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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.09. - 14.01.2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
RCC Cytotest Cell Research GmbH
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Appearance: yellow liquid
- Storage condition of test material: room temperature

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimum essential medium) supplemented with 10 % FCS (foetal calf serum).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital and ß-naphthoflavone induced liver of male Wistar rats.
Test concentrations with justification for top dose:
Cytotoxicity test
4hrs, with metabolic activation: 3.5, 7.0, 14.1, 28.1, 56.3, 112.5, 225.0, 450 µg/mL
4hrs, without metabolic activation: 3.5, 7.0, 14.1, 28.1, 56.3, 112.5, 225.0, 450 µg/mL
24hrs, without metabolic activation: 3.5, 7.0, 14.1, 28.1, 56.3, 112.5, 225.0, 450 µg/mL

Mutagenicity test
Experiment I (with and without metabolic activation):
4hrs: 14.1, 28.1, 56.3, 112.5, 225, 450 µg/mL (14.1 µg/ml was not continued)

Experiment II (without metabolic activation):
24 hrs: 14.1, 28.1, 56.3, 112.5, 225, 450 µg/mL (14.1 µg/ml was not continued)
Vehicle / solvent:
- Vehicle/solvent used: ethanol (0.5 % v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Ethylmethane sulfonate (0.3 mg/mL); with metabolic activation: 7,12-dimethylbenz(a)anthracene (2.5 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hrs (with and without metabolic activation) and 24 hrs (without metabolic activation).
- Expression time (cells in growth medium): approx. 7 days
- Selection time (if incubation with a selection agent): 7-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): after 15-16 days

SELECTION AGENT (mutation assays): 6-Thioguanin (11 µg/mL)

STAIN: 10 % methylene blue in 0.01 % KOH solution

NUMBER OF INDEPENDENT EXPERIMENTS: 2

NUMBER OF REPLICATIONS:
In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control.

EVALUATION: Counting of colonies formed after seeding of 3 - 5x10E5 cells in selective medium

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The test item will be considered to be mutagenic if:
1. A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.

2. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Assay evaluation criteria
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 10E6 cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 1996 - 1997.
b) the positive control substances must produce a significant increase in mutant colony frequencies.
c) the cloning efficiency (absolute value) of the negative and/or solvent controls must exceed 50 %.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item formed a fine emulsion in the medium at 56.3 µg/mL and above in the absence and presence of metabolic activation in the first experiment and at 14.1 µg/mL and above in the second experiment. The maximal applicable concentration was 450 µg/mL since higher concentrations led to inhomogeneous emulsions with phase separation in the medium.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects were observed up to the maximal concentration of the test item. The concentration range of the test item was limited by solubility rather than toxicity.

Any other information on results incl. tables

EXPERIMENTAL RESULT

concentration (µg/ml) S9 Mix relative cloning efficiency (%)* relative cell density (% of control) viability factor** mutant colonies / 106 cells relative cloning efficiency (%)* relative cell density (% of control) viability factor** mutant colonies / 106 cells
Experiment I, 4h treatment culture I culture II
Negative control - 100 100 0.82 7.8 100 100 0.75 8.9
Solvent control with Ethanol - 100 100 0.79 3.9 100 100 0.73 6.3
Positive control EMS 300 - 97.1 67.4 0.8 109.2 89.8 84.6 0.94 119.9
Test item 14.1 - 95.2 92.8 culture was not continued 87.5 97.6 culture was not continued
Test item 28.1 - 99.5 96.6 0.57 8.8 95 82.6 0.66 10.5
Test item 56.3 - 84.9 90.5 0.63 2.3 80.2 79.5 0.78 4.3
Test item 112.5 - 86.8 109.5 1.03 5.1 87.6 75.9 0.69 12.7
Test item 225 - 91.1 81 0.64 9.8 76.1 75.2 0.77 5.6
Test item 450 - 90.4 111 0.8 19.3 81.9 71.2 0.7 7
Negative control + 100 100 0.67 6.4 100 100 0.47 9
Solvent control with DMSO + 100 100 0.84 6.7 100 100 0.76 8.8
Solvent control with Ethanol + 100 100 0.79 1.3 100 100 0.65 4
Positive control DMBA 2.5 + 52 86.4 0.85 453.6 50.6 62.9 0.63 638.3
Test item 14.1 + 112.8 120.3 culture was not continued 123.4 69.3 culture was not continued
Test item 28.1 + 110 122.4 0.75 10.7 107.5 103.6 0.76 3.4
Test item 56.3 + 107.3 100.9 0.7 1.5 115.9 113.5 0.69 14.4
Test item 112.5 + 93.5 117.4 0.65 4 110.7 87.7 0.67 6.5
Test item 225 + 109.8 147.5 0.93 1.6 119.2 91.7 0.68 5.8
Test item 450 110.8 137.4 0.8 3.2 113.4 148.4 0.64 4.9
 Experiment II, 24 h treatment culture I culture II
Negative control - 100 100 0.72 7.5 100 100 0.68 5.2
Solvent control with Ethanol - 100 100 0.9 0.6 100 100 0.6 6.9
Positive control EMS 300 - 35.5 41.8 0.52 578 31.1 83.5 0.26 606.9
Test item 14.1 - 98.8 101.4 culture was not continued 99.5 101.1 culture was not continued
Test item 28.1 - 82.3 83 0.67 7 82.6 135.5 0.6 7.7
Test item 56.3 - 93.2 57.2 0.65 1.7 73.3 113.1 0.57 8.3
Test item 112.5 - 79 85.8 0.93 3.2 77.2 104.8 0.79 7.9
Test item 225 - 93.8 81.1 0.97 3.4 85.1 104 0.68 0
Test item 450 - 87 74.8 0.87 7.3 84.4 112.2 0.57 14.3

* mean number of cells per flask divided by mean number of cells per flask of the negative control multiplied with 100 (from cultures seeded with 500 cells each before treatment with the test substance)

** mean number of cells per flask divided by number of cells seeded (from cultures seeded with 500 cells each after expression)

Up to the highest investigated concentrations no relevant increase in mutant colony numbers was observed in both independent experiments. An isolated increase (19.3 colonies per 106cells) exceeding the threshold of three times the corresponding solvent control and the historical range of our negative and solvent controls was observed at 450.0 µg/ml in experiment I, culture I without metabolic activation. This increase was judged as biologically irrelevant since it was not reproduced in the parallel culture (culture II) under identical conditions. This effect is probably induced by inhomogeneity of the emulsion of the test item in medium at the maximal concentration. The factor of three times the corresponding solvent control was exceeded at almost all concentrations in the first culture of the second experiment. This effect however, is based upon the very low solvent control (0.6 colonies per 106cells) and represents statistical fluctuations at such low absolute numbers. The absolute numbers of colonies remained well within our range of historical negative and solvent controls and are below the corresponding negative control.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

In an HPRT study according to OECD test guideline 476 and in compliance with GLP principles, the test article’s potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. In order to determine the concentration range for the mutagenicity, a pre-test was performed with eight concentrations ranging from 3.5 to 450.0 µg/ml without metabolic activation (4 h and 24 h treatment) and with metabolic activation (4 h treatment). The maximal concentration was limited by the solubility of the test item. Concentrations above 450 µg/ml formed unstable emulsions leading to phase separation. No relevant toxic effects occurred in the pre-experiment up to the maximal concentration. Therefore, the following concentrations were evaluated in the main study: 28.1; 56.3; 112.5; 225.0; and 450.0 µg/ml. In the man test, the test item formed a fine emulsion in the medium at 56.3 µg/ml and above in the absence and presence of metabolic activation in the first experiment and at 14.1 µg/ml and above in the second experiment. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies. Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in both independent experiments. An isolated increase exceeding the threshold of three times the corresponding solvent control and the historical range of our negative and solvent controls was observed at 450.0 µg/ml in experiment I, culture I without metabolic activation. This increase was judged as biologically irrelevant since it was not reproduced in the parallel culture (culture II) under identical conditions and based on a very low solvent control value. In conclusion, the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.