Paraffin waxes and Hydrocarbon waxes

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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 2005-07-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restriction because it was carried out in accordance with OECD Test Guideline 473 and is GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

The growth rate and viability of cells on the day of exposure did not meet the criteria as mentioned in the study plan. The doubling time was 14.4 hours instead of between 9 and 14 hours, and the viability was 89.4% instead of above 90%.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

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:

Paraffin waxes and Hydrocarbon waxes

IUPAC Name:

Paraffin waxes and Hydrocarbon waxes

Test material form:

liquid: viscous

Details on test material:

-Test Substance: CAS number: 8002-74-2
Name: Sasolwax 5203
Chemical Name: aliphatic alkane
Other Name: hydrocarbon wax
Purity: 100%
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 (mean of mixture)
Melting point: 54°C
Viscosity 3.5 mm2/s at 100°C
Colour Saybolt ASTM D 156: 30
Oil content: 0.2 %
Congealing Point: 54 °C

Prior to administration, test substance was melted and extracted in DMSO at a concentration of 360 mg/ml (1 mol/l) Sasolwax 5203 for approximately 60 minutes at 60±2°C under agitation. From this stock solution, serial dilutions in DMSO were prepared, and 100 μl of each of these were added to a final volume of 10 ml culture medium.

Method

Target gene:

tyrosine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y tk +/- 3.7.2C line
Chromosome number is 40 (stable aneuploid karyotype, 2n=40)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male wistar rat S9 homogenate

Test concentrations with justification for top dose:

Nominal test concentrations of 0.018, 0.037, 0.074, 0.15, 0.29, 0.59, 1.2, 1.7, 2.4, 3.4, 4.9, 7, 10 mmol/l.
Cell viability at the highest concentration (10 mmol/l) was above 90% at test termination.

Vehicle / solvent:

dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Five to seven days prior to treatment, cells were generated from a frozen stock.
- Exposure duration: 24 hours

NUMBER OF CELLS EVALUATED: 3 million L5178Y cells in 5 millilitres culture medium

DETERMINATION OF CYTOTOXICITY
- relative initial cell yield, relative suspension growth, and relative total growth.

Evaluation criteria:

A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 100 mutants per 1,000,000 clonable cells (Aaron et al, 1994; Clive et al., 1995). A response was considered to be equivocal if the induced mutant frequency was more than 50 mutants per 1,000,000 c1onable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity. The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed or if a reproducible positive response for at least one of the test substance concentrations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test points.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

greater than 90% viability at highest dose tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Increased mutant frequency was observed at a single dose level of 2.4 mmol/l in the absence of S-9 mix. Study authors concluded that the reading was caused by an unaccountably low value of the cloning efficiency and is not indicative of mutagenicity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results
Dose (mmol/l)	Absence of S9		Presence of S9
	Mutant Frequency	Relative Total Growth	Mutant Frequency	Relative Total Growth
10	159	121	81	66
7	124	147	67	73
4.9	138	133	59	68
3.4	130	126	56	83
2.4	221	86	73	74
1.2	123	148	101	71
0.59	132	210	60	88
0.29	130	139	93	76
0.15	164	126	85	111
0	119*	100	54*	100
*Mean of duplicate cultures

Applicant's summary and conclusion

Conclusions:

In both the absence and presence of S9-mix, Sasolwax 5203 was not cytotoxic. Neither the initial cell yield nor the relative total growth (RTG) at the highest concentration tested were decreased.

In the absence of S9-mix at a single dose level of 24% extract, the mutant frequency was increased by 102 mutants per 1,000,000 clonable cells compared to the negative control. The cloning efficiency at this concentration was remarkably low (0.48) compared to the overall mean cloning efficiency (0.75), and the mutant cloning efficiency was not increased compared to the overall mean mutant cloning efficiency (107 compared to 101). At no other dose level, in both the absence and presence of S9-mix, any increases of the mean mutant frequency (MF) by more than 50 mutants per 1,000,000 clonable cells compared to the negative control were observed.

Executive summary:

In a mammalian cell gene mutation assay of the TK-locus, mouse lymphoma L5178Y cells were exposed to Sasolwax 5203 in DMSO at nominal concentrations of 0.018, 0.037, 0.074, 0.15, 0.29, 0.59, 1.2, 1.7, 2.4, 3.4, 4.9, 7.0, or 10 mmol/l in the presence or absence of mammalian metabolic activation by Aroclor 1254-induced male Wistar rat liver S-9 fraction for 24 hours at 37°C under 5% carbon dioxide.

 

Sasolwax 5203 was tested up to 10 mmol/l. The positive controls methyl methanesulphonate (MMS) and 3 -methylcholanthrene (MCA) induced the appropriate response. The negative control (DMSO) was within acceptable range.Increased mutant frequency was observed at a single dose level of 2.4 mmol/l in the absence of S-9 mix. Study authors concluded that the reading was caused by an unaccountably low value of the cloning efficiency and is not indicative of mutagenicity.There was no evidence that Sasolwax 5203 induced mutant colonies over background.

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was carried out in accordance with OECD Test Guideline 473 and is GLP compliant.