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In vitro tests

6 strains of histidin auxotroph Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100, TA 92, TA 94) were exposed to different concentrations (applied concentrations not specified) of sodium hypochlorite in absence and presence of S9-mix. Duplicate plates were used for each of the six concentrations (Ishidate 1984). The number of revertant colonies was scored after incubation at 37 °C for 2 days. There was no increased incidence of revertant colonies without S9-mix in any strain. With S9-mix there were 330 revertant colonies per plate at a concentration of 5 mg sodium hypochlorite/plate in the TA100 strain. The respective negative control showed 98 revertants. All other assays were negative.

Sodium hypochlorite (applied concentrations not specified) was tested for mutagenicity in a second Ames test (Kawachi et al. 1980) with and without metabolic activation. The Salmonella typhimurium strains used were TA100 and TA 98. There was no increased incidence of revertant colonies without metabolic activation in both strains and with metabolic activation in the TA98 strain. With S9-mix there were 35 revertant colonies per mg in the TA100 strain.

In a modified Ames test, the fluctuation test (LeCurieux et al. 1993), sodium hypochlorite (0.1 100 µg/mL) was exposed to Ames TA 100. 98 and 102 strains in a liquid medium in many replicate cultures (96-well microplate). After 3 days incubation bromothymol blue was added, which stains positive wells (containing prototrophic mutants) yellow, negative wells remain green. All assays were performed twice using triplicate microplates for every concentration. There was no detectable mutation.

In a cytogenicity test (Ishidate 1984) cells from the lung of a Chinese hamster were exposed to three concentrations (max. concentration: 0.5 mg/mL; other concentrations not specified) of sodium hypochlorite for 24 and 48 hours. 100 well-spread metaphases were observed under the microscope. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls. For a quantitative evaluation of the clastogenic potential the D20 was calculated which is the dose at which structural aberrations including gaps were detected in 20 % of the metaphases observed. In addition, the TR value was calculated which indicates the frequency of cells with exchange-type aberrations per unit dose. After 48 hours, 2 % of the evaluated cells were polyploid and 21 % showed structural aberrations at the maximum dose of 0.5 mg/mL. The D20 value was therefore 0.5 mg/mL, the TR value 18. At 24 hours the results were all negative, all other test material concentrations gave also negative results. The findings when applying metabolic activation in the form of S-9 mix were equivocal. Overall, the results are considered to be equivocal.

Sodium hypochlorite was tested for clastogenicity in mammalian cells in a second in vitro cytogenicity study (Matsuoka 1979). Cells from a clonal sub-live derived from the lung of a male Chinese hamster (CHL) were incubated for three hours with the test substance (0.5 mg/mL) and with or without S9-mix as metabolic activation system. Cells were cultured for 24 days and after fixation 100 well-spread metaphases were analysed for chromosomal aberrations. There were no results for the test without metabolic activation as the concentration used was cytotoxic. With S9-mix sodium hypochlorite induced gaps, breaks, exchanges and rings 11 % of the metaphases. It was therefore considered to be weakly positive in this assay.

Sodium hypochlorite was also tested for its ability to induce chromosome breakages, sister chromatid exchanges and micronuclei in human fibroblasts (Sasaki et al. 1989). The cells were grown and mixed with the test material solution in different concentrations (0.0744, 0.1488 mg/mL) and 5-bromodeoxyuridine. After incubation for 40-48 hours, chromosome slides were prepared and 100 metaphases were evaluated for the presence of chromosome breakages, sister chromatid exchanges and micronuclei. Mean values for sister chromatid exchanges were 8.00 and 10.12 per cell for 0.0744 and 0.1488 mg/mL, respectively. The negative control value (solvent) lead to 4.92 sister chromatid exchanges per cell. Therefore the trigger value of 2 x control value is reached for the high concentration. As this value is only just above the trigger and there are no chromosome breakages and micronuclei this result is considered to be equivocal and not to represent a clear indication of genotoxicity.

In a reverse gene mutation assay in yeast (Buschini, 2004) , strains of S. cerevisiae were exposed to Sodium hypochlorite at concentrations of 0, 0.2, 1, 5, 10, 20, 30 ppm in the presence and absence of mammalian metabolic activation (Cyt. P450 enzymes).

Sodium Hypochlorite was tested up to cytotoxic concentrations. A dose-dependent cell mortality rate was observed with 1 and 5 p.p.m. as the lowest effective doses (LED) in stationary (stat) and logarithmic (log) growth phase S. cerevisiae cells, respectively. Significant genotoxic effects were only observed in stat cells for gene conversion (>30 ppm.), reversion of ilv92 mutant (>10 ppm.) and respiration- deficient colonies (>20 ppm). Both convertant and RD frequencies increased with disinfectant concentration, whereas revertant induction up to 10 ppm appeared to be limited by the corresponding high cytotoxicity. The positive controls induced the appropriate responses in the corresponding strains. There was a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.

In a Comet assay (Buschini, 2004) human leucocytes were exposed to Sodium hypochlorite at concentrations of 0, 0.1, 0.2, 0.5, 1, 5 ppm in the absence of mammalian metabolic activation.

Sosium hypochlorite was tested up to cytotoxic concentrations. A significant effect (P < 0.001) on cell survival was detected at 1 ppm (16% of cell mortality) in the Comet assay. The LED (lowest effect dose) for induction of a significant increase (P < 0.05) in DNA migration is 0.5 p.p.m. However, the slope of the dose-response curve appears very slight (TL increase/p.p.m. = 0.061 mm, R2 = 0.94). The positive controls did induce the appropriate response. There was a weak concentration related positive response of cells with DNA strand breaks over background.

This study is classified as acceptable.

In vivo tests

Groups of eight weeks old, male ddY mice were intraperitoneally administered a single dose of sodium hypochlorite at concentrations of 0, 312.5, 625, 1250, 2500 mg av Cl/kg bw in a first test run (Hayashi et al. 1988). In a second trial 4 doses of 300 mg av Cl/kg bw spaced by 24 hours were similarly administered. The animals were killed by cervical dislocation 24 hours after the (last) administration. Femoral marrow cells were flushed out with foetal bovine serum and smeared on clean glass slides. 1000 polychromatic erythrocytes were scored and the number of micronucleated polychromatic erythrocytes was recorded. The proportion of polychromatic erythrocytes among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide. There was no sign of genotoxicity under the conditions described in the study.

In a second micronucleus test (Meier et al. 1985), groups of 8-11 weeks old, male/female CD-1 mice were orally (gavage) administered five doses of sodium hypochlorite at concentrations of 0, 1.6, 4.0 and 8.0 mg av Cl/kg bw daily on five subsequent days. The animals were killed by CO2 or cervical dislocation 6 hours after the (last) administration. Marrow cells were flushed out with foetal bovine serum, centrifuged, spread on slides, air-dried and stained in May-Gruenwald solution and Giemsa. 1000 polychromatic erythrocytes were scored and the number of micronucleated polychromatic erythrocytes was recorded. Sodium hypochlorite did no induce micronuclei.

Two cytogenetic bone marrow aberration assays were conducted:

- A. acute: 8-11 weeks old, male/female CD-1 mice were dosed acutely with sodium hypochlorite (orally, concentration: 0, 1.6, 4.0 and 8.0 mg/kg bw) and sacrificed after 6, 24 and 48 hours.

- B. Repeated dose test: 8-11 weeks old, male/female CD-1 mice were orally (gavage) administered five doses of sodium hypochlorite at concentrations of 0, 1.6, 4.0 and 8.0 mg av Cl/kg bw daily on five subsequent days and killed 6 hours after the last administration.

3 hours prior to sacrifice, animals were injected i.p. with 4.0 mg/kg bw colchicine to collect metaphases. The prepared slides of marrow cells were stained with 5-10 % Giemsa. The mitotic index was determined by scoring the number of cells in mitosis based on at least 500 cells. The endpoints were number of structural and numerical aberrations. Sodium hypochlorite did not induce chromosomal aberrations in the bone marrow of mice which shows a lack of clastogenic activity.

The oral administration of renal carcinogens is often associated with increased levels of 8-hydroxyguanosine in the target organ. To evaluate the carcinogenic potential of sodium hypochlorite, rats were orally administered 900 mg/kg bw of a sodium hypochlorite solution by gavage (Kasai et al. 1987). The animals were killed 0, 3, 6, 12, 24 and 48 hours after treatment and liver and kidneys were removed. After homogenisation the DNA was isolated, digested and analysed by HPLC. The amount of 8-hydroxyguanosine was compared to control samples from untreated animals. There was no significant increase in 8-hydroxyguanosine levels in the liver and kidney of treated animals when compared to control samples. This negative result was considered to confirm the lack of genotoxicity/mutagenicity and carcinogenicity of sodium hypochlorite.

Germ cell effects

Groups of 8-11 weeks old male B6C3F1 mice were orally (gavage) administered five doses of sodium hypochlorite at concentrations of 0, 1.6, 4.0 and 8.0 mg av Cl/kg bw daily on five subsequent days (Meier et al. 1985). The animals were killed by CO2 or cervical dislocation 1, 3 and 5 weeks after the (last) administration. The caudae epdidymides were dissected and suspended in 0.9 % saline. The solution was filtered and stained with 1 % Eosin Y. Slides were prepared from this solution and 1000 sperm-heads per animal were analysed. Observations of increased sperm-head abnormalities at 3 weeks following treatment, but not 1 or 5 weeks, suggest that the effect may be specific to late primary spermatocytes. In relation to levels of expected human exposure (app. 0.003 to 0.3 mg av Cl/kg bw/d), the dose levels employed in this study were high (1.6 to 8 mg av Cl/kg bw/d). The lowest dose level applied to mice was 5 times higher than a possible human exposition would be. Furthermore, no alterations in sperm count, sperm direct progressive movement, percent motility or sperm morphology were observed among adult male rats in a one-generation reprotoxicity study.

There are no studies on the in vitro gene mutation assays in mammalian cells of sodium hypochlorite available. However, chapter 1.4.3 of the TNsG on data requirements states that it is acceptable to waive a study if “the study is not scientifically necessary”, i.e. if “ the result may be predicted reliably from the intrinsic properties of the chemical“. Moreover, from substances like NaOH, which is generated by the contact of sodium hypochlorite with water/wetness, it is known that mutagenic effects are exerted due to high, non-physiological pH-values which can cause genotoxic effects in cultured mammalian cells as well.

The only constituents of sodium hypochlorite, sodium cations, chloride and hydroxy anions, are known to be essential substances of the human body which do not possess any genotoxic potential and do not undergo any bioactivation in the body.

The available in vivo micronucleus tests (Hayashi et al, 1988 and Meier et al., 1985) are clearly negative. The conducted bone marrow aberration assay also showed no significant effects (Meier et al., 1985). Furthermore, the results of the carcinogenicity study in rats and mice did not raise any concerns about the carcinogenic potential of sodium hypochlorite (Kurokawa et al, 1986). The increased incidence of lymphomas and leukaemias (8 to 12 %) in females observed in a carcinogenicity study in rats (Soffritti, 1997) are within the historical control data of 14.67 % (please refer to “Compilation of Spontaneous Neoplastic Lesions and Survival in Crl:CD(R) (SD) Rats from Control Groups” 2004, Charles River Laboratories).

In addition, the mutagenic potential of sodium hypochlorite was investigated in the reverse mutation test with bacteria (S. typhimurium TA100, TA98, TA94, TA92, TA1535 and TA1537) in the presence and absence of metabolic activation. Except in one of two experiments conducted with TA100 in presence of S9 mix sodium hypochlorite did not induce an increase in the number of revertants in any of the bacteria strains tested with or without S9 mix (Ishidate et al (1984); Kawachi et al (1980) and Le Curieux et al. (1993)).

Sodium hypochlorite was investigated in three studies for its potential to exert clastogenic effects in the chromosomal aberration test and sister chromatid exchange study using Chinese hamster lung fibroblasts (CHL) and Human HE2144 fibroblasts, respectively. Under the experimental conditions employed, sodium hypochlorite equivocal results in Chinese hamster lung fibroblasts/Human fibroblasts (21% structural aberrations after 48 h exposure to 0.5 mg/mL and negative after 24 h exposure; Isihidate, 1984; 10.12 sister chromatid exchanges per cell were observed at the highest concentration tested, 0.1488 mg/mL, compared to 4.92 in case of negative control in the Human fibroblast study; no chromosome breakages and micronuclei were observed; Sasaki, 1980). In the second study conducted in Chinese Hamster fibroblasts in presence of S9 mix chromosomal gaps, breaks, and exchanges were observed in 11 % of the metaphases which mean a weak positive result (Matsuoka, 1979). The positive response of sodium hypochlorite must be seen as a secondary effect of the oxidising hypochlorite ion leading to the generation of reactive (oxygen) species and causing an increased incidence in the numbers of structural chromosomal aberrations.

Based on the weight of evidence (negative results in in vivo mutagenicity tests, and bioavailability shown by the acute oral toxicity study) the results of all of these tests are representative for demonstrating the absence of a mutagenic potential of sodium hypochlorite since this substance will dissociate under physiological (i.e. aqueous) conditions and release Na+ and Cl-.

Since the available in vivo studies clearly demonstrate that neither sodium nor chloride reveal a mutagenic potential, further testing of the genotoxic potential of sodium hypochlorite is scientifically unjustified and should not be performed.

Summary

Sodium hypochlorite showed positive results in one of three available in vitro tests in bacteria, but only in strain TA100. In the two other tests, negative results were obtained in all strains used, including TA100. Equivocal or positive results were found in cytogenetic tests in mammalian cells. However, all in vivo tests (two micronucleus tests and one cytogenetic test) were clearly negative. Equivocal results were obtained in the germ cell assay. Positive effects were observed only 3 weeks after the last dose, but not at week 1 and 5. Based on the mechanism of action, the weight of evidence and taking into account the results of the carcinogenicity and reprotoxicity studies sodium hypochlorite / hypochlorous acid is not considered to be genotoxic/mutagenic or clastogenic.


Short description of key information:
Sodium hypochlorite showed positive results in one of three available in vitro tests in bacteria, but only in strain TA100. In the two other tests, negative results were obtained in all strains used, including TA100. Equivocal or positive results were found in cytogenetic tests in mammalian cells. However, all in vivo tests (two micronucleus tests and one cytogenetic test) were clearly negative. Equivocal results were obtained in the germ cell assay. Positive effects were observed only 3 weeks after the last dose, but not at week 1 and 5. Based on the mechanism of action, the weight of evidence and taking into account the results of the carcinogenicity and reprotoxicity studies sodium hypochlorite / hypochlorous acid is not considered to be genotoxic/mutagenic or clastogenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results obtained in in vitro, in vivo and germ cell mutagenicity studies and taking into account the mechanism of action, the weight of evidence and the results of the carcinogenicity and reprotoxicity studies sodium hypochlorite/hypochlorous acid is not considered to be genotoxic/mutagenic or clastogenic and thus has not to be classified mutagenic according to Council Directive 67/548/EEC and CLP.

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