2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key studies provided were conducted to recognised testing guidelines (or are comparable with standard guidelines) and with GLP certification.

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:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His: Salmonella
Trp: E. coli

Species / strain / cell type:

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

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: "Details on test system and conditions"

Details on test system and experimental conditions:

DOSE SELECTION
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation
test (experiment II). The assay was performed in two independent experiments both with and without liver microsomal activation.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

-POSITIVE CONTROLS:
Without metabolic activation

Strains: TA 1535, TA 100
Name: sodium azide, NaN3
Purity: at least 99 %
Dissolved in: water deionised
Concentration: 10 µg/plate

Strains: TA1537,TA98
Name: 4-nitro-o-phenylene-diamine, 4-NOPD
Purity: > 99.9 %
Dissolved in: DMSO (MERCK, D-64293 Darmstadt; purity > 99 %)
Concentration: 10 µg/plate in TA 98, 50 µg/plate in TA 1537

Strains: WP2 uvrA
Name: methyl methane sulfonate, MMS
Purity: > 99.0 %
Dissolved in: water deionised
Concentration: 4 µI/plate

With metabolic activation

Strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Name: 2-aminoanthracene, 2-AA
Purity: 97.5 %
Dissolved in: DMSO (MERCK, D-64293 Darmstadt; purity > 99 %)
Concentration: 2.5 µg/plate, 10 µg/plate in strain WP2 uvrA

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded
at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: No relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

PRECIPITATION: The test item precipitated in the overlay agar at 3 µg/plate and above. The undissolved particles had no influence on the data recording.

MUTAGENICITY: No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table of Test Results: Number of revertants in the control or after treatment with the testsubstance:

Standard plate test (33 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	16	0.7	no	negative
	yes	13	1.1	no	negative
TA 1537	no	8	0.8	no	negative
	yes	14	09.	no	negative
TA 98	no	19	0.9	no	negative
	yes	31	1.0	no	negative
TA 100	no	138	1.0	no	negative
	yes	163	1.1	no	negative
E. coli WP2 uvrA	no	50	1.0	no	negative
	yes	41	1.2	no	negative
Preincubation test (33 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	12	1.4	no	negative
	yes	14	1.0	no	negative
TA 1537	no	6	1.1	no	negative
	yes	7	1.0	no	negative
TA 98	no	22	0.5	no	negative
	yes	25	1.0	no	negative
TA 100	no	116	0.9	no	negative
	yes	148	1.1	no	negative
E. coli WP2 uvrA	no	30	0.9	no	negative
	yes	35	1.3	no	negative

Conclusions:

The test material failed to induce statistically significant reverse mutations in bacteria under the conditions of the test.

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:

2002

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)

GLP compliance:

yes (incl. QA statement)

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):

The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone V79/T5 (sub-clone V79/D3) in stock cultures: 12 hrs) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of
22.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Concentration range in the main test (without metabolic activation, preparation interval 18 h, exposure period 4 h): 10, 20, 40, 60, 80, 100 µg/ml
Concentration range in the main test (without metabolic activation, preparation interval 18 h, exposure period 18 h): 5, 10, 20, 40, 60, 80 µg/ml
Concentration range in the main test (without metabolic activation, preparation interval 28 h, exposure period 28 h): 20, 40, 60 80 µg/ml
Concentration range in the main test (with metabolic activation, preparation interval 18 h, exposure period 4 h): 12.5, 25, 50, 75, 100, 150 µg/ml
Concentration range in the main test (with metabolic activation, preparation interval 28 h, exposure period 4 h): 25, 25, 50, 75, 100, 150 200 µg/ml

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: "Details on test system and conditions"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 18 and 28 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)

POSITIVE CONTROLS:
Without metabolic activation
Name: EMS; Ethylmethane sulfonate
Purity: > 98 %
Dissolved in: nutrient medium
Final Concentration: 100 - 200 µg/ml (0.8-1.6 mM)

With metabolic activation
Name: CPA; Cyclophosphamide
Purity: 98 %
Dissolved in: nutrient medium
Final Concentration: 0.7 - 1.0 µg/ml (2.5 - 3.5 µM)

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see: "Additional information on results"

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: of the test item after 4 hrs and 24 hrs treatment was observed at 173.8 µg/ml and above.

RANGE-FINDING/SCREENING STUDIES: Test item concentrations between 10.9 and 1390 µg/ml (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 43.4 µg/ml and above.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after treatment with 86.9 µg/ml and above in the absence of S9 mix. In addition, 4 hrs treatment with 173 µg/ml and above in the presence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 100 µg/ml (without S9 mix) and 150 µg/ml (with S9 mix) were chosen as top concentrations in the main experiment I. In experiment II 80 µg/ml were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix at the 28 hrs preparation interval, 200 µg/ml were chosen as top treatment concentration with respect to the results obtained in experiment I.

MUTAGENICITY: No biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. A single statistically significant increase (3 %) was observed in experiment II, but was within our historical control range (0.0 - 4.0 % aberrant cells, exclusive gaps) and is regarded as being biologically irrelevant.
No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test material failed to induce statistically significant chromesome aberrations under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The study provided was conducted to in a comparable way to recognised testing guidelines and with GLP certification.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

other: Chinese hamster (Cricetulus griseus) random outbred strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln, Switzerland
- Age at study initiation: females: 6-10 weeks, males: 4-9 weeks
- Weight at study initiation: females: 20-30 g; males: 21-31 g
- Housing: individually
- Diet (e.g. ad libitum): NAFAG No.924, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 51-54
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours

Route of administration:

oral: gavage

Vehicle:

0.5 % aqueous solution of sodium carboxymethylcellulose (CMC) plus 0.1 % Tween 80 (Tw 80)

Duration of treatment / exposure:

daily on 2 consecutive days

Frequency of treatment:

once a day

Post exposure period:

24 hours

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (128 mg/kg)

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µl rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Gruenwald solution for 2 min then in May-Gruenwald solution/water 1/1 for
2 min and then in Giemsa's, 40 % for 20 min. After being rinsed in methanol 55 % for 5-8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylol and mounted in Eukitt.

METHOD OF ANALYSIS: The slides of three female and three male animals were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.

Statistics:

The significance of difference was assessed by x²-test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells
- Induction of micronuclei (for Micronucleus assay): In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control.
- Statistical evaluation: The significance of difference was assessed by χ2-test

Conclusions:

The test substance failed to induce statistically significant genome mutations under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are reliable in vitro and in vivo studies available to assess the potential of the test substance for gene mutations in bacteria, cytogenicity in mammalian cells and micronucleus in Chinese hamster.

 

Gene mutation in bacteria:

In a GLP conform study according to OECD guideline 471, the test substance (purity: 99.5 weight-%) was tested for its mutagenic potential based on its ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The assays were performed according to the plate incorporation test (experiment I) and the preincubation test (experiment II) (RCC Cytotest Cell Research GmbH, 2002). The assay was performed in two independent experiments with and without liver microsomal activation(Phenobarbital/β-Naphthoflavone induced rat liver S9-mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 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.

Toxic effects (below the factor of 0.5), evident as a reduction in the number of revertants, were observed in strain TA 1535 without S9 mix at the dose of 5000 µg/plate and in strain TA 1537 with S9 mix at a dose of 2500 and 5000 µg/plate and without S9 mix at doses from 1000 to 5000 µg/plate in experiment I. In experiment II, toxic effects were observed in strain TA 1537 without S9 mix (2500 and 5000 µg/plate) and in strains TA 98 (without S9 mix: 333 -5000 µg/plate; with S9 mix: 5000 µg/plate) and TA 100 (with and without S9 mix: 5000 µg/plate).

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

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

In conclusion, 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.

Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

In a study according to Ames et al. (1973) the test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium (CIBA-Geigy, 1982). The investigations were performed with strains TA 98, TA 100, TA 1535 and TA 1537. The following concentrations of the trial substance were tested without and with microsomal activation: 25, 75, 225, 675 and 2025 µg/plate.

In order to confirm the results, additional experiments were performed on strain TA 100 at the concentrations of 250, 500, 1000, 2000 and 4000 µg/plate.

These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).

In the experiments performed without microsomal activation no marked deviations were observed between controls and treated colonies.

In the experiments in which activation mixture was added to the cultures, treatment with the test substance led to an increase in the number of back-mutant colonies of strain TA 100.

The metabolites of the test substance formed as a result of microsomal activation thus displayed a mutagenic effect in this test system.

 

Another study according to Ames et al. (1973) was carried out in 1983. The test substance (purity: > 98 weight-%) was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium (CIBA-Geigy, 1983). The investigations were performed without and with microsomal activation on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the test material : 20, 80, 320, 1280 and 5120 µg/plate. In order to confirm the results, the experiments were repeated. Additional experiments were carried out with the concentrations of 250, 500, 1000, 2000 and 4000 µg/plate.

In the experiments performed without and with microsomal activation on strains TA 1535 and TA 1537 comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed a reduction in the colony count due to a growth-inhibiting effect of the test material at the upper concentrations. This effect was also observed in the experiments without microsomal activation and in the first experiment with microsomal activation on strain TA 100.

In the experiments performed with microsomal activation treatment with the test substance led to a slight increase in the number of back-mutant colonies of strain TA 100 at the concentrations of 320 µg/plate and above.

The metabolites of the test substance thus displayed a weak albeit mutagenic effect in this test system.

 

The same method (Ames et al., 1973) was carried out in 1986. The test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium (CIBA-Geigy, 1986). The investigations were performed without and with microsomal activation on strains TA 98, TA 100 and TA 1537 with the following concentrations of the trial substance : 20, 78, 313, 1250 and 5000 µg/plate.

In the experiments performed without and with microsomal activation, none of the tested concentrations of the test substance led to an increase in the incidence of histidine-prototrophic mutants in comparison to the negative control.

Owing to a growth-inhibiting effect of the substance in the experiments without microsomal activation a reduction in the colony count was observed at the upper concentrations. In the experiments with activation this effect was perceptible at the highest concentration only.

No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

 

Another Ames test according to Ames et al. (1973) was made by Hattori 1983. The test compound was treated for mutagenic effects on histidine-auxotrophic strains of Salmonella typhimurium, TA 100, TA 98, TA 1535, TA 1537 and TA 1538 and on a tryptophan-auxotrophic strain of E. coli (WP 2 uvr A ) (Hattori M., 1983). The investigations were performed with the following concentrations of the test substance without and with microsomal activation: 0.05, 0.1, 0.5, 1, 5, 10, 50, 100,- 500, 1000 and 5000 µg/plate.

In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations revealed no marked deviations. No evidence of the induction of point mutations by the compound or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments.

Overall conclusion: The majority and the guideline conform gene mutation tests in bacteria indicate negative results. Therefore, the test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays neither with nor without S9 mix.

Gene mutation in mammalian cells:

The test substance was tested for mutagenic effects on L5178Y/TK mouse lymphoma cells in vitro (CIBA-Geigy, 1983). The investigations were performed without microsomal activation at concentrations of 11.25, 22.5, 45.0, 90.0 and 180.0 µg/ml and with microsomal activation at concentrations of 8.75, 17.5, 35.0, 70.0 and 140.0 µg/ml.

This test system permits the detection of forward point mutations in mammalian cells induced by chemical substances or by their metabolites. Mutagenic effects manifest themselves in the occurrence of mutants insensitive to 5-bromodeoxyuridine (BUdR). A mutagenic effect of a substance is demonstrable on comparison of the number of colonies in the treated and control cultures.

To ensure that mutagenic effects of potential metabolites of the test substance formed in mammals are also detected, experiments are performed in which the cultures are treated with the compound in the presence of an activation mixture (rat-liver microsomes and co-factors).

In the experiment performed without microsomal activation, the highest concentration (180.0 µg/ml) proved to be unsuitable because of cytotoxicity. The next four concentrations did not produce a marked increase in the mutant frequency. Comparison of the mutant-frequency values with the control gave factors of 1.56, 1.44, 0.94 and 0.95 down to the lowest concentration. In the investigation in which the cultures were additionally treated with an activation mixture, comparison of the mutant-frequency values with the controls gave a factor of 1.04 at the highest concentration and factors of 1.21, 1.07, 1.27 and 1.18 down to the lowest concentration.

Comparison of the concurrent positive controls with ethylmethane sulfonate (EMS: 0.75 µl/ ml) and the negative controls in the experiment without activation gave a factor of 59.0. In the experiment with metabolic activation, comparison of the mutant frequency values in dimethylnitrosamine (DMN: 0.75 µl/ml)-treated positive controls and negative controls revealed a factor of 45.4.

It is concluded that under the given experimental conditions no evidence of mutagenic effects of the test substance was observed in this mammalian forward mutation system.

 

Cytogenicity in mammalian cells:

In a GLP study performed according to OECD guideline 473, the substance (purity: 99.5 weight-%) was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments (RCC Cytotest Cell Research GmbH, 2002).

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (1390 µg/ml) was chosen with regard to the solubility properties of the test item in an appropriate solvent.

Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

The treatment concentrations ranged from 5 – 200 µg/ml.

The following study design was performed:

Experiment I: with and without S9 mix; exposure period 4 hours, recovery period 14 hours, preparation interval 18 hours.

Experiment II: without S9 mix; exposure period 18 hours, preparation interval 18 hours.

Experiment II: without S9 mix; exposure period 28 hours, preparation interval 28 hours.

Experiment II: with S9 mix; exposure period 4 hours, recovery period 24 hours, preparation interval 28 hours

Toxic effects indicated by reduced cell numbers or mitotic indices of below 50 % of control were observed in experiment II in the absence of S9 mix at both treatment intervals.

In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

A single statistically significant increase (3 %) was observed in experiment II, but was within the historical control range (0.0 - 4.0 % aberrant cells, exclusive gaps) and is thus regarded as being biologically irrelevant.

No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

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

 

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test neither with nor without S9 mix.

 

Cytogenicity in vivo

 

A micronucleus assay was performed to evaluate mutagenic effects of the test substance on somatic interphase cells in vivo.Mutagenic effects present themselves in interphase cells in form of nucleus anomalies of bone marrow cells. These anomalies occur in interphase cells as a consequence of damage during the mitotic process. The increase in anomalies shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations.

The test substance(purity: > 98 weight-%)was administered by gavage. Treatment consisted of one daily dose of 750, 1500 or 3000 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application. Smears were made from the bone marrow

The bone marrow smears from animals treated with various doses of the test substance showed no significant difference from the control.

The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group.

By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 9.93 % cells with nuclei anomalies. This is significantly different from the controls treated with the vehicle (0.5 % carboxymethylcellulose + 0.1 % Tween 80) alone.

It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

A study to assess sister chromatid exchange was carried out according to Perrx and Evans (1975). The test substance was administered by gavage. Treatment consisted of a single dose (CIBA-Geigy, 1983). The animals were sacrificed 24 h after the application and 2 h after an intraperitoneal injection, of colcemide (10 mg/kg). Bone marrow drop-preparations were made and stained according to a modified fluorochrome plus Giemsa technique.

The number of SCE's in animals treated with the various doses of the test substance showed no significant increase in comparison with the concurrent negative control group. The incidence of SCE's in the control group and in the dosage groups corresponds to the frequency observed in animals of the breed used.

A "positive control" experiment with DMBA (100 mg/kg) yielded a mean value of 10.6 SCE's per cell. This is significantly different from the controls treated with the vehicle (0.5% CMC + 0.1% Tween 80) alone.

It is concluded that under the condition of this experiment no effects were obtained that are suggestive of a clastogenic property of the test substance.

Short description of key information:
in vitro:
Gene mutation in bacteria:
key study, Ames test, S. typhimurium/ E.coli, with and without metabolic activation: negative (GLP, OECD 471; RCC Cytotest Cell Research GmbH, 2002)

supporting study, Ames test, S. typhimurium, with and without metabolic activation: positive with metabolic activation; negative without metabolic activation (in accordance with the method described by AMES et al., 1973; CIBA-Geigy, 1982)

supporting study, Ames test, S. typhimurium, with and without metabolic activation: positive with metabolic activation; negative without metabolic activation (GLP, in accordance with the method described by AMES et al., 1973; CIBA-Geigy, 1983)

supporting study, Ames test, S. typhimurium, with and without metabolic activation: negative (in accordance with the method described by AMES et al., 1973; CIBA-Geigy, 1986)

supporting study, Ames test, S. typhimurium/E. coli, with and without metabolic activation: negative (in accordance with the method described by AMES et al., 1973; Hattori M., 1983)

Gene mutation in mammalian cells:
supporting study, mouse lymphoma assay (MLA), L5178Y cells, with and without metabolic activation: negative (based on CLIVE and SPECTOR (1975); CIBA-Geigy, 1983)

Cytogenicity in mammalian cells:
Chromosome aberration, chinese hamster V79 cells, in vitro: negative (GLP, OECD 473; RCC Cytotest Cell Research GmbH, 2002)

in vivo:
key study, micronucleus test, chinese hamster, in vivo: negative (comparable to guideline study; CIBA-Geigy, 1983)

supporting study, sister chromatid exchange, chinese hamster, in vivo: negative (GLP, according to P. PERRY and H. J. EVANS, (1975); CIBA-Geigy, 1983)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.