Bacterial Reverse Mutation Assay for Mainstream Tobacco Smoke

Health Canada
T-501, 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. Tester Strains
10. Collection of TPM
11. Processing of TPM
12. Mutagenicity Testing
13. Quality Control and Documentation
14. Statistical Analysis and Interpretation of Results
15. Reporting of Assay Results
16. References
17. Appendices
    

1 Scope Of Applications

1. This document sets out how to test the total particulate matter (TPM) from mainstream tobacco smoke in the Bacterial Reverse Mutation Assay (Ames Assay) with the pre-incubation method.

2 Normative References

1. Organization for Economic Co-operation and Development. Guideline for testing chemicals OECD No. 471: Bacterial Reverse Mutation Assay. July 21, 1997.
2. 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)
3. Health Canada Official Method T-304. Determination of Humectants in Whole Tobacco. Second Edition. 2003.
4. International Organization for Standardization. Standard ISO 4387. Cigarettes - Determination of total and nicotine-free dry matter using a routine analytical smoking machine. Third Edition. 2000.
5. Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research, 113:173-215, 1983.
6. Mortelmans, K. and Zeiger, E. The Ames Salmonella/Microsome Mutagenicity Assay. Mutation Research, 455: 29-60, 2000.

3 Definitions and Abbrevations

1. Ames Assay: A bacterial mutagenicity assay, also known as the “Bacterial Reverse Mutation Assay”, “Salmonella Mutagenicity Assay”, “Salmonella Mammalian Microsome Mutagenicity Assay” or “Salmonella typhimurium Reverse Mutation Assay”. The Salmonella typhimurium histidine (his) reversion system is a microbial assay that detects mutation in a gene of a histidine auxotroph to produce a wild type via base-pair and/or reading-frame changes in the DNA sequence of the Salmonella typhimurium bacterium.
2. Aseptic technique: Procedures used to keep cultures, instruments, media and samples free of microbial contamination.
3. Base-pair substitution: A substitution of one pair of nitrogenous bases for another in a DNA sequence.
4. Cofactor: A non-protein organic substance that binds to a protein in order to form an active enzyme.
5. DMSO: Dimethylsulphoxide
6. Glass fibre filter disc (pad): A pad used to collect particulate matter from tobacco smoke.
7. Histidine (his): An essential amino acid required, in the case of this bioassay, for the growth of Salmonella bacteria.
8. Mutagen: A substance that has been shown to cause permanent changes in the coding sequence of DNA and/or permanent changes in the chromosomal structure.
9. Mutant: An organism differing from the parental strain as a result of mutation.
10. Mutation: The process producing a gene or a chromosome differing from the wild type.
11. Pre-incubation assay: This is the modified Ames assay procedure, in which the mixture of bacterial tester strains, the test compound and the S9 metabolic activation mix or phosphate buffer is incubated at 37°C for 20 minutes before being added to the top agar and over-layered on the minimal glucose agar plates.
12. R-factor: A plasmid containing the damage-inducible genes MucA/B, as well as a gene for antibiotic resistance. The MucA/B genes increase the susceptibility of Salmonella to DNA – damaging agents.
13. Replicate:  A TPM preparation generated by an independent smoking of cigarettes taken from the same cigarette sample.
14. Revertant: A wild type bacterium that was formerly an auxotroph mutant.
15. rfa mutation: A mutation that results in increased permeability of the bacterial cell wall to large molecules.
16. Salmonella typhimurium tester strain: The genetically altered Salmonella typhimurium bacteria used in this assay.
17. 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.
18. S9 mix: The combined mixture of S9 rat liver fraction and other co-factors, which provides a metabolic activation system in this assay.
19. Spontaneous mutation: A mutation not caused by addition of the test or control substance.
20. Total particulate matter:  That portion of the mainstream smoke which is trapped on the glass fibre filter disc (pad).
21. uvrB mutation: A mutation resulting in bacteria that are abnormally sensitive to a variety of physical and chemical DNA-damaging agents, including ultraviolet light.

4 Method Summary

1. Mainstream smoke of cigarettes (e.g. 20 cigarettes), smoked on a rotary smoking machine under modified (intense) ISO conditions, is trapped onto either a 44 mm or 92 mm diameter glass fibre filter disc (pad), as per Appendix 1. (See note in Appendix regarding the size of the filter pad.) The number of replicates is as per the applicable regulations.
2. The total particulate matter trapped on a pad is extracted with dimethylsulphoxide (DMSO) to achieve a target concentration of 10 mg TPM/mL of DMSO, as per Appendix 2.
3. Samples of TPM are further characterized (water, nicotine, tar, glycerol, menthol and propylene glycol, where applicable), as per Appendix 3.
4. In the pre-incubation method, developed by Yahagi et al (1975), a histidine-dependent (his-) mutant strain of Salmonella typhimurium is mixed with a testing chemical (which in this case is the TPM mixture) in the presence or absence of rat liver enzymes (S-9 mix). Molten soft agar (top agar) is then added and the mixture is over-layered on top of minimal glucose agar. After the agar solidifies, the plate is incubated at 37 ± 1°C for 48 - 72 hours.
   
   Note: The pre-incubation modification of Ames Assay shows increased sensitivity for some chemicals over the plate incorporation method.
5. The colonies, which are histidine independent (his⁺) wild type revertants, are counted.
6. The number of replicates is as per the applicable regulations.

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 as per Appendix 1.
2. Equipment needed for preparation of TPM as per Appendix 2.
3. Equipment needed for further characterization of TPM as per Appendix 3.
4. All appropriate apparatus and equipment needed to perform the Ames Assay. Refer to Maron and Ames (1983) and Mortelmans and Zeiger (2000) for details.

6 Reagents and Supplies

Note: All reagents shall be free of mutagens or, at least, should not increase the rate of spontaneous mutation. Use analytical grade chemicals whenever possible.

1. For the requirement of chemicals, refer to Maron and Ames (1983) and Mortelmans and Zeiger (2000).
2. Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535, TA1537
3. Reagents and Supplies needed for collection of TPM as per Appendix 1
4. Reagents and Supplies needed for preparation of TPM as per Appendix 2.
5. Reagents and Supplies needed for further characterization of TPM as per Appendix 3.

7 Preparation of Glassware and Plastic Ware

1. Glassware and plastic ware should be cleaned and sterilized.
2. Cleaning required for collection of TPM as per Appendix 1
3. Cleaning required for preparation of TPM as per Appendix 2.
4. Cleaning required for further characterization of TPM as per Appendix 3.

8 Preparation of Solutions and Media

1. Potential carcinogens should be prepared in a fume hood with caution appropriate for this type of hazardous material.
2. Refer to Maron and Ames (1983) for the preparation of solutions and media required for the assay.
3. Prepare positive control solutions required for the analysis. For example, refer to Mortelmans and Zeiger (2000).
4. For solutions needed for the further characterization of TPM, refer to Appendix 3.

9 Tester Strains

1. Bacterial Tester Strain Cultures
   1. Obtain Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535, TA1537.
2. Preparation of Tester Strain Cultures
   1. Refer to Mortelmans and Zeiger (2000) for culture preparation, storage, testing, purification and quality control procedures for tester strains.
   2. Refer to Maron and Ames (1983) for the methodology to confirm the genotypes of the tester strains with respect to the following:
      • Histidine Requirement
      • rfa Mutation
      • uvrB Mutation
      • Presence of R-factor (Ampicillin and Ampicillin-Tetracycline Resistance)

10 Collection of TPM

1. Refer to Appendix 1: Collection of TPM on glass fibre filter disc.

11 Processing of TPM

1. Refer to Appendix 2: Preparation of TPM from glass fibre filter disc.
2. Samples of TPM are further characterized as per Appendix 3.

12 Mutagenicity Testing

1. Follow standard aseptic procedures for microbiological investigations.
2. Preparation of Fresh Overnight Bacterial Culture for Mutagenicity Assay
   1. Prepare tester strain culture to a density of 1-2 x 109 bacteria per mL as described by Maron and Ames (1983).
   2. The bacterial density of the culture used should be estimated by measuring the optical density at 650 nm and by bacterial viability count.
   3. Confirm the tester strain culture density by bacterial viability count as follows:
      • Perform serial dilutions by mixing appropriate quantity of bacterial culture (e.g. 100 µL of bacterial culture) and nutrient broth (e.g. 900 µL of nutrient broth) and plate the appropriate dilution on nutrient agar plates.
      • Incubate the plates at 37 ± 1°C and, after 24 hours, count the number of colonies either manually or using an appropriate automated counting device.
   4. Maintain the culture(s) at room temperature (i.e. 22 ± 2°C) during the assay.
3. Assay (Pre-incubation Method):
   1. To prepare for the assay, perform the following on the day of the assay:
      • Prepare 5% S9 mixture as follows, adding the ingredients in the order indicated:
        

        Ingredients	(per 50 mL)
        Sterile deionized water	19.25 mL
        0.2M Phosphate Buffer, pH 7.4	25.00 mL
        0.1M Nicotine Adenine Dinucleotide Phosphate (NADP) solution	2.00 mL
        1M Glucose-6-Phosphate solution	0.25 mL
        0.4M Magnesium Chloride (MgCl2)+1.65M Potassium Chloride (KCl) solution	1.00 mL
        S9 rat liver fraction	2.50 mL

      • Prepare Top Agar as per Maron and Ames (1983).
      • Remove samples from the cryofreezer (-70°C or below) and thaw at room temperature.
      • Remove required controls from storage (freezer/refrigerator) and equilibrate to room temperature.
      • Label all tubes and plates needed for samples and controls for the assay.
   2. Prepare reaction mixture in a sterile pre-labelled reaction tube by adding appropriate amount of phosphate buffer or S9 Mix, appropriate amount of either solvent or TPM sample or positive control solution and fresh overnight bacterial culture. Refer to OECD No. 471 (1997) for details.
      
      Note: The dosing protocol must include at least one concentration which is in the toxicity range. At least seven (7) non-zero concentrations are to be employed. The actual concentrations cannot be specified in advance, as they are dependent on the nature of the sample under investigation.
      
      Note: It has been observed that for TPM mixture prepared from “typical” commercially available Canadian flue-cured tobacco cigarettes, concentrations of 0, 25, 50, 75, 100, 125, 250, 500 mg per plate will generally give a satisfactory response. For atypical cigarettes, a range finding experiment will be necessary.
   3. Incubate the tubes in the shaker incubator (100-120 rpm) for 20 ± 2 min. at 37± 1°C.
   4. Record the presence of precipitates in the reaction mixture, if any.
   5. After incubation, add 2 mL of top agar to each reaction mixture tube and vortex to mix.
   6. Overlay the mixture of top agar and reaction mixture on top of minimal glucose agar.
      
      Note: Complete the above two steps within 20 seconds.
   7. Prepare three plates for each concentration.
   8. After the overlay has solidified, invert the plates and incubate at 37 ± 1°C for 48 – 72 hrs.
4. Procedure for Counting Revertant Colonies:
   
   Note: Normal practice is to consider the colony counts obtained from three plates at each concentration when samples are examined for evidence of mutagenicity.
   1. Examine the plate after incubation for background lawn and count the number of revertant colonies.
   2. Record the number of revertant colonies for individual plates.
   3. Record any evidence of thinning or scarcity of the bacterial lawn.
   4. Record the presence/absence of any precipitation found on assay plate.

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 microbiology laboratories. Verify the performance characteristics of the control solutions.
2. Genotypes of the Tester Strains
   1. All the genotypic characteristics of a strain as indicated in this Official Method must be present. Please refer to 9.2.2.
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. Each analysis should include positive and solvent controls in triplicate for each tester strain used.
   3. Triplicate plates treated with solvent control solution, with and/or without metabolic activation, are used to define background counts (i.e. negative controls) as per OECD No. 471 (1997).
      
      Note: For example, where DMSO is used the concentration of DMSO should not exceed 4% v/v.
   4. When the S9 mix is used, a control plate without the S9 mix must also be included.
4. Evaluation of Negative controls
   1. The tester strain is mixed with the top agar and seeded on a minimal glucose agar in triplicate at the same time that the assay is performed. The number of spontaneous revertant colonies is recorded. This number should be checked frequently.
      
      Note: The average colony count for the triplicate plates must fall within a pre-defined range determined in an appropriate manner (see table 1 below for an example).
      
      Note: It is incumbent upon each laboratory to determine and to communicate acceptability ranges for each bacterial culture using appropriate statistical methodologies. One possibility would be to determine the 5th and 95th percentiles from control data accumulated over several months of testing. These would then become the lower (5th percentile) and the upper (95th percentile) bounds for acceptability.
      
      The following table describes typical ranges for the background colony counts, for 5 different strains of salmonella typhimurium. The typical colony count range for negative control was 20-50 for TA98, 70-200 for TA100 strain, 150-400 for TA102 strain, 5-20 for TA1535 strain, and 5-20 for TA1537 strain.
      
      

      Table 1: Typical Ranges for Background Colony Counts

      Salmonella typhimurium Strain	Typical Colony Count Range for Negative (solvent) Control
      TA98	20 - 50
      TA100	70 - 200
      TA102	150 - 400
      TA1535	5 - 20
      TA1537	5 - 20

5. Evaluation of Positive Controls
   1. Triplicate culture plates are also to be prepared and treated with a specific concentration of a chemical known to give a demonstrable response as per OECD No. 471 (1997) when evaluated at the same time as test samples (see Table 2 below). The average colony count for positive control replicate plates must exhibit at least a three- fold increase over that of the respective average negative control for each bacterial strain. Table 2 also lists some ranges for typical revertant counts per plate that would be seen under normal practice of the method using the selected strain and chemical control combinations.
      
      The following table describes the typical response ranges for positive chemical controls. These include the S9 activation, positive control substance used, the concentration of the bacteria and the typical range for the following bacterial strains: TA98, TA100, TA1535, TA 1537, TA102, TA98, TA100, TA1535, TA1537 and TA102.
      
      

      Table 2: Typical Response Ranges for Positive Chemical Controls

      Bacteria Strain	S9 Activation	Positive Control Substance	Concentration (µg/plate)	Typical Range (revertants/plate )
      TA98	+S9	2-aminoanthracene	2	1250 - 2100
      TA100	+S9	2-aminoanthracene	2	1250 - 2100
      TA1535	+S9	2-aminoanthracene	4	300 - 600
      TA1537	+S9	2-aminoanthracene	4	300 - 600
      TA102	+S9	2-aminoanthracene	7.5	1450 – 2100
      TA98	-S9	2-nitrofluorene	4	500 - 1750
      TA100	-S9	sodium azide	1	400 - 600
      TA1535	-S9	sodium azide	1	400 - 600
      TA1537	-S9	9-aminoacridine	100	400 - 750
      TA102	-S9	Mitomycin C	0.5	1150 - 1600

      
      
6. Bacterial Growth
   1. The density of the bacterial culture used for the assay should be a minimum of 1x109 viable bacteria per mL of nutrient broth.
   2. The bacterial background lawn should be routinely examined using a microscope under ‘low power’ as an aid to the identification of toxicity. Scarcity of the bacterial lawn usually indicates that the testing concentration is beyond an acceptable range.

14 Statistical Analysis and Interpretation of Results

1. Evaluate Plate-to-Plate Variation within an Assay
   1. The criteria used for this test is based on the properties of the chi-square distribution:
      (χ²), where (n - 1) [s²/σ²] ~ χ²_n-1
   2. In the above equation, n = 3 represents the triplicate plate results at each concentration, s2 is the observed variance of the triplicate plates at each concentration and σ2 is the expected variance at each concentration.
   3. Table 3 lists some typical expected variance statistic (σ2) estimates for some typical TPM concentration/bacterial strain/S9 activation combinations based on normal practice of the method.
      
      The following table displays the typical variances estimates for triplicate plate revertant counts. These include the variance taken for various strains of bacteria at different concentration (ranging from 0 to 0.50 mg/plate). The bacterial strains include TA 98, TA100, TA 1535, TA 1537, and TA102. Each strain was tested with positive and negative S9 activation.
      
      Table 3: Typical Variance Estimates for Triplicate Plate Revertant Counts
      

      Strain and S9 Activation	Estimates of σ2 for triplicate plates at TPM concentrations
      	0	0.025	0.050	0.075	0.100	0.125	0.250	0.500
      TA98 (+S9)	17	71	103	152	220	296	529	619
      TA98 (-S9)	5	6	10	7	10	10	10	12
      TA100 (+S9)	36	34	43	48	64	74	108	212
      TA100 (-S9)	57	55	50	58	51	71	125	100
      TA1535 (+S9)	2	2	3	3	3	5	4	7
      TA1535 (-S9)	2	4	4	5	4	4	5	8
      TA1537 (+S9)	4	7	10	11	16	18	22	32
      TA1537 (-S9)	4	5	6	6	6	6	8	3
      TA102 (+S9)	143	108	144	129	154	210	240	236
      TA102 (-S9)	115	124	120	151	162	95	143	160

15 Reporting of Assay Results

1. Reports of mutagenicity data must include the following elements, as per Appendix 4:
   • Sample ID (for reference to cigarette brand)
   • Smoking data (smoking machine identity, smoking date, puff count, number of test samples smoked, amount of total particulate matter)
   • Control data
   • Chemical data (including water, nicotine, propylene glycol and glycerol contents, where applicable)
   • Assay observations: sample ID, strain, concentration, with/without S9, count (for each replicate), mean, standard deviation.

16 References

1. Ames, B.N., McCann, J., Yamasaki, E., Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-microsome Mutagenicity Test. Mutation Research 31: 347-364, 1975.
2. Bernstein, L., Kaldor, J., McCann, J., Pike, M.C., An Empirical Approach to the Statistical Analysis of Mutagenesis Data from the Salmonella Test. Mutation Research, 97: 267-281, 1982.
3. Edler, L., Statistical Methods for Short-term Tests in Genetic Toxicology: The First Fifteen Years. Mutation Research, 277: 11-33, 1992.
4. Sokal, R.R., and Rohlf, F.J., Biometry. 3rd edition. W.H. Freeman and Co, San Francisco. pp. 493-521, 1995.
5. Yahagi, T., Degawa, M., Seino Y., Mat Sishima, T., Nagoa, M., Sugimura, T. and Hashimoto, Y., Mutagenicity of Carcinogenic Azo Dyes and Their Derivatives. Cancer Letter 1, 91-96, 1975.

Appendices

Appendix 1
Collection of total particulate matter (tpm) on glass fibre filter disc

1 Summary

1. Mainstream smoke of cigarettes (e.g. 20 cigarettes), smoked on a rotary smoking machine under modified (intense) ISO conditions, 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 test samples may also need to be adjusted to provide a minimum of 180 mg of TPM per 92-mm collection pad, or 100 mg of 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 cigarette 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 reagent 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 121oC for 30 minutes at 15 pounds per square inch (psi).

5 Sampling

1. The sampling of tobacco products 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 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:
   • 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:
   • Ash plate
   • Ports
   • Pad holders
   • All work surfaces, including the exterior of extraction flasks and stoppers
   • 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 cigarettes, perform three clearing puffs and remove the pad holder from the smoking machine.
2. The number of replicates to be generated is as per applicable regulations. All replicates must be analyzed on the same day.
3. Determine the TPM as specified in Health Canada Official Method T-115.
4. 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.
5. Perform sample extraction as per Appendix 2.

Appendix 2
Preparation Of Total Particulate Matter (Tpm) From Glass Fibre Filter Disc

1 Summary

1. Total particulate matter smoke 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 reagent 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:   All procedures are to be performed such that background contamination is minimized by sterilizing/disinfecting equipment and work surfaces.

Note: 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 44mm 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°C for 48 hours.
7. Store all solutions at -70°C or below until used.

Appendix 3
Determination Of Glycerol, Menthol, Nicotine, Propylene Glycol, Water And Tar In Conjunction With The Analysis Of Total Particulate Matter (TPM)

1 Method Summary

1. Total particulate matter (TPM) from either parallel smoking runs (alternating nicotine/water smoking with Ames smoking), or a sub-sample of the TPM used for the Ames assay, is extracted following Health Canada Official Method T-115.
   
   Note: When using parallel runs, the same number of cigarettes will be smoked as used for the generation of TPM of the Ames assay.
2. An aliquot of the extract is analyzed for nicotine and water as per Health Canada Official Method T-115.
3. A second aliquot is used to determine glycerol, menthol and propylene glycol as per Health Canada Official Method T-304. Nicotine data is also available from this analysis.
4. Two blank pads will be analyzed for water with each sample for chemical analysis in order to perform the proper sample blank determination and correction for water.
5. Quantification is achieved using an internal standard calibration by comparing the FID or TCD response of the analytes in the samples against a multi-point calibration of the corresponding standards.

2 Apparatus and Equipment

1. Equipment as described in Health Canada Official Method T-115.
2. Equipment as described in Health Canada Official Method T-304.

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 reagent grade.

1. Reagents as described in Health Canada Official Method T-115.
2. Reagents as described in Health Canada Official Method T-304.
3. Menthol [89-78-1] Distilled-in-Glass

4 Preparation of Solutions

1. Prepare solutions as described in Health Canada Official Method T-115.

5 Preparation of Standards

1. Prepare standards as described in Health Canada Official Methods T-115 and T-304 using trans-anethole as the internal standard for T-304.
2. Preparation of the Menthol Standard
   1. Menthol Primary Stock – Dissolve 1 g of menthol in a 100 mL volumetric flask with extraction solution.
   2. Secondary Stock - Dilute to 100 mL in a volumetric flask with extraction solution 5 mL of menthol primary stock along with 5 mL each of glycerol, propylene glycol and nicotine primary stock solutions.
   3. Low Standard - Dilute 1 mL of secondary stock solution to 50 mL with extraction solution (menthol 0.01 mg/mL).
   4. Medium Standard - Dilute 10 mL of secondary stock solution to 50 mL with extraction solution (menthol 0.1 mg/mL).
   5. High Standard - Dilute 25 mL of secondary stock solution to 50 mL with extraction solution (menthol 0.5 mg/mL).

6 Preparation of Glassware

1. Clean and dry glassware in a manner to ensure that contamination from residues on glassware does not occur.

7 Sample Preparation

1. Collect TPM as per Appendix 1.
   
   Note: Two separate collections of TPM may be required; one used for the Ames assay, the second to be analyzed for chemical compounds.  When using parallel runs, the same number of test samples will be smoked as used for the generation of TPM of the Ames assay.
2. Preparation of the TPM for chemistry analysis is as described in Health Canada Official Method T-115 with the following modifications:
   1. Cut the pad into quarters instead of folding.
   2. Use 125 mL Erlenmeyer flasks.
   3. Prepare two laboratory reagent blanks with each smoked sample.
   4. Add 80 mL of extraction solution to the flasks.
   5. Wrap the flasks in foil after the addition of extraction solution.
   6. Shake the sample and blanks for 30 minutes using a wrist-action shaker.

8 Sample Analysis

1. Determine the tar, water content and nicotine as per Health Canada Official Method T-115.
2. Determination of Glycerol, Propylene Glycol and Menthol
   1. Samples are analyzed for glycerol, nicotine, propylene glycol, and menthol as per Health Canada Official Method T-304 with the following modifications:
      • Extraction solution used is as per Health Canada Official Method T-115
      • The internal standard is trans-anethole as per Health Canada Official Method T- 115
      • Two blanks are analyzed per set of samples
      • Prepare the menthol standard as per 5.2
      • Quantification of menthol and nicotine is achieved in the same manner as the humectants described in T-304 with trans-anethole as the internal standard.

9 Calculations

1. Calculations are done as described in Health Canada Official Methods T-115 and T-304 (using the appropriate internal standard).
2. Combine the calculated contents from the independent runs.

10 Quality Control

1. For quality control measures, refer to Health Canada Official Methods T-115 and T-304.

Appendix 4
Sample Reporting Formats For Ames Reverse Mutation Assay Data

1 Sample ID

The following table contains the tobacco cigarette sample description for various laboratory sample identification.

2 Smoking Data

The following table includes the smoking data for various runs performed on set 1. Four samples (030001, 030002, 030003, and 030004) were tested with 3 replicates each. The table specifies smoking date, number of cigarettes smoked, and rotary smoking machine use. Between 6.9 to 8.7 puffs were used per cigarette. The weight of the mainstream total particulate matter was recorded in mg. 

3 Control Data

The table below displays the control data for various bacterial strains including a negative control and three positive controls. For each sample, the assay date, concentration of positive control used as well as colony counts have been recorded.

4 Chemical Data

The following table displays the chemical data for runs performed through set 1-5. These were all performed as 5 replicates of sample 030001. The puff count ranges between 0.1-7.3 per cigarette, the mainstream total particulate matter ranged between 0.3- 13.2 mg/ cigarette, the nicotine concentration ranged from 0.38 mg to 0.912 mg/cigarette, the tar concentration is between 0.2 to 10.2 mg/ cigarette, and the glycerol concentration ranged from 0.03 to 1.14 mg/ cigarette.  The propylene glycol concentration among all samples, ranges between not quantified to <0.0008 mg/cigarette.

5 Mean Response

The following table displayed the mean response for sample 030001, including three replicates. Each replicate was performed with different doses of testing chemical in ug/plate ranges. The mean and standard deviation of 5 strains (TA 98, TA 100, TA1535, TA 537, and TA102) with S9 activation was recorded.

6 Observations (Example With S9)

The following table displays the observations obtained for sample ID 030001, with three replicates. Each replicate was performed with different doses of ug/plate. The counts in triplicates of 5 strains (TA 98, TA 100, TA1535, TA 537, and TA102) with S9 activation were recorded.