Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 6, 1995 to Jun. 27, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Remarks:
CIBA-GEIGY Limited, Genetic Toxicology
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white solid
- Storage condition of test material: Room temperature

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F10 medium supplemented with 10% pre-tested foetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin in tissue culture.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the original test:
with S9 mix: 7.41, 22.22, 66.67 and 200.00 µg/ml
without S9 mix: 0.19, 0.56, 1.67 and 5.0 µg/ml

Concentration range in the confirmatory test:
with S9 mix: 7.50, 15.0, 30.0 and 60.0 µg/ml
without S9 mix: 0.75, 1.50, 3:00 and 6.00 µg/ml

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with S9 mix
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours (with S9 mix), 21 hours (without S9 mix)
- Expression time (cells in growth medium): 7 to 8 days
- Selection time (if incubation with a selection agent): 7 to 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2.5-5.0 x 10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency


Evaluation criteria:
The test substance will be considered to be mutagenic if:
- The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20;
-There is a significant dose-relationship as indicated by the linear trend analysis; and
-The effects described above are reproducible.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
200.0 µg/ml with metabolic activation and 5.0 µg/ml without metabolic activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test with and without metabolic activation 12 concentrations of the test substance were tested. The concentrations selected ranged from 0.195 to 400.00 µg/ml and separated by 2-fold intervals. In the part with metabolic activation, at the highest concentration of 400.0 µg/ml, an acute growth inhibiting effect of 91.0% could be seen, while the next lower concentration of 200.0 µg/ml inhibited 76.0%. Without metabolic activation treatment with the test substance proved fully growth inhibiting down to the concentration of 25.0 µg/ml. The next two lower concentrations of 12.5 and 6.25 µg/ml revealed an acute inhibition of growth of 99.3 and 96.7%, respectively. Accordingly, four concentrations were selected for the original experiment ranging from 7.41 to 200.0 µg/ml and from 0.19 to 5.0 µg/ml in the presence and absence of metabolic activation, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity test with metabolic activation (Table 1):

The original experiment was performed at the following concentrations: 7.41, 22.22, 66.67 and 200.00 µg/ml. The highest concentration proved toxic after treatment. The mean growth inhibiting values found at the next lower concentration after treatment and expression were 98.9%* and 57.4%* respectively. In the confirmatory experiment the concentrations applied were 7.50, 15.0, 30.0 and 60.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 99.7%*. The mean growth inhibitory effect after the expression period was 65.5%*. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation (Table 2):

The original experiment was performed at the following concentrations: 0.19, 0.56, 1.67 and 5.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 95.0%* and 31.9%* respectively. In the confirmatory experiment the concentrations applied were 0.75, 1.50, 3.00 and 6.00 µg/ml. The highest concentration proved toxic. The next lower concentration revealed a mean acute growth inhibitory effect of 70.5%*. The mean growth inhibition after the expression period was 11.6%*. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

*Mean of two independent cultures

Table 1: With metabolic activation

Treatment

Mean mutant frequency

(x 10E-6)

Mean mutant factor

Significance (P)

Original Experiment

Vehicle control

13.17

-

-

Positive control

172.14

13.07

P<0.001

TS, 200.00 µg/ml

T

T

T

TS, 66.67 µg/ml

19.60

1.49

0.001<p<0.002

TS, 22.22 µg/ml

14.35

1.09

Ns

TS, 7.41 µg/ml

12.46

0.95

Ns

Confirmatory Experiment

Vehicle control

7.26

-

-

Positive control

115.86

15.96

P<0.001

TS, 60.00 µg/ml

12.11

1.67

0.02<p<0.05

TS, 30.00 µg/ml

9.92

1.37

Ns

TS, 15.00 µg/ml

7.35

1.01

Ns

TS, 7.50 µg/ml

7.88

1.09

Ns

 

Table 2: Without metabolic activation

Treatment

Mean mutant frequency

(x 10E-6)

Mean mutant factor

Significance (P)

Original Experiment

Vehicle control

13.78

-

-

Positive control

2203.81

159.90

P<0.001

TS, 5.00 µg/ml

19.56

1.42

P<0.001

TS, 1.67 µg/ml

13.52

0.98

Ns

TS, 0.56 µg/ml

13.35

0.97

Ns

TS, 0.18 µg/ml

11.57

0.84

Ns

Confirmatory Experiment

Vehicle control

7.48

-

-

Positive control

1147.38

153.46

P<0.001

TS, 6.00 µg/ml

T

T

T

TS, 3.00 µg/ml

5.78

0.77

Ns

TS, 1.50 µg/ml

2.51

0.34

Ns

TS, 0.75 µg/ml

5.35

0.72

Ns

TS: test substance; T: toxic

Applicant's summary and conclusion

Conclusions:
Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test substance and its metabolites did not show any mutagenic activity.
Executive summary:

The test substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The test substance was dissolved in ethanol. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment. In a preliminary cytotoxicity test the concentrations for the main test were determined at 200.0 µg/ml and 5.0 µg/ml, with and without metabolic activation, respectively. The original experiment with S9-mix was performed at the following concentrations: 7.41, 22.22, 66.67 and 200.00 µg/ml. The highest concentration proved toxic after treatment. The mean growth inhibiting values found at the next lower concentration after treatment and expression were 98.9% and 57.4%, respectively. In the confirmatory experiment the concentrations applied were 7.50, 15.0, 30.0 and 60.0 µg/ml. The original experiment without S9-mix was performed at the following concentrations: 0.19, 0.56, 1.67 and 5.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 95.0% and 31.9%, respectively. In the confirmatory experiment the concentrations applied were 0.75, 1.50, 3.00 and 6.00 µg/ml. The highest concentration proved toxic. In both experiments with and without S9-mix, comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG). Therefore, based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test article and its metabolites did not show any mutagenic activity in this forward mutation system.