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Genetic toxicity: in vitro

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genetic toxicity in vitro
Type of genotoxicity: other: cytotoxicity
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)

Data source

Reference Type:
Evaluation of the potential effects of ingredients added to cigarettes. Part 3: In vitro genotoxicity and cytotoxicity
E. Roemer
Bibliographic source:
Food and Chemical Toxicology; 40, (2002), 105-111

Materials and methods

Test guideline
according to guideline
Principles of method if other than guideline:
The cytotoxicity of the gas/vapor phase and the particulate phase was determined in the neutral red uptake assay with mouse embryo BALB/c 3T3 cells.
GLP compliance:
Type of assay:
other: neutral red uptake assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Benzyl propionate
EC Number:
EC Name:
Benzyl propionate
Cas Number:
Molecular formula:
benzyl propionate
Details on test material:
- Name of test material: benzyl propionate
- Molecular formula: C10H12O2
- Molecular weight: 164.20 g/mole
- Substance type: Organic
- Physical state: Liquid


Species / strain
Species / strain / cell type:
mammalian cell line, other: mouse embryo BALB/c 3T3 cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
eight concentrations up to approximately 160 µg TPM/ml medium
Vehicle / solvent:
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
Three TPM batches and three GVP batches were assayed for each cigarette type. For each batch, four replicate 96-well microtiter plates were used. Each concentration was replicated six times per microtiter plate. In each well, 10(4) cells were seeded and cultivated in culture medium containing 10% fetal bovine serum
(FBS). After 24 h the cells were exposed for 24 h to the smoke fractions dissolved in culture medium containing 4.8% FBS. At the end of the exposure period, the medium containing the smoke fractions was replaced with culture medium containing neutral red (25 mg/ml). Following a 3-h incubation period, 100 ml of an extraction solution (1% acetic acid in 50% ethanol) was added to each well and the absorbance of each well (directly correlated with the number of viable cells) was measured at 540 nm on a microtiter plate reader. To ensure the validity of the assay, TPM from the Reference Cigarette 1R4F and pure acrolein were both assayed in parallel.
Evaluation criteria:
The cytotoxic response was characterized as the EC50 value; that is, the effective concentration in mg TPM/ml culture medium that reduced the number of viable cells in the exposed culture by 50% compared to the untreated control. The measure of dosing, mg TPM/ml, refers either to the mass of the particulate phase itself or to the trapped gas/vapor phase constituents accompanying that particle mass.
Arithmetic means and measures of variance were calculated as descriptive statistics. The one-way analysis of variance was used to compare the results obtained for the control cigarette and those obtained for the test cigarettes containing the same group of ingredients. In those cases where this overall comparison showed a significant difference between the cigarettes, the Duncan test for pairwise comparison was applied. Results were considered to be statistically significant at P40.05 without adjustment for multiple testing

Results and discussion

Test results
Species / strain:
mammalian cell line, other: mouse embryo BALB/c 3T3 cells
Metabolic activation:
not specified
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
This study demonstrates that within the sensitivity and the specificity of the test systems, the addition of the commonly used ingredients added did not increase the or cytotoxic activity of the resulting mainstream smoke, even at the exaggerated levels used.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):

The test substance, benzyl propionate did not induce cytotoxicity in an neutral red uptake assay conducted on mouse embryo BALB/c 3T3 cells.
Executive summary:

The test substance, benzyl propionate did not induce cytotoxicity in an neutral red uptake assay conducted on mouse embryo BALB/c 3T3 cells.

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