Paraffin waxes and Hydrocarbon waxes

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2005-06-21 to 2005-08-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restriction because it closely adhered to OECD Test Guideline 476 using cell line L5178Y, with and without metabolic activation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

8002-74-2

Cas Number:

8002-74-2

IUPAC Name:

8002-74-2

Constituent 2

Reference substance name:

Sasolwax 5203, hydrocarbon wax

IUPAC Name:

Sasolwax 5203, hydrocarbon wax

Test material form:

liquid: viscous

Details on test material:

100% purity
Chemical name: aliphatic alkane
Other name: hydrocarbon wax
Appearance: solid, white, waxy
Batch/Lot Number: A053603
Molecular Formula: C(n)H(2n+2); Sasolwax 5203 contains alkanes ranging from C19 to C36. The man component is C26
Average Molecular Weight: 360
Melting point: 54°C
Supplier: Sasol Wax GmbH, Hamburg, Germany
TNO Test Substance Number: 0500A9

Just before use, the test material was melted and extracted once in dimethylsulphoxide (DMSO), at a concentration of 360 mg/ml (1: 1 w/w). The extraction was performed for approximately 60 minutes at 60 ± 5°C, under constant agitation. Thereafter, the clear extract (DMSO aliquot) was collected and used for the preparation of the serial dilutions in DMSO. Fifty microlitres of the stock concentration and its serial dilutions were added to a final volume of 5 millilitres culture medium.

- Viscosity (3.5 mm2/s at 100 oC)
- Congealing point (54 oC)
- Color saybolt ASTM D156 (30)
- Oil content (0.2%)

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12 with Glutamax-I supplemented with heat-inactivated foetal calf serum (10%), penicillin (100IU/ml medium), and streptomycin (100 μg/ml medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The first chromosomal aberration assay used concentrations of 0.034, 0.069, 0.138, 0.277, 0.625, 1.25, 2.5, 5, or 10 mmol/l of the test material in final culture medium. The second chromosomal aberration test used concentrations of 2.78, 4.17, 5.56, 6.94, 8.33, or 10 mmol/l of the test material in final culture medium.

Vehicle / solvent:

Dimethyl sulphoxide was used as the vehicle because it is miscible with the test material and is a known extraction solvent for polyaromatic substances.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: 50μl of extract at various concentrations was added to a final volume of 5 millilitres of culture medium.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours in the absence or presence of S9 in the first test; 4 hours in the presence of S9 or 18 hours in the absence of S9 in the second test
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours

At least 100 nuclei in each culture were examined to determine mitotic index.

Evaluation criteria:

The study was considered valid because the positive controls gave the statistically significant increases in the number of aberrant cells and the negative controls were within the historical range. A response is considered to be positive if a concentration-related increase or a reproducible increase in the number of cells with structural chromosomal aberrations is observed.
A response is considered to be equivocal if the percentage of cells with structural chromosomal aberrations is statistically marginal higher than that of the negative control (0.05A test material is considered to be clastogenic if a concentration-related increase in the percentage of cells with structural chromosomal aberrations over the concurrent control frequencies is observed, or if a single positive test point is observed in both tests.
A test material is considered to be negative in the chromosomal aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points. Cells with only gaps (achromatic lesions), heavily damaged cells (cells with multiple aberrations) and cells with polyploidy and endoreduplication were recorded separately and not included in the final assessment of clastogenic activity.

Statistics:

Data were analysed statistically by Fisher's exact probability test (two-sided) to determine significant differences between treated and control cultures.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the second chromosomal aberration test, in the single treatment group with metabolic activation (S9-mix), the mitotic index of the highest extract concentration analysed (l00%) was slightly reduced to 82% of that of the concurrent control. The mitotic indices of the two lower extract concentrations analysed (83.3% and 69.4%) were not reduced when compared to the concurrent control. None of the extract concentrations analysed induced a statistically significant increase in the number of aberrant cells. In the continuous treatment group without metabolic activation (S9-mix), the mitotic indices of none of the extract concentration analysed (l00%, 83.3%, and 69.4 %) were reduced when compared to the concurrent control. None of the extract concentrations analysed induced a statistically significant increase in the number of aberrant cells. In both chromosomal aberration tests, the positive control substances mitomycin C (in the absence of a metabolic activation system) and cyclophosphamide (in the presence of a metabolic activation system) induced the expected statistically significant increases in the incidence of structural chromosomal aberrations.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The data obtained in both chromosomal aberration tests, support the conclusion that, under the conditions used in this study, the extract of SASOLWAX 5203 was not clastogenic for CHO cells.

Executive summary:

Two mammalian cell chromosome aberration tests were performed. In the first test, Chinese hamster ovary (CHO) cells were exposed to extracts of Sasolwax 5203 at concentrations of 0.034, 0.069, 0.138, 0.277, 0.625, 1.25, 2.5, 5, or 10 mmol/l for four hours with or without metabolic activation at 37°C under 5% CO₂. In the second chromosome aberration test, extracts of Sasolwax 5203 were tested at concentrations of 2.78, 4.17, 5.56, 6.94, 8.33, or 10 mmol/l for 18 hours continuous treatment without metabolic activation or 4 hours pulse treatment with metabolic activation at 37°C under 5% CO₂. None of the extract concentrations analysed induced a statistically significant increase in the number of aberrant cells with or without metabolic activation or at pulse or continuous exposure. In both chromosomal aberration tests, the positive control substances mitomycin C (in the absence of a metabolic activation system) and cyclophosphamide (in the presence of a metabolic activation system) induced the expected statistically significant increases in the incidence of structural chromosomal aberrations.

This study received a Klimisch score of 1 and is classified as reliable without restriction because it closely adhered to OECD Test Guideline 476 using cell line L5178Y, with and without metabolic activation.