In Vitro Micronucleus Assay for Mainstream Tobacco Smoke

Health Canada
T-503, Second Edition
November 1, 2004

Table of Contents

1. Scope Of Applications
2. Normative references
3. Definitions and Abbrevations
4. Method Summary
5. Apparatus and Equipment
6. Reagents and Supplies
7. Preparation of Glassware and Plastic Ware
8. Preparation of Solutions and Media
9. Preparation Of Cho Cell Culture Suspension
10. Collection Of Total Particulate Matter of the Smoke
11. Preparation Of Test Sample
12. The Clastogenicity/genotoxicity Test (In Vitro Micronucleus Assay)
13. Quality Control and Documentation
14. Reporting of Assay Results
15. References
16. Appendices
    

1. Scope Of Applications

1. This document sets out how to test the total particulate matter from mainstream tobacco smoke on Chinese Hamster Ovary cells using the in vitro Micronucleus Assay.

2 Normative References

1. Health Canada Official Method T-115. Determination of Tar, Water, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke. Second Edition. 2003. (Parts applicable to manufactured cigarettes only)
2. International Organization for Standardization. Standard ISO 4387: Cigarettes – Determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine. Third Edition. 2000. 

3 Definitions and Abbreviations

1. CHO cells: Chinese hamster ovary cells.
2. CMF-PBS: Phosphate buffered saline without calcium chloride and magnesium chloride.
3. CP: Cyclophosphamide.
4. DMSO: Dimethylsulphoxide.
5. Glass fibre filter disc (pad): A pad used to collect particulate matter from tobacco smoke.
6. Hemocytometer (hemacytometer): A specialized microscopic grid used to count the number of cells in a suspension.
7. Micronucleus (MN): A small extra-nuclear body formed at mitosis from acentric chromosome fragments or whole chromosomes that are not incorporated into either daughter nucleus.
8. MMC: Mitomycin C.
9. NADP: Nicotinamide adenine dinucleotide phosphate.
10. S9 rat liver fraction: The supernatant of liver homogenates prepared from male rats exposed to an enzyme-activity-inducing substance (such as Aroclor-1254, phenobarbitone or ß-naphthoflavone). The fraction permits in vitro simulations of the metabolic activation reactions that are ordinarily restricted to mammalian liver.
11. TPM: That portion of the mainstream smoke that is trapped on the glass fibre filter disc (pad).

4 Method Summary

1. Cigarettes (e.g. 20 cigarettes) are smoked under modified (intense) ISO conditions using a 20-port rotary smoking machine.
2. Mainstream smoke is passed through a 44-mm or a 92-mm glass fibre filter disc for TPM collection.
3. A solution of TPM is prepared to a concentration of approximately 10 mg of TPM/mL DMSO.
4. Chinese hamster ovary (CHO) cells are treated with different doses of the TPM solution in a tissue culture vessel (in duplicate). It is incumbent on each laboratory to determine cell cycle time and adjust treatment and recovery schedules accordingly. An example treatment schedule is (i) short-term exposure (e.g. 3 hour) without metabolic activation followed by recovery (e.g. 27 hour), (ii) short-term exposure (e.g. 3 hour) with metabolic activation followed by recovery (e.g. 27 hour), and (iii) long-term continuous exposure (e.g. 30 hour) without metabolic activation.
   
   Note 1: Negative (solvent) and positive controls are assayed concurrently.
   
   Note 2: All treatment schedules may be run concurrently, or schedules (i) and (ii) may be performed first, using schedule (iii) as a confirmatory test following negative or equivocal results in both schedules (i) and (ii).
5. The in vitro micronucleus assay is performed without cytochalasin-B.
6. After treatment, cells are harvested by trypsinization followed by fixation.
7. Slides are prepared using a cytospin centrifuge. Alternatively, the cells may be dropped, spread or smeared on clean slides.
8. Staining of cells is performed with Acridine Orange to show micronucleus (MN).
9. A minimum of 1000 randomly selected interphase cells are scored from each flask for the presence of micronuclei.

Warning: The testing and evaluation of certain products against this test method may require the use of materials and or equipment that are potentially hazardous and this document does not purport to address all the safety aspects associated with its use.  Anyone using this test method has the responsibility to consult with the appropriate authorities and to establish health and safety practices in conjunction with all existing applicable regulatory requirements prior to its use.

5 Apparatus and Equipment

1. Equipment needed for collection of TPM and sample preparation as per Appendices 1 and 2.
2. Inverted microscope (or equivalent)
3. Laminar flow biological containment cabinet – Class II, type B
4. CO₂ incubator
5. Automatic pipettes
6. Cytospin centrifuge
7. Slide dryer
8. Fluorescence microscope equipped with relevant filters for Acridine Orange stain
9. Key counter
10. Tissue culture flask
11. 10 mL sterile disposable pipettes
12. 5 mL sterile disposable pipettes
13. Hemocytometer
14. 15 mL sterile disposable tubes
15. Water bath
16. Glass slides and cover-slips
17. Balance
18. pH meter

6 Reagents and Supplies

Note: Wherever possible, reagents are identified by their Chemical Abstract Service [CAS] registry numbers in square brackets. Use analytical grade chemicals whenever possible.

1. Fetal Bovine Serum
2. CHO cells
3. Nutrient mixture (F-12 Ham with L-glutamine and NaHCO₃)
4. Calcium-magnesium free phosphate buffered saline (CMF-PBS)
5. Penicillin-streptomycin (e.g. in solution or powder form)
6. Trypan blue solution (0.4%) [72-57-1]
7. Trypsin [9002-07-7] or equivalent
8. Dimethyl sulphoxide [67-68-9]
9. Mitomycin C [50-07-7]
10. Colchicine [64-86-8]
11. Cyclophosphamide [6055-19-2]
12. S9 rat liver fraction
13. NADP [1184-16-3]
14. Isocitric acid [1637-73-6]
15. Methanol [67-56-1]
16. Glacial acetic acid [64-19-7]
17. Phosphate buffer (e. g. Sorenson)
18. Acridine Orange
19. Mounting media
20. Sterile, deionised water
21. 0.2 µm sterile filter
22. Reagents and supplies needed for collection and preparation of test samples as per Appendices 1 and 2.

7 Preparation of Glassware and Plastic Ware

1. Glassware and plastic ware should be sterile, clean and, when necessary, disposable.
2. Cleaning required for collection and preparation of test samples as per Appendices 1 and 2.

8 Preparation of Solutions and Media

Note 1: All reagent and media solutions preparation should follow standard aseptic procedures and, whenever applicable, follow the manufacturer’s instructions for the preparation of solutions.

Note 2: Potential carcinogen(s)/toxin(s) should be prepared in a laminar flow biological containment cabinet with caution appropriate for this type of hazardous material.

1. Prepare the complete growth media by mixing 90% nutrient mixture with 10% fetal bovine serum, 100 units/mL penicillin and 100 mg/mL streptomycin.
   
   Note:Growth media is also used as the diluent in this assay in absence of metabolic activation.
2. Prepare CMF-PBS following manufacturer’s instructions.
3. Prepare 0.25% (w/v) trypsin solution in CMF-PBS prior to performing the assay.
4. Prepare fixative solution by mixing glacial acetic acid with methanol (1:3 v/v).
   
   Note: Other fixative solutions may be used, as applicable.
5. Prepare sufficient quantity of S9 mixture on the day of the assay [section 12.3.2] by mixing the following:

   Ingredients	(for 0.275 mL of S9 mixture)
   DL-Isocitric acid trisodium salt solution (13.5 %, w/v, FW 258.1)	0.100 mL
   NADP solution (75 mg/mL, FW 765.4)	0.100 mL
   S9 rat liver fraction	0.075 mL

6. Preparation of Negative Control Solutions
   1. Use DMSO in growth media as the negative control. Use the same volume of DMSO as that used in the maximum dose level of test sample.
7. Preparation of Positive Control Solutions
   1. Mitomycin C (MMC) solution
      1. Prepare a stock solution of MMC (e.g. 2 mg MMC/4 mL sterile deionised water).
      2. At the start of an assay, prepare appropriate working concentrations of MMC solution (e.g. in absence of metabolic activation system: 2.0 μg/mL of growth media for short-term treatment or 0.5 μg/mL in growth media for long-term continuous treatment)
   2. Colchicine solution
      1. Prepare a stock solution of Colchicine (e.g. 10 mg Colchicine/10mL sterile deionised water).
      2. At the start of an assay, prepare appropriate working concentrations of Colchicine solution (e.g. in absence of metabolic activation system: 2.0 μg/mL of growth media for short-term treatment or 0.5 μg/mL in growth media for long-term continuous treatment)
   3. Cyclophosphamide (CP) solution
      1. Prepare a stock solution of cyclophosphamide (e.g. 75 mg CP/10 mL sterile, deionised water).
      2. At the start of an assay, prepare an appropriate working concentration of CP solution (e.g. in presence of metabolic activation system: 7.5 μg/mL in growth media for short-term treatment)
8. Preparation of staining solution
   1. Prepare a stock solution of Acridine Orange (e.g. 10 mg Acridine Orange/4 mL Phosphate buffer)
   2. Prepare an appropriate working concentration of Acridine Orange (e.g. 20 μL stock Acridine Orange solution in 50 mL phosphate buffer)

9 Preparation of CHO Cell Suspension

1. Calculate the amount of CHO cell suspension to harvest in order to conduct the assay. Five mL of a 100,000 viable cell/mL culture is required per 25 cm² (50 mL) culture flask.
   
   Note: Set up and use 2 flasks for each dose of the sample, negative (solvent) control and positive control.
2. CHO cells are grown as a monolayer in tissue culture grade flasks at 37 ± 1°C in a humidified atmosphere of 5% CO₂. When cells approach confluence they can be harvested or sub-cultured by trypsinization as follows:
   1. Remove the growth medium.
   2. Add CMF-PBS to rinse.
   3. Discard the washing solution.
   4. Add CMF-PBS for a second time to rinse.
   5. Discard the washing solution.
   6. Add 0.25% trypsin solution to the monolayer for an appropriate amount of time, and then remove solution.  The amount will vary depending on the age of the trypsin solution.
   7. Add fresh growth medium to the flask and mix well to make a single cell suspension.
3. Counting the Number of CHO Cells in Single Cell Suspension
   1. Mix 0.5 mL of cell suspension with 0.5 mL of 0.4% Trypan Blue solution.
      
      Note:  Care should be exercised when working with Trypan Blue, as it is toxic and may be carcinogenic.
   2. Allow this mixture to stand for at least 5 minutes, but no more than 15 minutes.
   3. Make sure that the hemocytometer is clean and dry prior to use. Keep the cover glass in place on the hemocytometer.
      
      Note:  Alternatively, one can use automated cell counting.
   4. Transfer the cell/trypan blue suspension to the chamber of the hemocytometer with a capillary tube or other suitable device. Do not overfill or underfill the chambers.
   5. Count the number of viable and non-viable cells from 4 corner squares and 1 central square. Non-viable cells will stain blue.
   6. Calculate average viable cell count per square.
   7. Calculate the number of viable cells per mL of cell suspension as follows:
      Viable cells/mL = (Average cell count/square) x (Dilution factor) x 10⁴ (Chamber conversion factor).
      Example: (125 cells) x (2) x 10⁴ = 2.5 x 10⁶ cells/mL.
   8. After determination of cell number, the cell suspension can be diluted with growth media and seeded into tissue culture flasks at a density of 1 x 10⁵ cells/mL. Five mL of a 100,000 viable cell/mL culture is required per 25 cm² (50 mL) culture flask.
      
      Note:  Set up and use 2 flasks for each dose of the sample, negative (solvent) control and positive control.

10 Collection of Total Particulate Matter of the Smoke

1. Refer to Appendix 1: Collection of Total Particulate Matter on Glass Fibre Filter Disc.

11 Preparation of Test Sample

1. Refer to Appendix 2: Preparation of Total Particulate Matter from Glass Fibre Filter Disc.

12 The Clastogenicity/genotoxicity Test (in Vitro Micronucleus Assay)

1. Preparation of Tissue Culture Flask
   1. Dispense 5 mL of the cell suspension into each of the 25 cm² (50 mL) tissue culture flasks.
      
      Note:  If volumes other than 5 mL are to be used, then all other volumes must be adjusted accordingly.
   2. Confirm presence or absence of CHO cells using an inverted microscope.
   3. Incubate the flasks at 37 ± 1°C in a humidified, 5% CO₂ atmosphere for 24 ± 3 hours.
2. Preparing Desired Concentrations of Sample and Controls for the Test
   1. Prepare the desired concentrations of the negative, TPM or positive control solution by mixing with the appropriate amount of growth medium.
      
      Note 1: A range finder experiment may be required to ensure that the highest concentration of the test sample produces approximately 60% toxicity as compared to solvent control culture (see 12.4.3 for toxicity determination). 
      
      Note 2: Experience with typical Canadian flue-cured cigarettes shows that concentrations of 0, 75, 100, 150, and 200 mg TPM/mL will generally give a satisfactory response with short-term treatment.
      
      Note 3:Solvent used to prepare dilutions shall correspond to solvent used in preparation of test sample (e.g. DMSO).
3. Short and Long-Term Exposure of CHO Cells
   1. Short-term Treatment in Absence of Metabolic Activation Followed by Recovery – Schedule (i)
      1. Remove the culture medium from each flask.
      2. Treat the flasks by adding 5 mL of growth medium containing either negative control/positive control/differing concentrations of TPM to each flask.
         
         Note: Suggested TPM concentration range: 0, 75, 100, 150 and 200 µg TPM/mL growth medium. (This typical range is likely to include the TPM concentration that can produce approximately 60% cytotoxicity as compared to solvent/negative control.)
      3. Incubate the flask(s) at 37 ± 1°C in a humidified, 5% CO₂ atmosphere for desired time (e.g. 3 hours)
      4. Remove the medium with solvent, positive control or different concentrations of tobacco smoke sample from the flask(s).
      5. Rinse the flask(s) with CMF-PBS.
      6. Add 5 mL of fresh growth medium only to each flask.
      7. Incubate the flask(s) at 37 ± 1°C in a humidified, 5% CO₂ atmosphere for approximately a period of 2 cell cycles after beginning of the treatment (e.g. 27 hours).
   2. Short-term Treatment in Presence of Metabolic Activation Followed by Recovery  - Schedule (ii)
      1. Remove the culture medium from each flask.
      2. Treat the flasks by adding 4.625 mL of nutrient mixture F-12 Ham without serum, plus 0.1 mL either negative control/positive control/differing concentrations of TPM and 0.275 mL of S9 mixture [as per section 8.5] to each flask.
         
         Note: Suggested TPM concentration range: 0, 75, 100, 150 and 200 µg TPM/mL growth medium. (This typical range is likely to include the TPM concentration that can produce approximately 60% cytotoxicity as compared to solvent/negative control.)
      3. Incubate the flask(s) at 37 ± 1°C in a humidified, 5% CO₂ atmosphere for desired time (e.g. 3 hours)
      4. Remove the medium with solvent, positive control or different concentrations of TPM sample from the flask(s).
      5. Rinse the flask(s) with CMF-PBS.
      6. Add 5 mL of fresh growth medium only (nutrient mixture F-12 Ham with serum) to each flask.
      7. Incubate the flask(s) at 37 ± 1°C in a humidified, 5% CO₂ atmosphere for approximately a period of 2 cell cycles after beginning of the treatment (e.g. 27 hours).
   3. Long-term Continuous Treatment in Absence of Metabolic Activation – Schedule (iii)
      1. Remove the culture medium from each flask.
      2. Treat the flasks by adding 5 mL of growth medium containing either negative control/positive control/differing concentrations of TPM to each flask.
         
         Note: Suggested TPM concentration range: 0, 75, 100, 150 and 200 µg TPM/mL growth medium. (This typical range is likely to include the TPM concentration that can produce approximately 60% cytotoxicity as compared to solvent/negative control.)
      3. Incubate the flask(s) at 37 ± 1°C in a humidified, 5% CO₂ atmosphere for approximately a period of 2 cell cycles after beginning of the treatment (e.g. 30 hours).
4. Harvesting and Counting of Cells
   1. Cells may be detached from the flask surface by trypsinization process as per Section 9.2.
   2. Count the total number of cells from both negative (solvent) control as well as treated cultures as per Section 9.3.
   3. Cell proliferation and toxicity may be determined by comparing cell counts at seeding or at start of treatment to cell counts at the time of harvest for both treated and control cultures. Refer to Sections 9.3 and 14.
5. Micronucleus Staining
   1. Fix the cells with fixative (e.g. 1:3 v/v glacial acetic acid:methanol) and prepare the slides by using a cytospin centrifuge. Alternatively, the cells may be dropped, spread, or smeared on slides.
   2. Perform staining of cells with Acridine Orange, using the following method if desired:
      1. Immerse the slide in working solution of Acridine Orange [section 8.8.2].
      2. Stain for maximum of 5 minutes.
      3. Rinse the slide immediately by immersing in sterile deionised water.
      4. Cover cells using a micro cover glass and mounting medium.
6. Scoring for the Presence of Micronuclei
   1. Scan a minimum of randomly selected 1000 cells for the presence of micronucleus from each culture flask. A total of 2000 cells per concentration (1000 cells per culture; 2 cultures per concentration) will be scored for the presence of micronuclei. Micronuclei not exceeding 1/3 of the main nucleus diameter, not overlapping with the main nucleus and with distinct borders, will be included in the scoring.

13 Quality Control and Documentation

1. Chemicals and Media
   1. Verify and record the sterility of media, reagents and solutions as per good laboratory practice for tissue culture laboratories. Verify the performance characteristics of the control solutions.
2. Cell Culture Maintenance
   1. The cell culture should be examined daily under an inverted microscope and any changes in morphology or adhesive properties noted.
   2. Cells should be checked regularly for the absence of mycoplasma contamination and only used if none is found.
   3. The background frequency of micronuclei for the cell line should be lower than 25/1000 cells.
3. Laboratory Controls
   1. To assess the overall performance of the analysis, a Kentucky Reference 2R4F control cigarette must be included in the sample. (The results of the control cigarette may be compared, using appropriate statistical techniques, to “expected values” generated by the laboratory or, if none exist, to values found in literature. This will provide information to the laboratory on test accuracy and precision.)
   2. There should be at least 90% increase in the number of cells at the time of harvesting in solvent (negative) control cultures as per 12.4.3.
   3. The average relative %MNC and %MN of the positive control must pass appropriate acceptance criteria defined by the individual laboratory.
   4. Coding of slides must be performed prior to analysis to minimize potential operator bias.
4. Toxicity
   1. The highest concentration of the test article should exhibit less than or equal to approximately 60% toxicity.

14 Reporting of Assay Results

1. Reports of results must include the following elements (see examples in Appendix 3):
   • Sample ID (for reference to cigarette brand)
   • Smoking data (smoking machine identity, smoking date, puff count, number of test samples smoked, total particulate matter)
   • Raw micronucleus assay cell counts (normal cells per 1000 observed cells, micronucleated cells per 1000 observed cells, total micronuclei per 1000 observed cells and number of viable cells per mL of cell suspension) for all positive control and TPM treatment doses.
   • Number of viable cells per mL of cell suspension at the time of seeding as well as at the time of harvesting.

15 References

1. Fenech, M. (2000) The in vitro micronucleus technique. Mutation Research 455:81-95.
2. Garriott, M.L., Barry Phelps, J., and Hoffman, W.P. (2002) A protocol for the in vitro micronucleus test I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity. Mutation Research 517:123-134.
3. Kirsch-Volders, M., Sofuni, T., Aardema, M., Albertini, S., Eastmond, D., Fenech, M., Ishidate, M. Jr., Kirchner, S., Lorge E., Morita, T., Norppa, H., Surralles, J., Vanhauwaert, A., and Wakata, A. (2003) Report from the in vitro micronucleus working group. Mutation Research 540:153-163.
4. Matshushima, T., Hayashi, M., Matsuoka, A., Ishidate, M. Jr., Miura, K.F., Shimizu, H., Suzuki, Y., Morimoto, K., Ogura, H., Mure, K., Koshi, K., and Sofuni, T. (1999) Validation study of the in vitro micronucleus test in Chinese hamster lung cell line (CHL/IU). Mutagenesis 14(6):569-580.
5. Massey, E., Aufderheide, M., Koch, W., Lodding, H., Pohlmann, G., Windt, H., Jarck, P., and Knebel, J.W. (1998) Micronucleus induction in V79 cells after direct exposure to whole cigarette smoke. Mutagenesis 13(2):145-149.

Appendices

Appendix 1
Collection of Total Particulate Matter (Tpm) on Glass Fibre Filter Disc

1 Summary

1. Cigarettes (e.g. 20 cigarettes), are smoked on a rotary smoking machine under modified (intense) ISO conditions. TPM is trapped onto either a 44-mm or 92-mm diameter glass fibre filter disc.
   
   Note:Smoke a sufficient amount of cigarettes such that breakthrough of TPM does not occur, and the limits of TPM, defined in ISO 4387, are not exceeded. The number of cigarettes samples may also need to be adjusted to provide a minimum of 180 mg TPM per 92-mm collection pad, or 100 mg TPM per 44-mm collection pad.

2 Apparatus and Equipment

1. Equipment needed to perform conditioning as specified in Health Canada Official Method T-115.
2. Equipment needed to perform marking for butt length as specified in Health Canada Official Method T-115.
3. Equipment needed to perform smoking of cigarettes as specified in Health Canada Official Method T-115.

3 Reagents and Supplies

Note:Wherever possible, reagents are identified by their Chemical Abstract Service [CAS] registry numbers in square brackets. All reagents shall be at least analytical grade.

1. Ethanol [67-17-5], 70% (v/v)
2. Reagents and supplies as specified in Health Canada Official Method T-115.

4 Preparation of Glassware

1. Clean and dry glassware in a manner to ensure that contamination from residues on glassware does not occur.
2. Sterilize all lab ware by autoclaving at 121^oC for 30 minutes at 15 pounds per square inch (psi).

5 Sampling

1. The sampling of cigarette for the purpose of testing shall be as specified in Health Canada Official Method T-115.

6 Cigarette Preparation

1. Mark cigarettes for butt length as specified in Health Canada Official Method T-115.
2. Prepare cigarettes to be smoked as specified in Health Canada Official Method T-115.
3. Condition cigarettes as specified in Health Canada Official Method T-115.

7 Smoking Machine Preparation

1. The ambient conditions for smoking shall be as specified in Health Canada Official Method T-115.
2. Use only non-UV lighting in the rooms in which the sample generation and sample analyses are conducted.
3. The machine conditions for a rotary machine shall be as specified in Health Canada Official Method T-115, noting the following:
   1. To reduce bacterial contamination, all neoprene washers and labyrinth seals are to be cleaned with 70% ethanol.
4. To reduce bacterial contamination, various smoking machine parts are cleaned with a 70% ethanol solution prior to smoking. These parts are as follows:
   1. Ash plate
   2. Ports
   3. Pad holders
   4. All work surfaces, including the exterior of extraction flasks and stoppers
   5. Any other items, such as gloves, that may come in contact with the sample or cleaned work surfaces

8 TPM Generation

1. Smoke the cigarettes and collect the TPM as specified in Health Canada Official method T-115 with the following modifications:
   • The smoking conditions are modified in the following manner:
     • puff volume is increased from 35 mL to 55 mL,
     • puff interval is decreased from 60 s to 30 s, and
     • all ventilation holes are blocked by placing over them a strip of Mylar adhesive tape, Scotch Brand product no. 600 Transparent Tape, and the tape must be cut so that it covers the circumference and is tightly secured from the end of the filter to the tipping over-wrap seam, or by another method of equivalent efficiency.  
   • After smoking the required number of test samples, perform three clearing puffs and remove the pad holder from the smoking machine.
2. Determine the TPM as specified in Health Canada Official method T-115.
3. Upon completion of TPM determination, the pad is to be removed from the pad holder, folded into quarters (TPM side in), and the pad holder wiped with the folded pad.
4. Perform sample extraction as per Appendix 2.

Appendix 2
Preparation of Total Particulate Matter (TPM) From Glass Fibre Filter Disc

1 Summary

1. TPM trapped on a glass fibre filter disc is extracted with DMSO to achieve a target concentration of 10 mg TPM/mL of DMSO.

2 Apparatus and Equipment

1. Centrifuge
2. Pipettors and sterile tips (various sizes)
3. Freezer, -20°C to -86°C
4. Fume hood, class II, type B
5. Rotary shaker at 200 rpm
6. Wrist action shaker
7. Thermometer
8. Timers
9. Incubator

3 Reagents and Supplies

Note: Wherever possible, reagents are identified by their Chemical Abstract Service [CAS] registry numbers in square brackets. All reagents shall be at least analytical grade.

1. Glass fibre filter disc and holder
2. Polymethylpentene (PMP) Erlenmeyer flask (125 mL) or equivalent
3. Dimethylsulphoxide (DMSO) [67-68-5]
4. Sterile 25 mL amber bottles
5. Sterile amber vials
6. Sterile cheesecloth
7. Sterile disposable graduated pipettes (5 mL and 10 mL)
8. Sterile disposable plastic conical tubes (50 mL capacity)
9. Sterile forceps
10. Sterile funnels
11. Sterile graduated centrifuge tubes (15 mL and 50 mL)
12. Sterile nylon mesh (e.g. Tissue specimen bags from Thermo Shandon)

4 Preparation of Glassware

1. Sterilize all lab ware to be used by autoclaving at 121°C at 15 psi until sterility is achieved.

5 Sample Preparation

Note 1: All procedures are to be performed such that background contamination is minimized by sterilizing/disinfecting equipment and work surfaces.

Note 2:  If the glass fibre filter disc prepared as in Appendix 1 is not extracted immediately, it can be stored in an airtight flask at –70°C or below.  The pads must be allowed to come to room temperature before extraction with DMSO.

1. Transfer the glass fibre filter disc prepared as in Appendix 1 to a sterile 125 mL PMP Erlenmeyer flask.
2. Pipette the appropriate amount of DMSO to the flask to achieve a target concentration of 10 mg TPM/mL DMSO. The volume of DMSO required to prepare a 10mg/mL solution of TPM is determined by the following calculation:
   Volume (mL) = Total Weight TPM/10
   
   Note:The volume of DMSO to be added is to be determined to two decimal places.
3. Shake the PMP Erlenmeyer flask for 20 minutes on a wrist action shaker.
4. Filter the DMSO extract into a 50 mL sterile centrifuge tube through sterile cheesecloth to remove the filter disc material.
   
   Note:If TPM was collected on a 44-mm glass fibre filter disc, removal of the filter disc material may also be performed as follows:
   • Shake the filter disc with the appropriate amount of DMSO in a 25-mL amber bottle on a rotary shaker.
   • Centrifuge at 1500 rpm for 5 minutes using a sterilized mesh bag placed in a conical centrifugation tube.
5. Dispense an aliquot/aliquots of DMSO extract into appropriately pre-labelled sterile amber vial(s). This extract is the TPM stock sample solution.
6. Check the sterility of the TPM stock sample solution by plating onto and incubating a nutrient agar plate at 37 ± 1°C for 48 hours.
7. Store all solutions at -70°C or below until used.

Appendix 3
Examples of Sample Reporting Formats for in Vitro Micronucleus Assay Data

1 Sample ID

The following table displays a list of sample description for ID 30001, 30002, and 30003.

2 Smoking Data

The following table displays smoking data for run 2 and 3 of set number 1, with sample ID30003 on rotary machine.

1. Samples extracted in appropriate solvent control to give a final concentration of 10.0 mg/mL

3 Positive Control Data at Cell Harvest

The following table displays positive control data at cell harvest. The positive control substance is mitomycin C, and the assays date is recorded. The treatment time is for 3 hour, and the S9 was removed as an option for metabolic activation. The concentration, normal cell counts, micronucleus (MN) cells and number of micronucleus (MN) for both slides 1 and 2 were recorded.

4 Cell Counts Prior to Cell Treatment

The following table displays the cell counts prior to treatment for assays performed on two different dates. 

5 Raw Micronucleus Data at Cell Harvest (Observations per slide)

The following table displays the raw macronucleus data at cell harvest. This includes the set number, run number, sample ID, metabolic activation, concentration, as well as treatment time for each cell count. The observations include normal cells, MN cells, Number of micronucleus (MN), Cells (x10s) for both slide 1 and 2.