Reaction mass of 5-[(Disubstituted-dihydrocarbopolycyclyl)diazenyl]-6-hydroxy-4-methyl-2-substituted-1-propyl-1,2-dihydropyridine-3-carbonitrile and 1-Butyl-5-[(disubstituted-dihydrocarbopolycyclyl)diazenyl]-6-hydroxy-4-methyl-2-substituted-1,2-dihydropyridine-3-carbonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 41048 is considered to be neither mutagenic nor clastogenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

exception in strain TA 1537 with metabolic activation in experiment I at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Discussion of Results:

- 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, with the exception in strain TA 1537 with metabolic activation in experiment I, reduced background growth was observed at 5000 µg/plate.

- No toxic effects, evident as a reduction in the number of revendants, occurred in the test groups with and without metabolic activation with the exception in strain TA 1537 with metabolic activation in experiment I, a reduction in the number of revendants was observed at 5000 µg/plate.

- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41039/A 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. They showed a distinct increase of induced revertant colonies.

Conclusions:

FAT 41039/A, a structural analogue of FAT 41048, did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used in this bacterial reverse mutation assay.

Executive summary:

FAT 41039/A, a structural analogue of FAT 41048, was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

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, with the exception in strain TA 1537 with metabolic activation in experiment I, reduced background growth was observed at 5000 µg/plate. No toxic effects, evident as a reduction in the number of revendants, occurred in the test groups with and without metabolic activation with the exception in strain TA 1537 with metabolic activation in experiment I, a reduction in the number of revendants was observed at 5000 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41039/A 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. They 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 1055 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 1055 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 40 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 40 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

- In the range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 8.2 and 1055 µg/mL were applied. No clear toxic effects were observed after treatment up to the highest applied test item concentration.

- In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 8.2 µg/mL and above in the absence and the presence of S9 mix. In both cytogenetic experiments no relevant influence of the test item on the pH value or osmolality was observed (e.g. Exp. I: solvent control 374 mOsm, pH 7.5 versus 380 mOsm and pH 7.4 at 40 µg/mL).

- In all experimental parts test item precipitation in culture medium was observed 4 hrs after start of treatment with 10µg/mL and above, except in Experiment II at preparation interval 28 hrs, in the presence of S9 mix, where precipitation occurred at 5 µg/mL and above.

- In this study, neither reduced mitotic indices nor reduced cell numbers of below 50 % of control was observed up to the highest applied test item concentrations being far in the range of test item precipitation.

- In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. A single statistical significant (p < 0.05) increase was observed in Experiment I, in the presence of S9 mix, after 4 hrs treatment with 5 µg/mL Although this increase of 3.0 % aberrant cells was statistically significant compared to the low response (0.5 % aberrant cells, exclusive gaps) in the solvent control data, the response is within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significance has to be regarded as being biologically irrelevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.2- 3.5%) as compared to the rates of the solvent controls (1.3-2.3%).

- In both experiments, EMS (300 and 400µg/mL, respectively) and CPA (1.4 and 2.0µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

FAT 41039/A, a structural analgue of FAT 41048, did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations.

Executive summary:

FAT 41039/A, a structural analogue of FAT 41048, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hrs with and without metabolic activation. In Experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (Exp. I and II) and 28 hrs (Exp. II) after start of treatment with the test item. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

- In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 8.2 and 1055 µg/mL were applied. No clear toxic effects were observed after treatment up to the highest applied test item concentration.

- In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 8.2 µg/mL and above in the absence and the presence of S9 mix. In both cytogenetic experiments no relevant influence of the test item on the pH value or osmolality was observed (e.g. Exp. I: solvent control 374 mOsm, pH 7.5 versus 380 mOsm and pH 7.4 at 40 µg/mL).

- In all experimental parts test item precipitation in culture medium was observed 4 hrs after start of treatment with 10 µg/mL and above, except in Experiment II at preparation interval 28 hrs, in the presence of S9 mix, where precipitation occurred at 5 µg/mL and above.

- In this study, neither reduced mitotic indices nor reduced cell numbers of below 50 % of control was observed up to the highest applied test item concentrations being far in the range of test item precipitation.

- In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. A single statistical significant (p < 0.05) increase was observed in Experiment I, in the presence of S9 mix, after 4 hrs treatment with 5 µg/mL Although this increase of 3.0 % aberrant cells was statistically significant compared to the low response (0.5 % aberrant cells, exclusive gaps) in the solvent control data, the response is within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significance has to be regarded as being biologically irrelevant.

- In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.2- 3.5%) as compared to the rates of the solvent controls (1.3-2.3%).

- In both experiments, EMS (300 and 400 µg/mL, respectively) and CPA (1.4 and 2.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item FAT 41039/A did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations.

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

BACTERIAL REVERSE MUTATION ASSAY:

FAT 41039/A, a structural analogue to FAT 41048, was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000µg/plate with and without metabolic activation in both independent experiments, with the exception in strain TA 1537 with metabolic activation in experiment I, reduced background growth was observed at 5000µg/plate. No toxic effects, evident as a reduction in the number of revendants, occurred in the test groups with and without metabolic activation with the exception in strain TA 1537 with metabolic activation in experiment I, a reduction in the number of revendants was observed at 5000µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41039/A 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. They 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.

IN VITRO CHROMOSOMAL ABERRATION ASSAY

FAT 41039/A, a structural analogue to FAT 41048, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hrs with and without metabolic activation. In Experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (Exp. I and II) and 28 hrs (Exp. II) after start of treatment with the test item. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

- In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as an indicator for cytotoxicity. Concentrations between 8.2 and 1055µg/mL were applied. No clear toxic effects were observed after treatment up to the highest applied test item concentration.

- In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 8.2µg/mL and above in the absence and the presence of S9 mix. In both cytogenetic experiments no relevant influence of the test item on the pH value or osmolality was observed (e.g. Exp. I: solvent control 374 mOsm, pH 7.5 versus 380 mOsm and pH 7.4 at 40µg/mL).

- In all experimental parts test item precipitation in culture medium was observed 4 hrs after start of treatment with 10µg/mL and above, except in Experiment II at preparation interval 28 hrs, in the presence of S9 mix, where precipitation occurred at 5µg/mL and above.

- In this study, neither reduced mitotic indices nor reduced cell numbers of below 50 % of control was observed up to the highest applied test item concentrations being far in the range of test item precipitation.

- In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.0 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. A single statistical significant (p < 0.05) increase was observed in Experiment I, in the presence of S9 mix, after 4 hrs treatment with 5µg/mL Although this increase of 3.0 % aberrant cells was statistically significant compared to the low response (0.5 % aberrant cells, exclusive gaps) in the solvent control data, the response is within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significance has to be regarded as being biologically irrelevant.

- In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.2- 3.5%) as compared to the rates of the solvent controls (1.3-2.3%).

- In both experiments, EMS (300 and 400µg/mL, respectively) and CPA (1.4 and 2.0µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item FAT 41039/A did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations.

Justification for classification or non-classification

The substance was considered to be neither mutagenic nor clastogenic, hence does not warrant classification for mutagenicity according to Regulation (EC) No. 1272/2008 (CLP) criteria.