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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:
experimental study
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
lot/batch No.of test material: 2494
Expiration date of the lot/batch: Jan 1st, 2000
Purity: > 99%

Method

Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Without metabolic activation: 1.0, 2.5, 5.0 and 10.0 µg/ml
With metabolic activation: 1.0, 5.0, 10.0 and 20.0 µg/ml
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
With metabolic activation
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours

NUMBER OF REPLICATIONS:2

DETERMINATION OF CYTOTOXICITY
- relative total growth and plating efficiency.

Evaluation criteria:
The substance is mutagenic, if the mutation fequency is three times higher than the spontaneous mutation frequency and is reproducible.
The test substance is mutagenic, if there is a reproducible concentration related increase in the mutation frequency.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
In the absense of S9-mix, statistically significant increases of the mutant frequency were observed at concentrations of 2.5 µg/ml and 15 µg/ml in the repeat experiment. These effects were not reproducible and were due to the low mutant frequency of the solvent control. Additionally, the enhancement of the 15.0 µg/mL concentration was found in a high cytotoxic range.
In the presence of S9-mix, statistically significant enhancements were observed at concentration of 10.0 µg/ml in the first experiment and at concentration of 5.0 µg/ml in the second experiment. These increases were neither dose dependent nor reproducible and were also caused by low mutant frequency of the solvent control.
All observed enhancements were within the historical control range of the used cell line and are therefore of no toxicological relevance.

Any other information on results incl. tables

First main experiment:

   Dose [µg/ml]  S9 - mix  Cytotoxicity: relative survival [%]  Mutation freuqueny
Negative control  0.0  -  110.0  8.3
Solvent control  0.0  -  100  19.7
Positive control (EMS)  1000  - 81.7   1011.4
Test item  1.0  -  102.9  25.5
   2.5  -  88.5  28.7
   5.0  - 77.4   9.2
   10.0  -  42.3  21.4
   15.0  -  0.9  not determined
             
Negative control  0.0  +  104.8  6.8
Solvent control   0.0  +  100.0  10.6
Positive control (DMBA)  7.7  +  64.3  260.9
Test item  1.0  +  99.1  22.9
   5.0  +  92.2  10.4
   10.0  +  93.5  25.6
   20.0  +  21.8  27.5
   25.0  +  1.1  not determined

Repeat experiment:

   Dose [µg/ml]  S9 - mix  Cytotoxicity: relative survival [%]  Mutation freuqueny
Negative control  0.0  -  114.8 21.4
Solvent control  0.0  -  100.0 7.1
Positive control (EMS)  1000  - 94.2 606.3
Test item  1.0  -  104.2 9.1
   2.5  -  91.9 22.4
   5.0  - 87.2 5.3
   10.0  - 35.0 13.3
   15.0  -  0.5 26.9
             
Negative control  0.0  +  105.3  6.3
Solvent control   0.0  +  100.0  5.0
Positive control (DMBA)  7.7  +  70.4  162.3
Test item  1.0  +  98.1  6.0
   5.0  +  97.7  22.0
   10.0  + 84.4  3.4
   20.0  +  22.6  6.4
   25.0  +  2.5  4.8

Applicant's summary and conclusion

Conclusions:
The test item is not mutagenic in HGPRT assay up to the concentrations accompanied by the cytotoxic effect of the test compound.
Executive summary:

The gene mutation potential of the test item was investigated in the HGPRT assay.

The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activaion.

The test substance was dissolived in ethanol and tested at the concentrations of 1.0, 2.5, 5.0 and 10.0 µg/ml without metabolic activation and at the concentrations of 1.0, 5.0, 10.0, and 20.0 µg/ml with metabolic activation. The concentration ranges were based on the cytotoxic effect of the test substance.

Up to the highest concentrations tested no reproducible increase in mutant colony numbers was obtained.

The test substance is not mutagenic in HGPRT assay.