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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
09/1976 to 03/1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report similar or equivalent to OECD TG 476: pre-GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1977

Materials and methods

Principles of method if other than guideline:
Method: other: API procedure (see Reference). Similar to OECD test guideline 476.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: low viscosity liquid hydrocarbon

Method

Target gene:
Thymidine kinase
Species / strain
Species / strain / cell type:
other: Forward mutation assay using cell line L5178Y
Metabolic activation:
with and without
Metabolic activation system:
S-9 metabolic fraction from Aroclor 1254-induced Sprague-Dawley rats
Test concentrations with justification for top dose:
0.065 to 1.004 microliters/ml
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
yes
Remarks:
Liver homogenate only
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Remarks:
Without activation: ethylmethanesulfonate (EMS). With activation: dimethylnitrosamine (DMN).
Details on test system and experimental conditions:
DURATION
- Exposure duration: Five hours
- Expression time (cells in growth medium): Three days
- Selection medium: Selection medium was made from Fischer's medium supplemented with 20% horse serum, sodium pyruvate, 0.37% agar, and 5% (w/v) of bromodeoxyuridine (BUdR).
- Selection time (if incubation with a selection agent): Ten days


DETERMINATION OF CYTOTOXICITY
- Toxicity was measured as loss in growth potential of the cells induced by a five-hour exposure to the chemical followed by a 24-hour expression period in growth medium.

Evaluation criteria:
A compound is considered mutagenic in the mouse lymphoma assay if:
a. A dose response relationship is observed over three of the four dose levels employed.
b. The minimum increase at the high level of the dose response curve is at least 2.5 times greater than the solvent control value.
c. The solvent control data are within the normal range of the spontaneous background for the TK locus.

A mutation index was derived by dividing the number of clones formed in the BUdR-containing selection medium by the number found in the same medium without BUdR. The ratio was then compared to that obtained from other dose levels and from positive and negative controls. Colonies were counted on an electronic colony counter that resolves all colonies greater than 200 microns in diameter.
Statistics:
None specified.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Without activation: unambiguously negative. With activation: the number of mutants was increased at the 0.52 microliters/ml dose, but appeared to result from a slight decrease in the number of viable colonies.
Cytotoxicity / choice of top concentrations:
other: Little toxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: mouse lymphoma L5178Y cells

Any other information on results incl. tables

With activation, there was no trend indicating a dose-related response, and therefore, the increase in number of mutations was not thought to be compound related.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The in vitro forward mutation assay in mammalian cells to assess the genotoxicity of unleaded gasoline was negative. This finding does not warrant the classification of unleaded gasoline as a genotoxin under Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

API PS-6 (unleaded gasoline) was examined for its potential to induce mutations in the L5178Y mouse lymphoma cell line, in both the presence and absence of an S9 metabolic activation system. The doses were: 0.065, 0.13, 0.26, 0.52, and 1.04 microliters/ml. API PS-6 did not induce a statistically significant increase in the number of mutant colonies at any of the doses with or without metabolic activation; little cytotoxicity was observed. Both the positive and negative controls responded appropriately. Under the conditions of this study, API PS-6 was not mutagenic. This finding does not warrant classification of unleaded gasoline as a genotoxin under Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.