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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Ozone Is Mutagenic in Salmonella
Dillon D. et al.
Bibliographic source:
Environmental and Molecular Mutagenesis 19, 331-337

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
see box " Principles of method if other than guideline"
Principles of method if other than guideline:
A standard Ames test was modified for gasvapour exposure. The petri dishes were exposed to an atmosphere containing ozone for 35 minutes.
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
Ozone was produced using an Ozone Generator, Type GLX, machine (Argentox, Hamburg, Germany) operating via an electrical discharge in dry oxygen. Different concentrations of ozone were achieved by regulating the flow rate of oxygen used as support gas and by varying the voltage. Ozone output was determined by titrating the iodine liberated from a solution of KI buffered with Na2HPO4 and KH2PO4 using standardised sodium thiosulphate.


Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 98, TA 100, TA102, TA104
Details on mammalian cell type (if applicable):
- Source of cells: Prof. Bruce N. Ames (University of California, Berkeley)

- Nutrient medium: Oxoid No.2
- Top agar used to overlay Vogel-Bonner plates
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
6 concentrations per experiment ranging between 0.019 and 9 ppm.
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
oxygen gas
True negative controls:
Positive controls:
Positive control substance:
sodium azide
mitomycin C
other: 2-Aminoanthracene (all strains, 1µg/plate, +S9) and formaldehyde (TA104, 50 µg/plate, -S9)
Details on test system and experimental conditions:
The standard plate incorporation protocol by Maron and Ames, 1983 were followed. Overnight bacterial culture (0.1 mL) and S9 mix or 0.05 M phosphate buffer, pH 7.4 (0.5 mL) were added to top agar (2.0 mL), mixed, and used to overlay Vogel-Bonner plates (3 plates per concentration). Cells were exposed on the plates stacked in glass jars of known volume with tapped, ground glass lids. Petri plate lids were slightly raised by the insertion of foil clips to facilitate circulation of the gas. All treatments comprised application of generator voltage for 5 min, after which time the jars were sealed to maintain ozone atmospheres for an additional 30 min. At the conclusion of this period, the residual ozone was purged with air. Plates were incubated at 37 °C for 2 days in the jars and then for an additional day outside the jars. Colonies were counted with a Biotran III colony counter. Qualitative indications of toxicity were obtained from the extent of background growth. Concurrent negative controls were performed by exposing cells, as for ozone, to the oxygen support gas with the generator switched off, or to air, with the gas flow set to zero. In total 6 experiments were conducted. Experiment 1-4 were conducted with an flow rate of 5 L/min and experiments 5 and 6 were conducted at flow rates of 7 L/min. Dosimetry was undertaken concurrently with each experiment.
Evaluation criteria:
Values significantly different (p< 0.01) from respective air controls (Dunnett's t-test) were regarded a positive response.
The parametric method of Dunnett (1955), involving calculation of the Student's t-statistic and a nonparametric, ranking procedure [Wahrendorf et al., 1985] were applied.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA1535, TA98 and TA100 and TA 104
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Species / strain:
S. typhimurium, other: TA102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
at the two top doses (7.04 and 3.62 ppm)
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
Ozone was consistently non-mutagenic to strains TA1535, TA98, and TA100 and induced a slight but non reproducible and generally statistically non-significant increase in revertants of TA104 at a single concentration (not the high dose) and only in one experiment (maximum fold increase of 1.2 in comparison to control). Reproducible and statistically significant increases (p< 0.01) in revertant counts with TA102 were, induced at 0.022, 0.036, and 0.59 ppm ozone (Fig 2 below). In most experiments, mutagenicity was unaffected by the presence or absence of S9. Maximum fold increases of 3.1 over air controls were obtained with 0.19 ppm ozone, and small increases in revertants were recorded in separate experiments for the mean dose of 0.022 ppm, and over the range 0.019 to 0.22 ppm ozone. With higher concentrations, however, almost linear decreases in revertants occurred, probably due to toxicity, which was manifest as a reduction in the number of his+ revertants per plate and/or as a thinning of the background lawn at the highest dose levels. However, dose-related responses were not always obtained.
Remarks on result:
other: no dose response occurred

Applicant's summary and conclusion

In conclusion, the test item is considered overall as not genotoxic in the bacterial reverse mutation assay in the presence and absence of mammalian metabolic activation.
Executive summary:

In a modified reverse gene mutation assay in bacteria (similar to OECD 471) strains of S. typhimurium (TA1535, TA104, TA102, TA100 and TA98) were exposed to Ozone at six concentrations/experiment ranging from of 0.019 to 9 ppm in the presence and absence of mammalian metabolic activation for a total exposure time of 35 minutes. Positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background, except for strain 102. This strain is known for its sensitivity against oxygen radicals. Ozone, at two to three consecutive doses, induced weak, albeit statistically significant mutagenic responses with and without S9. But, these effects were not dose related and occurred only at the lower concentrations. Higher concentrations, which were not identified as toxic, showed revertant levels similar to the concurrent control. Moreover, a certain variance occured between the different experiments. Based on the results, ozone is not genotoxic in tester strains TA98, TA100, TA104 and TA1535 with and without metabolic activation. Ambigious results were obtained with tester strain TA102.