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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only one strain of Salmonella was used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions from Aroclor exposed rats
Test concentrations with justification for top dose:
Tests with and without Metabolic Activation: 0, 1, 5, 10, 25, 50, and 100 uL/plate (2mL test material dissolved in 3mL cyclohexane)
Control Plate: 10uL DMSO
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO/ cyclohexane
- Justification for choice of solvent/vehicle: Positive controls dissolved into DMSO, Test substance soluble in acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
10uL DMSO Plate
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
DURATION
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS:
- triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants and/or clearing of the background lawn of bacterial growth
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the positive controls meet the criteria for a positive response and if no more than 5% of the plates are lost through contamination or other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increases in a concentration-related manner and/or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitution for frameshifts in the genome of Salmonella typhimurium. Negative results indicate that under the test conditions, the test substance is not mutagenic.
Statistics:
The mean plate count and standard deviation for each dose point were determined. Any test value that was equal to or greater than two times the mean value of the concurrent vehicle control was considered to be a positive dose.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
not cytotoxic up to 50uL/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity was noted at doses above 25 uL/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results: negative

It is concluded in this study that the test material is not a mutagenic agent. Classification is not warranted under the new 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:

The test material was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strain TA 98 in the absence and presence of a liver S9 fraction for metabolic activation. The test was performed in triplicate using doses of 0, 1, 5, 10, 50, 100 uL/plate.  Concentrations above 25 uL/plate were found to be cytotoxic. In all cases, the test material did not induce any significant changes in the number of revertant colonies.  It is concluded in this study that the test material is not a mutagenic agent. Classification is not warranted under the new 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only one strain of Salmonella was used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test substance was identified as a hydrocarbon solvent with the following characterization: density at 15 deg C: 0.831 kg/L; aromatics : 28.7% v/v; flash point 128 deg.C; BP range 264-287deg C.

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions from Aroclor exposed rats
Test concentrations with justification for top dose:
Tests with and without Metabolic Activation: 0, 1, 5, 10, 25, 50, and 100 uL/plate (2mL test material dissolved in 3mL cyclohexane)
Control Plate: 10uL DMSO
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO/ cyclohexane
- Justification for choice of solvent/vehicle: Positive controls dissolved into DMSO, Test substance soluble in acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
10uL DMSO Plate
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
DURATION
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS:
- triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants and/or clearing of the background lawn of bacterial growth
Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the positive controls meet the criteria for a positive response and if no more than 5% of the plates are lost through contamination or other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increases in a concentration-related manner and/or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitution for frameshifts in the genome of Salmonella typhimurium. Negative results indicate that under the test conditions, the test substance is not mutagenic.
Statistics:
The mean plate count and standard deviation for each dose point were determined. Any test value that was equal to or greater than two times the mean value of the concurrent vehicle control was considered to be a positive dose.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
not cytotoxic up to 50uL/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity was noted at doses above 25 uL/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

It is concluded in this study that the test material is not a mutagenic agent. Classification is not warranted under the new 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:

The test material was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strain TA 98 in the absence and presence of a liver S9 fraction for metabolic activation. The test was performed in triplicate using doses of 0, 1, 5, 10, 50, 100 uL/plate.  Concentrations above 25 uL/plate were found to be cytotoxic. In all cases, the test material did not induce any significant changes in the number of revertant colonies.  It is concluded in this study that the test material is not a mutagenic agent. Classification is not warranted under the new 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.