M. Radice ⁽¹⁾, G. Talamini ⁽²⁾, F. Faccioli ⁽³⁾, and G. Coppi ⁽⁴⁾

⁽¹⁾ Industriale Chimica, 21047 Saronno (VA), Italy; ⁽²⁾ Toxicological Work Group, IRCCS, Fond. Maugeri, University of Pavia (Italy); ⁽³⁾ Eurofins Biolab S.r.l., 20090 Vimodrone (MI), Italy; ⁽⁴⁾ Consultant AFI, Assoc. Farmaceut. Indust., (MI), Italy

Genotoxicity testing of therapeutic drugs is generally used for hazard identification with respect to DNA damage and its fixation (1). These damages can be manifested in the form of gene mutation, and these changes are responsible for heritable effects on germ cells and impose risks to future generations (2). In addition, it has been well documented that somatic mutations can also play an important role in malignancy (3).

The Ames Salmonella mutagenicity assays is a short-term bacterial reverse-mutation assay specifically designed to detect mutagenicity in a wide range of chemical substances that can produce genetic damage leading to gene mutation (4).

The Sulfamethylthiazole is a well known sulfamidic drug with a great antimicrobial activity against many bacteria; it is an analogue to Sulfamethoxazole and is used as antibacterial substance particularly active in ophthalmic preparations.

The synthesis of this API pass throught the intermediate 2-amino-4-methyl thiazole (figure 1):

The presence of a free amine group on an aromatic ring trig an alert for the potential genotoxicity of this intermediate. Nevertheless it is reduced to a non objectionable amount in the final product ( less than 100 ppm) by the current manufacturing process, a study on this substance was started in order to support current specification already in place since mid’80’s, far before the existence of the ICH guidelines

Methods

Test substance Sulfamethythiazole

Sulfamethylthiazole (CAS no: 515-59-3) was obtained from the in-house chemistry department of Industriale Chimica and analyzed in the in-house analytical department.

The I.R. spectrum (Ph. Eur. 2.2.24) recorded in KBr 1% (w/w) corresponds to sulfamethylthiazole reference spectrum, recorded in the same conditions.

The U.V. spectrum shows a maximum at 286 + 2 nm and E^1%_1cm equal to 414 + 20.7.

Melting point (Ph. Eur. 2.2.15) is 239,4 °C.

Loss of drying: 0.08%.

Water: 0.07%

Sulphated ash: 0.03%

Sulphates: < 50 ppm

Chlorides: < 25 ppm.

Assay (NaNO₂) : 100.1%

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

Mass Spectroscopy: The exact isotopic mass of the test was calculated to 269.03 from (C₁₆H₁₁ N₃O₂S₂). The +ESI mass spectrum shows a molecular ion [M+H]+ at m/z 270. The adduct ions and cluster are in accordance with the proposed structure of Sulfamethylthiazole reference standard.