4-methylstyrene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

no

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

4-methylstyrene

EC Number:

210-762-8

EC Name:

4-methylstyrene

Cas Number:

622-97-9

Molecular formula:

C9H10

IUPAC Name:

1-ethenyl-4-methylbenzene

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

Trial 1: 1, 10, 25, 50 and 100 nL/mL
Trial 2: 25, 50, 65, 80 and 100 nL/mL (confirmatory assay)
Trial 3: 25, 50, 65, 80 and 100 nL/mL (repeating Trial 2 with new lot of test substance due to suspected improper storage of lot used in both earlier
assays)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, 1%

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0.1 µgmL

Details on test system and experimental conditions:

Whole blood cultures
Human venous blood was drawn into a sterile syringe that contained heparin to prevent clotting. Cultures were initiated by adding 0.6 ml of blood to 9.4 ml pf medium in 25 cm2 plastic flasks standing upright to minimize the surface area. The medium was RPMI 1640 supplemented with 15 % fetal calf serum (FCS), 1% penicillin-streptomycin, and phytohaemagglutinin (PHA-M).
Treatment of cultures with the test substance
Blood was added to culture medium containing PHA and incubated at 37 °C in the dark for 24 h. At this time cells are beginning to grow due to stimulation by PHA. Solutions of the test substance EMS or the solvent control were then added to appreciate cultures, followed by BrdU (final concentration 25 uM) and cultures were re-incubated for a further 46-48 hours (total culture time: 70-72 h). Colcemid was added 2.5 hours before harvest of dividing lymphocytes. Two replicate cultures were used for each treatment in assay.
Lymphocyte fixation
Thee cell suspension was centrifuged, the supernatant discarded, and cells treated with hypotonic KCl (0.075 M) for 3 minutes to swell the cells and get rid of red blood cells. Cells were then washed three times with fixative (methanol/glacial acetic acid, (3:1)) and dropped on to slides to air day. In trial 3 BrdU was added at initiation of cultures, not after the test compound.
Staining for detection of SCE was accomplished by a modified fluorescent plus Giemsa technique (Perry and Wolff 1974 and Goto et al. 1978).

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The solvent control did not show any mutagenic activity. Results for the positive control showed expected mutagenic activity.
Trial 1: A slight, but statistically significant (P<0.001) higher number of sister chromatid exchanges (SCEs) per cell was noted at 50 and 100 nL/mL compared to solvent control, with some evidence of a dose-response.
Trial 2: This assay repeated the first trial, using more concentrations in the range of ‘positives’ from Trial 1. In this assay a slight, but statistically significant (P<0.25 or P<0.001) higher number of SCEs per cell was observed at 50 through 80 nL/mL. However, the difference from solvent control at the highest concentration of 100 nL/mL was not statistically significant and, overall, there was no evidence of a dose-response.
Trial 3: A new lot of the test substance, stored at -20⁰C (instead of room temperature), was used in this trial, using the same concentrations of test substance as in Trial 2 and following essentially the same methodology. A slight, but statistically significant (P<0.001) higher number of SCEs per cell was noted in this assay at concentrations of 65 through 100 nL/mL, without a clear dose-relationship.   
Dose-related cell toxicity was noted at all test substance concentrations, as evidenced by a reduction in the frequency of dividing cells (mitotic index) and cell cycle delay.

Any other information on results incl. tables

Treatment	Mitotic index (% 500 cells)	% of cells at first second and third metaphase
Solvent control	1.2 %	26	58	16
25 nl/ml	0.4 %	42	51	7
100 nl/ml	0.6 %	66	34	0

Applicant's summary and conclusion

Conclusions:

Under the study conditions, dose-related cell toxicity was noted at all test substance concentrations, as evidenced by a reduction in the frequency of dividing cells (mitotic index) and cell cycle delay. A slight but statistically significant and reproducible higher number of SCEs was observed in cultured human lymphocytes treated with test substance over a concentration range of 50 to 100 nL/mL. The maximum response in cultures treated with test material was approximately 84% over the solvent control. Given that these data did not meet the evaluation criteria for a ‘positive’ result, with a less than a 2-fold increase in SCEs over solvent control and without a clear dose-response, the test substance was designated as ‘weakly positive’.

Executive summary:

A study using cultured human lymphocytes was conducted to determine the potential of the test substance to induce sister chromatid exchanges (SCEs) in vitro, according to a method similar to OECD Guideline 479. DMSO (1%) was used as the solvent control and the positive control was ethylmethanesulfonate. Three trials were performed, using an overall concentration range of 1 to 100 nL/mL. The solvent control did not show any activity, results for the positive control showed expected increases in SCEs. Under the study conditions, dose-related cell toxicity was noted at all test substance concentrations, as evidenced by a reduction in the frequency of dividing cells (mitotic index) and cell cycle delay. A slight but statistically significant and reproducible higher number of SCEs was observed in cultured human lymphocytes treated with test substance over a concentration range of 50 to 100 nL/mL. The maximum response in cultures treated with test material was approximately 84% over the solvent control. Given that these data did not meet the evaluation criteria for a ‘positive’ result, with a less than a 2-fold increase in SCEs over solvent control and without a clear dose-response, the test substance was designated as ‘weakly positive’ (Galloway, 1980).