2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

Endpoint summary

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 TA 1538), in an in vitro gene mutation study in mammalian cells (Chinese Hamster V79 cells) and in one in vitro chromosome aberration study in Chinese Hamster V79 cells. Negative results were obtained in the mammalian cytogenicity study and the gene mutation test. In the Ames test (using

tester strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 153)

in which the mutagenic potential of the test item in bacteria was examined in vitro for back mutations, negative results were obtained in the absence of metabolic activation in tester strains S. thyphimurium TA 100, TA 1535 and TA 1537. The test item increased the number of revertant colonies in tester strains S. thyphimurium TA 98 and TA 1538 with an without metabolic activation (10% rat S9 mix): An increase in the number of revertants was also reported in Salmonella thyphimurium TA 98 with metabolic activation  (30% rat S9 mix and 30% hamster S9 mix). Therefore, the test substance is considered to be mutagenic under the conditions described. However, based on the detailed results the biological relevance is unclear.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Oct 1989

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 26 May 1983

Deviations:

no

Remarks:

E. coli or Salmonella th. TA102 is missing

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:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster S9 mix

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 and 5000 µg/plate (with 5000 µg/plate as highest test concentration required by guideline)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

other: 2-Aminoanthracene (Ames/Prival Test), Benzidine (Prival)

Details on test system and experimental conditions:

The mutagenicity tests were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and presence of a metabolising system derived from rat or hamster liver homogenate. A dose range from 8 different doses from 4 to 5000 µg/plate was used.

Two independent experiments for each test protocol (Ames; Prival; 3 plates per dose) were performed.

METHOD OF APPLICATION:
1. Ames Test:
in agar (plate incorporation)
2. Prival Test:
preincubation;
- Cell density at seeding (if applicable): 0.1 mL of an overnight broth culture of bacterial tester strain

DURATION
- Preincubation period: 30 min (Prival Test)

- Exposure duration: 48-72 h

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: surviving fraction
- Any supplementary information relevant to cytotoxicity:
preliminary toxicity tests were performed with five or four tester strains using three plates per dose. A reduced rate of spontaeously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity.

Rationale for test conditions:

based on pretest

Evaluation criteria:

according to OECD guideline

Statistics:

none

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial at 500 µg/plate

Vehicle 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

Remarks:

incomplete bacterial lawn at 500 µg/plate

Vehicle 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

Remarks:

incomplete bacterial lawn at 500 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial lawn at 300 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial lawn

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 2500 µg/plate and above

Remarks on result:

other:

Remarks:

only tested with rat S9 mix

Conclusions:

The test item increased the number of revertant colonies in tester strains Salmonella thyphimurium TA 98 and TA 1538 with an without metabolic activation (10% rat S9 mix): An increase in the number of revertants was also reported in Salmonella thyphimurium TA 98 with metabolic activation (30% rat S9 mix and 30% hamster S9 mix). Therefore, the test substance is considered to be mutagenic under the conditions described. However, based on the detailed results the biological relevance is unclear.

Executive summary:

The test substance was tested for mutagenicity with Salmonella thyphimurium strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98. The mutagenicity studies were conducted in the standard plate (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolising system derived from rat or hamster liver homogenate. A dose range of 8 different doses from 4 to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All positive control compounds gave the expected increase in the number of revertant colonies.

- Toxicity:

The test compound proved to be toxic to the bacterial strains at 2500 µg/plate. 5000 µg/plate was chosen as top dose level for the mutagencity study.

- Ames Test:

In the absence of metabolic activation the test item gave a dose dependent increase in the number of revertant colonies with the bacterial strains TA 1538 and TA 98. In the presence of metabolic activation (rat S9 mix, 10%) treatment of the cells with the test substance results in relevant increases in the number of revertant colonies with TA 98 and TA 1538.

- Prival Test:

In the presence of hamster liver S9 mix the preincubation method according to Prival and in the presence of rat liver S9 mix (30%) using the standard plate test the test compound resulted in increases in the number of revertant colonies with tester strain TA 98 (TA 1538 was not tested).

However, in case of TA 98 the increase in the number of revertants reached only barlely twice the value of the negative control while the revertant number in case of the positive coontrols was approx. 30fold (in case of 10% rat S9 mix) to 5.5 (in case of hamster S9 mix) depending on utilised positive control compound increased. Furthermore, it should be considered that the tester strain TA 98 contains the R-factor, the plasmid pKM 101, which increases error-prone DNA repair and therefore, artificially increases the susceptibility to DNA damages regardless if their origin. In contrast, no changes were observed in the number revertant colonies in case of the corresponding tester strain TA 1537 not containing the plasmid. Taking into account the before mentioned, the biological relevance and additionally the relevance for humans of the observed tendency to an increasing number of revertant colonies as reported for tester strains TA 98 and 1538 is unclear.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan - Feb 1991

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:

Adopted 26 May 1983

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: stored in liquid nitrogen in the Laboratory of Genetic Toxicologiy of Hoechst AG
- Suitability of cells: recommened by OECD guideline
- doubling time: 12-16 h
- Number of passages if applicable: Thawed stock cultures were propagated at 37°C in 175 cm2 plastic flasks. Seeding was done with about 8-10 x 10(exp5) cells per flask
- Methods for maintenance in cell culture if applicable: in 30 mL MEM-medium supplemented with 10% fetal calf serum. Cells were subcultured twice per week.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM + 10% fetal calf serum
- Properly maintained: yes

Cytokinesis block (if used):

Colcemide

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

without S9 mix
7h: 200 µg/mL
18 h: 20, 100, 200 µg/mL
28 h: 200 µg/mL

with S9 mix
7h: 200 µg/mL
18 h: 20, 100, 200 µg/mL
28 h: 200 µg/mL
based on results of a pretest (solubility, toxicity). The highest concentration with metabolic activation produced a slight decrease of the mitotic index 7h after treatment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
3-6 x 10(exp5) cells seeded into 25 cm2 plastic flasks (28 h preparation)
6-9 x 10(exp 5) cells seeded into 25 cm2 plastic flasks (18 h preparation)
1-3 x 10(exp 6) cells seeded into 75 cm2 plasic flasks (7 h preparation)

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 4.5, 15.5 and 25.5 h after the start of the treatment colcemide was added to the cultures
- Fixation time (start of exposure up to fixation or harvest of cells): 2.5 h after application of colcemide (7, 18 and 28 h preparation) the cells were trypsinised, the cells were washed and fixative was added

SPINDLE INHIBITOR (cytogenetic assays): 0.04 µg/mL colcemide

STAIN (for cytogenetic assays): 2% orcein solution

NUMBER OF REPLICATIONS: 2 cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After fiaxation a few drops of cell suspension were placed with a pasteur pipette onto clean microscopic slides which had been stored in distilled water at 4°C, the drops were then briefly passed through a Bunsen flame and aair-dried for 24 h. Staining was performed as follows:
- staining for 10 min in 2% orcein
- rinsing 3 imes 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
- embedding in Entellan or Eukitt

NUMBER OF CELLS EVALUATED: 1000 cells for mitotic index

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 1 00 metaphases per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: performed

Rationale for test conditions:

performed according to OECD guideline

Evaluation criteria:

- The test substance is classified as mutagenic if it induced a significantly increased aberration rate as compared with the solvent controls with one of the concentrations tested. The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry
- the test substance is classified as mutagenic if there is a reproducible concentration related increase in the aberration rate
- the test substance is classified as non mutagenic when it tests negatively both with and without metabolic activation

Statistics:

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

Remarks:

mitotitic index reduced to 65% at 200 µg/mL test item, with metabolic activation, 7h fixation interval

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY:
The highest concentrations from 300 up to 500 µg/mL showed a precipitation in cell culture medium after the treatment of 4 h. The highest concentration at which no visible precipitation was observed, was found to be 200 µg/mL.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cytotoxicity experiment proved the test item was not cytotoxic to the V79 cells in the presence of metabolic activation. Following treatment in the absence of metabolic activation, several cytotoxicity was observed. Survival declined in a dose related manner reaching 56% of the solvent control value at the dose level of 100 µg/mL. At a concentration of 200 µg/mL survival was reduced to 31.4% of the solvent control value.

TOXICITY AND MUTATION RESULTS:
In the main experiment the mitotic index was slightly reduced after treatment at the highest dose levels with metabolic activation at fixation intervals 7h up to 65%.
Without metabolic activation no indication of cytotoxicity (reduction of mitotic index) was observed.
After treatment with the test article there was no relevant increase in the number of polyploid cells as compared with the solvent controls. A weak enhancement of the aberration rate was observed incusive gaps 7 h after start of the treatment at 200 µg/mL without S9 mix. This effect is not considered to be of genotoxic relevance, since no reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with other concentrations/preparation times, neither with, nor without metabolic activity.

Conclusions:

The results of the chromosome aberration test in V79 Chinese hamster cells lead to the conclusion that the test item is not mutagenic in this test system.

Executive summary:

The in vitro chromosome aberration study was performed to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster.The assay was conducted in two parallel cultures, using identical procedures with and and without rat liver microsomal activation. The test article was tested with the following concentrations:

without S9 mix:

7h : 200 µg/mL

18h: 200, 100 and 20 µg/mL

28h: 200 µg/mL

with S9 mix:

7h: 200 µg/mL

18h: 200, 100 and 20 µg/mL

28h: 200 µg/mL

According to the preliminary experiment for solubility and cytotoxicity the concnetration ranges were selected.

In the main experiment the mitotic index was slightly reduced after treatment at the highest dose levels with metabolic activation at fixation intervals 7h up to 65%.

Without metabolic activation no indication of cytotoxicity (reduction of mitotic index) was observed.

After treatment with the test article there was no relevant increase in the number of polyploid cells as compared with the solvent controls. The test substance was assessed for its mutagenic potential in vitro in the chromosome aberration test with two independent cell cultures without and with metabolic activation, The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances. A weak enhancement of the aberration rate was observed incusive gaps 7 h after start of the treatment at 200 µg/mL without S9 mix. This effect is not considered to be of genotoxic relevance, since no reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with other concentrations, neither with, nor without metabolic activity.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Nov 1990

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)

Version / remarks:

adopted April 4, 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

HGPRT (hypoxanthine-guanine phosphoribosyl transferase) coding gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells:
recommended by OECD guideline
- Number of passages if applicable:
Thawed stock cultured are kept at 37°C and 5% CO2 in plastic flasks. Seeding was carried out with about 1x10(exp 6) cells per flask. The cells were subcultured twice a week. Two days old exponentielly growing cultures were subcultured for another day and subsequnetly exposed to the test item.
- Methods for maintenance in cell culture if applicable:
Large stocks of mycoplasma-free V79 cell line are stored in liquid nitrogen in the laboratory`s cell bank allowing the repeated use of the same cell culture batch for many experiments.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
MEM medium with 10% FCS containing 2 mM L-glutamine and 0.01% neomycisulfonate. For the selection of mutants the medium was supplemented with 0.11% thioguanine. 5% CO2
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix (Aroclor 1254 treated)

Test concentrations with justification for top dose:

10, 25, 50, 100 and 200 µg/mL based on the results of the cytotoxicity and solubility test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
Two days old, exponentially growing cultures which are more than 50% confluent are trypsinated and a single cell suspension is prepared.Subsequently the cellls are replaced for mutagenicity testing and for determination of plating efficiency.
Day 1:
a. Approx. 400 cells in 25 cm2 flasks with 5 mL medium for determination of the plating efficiency; in duplicate for each experimental point
b. 6 x 10(exp 6) cells in 175 cm2 flasks with 30 mL medium for the mutagenicity test, one flask per experimental point (three independent assays)

DURATION
- Exposure duration: 4 h (Day 2)
- Expression time (cells in growth medium):
a. determination of plating efficiency:
Days 2 to 8 (on Day 8 fixation and staining of the colonies for determination of the plating efficiency
b. mutagenicity test:
Days 2 to 5. On Day 5 subculturing in 175 cm2 flasks until Day 9. On Day 9 subculturing in five 75 cm2 flasks with culrure medium containing 6-thioguanine (mutant selection approx. 400000cells/flask) and subculturing in two 25 cm2 flasks for plating efficiency (approx. 400 cells per flask) until Day 16 (fixation and staining of colonies).
- Selection time (if incubation with a selection agent):
Day 9 to Day 16
- Fixation time (start of exposure up to fixation or harvest of cells): Day 16

SELECTION AGENT (mutation assays):
6-thioguanine

STAIN (for cytogenetic assays):
10% methylene blue in 0.01 n KOH

NUMBER OF REPLICATIONS:
3

CELLS EVALUATED:
Only colonies with more than 50 cells were counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (absolute and relative plating efficieny)

Rationale for test conditions:

According to OECD guideline 476.
For toxic substances a percentage survival rate relative to the solvent control is calculated for each treatment. The dose level which results in a predicted survival of approx. 30% is estimated from the results obtained. This dose is chosen as the highest dose level. The lowest doses are chosen from the level of the negative controls. If the test substance is not sufficiently toxic to reduce survival up to the 30% level (the maximum of 10 mM or the lowest concentration at which visible precipitation is observed) will be selected as maximal dose.

Evaluation criteria:

- the stest substance is classified as mutagenic if it reproducibly induces with one of the test substance concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- the test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from enhancemant factor for induced mutants. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

Mann-Whitney-U-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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first and third experiment no relevant reproducible enhancement of the mutant colonies or mutant frequency over the range of the negative control was found with any of the concentrations used either with or without metabolic activation. Under the test conditions of this study there was an indication of mutagenicity in the second main experiment. At 200 µg/mL of test item without metabolic activation and at 50 µg/mL with metabolic activation a statistically significant enhancement of the mutation rate over the negative controls was induced. However, the unequivocal negative results were found in the first and third experiment.

Conclusions:

The result of this HGPRT test in V79 Chinese hamster cells lead to the conclusion that the test item is not mutagenic under the conditions described.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations at the HGPRT locus in V79 cells of the Chinese hamster in vitro.

The assay was performed in three independent experimants, using identical procedures, both with and without metabolic activation (rat liver S9 mix). The test item was tested with the following concentrations:

without S9 mix: 10, 25, 50, 100 and 200 µg/mL

with S9 mix: 10, 25, 50, 100 and 200 µg/mL

According to the preliminary experiment for toxicity the concentration renges were selected. In the preliminary experiment the test substance precipitate at 300 µg/mL in the cell culture medium with and without S9 mix. No higher concentrations were used in the main experiments.

Up to the highest investigated dose no reproducible increase in mutant colony numbers was obtained in experiments 1 and 3. In the seceond experiment at 200 µg/mL of test item without metabolic activation and at 50 µg/mL with metabolic activation a statistically significant enhancement of the mutation rate over the negative controls was induecd. However, the was no dose response relation and unequivocal negative results were found in the first and third experiment.

Appropriate reference mutagenes were used as positive controls and showed a disitnct increase in induced mutant colonies.

In conclusion, the test item does not induce gene mutationds in the HGPRT test with V79 Chinese hamster cells, either with or without metabolic activation under the conditions discribed. Therefore, the test substance is considered to be non-mutagnic in this HGPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item was tested in the in vivo MNT. It was administered orally by gavage to male and female mice in doses of 0 and 5000 mg/kg bw.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test item was within the normal range of the negative control. The number of normochrmatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and was statistically not different from the control values.

Cyclophosphamid induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes showed a difference to the negative control values.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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/in vivo data are regarded as relevant for humans.

Additional information

Mutagenicity of the submission substance has been investigated in vitro in a bacterial reverse mutations assay. The result was as follows:

- Toxicity:

The test compound proved to be toxic to the bacterial strains at 2500 µg/plate. 5000 µg/plate was chosen as top dose level for the mutagencity study.

- Ames Test:

In the absence of metabolic activation the test item gave a dose dependent increase in the number of revertant colonies with the bacterial strains TA 1538 and TA 98. In the presence of metabolic activation (rat S9 mix, 10%) treatment of the cells with the test substance results in relevant increases in the number of revertant colonies with TA 98 and TA 1538.

- Prival Test:

In the presence of hamster liver S9 mix the preincubation method according to Prival and in the presence of rat liver S9 mix (30%) using the standard plate test the test compound resulted in increases in the number of revertant colonies with tester strain TA 98 (TA 1538 was not tested).

However, in case of TA 98 the increase in the number of revertants reached only barlely twice the value of the negative control while the revertant number in case of the positive coontrols was approx. 30fold (in case of 10% rat S9 mix) to 5.5 (in case of hamster S9 mix) depending on utilised positive control compound increased. Furthermore, it should be considered that the tester strain TA 98 contains the R-factor, the plasmid pKM 101, which increases error-prone DNA repair and therefore, artificially increases the susceptibility to DNA damages regardless if their origin. In contrast, no changes were observed in the number revertant colonies in case of the corresponding tester strain TA 1537 not containing the plasmid. Taking into account the before mentioned, the biological relevance and additionally the relevance for humans of the observed tendency to an increasing number of revertant colonies as reported for tester strains TA 98 and 1538 is unclear.

Negative results were obtained in a genotoxicity assay and a cytogenicity assay in mammalian cells in vitro (HGPRT test and chromosome aberration test, both in the presence and the absence of metabolic activation).

Furthermore, the results of an in vivo MNT in mice indicate that the test substance is not mutagnic in the micronucleus test.

Justification for classification or non-classification

Due to the findings in the performed in vitro assays as well as the result of an in vivo micronucleus test in mice no classification for mutagenicity is recommended according to the criteria of Regulation (EC) No 1272/2008.