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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Started: october 11, 2010. Ended: october 13, 2010
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
The test was performed using only one strain (strain TA 98) because it is reported to be the most sensitive to hydrocarbon mutagens. Deviations to the standard Salmonella assay allow a more reliable evaluation of the mutagenicity of a complex petroleum hydrocarbon mixture: therefore, this aspect was not considered to affect the study reliability.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
The test was performed according to ASTM E 1687 Guideline
Principles of method if other than guideline:
The method for mutagenicity testing of mineral oils provides two fundamental modifications to the Ames procedure:
- a preliminary solubilization of the oil in cyclohexane to facilitate extraction of highly viscous or solid samples, followed by DMSO extraction;
- substitution of rat liver S-9 with hamster liver S-9.
GLP compliance:
as reported in the section "test procedure" of the test report released by the Laboratory
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Lubricating oils, used, vacuum distd.
EC Number:
EC Name:
Lubricating oils, used, vacuum distd.
Cas Number:
Lubricating oils, used, vacuum distd.
Details on test material:
The sample used was identified as FLL-TDA.


Target gene:
TA 98 strain is a frameshift tester strain and it is ampicillin resistant. This strain carries, along with the defect in the histidine gene (His-) a deep rough (rfa) character and an uvrB deletion (uvrB-).
Species / strain
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
Metabolic activation system:
hamster liver S-9
Test concentrations with justification for top dose:
Three dosing solutions were prepared for the test material and reference oil by diluting the extract with DMSO to the appropriate concentrations in 60 mcl dosing aliquots. The test material extract was tested neat and dosed at 0, 12, 24, 36, 48 and 60 µl per plate in a 60 µl dosing aliquot. The reference oil was diluted 1:4 with DMSO before preparing the dosing solutions. The doses tested were 0, 3, 6, 9, 12, 15 mcl per plate in a 60 µl dosing aliquot.
Vehicle / solvent:
Dimethylsulphoxide (DMSO)
Three extracts of the test material were prepared according to the following procedure: one volume of sample was added to 5 volumes of DMSO in a glass tube and the resulting mixture vortexed continuously for 30 minutes. The phases were separated by centrifugation and the extract (lower phase) removed with a pipet and stored in an amber vial until tested. The test material extracts were tested in triplicate. A reference oil used as a positive control was extracted and tested only once.
Untreated negative controls:
Negative solvent / vehicle controls:
Dimethylsulphoxide (DMSO)
True negative controls:
Positive controls:
Positive control substance:
other: Reference oil
Evaluation criteria:
The test material is considered positive:
- if the increase in the number of reverted colonies is statistically significant in comparison with the number of control reversions (Student's "t" test);
- if a dose-response can be verified, that is, a positive correlation between the number of reversions and the dose over a range of at least 3 doses (linear regression test);
- even if the statistically significant increase is recorded at one dose only, when confirmed in independent assays.
- if the mutagenicity index (MI) is grater than 1.0.
The mean and standard deviation were calculated for reversions read in each dosage group.
The data for test material were analyzed using a suitable graphics-statistics program, and determining by linear regression analysis the slope of the dose-response curve. This value is the Mutagenicity Index (MI) of the test material.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 98
Metabolic activation:
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
not valid
Positive controls validity:
Additional information on results:
An appreciable increase in the number of reversions in comparison with the vehicle control was evident. The MI was calculated 1.7.
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 extracts of the substance tested with metabolic activation induced a significant increase in the number of reversions in TA 98 Salmonella typhimurium strain.
According to Directive 67/548/EEC and to Regulation (EC) n. 1272/2008, study results indicate that the substance should be classified for genetic toxicity as following:
- category 3 mutagen, R68;
- category 2 mutagen, H341.