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Peracetic acid was evaluated for its mutagenic and genotoxic potential in vitro and in vivo. Overall the data summarised below and in the results tables do not raise any concern with regard to the mutagenic and genotoxic potential of peracetic acid.

In vitro tests

Peracetic acid (given as P3 oxonia aktiv containing 4.6% peracetic acid) was tested for its in vitro mutagenicity in an bacterial reverse mutation test (Ames test) (Wallat, 1984a). 5 different concentrations (7, 36, 183, 915, 4576 µg/plate and 57, 114, 228, 457, 915 µg/plate in the first and second series of the test, respectively) of the test material were applied to 6 different strains of Salmonella typhimurium (TA 1535, TA 100, TA 102, TA 1537, TA 1538 and TA 98) in two independent experimental series. The tests were performed in the absence or presence of S9-mix. Peracetic acid (given as P3 oxonia aktiv) did not induce reverse mutations in the presence or absence of S9-mix in the tested strains of Salmonella typhimurium.

In a further study (Zeiger et al., 1988), peracetic acid (40%) was tested for mutagenicity in different strains of Salmonella typhimurium (TA 97, TA 98, TA 100, TA 1535), using a pre-incubation protocol. All tests were performed in the absence and presence of S9 mix from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Peracetic acid (0.3, 1.0, 3.3, 10.0, 20.0, 33.0, 100 and 200 µg/plate) did not induce reverse mutations in the tested strains of Salmonella typhimurium both in the absence and in the presence of exogenous metabolic activation.

In another in vitro test (Philips, 1994), Proxitane-0510 (containing 5.17% peracetic acid) was evaluated for its potential to induce chromosome aberrations with or without metabolic activation (S9-mix) in cultured human lymphocytes in concentrations up to 5 mg/mL. In the two independent tests performed, the results demonstrated a clastogenic effect of peracetic acid in the absence of metabolic activation. In the presence of metabolic activation, the increase in the incidence of aberrant metaphases wes significant at the high dose of 5 mg/mL only where also significant cytotoxicity to human lymphocytes was evident. At the lower dose levels, the increases in aberrant metaphases were neither statistically significant nor dose-related. It is therefore concluded that in the presence of metabolic activation, peracetic acid does possess no clastogenic potential at concentrations causing no high cytotoxity in human lymphocytes.

In a chromosome aberration test with cultured Chinese hamster V79 cells the test item Wofasteril SC 100 (peracetic acid 10.7%) was examined for the ability to cause chromosomal damage (Herold, 2002). Two independent assays were performed. Concentrations were 0.09, 0.13 and 0.2 µl/ml in the absence of metabolic activation (S9-mix) and 0.13, 0.2 and 0.3 µl/ml in the presence of S9-mix. Based on the results of the study it is concluded that the test item does not induce structural chromosomal aberrations under the experimental conditions described. The test item is considered to be non-mutagenic in the Chinese hamster V79 cytogenetic test in vitro.

In a gene mutation test in Chinese hamster lung fibroblasts (V79) the potential of the test material Wofasteril SC 100 to induce gene mutations in the absence and presence of S9 mix was evaluated (Herold, 2002). Two independent experiments were performed. Test concentrations were 0.16, 0.22, 0.31, 0.43, 0.5 and 0.6 µL/mL without metabolic activation and 0.5, 0.6, 0.7, 0.84, 0.98 and 1.18 µL/mL with metabolic activation. At the end of the expression period cells were cultured in selective medium (6-thioguanine) for determination of mutant colonies. The test material was considered to be non-mutagenic in the in vitro cell gene mutation test (HPRT-test).

In an in vitro unscheduled DNA synthesis (UDS) assay (Coppinger et al., 1983), human diploid foetal lung cells were exposed to different concentrations of peracetic acid. For the analysis two methods in two separate tests were used: liquid scintillation counting (LSC) and autoradiography (AR). In the LSC-assay slight increases in DNA specific activity were obtained at 8 and 16 µg/mL peracetic acid. The repeat experiment showed similar results for 16 and 32 µg/mL. The increases, however, where slight (below 1.6-fold of the solvent control) and did not show consistency with respect to the concentration applied. These results were considered to be negative for the induction of UDS by peracetic acid. The results obtained from the AR-assay were similar to the LSC-results. The first experiment yielded positive but highly variable results for UDS initiation. However, when the study was repeated with the same and a new batch of peracetic acid, no evidence of UDS was observed. In order to clarify the results obtained in these assays an in vitro-DNA repair assay based on the ultracentrifugation of density labelled DNA was conducted. This test gave unambiguous negative results for UDS induction by peracetic acid. It can be concluded that peracetic acid does not induce DNA repair in diploid foetal lung cells.

The potential of peracetic acid to cause mutagenic effects and DNA damage was investigated in the Comet assay in human leukocytes and in Saccharomyces cerevisia D7 (Buschini et al., 2004). However, because of relevant methodological deficiencies and insufficient documentation the test results are not considered reliable. The concentration of peracetic acid investigated was 0.1 - 5.0 ppm in the Comet assay. Peracetic acid was demonstrated to be weakly genotoxic in this assay at 0.5 ppm and higher. The degree of DNA damage induced was, however, only small and the significance of the results is not clear in view of missing criteria for a positive effect and due to the lack of historical control data. Moreover, the test material used was not specified.

In Saccharomyces cerevisia D7, mitotic gene conversion, point mutation and mitochondrial DNA mutability was assessed at peracetic acid concentrations of 0.2 - 15 ppm. Peracetic acid did not show a significant genotoxicity potential in Saccharomyces cerevisia D7 (mitotic gene conversion, point mutation and mitochondrial DNA mutability). The only positive response was observed for the endpoints mitotic gene conversion and point mutation without endogenous metabolic activation in stationary phase, but only at cytotoxic doses (significant reduction of cell survival). No effect was notable in the presence of metabolic activation which is considered to be the more relevant system under physiological conditions.

In vivo tests

In an in vivo micronucleus test (Wallat, 1984b), three concentrations (2 x 200, 2 x 400 and 2 x 800 mg/kg bw) of peracetic acid (tested as P3 oxonia aktiv containing 4.6% peracetic acid) were orally administered to 7 mice per group with an interval of 24 hours between the two equal doses and a total volume of 2 x 10 mL/kg. 6 hours after the second application animals were sacrificed and bone marrow smears from both femurs of each animal were prepared. The number of micronuclei and the polychromatic/normochromatic erythrocytes ratio were determined by light microscopy in 1000 cells per animal. There were no differences in the number of micronuclei between test groups and negative control animals. In the highest dose group the polychromatic/normochromatic erythrocytes ratio was elevated. Severe clinical signs were observed at this dose in the test and in a preliminary toxicity test. The elevated polychromatic/normochromatic erythrocytes ratio is therefore not considered to be the result of a direct genotoxic effect of P3 oxonia aktiv but rather considered to be secondary to irritating effects and tissue damage at the site of first contact resulting in alterations in haematology. This effect on haematology was already demonstrated in other studies performed with peracetic acid. It was, thus, concluded in this study that the test substance does not possess a mutagenic potential after oral administration in the described in vivo test system.

In a second micronucleus test in mice (Blowers, 1994a), groups of male and female mice were given a single oral dose of Proxitane-0510 (containing 5.17% peracetic acid) at dose levels of 0, 8, 35 or 150 mg/kg bw, respectively. A further group was given 100 mg cyclophosphamide/kg bw as positive control. 5 animals of each sex were killed 24, 48 and 72 hours after treatment and the femoral bone marrow was removed to be examined for the incidence of micronuclei in the polychromatic erythrocytes (PCEs), the proportion of polychromatic erythrocytes in the erythrocyte population and the incidence of micronuclei in the normochromatic erythrocytes (NCEs). There were no significant differences in the frequency of micronuclei in PCE or in NCE between mice treated with Proxitane-0510 and the untreated controls. This was true for all doses of Proxitane-0510 tested, and all three sampling times and both sexes. At the high dose level of 150 mg/kg bw, the percentage of PCE was decreased in females at the 48 hour sampling time point. In both male and female mice, cyclophosphamide induced a statistically significant increase in micronuclei in PCE at 24 and 48 h and in NCE at 48 and 72 h. This indicates that the system was capable of detecting the effects of a known genotoxin. Proxitane-0510 did not induce a dose-related decrease in the proportion of PCE, indicating a lack of toxicity of the bone marrow. However, the highest dose tested (150 mg/kg bw) was found to be the maximum tolerated dose in both sexes in preliminary toxicity studies. The results of the study indicate that Proxitane-0510 does not induce micronuclei in the bone marrow of mice. It can be concluded that Proxitane-0510, given by the oral route, does not cause chromosome damage in the bone marrow of mice.

In an in vivo UDS test in the rats (Nesslany, 2002), 3 animals per group were orally dosed with 1000 and 2000 mg peracetic acid (5%)/kg bw (= 52 and 104 mg peracetic acid/kg bw). After 2-4 and 12-16 hours the animals were killed and slides of primary hepatocytes were prepared. The viability, the net grain count, net grain count in cells in repair and the percentage of cells in S-phase was determined for at least 150 cells per animal. For both expression times the mean net nuclear grain count values were below the threshold value of 0 for a positive response. No significant increase in the percentage of cells in repair at any dose tested was observed when compared to the respective controls. Furthermore, cells in repair showed comparable net nuclear grain count values in cells from dosed animals and control animals. The frequency of cells in S-phase was low, indicating that the test material did not induce a proliferative effect in rat liver under the described experimental conditions. The test material did not induce unscheduled DNA synthesis under the conditions described in this study.

In a second unscheduled DNA synthesis (UDS) assay (Blowers, 1994b), Proxitane-0510 (containing 5.17% peracetic acid) was assessed for its ability to induce DNA repair in rat hepatocytes after an oral gavage treatment. The study was carried out in three separate parts, assessing two oral doses of Proxitane-0510 at 330 and 1000 mg/kg after two treatment times (2 and 16 h). After sacrifice of the animals 2 or 16 h after treatment the hepatocytes were isolated, cultivated and incubated with tritiated thymidine. Grains in the nucleus area and in 3 equal sized areas in the cytoplasm were counted. The percentage of cells in S-phase was additionally measured. No significant increases in UDS, measured as net grain increase, were observed in either of the treated groups. The positive control responses confirmed the validity of the assay. It was concluded that Proxitane-0510 did not induce unscheduled DNA repair in the in vivo UDS assay under the conditions described in the study.

Further genotoxicity tests

The evaluation of the genotoxic potential of different waters (surface water, sewage wastewater) disinfected with peracetic acid mostly in comparison to treatment with NaClO and ClO2, was investigated in a number of studies originating from a research project on potential genotoxic risk from the disinfection of surface water for human consumption supported by the Italian Ministry for University (MURST). The results from tests in bacteria and mammalian cells, fish and molluscs, most of which were non-validated, non-standard genotoxicity assays, provide a strong weight of evidence that disinfection of surface water with peracetic acid does not increase the genotoxic potential in comparison with the untreated water. This agrees with the observation that peracetic acid-derived disinfection by-products are non-genotoxic and furthermore corresponds to the results of genotoxicity tests performed in vitro and in vivo with peracetic acid.


Short description of key information:
1. Gene mutation:
- In vitro: negative in Ames tests with S. typhimurium TA 1535, TA 1537, TA97, TA 98, TA 100, TA102 with and without metabolic activation (similar to OECD Guideline 471 or from reliable source)
- In vitro: negative in mammalian cell gene mutation assay with Chinese hamster lung fibroblasts (V79) (HPRT) with and without metabolic activation (EU Method B.17)

2. Chromosome aberration
- In vitro: negative in mammalian chromosome aberration test with human lymphocytes with metabolic activation; ambiguous without metabolic activation (similar to EU Method B.10)
- In vitro: negative in mammalian chromosome aberration test with Chinese hamster lung fibroblasts (V79) with and without metabolic activation (similar to EU Method B.10)
- In vivo: negative in micronucleus assays with mouse CF1/W 68 and CD-1 (EU Method B.12 or similar to OECD Guideline 474)

3. DNA damage and/or repair
- In vitro: negative in unscheduled DNA synthesis assay with mammalian cells (Human lung fibroblasts, WI-38 CCL75) without metabolic activation (marginal positive results, but inconsistent within the concentrations applied and the test runs) (similar to EU Method B.18)
- In vitro: negative in DNA repair assay with mammalian cells (Human lung fibroblasts, WI-38 CCL75) without metabolic activation (similar to EU Method B.18)
- In vivo. negative in unscheduled DNA synthesis assay with rat Fischer 344 and Fischer 344/DuCrj (EU Method B.39 / OECD Guideline 486)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on mutagenicity of peracetic acid do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.