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Acid Green 016 has been included by ECHA into the Annex III inventory; one of the critical aspects indicated as reason for inclusion is that the substance has been estimated to be suspected mutagen. The estimation has been conducted using KNN Mutagenicity model in VEGA (Q)SAR platform. the prediction outcome has been indicated as moderate reliability.

The prediction performed using KNN model in VEGA platform involved six chemicals (details in the file attached on 'information panel'):

- compound 1 (CAS: 89923-62-6) Sodium 1-amino-9,10-dioxo-4-{[3-(propionylamino)phenyl]amino}-9,10-dihydro-2-anthracenesulfonate

- compound 2 (CAS: 2586-60-9) Disodium 5,5'-[4,4'-biphenyldiyldi(E)-2,1-diazenediyl]bis(6-amino-4-hydroxy-2-naphthalenesulfonate)

- compound 3 (CAS: 115767-74-3) 4-[(1E)-1-{4-[2-(Dimethylamino)ethoxy]phenyl}-2-(4-isopropylphenyl)-1-buten-1-yl]phenyl dihydrogen phosphate

- compound 4 (CAS: 6449-35-0) Disodium 3-[(E)-{4'-[(E)-(6-amino-1-hydroxy-3-sulfonato-2-naphthyl)diazenyl]-3,3'-dimethoxy-4-biphenylyl}diazenyl]-4-hydroxy-1-naphthalenesulfonate

- compound 5 (CAS:5102-83-0) (2E,2'E)-2,2'-[(3,3'-Dichloro-4,4'-biphenyldiyl)di(1E)-2-hydrazinyl-1-ylidene]bis[N-(2,4-dimethylphenyl)-3-oxobutanamide]

- compound 6 (CAS:5102-83-0)6-(Dimethylamino)-3,3-bis[4-(dimethylamino)phenyl]-2-benzofuran-1(3H)-one

In the prediction report (attached on 'information panel'), the similarity has been indicated in the range of 0.761 - 0.799; however, comparing the chemical structure of Acid green 016 with those of the abovementioned substances, it is evident that there are many significant differences which can impact the toxicological behaviour, determining different characteristics. Deeply analysing the results, substances indicated as mutagen, on the basis of experimental values, are the compound 1, 2 and 4: the first case is an anthrachinone derivative, compound 2 and 4 are both biphenylyl-diazenyl derivatives. In all of these cases, a read across approach with Acid Green 016 can be considered as unsuitable and not appropriate, thus the prediction can be considered as non-reliable.

In addition, all the mutagenicity prediction tools available in VEGA platform have been run and the results, focusing on the involved substances, have been analysed. In all cases, i.e. CAESAR 2.1.13, SarPy/IRFMN 1.0.7 and model (ISS) 1.0.2, the predicted compound has been indicated to be outside the Applicability Domain of the model and the substances involved in the estimations were deeply different from target chemical, from the structural and functionalisation point of view. Only one substance used by CAESAR 2.1.13 and SarPy/IRFMN 1.0.7 resulted to be appropriate and suitable for the read across approach: CAS 25317-10-6 shares the same scaffold structure of Acid Green 016, presenting only minor differences, which cannot impact the genotoxicity potential (details below). In this case, experimental value is indicated as non-mutagenic (details in the file attached on 'information panel').

CAS: 25317-10-6 is the acid form of Green S (E 142) (EC: 221-409-2; CAS: 3087-16-9), which can be considered another analogous of Acid Green 016.

Green S (E 142) is a triarylmethane food colour consisting of dark-green powder or granules, soluble in water and slightly soluble in ethanol.

Green S is authorised as a food additive in the EU; on 2010 the Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion re-evaluating the safety of Green S (E 142) (EFSA, 2010). Although the panel considered not adequate the available genotoxicity data, they stated that the lack of adequate genotoxicity information is balanced by the negative results from carcinogenicity and reproductive toxicity studies.

Green S (CAS: 3087-16-9) is indicated under other names, e.g. Brillant Acid Green BS and Acid Green 50, and some available data can be retrieved from Chemical Carcinogenesis Research Information System (CCRIS) and Genetic Toxicology Data Bank (Gene-Tox).

The CCRIS database reports negative results for the AMES Salmonella typhimurium assay performed on TA98, TA100, TA1535 and TA1538, with and without metabolic activation (Salas et al, 1987). Furthermore, results for in vivo investigatin performed on male and female mice are indicated as negative (Brantomet al., 1987).

In the Geno-Tox data bank a Mitotic recombination or gene conversion assay is indicates, reporting negative results (ref. EMICBACK/52546, 1984).


The target chemical Acid Green 016 and the structural analogues Green S and CAS 25317-10-6 share the same scaffold structure, characterized by two aniline rings and a naphthalene sulphonate moiety. One of the differences is that anilines bear methyl groups in case of Green S and CAS 25317-10-6, instead of the ethyl ones; this is expected to not determine a different reactivity and the steric indance for the target substance. A further difference regards the presence of a hydroxy group on the naphthalene sulphonate portion of Green S and CAS 25317-10-6. From the metabolic point of view, the substances involved in read across approach represent subsequent oxidative steps, i.e. Green S and CAS 25317-10-6 can be considered as possible oxidative products of Acid Green 016 and this lets to expect a comparable adsorption, distribution and excretion pathway.

A further difference between Green S and target chemical concerns the salification, which is expected to not impact the reliability and suitability of the read across approach proposed; in fact, a salification of Acid Green 016, due to biological processes, is likely to occur.


Brantom PG, Creasy DM and Gaunt IF (1987). Long-term toxicity study of green s in mice; food chem. toxicol. 25(12):977-983, 1987

EFSA (2010). EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS); Scientific Opinion on the reevaluation of Green S (E 142) as a food additive. EFSA Journal 2010;8(11):1851. [32 pp.]. doi:10.2903/j.efsa.2010.1851. Available online:

EMICBACK/52546 (1984); mutat res 133: 199 -224

Salas J, Pollastrini M, Barea M, Penafiel C, Sanz MD, Alcaraz B and Salas MA (1987). Safety of three food colors for human consumption; aliment., equipos tecnol. 6(2):171-175

Justification for classification or non-classification