Potassium iodide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxic effects of the test chemical was evaluated in vitro using the cytokinesis-block micronucleus assay on CHO cells. The study was performed using CHO cells. The test chemical was dissolved in culture medum and used at dose level of 0.625, 1.25, 2.5, 5 and 10 mM. In preliminary cytotoxicity assays, CHO cells were exposed for 1 h to the test compound at concentrations ranging from 0.001 to 5 mg/ml. The cells were exposed for 3 h to concentrations of the test compounds of 0.625, 1.25, 2.5, 5 and 10 mM. Test compound and MMS (30 mg/ml) were dissolved the culture medium. Exponentially growing CHO-K1 cells were plated in a six-well plate on glass coverslips (1.5 X 10⁵cells/well) and cultured 24 h prior to compound treatment. Duplicate coverslips were established for each experiment, and at least two independent experiments were performed. The cells were exposed to the chemicals at different concentrations for 3 h in a FCS free medium. At the end of treatment, cells were washed twice with PBS before a 20 h incubation in fresh medium containing 10% of FCS and 3 mg/ml of cytochalasin B. Thereafter, cells were washed twice with PBS and allowed to recover for 1.5 h in 10% FCS fresh medium. Cells were fixed with cold methanol, stained with acridine orange (62.5 mg/ml) for 5 min and mounted in Sorensen buffer. Slides were coded and blindly examined under an epifluorescence microscope at 1000X magnification under oil immersion. Briefly, the cells should be binucleated (BN) with an intact nuclear membrane and should be situated within the same cytoplasmic boundary. MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells. One thousand (1000) binucleated cells were scored for each slide. The frequencies of BN, of BN with MN (MNBN) and of apoptotic cells (AP) were estimated. MMS (30 mg/ml), a well known alkylating agent was used as positive control. Cytotoxicity was measured by the BN cell ratio between treated and control slides. Based on the observations made, the test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line and hence it is not liekly to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Genotoxic effects of the test chemical was evaluated in vitro using the cytokinesis-block micronucleus assay on CHO cells

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Details on mammalian cell line
- Type and identity of media: The cell line was grown at 37 C in a humidified atmosphere at
5% CO2 in air, in HAM’S F12 medium with L-glutamine supplemented with 10% fetal calf serum (FCS), penicillin (50 UI/ml) and streptomycine (50 µg/ml). Cells were subcultured 24 h before treatment.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

no data

Test concentrations with justification for top dose:

0.625, 1.25, 2.5, 5 and 10 mM

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

MMS (30 µg/ml)

Details on test system and experimental conditions:

Cell culture procedure
Chinese hamster ovary (CHO-K1) cells were purchased from Eurobio (France). They were routinely maintained from stocks stored in liquid nitrogen. CHO cells were grown at 37 C in a humidified atmosphere at 5% CO2 in air, in HAM’S F12 medium with l-glutamine supplemented with 10% fetal calf serum (FCS), penicillin (50 UI/ml) and streptomycine (50 mg/ml). Cells were subcultured 24 h before treatment.

Cell treatment
In preliminary cytotoxicity assays, CHO cells were exposed for 1 h to the test compounds at concentrations ranging from 0.001 to 5 mg/ml.
In the alkaline comet assay and in the cytokinesisblock micronucleus assay, cells were exposed for 3 h to concentrations of the test compounds of 0.625, 1.25, 2.5,5 and 10 mM. Tests compounds and MMS (30 mg/ml) were dissolved the culture medium. Etoposide (0.5 mg/ml) was dissolved in DMSO.

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 150000 cells/well

DURATION
- Preincubation period: No data
- Exposure duration: 3 hrs
- Expression time (cells in growth medium): 20 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): Acridine orange

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were fixed with cold methanol, stained with acridine orange (62.5 mg/ml) for 5 min and mounted in Sorensen buffer. Slides were coded and blindly examined under an epifluorescence microscope at 1000X magnification under oil immersion.

NUMBER OF CELLS EVALUATED: One thousand (1000) binucleated cells were scored
for each slide.

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, Cytotoxicity was measured by the Binucleate cell ratio between treated and control slides
- Any supplementary information relevant to cytotoxicity: No data

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

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells.

Statistics:

In the cytokinesis-block micronucleus assay, data were expressed as the percentage of binucleated cells with micronuclei. Comparisons between control and treated cell cultures were made using ANOVA and Dunnett’s one sided test.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

not specified

Genotoxicity:

negative

Remarks:

Potassium iodate did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: In preliminary cytotoxicity assays, CHO cells were
exposed for 1 h to the test compounds at concentrations ranging from 0.001 to 5 mg/ml.

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

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

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Conclusions:

The test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line and hence it is not liekly to classify as a gene mutant in vitro.

Executive summary:

Genotoxic effects of the test chemical was evaluated in vitro using the cytokinesis-block micronucleus assay on CHO cells. The study was performed using CHO cells. The test chemical was dissolved in culture medum and used at dose level of 0.625, 1.25, 2.5, 5 and 10 mM. In preliminary cytotoxicity assays, CHO cells were exposed for 1 h to the test compound at concentrations ranging from 0.001 to 5 mg/ml. The cells were exposed for 3 h to concentrations of the test compounds of 0.625, 1.25, 2.5, 5 and 10 mM. Test compound and MMS (30 mg/ml) were dissolved the culture medium. Exponentially growing CHO-K1 cells were plated in a six-well plate on glass coverslips (1.5 X 10⁵ cells/well) and cultured 24 h prior to compound treatment. Duplicate coverslips were established for each experiment, and at least two independent experiments were performed. The cells were exposed to the chemicals at different concentrations for 3 h in a FCS free medium. At the end of treatment, cells were washed twice with PBS before a 20 h incubation in fresh medium containing 10% of FCS and 3 mg/ml of cytochalasin B. Thereafter, cells were washed twice with PBS and allowed to recover for 1.5 h in 10% FCS fresh medium. Cells were fixed with cold methanol, stained with acridine orange (62.5 mg/ml) for 5 min and mounted in Sorensen buffer. Slides were coded and blindly examined under an epifluorescence microscope at 1000X magnification under oil immersion. Briefly, the cells should be binucleated (BN) with an intact nuclear membrane and should be situated within the same cytoplasmic boundary. MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells. One thousand (1000) binucleated cells were scored for each slide. The frequencies of BN, of BN with MN (MNBN) and of apoptotic cells (AP) were estimated. MMS (30 mg/ml), a well known alkylating agent was used as positive control. Cytotoxicity was measured by the BN cell ratio between treated and control slides. Based on the observations made, the test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line and hence it is not liekly to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no study available

Genetic toxicity in vivo

Description of key information

Drosophila lethal mutation assay was performed to determine the mutagenic nature of the test chemical in vivo. The study was performed using male Oregon-R stock Drosophila flies. The test chemical was injected into larvae by use of a micro-injectionfollowing the method of Beadle and Ephrussi.As per this method, 0.5-1.0 cubic mm od substance may be injected into each larva. The gonads were thus bathed in the solution. The treatment after injection of 0.038 per cent I2 in 0.075 per cent KI did not produce a significant increase in the lethal mutation rate of the X-chromosome. Based on the observations made, thetest chemical did not induce lethal mutations in X- chromosomes of Drosophila Oregon-R stock and is hence it is not likely to be mutagenic in vivo.

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

Drosophila lethal mutation test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vivo Drosophila lethal mutation test was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Strain:

other: Oregon-R stock

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: No data
- Weight at study initiation: No data
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: The flies were raised in ½ pint milk bottles
- Diet (e.g. ad libitum): Standard food of corn meal, molasses and agar, with brewers' yeast added,
- Water (e.g. ad libitum): No data
- Acclimation period: No data

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

IN-LIFE DATES: From: To: No data

Route of administration:

other: Micro-injection

Vehicle:

No data

Details on exposure:

No data

Duration of treatment / exposure:

No data

Frequency of treatment:

No data

Post exposure period:

No data

Remarks:

0.5-1.0 cubic mm

No. of animals per sex per dose:

6 males

Control animals:

not specified

Positive control(s):

No data

Tissues and cell types examined:

X-chromosome

Details of tissue and slide preparation:

Other:
The lethals were detected by the usual C1B method:
(1) C1B/x-ple* female by + (treated) male
(2) F1 of C1B/+ female by x-ple male
(3) Females from cultures indicating lethal or semi-lethal mutations were mated by x-ple males.

Evaluation criteria:

No data

Statistics:

No data

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: No mutagenic potential

Additional information on results:

No data

Table: Effect of various test chemical on the lethal mutation rate in the x-chromosome of the Oregon-R race of Drosophila Melanogater

 

Chemical	Treatment	Time	No. of animals	No, of chromosomes tested	No. of lethals	% lethals	Location of lethals	Mutation rate
Test chemical	Injection	-	6	386	2	0.52	-	-

 

Conclusions:

The test chemical did not induce lethal mutations in X- chromosomes of Drosophila Oregon-R stock and is hence it is not likely to be mutagenic in vivo.

Executive summary:

Drosophila lethal mutation assay was performed to determine the mutagenic nature of the test chemical in vivo. The study was performed using male Oregon-R stock Drosophila flies. The test chemical was injected into larvae by use of a micro-injectionfollowing the method of Beadle and Ephrussi.As per this method, 0.5-1.0 cubic mm od substance may be injected into each larva. The gonads were thus bathed in the solution. The treatment after injection of 0.038 per cent I2 in 0.075 per cent KI did not produce a significant increase in the lethal mutation rate of the X-chromosome. Based on the observations made, thetest chemical did not induce lethal mutations in X- chromosomes of Drosophila Oregon-R stock and is hence it is not likely to be mutagenic in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

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

Gene mutation in vitro:

Genotoxic effects of the test chemical was evaluated in vitro using the cytokinesis-block micronucleus assay on CHO cells. The study was performed using CHO cells. The test chemical was dissolved in culture medum and used at dose level of 0.625, 1.25, 2.5, 5 and 10 mM. In preliminary cytotoxicity assays, CHO cells were exposed for 1 h to the test compound at concentrations ranging from 0.001 to 5 mg/ml. The cells were exposed for 3 h to concentrations of the test compounds of 0.625, 1.25, 2.5, 5 and 10 mM. Test compound and MMS (30 mg/ml) were dissolved the culture medium. Exponentially growing CHO-K1 cells were plated in a six-well plate on glass coverslips (1.5 X 10⁵cells/well) and cultured 24 h prior to compound treatment. Duplicate coverslips were established for each experiment, and at least two independent experiments were performed. The cells were exposed to the chemicals at different concentrations for 3 h in a FCS free medium. At the end of treatment, cells were washed twice with PBS before a 20 h incubation in fresh medium containing 10% of FCS and 3 mg/ml of cytochalasin B. Thereafter, cells were washed twice with PBS and allowed to recover for 1.5 h in 10% FCS fresh medium. Cells were fixed with cold methanol, stained with acridine orange (62.5 mg/ml) for 5 min and mounted in Sorensen buffer. Slides were coded and blindly examined under an epifluorescence microscope at 1000X magnification under oil immersion. Briefly, the cells should be binucleated (BN) with an intact nuclear membrane and should be situated within the same cytoplasmic boundary. MN should be morphologically identical to but smaller than nuclei, their diameter usually varied between 1/6th and 1/3rd of the mean diameter of the main nuclei. MN should be readily distinguished and not be linked to the main nuclei via nucleoplasmic bridges. Cells showing chromatin condensation or nuclear fragmentation with an intact cytoplasmic membrane were classified as apoptotic cells. One thousand (1000) binucleated cells were scored for each slide. The frequencies of BN, of BN with MN (MNBN) and of apoptotic cells (AP) were estimated. MMS (30 mg/ml), a well known alkylating agent was used as positive control. Cytotoxicity was measured by the BN cell ratio between treated and control slides. Based on the observations made, the test chemical did not induce any increase in the frequency of MNBN cells for doses ranging from 0.625 to 10 mM in the micronucleus assay in the CHO cell line and hence it is not liekly to classify as a gene mutant in vitro.

In the same study, genotoxic effects of the test chemical was evaluated in vitro using the alkaline comet assay using CHO cells. The test chemical was dissolved in culture medium and used at dose level of 0.625, 1.25, 2.5, 5 and 10 mM. Cells were collected by trypsination,suspended in prewarmed low melting point (LMP) agarose (0.5% in PBS) and deposited on a conventional microscope slide (initially dipped in 1% agarose and dried) precoated with normal agarose (0.8% in PBS). Slides were put in a lysis solution (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris pH10, 10% DMSO and 1% Triton X 100) for 1 h at about 5 C. DNA was allowed to unwind in electrophoresis buffer (0.3 M NaOH, 1 mM EDTA, pH13.6) for 40 min at room temperature. Slides were then placed into a horizontal electrophoresis tank and exposed to 0.7 V/cm (300 mA) for 24 min. After electrophoresis, slides were washed twice in neutralization buffer (0.4 M Tris, pH7.5) and dehydrated in ethanol for 5 min. After staining with ethidium bromide, 50 randomly selected cells per slide were submitted to image analysis. Olive tail moment was used to evaluate the extent of DNA damage in individual cells. Median values of OTM were calculated without taking HDC into account. Each dose was tested in duplicate and at least two independent assays were performed. Etoposide (0.5 mg/ml), a well known inhibitor of topoisomerase II inducing DNA double strand breaks, was used as positive control. In parallel to the assessment of DNA damage, cell viability was measured using the Trypan blue exclusion method. Cell viability was expressed as proportion of total cells. No primary DNA damage was observed after cell exposure to the test chemical. The slight increase in tail moment observed for the 10 mM potassium iodate concentration was not statistically significant from control. The test chemical did not induce DNA damage in CHO cells when tested at concentrations up to 10 mM in alkaline comet assay and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation in vivo:

Drosophila lethal mutation assay was performed to determine the mutagenic nature of the test chemical in vivo. The study was performed using male Oregon-R stock Drosophila flies. The test chemical was injected into larvae by use of a micro-injectionfollowing the method of Beadle and Ephrussi.As per this method, 0.5-1.0 cubic mm od substance may be injected into each larva. The gonads were thus bathed in the solution. The treatment after injection of 0.038 per cent I2 in 0.075 per cent KI did not produce a significant increase in the lethal mutation rate of the X-chromosome. Based on the observations made, thetest chemical did not induce lethal mutations in X- chromosomes of Drosophila Oregon-R stock and is hence it is not likely to be mutagenic in vivo.

In the same study, Drosophila lethal mutation assay was also performed to determine the mutagenic nature of the test chemical in vivo. The study was performed using male Oregon-R stock Drosophila flies. The chorion was removed from fertilized eggs by gently stroking with a blunt glass needle. They were then immersed in sub lethal 50% concentration of the test chemical. The eggs used for the second series of experiments were approximately from 2 to 4 hours old when treated. For the injection series larvae were grown in culture dishes rich in yeast, and larvae just prior to pupation were used. The treatment immersion in 50% test chemical solution did not produce a significant increase in the lethal mutation rate of the X-chromosome. Based on the observations made, thetest chemical did not induce lethal mutations in X- chromosomes of Drosophila Oregon-R stockand hence it is not likely to be mutagenic in vivo.

In vivo mammalian chromosome aberration study was performed in another experiment to determine the mutagenic nature of the test chemical. The study was performed using male and female Wistar, Long-Evans, and Gifu-agouti strain rats.The MTK-sarcoma III, an ascites tumor of rats, was employed exclusively for the experiment. All the animals in which the tumor was transplanted died within 9 days. The test compounds were administered intraperitoneally to rats in each experimental group, starting on the 3rd or 4th day of tumor transfer, at dosages determined after several trials. Rats of control group received intraperitoneal injection of physiological saline solution. Cytological preparations were made according to the acetic-dahlia squash technique at appropriate intervals following application of the chemicals. A part of the preparations were fixed with absolute methanol or with acetic acid-ethanol (3:1) and stained with May-Grunwald-Giemsa or with Feulgen's reagent. The tumor cells were observed for pycnotic aggregation of chromatin, karyorrhexis, multinucleate cells, chromosome clumping, contraction or stickiness of chromosomes, irregular chromosome distribution, lagging or bridges of chromosomes at anaphase, multipolar spindle formation, vacuolization or blebbing of cytoplasm, and disintegration of cytoplasm. Based on the observations made, the test chemical induced chromatid aggregation, stickiness of chromosomes and contraction of chromosomes in the tumor cells of male and femaleWistar, Long-Evans, and Gifu-agouti rats and is likely to be mutagenic in vivo.

Based on the observations made, the test chemical does not enhibit gene mutation in vitro and in vivo. Hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the observations made, the test chemical does not enhibit gene mutation in vitro and in vivo. Hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.