Potassium nitrite

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial gene mutation study:

AMES test was performed according to OECD Guidelines to evaluate the mutagenic potential of the test chemical.The test chemical was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. There was no evidence of increased mutagenic activity in the urine of rats receiving 3000 mg/litre, either in the presence or in the absence of S-9 mix.Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Chromosomal Abberation study:

The test chemical is not mutagenic at the highest tested concentration of 1.0 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 gene mutation study in mammalian cells

The test substance did not induce gene mutation at the tk locus in the genome of L5178Y mouse (TK+/-) lymphoma cells and, consequently it was non-mutagenic (negative) in mammalian gene mutation test, under the experimental conditions described

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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 publication.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Principles of method if other than guideline:

AMES test was performed according to OECD Guidelines to evaluate the mutagenic potential of the test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella typhimurium and tryptophan Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S-9 mix with 10% S-9 prepared from livers of Aroclor 1254-treated rats

Test concentrations with justification for top dose:

Concentrates of the urine was used(from urine of rats receiving 3000 mg/litre of test chemical)

Vehicle / solvent:

- 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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Β-glucuronidase

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in medium; Standard plate method

Rationale for test conditions:

Not specified

Evaluation criteria:

Evaluation was done considering a dose dependent increase in the number of revertants /plate.

Statistics:

Not specified.

Key result

Species / strain:

bacteria, other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA

Metabolic activation:

with and 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:

valid

Additional information on results:

There was no evidence of increased mutagenic activity in the urine of rats receiving 3000 mg/litre, either in the presence or in the absence of S-9 mix .

Remarks on result:

other: No mutagenic effct were observed

Conclusions:

Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. The test result was considered to be negative in all strain in the presence and absence of metabolic activation S9.

Executive summary:

AMES test was performed according to OECD Guidelines to evaluate the mutagenic potential of the test chemical. The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. There was no evidence of increased mutagenic activity in the urine of rats receiving 3000 mg/litre, either in the presence or in the absence of S-9 mix.. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Weight of evidence approach based on the available data of the target chemical and of various read-across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

The test chemical was tested for chromosomal aberration effects in CHO cells in the presence and absence of metabolic activation.

GLP compliance:

yes

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: 27-32 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were 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

Remarks:

5.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Remarks:

6.

Cytokinesis block (if used):

5. No data
6. Colchicine

Metabolic activation:

with and without

Metabolic activation system:

5. S9 metabolic activation system
6. Rat liver S9 tissue homogenate

Test concentrations with justification for top dose:

5. 0.0, 0.25, 0.5 and 1.0 mg/mL
6. The chemical was tested in Chinese hamster ovary (CHO) cells at 0 (media), 37.5, 50, 75 and 100
mM in the absence of metabolic activation and at 0 (media), 50, 75, 100, 150 and 200 mm in the
presence of metabolic activation. No justification for top dose.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640
- Justification for choice of solvent/vehicle: The test chemical was soluble in RPMI 1640

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

5. 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 22-25 hrs (without metabolic activation system)
- Expression time: 16-21 hrs (with and without metabolic activation system- Phase I and II)
- 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 labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.

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: 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 vehicle control. In the absence of S9 mix, the mean mitotic index observed was 10.02 (NC), 8.37 (T1), 6.97 (T2), 4.17(T3) and 8.70 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.02 (NC), 8.18 (T1), 6.91 (T2), 4.16 (T3) and 8.52 (PC). In the cytotoxicity experiment, the highest test concentration 2 (T3) mg/mL of culture media showed more than 50% reduction in the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence the concentrations [0.25, 0.5 and 1.0 mg/mL] were selected for the main study. Hence, 1.0 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.

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


6. Before treatment an appropriate number of flasks were seeded with cells at a density of 20 000 cells/
cm2. In the presence of S9, S9 mix was added to minimal medium at a final concentration of 10%. Tr
eatment duration in the absence of S9 was 24 h; colchicine was added for the last 3 h at a concentr
ation of 8 ug/ml. The treatment period in the presence of S9 was 3 h, after which the cell cultures
were washed and fresh medium added for a 21 h recovery period; colchicine was added for the last 3
h at 8 ug/ml. 24 hours after the start of treatment both series of cultures were harvested, and the cells
were brought into suspension with trypsin. The cell suspension was centrifuged and re-suspended
in hypotonic solution. After this, the cells were fixed in freshly prepared methanol-acetic acid fixati
ve and washed 3 times in fixative. Air-dried slides were prepared from the cell suspension and stai
ned in 3% Giemsa. The mitotic index and the reduction in mitotic index compared with the controls
was calculated. Toxicity was also measured by parallel treatments of plates containing 200 cells; af
ter treatment these cultures were washed and allowed to grow for 7 days. The cultures were stained,the number of colonies counted, and survival was expressed as a percentage of the control values.
Where possible, 100 metaphases were scored per culture.

Rationale for test conditions:

No data

Evaluation criteria:

5. 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 vehicle 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 within 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.

6. 200 metaphase plates per culture were analysed microscopically for chromatid and chromosome
deletions and exchanges, and for isolocus events (gaps not included).

Statistics:

5. Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
6. Not specified

Species / strain:

lymphocytes: Human perpheral blood lymphocytes

Remarks:

5.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

6.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

5. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not 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: No precipitation was observed at 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 vehicle control.

In the absence of S9 mix, the mean mitotic index observed was 10.02 (NC), 8.37 (T1), 6.97 (T2), 4.17(T3) and 8.70 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.02 (NC), 8.18 (T1), 6.91 (T2), 4.16 (T3) and 8.52 (PC).

In the cytotoxicity experiment, the highest test concentration 2 (T3) mg/mL of culture media showed more than 50% reduction in the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence the concentrations [0.25, 0.5 and 1.0 mg/mL] were selected for the main study.

Hence, 1.0 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

6. In the absence of metabolic activation, the percentages of cells bearing aberrations were 0.5, 2.0,
4.0 and 19.0% at 0, 37.5, 50, 75 mM, respectively. Percent cell survival in the absence of metabolic
activation were 100, 96, 83 and 44 at 0, 37.5, 50, 75 mM, respectively. In the presence of metabolic
activation, the percentages of cells bearing aberrations were 0, 1.0, 0.5, 1.5, 7.5 and 39.6% at 0, 50,
75, 100, 150 and 200 mm. Percent survival percentages in the presence of metabolic activation were
100, 93, 79, 84, 28 and 22% at 0, 50, 75, 100, 150 and 200 mM, respectively. Both in the presence a
nd absence of metabolic activation, a dose-dependent increase in osmolality was observed at ≥37.5
mM (-S9) and at ≥50 mM (+S9). The marked increases in the number of aberrant cells at 75 mM (-
S9) and at 100 mM (+S9) were likely secondary to excessive cytotoxicity and/or increased osmolal
ity. The minimal increases in the number of aberrant cells at 37.5 mM (-S9) and 50 mM (+S9) were
also observed in the presence of increased osmolality. Beause the positive effects were considered
to be artefactual responses, the chemical was evaluated to be non-clastogenic in CHO cells in the
presence and absence of metabolic activation.

Remarks on result:

other: No mutagenic potential

Cytotoxicity results:

     

Before conducting the chromosomal aberration study,Sodium Iodide (CAS No. 7681-82-5)was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.5 (T1), 1 (T2) and 2 (T3) mg/mL of culture media. Cytotoxicity was observed in both absence and presence of metabolic activation (1%) at treated concentration T3 (2 mg/ml).

In the absence of S9 mix, the mean mitotic index observed was 10.02 (NC), 8.37 (T1), 6.97 (T2), 4.17(T3) and 8.70 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.02 (NC), 8.18 (T1), 6.91 (T2), 4.16 (T3) and 8.52 (PC).

In the cytotoxicity experiment, the highest test concentration 2 (T3) mg/mL of culture media showed more than 50% reduction in the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence the concentrations [0.25, 0.5 and 1.0 mg/mL] were selected for the main study.

Hence, 1.0 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.

The main study was performed in two independent phases;

Phase I

In the experiment, the cultures were exposed to Sodium Iodide (CAS No. 7681-82-5) 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), 0.667 (T2), 0.667 (T3) and 11.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.25 (T1), 0.5 (T2) and 1.0 (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 Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (1%) caused significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During the treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 10.16, 8.84, 8.25, 7.28 and 8.40 and in the presence of metabolic activation were 10.50, 9.18, 8.56, 7.99 and 8.81 for 0.0 (NC), 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL and 30 µg/mL (PC) concentrations respectively.

Phase II     

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), 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mLand 30µg/mL(PC)culture both in absence and presence 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), 0.667 (T3) and 9.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (T1), 0.667 (T2), 0.667 (T3) and 10.667 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 025 (T1), 0.5 (T2) and 1.0 (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, 9.13, 8.93, 7.44 and 8.49 andin the presence ofmetabolic activation were 9.84, 8.97, 8.74, 7.62 and 8.51 for0.0 (NC), 0.25 (T1), 0.5 (T2) and 1.0 (T3)  and 30 µg/mL(PC)concentrations respectively.

Conclusions:

The test chemical is not mutagenic at the highest tested concentration of 1.0 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:

Chromosomal Abberation studies were performed as follows:

5. This study was conducted to determine the chromosomal aberration induction potential of  the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 26-34 years age. Samples were collected in heparinized vials. 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 1640 and used at dose level of 0, 0.25, 0.5 and 1.0 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, 0.5, 1 and mg/mL) 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 proliferating blood 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, pulverization, 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. Based on the observations made, the test chemical is not mutagenic at the highest tested concentration of 1.0 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.

6.

The chemical was tested in Chinese hamster ovary (CHO) cells at 0 (media), 37.5, 50, 75 and 100 mM in the absence of metabolic activation and at 0 (media), 50, 75, 100, 150 and 200 mm in the presence of metabolic activation (rat liver S9 tissue homogenate). The cells were incubated with the test chemical for 24 hrs (absence of metabolic activation) or for 3 hrs (presence of metabolic activation). No positive control substances were used. Where possible, 200 metaphase plates per culture were analysed microscopically for chromatid and chromosome deletions and exchanges, and for isolocus events (gaps not included). In the absence of metabolic activation, the percentages of cells bearing aberrations were 0.5, 2.0, 4.0 and 19.0% at 0, 37.5, 50, 75 mM, respectively. Percent cell survival in the absence of metabolic activation were 100, 96, 83 and 44 at 0, 37.5, 50, 75 mM, respectively. In the presence of metabolic activation, the percentages of cells bearing aberrations were 0, 1.0, 0.5, 1.5, 7.5 and 39.6% at 0, 50, 75, 100, 150 and 200 mm. Percent survival percentages in the presence of metabolic activation were 100, 93, 79, 84, 28 and 22% at 0, 50, 75, 100, 150 and 200 mM, respectively. Both in the presence and absence of metabolic activation, a dose-dependent increase in osmolality was observed at ≥37.5 mM (-S9) and at ≥50 mM (+S9). The marked increases in the number of aberrant cells at 75 mM (-S9) and at 100 mM (+S9) were likely secondary to excessive cytotoxicity and/or increased osmolality. The minimal increases in the number of aberrant cells at 37.5 mM (-S9) and 50 mM (+S9) were also observed in the presence of increased osmolality. The test chemical was tested at exceedingly high concentrations compared OECD test guidelines. Based on the presented, the chemical was considered to induce clastogenic effects only by its virtue to alter osmolality at exceedingly high concentrations. For regulatory compliance, the chemical was therefore evaluated to be non-clastogenic in CHO cells in the presence and absence of metabolic activation.

Reference 3

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on experimental data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Not tested in the presence of metabolic activation

Principles of method if other than guideline:

WoE for the target chemical is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

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

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The cells were maintained in logarithmic growth by dilution three times a week to 3x10^5 cells/ml with Fisher's Medium for Leukemic Mice (FM0), supplemented with 10% horse serum and 1% 200mM glutamine (FM10).

Metabolic activation:

without

Metabolic activation system:

No metabolic activation system used.

Test concentrations with justification for top dose:

4. The chemical was tested at 0 (negative control), 0.1, 0.5, 1, 5, and 10 mg/ml
5. 22, 43, 82, 170, 340 ug/ml for 4 hours

Vehicle / solvent:

Not specified but pressumed to have been dissolved directly in culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other:

Remarks:

Not applicable: the test chemical was pressumed to have been dissolved in culture media

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell viability was assessed using trypan blue exclusion dye and by measurement of the cellular doubling time. L5178Y cells (1x10^6 cells/ml) in 50 ml sterile centrifuge tubes were incubated with the test chemical, the positive controls, or media, for four hours at 37°C. The cells were then washed three times in FM0 and incubated in a roller drum for 48 hours. This incubation period allowed for the expression of induced mutations. The cells were then plated in FM with 20% horse serum. To the media, 3.5% Noble agar and 100 μg/m1BUdR were added. Colony formation was scored after 10 days incubation at 37°C. In all of the experiments, the cloning efficiency of the control plates was found to be greater than 85%. The number of mutant colonies/10^5 cells was calculated for the media controls. The actual spontaneous mutation frequency was variable ranging for this series of experiments from 4-10 mutants/105 cells. For the purpose of calculations, this was designated as having a mutational frequency (MF) of one.

Rationale for test conditions:

No data

Evaluation criteria:

Values 2.5 greater than the mutational frequency (MF) of the negative control was considered as a positive mutational event.

Statistics:

Student t-test. P =< 0.05 was considered as statistically significant.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50% cytotoxicity at top dose based on pre-experimental data

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test substance did not induce gene mutation at the tk locus in the genome of L5178Y mouse (TK+/-) lymphoma cells and, consequently it was non-mutagenic (negative) in mammalian gene mutation test, under the experimental conditions described

Executive summary:

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

The mutagenic potential of the test chemical was assessed by mouse lymphoma assay (MLS) in the absence of metabolic activation using heterozygous L5178Y mouse (TK^+/-) lymphoma cells. The cytotoxicity induced by the test chemical was measured during a seven-day growth study. Doses resulted in 50 % cell viability compared to control values and the next four lower doses were used in the mutagenicity assay. Cell viability was assessed using trypan blue exclusion dye and by measurement of the cellular doubling time. L5178Y cells were incubated with the test substance at doses of 100, 500 µg/ml and 1, 5, or 10 mg/ml for 4 hours along with concurrent positive (ethyl methanesulfonat, EMS, N-methyl-N-nitro-N-nitrosoguanidine, MNNG) and vehicle (media) control substances. Colony formation was scored after 10 days incubation at 37°C. In all the experiments, the cloning efficiency of the control plates was found to be greater than 85%. The number of mutant colonies/105 cells was calculated for the media controls. The actual spontaneous mutation frequency was variable ranging from 4-10 mutants/105 cells. For the purpose of calculations, this was designated as having a mutational frequency (MF) of one. A mutation frequency of 2.5 or greater was considered a positive mutational event. The results of mutagenicity of the test chemical did not significantly alter the mutational frequency at doses tested. Positive control substances EMS and MNNG produced a MF of 12 and 15, respectively.The test substance was not tested in the presence of metabolic activation unlike some of the other compounds that were tested in the presence of metabolic activation in the study. The reason why the authors decided not to test potassium iodide for mutagenicity in the presence of metabolic activation was presumably due to the high solubility of the test chemical in water. That is, the solubility of potassium iodide is approx. 160 grams per 100 grams of water at 37 °C (Pawar et al. J Chem Eng Data 2009; 54: 1935-1937) meaning that this ionic compound will most likely dissociate spontaneously in the cytosol of cells up to saturated levels. The test chemical was therefore regarded to be non-mutagenic (negative) in L5178Y mouse lymphoma cells irrespective of the use of metabolic activation.

Above study is further supported by data for another test chemical. The mutagenic potential of the test chemical was evaluated using mouse lymphoma assay (MLS), heterozygous L5178Y mouse (TK+/-) lymphoma cells were exposed to 22, 43, 82, 170 or 340 µg/ml for 4 hours in the absence of metabolic activation. Concurrent positive control (ethyl methanesulfonat, EMS, N-methyl-N-nitro-N-nitrosoguanidine, MNNG) and vehicle (media) control substances were also included in the assay. Colony formation was scored after 10 days incubation at 37°C. In all the experiments, the cloning efficiency of the control plates was found to be greater than 85%. The number of mutant colonies/105 cells was calculated for the media controls. The actual spontaneous mutation frequency was variable ranging from 4-10 mutants/105 cells. For the purpose of calculations, this was designated as having a mutational frequency (MF) of one. A mutation frequency of 2.5 or greater was considered a positive mutational event. The results of mutagenicity of the test chemical indicate that it did not induce mutant colony formation and did not alter significantly the mutation frequency at any doses tested. Positive control substances EMS and MNNG produced a MF of 12 and 15, respectively. In conclusion, in the absence of metabolic activation the test chemical did not induce gene mutation at the tk locus in the genome of L5178Y mouse (TK+/-) lymphoma cells and, consequently it was non-mutagenic (negative) in mammalian gene mutation test, under the experimental conditions described.

Based on the data available and applying the weight of evidence approach, the test substance did not induce gene mutation at the tk locus in the genome of L5178Y mouse (TK+/-) lymphoma cells and, consequently it was non-mutagenic (negative) in mammalian gene mutation test, under the experimental conditions described

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria:

Various studies have been reviewed to ascertain the mutagenic potential of the test chemical. The results are as follows:

1. AMES test was performed according to OECD Guidelines to evaluate the mutagenic potential of the test chemical.The test chemical was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. There was no evidence of increased mutagenic activity in the urine of rats receiving 3000 mg/litre, either in the presence or in the absence of S-9 mix. Therefore test chemical was considered to be non mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

2. Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical (CAS no 7681-82-5) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative and positive controls are within the range of our historical data.The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

3. Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. 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 20 mg/plate being the maximum concentration. The chemical was dissolved in phosphate buffer. 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). 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.

Based on the available results, the test chemical is not likely to be mutagenic when tested in vitro on various bacterial strains both in the presence and absence of exogenous metabolic activation system. Hence, according to CLP Regulation, the test chemical can be classified under the category "Not Classified".

Chromosomal Abberation study:

Chromosomal Abberation studies were performed as follows:

5. This study was conducted to determine the chromosomal aberration induction potential of  the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 26-34 years age. Samples were collected in heparinized vials. 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 1640 and used at dose level of 0, 0.25, 0.5 and 1.0 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, 0.5, 1 and mg/mL) 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 proliferating blood 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, pulverization, 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. Based on the observations made, the test chemical is not mutagenic at the highest tested concentration of 1.0 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.

6.

The chemical was tested in Chinese hamster ovary (CHO) cells at 0 (media), 37.5, 50, 75 and 100 mM in the absence of metabolic activation and at 0 (media), 50, 75, 100, 150 and 200 mm in the presence of metabolic activation (rat liver S9 tissue homogenate). The cells were incubated with the test chemical for 24 hrs (absence of metabolic activation) or for 3 hrs (presence of metabolic activation). No positive control substances were used. Where possible, 200 metaphase plates per culture were analysed microscopically for chromatid and chromosome deletions and exchanges, and for isolocus events (gaps not included). In the absence of metabolic activation, the percentages of cells bearing aberrations were 0.5, 2.0, 4.0 and 19.0% at 0, 37.5, 50, 75 mM, respectively. Percent cell survival in the absence of metabolic activation were 100, 96, 83 and 44 at 0, 37.5, 50, 75 mM, respectively. In the presence of metabolic activation, the percentages of cells bearing aberrations were 0, 1.0, 0.5, 1.5, 7.5 and 39.6% at 0, 50, 75, 100, 150 and 200 mm. Percent survival percentages in the presence of metabolic activation were 100, 93, 79, 84, 28 and 22% at 0, 50, 75, 100, 150 and 200 mM, respectively. Both in the presence and absence of metabolic activation, a dose-dependent increase in osmolality was observed at ≥37.5 mM (-S9) and at ≥50 mM (+S9). The marked increases in the number of aberrant cells at 75 mM (-S9) and at 100 mM (+S9) were likely secondary to excessive cytotoxicity and/or increased osmolality. The minimal increases in the number of aberrant cells at 37.5 mM (-S9) and 50 mM (+S9) were also observed in the presence of increased osmolality. The test chemical was tested at exceedingly high concentrations compared OECD test guidelines. Based on the presented, the chemical was considered to induce clastogenic effects only by its virtue to alter osmolality at exceedingly high concentrations. For regulatory compliance, the chemical was therefore evaluated to be non-clastogenic in CHO cells in the presence and absence of metabolic activation.

Based on the available results, the test chemical is not likely to be mutagenic when tested in vitro on various bacterial strains both in the presence and absence of exogenous metabolic activation system. Hence, according to CLP Regulation, the test chemical can be classified under the category "Not Classified".

In vitro gene mutation study in mammalian cells

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

The mutagenic potential of the test chemical was assessed by mouse lymphoma assay (MLS) in the absence of metabolic activation using heterozygous L5178Y mouse (TK^+/-) lymphoma cells. The cytotoxicity induced by the test chemical was measured during a seven-day growth study. Doses resulted in 50 % cell viability compared to control values and the next four lower doses were used in the mutagenicity assay. Cell viability was assessed using trypan blue exclusion dye and by measurement of the cellular doubling time. L5178Y cells were incubated with the test substance at doses of 100, 500 µg/ml and 1, 5, or 10 mg/ml for 4 hours along with concurrent positive (ethyl methanesulfonat, EMS, N-methyl-N-nitro-N-nitrosoguanidine, MNNG) and vehicle (media) control substances. Colony formation was scored after 10 days incubation at 37°C. In all the experiments, the cloning efficiency of the control plates was found to be greater than 85%. The number of mutant colonies/105 cells was calculated for the media controls. The actual spontaneous mutation frequency was variable ranging from 4-10 mutants/105 cells. For the purpose of calculations, this was designated as having a mutational frequency (MF) of one. A mutation frequency of 2.5 or greater was considered a positive mutational event. The results of mutagenicity of the test chemical did not significantly alter the mutational frequency at doses tested. Positive control substances EMS and MNNG produced a MF of 12 and 15, respectively.The test substance was not tested in the presence of metabolic activation unlike some of the other compounds that were tested in the presence of metabolic activation in the study. The reason why the authors decided not to test potassium iodide for mutagenicity in the presence of metabolic activation was presumably due to the high solubility of the test chemical in water. That is, the solubility of potassium iodide is approx. 160 grams per 100 grams of water at 37 °C (Pawar et al. J Chem Eng Data 2009; 54: 1935-1937) meaning that this ionic compound will most likely dissociate spontaneously in the cytosol of cells up to saturated levels. The test chemical was therefore regarded to be non-mutagenic (negative) in L5178Y mouse lymphoma cells irrespective of the use of metabolic activation.

Above study is further supported by data for another test chemical. The mutagenic potential of the test chemical was evaluated using mouse lymphoma assay (MLS), heterozygous L5178Y mouse (TK+/-) lymphoma cells were exposed to 22, 43, 82, 170 or 340 µg/ml for 4 hours in the absence of metabolic activation. Concurrent positive control (ethyl methanesulfonat, EMS, N-methyl-N-nitro-N-nitrosoguanidine, MNNG) and vehicle (media) control substances were also included in the assay. Colony formation was scored after 10 days incubation at 37°C. In all the experiments, the cloning efficiency of the control plates was found to be greater than 85%. The number of mutant colonies/105 cells was calculated for the media controls. The actual spontaneous mutation frequency was variable ranging from 4-10 mutants/105 cells. For the purpose of calculations, this was designated as having a mutational frequency (MF) of one. A mutation frequency of 2.5 or greater was considered a positive mutational event. The results of mutagenicity of the test chemical indicate that it did not induce mutant colony formation and did not alter significantly the mutation frequency at any doses tested. Positive control substances EMS and MNNG produced a MF of 12 and 15, respectively. In conclusion, in the absence of metabolic activation the test chemical did not induce gene mutation at the tk locus in the genome of L5178Y mouse (TK+/-) lymphoma cells and, consequently it was non-mutagenic (negative) in mammalian gene mutation test, under the experimental conditions described.

Based on the data available and applying the weight of evidence approach, the test substance did not induce gene mutation at the tk locus in the genome of L5178Y mouse (TK+/-) lymphoma cells and, consequently it was non-mutagenic (negative) in mammalian gene mutation test, under the experimental conditions described

Justification for classification or non-classification

Based on the available results, the test chemical is not likely to be mutagenic when tested in vitro on various bacterial strains both in the presence and absence of exogenous metabolic activation system. Hence, according to CLP Regulation, the test chemical can be classified under the category "Not Classified".