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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 02 to August 30, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13

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
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Acid Blue 284 - Similar Substance 01
IUPAC Name:
Acid Blue 284 - Similar Substance 01
Test material form:
solid: particulate/powder

Method

Target gene:
Mutagenic effects are detected by the appearance of cells resistant to 6-TG
Species / strain
Species / strain / cell type:
other: CHINESE HAMSTER CELLS V79, clone 65/3
Details on mammalian cell type (if applicable):
Origin: Dr. D. Wild, Freiburg, Germany.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Cytotoxicity test
Range with metabolic activation:
0.24 to 500.0 ug/ml
Range without metabolic activation:
0.24 to 500.0 ug/ml
Mutagenicity test
Original experiment:
Range with metabolic activation:
18.52 to 500.0 ug/ml
Range without metabolic activation:
11.11 to 300.0 ug/ml
Confirmatory experiment:
Range with metabolic activation:
62.5 to 500.0 ug/ml
Range without metabolic activation:
37.5 to 300.0 ug/ml
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Nitrosodimethylamine
Remarks:
With metabolic activation
Untreated negative controls:
yes
Remarks:
vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethansulfonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
Preliminary cytotoxicity test
A cytotoxicity test was performed on V79 cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. For each concentration and the untreated controls, 2.5x10s V79 cells were seeded in 5 ml growth medium into a 25 cm2 tissue culture flask and incubated overnight. The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system. In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (DMSO) controls were tested. The highest concentration was determined in a preliminary solubility test. Lower concentrations were prepared by serial dilution by a factor of 0.5. The treatment was terminated by washing the cultures with phosphate buffered saline (PBS). Compound-induced cytotoxicity was estimated by cloning efficiency immediately after treatment. The cultures were counted and diluted so that 100 cells were seeded per 9.6 cm2 in 3 ml of growth medium. After seven to eight days of growth the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter (Artek Counter®, Fisher Scientific) or by the naked eye. The sensitivity of the colony counter was adjusted to detect clones of about twenty or more cells. The concentration to be selected as the highest for the mutagenicity assay was the one causing about 50-90% reduction of viable cells in comparison with the mean of the two negative controls or corresponds to the substance's solubility limit (precipitates in the culture).
Mutagenicity test
Depending on the toxicity of the test compound 2.5-5.0x10ʌ6 cells of passage 25 (original experiment) and passage 25 (confirmatory experiment) were plated in 30 ml growth medium into 175 cm2 flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone. In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen Ethylmethansulphonate (EMS) at a concentration of 0.3 ul/ml. In the part with metabolic activation the positive control was the promutagen N-Nitrosodimethylamine (DMN) at a concentration of 1.0 ul/ml. The treatment was terminated by washing the cell layer extensively with PBS. After washing, the cells were suspended by trypsinisation, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM), diluted with fresh growth medium and replated into flasks at 2x10ʌ6 cells. The cultures were incubated at 37°C for seven to eight days during which the cells could recover and divide to express the mutant phenotype. The cultures were subcultered after the second or third day transferring 2x10ʌ6 cells to a fresh flask to maintain exponential growth during the expression phase.
In parallel cytotoxicity of the compound was estimated from the cloning efficiency immediately after treatment. The counted cell suspension of each concentration level was further diluted so that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and incubated at 37 °C. The number of colonies which developed within seven to eight days in these cultures reflected the viability at the end of the treatment (survival values).
At the end of the expression period the cultures were trypsinised, pelleted, resuspended in fresh growth medium and counted with a haemocytometer or electronic coulter counter (Coulter Counter®, Model ZM). The cell suspension of each culture was diluted with fresh growth medium and an aliquot replated into four flasks (75 cm2 growth area) each containing 2x10ʌ6 cells for the mutant selection. The high-density cultures were subjected to the mutant selection procedure by supplementing the growth medium with 8 ug/ml 6-thioguanine (6-TG). Only cells mutated at the hprt locus could survive the 6-thioguanine treatment. The number of colonies formed in these flasks during the following days reflected the overall number of mutations induced by the treatment with the test substance or the mutagen (positive control). After seven to eight days incubation at 37 °C, the cultures were fixed and stained with Giemsa. The mutant clones were counted with the naked eye.
In parallel the viability at the end of the expression period was estimated from the cloning efficiency. The remaining cell suspensions from the various expression cultures were further diluted such that 100 cells were seeded per 9.6 cm2 in 2.5 ml of growth medium and were incubated at 37 °C. The number of colonies which developed within these low-density cultures reflected the viability at the end of the expression period (viability values).
Evaluation criteria:
Assay acceptance criteria
• The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.
• From each experiment, at least three concentrations of the test substance, one positive and one solvent control should be evaluated.
• The mutant frequency of the solvent controls (spontaneous mutant frequency) should not exceed 35xl0-6.
• The positive control should fulfil the criteria for a mutagenic substance.
• The highest concentration of the test substance applied in the mutagenicity test should either reduce the viable cells by about 50-90% or correspond to the test substance's solubility limit (precipitates in the culture). In case of non-toxic freely soluble compounds the highest tested concentration
will be 5 mg/ml. In special cases the highest concentration can be determined by the sponsor.
Assay evaluation criteria
All mutant frequencies are normalized to a virtual cloning efficiency of 100 % at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15 %, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the
result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.
Statistics:
Assessment of statistical significance of mutation frequency
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines.  

Results and discussion

Test results
Species / strain:
other: CHINESE HAMSTER CELLS V79, clone 65/3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Cytotoxicity test

A preliminary range finding test was run assessing cytotoxicity. The test substance was tested at concentrations up to 500.0 µg/ml. Higher concentrations could not be applied due to solubility limitations in the culture vehicle. In the part with metabolic activation, at the highest concentration of 500.0 µg/ml an acute growth inhibiting effect of 40.2 % could be seen. Without metabolic activation treatment with the test substance proved growth inhibiting by 99.98 % at the concentration of 500.0 µg/ml. The next lower concentration of 250.0 µg/ml revealed an acute inhibition of growth of 70.1 %. Accordingly, 500.0 µg/ml with and 300.0 µg/ml without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

Mutagenicity test with metabolic activation

The original experiment was performed at the following concentrations: 18.52, 55.56, 166.67 and 500.0 µg/ml. No toxicity was found at any concentration. In the confirmatory experiment the concentrations applied were 62.5, 125.0, 250.0 and 500.0 µg/ml. The highest concentration revealed a mean acute growth inhibition of 33.7%. N-Nitrosodimethylamine (DMN, 1.0 µl/ml) was used as positive control. 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 significant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

Mutagenicity test without metabolic activation

The original experiment was performed at the following concentrations: 11.11, 33.33, 100.0 and 300.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 82.0% and 19.5% respectively. In the confirmatory experiment the concentrations applied were 37.5, 75.0, 150.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3%. The mean growth inhibition after the expression period was 24.0%. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control. 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 significant increase of the mutant frequencies as determined by the screening with 6-TG.

Applicant's summary and conclusion

Conclusions:
Non mutagenic
Executive summary:

Method

The test substance was tested for its mutagenic effect by the gene mutation test with chines hamster cells V79, according to OECD Guideline 476.

The test system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-TG and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, an experiment is performed, in which the metabolic turnover of the test material is simulated in vitro by the addition of an activation mixture to the cell cultures containing rat-liver post mitochondrial supernatant (S9 fraction) and cofactors.

Observation

The original experiment was performed at the following concentrations: 11.11, 33.33, 100.0 and 300.0 µg/ml. The mean growth inhibition values found at the highest concentration after treatment and expression were 82.0 % and 19.5 % respectively. In the confirmatory experiment the concentrations applied were 37.5, 75.0, 150.0 and 300.0 µg/ml. The highest concentration revealed a mean acute growth inhibitory effect of 69.3 %. The mean growth inhibition after the expression period was 24.0 %. Ethylmethansulfonate (EMS, 0.3 µl/ml) was used as positive control.

Results

Non mutagenic