Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Following 2 Key in vitro genotoxicity studies were available for Chloramine B trihydrate:

- Chloramine B trihydrate was tested in a key mutagenicity by the Bacterial Reverse Mutation Test (Täublová, 2006). The test was performed according to OECD TG 471/ EU B13/14 in 4 Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2uvrA strain. Doses of 0.3-100 µg dissolved in water were applied to the plates in emulsion in amount of 0.05 mL. Two series of experiments were performed with each strain- without metabolic activation and with supernatant of rat liver and a mixture of cofactors. In the arrangement given above, the test substance Chloramin B trihydrate was nonmutagenic for all used bacterial strains with as well as without metabolic activation.

- Chloramine B trihydrate was tested in a key in Vitro Chromosome Aberration Test (Marhan, 2006). The test was carried out in human peripheral blood lymphocytes with and without in vitro metabolic activation in two separate assays. The test substance was dissolved in water and three doses -0.05, 0.1 and 0.2 mg/mL of culture were used. Under the test conditions used, Chloramin B trihydrate does not induce any structural chromosome aberrations in cultured mammalian somatic cells.

Further in vitro Weight of Evidence was available for potential DNA damage and mammalian gene mutation:

- Read across substance Chloramine T was tested, as Nitrogen-chlorine (N-Cl) derivatives are a class of long-lived oxidants produced by stimulated phagocytes which may be important mediators of the inflammatory response (Weitberg, 1986). Because other phagocyte-generated oxidants cause genetic damage in cultured mammalian cells, the ability of the Chloramine T was studied to produce sister-chromatid exchanges (SCEs) in cultured Chinese hamster ovary (CHO) cells. CHO cells were incubated for 30 h with Cl-T (10-8 M – 10-5 M) and genetic damage was analyzed utilizing the SCE assay. A significant (p<0.0005) dose-dependent increase in sister-chromatid exchanges was observed.

- Read across substance Benzenesulphonamide dissolved in dimethyl sulfoxide (DMSO) was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation (Flügge, 2011). The test was carried out employing two exposure times without S9-mix: 3 and 24 hours, and one exposure time with S9-mix: 3 hours; this experiment with S9-mix was carried out in two independent assays. In the preliminary experiment without and with metabolic activation cytotoxicity (decreased survival) was noted at the top concentration of 5000 µg/mL. Hence, in the experiment without or with metabolic activation the concentration range of 312.5 to 5000 µg/mL was used. In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 5000 µg/mL. Positive and negative controls showed mutation frequencies within the expected range. The requested evaluation criterion for a plating efficiency of ≥0.5 was accomplished. The mutation frequencies of Benzenesulphonamide were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation. In addition, no change was observed in the ratio of small to large mutant colonies. Under the present test conditions, Benzenesulphonamide, tested up to a cytotoxic concentration of 5000 µg/mL medium in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test.

Finally, in vivo studies were available, demonstrating absence of genotoxicity in Drosophila and Micronucleus test:

- A key in in vivo micronucleus test with Chloramine B trihydrate was done for the detection of chromosome damage induction (Čihák, 2009). The test was performed according to OECD Test Guideline No. 474 – Genetic Toxicology: Micronucleus Test.The test substance was administered to the mice of ICR strain intraperitoneally in multiple dose regime: 3 doses with 24-hour interval between dosing. The doses administered were as follows: 3 x 20 and 3 x 30 mg/kg in males, resp. 3 x 40 and 3 x 65 mg/kg in females. Cytogenetic effect was evaluated at two cell harvesting times: 24, 48 hours after last administration. Under the experimental design described above, the result of mouse micronucleus test with test substance Chloramine B trihydrate, was negative. The test substance did not induce the micronuclei in the immature erythrocytes in bone marrow of mice therefore it is considered non-mutagenic in this test.

- Further supporting information for absence of in vivo genotoxicity of Chloramine T was found in a secondary source on mutagenic effects of environmental compounds and ingredients of cosmetics, tested in the Salmonella test, Drosophila and the Micronucleus test on mouse bone marrow (Gocke et al, 1981). Chloramine T was negative in the Drosophila and Micronucleus test.

A read-across justification was worked out and separately attached in Section 13. Based on the data from the repeated dose toxicity studies, as well as the comparable molecular structure and similar physicochemical properties, it was concluded that both data from Chloramine B trihydrate as those of Chloramine T and metabolites such as BSA and p- and o-TSA can be used for read-across.

 

In conclusion, the available battery of genotoxicity studies including bacterial and mammalian mutation and chromosomal aberration demonstrated that Chloramine B trihydrate has no genotoxic potential. There is no further concern towards human safety on genotoxic potential.

 


Justification for selection of genetic toxicity endpoint
Although other endpoints are also important for genetic toxicity, the Ames test was selected as this test is the most sensitive.

Short description of key information:
Genotoxic potential of Chloramine B trihydrate was evaluated by means of a test battery, including key in vitro studies for bacterial reverse mutation and chromosome aberration, and an in vivo micronucleus test, all negative. Further weight of evidence was delivered by read across with Chloramine T, which although positive in a Sister Chromatid Exchange test, was negative in vivo in a Micronucleus test. Finally Benzenesulfonamide was also tested negative as a read across substance in the mammalian gene mutation test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Chloramine B trihydrate does not need to be classified for genetic toxicity according to the Directive 67/548/EEC, Annex VI and CLP regulation (No. 1272/2008 of 16 December 2008). There is no further concern towards human safety on genotoxic potential.