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Toxicological information

Genetic toxicity: in vitro

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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:
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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.