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Genetic toxicity in vitro

Description of key information
There are 4 studies available. in two Ames test in bacteria the test material produced positive results , which were not reproduced when mammalian cells were employed in a gene mutation assaThe negative result was supported by the negtive result of a chromosomal aberration study in vitro.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP, but standard guideline methods applied
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
no
Remarks:
but standard metod applied
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
0 - 6,3 - 12,5 - 25 - 50 - 100 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION

- Exposure duration: 4 h
- Expression time (cells in growth medium): 72 h
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 76 h

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative suspension and total growth (RSG and R TG)

OTHER EXAMINATIONS:
- Determination of Size Distribution of the Colonies
Evaluation criteria:
A mutation assay is considered acceptable if it meets the following criteria:
a) The absolute cloning efficiency of the negative and/or solvent controls was> 50%.
b) The spontaneous mutant frequency in the negative and/or solvent controls were in the range of our historical control data with manual evaluation of the plates: 22 - 115 mutants per 10E6 cells.
c) The positive controls (MMS and 3-MC) induced significant (at least 2-fold) increases in the mutant frequencies. The cloning efficiencies were greater than 10 % of the concurrent vehicle control group.

A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered nonmutagenic in this system.
A significant response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations a mutation frequency that is two times higher than the mean spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a concentration-related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if either, the relative total growth or relative suspension growth is less than 10% of the solvent control or the cloning efficiency after the expression period is less than 20 %.

 

 

 

experiment I

 

Conc. (µg/mL)

S9 mix

Relative cloning efficiency

Relative total growth

Mutant colonies/ 106cells

Induction factor

Neg. contr. with medium 

 

-

100.0

100.0

52

 

Neg. contr. with DMSO   

 

-

100.0

100.0

66

1.0

Pos. control with MMS

13.0

-

77.2

151.8

167

3.2

Test item

3.1

-

96.4

culture was not continued

Test item

6.3

-

91.5

111.1

51

0.8

Test item

12.5

-

88.4

98.5

74

1.1

Test item

25.0

-

105.9

86.3

110

1.7

Test item

50.0

-

78.9

77.0

108

1.6

Test item

100.0

-

74.2

84.5

85

1.3

 

 

 

 

 

 

 

Neg. contr. with medium 

 

+

100,0

100,0

72

 

Neg. contr. with DMSO   

 

+

100,0

100,0

70

1,0

Pos. control with 3-MC

3,0

+

95,3

100,7

205

2,9

Test item

3,1

+

118,9

culture was not continued

Test item

6,3

+

116,7

176,2

53

0,8

Test item

12,5

+

84,3

79,7

73

1,0

Test item

25,0

+

105,1

89,9

100

1,4

Test item

50,0

+

114,6

88,3

93

1,3

Test item

100,0

+

110,6

120,6

50

0,7

Conclusions:
Interpretation of results (migrated information):
negative

During the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y.
Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay.
Executive summary:

The study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in one experiment with and without metabolic activation. The test item was dissolved in DMSO and tested at the following concentrations: 3.1; 6.3; 12.5; 25.0; 50.0 and 100.0 µg/ml

Precipitation of the test item occurred at 50 µg/ml and above.

Strong toxic effects occurred in the pre-experiment at 50 µg/ml and above under precipitation of the test item. No relevant toxic effects were observed in the main experiment in the absence and, presence of metabolic activation up to the maximal concentration tested. Since precipitation occurred again at 50 µg/ml and above, the concentration range of the main experiment was limited by the solubility of the test item rather than by toxicity.

No substantial increase in mutant colony numbers was observed and the threshold of twice the colony numbers of the corresponding control was not reached or exceeded at any concentration. There was no relevant shift of the ratio of small versus large colonies as compared to the solvent control.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y. Therefore, the test item NS is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Two bacterial reverse mutation tests produced positive results, which were not supported by the results of a gene mutation assay in vitro in mammalian cells. The test material also did not show clastogenic effects in a mammalian chormosomal aberration test. Overall the test material is considered not mutagenic.


Justification for selection of genetic toxicity endpoint
In a weight of evidence approach the results of different studies were evaluated

Justification for classification or non-classification

no classification

positive results in a bacterial test alone do not justify a classification in the presence of additional negative results.