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Key value for chemical safety assessment

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According to the Column 1 of the REACH regulation annex VII, an in vitro gene mutation study in bacteria is required (section 8.4.1).

No reliable bacterial assays for chlorine dioxide were available. However, chlorine dioxide is a powerful oxidising agent and is inherently toxic to bacteria as shown by its use as disinfectant in the drinking water. Considering both the bacteriostatic effects of the chemical and the availability of the whole reliable in vitro and the in vivo tests (see below), it is concluded that the information from a bacterial mutagenic test like the Ames' test is not be required for the assessment of chlorine dioxide genotoxicity. Therefore, an in vitro gene mutation study in bacteria using chlorine dioxide is scientifically unjustified.

Regarding the in vitro genotoxicity, two studies were identified as key study:

-      The mouse lymphoma assay (Cifone, 1986) using mice L5178Y cells gave positive results for reverse gene mutations.

-      The chromosome aberration assay (Ivett, 1986) performed on CHO cell lines gave positive results.

The conclusion from the results obtained from both of the available in vitro studies was positive. However, considering the strong reactivity of the chlorine dioxide, the positive results observed in vitro were likely due to the in vitro formation of reactive oxygen species which were well known to be genotoxic. In addition, it is also possible that the chromosome aberration test included the evaluation of dose levels that would be considered to be excessively toxic using modern criteria, and the mouse lymphoma assay would be judged as borderline or equivocal positive using modern, globally accepted, evaluation criteria. In this case both results may be considered to be potential false positives caused by excessive toxicity. Therefore, it is not possible to conclude on the genotoxicity potential of chlorine dioxide regarding the in vitro tests only.

 

In vivo, three studies were identified as 'key study'.

-      The sister chromatid exchange (SCE) assay (Ivett, 1984) was performed by the administration of ClO2 intraperitoneally to male mice. SCE were observed 26 hrs after dosing in the bone marrow tissue of mice. No significant increase in SCE frequency was observed in any of the dose groups relative to the negative controls and the frequencies were within the range of historical controls.

-      The chromosome aberration assay (Ivett, 1984) was performed by the administration of ClO2 intraperitoneally to mice. Chromosome aberrations were observed 6, 24 and 48 hrs after dosing in the bone marrow tissue of mice. No significant increase in the frequency of chromosomal aberrations was observed.

-      The dominant Rodent lethal assay (Moore, 1984) was performed by the administration of ClO2 intraperitoneally to mice. Matings were conducted in order to study the consequences of chromosome aberrations in germ cells. Chlorine dioxide was not mutagenic, under the conditions tested, in the mouse dominant lethal assay.

 

The results obtained in vitro and in vivo are different. However according to the ECHA guidance on "information requirements and chemical safety assessment" (R.7a), the results of in vivo tests possess a higher degree of reliability. Indeed, the use of immortalized cell lines such as CHO or L5178Y can give false positive results because of impaired p53 checkpoint and repair pathway processes. In addition, the use of modern evaluation criteria for both of the in vitro assays would likely result in a conclusion of negative, equivocal, or borderline positive. The results obtained in vitro with chlorine dioxide can therefore be considered to be false positive, likely resulting from the reactive oxygen species or from the evaluation of dose levels exhibiting excessive toxicity. The toxicokinetic data on chlorine dioxide indicated that chlorine dioxide and its metabolite, the chlorite, were distributed in the bone marrow. Therefore, the negative results obtained in vivo after an intraperitoneal administration of chlorine dioxide cannot be attributed to a lack in target tissue exposure.

  

In conclusion, chlorine dioxide is not classified as genotoxic considering the overall results from the in vivo studies.

Short description of key information:

In vitro:

Mouse lymphoma assay: positive (K, reliability 2)

Chromosome aberration test: positive (K, reliability 2)

In vivo:

Sister chromatid exchange: no significant increase in SCE frequency (K, reliability 2)

Chromosome aberration test: no significant increase in the frequency of chromosomal aberrations (K, reliability 2)

Dominant rodent lethal assay: not mutagenic (K, reliability 2)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonised classification:

The classification entered in the Annex VI to the CLP Regulation for Chlorine dioxide is not harmonised for the mutagenicity category.

Self-classification:

Based on the above data no additional self-classification is proposed.