Vinyltoluene

Endpoint summary

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 negative response without S9  (Similar/equivalent to OECD 490/GLP).

Link to relevant study records

Reference

Reference 1

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

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

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.

Target gene:

Thymidine kinase

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:

cytotoxicity

Remarks:

100 ug/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-07-2016 to 23-09-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 471 and GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Vinyltoluene
- Physical state: liquid
- Analytical purity: ≥99.2%
- Isomers composition:
3-methylstyrene (CAS No. 100-80-1): 64.3 % w/w
4-methylstyrene (CAS No. 622-97-9): 35.7 % w/w
- Lot/batch No.:201605
- Expiration date of the lot/batch: May 2017

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Delor 106-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity tests ((plate incorporation; TA 98): 0, 10, 100, 500, 1000, 2500, 5000 µg per plate
First mutagenicity experiment (plate incorporation): 0, 3, 10, 30, 100, 300 µg per plate

Preliminary toxicity tests (pre-incubation; TA 98): 0, 10, 25, 50, 100, 200 µg per plate
Second mutagenicity experiment (pre-incubation): 0, 2.5, 5, 10, 25, 50 µg per plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine (NPD); 2-aminofluorene (2-AF); 2-aminoanthracene (2-AA); 9-aminoacridine hydrochloride monohydrate (9-AAc)

Remarks:

AS (1.5 µg/plate; TA 100 &TA 1535 –S9) NPD (20 µg/plate; TA 98 –S9) 2-AF (10 µg/plate; TA 100 & TA 98 –S9) 2-AA (1.0 µg/plate -TA 1535, 2.5 µg/plate -TA 1537, 25 µg/ plate E.coli +S9) 9-AAc (100 µg/plate; TA 1537 –S9) MNNG (20 µg/plate; E. coli –S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 mins
- Exposure duration: 48 - 72 hrs (both methods)

NUMBER OF REPLICATIONS: Triplicate plating was used at each dose level; Two mutagenicity experiments using plate incorporation and preincubation methods.

DETERMINATION OF CYTOTOXICITY: clearing or diminution of the background lawn

Evaluation criteria:

The main criterion for evaluation of results was the modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). According to this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as "biologically relevant“:

-if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
-if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;

A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 417

3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 – 91

Statistics:

The main criterion for evaluation of results was the modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3).

2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 417

3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 – 91

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300µg per plate in first experiment; none in second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300µg per plate in first experiment; none in second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300µg per plate in first experiment; none in second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300µg per plate in first experiment; none in second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

300µg per plate in first experiment; none in second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test (plate incorporation): All application forms were clear; precipitation occurred after adding to top agar starting with 500 µg per plate. Toxicity of the test substance was observed at 500 µg per plate (no background, a lot of colonies on plates). No signs of toxicity were observed at 100 µg per plate. (Table B)

First mutagenicity test: The dose of 300 µg per plate was partially toxic - it caused a diminution of bacterial background and precipitated in top agar (slight turbidity).

Preliminary toxicity test (pre-incubation):Two toxicity experiments were performed with various amount of DMSO (100 and 50 µL per plate) for dosing of the test substance. 100 µL of DMSO was toxic for bacterial background, 50 µL of DMSO was without toxicity of solvent. (Table C)

Second mutagenicity test: All application forms were clear; precipitation did not occur at any dose.

COMPARISON WITH HISTORICAL CONTROL DATA:Spontaneous reversions, negative (DMSO) and positive controls were compared with historical controls in our laboratory and were all acceptable. The current ranges are given in the Table A in the report.

Remarks on result:

other: other: Preliminary toxicity (plate incorporation)

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the above-described experimental design, the test substance, Vinyltoluene, was non-mutagenic for all the Salmonella typhimurium and Escherichia coli strains with and without metabolic activation. Pre-incubation modification had no influence on the non-mutagenic effect of the test substance.

Executive summary:

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 and/or precipitating 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.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to OECD 473 and GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix

Test concentrations with justification for top dose:

0, 1.5, 5, 16, 50 µg/mL (-S9)
0, 5, 16, 50 µg/mL (+S9)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C (-S9) : 0.125, 0.25 µg/mL; Cyclophosphamide (+S9): 15, 50 µg/mL

Details on test system and experimental conditions:

CHO cultures were handled under gold lights to prevent photolysis of bromodeoxyuridine (BrdU)-substituted DNA. Each test consisted of concurrent solvent and positive controls and of at least three doses of the study chemical; the high dose was limited by toxicity or solubility but did not exceed 5 mg/mL.

In the chromosomal aberration test without S9, cells were incubated in McCoy's 5A medium with the study chemical for 8 hours at 37C; colcemid was added, and incubation was continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa.

For the chromosomal aberration test with S9, cells were treated with the study chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9.

Evaluation criteria:

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. 100 first-division metaphase cells were scored at each dose for the chromosomal aberration test. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:

Statistical analyses were conducted on both the slopes of the dose-response curves and the individual dose points. Chromosomal aberration data are presented as percentage of cells with aberrations. As with SCEs, both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly increased dose point (P <0.05) was sufficient to indicate a chemical effect.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: other: Main test

Remarks:

Migrated from field 'Test system'.

Conclusions:

In cytogenetic tests with CHO cells, vinyl toluene did not induce or chromosomal aberrations in either the presence or the absence of Aroclor 1254-induced male Sprague Dawley rat liver S9.

Executive summary:

In a mammalian cell cytogenetics assay (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.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline [In vitro mammalian cytogenetics [Chromosome aberration] OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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.

 

Gene mutation (Bacterial Reverse Mutation Assay/Ames test)

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 and/or precipitating 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 vitro cytogenicity (chromosome aberration) study in mammalian cells

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.

 

Gene mutation (mammalian cell gene mutation assay)

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.

Justification for classification or non-classification

Based on the available information in the dossier, vinyl toluene (CAS No. 25013 -15 -4) does not need to be classified for germ cell mutagenicity in accordance with the criteria outlined in Annex I of 1272/2008/EC.