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Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Ames test was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in Dimethylsulfoxide (DMSO) at a dosage of 0, 3.15, 10, 31.5, 100, 315, 1000 or 3150 mg/plate (with S9) or 0, 100, 315, 945 or 3150 mg/plate (without S9). The test compound was dissolved in the incubation mixture up to the highest concentration. No indications of toxicity, such as a reduced his-background lawn, were observed. The test chemical did not induce gene mutation in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without S9 liver fractions and hence is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

In vitro mammalian chromosome aberration study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed was as per OECD guideline No. 473. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in RPMI medium and used at dose level of 0.0 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.5, 1 and 2mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferatingblood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pluverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) were determined. The test chemical did not induce an increase in the frequency of aberrations and is non-clastogenic up to 2 mg/mL, both in the presence (1 and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

In vitro mammalian cell gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. CHL cell line for the study was maintained in tissue-culture flasks RPMI 1640 medium containing glutamax TM and supplemented with 10% FCS, 100µg/mL penicillin and 100 µg/mL streptomycin. Range-finding experiments were used in order to determine suitable concentration ranges for testing and based on this 10 mM was considered as the highest test concentration. The test chemical was dissolved in sterile water or reagent-grade DMSO depending on solubility and the cell line was exposed to the test chemical at three concentration upto 10mM for 3 hrs in the presence and absence of S9 mix and for 24 hrs without S9 mix. The cell line was washed twice either by rinsing flasks containing cells growing in mono-layer. Cytochalasin B was added at wash-off and remained for the duration of the experiment. For the continuous treatments (i.e., 24 + 0 h) cytochalasin B was added 3 h after the start of treatment. After the desired treatment and recovery times, cells were harvested by centrifugation to collect TK6 cells. The cells were counted and re-suspended in an appropriate volume of culture medium to give a final concentration of 6 × 104cells/mL, and a 250-µL aliquot of each cell suspension was centrifuged in a Cytospin centrifuge onto a labeled a glass microscope slide at 1000 rpm [approximately 110Xg] for 5 min. Slides were allowed to dry in air prior to being fixed for 9 min in 90% methanol. Slides were stained for 10 s with a solution of 125 g/mL acridine orange and after further 10-min incubation in phosphate-buffered saline slides were allowed to air-dry in the dark. Prior to scoring, several drops of phosphate-buffered saline were added to the slides in order to activate the stain.The cell line was observed for gene mutation at the TK locus. Toxicity was not induced in the TK6 cells upon treatment. The test chemical did not induce gene mutation at the TK locus in CHL cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Ames test was performed to determine the mutagenic nature of the test chemical.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S-9 Mix (from liver homogenate of Aroclor 1254 treated rats)
Test concentrations with justification for top dose:
With S9 mix: 0, 3.15, 10, 31.5, 100, 315, 1000 or 3150 µg/plate
Without S9 mix: 0, 100, 315, 954 or 3150 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Dimethylsulphoxide)
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO but its role as solvent in unknown
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-methyl- N'- nitro- N- nitrosoguanidine and benzo(a)pyrene- 4,5-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): No data

DURATION
- Preincubation period: No data
- Exposure duration: 2-3 days
- Expression time (cells in growth medium): 2-3 days
- 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: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

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
Rationale for test conditions:
No data
Evaluation criteria:
Colonies with his+ revertants were counted.
Statistics:
No data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without S9 liver fractions and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

Ames test was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in Dimethylsulfoxide (DMSO) at a dosage of 0, 3.15, 10, 31.5, 100, 315, 1000 or 3150 mg/plate (with S9) or 0, 100, 315, 945 or 3150 mg/plate (without S9). The test compound was dissolved in the incubation mixture up to the highest concentration. No indications of toxicity, such as a reduced his-background lawn, were observed. The test chemical did not induce gene mutation in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without S9 liver fractions and hence is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
This in vitro assay was performed to assess the potential of the test chemical to induce structural / numerical chromosomal aberrations in one experiment (phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in phase I, a second experiment (phase II) was performed.
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration assay
Target gene:
No data
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 25-31 years age
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood was used
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- 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:
S9 metabolic activation system
Test concentrations with justification for top dose:
0.00 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI medium
- Justification for choice of solvent/vehicle: The test chemical was soluble in RPMI medium
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
RPMI medium
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time (cells in growth medium): Phase 1: 16-21 (with and without metabolic activation system)
Phase 2: 16-21 hrs (with metabolic activation system)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells):21-25 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives.

NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was assessed at the concentrations of 0.00 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL of culture media.

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

- OTHER: No data
Evaluation criteria:
A test item can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If the increase is dose-related
 Any of the results are outside the historical negative control range

A test item can be classified as non - clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If there is no dose-related increase
 All results are inside the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.
If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed
Statistics:
Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
Species / strain:
lymphocytes: Human perpheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In the cytotoxicity experiment, the highest test concentration 2 (T3) mg/ mL of culture media did not showed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: To assess the effect of test item on the pH of media at 0 h and 4 h exposure, the pH of test item in culture medium was assessed after incubation at 37 ± 2 °C. No significant change in pH was observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Precipitatio nwas not observed up to a dose leve of 2 mg/mL
- 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 cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations (0.5, 1 and 2 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding.

The cytotoxicity due to treatment with test item was assessed based on the mitotic index. The cytotoxicity experiment was conducted at the concentrations of 0.5 (T1), 1 (T2) and 2 (T3) mg/mL of culture media. A reduction in mitotic index was observed in the treated concentrations, both in the absence and in the presence of metabolic activation (1%), respectively.

In the absence of S9 mix, the mean mitotic index observed was 10.04 (NC), 9.14 (T1), 8.37 (T2), 8.69 (T3) and 8.04 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.32 (T1), 8.84 (T2), 8.84 (T3) and 7.98 (PC).

In the cytotoxicity experiment, the highest test concentration 2 (T3) mg/ mL of culture media did not showed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence these concentrations [0.5 (T1), 1 (T2) and 2 (T3) mg/mL] were selected for the main study.

Hence, 2 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

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: Please refer table remarks section

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Cytotoxicity results:

                

The cytotoxicity due to treatment with test item was assessed based on the mitotic index. The cytotoxicity experiment was conducted at the concentrations of 0.5 (T1), 1 (T2) and 2 (T3) mg/mL[pM1] of culture media. A reduction in mitotic index was observed in the treated concentrations, both in the absence and in the presence of metabolic activation (1%), respectively.

In the absence of S9 mix, the mean mitotic index observed was 10.04 (NC), 9.14 (T1), 8.37 (T2), 8.69 (T3) and 8.04 (PC). In the presence of S9 mix, the mean mitotic index observed was10.03 (NC), 9.32 (T1), 8.84 (T2), 8.84 (T3) and 7.98 (PC).

In the cytotoxicity experiment, the highest test concentration2 (T3)mg/ mLof culture mediadid not showed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence these concentrations [0.5 (T1), 1 (T2) and 2 (T3) mg/mL]were selected for the main study.

Hence, 2 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

Phase 1 results:           

In the experiment, the cultures were exposed to1,2-benzisothiazol-3(2H)-one 1,1-dioxide, sodium salt(CAS no. 128-44-9). for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%).

The mean percentage of aberrant cells was 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.333 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.333 (T3) and 10.667 (PC)in the presence of metabolic activation at the concentration of 0.00 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations.

The observed mean mitotic indexin the absence of metabolic activation were 10.03, 9.23, 8.74, 8.72 and 8.03 andin the presence ofmetabolic activation were 10.13, 9.28, 8.82, 8.83 and 8.04 for NC, T1, T2, T3 and PC concentrations respectively.

Phase 2 results:

            

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were 0.0 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL[pM2] culture both in presence and in absence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours.

The mean percent aberrant cells were 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.333 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.10, 9.53, 8.76, 8.88 and 8.03 andin the presence ofmetabolic activation were 10.04, 9.72, 8.74, 8.82 and 8.11 for NC, T1, T2, T3 and PC concentrations respectively

Conclusions:
The test chemical is non-clastogenic up to 2 mg/mL, both in the presence (1 and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

In vitro mammalian chromosome aberration study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed was as per OECD guideline No. 473. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in RPMI medium and used at dose level of 0.0 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.5, 1 and 2mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferatingblood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pluverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) were determined. The test chemical did not induce an increase in the frequency of aberrations and is non-clastogenic up to 2 mg/mL, both in the presence (1 and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mammalian cell line, other: CHL
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
containing glutamaxTM and supplemented with 10% FCS, 100 µg/mL penicillin and 100 µg/mL streptomycin
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes, TK6 cells were found to have an average generation time of 16 h and a modal chromosome number of 47.
- 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:
rat liver S-9 induced with Aroclor 1254
Test concentrations with justification for top dose:
Three concentration upto 10mM (2412 µg/mL)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile water or reagent-grade DMSO
- Justification for choice of solvent/vehicle: depending on solubility
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
sterile water or reagent-grade DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 3 hrs (With and without S9) and 24 hrs (without S9)
- Expression time (cells in growth medium): 21 hrs (with and without S9) and 24 hrs (Without S9)
- 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: All solvent-control cultures were done in quadruplicate and all treated cultures in duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity (by RI) was determined concurrently.

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

OTHER: After the desired treatment and recovery times, cells were harvested by centrifugation to collect TK6 cells. The cells were counted and re-suspended in an appropriate volume of culture medium to give a final concentration of 6 × 104 cells/mL, and a 250-µL aliquot of each cell suspension was centrifuged in a Cytospin centrifuge onto a labeled a glass microscope slide at 1000 rpm [approximately 110Xg] for 5 min. Slides were allowed to dry in air prior to being fixed for 9 min in 90% methanol. Slides were stained for 10 s with a solution of 125 g/mL acridine orange and after further 10-min incubation in phosphate-buffered saline slides were allowed to air-dry in the dark. Prior to scoring, several drops of phosphate-buffered saline were added to the slides in order to activate the stain.
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for gene mutation at the TK locus
Statistics:
The data analysed for statistical significance with Fisher’s exact test. Heterogeneity between replicate cultures was assessed by means of a binomial dispersion test.
Species / strain:
mammalian cell line, other: CHL
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
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
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Range-finding experiments were used in order to determine suitable concentration ranges for testing

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation at the TK locus in CHL cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro mammalian cell gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. CHL cell line for the study was maintained in tissue-culture flasks RPMI 1640 medium containing glutamax TM and supplemented with 10% FCS, 100µg/mL penicillin and 100 µg/mL streptomycin. Range-finding experiments were used in order to determine suitable concentration ranges for testing and based on this 10 mM was considered as the highest test concentration. The test chemical was dissolved in sterile water or reagent-grade DMSO depending on solubility and the cell line was exposed to the test chemical at three concentration upto 10mM for 3 hrs in the presence and absence of S9 mix and for 24 hrs without S9 mix. The cell line was washed twice either by rinsing flasks containing cells growing in mono-layer. Cytochalasin B was added at wash-off and remained for the duration of the experiment. For the continuous treatments (i.e., 24 + 0 h) cytochalasin B was added 3 h after the start of treatment. After the desired treatment and recovery times, cells were harvested by centrifugation to collect TK6 cells. The cells were counted and re-suspended in an appropriate volume of culture medium to give a final concentration of 6 × 104cells/mL, and a 250-µL aliquot of each cell suspension was centrifuged in a Cytospin centrifuge onto a labeled a glass microscope slide at 1000 rpm [approximately 110Xg] for 5 min. Slides were allowed to dry in air prior to being fixed for 9 min in 90% methanol. Slides were stained for 10 s with a solution of 125 g/mL acridine orange and after further 10-min incubation in phosphate-buffered saline slides were allowed to air-dry in the dark. Prior to scoring, several drops of phosphate-buffered saline were added to the slides in order to activate the stain.The cell line was observed for gene mutation at the TK locus. Toxicity was not induced in the TK6 cells upon treatment. The test chemical did not induce gene mutation at the TK locus in CHL cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo:

In vivo chromosome aberration assay was performed to determine the mutagenic nature of the test chemical. Chinese hamsters were treated with sodium saccharin at dose levels of 0 or 1.5 mg/Kg bw/day. Water was used as control. Animals in both groups were given an ip injection of pertussis vaccine, each animal receiving 0.25 ml of a suspension containing 16 x 109bacteria/ml. On days 2, 3 and 4 after the pertussis injection, water or saccharin dissolvedin water was administered by gastric intubation to animals of the control and test group, respectively.Bone-marrow cultures were taken on day 8 after the pertussis injection, a PHA injection having been given on day 6. In every culture, 50 metaphases were analysed. Selection of mitoses occurred with a low-power objective (x 10). No numerical or structural abnormalities can be seen at this magnification. The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted in every culture with a high-power oil-immersion objective. The number of polyploid cells was related to the total number of mitoses observed in the course of selection of the 50 metaphases for analysis. The total number of breaks was expressed in terms of the minimal number of breaks necessary for the formation of the total number of structural abnormalities in 50 metaphases. The test chemical did not induce a statistical significant increase in thechromosome aberrations between the control and treated group and hence the test chemical is not likely to classify as a gene mutant in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vivo chromosome aberration assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vivo chromosome aberration assay
Species:
hamster, Chinese
Strain:
not specified
Details on species / strain selection:
No data
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: No data
- Age at study initiation: 2-3 months
- Weight at study initiation: 20 g
- Assigned to test groups randomly: [no/yes, under following basis: ] No data
- Fasting period before study: No data
- Housing: No data
- Diet (e.g. ad libitum): No data
- Water (e.g. ad libitum): No data
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%):No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
- Concentration of test material in vehicle: 0 or 1.5 mg/Kg bw/day
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data
Details on exposure:
For oral route
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in water to give a dose level of 0 or 1.5 mg/Kg bw/day

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data
Duration of treatment / exposure:
Duration of exposure: 3 days
Duration of treatment: 8 days
Frequency of treatment:
3 days
Post exposure period:
5 days
Remarks:
0 or 1.5 mg/Kg bw/day
No. of animals per sex per dose:
Total: 40
0 mg/Kg bw/day: 20
1.5 mg/kg bw/day: 20
Control animals:
yes, concurrent vehicle
Positive control(s):
No data
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The absence of data on the acute toxicity of saccharin in the Chinese hamster, an LD50 equal to that in the mouse was assumed, and on this basis the dose level chosen was relatively high being 10% of the LD50 for the mouse.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: No data

METHOD OF ANALYSIS: Bone-marrow cultures were taken on day 8 after the pertussis injection, a PHA injection having been given on day 6. In every culture, 50 metaphases were analysed. Selection of mitoses occurred with a low-power objective (x 10). No numerical or structural abnormalities can be seen at this magnification. The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted in every culture with a high-power oil-immersion objective. The number of polyploid cells was related to the total number of mitoses observed in the course of selection of the 50 metaphases for analysis. The total number of breaks was expressed in terms of the minimal number of breaks necessary for the formation of the total number of structural abnormalities in 50 metaphases.

OTHER: No data
Evaluation criteria:
The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted
Statistics:
It was assumed that the observed frequencies would follow approximately a Poisson distribution. In order to obtain a normal distribution, the data were transformed according to the transformation of Freeman and Tukey. A student’s t test was applied to the transformed data. Because we were not interested in the possible decrease in chromosome aberrations by saccharin, only the probability of an increase in abnormalities by chance was tested. For this reason a one tailed test was used.
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: No mutagenic potential
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: No data
- Solubility: No data
- Clinical signs of toxicity in test animals: No data
- Evidence of cytotoxicity in tissue analyzed: No data
- Rationale for exposure: No data
- Harvest times: No data
- High dose with and without activation: No data
- Other: No data

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data
- Induction of micronuclei (for Micronucleus assay): No data
- Ratio of PCE/NCE (for Micronucleus assay): No data
- Appropriateness of dose levels and route: No data
- Statistical evaluation: No statistically significant differences were found between the control and treated groups.

Table: Number of polyploidy cells on 50 metaphases in the bone marrow of control and the test chemical treated Chinese hamsters

Polyploid cells*

No. of hamsters

X

Y

Controls

Saccharin treated

0

1

4

5

1

2.41

4

2

2

3.15

4

3

3

3.73

4

3

4

4.24

4

1

5

4.69

0

2

6

5.10

0

1

7

5.47

0

1

8

5.83

0

1

12

7.07

0

1

X: number found

Y: transformed values

The differences between the number of controls and treated hamsters with any given number of polyploidy cells were not significant: t: 0.9830 (38df); 0.10<PR<0.25

Table: Number of anueploid cells on 50 metaphases in the bone marrow of control and treated Chinese hamsters

Anueploid cells*

No. of hamsters

X

Y

Controls

Saccharin treated

0

1

7

6

1

2.41

4

1

2

3.15

4

3

3

3.73

2

3

4

4.24

3

3

5

4.69

0

4

X: number found

Y: transformed values

The differences between the number of controls and treated hamsters with any given number of anueploid cells were not significant: t: 1.2797 (38df); 0.10<PR<0.25

 

Table: Number of structural chromosome abnormalities in 50 metaphases in the bone marrow of control and treated Chinese hamsters

Structural abnormalities*

No. of hamsters

X

Y

Controls

Saccharin treated

0

1

6

7

1

2.41

3

7

2

3.15

8

3

3

3.73

0

2

4

4.24

1

1

5

4.69

2

0

X: number found

Y: transformed values

The differences between the number of controls and treated hamsters with any given number of structural chromosome abnormalities were not significant: t: 0.9576 (38df); PR<0.75

Conclusions:
The test chemical did not induce a statistical significant increase in the chromosome aberrations between the control and treated group and hence the test chemical is not likely to classify as a gene mutant in vivo.
Executive summary:

In vivo chromosome aberration assay was performed to determine the mutagenic nature of the test chemical. Chinese hamsters were treated with the test chemical at dose levels of 0 or 1.5 mg/Kg bw/day. Water was used as control. Animals in both groups were given an ip injection of pertussis vaccine, each animal receiving 0.25 ml of a suspension containing 16 x 109bacteria/ml. On days 2, 3 and 4 after the pertussis injection, water or saccharin dissolved in water was administered by gastric intubation to animals of the control and test group, respectively. Bone-marrow cultures were taken on day 8 after the pertussis injection, a PHA injection having been given on day 6. In every culture, 50 metaphases were analysed. Selection of mitoses occurred with a low-power objective (x 10). No numerical or structural abnormalities can be seen at this magnification. The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted in every culture with a high-power oil-immersion objective. The number of polyploid cells was related to the total number of mitoses observed in the course of selection of the 50 metaphases for analysis. The total number of breaks was expressed in terms of the minimal number of breaks necessary for the formation of the total number of structural abnormalities in 50 metaphases. The test chemical did not induce a statistical significant increase in thechromosome aberrations between the control and treated group and hence the test chemical is not likely to classify as a gene mutant in vivo.

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

Additional information

Gene mutation:

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

In vitro:

Ames test was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in Dimethylsulfoxide (DMSO) at a dosage of 0, 3.15, 10, 31.5, 100, 315, 1000 or 3150 mg/plate (with S9) or 0, 100, 315, 945 or 3150 mg/plate (without S9). The test compound was dissolved in the incubation mixture up to the highest concentration. No indications of toxicity, such as a reduced his-background lawn, were observed. The test chemical did not induce gene mutation in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 with and without S9 liver fractions and hence is not likely to classify as a gene mutant in vitro.

In another study by Sustainability Support Services (Europe) AB (2018), In vitro mammalian chromosome aberration study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed was as per OECD guideline No. 473. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in RPMI medium and used at dose level of 0.0 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.5, 1 and 2mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferatingblood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pluverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) were determined. 1,2-benzisothiazol-3(2H)-one 1,1-dioxide, sodium salt (CAS no. 128-44-9) did not induce an increase in the frequency of aberrations and is non-clastogenic up to 2 mg/mL, both in the presence (1 and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

In another study, Gene mutation toxicity study was performed to determine the mutagenic nature of Sodium saccharin. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 10.0 mg/plate being the maximum concentration. The chemical was dissolved in distilled water. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to negative control according to the plate incorporation test (Trial I) 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 triplicates. The test item was tested at the following concentrations i.e., 0.0 (NC), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in presence (+S9) and in absence (-S9) of metabolic activation. A significant substantial increase in revertant colony numbers were observed in the tester strain (TA 98 and TA 1537) following treatment with the test chemical both in the presence and absence of metabolic activation (S9 mix) except (TA 1535, TA 100 and TA 102). The spontaneous reversion rates in the negative and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in the Plate incorporation method. The test chemical did not induce gene mutations by base pair changes in the genome of the Salmonella typhimurium strains TA 1535, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system. It however induced gene mutation by frameshifts in the genome of strains TA 1537 and TA98 in the presence and absence of S9 metabolic activation system.

Also, In vitro mammalian chromosomal aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was mixed with HBSS and used at dose level of 0, 0.001, 0.005, 0.01, 0.02 or 0.05 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). The chromosome slide was stained in aqueous solution of 33258 Hoechst for 10 minutes, rinsed briefly in tap water, and mounted in phosphate buffer (pH 7.0) with a cover slip. The slide was exposed to an electric light (60 W, at 12-cm distance) for 1 hour. The cover slip was removed by tap water, and the slide was incubated in 1 M NaH2P04 (pH 8.0, 83-85° C) for 10 minutes, rinsed, and stained in 2.5% Giemsa (in phosphate buffer, 0.07 M, pH 7.0) for 5 minutes. Conventional Giemsa-stained slides were also prepared for scanning of chromosome aberrations. The frequency of aberrations, excluding gaps, was indicated by the number of breaks per cell. A ring, a dicentric, and a chromatid exchange were each scored as two breaks, a tricentric as four breaks, and an acentric fragment or an isochromatid break as one break. The test chemical did not induce chromosome aberration in pseudo-diploid Chinese hamster cell line (Don) upto a dose level of 0.01M. It however induced chromosome aberration at 0.05 M (10300 µg/mL).The aberrations at this dose included not only simple chromatid breaks but also a number of dicentrics and chromatid interchanges that yielded a threefold to six fold increased rate of breaks as compared to the control value. However, effective dose of the test chemical estimated is probably much higher than the doses taken in our daily life and hence is considered to be negative for gene mutation.

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 8.0 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical induced chromosomal aberration in chinese hamster fibroblast cell line CHL in the 48 hrs study. Also positive results were noted at 12.0 mg/mL at 24 hrs (25%).

In vitro mammalian cell gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. CHL cell line for the study was maintained in tissue-culture flasks RPMI 1640 medium containing glutamax TM and supplemented with 10% FCS, 100µg/mL penicillin and 100 µg/mL streptomycin. Range-finding experiments were used in order to determine suitable concentration ranges for testing and based on this 10 mM was considered as the highest test concentration. The test chemical was dissolved in sterile water or reagent-grade DMSO depending on solubility and the cell line was exposed to the test chemical at three concentration upto 10mM for 3 hrs in the presence and absence of S9 mix and for 24 hrs without S9 mix. The cell line was washed twice either by rinsing flasks containing cells growing in mono-layer. Cytochalasin B was added at wash-off and remained for the duration of the experiment. For the continuous treatments (i.e., 24 + 0 h) cytochalasin B was added 3 h after the start of treatment. After the desired treatment and recovery times, cells were harvested by centrifugation to collect TK6 cells. The cells were counted and re-suspended in an appropriate volume of culture medium to give a final concentration of 6 × 104cells/mL, and a 250-µL aliquot of each cell suspension was centrifuged in a Cytospin centrifuge onto a labeled a glass microscope slide at 1000 rpm [approximately 110Xg] for 5 min. Slides were allowed to dry in air prior to being fixed for 9 min in 90% methanol. Slides were stained for 10 s with a solution of 125 g/mL acridine orange and after further 10-min incubation in phosphate-buffered saline slides were allowed to air-dry in the dark. Prior to scoring, several drops of phosphate-buffered saline were added to the slides in order to activate the stain.The cell line was observed for gene mutation at the TK locus. Toxicity was not induced in the TK6 cells upon treatment. The test chemical did not induce gene mutation at the TK locus in CHL cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

To further support the non-mutagenic nature of the chemical, Unscheduled DNA synthesis was performed to determine the mutagenic nature of the test chemical. The study was performed using rat hepatocytes at dose levels of 1 X 10-4to 1 X 10-1M. The test chemical was toxic at dose levels of 3.16 X 103to 1 X 10-1M to the hepatocyte cells. Decreasing concentrations of sodium saccharin or 1-naphthalene sulphonic acid resulted in decreased toxicity and the culture exposed to 1 × 10-3to 1 × 10-4M concentrations resembled negative controls. The test chemical did not induce unscheduled DNA synthesis in cultured rat hepatocytes and hence the test chemical is not likely to be mutagenic in vitro.

In vitro cytogenicity study was performed to determine the mutagenic nature of the test chemical. CHL cell line for the study was maintained in tissue-culture flasks containingMcCoy’s 5A medium with 1% (v/v) l-glutamine, supplemented with 10% (v/v) foetal calf serum, and 100 U/mL penicillin and 100µg/mL streptomycin. Range-finding experiments were used in order to determine suitable concentration ranges for testing and based on this 10mM was considered as the highest test concentration. The test chemical was dissolved in sterile water or reagent-grade DMSO depending on solubility and the cell line was exposed to the test chemical at three concentration upto 10mM for 3 hrs in the presence and absence of S9 mix and for 24 hrs without S9 mix. The cell line was washed twice either by rinsing flasks containing cells growing in mono-layer. Cytochalasin B was added at wash-off and remained for the duration of the experiment. For the continuous treatments (i.e., 24 + 0 h) cytochalasin B was added 3 h after the start of treatment. After the desired treatment and recovery times, cells were harvested by either removing the monolayer with trypsin/EDTA. The cells were counted and re-suspended in an appropriate volume of culture medium to give a final concentration of 6 × 104cells/mL, and a 250-µL aliquot of each cell suspension was centrifuged in a Cytospin centrifuge onto a labeled a glass microscope slide at 1000 rpm [approximately 110Xg] for 5 min. Slides were allowed to dry in air prior to being fixed for 9 min in 90% methanol. Slides were stained for 10 s with a solution of 125 g/mL acridine orange and after further 10-min incubation in phosphate-buffered saline slides were allowed to air-dry in the dark. Prior to scoring, several drops of phosphate-buffered saline were added to the slides in order to activate the stain. One thousand binucleate cells per replicate (i.e., 2000 cells from each treatment condition) were analysed (where possible) for the presence of MN.Binucleate cells were only included in the analysis if the cytoplasm remained essentially intact and the daughter nuclei were of approximately equal size. A micronucleus was recorded if it had the same staining characteristics and a similar morphology to the main nucleus, was located separately in the cytoplasm, was smooth-edged, and had less than approximately one third the diameter of the main nucleus.An experiment was considered valid if there was no evidence of heterogeneity and if the ratio of binucleate and multinucleate cells vs mononucleate cells was at least 0.6 in solvent-control cultures.The test chemical did not induce micronucleus formation in CHL cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, treatment of WI-38 cells with the test chemical did not induce DNA repair synthesis nor did it specifically inhibit the repair of DNA damage induced by UV light. These results therefore do not support a mechanism involving the direct induction of DNA damage by sodium saccharin and hence the test chemical is not likely to classify as a gene mutant in vitro.

Sister chromatid exchange assay was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in HBSS and used at dose levels of 0.001, 0.002, 0.01, 0.02 or 0.05 M using pseudo-diploid Chinese hamster cell line (Don). Three hours after 1.0-1.2 X 106 cells per TD-40 culture bottle were seeded, BUdR (1µg/ml) and test chemical was added to the cultures under an ordinary yellow darkroom safety lamp. Concurrent solvent control was also included in the study. All cultures were kept in complete darkness at 37° C for 26 hours (this covered two rounds of cell cycle), and 0.25µg colchicine/ml was added for the final 2 hours. The cells were collected by scraping them with a rubber policeman and prepared air-dried slides following hypotonic treatment and fixation in ice-cold methanol: acetic acid (3: 1). Sister chromatids were differentiated by the fluorescence or Giemsa staining techniques. The acridine orange technique was used for fluorescence, and a modified FPG technique was used for Giemsa staining. Taking the existence of a dosage effect greater than two fold background as a criterion, the frequency of SCE remained at about twice the background value for all concentrations shown. Based on these considerations, the test chemical did not induce SCEs in pseudo-diploid Chinese hamster cell line (Don) and hence it is not likely to classify as a gene mutant in vitro.

In yet another Ames test , the test chemical in distilled water from doses 0.5-50 mg/plate was not mutagenic in Salmonella typhimurium strains TA97 and TA 102 with and without addition of S9 liver fractions from Aroclor induced rats.

In vitro micronucleus test was also performed to determine the mutagenic nature of the resr chemical. The study was performed using Chinese Hamster Ovary-K1 (CHO-K1). The test chemical was dissolved in aquabidest sterile and used at dose levels of 0, 100 or 500µM. The doses were based on the preliminary cytotoxicity study performed from 5-500µg/mL. CHO-K1 cells were grown in the 24-well plate for a 24-h before the treatments. The treatments were carried out as follows: (a) untreated, (b) DNA damage-inducing agents (DOXO), (c) treatment of SS.CHO-K1 cells were first stained with a photoactivated dye 1 EMA and then washed, lysed, and stained with lysis buffer containing RNase, nucleic acid dye 2 (SYTOX Green). DNA from apoptotic/ necrotic cells with compromised cell membranes was labeled with both EMA and SYTOX Green, which can be distinguished from EMA-negative and SYTOX Green-positive MN. Samples were analyzed with BD Accuri C6 flow cytometer. The incidence of MN was determined through the acquisition of 10.000 healthy nuclei (EMA-/SYTOX+) per well. In addition, the SYTOX fluorescence histogram showed the cell cycle progression profile and the hypodiploid gate was used to determine whether aneuploidy was induced. Analysis of micronucleus (MN) frequency was revealed by flow cytometric analysis based on the Microflow assay. However, the test chemical did not induce cytotoxic effect on proliferative cells and does not induce genotoxic effect on in vitro model system and hence it is not likely to classify as a gene mutant in vitro.

In vivo:

In vivo chromosome aberration assay was performed to determine the mutagenic nature of the test chemical. Chinese hamsters were treated with the test chemical at dose levels of 0 or 1.5 mg/Kg bw/day. Water was used as control. Animals in both groups were given an ip injection of pertussis vaccine, each animal receiving 0.25 ml of a suspension containing 16 x 109bacteria/ml. On days 2, 3 and 4 after the pertussis injection, water or saccharin dissolvedin water was administered by gastric intubation to animals of the control and test group, respectively.Bone-marrow cultures were taken on day 8 after the pertussis injection, a PHA injection having been given on day 6. In every culture, 50 metaphases were analysed. Selection of mitoses occurred with a low-power objective (x 10). No numerical or structural abnormalities can be seen at this magnification. The total numbers of aneuploidy cells, polyploid cells and structural abnormalities were noted in every culture with a high-power oil-immersion objective. The number of polyploid cells was related to the total number of mitoses observed in the course of selection of the 50 metaphases for analysis. The total number of breaks was expressed in terms of the minimal number of breaks necessary for the formation of the total number of structural abnormalities in 50 metaphases. The test chemical did not induce a statistical significant increase in the chromosome aberrations between the control and treated group and hence the test chemical is not likely to classify as a gene mutant in vivo.

In another study, Drosophila Wing Spot assay was performed to determine the mutagenic nature of the test chemical. ORR; mwh females and ORR; flr3/TM3 males were outcrossed. Eggs from optimally fertile parents were collected on standard Drosophila food for 8 h. After 2 and 3 days, i. e., at 48±4 hours and 72±4 hours of age, the larvae were washed out from the food with 20% NaCl solution. Approximately 100 larvae of each age group were transferred into vials containing 1.5 g instant medium, rehydrated with 5 mL of the solvent containing a known concentration of the test compound. Two independent experiments were conducted for each larval stage of 48 and 72 hrs respectively.The wings of the enclosing flies were mounted in Faure's solution and screened under a compound microscope at 400 X magnification. The wing spots (clones) were classified either as singles (mwh or flr3) or twins (mwh// flr3). The single spots were further classified as small single spots, 1-2 cells in size (S = 1-2), and large single spots, larger than 2 cells in size (S > 2). There was no significant increase above control leve1 in the spot frequency at any of the test concentrations of sodium saccharin. The test chemical did not induce a statistical significant increase in thespot frequencies between the control and treated group and hence the test chemical is not likely to classify as a gene mutant in vivo.

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutation in vitro and in vivo. Although mutagenic effects have been noted in the Ames test study mentioned above in the in vitro section, but the key study for chromosome aberration in vitro and the supporting in vitro and in vivo studies suggest the non-mutagenic nature of the test chemical in vitro and in vivo.

Justification for classification or non-classification

Based on the data available for the target chemical, the test chemical does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutation in vitro and in vivo. Although mutagenic effects have been noted in the Ames test study mentioned above in the in vitro section, but the key study for chromosome aberration in vitro and the supporting in vitro and in vivo studies suggest the non-mutagenic nature of the test chemical in vitro and in vivo.