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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
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:
From March 21 to June 24, 2011
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Eucarol AGE 91/S ( Sulfonation products of (esterification products of C9-11 (branched and linear) alkyl-(mono-, di- and tri-)glycosides with maleic anhydride) with disodium sulfite ) was used as supporting substance due to the structural analogy. It can be expected a similar structural features.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
according to
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
according to
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Details on test material:
Batch No.: PIC 484
CAS No.: 1228577-41-0
Colour: Yellow
Physical State: Liquid (50% in water)
Purity: 50% (in water)
Expiry Date: October 2012
Storage Conditions: at room temperature, protected from light


Target gene:
Thymidine Kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Test concentrations with justification for top dose:
A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL. A correction factor of 2 was comprised in order to test the test item as 100%.
Experiment I
with metabolic activation: 500, 540, 565, 590, 615 and 640 μg/mL
without metabolic activation: 100, 150, 200, 250, 300, 340, 380 and 420 μg/mL
Experiment II
with metabolic activation: 100, 200, 300, 400, 450, 500, 530 and 560 μg/mL
without metabolic activation: 5, 10, 25, 50, 100, 120, 150 and 180 μg/mL
Vehicle / solvent:
RPMI cell culture medium (RPMI + 5% HS). The solvent was compatible with the survival of the cells of the S9 activity.
Untreated negative controls:
treatment medium
Positive controls:
Positive control substance:
Migrated to IUCLID6: and Methylmethanesulfonate without S9; Benzo[a]pyrene with S9.
Details on test system and experimental conditions:
Mouse Lymphoma L5178Y cells have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10-12 h doubling time of the BSL BIOSERVICE stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40+/-2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus. To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640 supplemented with:
9.0 μg/mL hypoxanthine
15.0 μg/mL thymidine
22.5 μg/mL glycine
0.1 μg/mL methotrexate
The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for 1-3 days.
Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE. This allows the repeated use of the same cell batch in experiments. Each cell batch is routinely checked for mycoplasma infection.
Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
Evaluation criteria:
The test item is considered mutagenic if following criteria are met :
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 x 106
- A dose-dependent increase in mutant frequency is detected using an appropriate statistical analysis.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Untreated negative controls validity:
not valid
Positive controls validity:
Additional information on results:
No precipitation of the test item was noted in the main experiment
Growth inhibition was observed in experiment I and II with and without metabolic activation.
In experiment I with metabolic activation the relative total growth (RTG) was 10.8% for the highest concentration (640 μg/mL) evaluated. The highest concentration evaluated without metabolic activation was 420 μg/mL with a RTG of 15.7%.
In experiment II with metabolic activation the relative total growth (RTG) was 17.5% for the highest concentration (560 μg/mL) evaluated. The highest concentration evaluated without metabolic activation was 180 μg/mL with a RTG of 17.6%.

In experiment I and II without metabolic activation no biologically relevant increase of mutants was found after treatment with the test item. The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories [13, 14, 15]) was not exceeded by the induced mutant frequency at any concentration. In experiment I with metabolic activation increased mutant frequencies were found compared to the negative control.
Additionally a dose related effect was detected. However, as the GEF of 126 was not exceeded in any of the dose groups the biological relevance of this effect had to be proved in an independent repetition experiment: experiment II with metabolic activation did not confirm the effect found in the first experiment. The mutant frequencies showed no remarkably elevated mutant frequencies and no dose related effect was noted.
Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).
EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):

Under the experimental conditions reported, the test item "Sulfonation products of (esterification products of C9-11 (branched and linear) alkyl-(mono-, di- and tri-)glycosides with maleic anhydride) with disodium sulfite" is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.