Test substance 2-amino-4-methyl thiazole

2-Amino-4-methylthiazole (CAS 1603-91-4) was obtained from the in-house chemistry department, Industriale Chimica and analyzed from the in house analytical department.

Titration (NT) HClO₄ 0.1 M = 100.4%

The I.R spectrum (Ph. Eur. 2.2.24) recorded in KBr 1% (w/w) corresponds to 2-amino-4-methyl-tiazole reference spectrum, recorded in the same conditions.

NMR spectroscopy: multiplicity and chemical shift of the 1H-NMR signals are in accordance with the proposed structure of the 2-amino-4-methylthiazole reference standard recorded in the same conditions.

Mass Spectroscopy: The exact isotopic mass of the test was calculated to 114.17 from (C₄H₆ N₂S). The +ESI mass spectrum shows a molecular ion [M+1] at m/z 115. The adduct ions and cluster are in accordance with the proposed structure of 2-amino-4-methylthiazole reference standard.

Experimental procedures

The bacterial reverse mutation assay (Ames test) was performed on five mutant strains of Salmonella typhimurium (TA 1535, TA1537, TA 98, TA 100, TA 102) and Escherichia coli WP2pKM101 according to OECD 471:1997 (5). The presumed mutagenic activity of the test substances was determined by comparing number of reverting colonies in treated cultures with the number of the reverting organisms in the control cultures. The direct incorporation method in a plate was used both in presence of, and without, an enzymatic system for metabolic activation. As guideline recommends as maximum test concentration 50 mg/ml and 7 different dilutions of semi-log intervals between them; besides these, on the base of the physical state and use instruction of the sample the neat test substance has been tested.

The principle of this test is to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutagens which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. For this reason an appropriate minimal glucose agar and an overlay agar (Top agar) containing histidine and biotin (for Salmonella strains) or Tryptophan (for E. coli strain) in very small quantitative, to allow for a few cell divisions, is used, sufficient to permit the survival of the microorganisms and show a mutation when this appeared. Many of the test strains have several feature that make them more sensitive for the detection of mutations, including responsive DNA sequences at the reversion sites, increased cell permeability to large molecules and elimination of DNA repair systems or enhancement of error-prone DNA repair processes. The revertants bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.

The enzymatic system for metabolism activation (S9 mix) was prepared adding to S9 (an hepatic homogenate obtained from the liver of adult male rats which had previously been induced with “aroclor 1254” soybean oil solution) to Regensys A and to Regensys B containing respectively phosphate-buffered salt solution and glucose-6-phosphate and 153 mg NADP for the activation. S9 mix was subjected to a sterility control to exclude any possible contamination.

The test substance was tested at equivalent concentration of 50 mg/ml and 7 subsequent dilutions of semi-log intervals between equivalent to 15 mg/ml, 4.5 mg/ml, 1.35 mg/ml, 0.405 mg/ml, 0,122 mg/ml 0.037 mg/ml and 0.011 mg/ml. The positive controls were prepared at the following concentrations and were used for the sequent strains (Table 1).