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Key value for chemical safety assessment

Additional information

In a bacterial reverse mutation assay performed according to OECD TG 471 under GLP, the substance was tested at concentration levels of 0.8-2500 µg/plate against Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 with and without S9 mix [Muller W (1989)]. The concentration levels were selected based on toxicity seen in a preliminary test where the substance was very toxic to the bacterial strains at 20 ug/plate without metabolic activation and toxic at 2500 µg/plate with metabolic activation. Hence 2500 µg/plate was chosen as the highest dose in the second experiment. No precipitation was observed. The tests were negative, in both the presence and absence of S9 mix.

The substance was tested for mutagenic potential in an in vitro gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) with and without S9 mix according to EU Method B17 under GLP [Flugge C (2010)]. The duration of exposure was 4 or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. In a preliminary cytotoxicity experiment pronounced cytotoxicity was observed at concentrations of 5 or 10 ug AO/mL and above without and with metabolic activation, respectively. Hence high doses of 5 and 10 ug AO/mL were selected for the main tests without or with metabolic activation. Five concentrations ranging from 0.313 to 5.0 or 0.625 to 10.0 µg AO/mL were selected for the experiments without and with metabolic activation, respectively. Cytotoxicity in the form of decreased plating efficiency (PE1) and (PE2) was noted in both experiments each carried out without and with metabolic activation at the top concentrations of 5.0 or 10.0 µg AO/mL medium, respectively. In the experiments without metabolic activation (4-h or 24-h exposure) the mutation frequency of the cultures treated with AO were within the normal range of the controls. In the experiments with metabolic activation (4-h exposure) the mutation frequency of the cultures treated with AO were within the normal range of the controls. The mutation frequencies of the negative controls in both sets of experiments were well within the expected range. The positive controls also gave expected increases in mutation frequencies. It is concluded that the C12-14 AO was negative in this test, with and without metabolic activation.

No in vitro chromosome aberration test is available for the substance. However, in vivo tests are available in the literature on a close structural analogue, N,N-Dimethyl-1-methyldodecylamine oxide (CAS No. 60729-78-4), 1-methyl-C12 AO.

Szabova & Devinsky (1988) evaluated the effect of AO on micronuclei in mice exposed to 1-methyl-C12 AO. The substance, dissolved in water, was administered by oral gavage to 8 week old male and female mice weighing 25 -30 g at a dose of 250 mg/kg bw. The dose volume was 0.1 mL/10 g. The authors reported that the dose selection was based on 1/5 of the LD50. Harvests occurred at 6, 24, 48 and 72 hours post exposure. 500 PCE were analysed for the incidence of micronuclei. There was no statistical increase in micronuclei (t-test at p<0.05). The authors did not report a positive control for this test.

Karasova et al (1987) evaluated the effect of AO on micronuclei in Chinese hamsters exposed to 1-methyl-C12 AO. The substance, at doses of 160, 300 or 700 mg/kg i.p. was administered to animals 8-10 weeks old weighing 25-30 g. The study included two administrations, 24 hours apart, 6 hour harvest after last dose and included negative (water) and positive (cyclophosphamide) controls. Bone marrow was collected from both femurs and prepared separately for each animal. 1000 PCE were analysed for the incidence of micronuclei. There was no increase in micronuclei for the substance. The positive control showed an appropriate response.

 In a mammalian bone marrow chromosome aberration test Karasova et al (1987) evaluated the cytogenetic effect of AO in Chinese hamsters exposed to 1-methyl-C12 AO. The substance, at doses of 160, 300 or 700 mg/kg i.p. was administered to animals 8-10 weeks old weighing 25-30 g. Dose selection was based on the LD50 mentioned in the article (1004-1379 mg/kg p.o.). The high dose was approximately 50 % of the LD50. Colchicine was administered two hours prior to sacrifice. The authors analysed 250 metaphase cells/concentration. There was no increase in chromosome aberrations for the substance. The positive control showed an appropriate response.

An oral carcinogenicity study performed using C12-14 AO is available (See CSR Section 5.8) in which rats (Charles River CD) were exposed via the oral route for 2 years to C12-14 AO at concentrations of 0.01, 0.1 or 0.2% in feed (equivalent to 4.5, 45 or 90 mg AO/kg bw/day). No neoplastic or non-neoplastic treatment related effects were identified [International Research and Development Corporation (1983)]. A dermal carcinogenicity study is also available [Hazelton Laboratories Inc. (1982)]. In this study 0.1 ml of an aqueous solution of C12-14 AO at 0.05%, 0.13%, and 0.26% (active ingredient) was applied to the dorsal skin of mice (ICR Swiss mice, CD-1), 3 times/week, for 2 years. The study did not result in any carcinogenic response on the exposed skin or systemically.

In addition to the toxicity data that are available, it is also of value to examine the general chemical structure of C12-14 AO to determine whether it poses a potential carcinogenic or mutagenic risk. Lai & Woo (2001) identify ten general structural criteria of potential mutagenic and carcinogenic chemicals that are electrophiles or that may generate electrophiles after metabolic transformation. According to Lai and Woo these criteria were developed from a review of all structural classes of chemical carcinogens and SAR analysis of the effects of chemical reactivity, molecular geometry, and metabolism on carcinogenicity. These criteria are listed below:

1. Polycyclic structures with 3 to 6 aromatic rings that mimic the angular ring distribution of carcinogenic polycyclic aromatic hydrocarbons.

2. The presence of an amino, dimethylamino, nitroso or nitro group directly linked to a conjugated double bond system, particularly in instances where the amino or amine generating group is at the terminal end of the longest conjugated double bond system of the molecule.

3. Nitroso, hydrazo, aliphatic azo or aliphatic azoxy structures, 1-aryl-3,3-dialkyltriazenes and 1,1-diaryl-2-acetylenic carbamates.

4. The presence of sterically strained ring (eg epoxide, aziridine, gamma-lactones, and d-sultones) in any type of structure.

5. Low molecular weight carbamates, thiocarbamates and thiourea derivatives.

6.The presence of a haloalkyl (particularly 1,2-dihalo), haloalkenyl (both vinylic and allylic), alpha-haloether, alpha-haloalkanol or aloha-halocarbonyl grouping

7. Low molecular weight aliphatic structures containing conjugated double bonds or isolated double bonds situated at the terminal end of an aliphatic chain.

8. Low molecular weight aldehydes

9. Benzylic, allylic or pyrrolic esters if the acyloxy moiety is a good leaving group.

Amine oxides do not meet any of the above criteria used to classify electrophiles that are likely mutagens or carcinogens. The absence of these structural moieties or fragments on amine oxides provides additional evidence that amine oxides do not pose potential carcinogenic or mutagenic hazards.

On the basis of the in vitro studies available of the C12-14 AO itself, the in vivo studies available on the structural analogue, the carcinogenicity studies and the examination of structural alerts it can be concluded that the C12-14 AO is not genotoxic.


Justification for selection of genetic toxicity endpoint
No study was selected, since all the studies were negative.

Short description of key information:
The substance was negative in a bacterial reverse mutation assay and an in vitro gene mutation assay in cultured mammalian cells, with and without metabolic activation. A close structural analogue was negative in vivo in mammalian erythrocyte micronucleus assays using mice and Chinese hamsters and in a mammalian bone marrow chromosome aberration test using Chinese hamsters. The substance was negative in oral and dermal carcinogenicity studies, and provides no structural alerts for mutagenic or carcinogenic potential.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In a bacterial reverse mutation assay with multiple strains of Salmonella typhimurium [OECD TG 471], the substance was negative both with and without metabolic activation. In an in vitro gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) [EU Method B17] the substance was negative with and without metabolic activation. In vivo, a close structural analogue N,N-Dimethyl-1-methyldodecylamine oxide (CAS No. 60729-78-4) was negative in a mammalian erythrocyte micronucleus assay in which mice were dosed at 250 mg AO/kg bw ( 1/5 of the LD50) and in a similar assay in which Chinese hamsters were dosed i.p. at 160, 300 or 700 mg/kg bw. In a mammalian bone marrow chromosome aberration test the analogue substance was administered i.p. to Chinese hamsters at 160, 300 or 700 mg/kg bw the result was also negative. Two carcinogenicity studies are available for C12-14 AO, one via the oral route in rats and the other via the dermal route in mice. Both were negative. Examination of C12-14 AO for structural alerts indicating potential genotoxic or carcinogenic potential shows the absence of relevant structural moieties or fragments.

On the basis of all available evidence, it can be concluded that the C12-14 AO is not genotoxic.