Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance vinyl toluene (CAS No. 25013-15-4) was not mutagenic in the strains TA98, TA100, TA1535 and TA1537 of S. typhimurium and E. coli WP2 uvr A in the presence and absence of Delor 106-induced rat liver S9 metabolic activation (OECD 471/GLP).

Chromosome aberration (in vitro mammalian chromosome aberration): the substance vinyl toluene (CAS No. 25013-15-4) was concluded to be negative for the induction of chromosome aberrations in the presence and absence of Delor 106-induced rat liver S9 metabolic activation in CHO cells (Similar/equivalent to OECD 473/GLP).

Gene mutation (Mammalian cell gene mutation assay): the substance vinyl toluene (CAS No. 25013-15-4) gave a positive response in two out of three trials conducted without S9 at the highest doses tested; these doses also produced severe toxicity, as evidenced by a relative total growth of less than 10% (Similar/equivalent to OECD 476/GLP).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to OECD 476 and GLP compliant
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Vinyl toluene (Radian Corporation (Austin, TX))
- Physical state: liquid
- Analytical purity: approx. 99%
- Composition of test material, percentage of components: p-Vinyl toluene and m-vinyl toluene represented 31.6% and 68.4%, respectively, of the mixture
- Lot/batch No.:CH910
- Stability under test conditions: Results ofperiodic analysis by infrared spectroscopy, gas chromatography, determination of inhibitor concentration, and polymer concentration indicated no significant degradation of the study material throughout the studies.
- Storage condition of test material: Stability studies performed by gas chromatography indicated that vinyl toluene was stable as a bulk chemical when stored protected from light for 2 weeks at temperatures up to 25° C.

Refer to Appendix G and Table G1 for more details on specific chemical characterisation of the vinyl toluene used in the study.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:Fischer's medium supplemented with 2 mM L-glutamine, 110 pg/ml sodium pyruvate, 0.05% pluronic F68, antibiotics, and heat-inactivated horse serum. Normal cycling time was about 10 hours.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: Yes; subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day, to thymidine, hypoxanthine, and glycine for 1 day, and to normal medium for 3-5 days.
Metabolic activation:
without
Test concentrations with justification for top dose:
Trial 1: 12.5, 25, 50, 100 µg/mL

Trial 2: 10, 20, 40, 60, 80 µg/mL

Trial 3: 40, 45, 50, 55, 60, 65 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Methyl methanesulfonate:15 µg/mL
Details on test system and experimental conditions:
The highest dose of the study compound was determined by solubility or toxicity and did not exceed 100 ug/ml. All doses within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 X 106 cells in 10 ml of medium. Incubation with the study chemical continued for 4 hours, after which time the medium plus chemical was removed and the cells were re-suspended in 20 ml of fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 X 106 cells were plated in medium and soft agar supplemented with Tft for selection of Tft-resistant cells (TK +/+),and 600 cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37° C under 5% carbon dioxide for 10-12 days.

Replicates: Mean ± standard error from replicate trials of approximately 1 X 106 cells each.
Trials 2 & 3: See table H2 (e) Data presented are the results of four tests; (f) Data presented are the results of three tests.
Evaluation criteria:
Mutant fraction (frequency) is a ratio of the Tft-resistant cells to the cloning efficiency, divided by 3 (to arrive at MF per 1 X 106 cells treated);
MF = mutant fraction.

Significant positive response occurs when the relative mutant fraction (average MF of treated culture/average MF of solvent control) is greater than or equal to 1.6.

Both responses must be significantly (P< 0.05) positive for a chemical to be considered capable of inducing Tft resistance. If only one of these responses is significant, the call is "equivocal"; the absence of both trend and peak response results in a "negative" call.

Statistics:
Mean ± standard error from replicate trials of approximately 1 X 106 cells each. All data are evaluated statistically for both trend and peak response
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
60 µg only
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>40µg
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
60 µg only
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>50µg
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: other: Trial 1
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation

In the mouse lymphoma assay for induction of Tft resistance in L5178Y cells, vinyl toluene gave a positive response in two out of three trials conducted without S9 at the highest doses tested; these doses also produced severe toxicity, as evidenced by a relative total growth of less than 10%.
Executive summary:

In a mammalian cell gene mutation assay (90-2830), mouse lymphoma L5178Y cells cultured in vitro were exposed to vinyl toluene (approx. 99%; p-Vinyl toluene (31.6%) and m-vinyl toluene (68.4%)) in DMSO in 3 trials at concentrations of 12.5, 25, 50, 100 µg/mL; 10, 20, 40, 60, 80 µg/mL and 40, 45, 50, 55, 60, 65 µg/mL without metabolic activation.

Vinyl toluene was tested up to cytotoxic concentrations. The positive controls induced the appropriate response. In the first trial, the highest non-lethal concentration of vinyl toluene was 50 µg /mL. At this level there was no increase in mutant fraction, but the Relative Total Growth (RTG) remained high (69% ±7%), so the experiment was judged as inconclusive. The remaining two trials gave statistically significant responses at 60 µg /mL. This was the only significant dose level, even marginally lower doses (e.g., 55 µg g/mL) inducing considerably less toxicity and no significant increases in mutant fraction. The Relative Total Growth was below 10% at 60 µg /mL in both experiments. It appears, therefore, that appreciable toxicity is required before vinyl toluene is significantly mutagenic in this assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

One in vitro bacterial gene mutation assay, one mammalian cell cytogenetics assay (chromosome aberration) and one in vitro mammalian cell gene mutation test were available.

In a reverse gene mutation assay (OECD 471/GLP), strains TA98, TA100, TA1535 and TA1537 of S. typhimurium and E. coli WP2 uvr A were exposed to Vinyltoluene ( ≥99.2%; 3-methylstyrene (CAS No. 100-80-1): 64.3 % w/w, 4-methylstyrene (CAS No. 622-97-9): 35.7 % w/w) at concentrations of 0, 3, 10, 30, 100, 300 µg per plate (plate incorporation) and 0, 2.5, 5, 10, 25, 50 µg per plate (pre-incubation method) in the presence and absence of mammalian metabolic activation (Delor 106-induced rat liver S9). Vinyltoluene was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in either assay.

In a mammalian cell cytogenetics assay (Chromosome aberration; Similar to OECD 473/GLP), CHO cell cultures were exposed to vinyl toluene (approx. 99%; p-Vinyl toluene (31.6%) and m-vinyl toluene (68.4%)) in DMSO at concentrations of 0, 5, 16, 50 µg/mL (with Aroclor 1254-induced male Sprague Dawley rat liver S9 metabolic activation) and 0, 1.5, 5, 16, 50 µg/mL (without metabolic activation). Vinyl toluene was tested up to the highest dose not limited by toxicity or solubility. Positive controls induced the appropriate response. There was no evidence of chromosome aberrations induced over background.

In a mammalian cell gene mutation assay (Similar to OECD 476/GLP), mouse lymphoma L5178Y cells cultured in vitro were exposed to vinyl toluene (approx. 99%; p-Vinyl toluene (31.6%) and m-vinyl toluene (68.4%)) in DMSO in 3 trials at concentrations of 12.5, 25, 50, 100 µg/mL; 10, 20, 40, 60, 80 µg/mL and 40, 45, 50, 55, 60, 65 µg/mL without metabolic activation. Vinyl toluene was tested up to cytotoxic concentrations. The positive controls induced the appropriate response. In the first trial, the highest non-lethal concentration of vinyl toluene was 50 µg /mL. At this level there was no increase in mutant fraction, but the Relative Total Growth (RTG) remained high (69% ±7%), so the experiment was judged as inconclusive. The remaining two trials gave statistically significant responses at 60 µg /mL. This was the only significant dose level, even marginally lower doses (e.g., 55 µg g/mL) inducing considerably less toxicity and no significant increases in mutant fraction. The Relative Total Growth was below 10% at 60 µg /mL in both experiments. It appears, therefore, that appreciable toxicity is required before vinyl toluene is significantly mutagenic in this assay.

As the mammalian cell gene mutation assay gave a positive response in two out of three trials conducted without S9 at the highest doses tested (even though these doses also produced severe toxicity,), further in vivo studies will be considered.

Justification for classification or non-classification

Based on the available information in the dossier, it is not possible to conclude on the germ cell mutagenicity of vinyl toluene (CAS No. 25013 -15 -4) when the criteria outlined in Annex I of 1272/2008/EC are applied, as further in vivo data is required.