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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro genotoxicity studies with potassium superoxide have produced positive results, whereas the in vivo genotoxicity studies with the degradation product hydrogen peroxide have resulted in negative responses. Overall, due to rapid in vivo degradation of KO2, genotoxic responses are not anticipated. Also, in practice, exposure of workers and consumers to KO2 is not expected and is completely confined to closed containers or articles. Therefore, genotoxicity of KO2 is expected to be of low concern.

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA 92, TA 97, TA 100, TA 102, TA 104, TA 1535, and TA 1537
Metabolic activation:
without
Metabolic activation system:
S9 fractions from male Sprague-Dawley rat kidneys
Test concentrations with justification for top dose:
0, 0.33, 0.67, 1.33, 2.67 and 5.33 µmol/plate
Vehicle / solvent:
neutralized aqueous solution
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
no
Details on test system and experimental conditions:
Method of application: in agar (plate incorporation) + 'Preincubation assay' (20 min incubation in a glass tube)
Number of replications: 3
All strains/cell types tested
Statistics:
Linear regression analysis
Key result
Species / strain:
S. typhimurium, other: TA 92, TA 97, TA 100, TA 102, TA 104, TA 1535, and TA 1537
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Positive in S. typhimurium TA 100, TA 102, and TA 104
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
Salmonella typhymurium strain TA 104 was the most responsive strain. Marked effects were observed in TA 102 as well. In strain TA 100, the test substance only produced a weak increase in the number of mutants. No mutagenicity was observed in the other strains. The spectra of mutagenicity was similar for KO2, H2O2, and glucose oxidase, all being directly or indirectly generators of reactive oxygen species.
Conclusions:
Under the study conditions, the substance was found to be mutagenic in Salmonella typhimurium.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the substance according to OECD Guideline 471. Salmonella typhimurium strains TA 97, TA 100, TA 102, TA 104, TA 1535, and TA 1537 were treated with the test substance using the plate incorporation method with a 'preincubation assay' (20 min) at concentration ranges of 0 - 5.33 µmol/plate without metabolic activation. The exposure to the test substance produced mutagenic response in S. typhimurium strains TA 100, TA 102, and TA 104, with more marked effects in TA 102 and 104. Salmonella typhimurium strain TA 104 was the most responsive strain. Marked effects were observed in TA 102 as well. In strain TA 100, the test substance only produced a weak increase in the number of mutants. No mutagenicity was observed in the other strains. The strain spectra of the mutagenicity were similar for KO2, H2O2, and glucose oxidase, all being directly or indirectly generators of reactive oxygen species. Under the study conditions, the substance was found to be mutagenic in Salmonella typhimurium (Glatt, 1989).

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Homogenous T7 DNA was incubated with the test substance in 10 mM sodium phosphate buffer, pH 7, with subsequent size determination of DNA by band sedimentation through alkaline sodium chloride.
GLP compliance:
not specified
Type of assay:
other: single strand scissions
Species / strain / cell type:
other: T7 bacteriophage
Remarks:
extracted DNA (0.1 mM)
Test concentrations with justification for top dose:
Determined by measuring H2O2 concentration of stock KO2 (concentration of KO2 equals 2 x H2O2) (HO2° O2 + H+ --> H2O2 + O2, rate constant at neutral pH: k (M-1 s-1) = 10E05; McCord et al., 1977)
Vehicle / solvent:
10 mM sodium phosphate buffer at pH 7
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
dianisidine
Positive control substance:
other:
Details on test system and experimental conditions:
Exposure: 1 h at 37°C
The test substance was added to a solution of DNA.
SOD, catalase, metal chelators, exogenous metal ions, and OH. scavengers are used to probe the involvment of reactive reduced oxygen species and specific recations, e.g. Haber-Weiss or Fenton reaction
Rationale for test conditions:
Evaluation of the involvment of reactive reduced oxygen species (O2-, H2O2, and OH.) when KO2 is introduced to a solution of DNA
Evaluation criteria:
Number of strand scissions per intact strand (by band sedimentation through alkaline sodium chloride)
Statistics:
No data
Key result
Species / strain:
other: T7 bacteriophage DNA
Metabolic activation:
not applicable
Genotoxicity:
positive
Remarks:
OH* dependent
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Inhibition of strand scission by very small amount of the chelator DETAPA indicated that O2.and H2O2 by themsleves are not effective agents for breaking DNA. The complete protection observed with catalase and metal chelators strongly suggests that H2O2 (produced by dismutation of O2.-) and metals ions were directly involved in the strand scission observed with the test substance and test substance preincubation. The enhancement of H2O2 - induced strand scission by the addition of exogenous FeSO4 or CuSO4 suggests that H2O2 manifests its activity by being converted to OH. in a Fenton-type reaction. Moreover, OH. scavengers such as ethyl alcohol, tert-butyl alcohol and NaN3 inhibited H2O2 -induced DNA strand scission.

In the presence of both O2. and H2O2, additional OH. can be produced efficiently via a metal-catalyzed Haber-Weiss cycle, where O2. serves as a reducing agent to regenerate the reduced metal ion from oxidized metal ion. The increased strand break observed with the test substance as compared to the test substance preincubation was the result of additional OH. produced via this metal-catalyzed Haber-Weiss cycle. Strand breakage induced by the test substance was inhibited by SOD because the enzyme removed the source of reducing power, i.e., the O2., thereby removing the source of the reduced metal ion needed for OH. formation.
Conclusions:
Under the study conditions, the substance was considered to be mutagenic since it induced DNA strand scission via the generation of reactive OH..
Executive summary:

A study was performed to determine the in vitro mutagenic potential of the test substance in T7 bacteriophage DNA according to the DNA strand scission protocol in a KO2 -DNA system. The number of strand scissions per intact strand in T7 DNA was measured following a 1 h exposure to the test substance, in the presence or absence of metal ions and chelators. Inhibition of strand scission by very small amount of the chelator DETAPA indicated that O2.- and H2O2 by themselves are not effective agents for breaking DNA. The complete protection observed with catalase and metal chelators strongly suggests that H2O2 (produced by dismutation of O2.-) and metals ions were directly involved in the strand scission observed with the test substance or preincubation with the test substance. The enhancement of H2O2 - induced strand scission by the addition of exogenous FeSO4 or CuSO4 suggests that H2O2 manifests its activity by being converted to OH. in a Fenton-type reaction. Under the study conditions, the substance was considered to be mutagenic since it induced DNA strand scission via the generation of reactive OH.. (Lesko, 1980).

Endpoint:
genetic toxicity in vitro, other
Remarks:
Comet assay
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
Principles of method if other than guideline:
In vitro single cell gel/comet assay was conducted in mammalian cells for detection of DNA damage.
GLP compliance:
not specified
Type of assay:
comet assay
Species / strain / cell type:
mammalian cell line, other: V97 Chinese hamster cells (V79-UL)
Details on mammalian cell type (if applicable):
Cells were maintained in minimal essentil medium (MEM) with Earle's salts, supplemented with 10% fetalcalf serum, 2mM glutamine and antibiotics. Cells were cultivated in a humidified incubator at 37°C with 5% CO2 at pH 7.2 and harvested with 0.15% trypsin and 0.08% EDTA
Test concentrations with justification for top dose:
Between 0.15 and 0.6 mM
Vehicle / solvent:
Hank's balanced salt solution
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Statistics:
One-tailed t-test
.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
The substance caused a significant and concentration-related increase in DNA migration in the comet assay but FGP treatment did not enhance this effect.
Remarks on result:
other: cfr "additional information on results"
Conclusions:
Under the study conditions, the test substance was found to induce genotoxic effects by increased DNA migration in the alkaline comet assay.
Executive summary:

A study was conducted to determine the genotoxic potential of the test substance in an alkaline comet assay. The cells were exposed to a concentrations range of 0.15 - 6 mM test substance. The substance caused a significant and concentration-related increase in DNA migration in the comet assay but FGP treatment did not enhance this effect. Under the study conditions, the test substance was found to induce genotoxic effects by increased DNA migration in the alkaline comet assay (Speit, 1999).

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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-KL1
Details on mammalian cell type (if applicable):
Cells were passaged in antobiotic-free F12 media containing 10% fetal calf serum (heat activated) and 10% minimal essential media vitamins in a 5% CO2-in-air humidified incubator at 37°C. Subculture was performed using a 0.05% trypsin-EDTA solution in plastic tissue-flass.
Test concentrations with justification for top dose:
0, 10, 20, 40 and 80 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
no
Details on test system and experimental conditions:
Exposure: 7 d
Cell concentration: 10E+06
Measurement of superoxide anion (O2-) formation from the test substance and subsequent reduction of cytochrome C in the presence or absence of SOD (50 µg/ml)
Addition of superoxide dismutase (SOD) to confirm the involvment of O2- in KO2 induced cytotoxicity and mutagenesis
Rationale for test conditions:
Since O2- is implicated in mutagenic action, cytotoxicity and mutagenicity induced by O2- generated by KO2 were investigated in this study.
Evaluation criteria:
Cytochrome C reduction by O2-
CHO relative survival following exposure to O2- generated by KO2
Number of thioguanine-resistant mutants per 10E06 survivors following exposure to O2- generated by KO2
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
not specified
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
O2- generated by the test substance induced a decreased CHO cell survival and an increased number of HGPRT-deficient mutants resistant to the toxic effects of 6-thioguanine, both effects being reversed by the addition of SOD. Beyond a concentration of 20 µg/mL of the test substance, SOD could no longer protect the cells (cytotoxicity and genotoxicity) against O2- generated by the test substance.
Conclusions:
Under the study conditions, the substance was considered to be mutagenic in CHO cells.
Executive summary:

A study was conducted to determine the genotoxic potential of the substance according to a method similar to OECD Guideline 476. Chinese hamster cell lines (CHO-K1) were exposed for 7 d to the test substance at concentrations ranging from 0 to 80 µg/mL. O2 - generated by the test substance induced a decreased survival of CHO cells and an increased number of HGPRT-deficient mutants resistant to the toxic effects of 6-thioguanine, both effects being reversed by the addition of SOD. Beyond a concentration of 20 µg/mL of the test substance, SOD could no longer protect the cells (cytotoxicity and genotoxicity) against O2- generated by the test substance. Under the study conditions, the test substance was considered to be mutagenic in CHO cells (Cunningham, 1983).

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
Principles of method if other than guideline:
A quantitative protocol to determine cytotoxicity and the frequency of specific-locus mutants induced by chemical and physical mutagenic agents.
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO cell line K1-BH4 (mutation at the hprt locus) and its transformant, AS52 (mutation in the gpt gene)
Test concentrations with justification for top dose:
0, 10, 20, 30, 40, 60 and 80 µg/mL
Untreated negative controls:
yes
Remarks:
untreated
Positive controls:
yes
Remarks:
Streptonigrin and neutron-irradiations
Details on test system and experimental conditions:
Both AS52 cells and the parental K1-BH4 cells were exposed to the test substance, x-rays, UV-light, ethyl methanesulfate, ICR 191 or streptonigrin. Cytotoxicity as well as mutagenic activity were assessed and compared. Moreover, Southern Blot Hybridization Analysis and PCR were used to determine the nature of mutations at the level of restriction fragment length polymorphisms and to screen deletions, respectively.
Rationale for test conditions:
Reactive oxygen species (ROS) are known to be genotoxic to mammalian cells. They produce effects that include cytotoxicity, DNA strand breaks, specific-locus mutations, chromosome aberrations, sister chromatid exchanges, and tumor promotion (Vuillaume, 1987; and Meneghini, 1988).
Potent oxidizing agents such as KO2, which generates ROS, was tested regarding its potential to cause cytotoxicity and genotoxicity.
Evaluation criteria:
Cytotoxicity: cloning efficiency (expressed as the percent survival relative to the untreated control(s))
Mutant frequency: number of 6-thioguanine (TG)-resistant clonies per 10E06 clonable cells
Key result
Species / strain:
other: K1-BH4 and AS52 cells
Metabolic activation:
not applicable
Genotoxicity:
positive
Remarks:
only in AS52 cells
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance induced a similar toxicity in both cell types but was more mutagenic to AS52 cells. This difference in mutagenicity probably results from a higher recovery of multi-locus deletion mutants in the heterozygous gpt gene in AS52 cells in comparison to the hemizygous hprt gene in K1-BH4 cells, rather than a higher induction of mutations. This suggests that agents known to induce reactive oxygens generation should produce predominantly deletion mutations.
Conclusions:
Under the study conditions, the substance was considered to be mutagenic in CHO parental cells (K1-BH4) and transformant AS52 cells.
Executive summary:

A study was conducted to determine the genotoxic potential of the substance in Chinese ovary cell line K1 -BH4 (mutation at the hrpt locus) and its transformant, AS52 (mutation in the gpt gene). Cells were exposed to the test substance at concentrations of 0 - 80 µg/mL. The test substance induced a similar cytotoxicity in both cell types but was more mutagenic to AS52 cells. This differential mutagenecity probably results from a higher recovery of multi-locus deletion mutants in the heterozygous gpt gene in AS52 cells in comparison to the hemizygous hprt gene in K1-BH4 cells, rather than a higher induction of mutations. Under the study conditions, the substance was considered to be mutagenic in CHO parental cells (K1-BH4) and transformant AS52 cells (Hsie 1990).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vitro genotoxicity studies with potassium superoxide have produced positive results, whereas the in vivo genotoxicity studies with the degradation product hydrogen peroxide have resulted in negative responses. Overall, due to rapid in vivo degradation of KO2, genotoxic responses are not anticipated. Also, in practice, exposure of workers and consumers to KO2 is not expected and is completely confined to closed containers or articles. Therefore, genotoxicity of KO2 is expected to be of low concern.

No significant exposure occurs at the level of industrial/professional handling: KO2 is not manufactured in the EU. The substance is imported in the form of low dust tablets which are sealed and packed. Upon opening and before use, dust reduction measures are taken to avoid exposure via inhalation. The tablets are transferred from the barrels/bags into cartridges that are integrated into sealed Oxygen Self Rescuer units.

No significant exposure occurs at the level of consumer use: The self-rescuer device serves as a rescue device in emergency situations e.g. in mines or aircrafts. The user puts on the device and breathes through a mouthpiece and breathing tube. The moisture and CO2 from the breath react with KO2 to generate oxygen, potassium hydroxide and potassium carbonate in the cartridge. Due to the specific design of the device, the user is protected from any contact with KO2 or inhaling KO2 dust. When spent, the cartridges are sent back for recycling or waste incineration. Unused devices are disposed of in a similar way.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
other: evidence based on degradation product
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification. The in vivo genetic toxicity study with the degradation product is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
Test animals
- Source: Charles River Ltd. (UK), Margate, UK
- Age at study initiation: 42-52 days
- Weight at study initiation: 194-263 g
- Assigned to test groups randomly: yes (due to deaths some additional rats were dose which had not been included in the randomisation process)
- Fasting period before study:
- Housing: not more than 3 in polypropylene cages with wire mesh lids
- Diet (e.g. ad libitum): laboratory chow diet (Special Diet Services Ltd., RM1.[E].SQC) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 to 10 days

Environmental conditions
- Temperature (°C): 19-21
- Humidity (%): 48-59
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours darkness
Route of administration:
intravenous
Vehicle:
Water for injection (Phoenix Pharmaceuticals Ltd., Gloucester, UK, batch number 515004)
Details on exposure:
Dosing preparations were made by diluting the test substance in water for injection. Dilutions were made using water for injection. The test substance preparations were protected from light, stood on ice prior to use and were used within approximately 4 hours of initial formulation. Animals were administered the appropriate concentration dosing solution at a dose rate of 0.2 mL/min, to a total administered dose volue of approximately 25 mL/kg.
Duration of treatment / exposure:
25-33 minutes (at a dose rate of 0.2 mL/min)
Frequency of treatment:
Once by intravenous administration
Post exposure period:
2-4 or 12-14 hours
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
in water
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
in water
No. of animals per sex per dose:
Five to six
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetamidfluorene (2-AAF) was freshly suspended in corn oil using a Silverson homogeniser at 7.5 mg/mL to serve as the positive control for the 12-14 hour experiment.
Dimethylnitrosamine (DMN) dissolved in purified water at 1.0 mg/mL was used as the positive control for the 2-4 hour experiment.
Both positive controls were administered by oral gavage.
Tissues and cell types examined:
Liver, hepatocytes
Details of tissue and slide preparation:
Animals were anaesthetised. The abdominal surface was rinsed with 70 % ethanol, a V-shaped incision was made from the centre of the lower abdomen o the rib cage, and the skin and muscle removed to reveal the abdominal cavity. The inferior vena cava was then clamped superior to the kidney and a cotton tie was placed loosely around the hepatic portal vein. The vein was then cannulated using an appropriate catheter, the inner needle removed, and the ligature tightened. Cannulation of the superior vena cava was carried out via the right atrium, following cutting of the diaphragm and removal of the rib cage. The hepatic portal cannula was connected to a supply of calcium free Buffer 1 which had been gassed for at least 5-10 minutes with 5 % CO2 in air (v/v), and pumped at approximately 40 mL/min. The vena cava cannula was connected to a waste line and the liver washed free of blood, using approximately 400 mL Buffer 2 which was continually gassed with 5 % CO2 in air (v/v), at 40 mL/min until the reservoir volume had dropped from 400 mL to approximately 200 mL. The liver was then cut free and transferred to a sterile plastic petri dish with approximately 10 mL of the prewarmed (37 °C) Buffer 2. The liver capsule was removed and the hepatocytes carefully teased out using a combing method. The separated hepatocytes were gently washed through 150 micrometre nylon mesh with Williams E medium-Complete (WE-C) to a volume of 100 mL. Of this suspension, approximately 50 mL was taken and centrifuged at approximately 40 x g for 2-3 minutes. The resulting pellet was resuspended in approximately 40 mL WE-C. The centrifugation and resuspension procedure was repeated twice more and celles taken, diluted with an equal volume of 0.4 % (w/v) trypan blue and the proportion of viable cells (those with unstained nuclei) determined using a haemocytometer. The culture was then diluted to provide 1.5 x 10e5 viable cells/mL.
Medium was removed from the cells and the monolayers washed with 2 mL Williams E medium-Incomplete (WE-I) which was then replaced with 2 mL WE-I containing 10 microCi/mL [3H] thymidine. After 4 hours incubation at 37 °C in a 5 % CO2 in air (v/v) atmosphere, the medium was removed and the ceells washed with three changes of WE-I containing 0.25 mM thymidine. Cultures were then incubated overnight with 3 mL of the same medium. To prepare for autoradiography, coverslips were washed with 2 mL phosphate buffered saline and the cells fixed with three changes of 2 mL glacial acetic acid:ethanol (1:3 v/v). The coverslips were then washed four times with purified water, allowed to dry and mounted onto previously labelled microscope slides, cells side up, with DPX.
Evaluation criteria:
The study would be considered valid if:
1) the negative control animals had 0 net grains/nucleus (NNG) counts or less (i.e. a negative value, within historical range),
2) the positive control treatments had NNG values of five or more, with 50 % or more cells having NNG counts of five or greater.

The test substance would be considered as positive in the assay if, at any dose and at either time point,
1. the test substance yielded group mean NNG values greater than 0 NNG and 20 % or more of cells responding (mean NNG values similar to or greater than 5),
2. an increase was seen in both NNG and the percentage of cells in repair.
If the test substance failed to induce UDS at any dose tested after both 2-4 and 12-14 hours exposure, it would be considered as clearly negative in this system.
Statistics:
No data
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean net grain count for vehicle-control animals was less than zero and the positive control substances induced increases in group mean net grain counts of five or more (9.4 and 10.4 respectively), and 50% or more cells (84.7% and 83.7% respecively) had net grain counts of five or more.
Conclusions:
Under the study conditions, the substance was not genotoxic in vivo in the unscheduled DNA synthesis assay in rats.
Executive summary:

A study was conducted to determine the in vivo genotoxicity toxicity of the degradation product hydrogen peroxide according to OECD Guideline 486, in compliance with GLP. Male Wistar rats were administered intravenously the test solutions at concentrations of 1 or 2 mg/mL at a dose rate of 0.2 mL/min to achieve final doses of 25 mg/kg bw or 50 mg/kg bw. Animals were sacrificed either after 2 -4 hours or 12 -14 hours. In the 2 -4 hour experiment, dimethylnitrosamine (DMN) was used as the positive control and in the 12 -14 hour experiment, the positive control was 2 -acetamidfluorene (2 -AAF). Negative control animals were administered vehicle only (water). After sacrifice, hepatocytes from the liver were sampled and cultures of hepatocytes were treated with [3H] thymidine. Slides were prepared from each animals with fixed hepatocytes and dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically and the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm was determined (the net grain count, NNG). Negative controls produced a group mean NNG value of less than zero with 0 -0.3 % cells in repair. Group mean NNG values were increased by 2 -AAF and DMN treatment to at least 9.4 and more than 80% of cells were found to be in repair. In vivo treatment with 25 or 50 mg/kg bw did not produce a group mean NNG value greater than zero (-2.1- -2.7 respectively) nor were any more than 0.7% cells found in repair at either dose or time point. Under the study conditions, the substance was not genotoxic in vivo in the unscheduled DNA synthesis assay in rats (European Chemicals Bureau, 2003).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: evidence based on degradation product
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details. The genetic toxicity study with the degradation product is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Species:
mouse
Strain:
other: Swiss OF1/ICO:OF1 (IOPS Caw)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals:
- Source: Iffa Crédo, L'Arbresle, France
- Age at study initiation: approximately 6 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: five per sex in polycarbonate cages
- Diet (e.g. ad libitum): AO4 C pelleted diet (U.A.R., Villemoisson-sur-Orge, France) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

Environmental conditions:
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness
Route of administration:
intraperitoneal
Vehicle:
Water
Details on exposure:
The test substance was administered once by intraperitoneal route using a dose volume of 25 mL/kg, which allowed to test higher doses with less concentrated solutions. The quantitiy of the test substance administered to each animal was adjusted according to the body weight recorded at the time of dosing. The vehicle control animals received the vehicle alone, under the same conditions. The positive control animals received cyclophosphamide, by oral route, at a volume of 10 mL/kg.
Duration of treatment / exposure:
Once by intraperitoneal injection
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
Cyclophosphamide, administered by oral route in 10 mL/kg at a dose of 50 mg/kg body weight.
Tissues and cell types examined:
For each animal, the micronuclei were counted in 20000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Details of tissue and slide preparation:
At the time of sacrifce, all the animals were killed after CO2 inhalation in excess. The femurs of the mice were removed and the bone marrow eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with May-Grünwald-Giemsa. All the slides were coded for scoring.
Evaluation criteria:
A positive response was assumed if a statistically significant increase in the number of micronucleated polychromatic erythrocytes (MPE) when compared to the vehicle group occurred, which doubled the number of MPE of the historical control data, i.e. a number greater than 3.6/1000 PE. The results were considered as negative if the above criteria was not fully met.
Statistics:
The mean number of MPE and the PE/NE ratio from the treated groups were compared to simultaneous vehicle groups. The inter-group comparison was performed using: for MPE the X-square test, for PE/NE ratio the Student's t-test in which p = 0.05 was used as the lowest level of significance.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean values of MPE of all groups treated with the test substance were similar to those of their respective controls. A slight, statistically significant increase in the MPE number of the low-dose group after observed after 24 hours was considered as biologically insignificant, because the MPE value was within the range of historical controls, no dose-effect relationship was noted, and the increase was essentially attributed to one animal which had 13 MPE/2000 PE. The PE/NE ratio was statistically significant lower at the three doses at 24 hours and at 250 and 1000 mg/kg after 48 hours, showing that the test substance effectively affected the bone marrow cells.
Due to the marked mortality in the 2000 mg/kg dose group, a second cytogenetic study was carried out. In this second test, no mortality and no clinical signs were observed in the 250 and 500 mg/kg dose groups of both sexes and in the female 1000 mg/kg dose group. After treatment with 1000 mg/kg, one of sixteen males died, which was replaced by one of the supplementary group. Hypoactivity and piloerection was noted in the other treated males at 1000 mg/kg. No macroscopic abnormalities (abdominal cavity) were seen at necropsy at doses of 250, 500 and 1000 mg/kg except for a discolourated spleen in one male at 1000 mg/kg.
The mean values of micronucleated polychromatic erythrocytes were within the historical range in the two vehicle groups.
Cyclophosphamide induced a highly significant increase (p < 0.001) in the number of MPE. In addition, the PE/NE ratio decreased significantly (p < 0.05) showing the toxic effects of the positive control substance to bone marrow cells.
Conclusions:
Under the study conditions, the substance was not genotoxic in bone marrow erythrocyte micronucleus assay in mice.
Executive summary:

A study was conducted to determine the in vivo genetic toxicity of the degradation prouct hydrogen peroxide, according to OECD Guideline 474, in compliance with GLP. The test substance was tested for its potential to induce cytogenetic damage to the bone marrow cells of Swiss OF1 mice. Following preliminary toxicity testing, animals received one intraperitoneal injection of the test substance at concentrations of 0 to 2000 mg/kg bw (first cytogenicity test) or 0 to 1000 mg/kg bw (second cytogenicity test). The positive control animals received cyclophosphamide, by oral route, at a volume of 10 mL/kg. For each animal, bone marrow smears were prepared and the micronuclei were counted in 2000 polychromatic erythrocytes. The polychromatic (PE) to normochromatic (NE) erythrocyte ratio was established by scoring 1000 erythrocytes (PE + NE). The mean number of micronucleated polychromatic erythrocytes (MPE) and the PE/NE ratio from the treated groups were compared to simultaneous vehicle groups. The mean values of MPE of all groups treated with the test substance were similar to those of their respective controls. The mean values of micronucleated polychromatic erythrocytes were within the historical range in the two vehicle groups. Cyclophosphamide induced a highly significant increase in the number of MPE and a decrease in the PE/NE ratio showing the toxic effects to bone marrow cells. Under the study conditions, the test substance was not genotoxic in bone marrow erythrocyte micronucleus assay in mice (European Chemicals Bureau, 2003).

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

Mode of Action Analysis / Human Relevance Framework

In vivo, potassium superoxide reacts rapidly with water to produce potassium hydroxide (KOH), oxygen (O2) and potassium hydrogen peroxide (KHO2), which slowly degrades to KOH, H2O2 (hydrogen peroxide) and O2. KOH further dissociates into potassium and hydroxyl ions which constitute normal physiological ion pool. On the other hand, hydrogen peroxide is likely to degrade within a short time in in vivo conditions due to many alternative and competitive degradation pathways. Especially, in alkaline medium and in the presence of heavy and transition metals it is degraded rapidly.

Several in vitro studies have demonstrated that KO2 is genotoxic. This effect is mediated by reactive oxygen species (ROS), such as H2O2 and derived products. The results were confirmed by the presence of degrading enzymes, e.g. catalase. H2O2 is positive in a wide range of in vitro models whereas it had a negative outcome in in vivo studies. The key element seems to be the accessibility of ROS to the target. Indeed, in in vitro studies, the substance is in direct contact with the target whereas in in vivo studies, H2O2 can either decompose in the bowel before absorption or be degradated by catalase in red blood cells, thus decreasing its bioavailibility. Local toxicity at the site of injection following intraperitoneal administration, confirms that the contact between the ROS and the target is crucial for H2O2 - induced toxicity (Joint Assessment of Commodity Chemicals No. 22. Hydrogen Peroxide. ECETOC, January 1993). Based on these evidences, hydrogen peroxide was not classified as a mutagen. Similar argument can be presented for potassium superoxide. No significant exposure occurs at the level of industrial/professional handling or consumer use. In practice, KO2 is completely confined to closed containers or articles and hence not expected to come in contact with humans during normal use conditions.

Therefore, overall considering the in vivo degradation of KO2 and inaccessibility to human beings under normal use conditions, genotoxicity of KO2 is expected to be of low concern.

Additional information

In vitro studies:

A study was conducted to determine the in vitro genetic toxicity of the substance according to OECD Guideline 471. Salmonella typhimurium strains TA 97, TA 100, TA 102, TA 104, TA 1535, and TA 1537 were treated with the test substance using the plate incorporation method with a 'preincubation assay' (20 min) at concentration ranges of 0 - 5.33 µmol/plate without metabolic activation. The exposure to the test substance produced mutagenic response in S. typhimurium strains TA 100, TA 102, and TA 104, with more marked effects in TA 102 and 104. Salmonella typhimurium strain TA 104 was the most responsive strain. Marked effects were observed in TA 102 as well. In strain TA 100, the test substance only produced a weak increase in the number of mutants. No mutagenicity was observed in the other strains. The spectra of the mutagenicity are similar for KO2, H2O2, and glucose oxidase, all being directly or indirectly generators of reactive oxygen species. Under the study conditions, the substance was found to be mutagenic in Salmonella typhimurium (Glatt, 1989).

A study was performed to determine the in vitro mutagenic potential of the test substance in T7 bacteriophage DNA according to the DNA strand scission protocol in a KO2 -DNA system. The number of strand scissions per intact strand in T7 DNA was measured following a 1 h exposure to the test substance, in the presence or absence of metal ions and chelators. Inhibition of strand scission by very small amount of the chelator DETAPA indicated that O2.- and H2O2 by themselves are not effective agents for breaking DNA. The complete protection observed with catalase and metal chelators strongly suggests that H2O2 (produced by dismutation of O2.-) and metals ions were directly involved in the strand scission observed with the test substance or preincubation with the test substance. The enhancement of H2O2 - induced strand scission by the addition of exogenous FeSO4 or CuSO4 suggests that H2O2 manifests its activity by being converted to OH. in a Fenton-type reaction. Under the study conditions, the substance was considered to be mutagenic since it induced DNA strand scission via the generation of reactive OH.. (Lesko, 1980).

A study was conducted to determine the genotoxic potential of the test substance in an alkaline comet assay. The cells were exposed to a concentrations range of 0.15 - 6 mM test substance. The substance caused a significant and concentration-related increase in DNA migration in the comet assay but FGP treatment did not enhance this effect. Under the study conditions, the test substance was found to induce genotoxic effects by increased DNA migration in the alkaline comet assay (Speit, 1999).

A study was conducted to determine the genotoxic potential of the substance according to a method similar to OECD Guideline 476. Chinese hamster cell lines (CHO-K1) were exposed for 7 d to the test substance at concentrations ranging from 0 to 80 µg/mL. O2 - generated by the test substance induced a decreased survival of CHO cells and an increased number of HGPRT-deficient mutants resistant to the toxic effects of 6-thioguanine, both effects being reversed by the addition of SOD. Beyond a concentration of 20 µg/mL of the test substance, SOD could no longer protect the cells (cytotoxicity and genotoxicity) against O2- generated by the test substance. Under the study conditions, the test substance was considered to be mutagenic in CHO cells (Cunningham, 1983).

A study was conducted to determine the genotoxic potential of the substance in Chinese ovary cell line K1 -BH4 (mutation at the hrpt locus) and its transformant, AS52 (mutation in the gpt gene). Cells were exposed to the test substance at concentrations of 0 - 80 µg/mL. The test substance induced a similar cytotoxicity in both cell types but was more mutagenic to AS52 cells. This differential mutagenecity probably results from a higher recovery of multi-locus deletion mutants in the heterozygous gpt gene in AS52 cells in comparison to the hemizygous hprt gene in K1-BH4 cells, rather than a higher induction of mutations. Under the study conditions, the substance was considered to be mutagenic in CHO parental cells (K1-BH4) and transformant AS52 cells (Hsie 1990).

In vivo studies:

A study was conducted to determine the in vivo genetic toxicity of the degradation product hydrogen peroxide, according to OECD Guideline 474, in compliance with GLP. The test substance was tested for its potential to induce cytogenetic damage to the bone marrow cells of Swiss OF1 mice. Following preliminary toxicity testing, animals received one intraperitoneal injection of the test substance at concentrations of 0 to 2000 mg/kg bw (first cytogenicity test) or 0 to 1000 mg/kg bw (second cytogenicity test). The positive control animals received cyclophosphamide, by oral route, at a volume of 10 mL/kg. For each animal, bone marrow smears were prepared and the micronuclei were counted in 2000 polychromatic erythrocytes. The polychromatic (PE) to normochromatic (NE) erythrocyte ratio was established by scoring 1000 erythrocytes (PE + NE). The mean number of micronucleated polychromatic erythrocytes (MPE) and the PE/NE ratio from the treated groups were compared to simultaneous vehicle groups. The mean values of MPE of all groups treated with the test substance were similar to those of their respective controls. The mean values of micronucleated polychromatic erythrocytes were within the historical range in the two vehicle groups. Cyclophosphamide induced a highly significant increase in the number of MPE and a decrease in the PE/NE ratio showing the toxic effects to bone marrow cells. Under the study conditions, the test substance was not genotoxic in bone marrow erythrocyte micronucleus assay in mice (European Chemicals Bureau, 2003)

A study was conducted to determine the in vivo genotoxicity toxicity of the degradation product hydrogen peroxide according to OECD Guideline 486, in compliance with GLP. Male Wistar rats were administered intravenously the test solutions at concentrations of 1 or 2 mg/mL at a dose rate of 0.2 mL/min to achieve final doses of 25 mg/kg bw or 50 mg/kg bw. Animals were sacrificed either after 2 -4 hours or 12 -14 hours. In the 2 -4 hour experiment, dimethylnitrosamine (DMN) was used as the positive control and in the 12 -14 hour experiment, the positive control was 2 -acetamidfluorene (2 -AAF). Negative control animals were administered vehicle only (water). After sacrifice, hepatocytes from the liver were sampled and cultures of hepatocytes were treated with [3H] thymidine. Slides were prepared from each animals with fixed hepatocytes and dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically and the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm was determined (the net grain count, NNG). Negative controls produced a group mean NNG value of less than zero with 0 -0.3 % cells in repair. Group mean NNG values were increased by 2 -AAF and DMN treatment to at least 9.4 and more than 80% of cells were found to be in repair. In vivo treatment with 25 or 50 mg/kg bw did not produce a group mean NNG value greater than zero (-2.1- -2.7 respectively) nor were any more than 0.7% cells found in repair at either dose or time point. Under the study conditions, the substance was not genotoxic in vivo in the unscheduled DNA synthesis assay in rats (European Chemicals Bureau, 2003).

Justification for classification or non-classification

Based on the evidences presented above, potassium superoxide is not considered to warrant classification according to EU CLP (1272/2008) criteria.