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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:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Similar to OECD 490 and GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Vinyltoluene
EC Number:
246-562-2
EC Name:
Vinyltoluene
Cas Number:
25013-15-4
Molecular formula:
C9H10
IUPAC Name:
1-methyl-2-vinylbenzene
Test material form:
other: liquid
Details on test material:
- 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.
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.

Method

Target gene:
Thymidine kinase
Species / strain
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
Controls
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

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 ug/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In the mouse lymphoma assay in L5178Y cells, vinyl toluene was not mutagenic.
Executive summary:

In a mammalian cell gene mutation assay (similar to OECD 490/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.


 


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%). The remaining two experiments 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 (experiment 2: 5.5±0.5% and experiment 3: 8±1.0%). According to paragraph 67 of OECD 490 “If the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve between 20 and 10% RTG. The consensus is that care should be taken when interpreting positive results only found between 20 and 10% RTG and a result would not be considered positive if the increase in MF occurred only at or below 10% RTG (if evaluated).” Therefore, the mutagenic effects at 60 µg /mL in experiments 2 and 3 cannot be treated as positive results. The top dose with no cytotoxicity data in the first experiment is 50µg /mL, and there is no increase in mutant frequency compared to controls, therefore the substance is not mutagenic.