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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:
experimental study
Adequacy of study:
key study
Study period:
1992-01-20 - 1992-03-02
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
GLP compliance:
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Isophorone diamine of Hüls AG, purity 99.9 %, produced 25 Oct 1991; ID No. 3630/81 365


Target gene:
hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO (Chinese hamster ovary) K1 cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced Wistar rat liver S9
Test concentrations with justification for top dose:
0 - 2 mg/ml
Vehicle / solvent:
HO medium, dimethyl sulfoxide (DMSO)
Untreated negative controls:
= solvent/ vehicle control
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
positive control with metabolic activation: 3-Methylcholanthrene Migrated to IUCLID6: without metabolic activation
Details on test system and experimental conditions:
CHO cells that had been subcultured twice were seeded in HlO medium at a concentration of 2x10^5 cells/25 cm^2 flask. Two flasks were seeded for each concentration of the test substance. After incubation at 37 °c for approx. 20 hrs the medium was replaced by HO-medium containing the following concentrations of the test item:
0, 0.02, 0.03, 0.06, 0.12, 0.2, 0.3, 0.6, 1.2 and 2.0 mg/ml for HPRT #1 ± S9;
Exposure was for 4 hrs with and without S-9 mix. At the end of the treatment period the cells were harvested and cells of the same test substance concentration were pooled. Three dishes (60 mm) per culture were seeded with 200 cells/dish in H0 medium. After incubation for 6 days at 37 °c colonies were fixed with methanol, stained with Giemsa and counted. Cell survival is expressed as the cloning efficiency of treated cells relative to untreated cultures. The highest concentration to be used in the main study will be either the solubility limit of the test substance or the concentration resulting in a reduction of the cloning efficiency to approx. 80 %. In the absence of a solubility or toxicity limit, the highest concentration to be used will be 2.0 mg/ml.

To reduce the number of cells harboring spontaneous mutations of the HPRT locus, the cells for the main study were treated in HAT medium for approx. 1 week. Then the procedure described before was carried out using the following concentrations of the test item in the medium:
- HPRT #1 ±S9: 0, 0.02, 0.06, 0.2, 0.6, 2.0 mg/ml
- HPRT #2 ±S9: 0, 0.02, 0.06, 0.2, 0.6, 2.0 mg/ml
Positive controls demonstrating the sensitivity of the test system were ethyl methane sulfonate (EMS, without S9, 300 µg/ml) and 3-methylcholanthren (MCA, with S9, 10 µg/ml). Duplicate cultures were used for each treatment group. After an incubation time of 4 hrs the cells were washed 3 times with phosphate buffered saline (free of calcium and magnesium) and trypsinized. Duplicate cultures were pooled and eel ls were counted. The cells of each treatment groupwere divided into two subgroups. One subgroup was used to determine the cloning efficiency according to the procedure described before. The cells of the other subgroup were cultured in H1O medium for 7 days to allow for expression of the mutated phenotype. At the end of the expression period the cloning efficiency was determined. The selection of mutants was performed using five cultures for each treatment group. 1x10^6 cells were seeded per treatment group (5x10^5 cells/25 cm^2 flask, duplicate
cultures) in H6TG medium. After incubation for 6 days at 37 °C the colonies were fixed with methanol, stained with Giemsa and counted.
Evaluation criteria:
The validity of an experiment is determined by the following criteria:
- The cloning efficiency of the negative control at the end of the expression period must be at least 50 %.
- The mutation frequency of the negative control should not exceed the maximum spontaneous mutation frequency of approx. 20/10^6 cells
- The mutation frequencies of the positive controls must be significantly elevated.
- At least 4 dose levels must be examined, the highest dose being eather significantly toxic (cloning efficiency approx. 80 %) or being the solubility limit of the test substance. Concentrations higher than 2 mg/ml will not be tested.
- The results must be reproduced in a second independent experiment.

Criteria for evaluation of results:
- A test compound will be reported as being mutagenic in the HPRT test with CHO cells if it causes a statistically significant, dose related increase in mutant frequency at concentrations of the test substance resulting in greater than 50 % cell survival. In addition, a positive response is claimed only, if the mean mutant frequency in treated cultures reaches a value significantly above the maximum spontaneous mutant frequency (of approx. 20/10^6 viable cells)

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
none over test concentration range
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Remarks on result:
other: strain/cell type: CHO (Chinese hamster ovary) K1 cells

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

In this HPRT test with Chinese Hamster ovary (CHO) cells according to OECD TG 476 (1984), Isophorone diamine concentrations of
20 - 2,000 mg/l (+/- S9 mix from Aroclor 1254 induced rat livers) did not significantly increase the mutant frequency of treated cells. Cytotoxicity was not observed. It is concluded that Isophoron diamine, in the presence as well as in the absence of S9 mix, demonstrates no mutagenic potential in this in vitro mammalian cell assay at any of the concentrations tested.
Executive summary:

The test item Isophorone diamine was tested for its ability to induce forward mutation at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro both in the presence and the absence of exogenous metabolic activation(by Arochlor 1254 -induced rat liver S9). The substance was tested in concentrations of 20 mg/l to the solubility limit of 2000 mg/l with and without S9 mix. In this concentration range, Isophorone diamine did not induce any detectable cytotoxicity. Compared to the negative control treatment with Isophorone diamine in both assays (with and without metabolic activation) did not result in any reproducible statistically or biologically significant increase of the mutation frequency of the HPRT locus. It is concluded that Isophoron diamine, in the presence as well as in the absence of S9 mix, demonstrates no mutagenic potential in this in vitro mammalian cell assay.