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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
No genotoxicity of the test substance found.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from November 15, 1996 to March 26, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guidance test with GLP compliance
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: EEC Directive 92/69, L 383 A, Annexe V, B 10, dated December 29, 1992.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
V79 cells of the Chinese Hamster in vitro
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
s9 mix
Test concentrations with justification for top dose:
Without S9 mix:
-18h (Experiment I): 3.0, 5.0, 10.0, 30.0, 100.0, 500.0 μg/ml
-18h (Experiment II): 3.0, 5.0, 10.0, 30.0, 100.0, 300.0 μg/ml
-28h (Experiment I): 10.0, 30.0, 100.0, 500.0 μg/ml
-28h (Experiment II): 10.0, 30.0, 100.0, 300.0 μg/mll

with S9 mix:
-18h (Experiment I): 3.0, 5.0, 10.0, 30.0, 100.0, 500.0 μg/ml
-18h (Experiment II): 3.0, 5.0, 10.0, 30.0, 100.0, 500.0 μg/ml
-28h (Experiment I): 10.0, 30.0, 100.0, 500.0 μg/ml
-28h (Experiment II): 10.0, 30.0, 100.0, 500.0 μg/ml
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent solvent controls (MEM)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Ceil Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of C C R allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because
of standardized characteristics of the cells.
Thawed stock cultures were propagated at 37° C in 80 cm plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 105 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS, Boehringer Mannheim, D-68261 Mannheim). The cells were subcultured twice weekly. The cell cultures were incubated at 37 ° C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).
Evaluation criteria:
A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
Statistical significance was confirmed by means of the Fischer's exact test. However, both biological and statistical significance should be considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 45'176/A is considered to be non-mutagenic in this chromosome aberration test.
Executive summary:

The test article FAT 45'176/A, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test (5000 ug/ml) was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 3 and 5000 ug/ml (with and without S9 mix) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix toxic effects indicated by reduced cell numbers of about 50 % of control and below were observed after treatment with 100 ug/ml and above. In the presence of S9 mix reduced cell numbers were observed after treatment with 300 ug/ml and above. Precipitation of the test article was observed 4 h after start of treatment at concentrations of 30 ug/ml and above.

In experiment I in the absence of S9 mix and in both experiments in the presence of S9 mix, test article concentrations in a range of 3 - 500 ug/ml were applied for the investigation of the potential to induce cytogenetic damage. In absence of S9 mix in experiment II, the applied concen-tration range was 3 - 300 ug/ml. In the cytogenetic experiments, precipitation of the test article was observed 4 h after start of treatment at concentrations of 30 ug/ml and above.

In the absence and the presence of S9 mix, in both experiments no substantial reduction of the mitotic indices occurred at the evaluated experimental points, neither at concentrations with visible precipitation nor at concentrations without precipitation, except in experiment I in the presence of S9 mix at interval 18 h in cultures after treatment with 500 ug/ml.

In both experiments, in the absence and the presence of S9 mix, no biologically relevant increases in cells carrying structural chromosome aberrations were observed. Unreproduced significant increases in experiment II at interval 28 h in the absence of S9 mix were considered as biologically irrelevant.

In both experiments, no relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 45'176/A is considered to be non-mutagenic in this chromosome aberration test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Three in vitro tests were performed of the test substance, including Ames test, chromosome aberration test and gene mutation in vitro.

The Ames study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strains WP2 and WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicates. The test item was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate.

No toxic effects evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

The test article FAT 45176/A, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

In both experiments, in the absence and the presence of S9 mix, no biologically relevant increases in cells carrying structural chromosome aberrations were observed. Unreproduced significant increases in experiment II at interval 28 h in the absence of S9 mix were considered as biologically irrelevant.

In both experiments, no relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p<0.05) in cells with structural chromosome aberrations.

Under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 45176/A is considered to be non-mutagenic in this chromosome aberration test.

The gene mutation test in vitro was conducted to assess the potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiments. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay.

The test item was investigated at the following concentrations:

Experiment I without metabolic activation:

5, 10, 20, 40, 60, 80, 100 and 125 µg/mL and with metabolic activation:

10, 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL

Experiment II without metabolic activation:

25, 50, 100, 200, 250, 300, 350, 400, 450 and 500 µg/mL and with metabolic activation:

80, 120, 160, 180, 200, 220, 240, 260, 280 and 300 µg/mL

No precipitation of the test item was noted in the experiments.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 18.6% for the highest concentration (125 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 250 µg/mL with a relative growth of 14.2%. In experiment II without metabolic activation the relative growth was 19.2% for the highest concentration (500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 300 µg/mL with a relative growth of 19.8%.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Thus, it can be concluded that the test substance caused no genetic toxicity under the experiment condition of this in vitro mammalian cell gene mutation assay.

As a result, the substance FAT 45176/A was found negative in three independent in vitro mutagenicity assays and thus there is no further need to investigate the substance for mutagenicity in vivo.


Justification for selection of genetic toxicity endpoint
OECD guidance test with GLP compliance

Justification for classification or non-classification

Based on the available results of three in vitro tests, the test substance is not classified as per EU CLP (Regulation (EC) No 1272/2008) regulation for mutagenicity.