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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES Assay

In vitro genetic toxicity study was performed to determine the mutagenic nature of the test chemical. Strains TA100 and TA98 of Salmonella typhimirium were used as the tester strains. Assay was carried out as described by Ames et al. with some modifications. The test chemical was freshly dissolved in 100 µl of DMSO were pre-incubated at 37°C for 20 min with 0.5 ml of S-9 mix or 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 ml of bacterial culture. Two ml of molten soft agar at 45°C were added, and the resulting mixture was poured over 25 ml of minimal-glucose agar containing 0.1 µmol of L-histidine and 0.1 µmol of biotin. After 2-day incubation, colonies of histidine prototroph were counted as revertants.The test chemical was weakly mutagenic to both strains with S-9 mix and non mutagenic to both strains in the absence of S9 mix. Though there was weak mutagenic activity in the presence of S9 mix but dose dependent decrease in number of revertants was observed. Hence, the test chemical is considered to be non-mutagenic both in the presence and absence of S9 mix to S.Typhimurium TA 98 and TA 100 strains.

In vitro chromosomal aberration

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic when tested in an in vitro chromosomal aberration study.

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 mutagenicboth in the presence and absence of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic in nature.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 journals
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
Bacterial reverse mutation assay was performed to evaluate the toxic nature of the test chemical on Salmonella typhimurium TA98 and TA100 strains.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: Salmonella typhimurium TA100 and TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Base-pair-change mutant and frameshift mutant. Both strains have the R-factor of pKM101 as a plasmid
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: The S-9 fraction was prepared aseptically by centrifugation of the liver homogenate (25% in 0.15 M KCl) at 9000 g for 10 min as described by Ames et al.
- source of S9 : Male Sprague-Dawley rats weighing 100-120 g were injected intraperitoneally with polychlorinated biphenyl (Kanechlor 500) at a dose of 50 mg/100 g body weight, five days before they were killed
- concentration or volume of S9 mix and S9 in the final culture medium : S-9 mix contained 50 µmol of sodium phosphate buffer (pH 7.4), 4 µmol of MgCl2, 16.5 µmol of KCI, 2.5µmol of glucose-6-phosphate, 2 µmol of NADH, 2 µmol of NADPH, 2.5 µmol of ATP and 150µmol of S-9 fraction in a total volume of 0.5 ml.
Test concentrations with justification for top dose:
0, 0.5 or 1.0 µmoles/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was stable and soluble in DMSO.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : no data available
- Number of independent experiments : no data available

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: 2 days
- Harvest time after the end of treatment (sampling/recovery times):
Rationale for test conditions:
No data
Evaluation criteria:
After 2-day incubation, colonies of histidine prototroph were counted as revertants.
Statistics:
No data
Species / strain:
other: Salmonella typhimurium TA100 and TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: S. typhimurium TA 100, S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Ames test: The test chemical was weakly mutagenic to both strains with S-9 mix and non mutagenic to both strains in the absence of S9 mix. Though there was weak mutagenic activity in the presence of S9 mix but dose dependent decrease in number of revertants was observed. Hence, we consider the test chemical can be considered to be non-mutagenic both in the presence and absence of S9 mix to S.Typhimurium TA 98 and TA 100 strains.
Remarks on result:
other: non-mutagenic
Conclusions:
The test chemical was weakly mutagenic to both strains with S-9 mix and non mutagenic to both strains in the absence of S9 mix. Though there was weak mutagenic activity in the presence of S9 mix but dose dependent decrease in number of revertants was observed. Hence, the test chemical can be considered to be non-mutagenic both in the presence and absence of S9 mix to S.Typhimurium TA 98 and TA 100 strains.
Executive summary:

In vitro genetic toxicity study was performed to determine the mutagenic nature of the test chemical. Strains TA100 and TA98 of Salmonella typhimirium were used as the tester strains. Assay was carried out as described by Ames et al. with some modifications. The test chemical was freshly dissolved in 100 µl of DMSO were pre-incubated at 37°C for 20 min with 0.5 ml of S-9 mix or 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 ml of bacterial culture. Two ml of molten soft agar at 45°C were added, and the resulting mixture was poured over 25 ml of minimal-glucose agar containing 0.1 µmol of L-histidine and 0.1 µmol of biotin. After 2-day incubation, colonies of histidine prototroph were counted as revertants. The test chemical was weakly mutagenic to both strains with S-9 mix and non mutagenic to both strains in the absence of S9 mix. Though there was weak mutagenic activity in the presence of S9 mix but dose dependent decrease in number of revertants was observed. Hence, the test chemical can be considered to be non-mutagenic both in the presence and absence of S9 mix to S.Typhimurium TA 98 and TA 100 strains.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
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 chromosome aberration test
Target gene:
no data available
Species / strain / cell type:
lymphocytes: human
Remarks:
Study 6
Details on mammalian cell type (if applicable):
For lymphocytes:
- Sex, age and number of blood donors: Blood was obtained from a single individual
with no report of any infection or of exposure to drugs and radiation for at least 30 days prior to donating blood.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Study 7
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
CHO cells were cloned at Litton Bionetics Inc. and supplied to the other laboratories
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
Cells were routinely checked for mycoplasma contamination; the results of these analyses disclosed no evidence of mycoplasma contamination
- Number of passages if applicable:
Cells were not used beyond 15 passages after cloning.
- Methods for maintenance in cell culture: C
ells for experiments were thawed and grown in McCoys 5A medium supplemented with antibiotics and 10% fetal calf serum at 37°C using 5% CO2.
Metabolic activation:
with and without
Metabolic activation system:
6. Type and composition of metabolic activation system: Aroclor-1254-induced post-mitochondrial supernatant (S9) from the liver of Sprague-Dawley rat (protein content 38.5 mg/ml), obtained from Moltox, was used for metabolic activation
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium : For the in vitro chromosomal aberration test the final protein concentration of S9 mix in the culture was 0.75 mg/ml.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
7. Type and composition of metabolic activation system:
The mixture consisted of supernatant from the 9000g fraction of the livers from Aroclor 1254-induced male Sprague Dawley rats, NADP, and isocitrate in serum-free medium..
- source of S9
:
- method of preparation of S9 mix
:
- concentration or volume of S9 mix and S9 in the final culture medium
: The final concentration of S9 in the medium was either 15 (COL, LBI) or 20 (BSC, EHR) microliter/ml.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Test concentrations with justification for top dose:
6. Experiment 1= 0 (control), 156. 312, 625 microgram
Experiment 2= 0 (control), 78, 156, 312 microgram
Experiment 3 = 0 (control), 156, 312, 625 microgram
7. 0, 100, 125, 150 microgram/ml
LED 75, HDT- 150 microgram/ml
LED (lowest effective dose) is listed for positive and weak positive responses and HDT (highest dose tested) is listed for negative and equivocal responses.
Vehicle / solvent:
6. - 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)] : DMSO

- Justification for choice of solvent/vehicle:
7.
- 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)]
: DMSO

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium: The concentration of the solvent in the treatment flasks did not exceed 1%
- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Study 6
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Study 7
Details on test system and experimental conditions:
6. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicates
- Number of independent experiments: 3

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. : Colcemid (0.2 μg/ml) was added to each culture tube 3 h prior to harvesting. Cultures were centrifuged for 10 min at 1500 rpm, cell pellets were resuspended in hypotonic solution (0.0375 M KCl), and the tubes were left in a water bath at 37°C for 15 min.
- 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): Cells were dropped over clean, chilled slides, air dried, and stained with 5% Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): The culture volume was maintained at 5 ml and employed in duplicate for each exposure. Cells were centrifuged again and fixed 3–4-times in fresh, chilled Carnoy’s fixative (ethanol:acetic acid, 3:1). Cells were dropped over clean, chilled slides, air dried, and stained with 5% Giemsa.
7. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
: duplicates
- Number of independent experiments
: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Approximately 24 hr before treatment, cells were initiated at a density of 1.2-1.75 x106 cells/ 75 cm2 flask
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
:

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.
: For the trials without S9, the cells were incubated with the appropriate control or chemical for 8 hr. The cells were then washed and Colcemid added for a 2-2.5 hr exposure. For the trials with S9, the cells were treated with S9 and test
chemical in serum-free medium for 2 hr, washed, resuspended in medium containing serum, and incubated for an additional 8-10 hr, with Colcemid present for the final 2 hr.
- 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):
Cells were harvested by mitotic shake-off and stained with Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
Cells with good morphology and with a chromosome number of 21+/- 2 were selected for analysis. With the exception of the: high-dose positive controls, all slides were scored coded. 200 cells per dose were scored. Fewer cells were scored if a strong response was observed
or a dose was very toxic. For the ABS assay, cells were scored for “simple” (chromatid gaps and breaks, fragments, deletions, chromosome gaps and breaks, and double minutes), “complex” (interstitial deletions, triradials, quadriradials, rings, and dicentrics), and ‘other’ (pulverized, polyploids, and endoreduplications) aberrations. These categories were combined to form the category “total”.
- 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):
The percent of cells with aberrations, rather than aberrations per cell, were analyzed
so as to avoid distorting the data for cases where a small number of cells had a large number of aberrations.
- Determination of polyploidy:
- Determination of endoreplication:
Gaps and endoreduplications were scored but not tabulated in the totals or included in the statistical analyses.


- OTHER: The pH of the medium was monitored and if a change was observed, medium containing HEPES buffer was used and the cultures were then incubated in sealed flasks.
Evaluation criteria:
6. A hundred well-spread metaphase plates per culture were scored for cytogenetic damage. Only metaphases with 46 ± 2 centromere regions were included in the analysis. Breaks, fragments, deletions, exchanges, and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded, but separately included in the calculation.
7. if a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal (?); if a trial had significant trend and one significant dose it was judged weak positive (+ W); and if the trial had two significant doses it was judged positive (+), whether or not a positive trend was obtained. In the ABS trials, if only one dose was significant and the increase over the control was P < 0.0005 the trial was denoted (+ W*).
Statistics:
6. Statistical analysis was determined using GraphPad Prism 3.0 software wherever applicable. The mean numbers of revertant colonies for all treatment groups were compared with their respective control values. For chromosomal aberration assay, cells were observed using a Leica (Germany) microscope with 100× oil immersion objectives. One hundred well-spread metaphase plates per culture were scored for cytogenetic damage. Only metaphases with 46 ± 2 centromere regions were included in the analysis. Breaks, fragments, deletions, exchanges, and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded, but separately included in the calculation. To describe a cytotoxic effect the mitotic index (percentage of cells in mitosis) was determined in 1000 cells. In addition, the number of polyploid cells in 200 metaphase cells was scored. Statistical significance in structural aberrations was analyzed by Fisher’s exact test.
7. A binomial sampling assumption was used to evaluate an absolute increase in aberrations over the solvent control. Dose points with P values adjusted by Dunnett’s method were considered significant if (0.05, whereas a trend of P < 0.003 was significant.
Species / strain:
lymphocytes: human
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:
6. Chromosome aberration test (CA) in mammalian cells: The exposure to the test chemical could not induce significant structural aberrations, even with the highest concentration of 625 μg in 4 h and 300 μg in 22 h, with or without metabolic activation. In contrast significant (p < 0.01) increase in chromosomal aberrations were observed with positive controls, both in the absence and presence of metabolic activation.
7. Chromosome aberration test (CA) in mammalian cells: The test chemical can be considered to be non mutagenicboth in the presence and absence of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic in nature.
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 mutagenic both in the presence and absence of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic in nature.
Executive summary:

Various studies have been reviewed to determine the mutagenic potential of the test chemical in vitro. These include in vitro experimental studies performed on various mammalian cell lines for the test chemical. The results are mentioned below:

The genotoxicity profile of the test chemical was evaluated in an in vitro chromosomal aberration study.The method used for the in vitro chromosomal aberration assay was based on the OECD 473 Guidelines. The whole blood culture method (Moorehead et al. 1960) was used for the chromosomal aberration assay. Blood was obtained from a single individual with no report of any infection or of exposure to drugs and radiation for at least 30 days prior to donating blood. 0.5 ml blood, obtained by venipuncture through heparinized syringe, was added to DMEM+Ham-F12 medium supplemented with 20% FBS, 2% PHA, penicillin (100 IU/ml), and streptomycin (100 μg/ml). Cultures were gently mixed and incubated (CO2 5%) at 37°C for 72 h. Different sets of experimental cultures, 4 h and 22 h without metabolic activations and 4 h with metabolic activation were exposed to various test concentrations of the test chemical. The culture volume was maintained at 5 ml and employed in duplicate for each exposure. Colcemid (0.2 μg/ml) was added to each culture tube 3 h prior to harvesting. Cultures were centrifuged for 10 min at 1500 rpm, cell pellets were resuspended in hypotonic solution (0.0375 M KCl), and the tubes were left in a water bath at 37°C for 15 min. Cells were centrifuged again and fixed 3–4-times in fresh, chilled Carnoy’s fixative (ethanol:acetic acid, 3:1). Cells were dropped over clean, chilled slides, air dried, and stained. Statistical analysis was determined using GraphPad Prism 3.0 software wherever applicable. The mean numbers of revertant colonies for all treatment groups were compared with their respective control values. For chromosomal aberration assay, cells were observed using a Leica (Germany) microscope with 100× oil immersion objectives. One hundred well-spread metaphase plates per culture were scored for cytogenetic damage. Only metaphases with 46 ± 2 centromere regions were included in the analysis. Breaks, fragments, deletions, exchanges, and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded, but separately included in the calculation. To describe a cytotoxic effect the mitotic index (percentage of cells in mitosis) was determined in 1000 cells. In addition, the number of polyploid cells in 200 metaphase cells was scored. Statistical significance in structural aberrations was analyzed by Fisher’s exact test. The exposure to the test chemical could not induce significant structural aberrations, even with the highest concentration of 625 μg in 4 h and 300 μg in 22 h, with or without metabolic activation. In contrast significant (p < 0.01) increase in chromosomal aberrations were observed with positive controls, both in the absence and presence of metabolic activation.

This result is supported by another assay where the test chemical was tested for their ability to induce cytogenetic change in Chinese hamster ovary cells. CHO cells were cloned at Litton Bionetics Inc. and supplied to the other laboratories. Cells were not used beyond 15 passages after cloning. Cells for experiments were thawed and grown in McCoys 5A medium supplemented with antibiotics and 10% fetal calf serum at 37°C using 5% CO2. Cells were routinely checked for mycoplasma contamination; the results of these analyses disclosed no evidence of contamination. Approximately 24 hr before treatment, cells were initiated at a density of 1.2-1.75 x106 cells/ 75 cm2 flask. For the trials without S9, the cells were incubated with the appropriate control or chemical for 8 hr. The cells were then washed and Colcemid added for a 2-2.5 hr exposure. For  the trials with S9, the cells were treated with S9 and test chemical in serum-free medium for 2 hr, washed, resuspended in medium containing serum, and incubated for an additional 8-10 hr, with Colcemid present for the final 2 hr.Cells were harvested by mitotic shake-off and stained with Giemsa. Cells with good morphology and with a chromosome number of 21+/- 2 were selected for analysis. With the exception of the: high-dose positive controls, all slides were scored coded. 200 cells per dose were scored. Fewer cells were scored if a strong response was observed or a dose was very toxic. For the ABS assay, cells were scored for “simple” (chromatid gaps and breaks, fragments, deletions, chromosome gaps and breaks, and double minutes), “complex” (interstitial deletions, triradials, quadriradials, rings, and dicentrics), and ‘other’ (pulverized, polyploids, and endoreduplications) aberrations. These categories were combined to form the category “total”. The percent of cells with aberrations, rather than aberrations per cell, were analyzed so as to avoid distorting the data for cases where a small number of cells had a large number of aberrations. Mitomycin C (MMC) was the positive control in the trials without S9 and cyclophosphamide (CPA) was used in the trials with S9. A binomial sampling assumption was used to evaluate an absolute increase in aberrations over the solvent control. Dose points with P values adjusted by Dunnett’s method were considered significant if (0.05, whereas a trend of P < 0.003 was significant. if a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal (?); if a trial had significant trend and one significant dose it was judged weak positive (+ W); and if the trial had two significant doses it was judged positive (+), whether or not a positive trend was obtained. In the ABS trials, if only one dose was significant and the increase over the control was P < 0.0005 the trial was denoted (+ W*). The test chemical can be considered to be non mutagenicboth in the presence and absence of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic in nature.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenic both in the presence and absence of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic in nature.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
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:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
Study 9
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y mouse lymphoma cells, clone 3.7.2C, heterozygous at the tk locus, were originally obtained from Dr. Donald Clive (then of Burroughs Wellcome Laboratories, Research Triangle Park, NC).
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture: The stock cells were grown as a suspension culture under exponential growth conditions and treated with methotrexate (0.1 mg/ml) in the presence of THG (3 mg/ml thymidine, 5 mg/ml hypoxanthine, and 7.5 mg/ml glycine) prior to mutagenesis experiments to eliminate cells lacking thymidine kinase activity.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
Study 10
Details on mammalian cell type (if applicable):
- Species/cell type: CHO-K1-BH4
Metabolic activation:
with and without
Metabolic activation system:
9. Type and composition of metabolic activation system: Aroclor 1254–induced Sprague–Dawley rat liver S9 was obtained from Molecular Toxicology (Boone, NC) and contained approximately 42.5 microgram/ml protein.
- source of S9 : Aroclor 1254–induced Sprague–Dawley rat liver S9 was obtained from Molecular Toxicology (Boone, NC) and contained approximately 42.5 mg/ml protein.
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium : The Metabolic activation mixture was prepared with cofactors and S9 fraction at a concentration of either 4 or 10%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
10. Metabolic activation system: Arochlor-1254-induced male rat liver homogenate
Test concentrations with justification for top dose:
9. Cytotoxicity Assay - with and without S9- 0.38, 0.75, 1.5, 3.0, 6.0, 12, 24, 48,96,193, 385, 770, 1540, 3080, 6160 microgram/ml
Main study - Without and with S9 - 31, 39, 49, 61, 77, 96 microgram/ml
10. with and without S9 100, 125, 150, 200 and 250 ug/mL; additionally with S9 62.5 and 75 ug/mL
Vehicle / solvent:
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)] : 10% water for cytotoxicity assay as well as main study

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:
10. - 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)] : DMSO

- 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:
WATER
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
methylmethanesulfonate
Remarks:
study 9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
other: 5-Bromo 2'deoxyuridine (-S9), 3-methylcholanthrene (+S9) were used as positive controls
Remarks:
Study 10
Details on test system and experimental conditions:
9. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Duplicates
- Number of independent experiments : 2 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: 4 hours
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION
- Expression time (cells in growth medium between treatment and selection): Cell growth was monitored daily over a 2-day expression period by using a Coulter counter.
- Selection time (if incubation with a selective agent): On the second day of the expression period, about 3x106 cells were seeded in CM+ TFT for selection of TFT cells and about 600cells were seeded in nonselective CM to determine the percentage of viable cells.
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:
10. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
: one
- Number of independent experiments: single

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


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
After incubation for 7–8 days, the surviving colonies were counted, and the relative initial cell survival was calculated as compared to the untreated control (relative survival). For phenotypic expression of induced mutations, 1.5 × 106 treated cells were plated into one 150 mm dishes and were subcultured on day two or three and again on day five following the same procedure. At the end of the expression period (6–7 days), cultures were reseeded at 2 × 105 cells per 100mm dishes in mutant selection medium using twelve dishes per culture. In parallel, triplicate cultures of 200 cells were seeded in 60mm dishes to determine the cloning efficiency. Ten days later, the dishes were fixed, stained and scored.
Evaluation criteria:
10. Colonies were counted by eye. Mutant frequencies were calculated as TG-resistant mutant colonies per 106 clonable cells.
Statistics:
9. Statistical analyses were performed using the SAS analysis system (version 6.11; SAS Institute, Cary, NC), running on a Pentium 100 computer: Levene’s test [Levene, 1960] was performed to determine whether a significant difference existed among treatment variances; treatments were compared with controls by using a one-tailed Dunnett’s t-test [Dunnett, 1980]
10. Kastenbaum and Baumann method were used to evaluate the results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
150 microgram/ml.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
9. 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 cytotoxicity experiments were conducted using mouse lymphoma cell cultures treated for 4 hr with the test chemical. Dose-related increases in cytotoxicity were observed under both activation conditions. Based on the cytotoxicity observed in the range-finding experiments, concentrations of the test chemical ranged from 31 to 96 microgram/ml both with and without metabolic activation.

Gene mutation tests in mammalian cells: No statistically significant (P <0.05 by Dunnett’s analysis) increases in mutation frequency were observed at any acceptable concentration under either metabolic activation condition in any of the experiments. The numbers of small colonies in the cultures treated with the test chemical were similar to those in the solvent control cultures.

- Results from cytotoxicity measurements: In the first mutagenesis experiment with the test chemical, greater cytotoxicity was observed without metabolic activation than with metabolic activation. Therefore, the maximum concentration of the test chemical for the replicate mutagenesis experiment with the test chemical was increased to 150 microgram/ml.
10. 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):


STUDY RESULTS
- Concurrent vehicle negative and positive control data
: The number of mutants remained within (negative) control ranges with the exception of the number of mutants in the lowest dose tested with S9-mix. Positive controls gave the expected results

- Genotoxicity results
:
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
CYTOTOXICITY (% of control survival) at the highest tested concentration:
- With metabolic activation: 0.4% at 250 ug/mL ; - Without metabolic activation: 20% at 250 ug/mL
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
The increased number of mutants seen at 62.5 ug/mL in the assay with metabolic activation was considered to be not relevant, since no concentration effect relationship was observed.
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
Remarks on result:
other: not mutagenic

9. Confirmation of Test Chemical Concentration

All concentrations for all tests were confirmed before testing by high performance liquid chromotography (HPLC). Duplicate samplings of all dose solutions were diluted with water or methanol and HPLC mobile phase buffer to a concentration within the range of the standard curve. All dose preparations were found to be within 610% of their nominal labeled values.

Conclusions:
Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non-mutagenic when tested in vitro on mammalian cells.
Executive summary:

Various studies have been reviewed to determine the mutagenic potential of the test chemical in vitro. These include in vitro experimental studies performed on various mammalian cell lines for the test chemical. The results are mentioned below:

A mouse lymphoma assay was performed to determine the mutagenic potential of the test chemical. L5178Y mouse lymphoma cells, clone 3.7.2C, heterozygous at the tk locus, were used for the study. L5178Y mouse lymphoma cells were routinely cultivated in RPMI 1640 medium supplemented with 0.1% Pluronic F68, 0.22 mg/ml sodium pyruvate (R0P), and 10% heat-inactivated donor horse serum (R10P). The serum concentration was reduced to 5% (R5P) for the exposure medium. Cloning medium (CM) consisted of RPMI 1640 medium containing the same concentrations of sodium pyruvate and horse serum as R10P, plus BBLpurified- agar (final concentration ; 0.3%). The selective cloning medium contained 5 mg/ml trifluorothymidine (TFT). Antibiotics (31 microgram/ml penicillin and 50 microgram/ml streptomycin sulfate) were added to the culture media used for the mutagenesis assays but not to the stock cell cultures. All cells were incubated at 37°C in the presence of 5% CO2 in air. Preliminary cytotoxicity experiments were conducted using mouse lymphoma cell cultures treated for 4 hr with the test chemical.  Dose-related increases in cytotoxicity were observed under both activation conditions. Based on the cytotoxicity observed in the range-finding experiments, concentrations of  the test chemical ranged from 31 to 96 microgram/ml both with and without metabolic activation. Cultures with or without the metabolic activation mixture were treated with the appropriate chemical stock solution for a 4-hr exposure period, after which the treatment solutions were removed and replaced with fresh R10P. Cell growth was monitored daily over a 2-day expression period by using a Coulter counter. On the second day of the expression period, about 3x106 cells were seeded in CM+ TFT for selection of TFT cells and about 600 cells were seeded in non-selective culture medium to determine the percentage of viable cells. After incubation for 11 to 12 days in a humidified atmosphere containing 5% CO2 in air, colonies were counted using an Artek Model 880 automatic colony counter with a standard 50-mm lens. It was previously reported that the induction of gross aberrations correlates with the induction of small-colony tk mutants [Moore and Doerr, 1990]. To obtain information about the relative numbers of the large (>0.6 mm in diameter) and small (<0.6 mm in diameter) TFTr colonies, all dishes containing TFT were recounted using a machine setting that discriminates between the two populations. No statistically significant (P <0.05 by Dunnett’s analysis) increases in mutation frequency were observed at any acceptable concentration under either metabolic activation condition in any of the experiments. The numbers of small colonies in the cultures treated with the test chemical were similar to those in the solvent control cultures. Hence. the test chemical can be considered to be non-mutagenic when tested in vitro on mouse Lymphoma L5178Y Cells.

This result is supported by a HGPRT assay performed using CHO cells to evaluate the mutagenic potential of the test chemical. Cultures of CHO-K1-BH4 cells were maintained in Ham’s F10 medium, supplemented with 10% fetal bovine serum, l-glutamine, penicillin G, and streptomycin sulfate. For selection of mutants, hypoxanthine-free F10 medium containing 10mg/ml of thioguanine (TG) and 5% fetal bovine serum was used. Test chemical were dissolved in DMSO. The S9-mix was prepared from Arochlor-1254-induced male Sprague-Dawley rat liver homogenate (Litton-Bionetics, Inc.). The assay procedure was based on that reported by Hsie et al., as modified by Myhr and DiPaolo. Briefly, cultures of about 4×106 cells in 250ml flasks were treated for 4h at 37 degrees C in the presence and absence of S9-mix. Thereafter, cell monolayers were washed, trypsinized, and suspended in culture medium. Cytotoxicity was estimated by plating an aliquot of 200 cells in triplicate in 60mm dishes for each treated culture. After incubation for 7–8 days, the surviving colonies were counted, and the relative initial cell survival was calculated as compared to the untreated control (relative survival). For phenotypic expression of induced mutations, 1.5 × 106 treated cells were plated into one 150 mm dishes and were subcultured on day two or three and again on day five following the same procedure. At the end of the expression period (6–7 days), cultures were reseeded at 2 × 105 cells per 100mm dishes in mutant selection medium using twelve dishes per culture. In parallel, triplicate cultures of 200 cells were seeded in 60mm dishes to determine the cloning efficiency. Ten days later, the dishes were fixed, stained and scored. Colonies were counted by eye. Mutant frequencies were calculated as TG-resistant mutant colonies per 106 clonable cells. The increased number of mutants seen at 62.5 ug/mL in the assay with metabolic activation is considered to be not relevant, since no concentration effect relationship was observed.  Hence, the test chemical can be considered to be not mutagenic 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-mutagenic when tested in vitro on mammalian cells.

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

Genetic toxicity in vivo

Description of key information

Various studies have been reviewed to evaluate the genotoxic potential of the test chemical in vivo. These studies include in vivo experimental studies for the test chemical. The results are mentioned below:

In vivo micronucleus test was performed to evaluate the genotoxic potential of the test chemical. The study was performed according to OECD 474 Guidelines.Healthy male, C57BL/6J mice weighing 20 ± 2 g of age 5-7 weeks, bred and raised in the Animal facility of Glenmark Research Centre (Navi Mumbai, India) were used for the micronucleus test. The animals were housed in polycarbonate boxes (five per cage) with paddy husk for bedding, and maintained in a 12 h dark/light cycle at 22 ± 2°C and 50–65% humidity with access to pelleted diet (Nutrilab, Bangalore, India) and fresh RO water ad libitum. The mice were acclimatized for at least 5 days prior to the treatment. Groups of five male mice received single oral administration with the vehicle (0.5% methylcellulose) or the test chemical at 125 or 250 mg/kg, whereas the positive control group was injected intraperitoneally with Mitomycin-C at 5 mg/kg. The animals were killed and their bone marrow sampled at 24 or 48 h. The cells were centrifuged at 1500 rpm for 5 min and resuspended in fresh FBS. Smears of cells were made on microscope slides, which were then fixed and stained with May-Grunwald and Giemsa. Finally, the slides were air-dried, rinsed with xylene, and permanently mounted with DPX. A minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE). The ratio of PCE-to−normochromatic erythrocytes (NCE) was determined based on a total of at least 1000 PCE+NCE.There was no increase of MNPCE in both 24 h and 48 h after both 125 and 250 mg/kg duration exposure as compared to the corresponding control. Significant (p < 0.001) increases relative to the control were observed only in mice treated with the Mitomycin-C as positive control. The PCE/NCE ratio which was considerably reduced after the treatments with the high dose. Group mean values of micronucleated PCE were similar and not significantly different from the value for the vehicle control group, and fell within the laboratory historical negative control range. Hence, the test chemical can be considered to be non-genotoxic.

This result is supported by another study where the test chemical was evaluated for micronucleus induction in rat bone marrow in an in vivo micronucleus assay. Groups of 20 male and female Sprague Dawley rats were used for the study. Rats were orally administered the test chemical in water according to the dose regimen mentioned below.The loading dose was administered orally at time 0 on the first day of treatment. The maintenance dose was administered 12 hr after the loading dose, 24 hr after the loading dose, and every 24 hr thereafter through day 7. This regimen mimics that proposed for clinical use and results in rapid steady-state plasma levels of the test chemical. Animals were necropsied on day 8, approximately 24 hr after the final dose. Bone marrow cells were flushed from one femur into about 0.5 ml of fetal bovine serum (Gibco/BRL, Gaithersburg, MD) in a 2-ml conical polycarbonate tube. Cells were concentrated by centrifugation, spread on ethanol-cleaned microscope slides, air-dried, and fixed for 5 min in absolute methanol. One slide from each animal was stained with acridine orange and evaluated using epifluorescence microscopy at 630 or 1000 3 magnification; 200 red blood cells (RBC) were scored to determine the ratio of polychromatic erythrocytes (PCE) to RBC. Approximately 2000 PCE were evaluated to determine the proportion of PCE with micronuclei. Slight decreases in the PCE/RBC ratio were observed in male and female rats treated with the test chemical however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of the test chemical. Hence, the test chemical can be considered to be non genotoxic in nature.

Based on the various studies and applying the weight of evidence approach, the test chemical can be considered to be non genotoxic when tested in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
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:
mammalian erythrocyte micronucleus test
Species:
other: 2. mouse; 3. rats
Strain:
other: 2. C57BL/6J; 3. Sprague- Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
2. - Vehicle(s)/solvent(s) used: [none; no data; acetone; air; 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; water]: 0.5% methylcellulose
3. - Vehicle(s)/solvent(s) used: [none; no data; acetone; air; 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; water]: water
Details on exposure:
2. PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): 10 ml/kg
- Storage temperature of food:
3. no data available
Duration of treatment / exposure:
2. 24 and 48 hours
3. Rats were given a “loading dose” followed by maintenance doses of one-half the loading dose administered 12 hr later and daily thereafter for 6 additional days
Frequency of treatment:
2. daily
3. daily
Post exposure period:
no data available
Remarks:
Study 2: 125 and 250 mg/kgbw
Remarks:
Study 3: 0, 31.6, 62.5, 125 mg/kg
No. of animals per sex per dose:
2. 5 male mice
3. 5 male and 5 female rats/ dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
2. none; no data; 2-acetylaminofluorene; 2-nitrofluorene; 3-methylcholanthrene; 4-nitroquinoline-N-oxide; 7,12-dimethylbenzanthracene; 9,10-dimethylbenzanthracene; 9-aminoacridine; benzo(a)pyrene; congo red; cumene hydroperoxide; cyclohexylamine; cyclophosphamide; cyclophosphamide; ethylmethanesulphonate; ethylmethanesulphonate; ethylnitrosurea; ethylnitrosurea; furylfuramide; ICR 191; methylmethanesulfonate; mitomycin C; mitomycin C; monomeric acrylamide; N-dimethylnitrosamine; N-ethyl-N-nitro-N-nitrosoguanidine; 2-nitrofluorene; 4-nitroquinoline 1-oxide; sodium azide; triethylenemelamine: mitomycin C
- Justification for choice of positive control(s):
- Route of administration: the positive control group was injected intraperitoneally with Mitomycin-C at 5 mg/kg.
- Doses / concentrations: 5 mg/kg
Tissues and cell types examined:
2. The animals were killed and their bone marrow sampled at 24 or 48 h.
3. Bone marrow cells
Details of tissue and slide preparation:
2. CRITERIA FOR DOSE SELECTION: The doses of micronucleus test were based on the result of preliminary dose range finding experiments in which mice were given several doses of single administrations by oral gavage at a dose volume of 10 ml/kg. Signs of toxicity were then recorded. A dose of 250 mg/kg was selected as the highest dose for the micronucleus test, being close to the maximum tolerated dose for single administration.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):The animals were killed and their bone marrow sampled at 24 or 48 h. The cells were centrifuged at 1500 rpm for 5 min and resuspended in fresh FBS.
DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS: Smears of cells were made on microscope slides, which were then fixed and stained with May-Grunwald and Giemsa. Finally, the slides were air-dried, rinsed with xylene, and permanently mounted with DPX. A minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE). The ratio of PCE-to−normochromatic erythrocytes (NCE) was determined based on a total of at least 1000 PCE+NCE.


OTHER:
3. CRITERIA FOR DOSE SELECTION: Rats were given a “loading dose” followed by maintenance doses of one-half the loading dose administered 12 hr later and daily thereafter for 6 additional days. This regimen mimics that proposed for clinical use and results in rapid steady-state plasma levels of the test chemical. Animals were necropsied on day 8, approximately 24 hr after the final dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow cells were flushed from one femur into about 0.5 ml of fetal bovine serum (Gibco/BRL, Gaithersburg, MD) in a 2-ml conical polycarbonate tube. Cells were concentrated by centrifugation, spread on ethanol-cleaned microscope slides, air-dried, and fixed for 5 min in absolute methanol. One slide from each animal was stained with acridine orange and evaluated using epifluorescence microscopy at 630 or 1000 3 magnification; 200 red blood cells (RBC) were scored to determine the ratio of polychromatic erythrocytes (PCE) to RBC. Approximately 2000 PCE were evaluated to determine the proportion of PCE with micronuclei.

DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS:

OTHER:
Evaluation criteria:
2. minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE).
3. 200 red blood cells (RBC) were scored to determine the ratio of polychromatic erythrocytes (PCE) to RBC. Approximately 2000 PCE were evaluated to determine the proportion of PCE with micronuclei.
Statistics:
2. Statistical significance instructural aberrations was analyzed by Fisher’s exact test. In the micronucleus test the number of MNPCE in each treated group was compared with the number in the vehicle control group by one-way ANOVA followed by Dunnett’s test.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: non genotoxic
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not specified
Remarks on result:
other: non genotoxic
Additional information on results:
2 RESULTS OF RANGE-FINDING STUDY
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analysed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): There was no increase of MNPCE in both 24 h and 48 h after both 125 and 250 mg/kg duration exposure as compared to the corresponding control.
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): The PCE/NCE ratio which was considerably reduced after the treatments with the high dose. Group mean values of micronucleated PCE were similar and not significantly different from the value for the vehicle control group, and fell within the laboratory historical negative control range
- Appropriateness of dose levels and route:
- Statistical evaluation:
3. RESULTS OF RANGE-FINDING STUDY
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals:
- Evidence of cytotoxicity in tissue analysed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay):
Slight decreases in the PCE/RBC ratio were observed in male and female rats treated with the test chemicalhowever, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of the test chemical
- Appropriateness of dose levels and route:

- Statistical evaluation:
Conclusions:
Based on the various studies and applying the weight of evidence approach, the test chemical can be considered to be non genotoxic when tested in vivo.
Executive summary:

Various studies have been reviewed to evaluate the genotoxic potential of the test chemical in vivo. These studies include in vivo experimental studies for the test chemical. The results are mentioned below:

In vivo micronucleus test was performed to evaluate the genotoxic potential of the test chemical. The study was performed according to OECD 474 Guidelines.Healthy male, C57BL/6J mice weighing 20 ± 2 g of age 5-7 weeks, bred and raised in the Animal facility of Glenmark Research Centre (Navi Mumbai, India) were used for the micronucleus test. The animals were housed in polycarbonate boxes (five per cage) with paddy husk for bedding, and maintained in a 12 h dark/light cycle at 22 ± 2°C and 50–65% humidity with access to pelleted diet (Nutrilab, Bangalore, India) and fresh RO water ad libitum. The mice were acclimatized for at least 5 days prior to the treatment. Groups of five male mice received single oral administration with the vehicle (0.5% methylcellulose) or the test chemical at 125 or 250 mg/kg, whereas the positive control group was injected intraperitoneally with Mitomycin-C at 5 mg/kg. The animals were killed and their bone marrow sampled at 24 or 48 h. The cells were centrifuged at 1500 rpm for 5 min and resuspended in fresh FBS. Smears of cells were made on microscope slides, which were then fixed and stained with May-Grunwald and Giemsa. Finally, the slides were air-dried, rinsed with xylene, and permanently mounted with DPX. A minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE). The ratio of PCE-to−normochromatic erythrocytes (NCE) was determined based on a total of at least 1000 PCE+NCE.There was no increase of MNPCE in both 24 h and 48 h after both 125 and 250 mg/kg duration exposure as compared to the corresponding control. Significant (p < 0.001) increases relative to the control were observed only in mice treated with the Mitomycin-C as positive control. The PCE/NCE ratio which was considerably reduced after the treatments with the high dose. Group mean values of micronucleated PCE were similar and not significantly different from the value for the vehicle control group, and fell within the laboratory historical negative control range. Hence, the test chemical can be considered to be non-genotoxic.

This result is supported by another study where the test chemical was evaluated for micronucleus induction in rat bone marrow in an in vivo micronucleus assay. Groups of 20 male and female Sprague Dawley rats were used for the study. Rats were orally administered the test chemical in water according to the dose regimen mentioned below.The loading dose was administered orally at time 0 on the first day of treatment. The maintenance dose was administered 12 hr after the loading dose, 24 hr after the loading dose, and every 24 hr thereafter through day 7. This regimen mimics that proposed for clinical use and results in rapid steady-state plasma levels of the test chemical. Animals were necropsied on day 8, approximately 24 hr after the final dose. Bone marrow cells were flushed from one femur into about 0.5 ml of fetal bovine serum (Gibco/BRL, Gaithersburg, MD) in a 2-ml conical polycarbonate tube. Cells were concentrated by centrifugation, spread on ethanol-cleaned microscope slides, air-dried, and fixed for 5 min in absolute methanol. One slide from each animal was stained with acridine orange and evaluated using epifluorescence microscopy at 630 or 1000 3 magnification; 200 red blood cells (RBC) were scored to determine the ratio of polychromatic erythrocytes (PCE) to RBC. Approximately 2000 PCE were evaluated to determine the proportion of PCE with micronuclei. Slight decreases in the PCE/RBC ratio were observed in male and female rats treated with the test chemical however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of the test chemical. Hence, the test chemical can be considered to be non genotoxic in nature.

Based on the various studies and applying the weight of evidence approach, the test chemical can be considered to be non genotoxic when tested in vivo.

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

Additional information

Genetic toxicity in vitro

AMES Assay

Various studies have been investigated to determine the mutagenic potential of the test chemical when tested on various bacterial strains in vitro. The results are mentioned below:

In vitro genetic toxicity study was performed to determine the mutagenic nature of the test chemical. Strains TA100 and TA98 of Salmonella typhimirium were used as the tester strains. Assay was carried out as described by Ames et al. with some modifications. The test chemical was freshly dissolved in 100 µl of DMSO were pre-incubated at 37°C for 20 min with 0.5 ml of S-9 mix or 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 ml of bacterial culture. Two ml of molten soft agar at 45°C were added, and the resulting mixture was poured over 25 ml of minimal-glucose agar containing 0.1 µmol of L-histidine and 0.1 µmol of biotin. After 2-day incubation, colonies of histidine prototroph were counted as revertants.The test chemical was weakly mutagenic to both strains with S-9 mix and non mutagenic to both strains in the absence of S9 mix. Though there was weak mutagenic activity in the presence of S9 mix but dose dependent decrease in number of revertants was observed. Hence, we consider the test chemical to be non-mutagenic both in the presence and absence of S9 mix to S.Typhimurium TA 98 and TA 100 strains.

This result is supported by another Salmonella Mutagenicity assay was performed to evaluate the mutagenic potential of the test chemical. S.typhimurium strains TA98, TA100, TA1535 and TA1537; E.coli WP2uvrA were used for the study. They were exposed to 20 -5000 microgram/plate of the test chemical. The S9 mix contained 30% S9 liver homogenate. The concentration of the S9 mix was high as per the regular AMES assay protocols. The high concentration of the S9 in the S9 mix was justified as the study was performed in accordance with Tox Method No. 005 of the Ecological and Toxicological Association of the Dyestuffs Manufacturing Association. In addition the positive controls used in the study demonstrated that the concentration of S9 in the S9 mix is capable of activating mutagens. All strains responded to the mutagenic action of the appropriate non-activated positive controls, also all strains responded to the S9 activated control 2-aminoanthracene.The tested strains were observed for mutagenic response. The test chemical failed to give a mutagenic response in the presence and absence of S9 activation system. Hence, the test chemical can be considered to be non mutagenic to S. typhimurium strains TA98,TA100, TA1535 and TA1537; E.coli WP2uvrA.

The above results are further supported by a similar salmonella/mammalian microsome test developed by AMES was performed to evaluate the mutagenic potential of the test chemical. Salmonella typhimurium strains TA1535, TA1537, TA98, TA 97 and TA100 were obtained by the individual laboratories from Dr. Bruce Ames. Cultures of each tester strain were prepared for storage as described byAmes et al, 1975] supplied with the tester strains by Dr. Ames. Frozen cultures were stored in liquid nitrogen or in a -70°C freezer in 0.2-ml aliquots or in 1-ml aliquots in sterile, screw-cap vials. All overnight cultures (late log phase) were obtained by incubation at 37°C on a shaker for 12-15 hr and were routinely checked for genetic integrity as recommended by Ames et al [1975]. Liver S-9 was prepared from male Sprague-Dawley rats (RLI) and Syrian hamsters (HLI) that were induced with Aroclor 1254 were used as metabolic activation system. The test chemical was tested using the pre-incubation procedure of the Salmonella assay [Ames et al, 1975]. Briefly, 0.5 ml of S-9 mix or 0.1 M PO4 buffer was dispensed into an appropriate number of 13 X 100 mm culture tubes maintained at 37°C in a dry-bath. Then, 0.05 ml of cells and 0.05 ml of solvent or chemical dilution were added to each tube. The mixture was vortexed and allowed to incubate with shaking in the early tests (CWR, EGG), or standing for 20 min at 37°C. The protocol was later changed to eliminate the shaking procedure, because the commercial shakers available would not fit in the Class 11. Type B hoods and, for the purposes of laboratory safety, it was inadvisable to incubate the test chemical at 37°C in the open laboratory. Following the pre-incubation period, 2.5 ml or 2.0 ml of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar in a 15 x 100-mm plastic petri dish. After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h. At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer. Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test. The test chemical gave a negative response to Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100,TA 97 at all the doses tested both in the presence and absence of rat and hamster liver S9 metabolic activation system.

Even though the test chemical gave a positive response in the presence and absence of metabolic activation system to Salmonella typhimurium strains TA98 AND TA 100, but was non mutagenic to E.Coli. Hence the test chemical can be considered to be non-mutagenic to bacterial strains.

 

In vitro DNA damage and repair

The genotoxicity of the test chemical was also tested using the SOS Chromotest. E. coli PQ37 strain [F2 thr leu his-4 pyrD thi galE galK or galT lacDU169 srl300::Tn10 rpoB rpsL uvrA rfa trp::Muc1 sulA::Mud(Ap,lac)ts] was used for this study. This strain carries the sulA::lacZ fusion gene as a reporter gene for primary DNA damage induced during the SOS response. The cells were grown overnight at 37°C and shaken at 100 rpm in LB medium (10 g tryptone/L, 5 g yeast extract/L, 10 g sodium chloride/L, pH 7.4) supplemented with ampicillin (50 μg/mL) and tetracycline 17 μg/mL. Briefly, overnight cultures were grown in fresh LB medium to an optical density (OD600 nm) of 0.4. Then, 0.1 mL of the culture was diluted in either 0.9 mL of fresh LB medium for assay without metabolic activation or 0.9 mL of S9 mix for assay with metabolic activation. Fractions of 150 μL are distributed into a series of Eppendorf tubes containing 5 μL of test chemical to be tested. The mixtures are incubated first for 30 min at 8 degrees C and after 2 h at 37 degrees C with shaking in an Eppendorf  Termomixer apparatus (Sao Paulo, Brazil). Negative (distilled water) and positive (B[a]P and/or 4- NQO) controls were always included in each assay. A minimum of three independent experiments per treatment with four replicate each were conducted. b-Galactosidase and alkaline phosphatase were assayed in 96-well plates (Brand GMBH, Germany) as follows: for b-galactosidase activity, cell membranes were disrupted by mixing 135 μL of Z buffer (110 mM mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM Mg2SO4, 0.1% SDS, and 40 mM b-mercaptoethanol, pH 7.0) with 15 μL of cell culture for 20 min at 37 degrees C. The reaction was started by adding 30 μL of ONPG (4 mg/mL in phosphate buffer: 61 mL Na2HPO4 0.1 M and 39 mL NaH2PO4.H2O 0.1 M, pH 7). After 40 min, the enzymatic reaction was stopped by adding 100 μL of 1 M Na2CO3. For alkaline phosphatase activity, cell membranes were disrupted by adding 135 μl of T buffer (1 M Tris and 0.1% SDS, pH 8.8) to 15 μL of cell culture followed by mixing vigorously and incubation for 20 min at 37 degrees C. The enzyme reaction was started by adding 30 μL of PNPP solution (4 mg/mL in T buffer). After 40 min, the reaction was stopped by adding 50 μL of 2 M HCl. After 5 min, 50μl of 2 M Tris was added to restore the color. The final absorbances of the b-galactosidase and alkaline phosphatase assays were measured at 420 nm using a Multiskan Go microplate reader. The genotoxicity criterion used was the SOS induction factor (I) that represents the normalized induction data of the sulA gene in each treatment and was therefore considered to be an indirect measure of the primary DNA damage (genotoxicity) induced by the treatments. A substance was classified as not genotoxic if I was <1.5, inconclusive or weakly genotoxic if I was between 1.5 and 2.0, and genotoxic if I was >2.0, and a clear concentration–response relationship was observed. The genotoxic effect of the test chemical was investigated by means of the SOS chromotest. The test chemical didnot significantly increase the SOS induction factor (I) values in E. coli PQ37.The incorporation of chlorine at positions C-4 and C-7 in the quinoline base moiety did not modify molecule genotoxicity. The results showed that the test chemical failed to induce the SOS response in E. coli.

In vitro chromosomal aberration

Various studies have been reviewed to determine the mutagenic potential of the test chemical in vitro. These include in vitro experimental studies performed on various mammalian cell lines for the test chemical. The results are mentioned below:

The genotoxicity profile of the test chemical was evaluated in an in vitro chromosomal aberration study.The method used for the in vitro chromosomal aberration assay was based on the OECD 473 Guidelines. The whole blood culture method (Moorehead et al. 1960) was used for the chromosomal aberration assay. Blood was obtained from a single individual with no report of any infection or of exposure to drugs and radiation for at least 30 days prior to donating blood. 0.5 ml blood, obtained by venipuncture through heparinized syringe, was added to DMEM+Ham-F12 medium supplemented with 20% FBS, 2% PHA, penicillin (100 IU/ml), and streptomycin (100 μg/ml). Cultures were gently mixed and incubated (CO2 5%) at 37°C for 72 h. Different sets of experimental cultures, 4 h and 22 h without metabolic activations and 4 h with metabolic activation were exposed to various test concentrations of the test chemical. The culture volume was maintained at 5 ml and employed in duplicate for each exposure. Colcemid (0.2 μg/ml) was added to each culture tube 3 h prior to harvesting. Cultures were centrifuged for 10 min at 1500 rpm, cell pellets were resuspended in hypotonic solution (0.0375 M KCl), and the tubes were left in a water bath at 37°C for 15 min. Cells were centrifuged again and fixed 3–4-times in fresh, chilled Carnoy’s fixative (ethanol:acetic acid, 3:1). Cells were dropped over clean, chilled slides, air dried, and stained. Statistical analysis was determined using GraphPad Prism 3.0 software wherever applicable. The mean numbers of revertant colonies for all treatment groups were compared with their respective control values. For chromosomal aberration assay, cells were observed using a Leica (Germany) microscope with 100× oil immersion objectives. One hundred well-spread metaphase plates per culture were scored for cytogenetic damage. Only metaphases with 46 ± 2 centromere regions were included in the analysis. Breaks, fragments, deletions, exchanges, and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded, but separately included in the calculation. To describe a cytotoxic effect the mitotic index (percentage of cells in mitosis) was determined in 1000 cells. In addition, the number of polyploid cells in 200 metaphase cells was scored. Statistical significance in structural aberrations was analyzed by Fisher’s exact test. The exposure to the test chemical could not induce significant structural aberrations, even with the highest concentration of 625 μg in 4 h and 300 μg in 22 h, with or without metabolic activation. In contrast significant (p < 0.01) increase in chromosomal aberrations were observed with positive controls, both in the absence and presence of metabolic activation.

This result is supported by another assay where thetest chemical was tested for their ability to induce cytogenetic change in Chinese hamster ovary cells. CHO cells were cloned at Litton Bionetics Inc. and supplied to the other laboratories. Cells were not used beyond 15 passages after cloning. Cells for experiments were thawed and grown in McCoys 5A medium supplemented with antibiotics and 10% fetal calf serum at 37°C using 5% CO2. Cells were routinely checked for mycoplasma contamination; the results of these analyses disclosed no evidence of contamination. Approximately 24 hr before treatment, cells were initiated at a density of 1.2-1.75 x106 cells/ 75 cm2 flask. For the trials without S9, the cells were incubated with the appropriate control or chemical for 8 hr. The cells were then washed and Colcemid added for a 2-2.5 hr exposure. For  the trials with S9, the cells were treated with S9 and test chemical in serum-free medium for 2 hr, washed, resuspended in medium containing serum, and incubated for an additional 8-10 hr, with Colcemid present for the final 2 hr.Cells were harvested by mitotic shake-off and stained with Giemsa. Cells with good morphology and with a chromosome number of 21+/- 2 were selected for analysis. With the exception of the: high-dose positive controls, all slides were scored coded. 200 cells per dose were scored. Fewer cells were scored if a strong response was observed or a dose was very toxic. For the ABS assay, cells were scored for “simple” (chromatid gaps and breaks, fragments, deletions, chromosome gaps and breaks, and double minutes), “complex” (interstitial deletions, triradials, quadriradials, rings, and dicentrics), and ‘other’ (pulverized, polyploids, and endoreduplications) aberrations. These categories were combined to form the category “total”. The percent of cells with aberrations, rather than aberrations per cell, were analyzed so as to avoid distorting the data for cases where a small number of cells had a large number of aberrations. Mitomycin C (MMC) was the positive control in the trials without S9 and cyclophosphamide (CPA) was used in the trials with S9. A binomial sampling assumption was used to evaluate an absolute increase in aberrations over the solvent control. Dose points with P values adjusted by Dunnett’s method were considered significant if (0.05, whereas a trend of P < 0.003 was significant. if a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal (?); if a trial had significant trend and one significant dose it was judged weak positive (+ W); and if the trial had two significant doses it was judged positive (+), whether or not a positive trend was obtained. In the ABS trials, if only one dose was significant and the increase over the control was P < 0.0005 the trial was denoted (+ W*).The test chemical can be considered to be non mutagenicboth in the presence and absence of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic in nature.

Based on the available results and applying the weight of evidence approach, the test chemical can be considered to be non mutagenicboth in the presence and absence of metabolic activation system. Hence, the test chemical can be considered to be non mutagenic in nature.

In vitro mammalian cell gene mutation assay:

Various studies have been reviewed to determine the mutagenic potential of the test chemical in vitro. These include in vitro experimental studies performed on various mammalian cell lines for the test chemical. The results are mentioned below:

A mouse lymphoma assay was performed to determine the mutagenic potential of the test chemical. L5178Y mouse lymphoma cells, clone 3.7.2C, heterozygous at the tk locus, were used for the study. L5178Y mouse lymphoma cells were routinely cultivated in RPMI 1640 medium supplemented with 0.1% Pluronic F68, 0.22 mg/ml sodium pyruvate (R0P), and 10% heat-inactivated donor horse serum (R10P). The serum concentration was reduced to 5% (R5P) for the exposure medium. Cloning medium (CM) consisted of RPMI 1640 medium containing the same concentrations of sodium pyruvate and horse serum as R10P, plus BBLpurified- agar (final concentration ; 0.3%). The selective cloning medium contained 5 mg/ml trifluorothymidine (TFT). Antibiotics (31 microgram/ml penicillin and 50 microgram/ml streptomycin sulfate) were added to the culture media used for the mutagenesis assays but not to the stock cell cultures. All cells were incubated at 37°C in the presence of 5% CO2 in air. Preliminary cytotoxicity experiments were conducted using mouse lymphoma cell cultures treated for 4 hr with the test chemical.  Dose-related increases in cytotoxicity were observed under both activation conditions. Based on the cytotoxicity observed in the range-finding experiments, concentrations of  the test chemical ranged from 31 to 96 microgram/ml both with and without metabolic activation. Cultures with or without the metabolic activation mixture were treated with the appropriate chemical stock solution for a 4-hr exposure period, after which the treatment solutions were removed and replaced with fresh R10P. Cell growth was monitored daily over a 2-day expression period by using a Coulter counter. On the second day of the expression period, about 3x106 cells were seeded in CM+ TFT for selection of TFT cells and about 600 cells were seeded in non-selective culture medium to determine the percentage of viable cells. After incubation for 11 to 12 days in a humidified atmosphere containing 5% CO2 in air, colonies were counted using an Artek Model 880 automatic colony counter with a standard 50-mm lens. It was previously reported that the induction of gross aberrations correlates with the induction of small-colony tk mutants [Moore and Doerr, 1990]. To obtain information about the relative numbers of the large (>0.6 mm in diameter) and small (<0.6 mm in diameter) TFTr colonies, all dishes containing TFT were recounted using a machine setting that discriminates between the two populations. No statistically significant (P <0.05 by Dunnett’s analysis) increases in mutation frequency were observed at any acceptable concentration under either metabolic activation condition in any of the experiments. The numbers of small colonies in the cultures treated with the test chemical were similar to those in the solvent control cultures. Hence. the test chemical can be considered to be non-mutagenic when tested in vitro on mouse Lymphoma L5178Y Cells.

This result is supported by a HGPRT assay performed using CHO cells to evaluate the mutagenic potential of the test chemical. Cultures of CHO-K1-BH4 cells were maintained in Ham’s F10 medium, supplemented with 10% fetal bovine serum, l-glutamine, penicillin G, and streptomycin sulfate. For selection of mutants, hypoxanthine-free F10 medium containing 10mg/ml of thioguanine (TG) and 5% fetal bovine serum was used. Test chemical were dissolved in DMSO. The S9-mix was prepared from Arochlor-1254-induced male Sprague-Dawley rat liver homogenate (Litton-Bionetics, Inc.). The assay procedure was based on that reported by Hsie et al., as modified by Myhr and DiPaolo. Briefly, cultures of about 4×106cells in 250ml flasks were treated for 4h at 37 degrees C in the presence and absence of S9-mix. Thereafter, cell monolayers were washed, trypsinized, and suspended in culture medium. Cytotoxicity was estimated by plating an aliquot of 200 cells in triplicate in 60mm dishes for each treated culture. After incubation for 7–8 days, the surviving colonies were counted, and the relative initial cell survival was calculated as compared to the untreated control (relative survival). For phenotypic expression of induced mutations, 1.5 × 106treated cells were plated into one 150 mm dishes and were subcultured on day two or three and again on day five following the same procedure. At the end of the expression period (6–7 days), cultures were reseeded at 2 × 105cells per 100mm dishes in mutant selection medium using twelve dishes per culture. In parallel, triplicate cultures of 200 cells were seeded in 60mm dishes to determine the cloning efficiency. Ten days later, the dishes were fixed, stained and scored. Colonies were counted by eye. Mutant frequencies were calculated as TG-resistant mutant colonies per 106clonable cells. The increased number of mutants seen at 62.5 ug/mL in the assay with metabolic activation is considered to be not relevant, since no concentration effect relationship was observed.  Hence, the test chemical can be considered to be not mutagenic 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-mutagenic when tested in vitro on mammalian cells.

Genetic toxicity in vivo:

Various studies have been reviewed to evaluate the genotoxic potential of the test chemical in vivo. These studies include in vivo experimental studies for the test chemical. The results are mentioned below:

In vivo micronucleus test was performed to evaluate the genotoxic potential of the test chemical. The study was performed according to OECD 474 Guidelines.Healthy male, C57BL/6J mice weighing 20 ± 2 g of age 5-7 weeks, bred and raised in the Animal facility of Glenmark Research Centre (Navi Mumbai, India) were used for the micronucleus test. The animals were housed in polycarbonate boxes (five per cage) with paddy husk for bedding, and maintained in a 12 h dark/light cycle at 22 ± 2°C and 50–65% humidity with access to pelleted diet (Nutrilab, Bangalore, India) and fresh RO water ad libitum. The mice were acclimatized for at least 5 days prior to the treatment. Groups of five male mice received single oral administration with the vehicle (0.5% methylcellulose) or the test chemical at 125 or 250 mg/kg, whereas the positive control group was injected intraperitoneally with Mitomycin-C at 5 mg/kg. The animals were killed and their bone marrow sampled at 24 or 48 h. The cells were centrifuged at 1500 rpm for 5 min and resuspended in fresh FBS. Smears of cells were made on microscope slides, which were then fixed and stained with May-Grunwald and Giemsa. Finally, the slides were air-dried, rinsed with xylene, and permanently mounted with DPX. A minimum of 2000 polychromatic erythrocytes (PCE) per animal was scored in blindfold slides under 100× oil immersion for micronucleated polychromatic erythrocytes (MNPCE). The ratio of PCE-to−normochromatic erythrocytes (NCE) was determined based on a total of at least 1000 PCE+NCE.There was no increase of MNPCE in both 24 h and 48 h after both 125 and 250 mg/kg duration exposure as compared to the corresponding control. Significant (p < 0.001) increases relative to the control were observed only in mice treated with the Mitomycin-C as positive control. The PCE/NCE ratio which was considerably reduced after the treatments with the high dose. Group mean values of micronucleated PCE were similar and not significantly different from the value for the vehicle control group, and fell within the laboratory historical negative control range. Hence, the test chemical can be considered to be non-genotoxic.

This result is supported by another study where the test chemical was evaluated for micronucleus induction in rat bone marrow in an in vivo micronucleus assay. Groups of 20 male and female Sprague Dawley rats were used for the study. Rats were orally administered the test chemical in water according to the dose regimen mentioned below.The loading dose was administered orally at time 0 on the first day of treatment. The maintenance dose was administered 12 hr after the loading dose, 24 hr after the loading dose, and every 24 hr thereafter through day 7. This regimen mimics that proposed for clinical use and results in rapid steady-state plasma levels of the test chemical. Animals were necropsied on day 8, approximately 24 hr after the final dose. Bone marrow cells were flushed from one femur into about 0.5 ml of fetal bovine serum (Gibco/BRL, Gaithersburg, MD) in a 2-ml conical polycarbonate tube. Cells were concentrated by centrifugation, spread on ethanol-cleaned microscope slides, air-dried, and fixed for 5 min in absolute methanol. One slide from each animal was stained with acridine orange and evaluated using epifluorescence microscopy at 630 or 1000 3 magnification; 200 red blood cells (RBC) were scored to determine the ratio of polychromatic erythrocytes (PCE) to RBC. Approximately 2000 PCE were evaluated to determine the proportion of PCE with micronuclei. Slight decreases in the PCE/RBC ratio were observed in male and female rats treated with the test chemical however, none of these changes was statistically significant. No increases in the frequency of micronucleated PCE were observed at any dose level of the test chemical. Hence, the test chemical can be considered to be non genotoxic in nature.

Based on the various studies and applying the weight of evidence approach, the test chemical can be considered to be non genotoxic when tested in vivo.

Justification for classification or non-classification

Based on the available results, the test chemical can be considered to be non mutagenic in nature when tested under in vitro as well as in vivo conditions.It can be classified under the category "Not Classified" as per CLP Regulation.