Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be not mutagenic to base pair changes or frame shifts in the genome of the bacterial strains used. Hence, the test chemical can be considered to be non-genotoxic in nature.

In vitro chromosomal aberration study

Based on the observations and applying the weight of evidence approach, it can be considered the test chemical did not induce structural chromosomal aberrations in mammalian cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

In vitro mammalian cell gene mutation assay

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non- genotoxic to CHO cells both in the presence and absence of metabolic activation system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence approach based on various test chemicals
Justification for type of information:
Weight of evidence approach based on various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Weight of evidence approach based on various test chemicals
Principles of method if other than guideline:
Weight of evidence approach based on various test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and Tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
Study 2
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
Study 3
Metabolic activation:
with and without
Metabolic activation system:
2. Type and composition of metabolic activation system: Aroclor 1254 induced S9 metabolic activation system
3. Type and composition of metabolic activation system:
S9 in the S9 mix was prepared from the livers of Sprague-Dawley male rats treated with phenobarbital and 5,6-benzoflavone as inducers
- source of S9
: Kikkoman S9 mix manufactured within 6 months was used for the test.
- method of preparation of S9 mix
:The composition of S9 mix is ​​shown below.
Component Amount of S9 mix in 1 ml
S9 0.1 ml
MgCl2 8 μmol
KCl 33 μmol
G-6-P 5 μmol
NADPH 4 μmol
NADH 4 μmol
Phosphate buffered Na-solution (pH 7.4) 100 μmol

- concentration or volume of S9 mix and S9 in the final culture medium
:
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Test concentrations with justification for top dose:
2. 0.0, 0.050, 0.158, 0.501, 1.582 or 5 mg/plate
3. 6 doses of 156 to 5000 μg / plate
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Distilled water
- Justification for choice of solvent/vehicle: The test chemical was solulble in distilles water
3. - Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]
: Distilled water
- Justification for choice of solvent/vehicle:
A test substance was dissolved in distilled water for injection (Otsuka distilled water: Otsuka Pharmaceutical Factory Co., Ltd.) to prepare a stock solution. The stock solution was serially diluted with the solvent used to the specified concentration and then immediately processed (prepared before use).

- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Remarks:
Study 2
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide, Sodium azide, 9-aminoacridine, 2-Aminoanthracene were used as positive controls
Remarks:
Study 3
Details on test system and experimental conditions:
2. METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
3. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
:
- Number of independent experiments
: 2 independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
20 minutes
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times):

METHODS FOR MEASUREMENTS OF GENOTOXICITY
: After incubating each plate for 48 hours at 37 ° C, in order to confirm the growth inhibitory effect of the test substance on the test strain, the growth state of the test strain on the plate was observed using a stereoscopic microscope (× 60). Next, the colonies generated by the back mutation were counted. A colony analyzer (CA-11: System Science Co., Ltd.) was used for the measurement.
Evaluation criteria:
2.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 TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.
3. A positive result was obtained when the number of revertant colonies increased more than twice that of the solvent control and reproducibility or dose-dependence of the test substance was 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 control and vehicle control such an increase is not considered biologically relevant.
Species / strain:
S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
Study 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: Salmonella typhimurium TA100, TA98, TA1535 and TA1537
Remarks:
Study 3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Study 3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (N.C), 0.002(T1), 0.005(T2), 0.016(T3), 0.050(T4), 0.158(T5), 0.501(T6), 1.582(T7) and 5(T8) mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 - 5 mg/plate based on the solubility and precipitation test. In treated concentrations 5 - 0.002 mg/plate (T8-T1) no colony reduction or back ground lawn reduction was observed both, in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.050(T1), 0.158(T2), 0.501(T3), 1.582(T4) and 5(T5) mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
3. TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
- Data on osmolality:
- Possibility of evaporation from medium:
- Water solubility:
- Precipitation and time of the determination:
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):
Preliminary studies were conducted with doses of 8.00, 40.0, 200, 1000 and 5000 μg / plate. As a result, it was observed that TA100 and TA1535 of the direct method inhibited the growth of the test strains at 5000 μg / plate. Therefore, in this study, the maximum dose was 5000 μg / plate for each strain of the direct method and the metabolic activation method, and 6 doses (common ratio 2) were set for each strain.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
:The positive control substances in each system showed a clear mutagenic effect on each test strain.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
No clear increase in the number of revertant colonies was observed compared with the solvent control, and reproducibility was confirmed regardless of the presence or absence of addition of rat liver microsomes (S9)
Remarks on result:
other: not mutagenic
Conclusions:
Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be not mutagenic to base pair changes or frame shifts in the genome of the bacterial strains used. Hence, the test chemical can be considered to be non-genotoxic in nature.
Executive summary:

Various studies were reviewed to determine the mutagenic potential of the test chemical. These include the in vitro experimental results performed for the test chemicals. The results are mentioned below:

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (N.C), 0.002(T1), 0.005(T2), 0.016(T3), 0.050(T4), 0.158(T5), 0.501(T6), 1.582(T7) and 5(T8) mg/plate were selected for pre-experiment.Based on the pre-experiment results, the test item was tested with the following concentrations: 0.0 (NC), 0.050(T1), 0.158(T2), 0.501(T3), 1.582(T4) and 5(T5) mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, 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. The spontaneous reversion rates in the negative and positive controls were within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

This result is supported a bacterial reverse mutation test of the test chemical was carried out to determine its mutagenic potential. Five strains of histidine-requiring Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and tryptophan-requiring Escherichia coli WP2 uvrA 2 are used as test strains were selected for the study. The test dose was set based on the results of preliminary studies, i.e for each strain of the direct method (-S9 mix) and the metabolic activation method (+ S9 mix), 6 doses of 156 to 5000 μg / plate were set and tested. The test chemical was dissolved in distilled water for injection (Otsuka distilled water: Otsuka Pharmaceutical Factory Co., Ltd.) to prepare a stock solution. The stock solution was serially diluted with the solvent used to the specified concentration and then immediately processed (prepared before use).S9 in the S9 mix was prepared from the livers of Sprague-Dawley male rats treated with phenobarbital and 5,6 -benzoflavone as inducers. 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide, Sodium azide, 9-aminoacridine, 2-Aminoanthracene were used as positive controls. These positive control substances were dissolved in DMSO, divided into small aliquots, and stored frozen (-20 ° C). According to the pre-incubation method which is an improved method of the original method of Ames et al., tests were carried out for the direct method and the metabolic activation method. In a test tube, 100 μl of solvent, test chemical solution or positive control substance solution, then 500 μl of 0.1 M sodium phosphate buffer (pH 7.4) for direct method, S9 mix for metabolic activation method 500 μl and 100 μl of the test bacterial solution were added, and the mixture was incubated at 37 ° C for 20 minutes with shaking (pre-incubation). After the culture was completed, 2 ml of top agar was added and the mixture was layered on the plate. After incubating each plate for 48 hours at 37 ° C, in order to confirm the growth inhibitory effect of the test substance on the test strain, the growth state of the test strain on the plate was observed using a stereoscopic microscope (× 60). Next, the colonies generated by the back mutation were counted. A colony analyzer was used for the measurement. The test was conducted twice independently. A positive result was obtained when the number of revertant colonies increased more than twice that of the solvent control and reproducibility or dose-dependence of the test substance was observed. No clear increase in the number of revertant colonies was observed compared with the solvent control, and reproducibility was confirmed regardless of the presence or absence of addition of rat liver microsomes (S9). On the other hand, the positive control substances in each system showed a clear mutagenic effect on each test strain. Therefore, it was judged that the test chemical does not induce gene mutation in microorganisms under the conditions of this test.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be not mutagenic to base pair changes or frame shifts in the genome of the bacterial strains used. Hence, the test chemical can be considered to be non-genotoxic in nature.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence approach based on various test chemicals
Justification for type of information:
Weight of evidence approach based on various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Ninth Addendum, adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Annex 4A Directive 2000/32/EC: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test"
Deviations:
no
Principles of method if other than guideline:
Weight of evidence approach based on various test chemicals
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human lymphocytes
Remarks:
5
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; containing 10 % FCS (fetal calf serum). The antibiotic solution contains 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL, NATTERMANN, D-50829 Köln), and HEPES (final concentration 10 mM)
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
mammalian cell line, other: Fibroblast cell line (CHL) derived from Chinese hamster lung
Remarks:
6
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle-MEM medium was dissolved in 1000 ml of purified water, and then 2.2 g of sodium bicarbonate was added. The pH was adjusted to 7.2 with 1N hydrochloric acid, and then filtered under pressure by using a membrane filter. Calf serum that had been inactivated (56 ° C., 30 minutes) was added to a final concentration of 10%, and then used for the test.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
5. Type and composition of metabolic activation system: Phenobarbital/beta-naphthoflavone induced rat liver S9
6. Type and composition of metabolic activation system:
S9 in the S9 mix was prepared from the livers of Sprague-Dawley male rats treated with phenobarbital and 5,6-benzoflavone as inducers.
- source of S9
:Kikkoman S9 mix manufactured within 6 months was used for the test.
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
: The composition of S9 mix followed the method of Matsuoka et.al
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Test concentrations with justification for top dose:
5. Concentrations dosed:
- Experiment I: 4 hour exposure without metabolic activation: 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis.)
- Experiment I: 4 hour exposure with metabolic activation: 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis.)
- Experiment II: 22 hour continuous exposure without metabolic activation: 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis.)

The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation.
With respect to the molecular weight of the test item, 2280 μg/mL of T779 (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 14.8 and 2280 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item was observed before start of treatment. Since the cultures fulfilled the requirements for cytogenetic
evaluation, this preliminary test was designated Experiment I. Using reduced mitotic indices as an indicator for toxicity in the pre-test, no clear toxic effects were observed after 4 hrs treatment up to the highest applied concentration, in the absence and presence of S9 mix. Therefore, 2280 μg/mL was chosen as top treatment concentration for continuous exposure in Experiment II.

6. 24 and 48 hrs treatment: 0, 475, 950 or 1900 µg/mL

Confirmative test: 0, 1000, 1300, 1600 or 1900 µg/mL
Vehicle / solvent:
5. - Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

. - Vehicle(s)/solvent(s) used[none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Physiological saline
- Justification for choice of solvent/vehicle: The test chemical is soluble in Physiological saline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation: at a concentration of 770 µg/mL (Experiment I) and 550 µg/mL (Experiment II) (5)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation: at a concentration of 37.5 µg/mL (5)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Physiological saline
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
6
Details on test system and experimental conditions:
5. METHOD OF APPLICATION:
- in medium
- About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation, 50 µL S9 mix per mL medium were used. After 4 hrs, the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once. After washing the cells were resuspended in complete culture medium and cultured until preparation.
- In Experiment II, the culture medium was replaced with complete medium (with 10 % FCS) containing the test substance. The culture medium at continuous treatment was not changed until preparation of the cells.

All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

DURATION
- Exposure duration: 4 hours (Experiment I); 22 hours (Experiment II - continuous exposure)
- Expression time: 15 hours (Experiment I); 19 hours (Experiment II) (Start of exposure until introduction of spindle inhibitor)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours (Experiments I and II)

SPINDLE INHIBITOR (cytogenetic assays): Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 3°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide.
STAIN (for cytogenetic assays): Giemsa or fluorescent plus Giemsa technique

NUMBER OF REPLICATIONS:
- Replicates consisting of two primary cultures were tested.

NUMBER OF CELLS EVALUATED:
- One-hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromere regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (1000 cells per culture were counted.)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes. The number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.
- Determination of endoreplication: no data

6. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 24 and 48 hrs
- Expression time (cells in growth medium): 24 and 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): Colcemimd
STAIN (for cytogenetic assays): 1.2% Giemsa staining solution

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: One hundred per plate, that is, 200 mid-metaphase images per dose, were observed

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
5.
- The test substance was classified as non-mutagenic if: the number of induced structural chromosome aberrations in all evaluated dose groups was in the range of the laboratory historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) and no significant increase of the number of structural chromosome aberrations is observed.
- A test item is classified as mutagenic if: the number of induced structural chromosome aberrations was not in the range of the laboratory 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.

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory historical control data (0.0 – 1.5 % polyploid cells).

6.
The mid metaphase cells were observed for gaps, chromatid breaks (ctb), chromosome breaks (csb), chromosome breaks (csb), chromosome breaks They were classified as structural anomalies of exchange (cte), chromosome exchange (cse) and others (oth). At the same time, the incidence of ploidy cells was recorded. Chromosome analysis was carried out according to the classification method by Japan Environmental Mutagen Society and Mammal Test Subcommittee.

The frequency of occurrence of chromosomal structural abnormality was distinguished into cases where the cells possessing only the gap (+ gap) were included and when not included (-gap).
Frequency of occurrence of cells having structural abnormality in each test group or ploidy cells was judged according to the criteria of Ishikan et al. Negative (-) for cells with chromosomal abnormality negative (-), false positive (±) from 5% to less than 10%, positive (+) above 10%. Ultimately, it was judged as positive when reproducibility or dose dependence was observed.
Statistics:
5. Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test substance was not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

6. No statistical test was used
Species / strain:
lymphocytes: human
Remarks:
5
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Remarks:
5
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the mitotic index was clearly reuced at the highest concentration evaluated.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mammalian cell line, other: Fibroblast cell line (CHL) derived from Chinese hamster lung
Remarks:
6
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
5.
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No relevant increase in the pH and osmolality (Exp. I: solvent control: 276 mOsm, pH 7.3 versus 298 mOsm and pH 7.4 at 2280 µg/mL)
- Evaporation from medium: no data
- Water solubility: 630 g/L
- Precipitation: In both experiments, no visible precipitation of the test item in the culture medium could be observed

RANGE-FINDING/SCREENING STUDIES:
- The pre-test phase was performed with 10 concentrations of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times were 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 ug/mL) to reassure the replication time of the cultured lymphocytes. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA:
- EMS (550 and 770 µg/mL, respectively) and CPA (37.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations
- The proliferation index of the lymphocytes in solvent control cultures in the 22 hrs preparation interval with and without S9 mix (4 hrs treatment; both 1.00), in the 22 hours preparation interval without S9 mix (continuous treatment; 1.35), was checked by analysing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (M1, M1+, M2 and M3) indicating that the lymphocytes divided about 1.5 times within the early preparation interval. This is also proven by the occurrence of sufficient numbers of mitotic cellsand by a clear clastogenicity observed after treatment with the positive control substances.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In experiment I in the presence and absence of S9 mix, no cytotoxicity was observed up to the highest applied concnetration. In Experiment II, after continuous exposure, in the absence of S9 mix, the mitotic index was clearly reduced at the highest concentration evaluated (44.9% of the control).

6.
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: Cells were seeded on a multi-plate for cell culture, and the test substance solution was treated 3 days after the culture. In the case of the continuous treatment method, the treatment is carried out continuously for 24 or 48 hours, and in the short treatment method, after treatment for 6 hours in the presence of S9 mix (+ S 9 mix) or absence (-S 9 mix), fresh The medium was replaced with a culture medium, and the culture was continued for 18 hours. After fixation of the cells with 10% neutral buffered formalin solution, the cells were stained with 0.1% crystal violet aqueous solution for 10 minutes. An appropriate amount of pigment eluate (30% ethanol, 1% acetic acid aqueous solution) was added and allowed to stand for about 5 minutes to elute the dye, and the absorbance at 580 nm was measured. For each dose group, the ratio to the absorbance in the solvent control group, i.e. the cell viability was calculated. As a result, the cell proliferation inhibitory action was observed in the continuous treatment method, but clear action was not observed in the short-time treatment method (Fig. 1). The 50% cell proliferation inhibitory concentration calculated using the probit method was calculated as 1311 mg / ml in continuous 24-hour treatment and 585 μg / ml in the same 48-hour treatment. Moreover, it was considered to be 1900 μg / ml or more in the short time treatment.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
Based on the observations and applying the weight of evidence approach, it can be considered the test chemical did not induce structural chromosomal aberrations in mammalian cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.
Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using human lymphocytes in the presence and absence of metabolic activation system in experiment 1 of 4 hrs duration and in the absence of metabolic activation sytem in experiment 2 of 22 hours continuous duration. The test chemical was dissolved with deionized water and used at dose levels of 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 1 and 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 2. About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation, 50 µL S9 mix per mL medium were used. After 4 hrs, the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once. After washing the cells were resuspended in complete culture medium and cultured until preparation. In Experiment II, the culture medium was replaced with complete medium (with 10 % FCS) containing the test substance. The culture medium at continuous treatment was not changed until preparation of the cells. All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air). Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 3°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. One-hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromere regions were included in the analysis. The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. In experiment I in the presence and absence of  S9 mix, no cytotoxicity was observed up to the highest applied concnetration. In Experiment II, after continuous exposure, in the absence of S9 mix, the mitotic index was clearly reduced at the highest concentration evaluated (44.9% of the control). No chromosome aberration was however observed in both the experiments. Based on the observations made, the test chemical did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

In another study, the test chemical was studied for its ability to induce chromosomal aberrations in the mammalian cell line used. The study was performed using Fibroblast cell line (CHL) derived from Chinese hamster lung in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in physiological saline and treated for 24 hrs, 48 hrs. Two confirmatory tests were further carried out to confirm the outcome the study. The doses for 24 and 48 hrs treatment were 0, 475, 950 or 1900µg/mL and for the confirmative test it was 0, 1000, 1300, 1600 or 1900µg/mL. Cells were seeded on a multi-plate for cell culture, and the test substance solution was treated 3 days after the culture. In the case of the continuous treatment method, the treatment is carried out continuously for 24 or 48 hours. Two hours before the end of the culture, colcemid was added to a final concentration of 0.2 μg / ml. Cells were detached by trypsin treatment, and cells were collected by centrifugation. Afterhypotonic treatment with a 75 mM potassium chloride aqueous solution, the cells were fixed with a fixing solution (3 volumes of methanol: 1 volume of acetic acid). Chromosome specimens were prepared by air drying method and then stained with 1.2% Giemsa staining solution for 12 minutes. Following treatment, 200 mid-metaphase cells were observed for gaps, chromatid breaks (ctb), chromosome breaks (csb), chromosome breaks (csb), chromosome breaks. In the case of the test chemical treated group, chromosomal structural abnormality and polyploid cell induction tendency were not observed at both the 24 hour and 48 hour treatment doses. On the other hand, marked induction of chromosomal structural abnormality was observed in the cells treated with the positive control substance MMC. In the case of the test substance treated group, the appearance frequency of chromosome structural abnormality was 5% only at high dose of 1900 μg / ml in + S 9 mix treatment, and it was judged to be false positive. As a result of conducting the confirmatory test to investigate the reproducibility, the frequency of induction of chromosomal structural abnormality was 2.5 to 3.5% in the first test, and it did not exceed 5% which is the criterion of false positives. In the second confirmatory test, a slight tendency of structural abnormality of chromosomes was observed in the test substance treated group. Based on the above test results, it was judged to be false positive with respect to chromosomal aberration induction of the test chemical to mammalian cultured cells under the present test conditions.

Based on the observations and applying the weight of evidence approach, it can be considered the test chemical did not induce structural chromosomal aberrations in mammalian cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Weight of evidence approach based on various test chemicals
Justification for type of information:
Weight of evidence approach based on various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Weight of evidence approach based on various test chemicals
Principles of method if other than guideline:
Weight of evidence approach based on various test chemicals
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
8. Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Study 8,9
Details on mammalian cell type (if applicable):
8. - Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid Modal 20
Cytokinesis block (if used):
no data available
Metabolic activation:
with and without
Metabolic activation system:
8. Type and composition of metabolic activation system: S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
8. 0, 0.625, 1.25, 2.5 or 5 uM
9. 0 (vehicle = deionized water), 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 10.0 ug/ml
Vehicle / solvent:
8. Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle: The test chemical was easily dissolved in PBS.
9. - Vehicle(s)/solvent(s) used: [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]
: DEIONIZED WATER

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Phosphate buffeer saline
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
Remarks:
Study 8
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionized water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Remarks:
Study 9
Details on test system and experimental conditions:
8. METHOD OF APPLICATION: In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays): Not applicable

STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

9. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments
: 2 independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:
Evaluation criteria:
8. Chinese Hamster Ovary Cells (CHO) were observed for gene mutation caused by the test compound
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Study 8
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
Study 9
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
8. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Completed without S9 metabolic activation. A range of test concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 or 20mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assayMTTand the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since cytotoxicity was evident at the tested concentration in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 5mM.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable:No data
9. Gene mutation tests in mammalian cells:
In the first trial, the survival ranged from 105.9% to 10.7%. The second trial gave survivals ranging from 106.6% to 5.7%. Neither assay showed an increase in mutant frequency. Trials with metabolic activation were conducted at concentrations of 0, 1.0, 5.0, 10.0, 13.0, 15.0, 18.0, 20.0, 22.0 and 25.0 ug/ml. The first trial yielded survivals of 105.1% to 2.8% (at doses from 5.0 to 25.0 ug/ml). The repeat trial yielded survivals from 102.4% to 19.3% (at doses from 10.0 to 22.0 ug/ml). Neither trial showed an increase in mutant frequency.
Remarks on result:
other: not mutagenic
Conclusions:
Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non- genotoxic to CHO cells both in the presence and absence of metabolic activation system.
Executive summary:

The non- mutagenic nature of the test chemical was further confirmed with the help of experimental studies performed on mammalian cells. The results are as follows:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. The study was performed according to OECD 476 Guidelines. Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5 microM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested Tetrabutylammonium bromide concentration, the results showed no evidence of gene toxicity. In the concentrations 1.25 or 2.5mM of test chemical concentration, the results showed evidence of gene toxicity.It was considered that the test chemical in the concentration of 1.25 or 2.5 microM may cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation. Therefore, it was considered that the test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5 microM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

This is supported by a HGPRT assay forward assay performed to evaluate the genetic toxicity potential of the test chemical. Test material was assayed at concentrations of 0 (vehicle = deionized water), 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 10.0 ug/ml without and with metabolic activation on CHO Cells. In the first trial, the survival ranged from 105.9% to 10.7%. The second trial gave survivals ranging from 106.6% to 5.7%. Neither assay showed an increase in mutant frequency. Trials with metabolic activation were conducted at concentrations of 0, 1.0, 5.0, 10.0, 13.0, 15.0, 18.0, 20.0, 22.0 and 25.0 ug/ml. The first trial yielded survivals of 105.1% to 2.8% (at doses from 5.0 to 25.0 ug/ml). The repeat trial yielded survivals from 102.4% to 19.3% (at doses from 10.0 to 22.0 ug/ml). Neither trial showed an increase in mutant frequency. Hence, the test chemical can be considered to non- genotoxic to CHO cells both in the presence and absence of metabolic activation system.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non- genotoxic to CHO cells both in the presence and absence of metabolic activation system.

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

Additional information

Ames Assay

Various studies were reviewed to determine the mutagenic potential of the test chemical. These include the in vitro experimental results performed for the test chemicals. The results are mentioned below:

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (N.C), 0.002(T1), 0.005(T2), 0.016(T3), 0.050(T4), 0.158(T5), 0.501(T6), 1.582(T7) and 5(T8) mg/plate were selected for pre-experiment.Based on the pre-experiment results, the test item was tested with the following concentrations: 0.0 (NC), 0.050(T1), 0.158(T2), 0.501(T3), 1.582(T4) and 5(T5) mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, 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. The spontaneous reversion rates in the negative and positive controls were within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

This result is supported a bacterial reverse mutation test of the test chemical was carried out to determine its mutagenic potential. Five strains of histidine-requiring Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and tryptophan-requiring Escherichia coli WP2 uvrA 2 are used as test strains were selected for the study. The test dose was set based on the results of preliminary studies, i.e for each strain of the direct method (-S9 mix) and the metabolic activation method (+ S9 mix), 6 doses of 156 to 5000 μg / plate were set and tested. The test chemical was dissolved in distilled water for injection (Otsuka distilled water: Otsuka Pharmaceutical Factory Co., Ltd.) to prepare a stock solution. The stock solution was serially diluted with the solvent used to the specified concentration and then immediately processed (prepared before use).S9 in the S9 mix was prepared from the livers of Sprague-Dawley male rats treated with phenobarbital and 5,6-benzoflavone as inducers. 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide, Sodium azide, 9-aminoacridine, 2-Aminoanthracene were used as positive controls. These positive control substances were dissolved in DMSO, divided into small aliquots, and stored frozen (-20 ° C). According to the pre-incubation method which is an improved method of the original method of Ames et al., tests were carried out for the direct method and the metabolic activation method. In a test tube, 100 μl of solvent, test chemical solution or positive control substance solution, then 500 μl of 0.1 M sodium phosphate buffer (pH 7.4) for direct method, S9 mix for metabolic activation method 500 μl and 100 μl of the test bacterial solution were added, and the mixture was incubated at 37 ° C for 20 minutes with shaking (pre-incubation). After the culture was completed, 2 ml of top agar was added and the mixture was layered on the plate. After incubating each plate for 48 hours at 37 ° C, in order to confirm the growth inhibitory effect of the test substance on the test strain, the growth state of the test strain on the plate was observed using a stereoscopic microscope (× 60). Next, the colonies generated by the back mutation were counted. A colony analyzer was used for the measurement. The test was conducted twice independently. A positive result was obtained when the number of revertant colonies increased more than twice that of the solvent control and reproducibility or dose-dependence of the test substance was observed. No clear increase in the number of revertant colonies was observed compared with the solvent control, and reproducibility was confirmed regardless of the presence or absence of addition of rat liver microsomes (S9). On the other hand, the positive control substances in each system showed a clear mutagenic effect on each test strain. Therefore, it was judged that the test chemical does not induce gene mutation in microorganisms under the conditions of this test.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be not mutagenic to base pair changes or frame shifts in the genome of the bacterial strains used. Hence, the test chemical can be considered to be non-genotoxic in nature.

In vitro chromosomal aberration study

 

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using human lymphocytes in the presence and absence of metabolic activation system in experiment 1 of 4 hrs duration and in the absence of metabolic activation sytem in experiment 2 of 22 hours continuous duration. The test chemical was dissolved with deionized water and used at dose levels of 14.8, 25.9, 45.4, 79.4, 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 1 and 138.9, 243.1, 425.4, 744.5, 1302.9, and 2280.0 µg/mL (744.5, 1302.9, and 2280.0 µg/mL were selected for metaphase analysis) in experiment 2. About 80 hrs after seeding for each test group, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation, 50 µL S9 mix per mL medium were used. After 4 hrs, the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in "saline G". The washing procedure was repeated once. After washing the cells were resuspended in complete culture medium and cultured until preparation. In Experiment II, the culture medium was replaced with complete medium (with 10 % FCS) containing the test substance. The culture medium at continuous treatment was not changed until preparation of the cells. All cultures were incubated at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air). Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 3°C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. One-hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 ± 1 centromere regions were included in the analysis. The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. In experiment I in the presence and absence of  S9 mix, no cytotoxicity was observed up to the highest applied concnetration. In Experiment II, after continuous exposure, in the absence of S9 mix, the mitotic index was clearly reduced at the highest concentration evaluated (44.9% of the control). No chromosome aberration was however observed in both the experiments. Based on the observations made, the test chemical did not induce structural chromosomal aberrations in human lymphocytes in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

 

In another study, the test chemical was studied for its ability to induce chromosomal aberrations in the mammalian cell line used. The study was performed using Fibroblast cell line (CHL) derived from Chinese hamster lung in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in physiological saline and treated for 24 hrs, 48 hrs. Two confirmatory tests were further carried out to confirm the outcome the study. The doses for 24 and 48 hrs treatment were 0, 475, 950 or 1900µg/mL and for the confirmative test it was 0, 1000, 1300, 1600 or 1900µg/mL. Cells were seeded on a multi-plate for cell culture, and the test substance solution was treated 3 days after the culture. In the case of the continuous treatment method, the treatment is carried out continuously for 24 or 48 hours. Two hours before the end of the culture, colcemid was added to a final concentration of 0.2 μg / ml. Cells were detached by trypsin treatment, and cells were collected by centrifugation. Afterhypotonic treatment with a 75 mM potassium chloride aqueous solution, the cells were fixed with a fixing solution (3 volumes of methanol: 1 volume of acetic acid). Chromosome specimens were prepared by air drying method and then stained with 1.2% Giemsa staining solution for 12 minutes. Following treatment, 200 mid-metaphase cells were observed for gaps, chromatid breaks (ctb), chromosome breaks (csb), chromosome breaks (csb), chromosome breaks. In the case of the test chemical treated group, chromosomal structural abnormality and polyploid cell induction tendency were not observed at both the 24 hour and 48 hour treatment doses. On the other hand, marked induction of chromosomal structural abnormality was observed in the cells treated with the positive control substance MMC. In the case of the test substance treated group, the appearance frequency of chromosome structural abnormality was 5% only at high dose of 1900 μg / ml in + S 9 mix treatment, and it was judged to be false positive. As a result of conducting the confirmatory test to investigate the reproducibility, the frequency of induction of chromosomal structural abnormality was 2.5 to 3.5% in the first test, and it did not exceed 5% which is the criterion of false positives. In the second confirmatory test, a slight tendency of structural abnormality of chromosomes was observed in the test substance treated group. Based on the above test results, it was judged to be false positive with respect to chromosomal aberration induction of the test chemical to mammalian cultured cells under the present test conditions.

 

Based on the observations and applying the weight of evidence approach, it can be considered the test chemical did not induce structural chromosomal aberrations in mammalian cell line used in vitro when tested up to the highest required concentration in the absence and presence of metabolic activation.

In vitro mammalian cell gene mutation assay

The non- mutagenic nature of the test chemical was further confirmed with the help of experimental studies performed on mammalian cells. The results are as follows:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. The study was performed according to OECD 476 Guidelines. Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5 microM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested test chemical concentration, the results showed no evidence of gene toxicity. In the concentrations 1.25 or 2.5mM of test chemical concentration, the results showed evidence of gene toxicity.It was considered that the test chemical in the concentration of 1.25 or 2.5 microM may cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation. Therefore, it was considered that the test chemical in the concentration of 0, 0.625, 1.25, 2.5 or 5 microM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

This is supported by a HGPRT assay forward assay performed to evaluate the genetic toxicity potential of the test chemical. Test material was assayed at concentrations of 0 (vehicle = deionized water), 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 10.0 ug/ml without and with metabolic activation on CHO Cells. In the first trial, the survival ranged from 105.9% to 10.7%. The second trial gave survivals ranging from 106.6% to 5.7%. Neither assay showed an increase in mutant frequency. Trials with metabolic activation were conducted at concentrations of 0, 1.0, 5.0, 10.0, 13.0, 15.0, 18.0, 20.0, 22.0 and 25.0 ug/ml. The first trial yielded survivals of 105.1% to 2.8% (at doses from 5.0 to 25.0 ug/ml). The repeat trial yielded survivals from 102.4% to 19.3% (at doses from 10.0 to 22.0 ug/ml). Neither trial showed an increase in mutant frequency. Hence, the test chemical can be considered to non- genotoxic to CHO cells both in the presence and absence of metabolic activation system.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non- genotoxic to CHO cells both in the presence and absence of metabolic activation system.

Justification for classification or non-classification

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non- genotoxic when tested in vitro both in the presence and absence of exogenous metalbolic activation system. Hence, the test chemical can be classified under the category "Not Classified"as per CLP Regulation.