 100 1 0 0 1 0 0 1 0 0 0

25.6 6.0 100 4 2 0 3 0 0 0 0 0 1

5.5 100 2 0 0 2 0 0 2 0 0 0

50.0 5.6 100 2 2 0 0 0 0 2 1 0 0

7.8 100 2 2 0 0 0 0 1 0 0 0

ETOX 0.5 3.5 100 9 8 2 3 0 0 1 0 0 0

5.3 100 10 8 0 2 0 0 1 0 0 1

MMC 0.05 a 8.0 100 16 8 3 7 0 0 0 0 0 0

6.9 100 13 4 2 9 0 0 0 1 0 0

For explanatory footnotes, please see Table 1.
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Appendix 3 Protocol and Amendments
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Page 3 

 Testing Facility Study No. 

1. STUDY SPECIFIC DETAILS 

Purpose To evaluate the genotoxicity of the test substance using an in vitro 

mammalian chromosome aberration 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 473 for the testing of 

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

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Page 4 

 Testing Facility Study No. 

OPPTS 870.5375) 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 

Action (intended use) Industrial chemical 

Lot number

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 

Substance Atmosphere 

Will not be determined in this study. 

Formulation Analysis 

for Achieved 

Concentration

Will not be determined in this study

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Page 5 

 Testing Facility Study No. 

Study Design 

Dose Culture numbers 

Number 

Material Exposure 

conc. % 

v/v

4 Hours 

(0S9) 

4 Hours 

(+S9) 

21 Hours 

(0S9) 

0 Vehicle control - a01, a02 b01, b02 c01, c02 

1 Test substance 0.1 a11, a12 b11, b12 c11, c12 

2 0.2 a21, a22 b21, b22 c21, c22 

3 0.4 a31, a32 b31, b32 c31, c32 

4 0.8 a41, a42 b41, b42 c41, c42 

5 1.6 a51, a52 b51, b52 c51, c52 

6 3.2 a61, a62 b61, b62 c61, c62 

7 6.4 a71, a72 b71, b72 c71, c72 

8 12.8 a81, a82 b81, b82 c81, c82 

9 25.6 a91, a92 b91, b92 c91, c92 

10 50.0 a101, a102 b101, b102 c101, c102 

1 ETOX 0.5 a111, a112 b111, b112 c111, c112 

2 1.0 a113, a114 b113, b114 c113, c114 

3 2.0 a115, a116 b115, b116 c115, c116 

1 Mitomycin Ca 0.05 a121, a122 - c121, c122 

2 0.10 a123, a124 - c123, c124 

3 0.20 a125, a126 - c125, c126 

1 Cyclophosphamidea 8.0 - b121, b122 - 

2 12 - b123, b124 - 

3 16 - b125, b126 - 

Where high level = maximum practical without resulting in anoxia. The dose interval may be modified slightly 

at the time of the experiment for practical reasons. The 21 hour incubation ('confirmatory phase') is required by 

regulatory authorities where the initial phase does not show any indication of genotoxicity for the test substance; 

however, it is routinely performed at the same time as the initial phase to minimize potential delays in 

performance of the study. a

 Standard positive controls dosed as solutions; in units of μg/mL 

Modifications and Notes Gas testing procedures as per Appendix 6 of Testing Facility SOP 

 

2. REASON FOR CHOICE OF TEST SYSTEM 

Primary cultures of human peripheral lymphocytes are preferred because of their stable 

chromosome number and structure (karyotype) compared with cell lines, and their low and stable 

background rate of aberrations. 

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3. REASON FOR DOSE LEVEL SELECTION 

The highest dose chosen is approximately the maximum without causing anoxia. The dose 

interval of 2 is the minimum standard recommended by the OECD guideline. 

4. PARTICIPATING PERSONNEL 

Study Director 

Participating Scientist(s)

 

5. SCHEDULE 

Start 11 March 2010 

Draft report (non-audited) 29 April 2010 

Suggested date for client comments Within 3 weeks of receipt of draft report (see below) 

Final report 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. 

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6. COMPUTER SYSTEMS 

3⁄4 Statistical analysis involves look-up tables generated using validated statistical methods in 

SAS version 8.1. 

3⁄4 Arithmetic calculations of mean values etc for presentation in reports are made using 

Microsoft Excel 2000/2003. 

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 

Any solubility or compatibility testing for vehicle selection in this test system is normally 

performed in accordance with Testing Facility SOPs but, since it is not protocol-driven, is 

necessarily non-GLP. Records (or true copies) of the solubility testing, if done, will be filed 

alongside the GLP study raw data. 

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. 

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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. Routine Positive Controls 

Mitomycin C and Cyclophosphamide will be used as positive control agents. 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. 

7.5. Gaseous Positive Controls 

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

air in situ in the culture tubes. 

Identity: Ethylene oxide (ETOX) 

Vehicle: Ambient laboratory air 

Concentration: 0.5, 1.0 and 2.0% v/v 

8. EXPERIMENTAL PROCEDURES 

8.1. Test System 

8.1.1. Blood Sampling 

A peripheral blood sample will be taken from healthy, non-smoking, male donor(s) into blood 

collecting tubes containing sodium heparin and will be held at room temperature for up to 

2 hours prior to culture initiation. 

8.1.2. Culture Medium 

Complete RPMI 1640 medium will be prepared by supplementing RPMI 1640 medium with the 

following filter-sterilized components: 10% (v/v) fetal calf serum, 50 μg gentamycin per mL and 

4 units heparin per mL. 

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8.1.3. Lymphocyte Culture 

Phytohemagglutinin will be added to whole blood mixed with medium to stimulate lymphocyte 

division. Aliquots of 5 mL cell suspension will be incubated in flat-sided culture tubes (Nunc) at 

approximately 37°C in a humidified atmosphere containing 5% CO2. 

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 

Sprague-Dawley rat liver fraction supplied by MoltoxTM) 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 kept in a refrigerator set to maintain 

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. Treatment 

Treatments will be performed approximately 48 hours after culture initiation. For the S9 

cultures, 1 mL of medium will be removed and replaced with 1 mL S9 mix. The cell 

suspensions to be treated with gaseous test substances will be transferred to gas-tight tubes 

(24 mL glass anaerobic culture tubes supplied by Bellco Glass) and each tube will be sealed with 

a gas impermeable, butyl rubber septum (injectable) stopper and an aluminum seal. Using a 

gas-tight syringe and needle, the appropriate volume of air will be removed from each treatment 

tube, then the appropriate volume of treatment gas will be added. For the cell suspensions to be 

treated with the routine positive controls, the appropriate volume of liquid test substance will be 

added to the cultures in the original 5 mL flat-sided tubes. All the cultures will be incubated at 

ca.37°C in a humidified atmosphere containing 5% v/v CO2. Several concentrations of the test 

substance will be tested using all treatment regimes (4 hour treatment in the absence and 

presence of S9 mix and a 21 hour treatment in the absence of S9). The 21 hour incubation 

(‘confirmatory phase’) is required by regulatory authorities where the initial phase does not show 

any indication of genotoxicity for the test substance; however, it is routinely performed at the 

same time as the initial phase to minimize potential delays in performance of the study. 

Duplicate cultures will be treated at each experimental point. 

8.4. Medium Change (4-hour Treatment only) 

After the 4-hour treatment, the 4-hour gas-treated cultures will be opened in a laminar flow hood 

to allow the gas to dissipate and the cells will be transferred to flat-sided culture tubes (Nunc). 

All 4-hour treatment cultures will then be centrifuged at 1000 rpm for 5 minutes. The 

supernatant will be discarded and replaced with fresh complete medium. The cultures will be 

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returned to the incubator for another 17 hours of incubation before metaphase harvesting. For 

21-hour treated cultures (i.e. cultures in the confirmatory phase), no replacement of medium will 

be performed. 

8.5. Harvesting, Metaphase Preparation and Staining 

After colcemid addition (0.1 μg/mL), the cultures will be incubated for approximately 2 hours 

prior to harvesting. After 2 h, the gas-treated cultures from the 21h0S9 regime will be opened in 

a laminar flow hood to allow the gas to dissipate and the cells will be transferred to flat-sided 

culture tubes (Nunc). Cultures will be harvested by centrifugation. The cell pellet will be 

resuspended in hypotonic solution (0.075M KCl) and incubated at ca. 37°C. Fixative will be 

mixed with the suspended cells and following another centrifugation, the cells will be treated 

with 3 changes of fixative. After the third change of fixative, the cell pellet will be resuspended 

in fixative at an appropriate density for slide preparation. 

The fixed cells will be dropped onto clean slides and air-dried before staining. At least two 

slides will be prepared from each culture. Fixed cells not used for slide preparation will be 

discarded after completion of the experimental phase of study. Slides will be washed, stained 

with Giemsa, rinsed, air-dried then mounted with coverslips. 

9. SLIDE EXAMINATION 

9.1. Mitotic Index (MI) 

Lymphocyte toxicity is normally indicated by a decreased MI compared to the concurrent control 

group. The MI will be determined by examination of at least 500 cells (if available) from 

selected treatment groups, i.e. relevant dose levels not showing extreme toxic effects. The 

relative mitotic index (RMI) for each treated group will be calculated as a percentage compared 

with the concurrent vehicle control group. 

9.2. Selection of Slides for Detailed Examination 

Justification for selection of dose levels for examination will be presented in the results section 

of the report. Routinely, slides for examination of aberrations are selected primarily on the basis 

of the RMI results. For each treatment regime and phase, the highest dose level tested is selected 

for examination of aberrations unless there is toxicity (in which case, the highest level examined 

is the lowest concentration which causes a reduction in the RMI to below 50%). 

On occasion the treatment procedure may have a clear effect on the quality of metaphases or 

cause an extreme reduction in the absolute number of metaphases, in which case a different 

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maximal dose for examination may be justified. At least two additional lower dose levels of the 

test substance will be assessed. Usually these are the two adjacent lower dose levels, but this 

range may be extended at the Study Director's discretion if necessary to include a least one level 

showing little or no toxicity. 

In addition, one relatively non-toxic dose level of positive control agent will be selected for 

examination from each of the three treatment regimes/phases. 

9.3. Detailed Examination for Chromosome Aberrations 

Slides selected for examination will be randomized using Microsoft® Excel 2000/2003 then 

encoded to minimize potential operator bias. They will be examined by light microscopy, and 

(where practical) a total of 200 readable metaphases per experimental point will be examined for 

the presence of chromosome aberrations using oil-immersion optics. Slides from cultures not 

subjected to analysis for chromosome aberrations or needed for archiving, will be discarded after 

completion of the experimental phase of study. 

Readable metaphases are identified by the following criteria: 

■ chromosome number between 44 and 48 in a single stage of condensation 

■ well-spread with minimal overlap of chromosomes and chromosome arms 

■ chromatids separate with centromere intact 

■ structure of chromosomes clear and well-defined 

The International System for Chromosome Aberration Nomenclature (1995) will be followed to 

designate the observed aberrations. Since the nature of chromosomal and chromatid gaps is 

uncertain (they may or may not represent true breaks in chromatid structure), these two types of 

aberration will be recorded but will not be included in statistical analysis of aberrations. The 

incidences of numerical types of aberration, such as polyploidy and endoreduplication, will be 

recorded. As required by OECD guideline 473, obvious treatment-related increases in the 

incidence of polyploidy (if present) will be described in the report, although the biological 

relevance of such effects in an in vitro test is unknown. 

The location (Vernier reading) of observed aberrant metaphases will be recorded for potential 

peer review. 

10. EVALUATION AND INTERPRETATION OF RESULTS 

Results from replicate cultures will be combined to facilitate interpretation and maximize the 

power of statistical analysis. The results obtained for each treatment group will be compared 

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with the results obtained for the concurrent vehicle control group from the same treatment 

regime using Fisher’s Exact Test (SAS version 8.1). 

A positive response is normally indicated by a statistically significant (dose-related, if 

applicable) increase in the incidence of aberrant cells for the treatment group compared with the 

concurrent control group (p ≤ 0.01); individual and/or group mean values should exceed the 

laboratory historical control range (99% limit). A negative result is indicated where group mean 

incidences of aberrant metaphase cells for the group treated with the test substance are not 

significantly greater than incidences for the concurrent control group (p > 0.01) and where these 

values fall within or close to the historical control range. An equivocal response is obtained 

when the results do not meet the criteria specified for a positive or negative response. Additional 

slide reading or experimental work may be required by the Study Director to clarify results. 

For an assay to be considered valid, the vehicle/negative control results should lie within or close 

to the historical control range, while the positive control should produce a significant increase in 

the incidence of aberrant cells compared with the concurrent control. 

11. 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. 

12. 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: 

All raw data

Permanent specimens 

Documents generated at during this study 

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

The final report 

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Subsequent disposition will be documented at the Testing Facility. 

13. 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 

14. 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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Protocol Amendment 1 Page 2 

 

 Testing Facility Study No. 

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

below) 

Final report June 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. 

Justification: To include the experimental completion date and specify final report issuance 

timeline as requested by the Sponsor. 

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