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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP status unknown, near guideline study, published in peer reviewed literature, minor restrictions in design and/or reporting but otherwise adequate for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4 strains (not including TA1535) of S. typhimurium used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
95-63-6
Molecular formula:
C9-H12
Constituent 2
Reference substance name:
pseudocumene
IUPAC Name:
pseudocumene
Details on test material:
Obtained from Fluka

Method

Species / strain
Species / strain / cell type:
other: TA97a, TA98, TA100, TA102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 fraction prepared from Aroclor 1254-induced male outbred Imp:Lodz rat liver
Test concentrations with justification for top dose:
The compounds were tested up to the cytotoxic concentrations - 1, 5, 10, 20 and 30 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: mineral oil (light white oil) obtained from Sigma Chemical

- Justification for choice of solvent/vehicle: in previous studies when dimethylsulfoxide (DMSO) or ethanol were used for in vitro experiments, the reaction of the compounds appeared to be more toxic for the tester strains of bacteria.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
mineral oil (100 µL)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: 0.5 µL or µg
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 1.5 µL or µg
Positive control substance:
other: 2, aminofluorene
Remarks:
5.0 µL or µg
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
3.0 µL or µg
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37ºC

SELECTION AGENT (mutation assays): His+ revertant colonies

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Increase in the number of revertants per plate as compared to the revertants per solvent control plate
Evaluation criteria:
A chemical was considered to be a mutagen if it induced at least a 2-fold increase in the number of revertants per plate as compared to the revertants per solvent control plate, with accompanying dose-effect relationship in at least one tester strain.
Statistics:
Not applicable

Results and discussion

Test results
Key result
Species / strain:
other: TA97a, TA98, TA100, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
all except TA97a
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the presence and absence of S9-mix the highest dose, 30 µL/plate 1,2,4-trimethylbenzene, produced a decrease in the number of revertants to the level below 75%.

A similar, negative effect on the Salmonella tester strains was obtained in an experiment in which the compound was tested using another technique (data not shown). In the preincubation modification of the standard mutagenicity assay (20 min at 37°C) on S. typhimurium TA98 and TA 100 (- S9; + S9) pseudocumene (at doses from 1-20 µL/plate, dissolved in DMSO) induced no gene mutations.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

1,2,4-trimethylbenzene is negative in the Ames test, with and without metabolic activation, when tested in S. typhimurium TA97a, TA98, TA100 and TA102
Executive summary:

1,2,4 -trimethylbenzene was tested in vitro in the Ames test, plate incorporation method, with Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains in the presence and absence of rat liver S9 metabolic activation.  1,2,4 -trimethylbenzene gave negative results both with and without activation at the highest dose tested, 30 µL/plate.