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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic activity of the test substance was investigated in one bacterial reverse mutation assay (Ames test; tester strains used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2uvrA), and in one in vitro chromosome aberration study in Chinese Hamster V79 cells. Negative results were obtained in both tests.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July -- Aug 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted 21July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat S9 mix
Test concentrations with justification for top dose:
plate incorporation and preincubation test:
50, 160, 500, 1600 and 5000 µg/plate (according to guideline)

repeated preincubation test (based on observed cytotoxicity):
with metabolic activation
160, 300, 500, 750, 1000 and 1600 µg/plate (TA 1535)
500, 800, 1200, 1600, 2500 and 5000 µg/plate (TA 1537)

without metabolic activation
5, 16, 50, 160, 500, 1600 and 5000 µg/plate (all Sallmonella strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility/homogeneity
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) and preincubation; i
- Cell density at seeding (if applicable):
0.1 mL of overnight nutrient broth culture of the bacterial tester strain

DURATION
- Preincubation period: 20 - 30 min
- Exposure duration: approx. 48 h

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxixity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control

Rationale for test conditions:
based on observed cytotoxicity
Evaluation criteria:
Criteria for a positive response:
A test compound is classified as mutagenic if it has either of the following effects:
a. it produced 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. it induces 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.

The assays is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls innuce increases in the mutation frequency which are significant and within the laboratory's normal range.
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
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
Key result
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
Key result
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
Key result
Species / strain:
E. coli WP2 uvr A
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:
- 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.

- Solubility and toxicity
The test item was dissolved in DMSO and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 2500, 1600, 1200, 1000, 800, 750, 500, 300, 160, 50, 16 and 5 µg/plate were used in the different mutagenic experiments. Visible precipitation of the test substance on the plates was observed at 160 µg/plate and above. Because of the strong precipitation of the test item the bacterial lawn could not be evaluated at the dose level of 5000 µg/plate in the plate incorporation test. In the palte incorporation test toxicity was not observed either in the presence or absence of metabolic activation.
In the preincubation test the test item proved to be toxic to all Salmonella strains at dose levels of 1600 µg/plate and above and to E. coli WP2uvrA at a dose level of 5000 µg/plate in the presence of metabolic activation. In the absence of metabolic activation toxicity was observed with all Salmonella strains at all concentrations (50 µg/plate and above) and with E. coli WP2uvrA at concentrations of 160 µg/plate and above.
In the repetition of the preincubation test the test compound proved to be toxic to all Salmonella strains at dose levels of 16 µg/plate and above without metabolic activation. In the presence of metabolic activation toxicity was observed at a concentration of 1600 µg/plöate with the tester strain TA 1535 and at concentrations of 1600 µg/plate and above with tester strain TA 1537. Thinning of bacterial lawns and/or a reduction in the number of colonies was observed at these dose levels.

- Mutagenicity
Thr test item did not cause a significant increase in the number of revertant colonies at any dose level with any tester strains either in the absence or in the presence of S9-mix in each mutationtest. No dose-dependent effect was obtained. In the first preincubation test a dose/control ration of 1.8 - 2.0 was obtained with the strains TA 1535 and TA 1537 in the presnece of metabolic activation with individual doses. Although mean revertant values did not differ from the historical solvent control data range and no dose-dependency was observed, the preincubation test with these strains was repeated using a narrower dose range for clarification. No significant or dose-dependent increase in the number of revertants was observed in the repeat test confirming that the test substance is not mutagenic.
All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficicay of the exogeneous metabolic activation system were deminstrated.
Conclusions:
The results of this Ames test lead to the conclusion that the test substance is not mutagenic either in the absence or in the presence of an exogeneous metabolising system.
Executive summary:

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella thyphimurium and with E. coli WP2uvrA.

Two independent mutagenicity studies were conducted (one plate incorporation and one preincubation test), each in the absence and in the presence of a metabolising system derived from a rat liver homogenate. Additionally a repetition of the preincubation test was performed with the strains TA 1535 and TA 1537 in the presence of S9 -mix due to equivocal results and with all Salmonella strains in the absence of S9 -mix due to high toxicity.

For all studies, the test substance was dissolved in DMSO and each bacterial strain exposed to 5 dose levels in the plate incorporation and in the preincubation test. In the repetition of the preincubation test the tester strains were exposed to 6 dose levels with metabolic activation and to 7 dose levels without metabolic activation. Concentrations used for plate incorporation and preincubation test were 50, 160, 500, 1600 and 5000 µg/plate. For the repetition of the preincubation test in the absence of metabolic activation dose levels from 5 to 5000 µg/plate were chosen, due to high toxicity in the first preincubation test. In the presence of metabolic activation dose ranges were variable across the bacterial strains because of equivocal results. For tester strain TA 1535 concentrations of 160, 300, 500, 750, 1000 and 1600 µg/plate and for strain TA 1537 concentrations of 500, 800, 1200, 1600, 2500 and 5000 µg/plate were chosen.

Visible precipitation of the test substance on the plates was obserced at 160 µg/plate and above. Because of the strong precipitation of the test item the bactrial lawn could not be evaluated at the dose level of 5000 µg/plate in the plate incorporation test.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All positive controls showed the expected increase in the number of revertant colonies.

Toxicity:

In the plate incorporation test toxicity was not observed either in the presence or in the absence of metabolic actiovation. In the preincubation the test item proved to be toxic to all Salmonella strain at dose levels of 1600 µg/plate and above and to E. coli WP2uvrA at a dose level of 5000 µg/plate in the presence of metabolic activation. Inthe absence of metabolic activation toxicity was observed with all Salmonella strains at all concentrations (50 µg/plate and above) and wit E. coli WP2uvrA at concentrations of 160 µg/plate and above.

In the repetition of the preincubation test the test compound proved to be toxic to all Salmonella strains at dose levels of 16 µg/plate and above without metabolic activation. In the presence of metabolic activation toxicity was observed at a concentration of 1600 µg/plate with the trster strain TA 1535 and at concentrations of 1600 µg/plate and above with tester strain TA 1537.

Thinning of bacterial lawns ansd /or reduction in the number of colonies was observed at these dose levels.

Mutagenicity:

In the presence and in the absence of the metabolic activation system the test item did not result in relevant increases in th number of revertants in any of the bacterial strains. In the first preincubation test a dose/control ratio of 1.8 - 2.0 was obtained with the strains TA 1535 and TA 1537 in the presence of metabolic activation with individual doses. Although mean revertant values did not differ from the historical solvent control data range and no dose-dependency was observed, the preincubation test with these strains was repeated using a narrower dose range for clarification. No significant or dose-dependent increase in the number of revertants was observed in the repetition test confirming that the test sbstance is not mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun - Oct 2003
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)
Version / remarks:
Aopted 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
Aug 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
19 May 2000
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):
CELLS USED
- Source of cells: cell baank of "Genetic Toxicology", Aventis Pharma Germany
- Suitability of cells: recommended by guideline
- Cell cycle length, doubling time or proliferation index: 12-16 h
- Number of passages/Methods for maintenance in cell culture if applicable:
Thawed stock cultures were kept at approx. 37°C and 4% CO2 in 175 cm2 plastic flasks. About 5 x 10(exp 5) to 1 x 10(exp6) were seeded into each flask in 30 mL of MEM-medium supplement with approx. 10% (v/v) FCS containing approx. 2 mL L-glutamine. The cells were subcultured twice a week.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM with Earle's salts and L-glutamine, 4% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Cytokinesis block (if used):
Colcemide
Metabolic activation:
with and without
Metabolic activation system:
rat S9 mix
Test concentrations with justification for top dose:
First experiment:
- 3/20 h treatment/sampling time (with and without S9 mix): 39.1, 78.2, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL

Second experiment:
- 20/20 h treatment/sampling time (without S9 mix): 39.1, 78.2, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL
- 3/28 h treatment/sampling time (with S9 mix): 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL
- 28/28 h treatment/sampling time (without S9 mix): 78.2, 156.3, 312.5, 625, 1250, 2500 µg/mL
doses based on results of dose range finding tests
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:MEM cell culture medium
- Justification for choice of solvent/vehicle: homogeneity
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): approx. 5 x 10(exp5) to 1 x 10(exp6) cells in 30 mL; two slides were seeded with cells to yield 3-4 x 10(exp4) cells/slide and incubated for 48 h

DURATION
- Exposure duration: 3 (with and without S9 mix), 20 and 28 h (without S9 mix)
- Expression time (cells in growth medium): 20 and 28 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemide

STAIN (for cytogenetic assays): 2% orcein solution

NUMBER OF REPLICATIONS: 2 cultures/experiment

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colcemide was added for 2 h and metaphse spreads were prepared as follows:
The cultures were made hypotonic by adding approx. 5 mL 0.075 M potassium chloride solution at 37°C. The cells were then incubated for 20 min. at 37°C. The next step was the addition of 2 mL fixative. Then the liquid was replaced by 6 mL fixative (methanol: glacial acid, 3:1). After 10 min. the procedure was repeated. After at least 10 min., the slides were taken out and airdried for 24 h.
The chromosomes were stained as follows:
staining for 10 min. in 2% orcein solution, rinsing 3 times in distilled water, rinsing twice in acetone, brief rinsing in acetone/xylene, 2 min. in acetone/xylene, 5 min. in xylene, 10 min. in xylene and embedding

NUMBER OF CELLS EVALUATED:

1000 cells for determination of mitotic index

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
25-200 metaphases per experimental group and cell culture
500 metaphase cells for determination of incidence of polyploid metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: 500 metaphase cells for determination of incidence of polyploid metaphases
Rationale for test conditions:
based on guideline
Evaluation criteria:
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are statisitcally significant and within the laboratory's normal control range

A test substance is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the labory's historical control data
and/or
- no significant increase in the number of strucutral chromosome aberration is observed.

A test substance is classified as clastogenic if:
- the number of induced structural chromosome aberrations is above the range of the laboratory's historical control data
and
- either a concentration-related or significant increase in the number of strucutral chromosome aberrrations is observed.
Statistics:
one-sided Fisher's exact test
Key result
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:
- Solubility:
Evaluation of the solubility of the test item suspension in MEM Earle's salts cell culture medium showed that the limit dose 5000 µg/mL was a practicable concentration and produced macroscopic preciptitate down to the concentration of 625 µg/mL. Microscopic precipitation was noted down to the lowest concentration tested, 39.1 µg/mL. In order to cover concentrations with no macroscopic precipitation, doses down to at least 312.5 µg/mL had to be evaluated.

- Cytotoxicity:
In the absence of metabolic activation 3 h treatment produced moderate toxicity in form of a decrease in the mitotic index (59.4 % of the control) with the highest concentration (5000 µg/mL). The dose-toxicity course was slightly inconsistent with the low concentrations, which is considered as incidental finding. Treatment for 20 h and 28 h produced severe toxicity in high concentrations. Cell survival was reduced below 50% in the highest evaluated concentrations 1250 µg/mL (20 h treatemtn) and 625 µg/mL (28 h treatment). These concentrations showed also a reduction in the mitotic index reaching 55.4% with the dose level of 1250 µg/mL (20 h treatment) and 44.6% with the concentration of 625 µg/mL (28 h treatment).
In the presence of metabolic activation toxicity in form of mitosis inhibition was noticed with the highest concentration, 5000 µg/mL, in the first experiment. Because of the lack of metaphases, this dose could not be evaluated. In contrast, the second experiment with S9 mix (with prolonged recovery period) dhowed no relevant toxicity up to the highest dose. Obviously, mitosis was only temporarily inhibited and surviving cells continued mitosis after a certain recovery time.
Before treatment of the cells, the pH values and osmolarity of the treatment media were determined. The addition of the test item suspension did not have any effect on these parameters.

- Mutagnicity.
After treatment with the test item there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
No relevant or dose-dependent increase in the number of metaphases with aberrations was detected with any of the concentrations used, either with or without metabolic activation by S9 mix.
The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
Conclusions:
The test substance is not clastogenic in this in vitro chromosome aberration assay with V79 cells.
Executive summary:

In this study the potential of the test substance to induce chromosome aberrations was investigated in V79 cells of the Chinese hamster lung in vitro. For each experiment duplicate cultures were used for each concentration. The test compound was suspended in cell culture medium (MEM) and tested at the folliwing concentrations:

First experiment with 3/20 h treatment/sampling time:

without S9 mix: 39.1, 78.2, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL

with S9 mix: 39.1, 78.2, 156.3, 312.5, 625, 1250 and 5000 µg/mL

Second experiment with 20/20 h treatment/sampling time:

without S9 mix: 39.1, 78.2, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL

Second experiment with 3/28 h treatment/sampling time:

with S9 mix: 156.3, 312.5, 625, 1250 and 5000 µg/mL

Second experiment with 28/28 h treatment/sampling time:

Without S9 mix: 78.2, 156.3, 312.5, 625, 1250 and 2500 µg/mL

The maximum concentration based on the regulations of the corresponding guideline.

In the absence of metabolic activation 3 h treatment produced moderate toxicity in form of a decrease in the mitotic index (59.4% of the control) with the highest concentration (5000 µg/mL), while 20 h and 28 h treatment produced severe toxicity in high concentrations. Cell survival was reduced below 50% in the highest evaluated concentrations 1250 µg/mL (20 h treatment) and 625 µg/mL (28 h treatment). These concentrations showed also a reduction of the mitotic index reaching 55.4 % with the dose level of 1250 µg/mL (20 h treatment) and 44.6% with the concentration of 625 µg/mL (28 h treatment).

In the presence of metabolic activation toxicity was noticed in form of mitosis inhibition at the highest concentration, 5000 µg/mL, in the first experiment. Because of the lack of metaphases, this dose could not be evaluated. In contrast, the second experiment with S9 mix (with prolonged recovery period) showed no relevant toxicity up to the highest dose. Obviously, mitosis was only temporaily inhibited and surviving cells continued mitosis after a certain recovery time.

The test item produced macroscopic precipitation down to the concentration of 625 µg/mL and microscopic precipitation down to the lowest tested dose, 39.1µg/mL. In order to cover concentrations with no macroscopic precipitation, doses down to at least 312.5 µg/mL had to be evaluated in the study.

Up to the highest investigated dose the test compound induced no toxicologically relevant or dose-dependent increase in the number of aberrant metaphases. Appropriate reference mutagens used as positve controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9 mix.

In conclusion, the test substance did not induce chromosome aberrations in V79 Chinese hamster cell, either in the presence or in the absence of a metabolic activation system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans.

Additional information

The test item was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella thyphimurium and with E. coli WP2uvrA with and without netabolic activation (5 to 5000 µg/plate).

In the presence and in the absence of the metabolic activation system the test item did not result in relevant increases in th number of revertants in any of the bacterial strains. In the first preincubation test a dose/control ratio of 1.8 - 2.0 was obtained with the strains TA 1535 and TA 1537 in the presence of metabolic activation with individual doses. Although mean revertant values did not differ from the historical solvent control data range and no dose-dependency was observed, the preincubation test with these strains was repeated using a narrower dose range for clarification. No significant or dose-dependent increase in the number of revertants was observed in the repetition test confirming that the test sbstance is not mutagenic.

Furthermore, the potential of the test substance to induce chromosome aberrations was investigated in V79 cells of the Chinese hamster lung in vitro.

Up to the highest investigated dose the test compound induced no toxicologically relevant or dose-dependent increase in the number of aberrant metaphases. Appropriate reference mutagens used as positve controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9 mix.

In conclusion, the test substance did not induce chromosome aberrations in V79 Chinese hamster cell, either in the presence or in the absence of a metabolic activation system.

Justification for classification or non-classification

Due to the findings in the performed in vitro assays no classification for mutagenicity is recommended according to the criteria of Regulation (EC) No 1272/2008.