Pentyl 2-cyano-(3-(6-methoxybenzothiazol-2-ylimino)-2,3-dihydro-1H-isoindol-1-ylidene)acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was negative in in-vitro mammalian genetic toxicity tests in Chinese hamster lung fibroblasts (V79). However, it was positive for genotoxicity in bacterial reverse mutation test.

Based on the overall weight of evidence test substance, is expected not to have any genotoxic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Sep. 13, 2000 to Jan. 21, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 92/69, L 383 A, Annex B 14

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Plate incorporation test:
a: without metabolic activation:
50,160, 500, 1600 and 5000 µg/plate
b: with metabolic activation:
50,160, 500, 1600 and 5000 µg /plate

Preincubation test:
a: without metabolic activation:
16, 50, 160, 500, 1600 and 5000 µg/plate
b: with metabolic activation:
16, 50, 160, 500, 1600 and 5000 µg /plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthrecene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 to 30 min at approx. 30 °C
- Exposure duration: 48 h at approx. 37 °C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Microscopic thinning of the bacterial lawn and at least halving of the number of spontaneously occurring mutants compared to the corresponding solvent control value

Evaluation criteria:

The test item was considered positive if (a) at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
(b) a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system

Statistics:

According to the OECD guideline 471, a statistical analysis of the data was not mandatory

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at cytotoxic concentrations without dose-dependency

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Visible precipitation of the test compound on the plates was observed at 500 µg/plate and above.
- Other confounding effects: Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 µg /plate and lower concentrations in the plate incorporation test.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity: In the plate incorporation test toxicity was observed in a dose range of 1600 µg/plate and above in the absence and in the presence of metabolic activation. In the preincubation test toxicity was not observed either with or without metabolic activation.

Sterility checks and control plates:

Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

Conclusions:

Under the test conditions, the test substance is considered to be mutagenic in Salmonella typhimurium strain TA 98 in the presence of exogenous metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 870.5100 and EU method B.14. in compliance with GLP.

 

Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 were used in the mutagenicity assay. Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. For both studies, the test substance was suspended in DMSO, and each bacterial strain was exposed to 5 dose levels, in the preincubation test to 6 dose levels. Visible precipitation of the test compound on the plates was observed at 500 µg/plate and above.

Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 µg /plate and lower concentrations in the plate incorporation test.

 

The concentrations for the plate incorporation test were 50, 160,500, 1600 and 5000 µg/plate. Because of toxicity in the plate incorporation test dose levels from 16 to 5000 µg/plate were chosen for the preincubation test. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

 

In the plate incorporation test toxicity was observed in a dose range of 1600 µg/plate and above in the absence and in the presence of metabolic activation. In the preincubation test toxicity was not observed either with or without metabolic activation.

 

In the presence of metabolic activation, treatment of the bacterial strains with test substance resulted in relevant and dose-dependent increases in the number of revertant colonies with the strain TA 98.

 

Under the test conditions, the test substance is considered to be mutagenic in Salmonella typhimurium strain TA 98 in the presence of exogenous metabolic activation.

Reference 2

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 Oct. 09, 2000 to Mar. 15, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Cell culture medium: MEM (minimal essential medium) with Hankssalts and 25 mM Hepes-buffer
pH values and osmolality of the treatment media: Determined before treatment

Cytokinesis block (if used):

Colcemide was added to each culture 2 hours before sampling

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First experiment with 3 h treatment time:
without S9-mix: 500, 1000, 2500 and 3000 µg/mL
with S9-mix: 500, 1000, 2500 and 3000 µg/mL

Second experiment with 20 h treatment time:
without S9-mix: 50, 100, 250, 500, 750, 1000 and 2500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation )

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation )

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h (Experiment 1, with and without S9-mix), 20 h (Experiment 2 without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemide (approx. 0.05 µg/mL /culture medium)
STAIN (for cytogenetic assays): 2 % (w/v) orcein solution

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 1000 cells of each cell culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

The test substance is classified as clastogenic
(a) If it induces a statistically significant increase in the number of phases with aberrations (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
(b) If there is a concentration-related increase in the number of phases with aberrations (Without gaps).

The test substance is classified as non-clastogenic if the tests are negative both with and without metabolic activation.

Statistics:

-The Biometry of the results was performed with a one-sided Fisher - Exact test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects


RANGE-FINDING/SCREENING STUDIES:
Evaluation of the solubility of the suspension in cell culture medium showed that 4466 µg/mL was the highest practicable concentration and produced precipitate. This concentration corresponds to 10 mM, which is the highest dose level tolerated by the test system.

Preliminary toxicity study: Was carried out using a maximum concentration of 4466 µg/mL and a range of lower dose levels down to 50 µg/mL.
Precipitation of the test substance was observed after all treatment times in a dose range 50- 3000 µg/mL.


COMPARISON WITH HISTORICAL CONTROL DATA: Yes

The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

In the first and in the second main experiment the mitotic index was reduced (indication of toxicity) after treatment with the highest dose levels in the absence and the presence of S9-mix.

No relevant reproducible enhancement of metaphases with aberrations outside the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.

There was only an enhancement in the number of aberrations and in the number of phases excl. gaps at the 3 h treatment time with the high toxic dose level of 3000 µg/mL with S9-mix.

These data were found statistically significant enhanced in the Fisher's exact-test. However no dose-dependency was observed. These results indicate a possible mutagenic potential of the compound at best at a high toxic dose level.

Therefore in compliance with the criteria for a positive response, these results should not be used for the definitive ranking of this substance as a mutagenic compound.

Conclusions:

Under the test conditions, the test substance was not clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

Executive summary:

A study was conducted to investigate the potential of test substance to induce chromosome aberrations in V 79 cells of the Chinese hamster lung in vitro according to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10. in compliance with GLP.

Two experiments with duplicate cultures were used for each concentration. The test substance was suspended in DMSO and tested at the following concentrations:

First experiment with 3 h treatment time:

without S9-mix: 500,1000, 2500 and 3000 µg/mL

with S9-mix: 500, 1000, 2500 and 3000 µg/mL

Second experiment with 20 h treatment time:

without S9-mix: 50, 100, 250, 500, 750, 1000 and 2500 µg/mL

 

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. In the presence and absence of S9-mix an indication of cytotoxicity as reduction of mitotic index was observed.

Precipitation of the test compound was observed after all treatment times in a dose range of 50 to 3000 µg/mL

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix. There was only an enhancement in the number of aberrations and in the number of phases excl. gaps at the 3 h treatment time with the high toxic dose level of 3000 µg/mL with S9-mix.

These data were found statistically significant enhanced in the Fisher's exact-test. Beside this no dose-dependency was observed.

Therefore in compliance with the criteria for a positive response, these results should not be used for the definitive ranking of this substance as a mutagenic compound.

Test substance did not induce chromosome aberrations in V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation.

Under the test conditions, the test substancewas not clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Nov. 20, 2000 to Mar. 15, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, L133, pp. 61 - 63, March 1987

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HRPT locus (V79 Chinese hamster cells)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Cell culture medium: MEM (minimal essential medium) with Hankssalts and 25 mM Hepes-buffer
pH values and osmolality of the treatment media: Determined before treatment

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Main mutation experiment (First):
- without S9-mix: 31.25, 62.5, 125, 250, 500, 1000, 2000 and 3000 µg/mL
- with S9-mix: 62.5, 125, 250, 500, 1000, 2000, 3000 and 4000 µg/mL
Main mutation experiment (Second):
-without S9-mix: 31.25, 62.5, 125, 250, 500, 1000, 2000, 3000, 4000 and 4466* µg/mL
-with S9-mix: 31.25, 62.5, 125, 250, 500, 1000, 2000, 3000, 4000 and 4466* µg/mL
* = 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(without metabolic activation )

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(with metabolic activation )

Positive control substance:

9,10-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Seeded in each well of a microtiter plate

DURATION (Preliminary toxicity)
- Preincubation period: 12 h (overnight)
- Exposure duration: 4 h
- Incubation period: 4-5 d
- Fixation and staining: Crystal violet
NUMBER OF REPLICATIONS: 6 (For each concentration at least 6 wells)

DURATION (Main experiment)
- Preincubation period: 24 h
- Exposure duration: 4 h (Medium+ test substance+ buffer/S9-mix)
- Incubation period: 4-5 d
- Fixation and staining: Crystal violet

Evaluation criteria:

The test item was considered positive if
(a) It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
(b) There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
(c) Survival of the responding dose group is at least 30 %.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

- The biometry of the results for the test compound is performed off-line with the Mann-Whitney- U-Test

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects


RANGE-FINDING/SCREENING STUDIES:
Evaluation of the solubility of the suspension in cell culture medium showed that 4466 µg/mL was the highest practicable concentration and produced precipitate. This concentration corresponds to 10 mM, which is the highest dose level tolerated by the test system.

Preliminary toxicity study: Was carried out using a maximum concentration of 4466 µg/mL and a range of lower dose levels down to 50 µg/mL.

Based on the preliminary study results, 3000 µg/mL in the absence of S9-mix and 4000 µg/mL in the presence of S9-mix were selected as the maximum dose levels for the first main mutation experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

 

Mutation results:

First main experiment: The concentration 500 µg/mL of the test compound a statistically significant enhancement of the mutation rate over the range of the solvent controls was induced with the concentration 500 µg/mL of the test substance in the presence of metabolic activation.

 

Second main experiment: A statistically significant enhancement of the mutation rate over the range of the solvent controls was induced with the concentrations 1000 and 4000 µg/mL of the test substance without metabolic activation and with the concentrations 1000 and 4466 µg/mL of the test substance with metabolic activation.

These enhancements were neither dose-dependent nor reproducible and therefore of no biological relevance.

Conclusions:

Under the test conditions, the test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of test substance to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro according to OECD Guideline 476, EPA OPPTS 870.5300 and EEC Directive 87/302, L133 in compliance with GLP.

 

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The test substance was suspended in DMSO and tested at the following concentrations:

Main mutation experiment (First):

- without S9-mix: 31.25, 62.5, 125, 250, 500, 1000, 2000 and 3000 µg /mL

- with S9-mix: 62.5, 125, 250,500, 1000, 2000, 3000 and 4000 µg/mL

Main mutation experiment (Second):

-without S9-mix: 31.25, 62.5, 125,250, 500, 1000, 2000, 3000, 4000 and 4466* µg/mL

-with S9-mix: 31.25, 62.5, 125, 250, 500, 1000, 2000, 3000, 4000 and 4466* µg/mL

* = 10 mM

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed toxic effects with and without metabolic activation. Higher concentrations were not used because of the 10 mM limitation (OECD guideline). Up to the highest investigated dose statistical significant increases, were obtained in two independent experiments, but these increases were either reproducible or dose dependent and therefore of no biological relevance.

 

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

 

The test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system.

 

Under the test conditions, the test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Mutagenicity:

A study was conducted to determine the mutagenic potential of test substance according to OECD Guideline 471, EPA OPPTS 870.5100 and EU method B.14.

Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 were used in the mutagenicity assay. Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. For both studies, the test substance was suspended in DMSO, and each bacterial strain was exposed to 5 dose levels, in the preincubation test to 6 dose levels. Visible precipitation of the test compound on the plates was observed at 500 µg/plate and above. Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 µg /plate and lower concentrations in the plate incorporation test. The concentrations for the plate incorporation test were 50, 160,500, 1600 and 5000 µg/plate. Because of toxicity in the plate incorporation test dose levels from 16 to 5000 µg/plate were chosen for the preincubation test. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies. In the presence of metabolic activation, treatment of the bacterial strains with test substance resulted in relevant and dose-dependent increases in the number of revertant colonies with the strain TA 98. Under the test conditions, the test substance is considered to be mutagenic in Salmonella typhimurium strain TA 98 in the presence of exogenous metabolic activation. (Dr. Stammberger I and Braun K DI, 2001).

 

 

A study was performed to investigate the potential of test substance to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro according to OECD Guideline 476, EPA OPPTS 870.5300 and EEC Directive 87/302, L133.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix). The test substance was suspended in DMSO and tested at the following concentrations:

Main mutation experiment (First):

- without S9-mix: 31.25, 62.5, 125, 250, 500, 1000, 2000 and 3000 µg /mL

- with S9-mix: 62.5, 125, 250,500, 1000, 2000, 3000 and 4000 µg/mL

Main mutation experiment (Second):

-without S9-mix: 31.25, 62.5, 125,250, 500, 1000, 2000, 3000, 4000 and 4466* µg/mL

-with S9-mix: 31.25, 62.5, 125, 250, 500, 1000, 2000, 3000, 4000 and 4466* µg/mL

* = 10 mM

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed toxic effects with and without metabolic activation. Higher concentrations were not used because of the 10 mM limitation (OECD guideline). Up to the highest investigated dose statistical significant increases, were obtained in two independent experiments, but these increases were either reproducible or dose dependent and therefore of no biological relevance.Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. Test substance does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system. The test substance is considered to be non-mutagenic in the HPRT-test with V79 Chinese hamster cells, either in the presence or absence of metabolic activation (Dr. Stammberger I and Gräser H DI, 2001).

 

 

Clastogenicity:

A study was conducted to investigate the potential of test substance to induce chromosome aberrations in V 79 cells of the Chinese hamster lung in vitro according to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10.

Two experiments with duplicate cultures were used for each concentration. The test substance was suspended in DMSO and tested at the following concentrations:

First experiment with 3 h treatment time:

without S9-mix: 500,1000, 2500 and 3000 µg/mL

with S9-mix: 500, 1000, 2500 and 3000 µg/mL

Second experiment with 20 h treatment time:

without S9-mix: 50, 100, 250, 500, 750, 1000 and 2500 µg/mL

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. In the presence and absence of S9-mix an indication of cytotoxicity as reduction of mitotic index was observed.

Precipitation of the test compound was observed after all treatment times in a dose range of 50 to 3000 µg/mL. Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix. There was only an enhancement in the number of aberrations and in the number of phases excl. gaps at the 3 h treatment time with the high toxic dose level of 3000 µg/mL with S9-mix.

These data were found statistically significant enhanced in the Fisher's exact-test. Beside this no dose-dependency was observed. Therefore in compliance with the criteria for a positive response, these results should not be used for the definitive ranking of this substance as a mutagenic compound.

Test substance did not induce chromosome aberrations in V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation. The test substance was not clastogenic in the in vitro chromosome aberration assay with V79 Chinese hamster lung cells.

Justification for classification or non-classification

The test substance was negative in in-vitro mammalian genetic toxicity tests in Chinese hamster lung fibroblasts (V79). However, it was positive for genotoxicity in bacterial reverse mutation test.

Based on the overall weight of evidence test substance, is expected not to have any genotoxic potential. Therefore no classification is required for genotoxicity according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).