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Genetic toxicity in vitro

Description of key information

Ames test (direct plate incorporation assay): negative

Ames test (pre-incubation assay): positive

Genetic toxicity in vivo

Description of key information

micronucleus test: negative

Additional information

The assessment of genetic toxicity of test substance was based on in vitro and in vivo experimental studies as well as on QSAR data.  

Genetic toxicity of test substance in vitro was assessed by 2 Ames assays. Both were run using only 4 Salmonella typhimurium strains: TA535, TA1537, TA98 and TA100.  

In the first Ames assay, the direct plate incorporation method was used. Two independent experiments were run, both with and without metabolic activation by S9 mix from rat liver; each strain was tested in triplicate; the highest tested concentration was 100 µg/plate without S9 mix and 333 µg/plate with S9 mix, based on a pre-experiment on toxicity. No mutagenic effect, in terms of increase of mean number of revertants was noted. Positive and negative controls were valid.  

In the second Ames test, the pre-incubation method was used. This method is more suitable for azo-dyes. Two independent experiments were run, both with and without metabolic activation by S9 mix from hamster liver; each strain was tested in triplicate; the highest tested concentration was 5000 µg/plate, based on a pre-experiment on toxicity. Mutagenic effect, in terms of increase of mean number of revertants, was noted in TA1537 and TA98 strains with metabolic activation. Positive and negative controls were valid.  

An in vitro cytogenicity study was not run, as adequate data from an in vivo cytogenicity study is available.  

An in vitro gene mutation assay was not run, as a positive response was found in a bacteria gene mutation assay.

Genetic toxicity of test substance in vivo was assessed by the micronucleus assay according to OECD guideline 474. Three groups of 5 mice per sex were dosed by gavage at 2000 mg/kg bw as a single dose. Bone marrow was sampled at 24, 48 and 72 h after dosing.  Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide at 50 mg/kg bw, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the micronucleus test, whereas the positive control substance produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes. It is concluded that the test substance can be considered as not mutagenic in this assay.  

In case of a positive result in an in vitro gene mutation test in bacteria, such as that obtained using the pre-incubation method, appropriate in vivo mammalian gene mutation assay should be considered.

Before proposing further in vivo tests, predictions by QSAR OECD Toolbox (v. were analysed. Structural alerts related to in vitro mutagenicity (Ames test) and in vivo mutagenicity (micronucleus test) were identified by the ISS model for the main constituents, in particular: alpha,beta-unsaturated carbonyls and aromatic diazo groups. The following SMILE notations were used:

[Cl-].[Cl-].Cl.Cl.CN(C)CCCN1C(=O)C([n+]2ccccc2)=C(C)C(/N=N\c2cccc(c2)/N=N\c2cc(O)c(/N=N/c3cccc(c3)/N=N\C3=C(O)N(CCCN(C)C)C(=O)C(=C3C)[n+]3ccccc3)c(O)c2)=C1O  and


It is known that azo compounds are biologically active through their metabolites, i.e. the reduction of azo bond, via the enzyme azoreductase in the intestinal microflora, produces the release of aromatic amines. Experimentally, the key role of the aromatic amines in causing the genotoxic effect was confirmed by a negative finding in the Ames assay with the direct plate incubation, where reasonably no release of aromatic amines occurred, and a positive finding in the Ames assay with the pre-incubation method, more appropriate to azo dyes and likely able to generate aromatic amines.  

The experimental in vivo micronucleus test gave a negative response, thus suggesting that reduction of azo bond and release of aromatic amines likely did not occur under test conditions.  It should be outlined that aryl azo compounds (Aryl-N=N-Aryl) with no free amino group are not expected to be genotoxic without metabolic azo-reduction to yield aromatic amines. In particular, the ability to undergo azo-reduction in the body is dependent on the water solubility of the chemical and may be affected by the mass weight, size, extent of ring substitution and presence of metal chelation. The lack of a positive evidence in vivo suggests that test substance may not release free aromatic amines upon in vivo rodent metabolism.  

As for the alpha,beta-unsaturated carbonyls, QSAR OECD Toolbox reports that these are bis-electrophiles reactive molecules that may interact with electron-rich biological macromolecules. Because of conjugation with the carbonyl group, the beta-carbon is positively polarized and becomes the preferred site of nucleophilic attack, as is in a classic Michael type addition. In spite of a common structural feature, alpha,beta-unsaturated carbonyl compounds can undergo different interactions with DNA, which lead to different genotoxic and mutagenic responses. The following genotoxic mechanisms are conceivable: formation of cyclic adducts, frameshift interaction, strand breaks, and crosslinking. In addition to direct interactions, other metabolic activations are conceivable, such as metabolic epoxidation and formation of radicals. The predominant interaction of alpha,beta-unsaturated carbonyl compounds with DNA components is the formation of cyclic 1,N-deoxyguanosine adducts. This reaction occurs through an initial Michael addition to the exocyclic nitrogen of deoxyguanosine (dG), followed by ring closure and formation of the 8-hydroxypropano adduct. Further reactions are also possible, including formation of cross-links with proteins and nucleic acids.  

It should be noted that, in the case of test substance, the alpha,beta unsaturated structure is a limit structure in resonance with the aromatic ring; moreover, the beta carbon is substituted, thus likely hindered towards reactions. Accordingly, the reactivity of the beta position towards reaction with electrophiles is likely lowered. As expected, such alert for mutagenicity was not confirmed experimentally.  

In summary, available data showed that:

- test substance has a relevant structural alert related to the possible release of aromatic amines upon reduction of the azo bond; the ISS model, implemented by QSAR Toolbox, finds such alert for both Ames test and micronucleus test;

- gene mutation in bacteria only occurs in case of actual release of aromatic amines, as by running the Ames test with the pre-incubation method, thus confirming the relevance of the structural alert;

- no genotoxic effects on micronuclei are seen in vivo, thus suggesting that no aromatic amines are generated under the test conditions (OECD 474).

Therefore, it is assumed that:

- the potential of test substance to induce gene mutation in mammalians is also related to the possible release of aromatic amines,

- in an in vivo gene mutation assay, reasonably performed by oral route, same metabolism of test substance might be expected as in an in vivo micronucleus test, i.e. if no release of aromatic amines occurs in one case, neither it occurs in the other case.

On these bases, it is considered as reasonable to exclude a potential of test substance to be genotoxic in an in vivo gene mutation assay and no further studies are deemed as necessary.  

Justification for classification or non-classification

Based on available experimental and QSAR evidences, test substance is considered as devoid of a genotoxic potential and no classification within the CLP Regulation (EC 1272/2008) is applied.