Styrene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication meeting basic scientific principles.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Dose-dependent genotoxic effects of styrene on human blood lymphocytes and the relationship to its oxidative and metabolic effects.

Author:

Chakrabarti, S. et al.

Year:

1993

Bibliographic source:

Environmental and Molecular Mutagenesis 22: 85-92

Reference 2

Reference Type:

secondary source

Title:

European risk assessment report, Styrene CAS No. 100-42-5, EINECS No. 202-851-5, Draft for submission to SCHER, November 2007.

Author:

European Union

Year:

2007

Bibliographic source:

European risk assessment report, Styrene CAS No. 100-42-5, EINECS No. 202-851-5, Draft for submission to SCHER, November 2007.

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:

yes

Remarks:

- An exogenous mammalian metabolic activation system is missing; no positive control

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

Styrene

EC Number:

202-851-5

EC Name:

Styrene

Cas Number:

100-42-5

Molecular formula:

C8H8

IUPAC Name:

ethenylbenzene

Specific details on test material used for the study:

- Name of test material (as cited in study report): Styrene (Aldrich Chemical Co., Milwaukee, WI)
- Analytical purity: > 99.8%
- Other: Styrene was purified by repeated distillation and purity was verified by gas chromatorgraphy. Stock solutions of styrene were freshly made.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human blood lymphocyte cultures

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-1640 medium (Flow) containing 10% fetal calf serum (Gibco) and 1% phytohaemagglutinin (Wellcome)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

0.001, 0.00025, 0.005, 0.01, 0.02, 0.05, 0.1 mg/ml (corresponding to 10, 25, 50, 100, 200, 500 and 1000 µM)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Positive control substance:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 72 h


SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.1 µg/mL)


DETERMINATION OF CYTOTOXICITY
- Method: At the end of lymphocyte culture, a 0.5 mL culture suspension was mixed with 0.5 mL of trypan blue solution (0.4%), and the viability was estimated by measuring the fraction of cells that excluded trypan blue taking into account of the integrity of the cell membrane.

Statistics:

The standard error of the mean for the data was determined by the use of binomial variances. Results were also submitted to statistical randomised design analysis of variance. Difference between treatment means was compared by a Student´s paired t-test. Difference between treatment means was also compared by Dunnett´s t-test. significance: P < 0.05

Results and discussion

Test results

Species / strain:

lymphocytes: human blood lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Styrene is able to induce a characteristic genotoxic response in vitro, as measured by the cell cycle delay and induction of SCEs. The induction of SCEs showed a significant increase with rising concentrations of styrene up to 200 µM.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µM styrene enormous cellular mortality

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was no significant impairment of the viability of the cells observed with styrene concentration up to 500 µM.
Counts of the first, second and third generation cells showed that the cell cycle was delayed in proportion to the concentration of styrene. Third generation cells were extremely rare. With increasing styrene concentrations, fewer cells are able to reach the second generation, whereas an increasing number of cells remain at the first generation.

Remarks on result:

other: strain/cell type: human blood lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The SCE frequency and the cell cycle length were increased linearly with increasing concnetrations of styrene up to 200 µM.

Table: In vitro genotoxic effects of styrene on human blood lymphocytes^§

Treatment [µM]	No. of subjects	Mean SCE/cell ± SE	% of cells (a)
			M1 (mean ± SE)	M2 (mean ± SE)	M3 (mean ± SE)
Styrene 10	12	13.0 ± 0.2*	56 ± 3	44 ± 2	0 ± 0
Styrene 25	12	13.9 ± 0.6*	60 ± 3	40 ± 2	0 ± 0
Styrene 50	12	14.7 ± 0.7*	63 ± 2	37 ± 2	0 ± 0
Styrene 100	12	17.4 ± 0.8*	N.D.	N.D.	N.D.
Styrene 200	12	22.6 ± 0.7*	75 ± 4	25 ± 1	0 ± 0
Styrene 500	12	21.6 ± 0.7*	78 ± 4	22 ± 1	0 ± 0
Styrene 1000	12	N.D.	N.D.	N.D.	N.D.
DMSO	12	6.3 ± 0.2 (b)	51 ± 2	47 ± 2	2 ± 0.4

N.D.: not determined. At 1000 µM styrene, no parameters could be measured because of huge cellular mortality.

(a): Results are averages of 500 cells per individual for a total of 6000 cells.

(b): Results are averages of 200 cells per individual for a total of 2400 cells.

§: Results of each concentration were compared to those of the control using Dunnett´s t-test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive