Phenethyl phenylacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial cell gene mutation

The mutagenicity of total particulate matter (TPM) containing the test material in cigarettes was investigated using an Ames assay protocol that conformed to OECD Guideline 471. For this purpose, prototype cigarettes containing a mixture of ingredients, reference cigarettes without these ingredients, and 2R4F cigarettes (a standard reference cigarette). 0-300 µg/ml (TPM Dose levels) of the test cigarette in which the concentration of the test chemical was around 6.5 ppm.The test chemical was tested with and without S9 metabolic activation in five strains of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537. Based on the observations made, test chemical did not induce reversion of gene mutation in the Salmonella typhimrium strains TA98, TA100, TA102, TA1535 and TA1537 with and without S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro chromosomal aberration study

Based on the available results and applying the weight of evidence approach, the test chemical failed to induce chromosomal aberrations in mammalian cells when tested in-vitro both in the presence and absence of S9 activation system. Hence the test chemical can be considered to be considered to be non mutagenic in-vitro and can be classified under the category "Not Classified" as per CLP Regulation.

In vitro mammalian cell gene mutation study

Based on the available results, the test chemical can be considered to be not mutagenic to mouse lymphoma cells both in the presence and absence of metabolic activation system. Hence, it can be classified under the category "Not Classified" as per CLP Regulation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

In vitro Ames Salmonella typhimurium assay was performed to determine the mutagenic nature of test chemical using smoke condensate.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

Type and composition of metabolic activation system: Each sample was tested with and without S9 metabolic activation in five strains of Salmonella typhimurium: TA98, TA100, TA102, TA1535, and TA1537

Test concentrations with justification for top dose:

0-300 µg/ml (TPM Dose levels) of the test cigarette in which the concentration of the test chemical was around 6.5 ppm

Vehicle / solvent:

- Vehicle(s)/solvent(s) [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Remarks:

Positive controls used but names not mentioned

Details on test system and experimental conditions:

METHOD OF APPLICATION : In agar

DURATION: No data

SELECTION AGENT (mutation assays):No data

SPINDLE INHIBITOR (cytogenetic assays):No data

STAIN (for cytogenetic assays):No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

Rationale for test conditions:

No data available

Evaluation criteria:

Evaluation of the Ames assay data was carried out in terms of the mutagenic response, taking into consideration the reproducibly dose-related increase in number of revertants, even if the increase was less than two fold.

Statistics:

No data available

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Ames test: From the comparison of the data obtained for the test and reference cigarettes, it was concluded that the addition of the test chemical did not result in a positive mutagenic response in any of the strains under the conditions already described.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical can be considered to be non mutagenic under the test conditions. Based on the observations made, test chemical did not induce reversion of gene mutation in the Salmonella typhimrium strains TA98, TA100, TA102, TA1535 and TA1537 with and without S9 metabolic activation system and hene it is not likely to classify as a gene mutant in vitro.

Executive summary:

The mutagenicity of total particulate matter (TPM) containing the test material in cigarettes was investigated using an Ames assay protocol that conformed to OECD Guideline 471. For this purpose, prototype cigarettes containing a mixture of ingredients, reference cigarettes without these ingredients, and 2R4F cigarettes (a standard reference cigarette). 0-300 µg/ml (TPM Dose levels) of the test cigarette in which the concentration of the test chemical was around 6.5 ppm.The test chemical was tested with and without S9 metabolic activation in five strains of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537. Evaluation of the Ames assay data was carried out in terms of the mutagenic response, No sporadic responses in revertants were recorded. The highest sensitivity and specificity of the mutagenic response were observed using TA98 with metabolic activation. From the comparison of the data obtained for the test and reference cigarettes, it was concluded that the addition of test chemical did not result in a positive mutagenic response in any of the strains under the conditions already described.The test chemical can be considered to be non mutagenic at a test concentration of 0.0065mg/l under the test conditions. Based on the observations made, test chemical did not induce reversion of gene mutation in the Salmonella typhimrium strains TA98, TA100, TA102, TA1535 and TA1537 with and without S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

To evaluate the mutagenic nature of the test chemical.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5a medium with 10% fetal calf serum, L-glutamine, and
antibiotics
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No Data Available

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: The S9 mix consisted of 15 microliter/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/
ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium

Test concentrations with justification for top dose:

without S9 160- 1600 µg/mL
with S9 500-5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: DMSO or Ethanol or acetone solvent was not specified.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Cyclophosphamide with S9, mitomycin C without S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments : without S9 - 3 trials and with S9 -2 trials

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: C ells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9
- Harvest time after the end of treatment (sampling/recovery times): For aberrations, standard harvest time was 14 hr

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive controls
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

Rationale for test conditions:

No data available

Evaluation criteria:

Chromosomal aberrations were noted, where cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes)

Statistics:

For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et a1 [1983, pp 714-7151. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment. Doses were based on observations of cell confluence and mitotic cell availability in the SCE test.
STUDY RESULTS: In the aberration test, slight increases were seen, but these were not statistically significant

Remarks on result:

other: No mutagenic potential of the test chemical was observed.

Trial 1 of 3, without S9

DDSE (µg/ml)	Cells	Percent cells with aberration
		Total	Simple	Complex
0	100	5	4	1
160	100	3	3	0
500	100	3	3	0
1600	100	12	12	0
Positive control - MMC
0.15	100	30	21	12

 

 

	Total
Trend statistic: negative	0.20E^+01
Trend probability: negative	0.21E^+01

 

Trial 2 of 3, without S9

DDSE (µg/ml)	Cells	Percent cells with aberration
		Total	Simple	Complex
0	100	8	6	2
800	100	12	12	1
1200	100	8	8	0
1600	100	6	6	0
Positive control - MMC
0.15	50	40	32	10

	Total
Trend statistic: negative	0.8E^+00
Trend probability: negative	0.79E^+00

 

Trial 3 of 3, without S9

 

DDSE (µg/ml)	Cells	Percent cells with aberration
		Total	Simple	Complex
0	100	3	3	1
500	100	5	4	1
1000	100	5	3	2
1500	100	9	7	2
Positive control - MMC
0.15	50	30	16	16

 

	Total
Trend statistic: negative	0.17E+01
Trend probability: negative	0.46E-01

 

Trial 1 of 2, with S9

DDSE (µg/ml)	Cells	Percent cells with aberration
		Total	Simple	Complex
0	100	7	6	1
500	100	5	4	1
1600	100	11	7	4
5000	100	7	6	1
Positive control - CP
15.00	100	20	10	10

 

	Total
Trend statistic: negative	0.51E^+00
Trend probability: negative	0.30E^+00

 

 

Trial 2 of 2, with S9

 

DDSE (µg/ml)	Cells	Percent cells with aberration
		Total	Simple	Complex
0	100	4	4	0
3000	100	9	6	3
4000	100	8	6	2
5000	100	9	5	4
Positive control - CP
15.00	100	14	8	8

 

	Total
Trend statistic: negative	0,12E^+01
Trend probability: negative	0,12E^+00

 

Conclusions:

In the aberration test, slight increases were seen, but these were not statistically significant. Hence, the test chemical can be considered to be non mutagenic when tested in-vitro in CHO cells in the presence or absence of S9 metabolic activation system.

Executive summary:

The test chemical was tested for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells.Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc- Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The S9 mix consisted of 15 microliter/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium. Doses were chosen for the aberration assay based on a preliminary test of cell survival 24 hours after treatment or on observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of aberrations. The chromosome aberration assay was performed using both short and long-term incubation periods and test chemical was tested at doses up to the cytotoxic level. In the presence of S9 mix ,cells were exposed to the test chemical at doses ranging from 500-5000 µg/mlfor 2 hours at the beginning of the test period.In tests without S9 mix,the test chemical was added at concentration range 160-1600 µg/ml and was left in culture until colcemid addition. The cells were harvested 14 hours after the beginning of treatment.Cells were collected by mitotic shake-off and slides were stained with Giemsa. Hundred cells were scored from each of the three highest dose groups and from positive (mitomycin C without S9, or cyclophosphamide with S9) and solvent controls. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaksand terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. Gaps and endore duplications were recorded but were not included in the totals. In the aberration test, slight increases were seen, but these were not statistically significant. Hence, the test chemical can be considered to be non mutagenic when tested in-vitro in CHO cells in the presence or absence of S9 metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of the given test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)

Test concentrations with justification for top dose:

0, 0.5, 1.0, 2.5 or 5.0 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle:Phenethyl phenylacetate was easily dissolved in ethanol.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: N-ethyl-N-nitrosourea (ENU)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period:One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration:3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays): Not applicable

STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.


NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.



- OTHER:

Rationale for test conditions:

No data

Evaluation criteria:

The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated

Statistics:

Mean were observed.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic effect were observed.

Effect of Phenethyl phenylacetate exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	660/4	0/4	794/4
Pos. control	2/4	631/4	15/4	644/4
0.5 mM	0/4	600/4	0/4	686/4
1.0 mM	0/4	575/4	0/4	665/4
2.5 mM	0/4	579/4	0/4	732/4
5.0 mM	0/4	578/4	0/4	725/4

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to Phenethyl phenylacetate in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	-1.37 x10^-4	2.60x10^-4
0.5 mM	N/A	N/A
1.0 mM	N/A	N/A
2.5 mM	N/A	N/A
5.0 mM	N/A	N/A

Conclusions:

No cytotoxic effects were observed when CHO cells were exposed to the test chemical for 3 hrs. Based on the results of the current study, it can be concluded that the test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and hence it does not induce cytotoxic effects at concentrations of ≤ 5.0mM.

Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. No cytotoxic effects were observed when CHO cells were exposed to the test chemical for 3 hrs. Based on the results of the current study, it can be concluded that the test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and hence it does not induce cytotoxic effects at concentrations of ≤ 5.0mM.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro bacterial cell gene mutation

Data from various experimental studies were reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

 

The mutagenicity of total particulate matter (TPM) containing the test material in cigarettes was investigated using an Ames assay protocol that conformed to OECD Guideline 471. For this purpose, prototype cigarettes containing a mixture of ingredients, reference cigarettes without these ingredients, and 2R4F cigarettes (a standard reference cigarette). 0-300 µg/ml (TPM Dose levels) of the test cigarette in which the concentration of the test chemical was around 6.5 ppm.The test chemical was tested with and without S9 metabolic activation in five strains of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537. Evaluation of the Ames assay data was carried out in terms of the mutagenic response, No sporadic responses in revertants were recorded. The highest sensitivity and specificity of the mutagenic response were observed using TA98 with metabolic activation. From the comparison of the data obtained for the test and reference cigarettes, it was concluded that the addition of test chemical did not result in a positive mutagenic response in any of the strains under the conditions already described.The test chemical can be considered to be non mutagenic at a test concentration of 0.0065mg/l under the test conditions. Based on the observations made, test chemical did not induce reversion of gene mutation in the Salmonella typhimrium strains TA98, TA100, TA102, TA1535 and TA1537 with and without S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

This result is supported by another study where the mutagenicity of the particulate phase of the resulting cigarette smoke was assayed in the Salmonella plate incorporation (Ames) assay with tester strains TA98, TA100, TA102, TA1535 and TA1537. The assay was performed as described by Maron and Ames fulfilling the criteria specified in OECD Guideline 471.For the Salmonella reverse mutation assay, the total particulate matter (TPM) was collected in a glass impaction trap, suspended in dimethyl sulfoxide (DMSO), and stored immediately after preparation at -75 °C for up to l month until use. Determinations were performed with the tester strains TA98, TAI00, TA102, TA1537 and TA1535 in the presence and in the absence of a metabolic activation system consisting of the postmitochondrial fraction of the livers from rats treated with Aroclor 1254. For each cigarette type, four MSC batches were prepared and assayed - at least five doses up to 5 mg MSC/plate . Each dose was plated in triplicate . For plating, approximately 108 bacteria suspended in 100 μl culture medium, 120 pl of the MSC dissolved in DMSO or DMSO alone, 500 μl S9 mix or 0.1 mol/l phosphate buffer, pH 7 .4, were added to 2 ml of top agar supplemented with histidine and biotin (0 .05 nmol each) . The components were mixed and spread evenly on minimal glucose agar plates . After the top agar hardened, the plates were incubated in the dark at 36°C for 44-48 h. The number of His+ revertant colonies was determined with an automatic colony counter. In all experiments several negative and positive strain-specific and S9-specific control substances were assayed concurrently .The normal mutagenic response to MSC is characterized by a linear increase in the number of revertants as the amount of MSC(main smoke condensate) was increased . This is followed by a leveling or decrease in response due to toxicity . The mutagenic response reported here was calculated as the slope (revertants/mg TPM) of the linear portion of the dose-response curve fitted with Poisson-weights to the data (Edler, 1992) . A single slope was calculated for each of the four batches. Tester strains TA102, TA1535 and TA1537 without metabolic activation showed no response, or only a marginal response to the TPM . Equally, tester strains TA102 and TA1535 with metabolic activation were also non-responsive. This has been found for all test cigarettes that have been evaluated. Therefore, it can be concluded that within the sensitivity and the specificity of these tester strains, the addition of the test chemical did not produce mutagenic response that are not normally found in TPM. Hence, it can be considered that the test chemical did not induce mutagenic response either in the presence or absence of metabolic activation system in S. typhimurium TA 1535, TA 100, TA 98 and TA 102 strains.

 

The above results are strengthened by a gene mutation toxicity study was performed to determine the mutagenic nature of test chemical using preincubation assay. The study was performed using Salmonella typhimurium strain TA97, TA98, TA1535 and TA100 with and without 10% and 30% rat liver and hamster liver S9 metabolic activation system. The study was performed at dose level of 0, 0.3, 1, 3, 10, 33, 66, 100, 333, 1000, 3333 or 10000 µg/plate for TA100 and TA98 and at a dose level of 0, 0.3, 1, 3, 10, 33, 100, 333, 1000, 3333 or 10000 µg/plate for TA1535 and TA97 with DMSO as the solvent.The positive controls used were 2-Aminoanthracene (0.5,1, 2.5, 5 microgram/plate); Sodium Azide (5 microgram/plate); 9-Aminoacridine (50 microgram/plate); 4-Nitro-O-Phenylenediamine (2.5 microgram/plate). The plates were observed for a dose dependent increase in the number of revertants/plate. Test chemical did not induce a dose dependent increase in the number of revertants per plate and hence exhibited no mutagenic activity under the given test conditions.

 

These results are also supported by a gene mutation toxicity study performed to determine the mutagenic nature of the test chemical. The 4 bacterial strains used (TA 98, TA 100, TA 1535 and TA 1537) were histidine-requiring mutants of S. typhimurium LT-2. Initially, cultures were grown in Difco nutrient broth. Since this medium was suspected to have a weak mutagenic activity, it was substituted for Oxoid nutrient broth No. 2 in later experiments. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 micromole histidine and 0.05 micromole biotin. Plates used for viable counts contained 10 micromole histidine (and 0.05 micromole biotin). The experiments were carried out essentially as described by Ames. The following controls were made for each experiment: the viable count was determined; the number of spontaneous revertants was measured; the presence of the rfa-mutation was checked by crystal violet inhibition; the presence of the plasmid pKM 101 in strains TA 98 and TA 100 was checked by resistance to ampicillin;the response to the positive controls: N-methyl-N'-nitro-N-nitrosoguanidine (not requiring metabolic activation) and 2-aminoanthracene (requiring activation) was checked. In this study, 4 different preparations of S-9 from Aroclor-induced rats, each derived from a pool of 3 animals, while the same preparation of S-9 from 3 -methylcholanthrene induced rats was used throughout the experiments. The test chemical was not mutagenic in the bacterium Salmonella typhimurium LT-2 strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.

 

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be not mutagenic to bacterial cell lines both in the presence and absence of metabolic activation system. Hence, it can be classified under the category "Not Classified" as per CLP Regulation.

 

In vitro chromosomal aberration study

 

Available data on test chemical was reviewed to determine the clastogenic nature of the test chemical. The studies are the follows.

 

Study 1: The test chemical was tested for its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells.Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc- Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The S9 mix consisted of 15 microliter/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium. Doses were chosen for the aberration assay based on a preliminary test of cell survival 24 hours after treatment or on observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of aberrations. The chromosome aberration assay was performed using both short and long-term incubation periods and test chemical was tested at doses up to the cytotoxic level. In the presence of S9 mix ,cells were exposed to the test chemical at doses ranging from 500-5000 µg/mlfor 2 hours at the beginning of the test period.In tests without S9 mix,the test chemical was added at concentration range 160-1600 µg/ml and was left in culture until colcemid addition. The cells were harvested 14 hours after the beginning of treatment.Cells were collected by mitotic shake-off and slides were stained with Giemsa. Hundred cells were scored from each of the three highest dose groups and from positive (mitomycin C without S9, or cyclophosphamide with S9) and solvent controls. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaksand terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. Gaps and endore duplications were recorded but were not included in the totals. In the aberration test, slight increases were seen, but these were not statistically significant. Hence, the test chemical can be considered to be non mutagenic when tested in-vitro in CHO cells in the presence or absence of S9 metabolic activation system.

 

Study 2: An in vitro chromosome aberration assay was conducted according to the OECD test guideline (OECD TG 473) to assess the clastogenic potential of the test chemical either with or without metabolic activation (S9 mix) in Chinese hamster lung cells. The cytotoxic nature of the test chemical was assessed in a preliminary test, and the highest test dose was selected as the one which caused 50% cytotoxicity. A cytotoxic index was produced by calculating the relative population doubling (RPD) at each dose compared with the PDs in negative control groups. The preliminary test indicated that the concentration at which approximately 50% cell growth was inhibited was 92 μg/ml for the 6-hours treatment without metabolic activation, 178 μg/ml for the metabolic activation method, and 97 μg/ml for the continuous treatment. Consequently, the main test was carried out at doses of 0–170 μg/ml. The aberration assay was performed employing either a direct (short-term, 6 hours) or a continuous (24 hours) treatment method. In the direct method, CHL cells were exposed to 40, 60, 80 or 100 µg/ml of test chemical in the absence of S9 mix or to 80, 110, 140 or 170 µg/ml in the presence of S9 mix for 6 hours, then the cells were washed and further cultured for 18 hours. In the 24-hours continuous treatment, the cells were incubated with 40, 60, 80 or 100 µg/ml test chemical for 24 hours in the absence of metabolic activation. Vehicle (DMSO) and positive control (mitomycin C without S9 and cyclophosphamide with S9) substances were also included in the assay. Chromosome specimens were prepared using the conventional method and the cells were stained with Giemsa. Three-hundred metaphases (150 metaphases/dish) were examined for structural aberrations (excluding gaps) and polyploidy. The significant differences in the frequencies of structural aberrations and polyploid cells were tested using Fisher’s exact test and the Cochran-Armitage tests. The frequencies of chromosomal aberration in the negative and positive control groups were within the 95% probability distribution of background data, consequently those in the positive control groups were significantly increased. All the treatment groups had ≤ 1.0% structural aberrations and ≤ 1.3% polyploidy, and exhibited no significant differences in the frequencies of chromosomal aberrations from the negative controls or dose-dependency. The RPDs of the 6-h and 24-h treatments were 21% and 23%, respectively. However, since no significant increases in chromosomal aberrations were seen at these doses, it was considered that the test chemical does not induce chromosomal aberrations in vitro.

 

Based on the available results and applying the weight of evidence approach, the test chemical failed to induce chromosomal aberrations in mammalian cells when tested in-vitro both in the presence and absence of S9 activation system. Hence the test chemical can be considered to be considered to be non mutagenic in-vitro and can be classified under the category "Not Classified" as per CLP Regulation.

 

In vitro mammalian cell gene mutation study

 

The non-mutagenic nature of the test chemical is being further confirmed by the results of in vitro mammalian cell gene mutation assay. Various studies have been reviewed to determine the mutagenic potential of the test chemical. The results are as follows:

 

Study 1: An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. No cytotoxic effects were observed when CHO cells were exposed to the test chemical for 3 hrs. Based on the results of the current study, it can be concluded that the test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and hence it does not induce cytotoxic effects at concentrations of ≤ 5.0mM.

 

Study 2: Mouse Lymphoma cell mutagenesis (MLY) assay was performed on TK+/- -3 .7 .2C heterozygote of the L5178Y mouse lymphoma cell line to evaluate the mutagenic potential of the test chemical. TK+/- -3 .7 .2C heterozygote of the L5178Y mouse lymphoma cell line was maintained in Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate, and Pluronic F68 with and without Aroclor 1254-induced rat liver S9 activation system at 1000, 1500 μg/ml. In a typical assay procedure, the thymidine kinase competent heterozygote was exposed to the test chemical in both the presence and absence of an induced rat liver S9 and cofactors (CORE). After a 4 hour exposure period, the cells were washed and incubated at 37° C for 48 hours to allow phenotypic expression before cloning 3x10^6 cells in Noble agar containing the selective agent trifluorothymidine or bromodeoxyuridine. Colonies were counted after 10-14 days growth using an automatic colony counter. Mutant frequency was determined by calculating the ratio of mutant to viable colonies cloned without selective medium. The test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the test chemical was not mutagenic in nature.

 

Based on the available results, the test chemical can be considered to be not mutagenic to mouse lymphoma cells both in the presence and absence of metabolic activation system. Hence, it can be classified under the category "Not Classified" as per CLP Regulation.

Justification for classification or non-classification

Based on the available results, the test chemical can be considered to be not mutagenic both in the presence and absence of metabolic activation system. Hence, it can be classified under the category "Not Classified" as per CLP Regulation.