2-Amino-4-thiazole acetic acid is a building block used in the synthesis of different active pharmaceutical products, specifically in the process of manufacture of Cefotiam. 2-Amino-4-thiazole acetic acid is incorporated in Cefotiam as amide, but it could be re-obtained by hydrolysis, and so it becomes an obvious potential degradation impurity. Following the possibility to have this compound as by-process or degradation impurity, above the identification thresholds outlined by ICHQ3A (1), as foreseen by ICH M7 (2), it needs to be evaluated for its potential genotoxicity.
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 (3).
The presence of a free amine group on an aromatic ring trigger an alert for the potential genotoxicity of this intermediate; a study on this substance was started in order to support the specification now reported by ICH M7
Methods
Test substance 2-amino-4-thiazole acetic acid
2-Amino-4-thiazole acetic acid (CAS 29676-71-9) was obtained from the in-house Chemistry Department, Industriale Chimica and analyzed from the in house Analytical Department. The purity was tested by I.R. spectrum, NMR spectroscopic, and mass spectroscopic analysis.
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 (4) and GLP.
It is noted that the presumed mutagenic activity was determined by comparing number of revertant colonies in treated plates with those of negative controls; a response is positive if the number of colonies in the treated plates is double or more in respect to control plates.
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. S 9 mix was subjected to a sterility control to exclude any possible contamination.
The test substance was tested at the concentration of 50 mg/ml and 7 subsequent dilutions of semi-log intervals 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 concentrations and were used for the strains as reported in Table 1.
Table 1. Positive control for the various strains with and without S9 mix.
| Compound | (µg/plate) | Strain | S9 |
|---|---|---|---|
| Sodium azide (NaN 3 ) | 1.5 | S.typhimurium TA 1535 | without |
| S. typhumurium TA 100 | |||
| 9-Aminoacridine (9-AAc) | 100 | S. typhimurium TA 1537 | without |
| 2-Nitrofluorene (2NF) | 10 | S. typhumurium TA 98 | without |
| Mitomycin C (MMC) | 0.5 | S. typhimurium TA 102 | without |
| 4-Nitroquinoline (4NQ) | 50 | E. coli WP2pKM101 | without |
| 2-Aminoantracene (2AAn) | 10 | S. typhimurium TA 1537 | with |
| S. typhimurium TA 98 | |||
| S. typhimurium TA 102 | |||
| E. coli WP2pKM101 | |||
| Ciclophosphamide CP) | 100 | S. typhimurium TA 1535 | with |
| S. typhimurium TA 100 |
All positive controls and other reagents were obtained from Sigma-Aldrich (St. Louis, Missoury, USA). All experiments were run with concurrent controls and in triplicate.
Results
Results obtained with different concentrations of 2-ammino-4-thiazole acetic acid in the Ames test are shown in Tables 2-5.
Table 2. Bacterial reverse-mutation test of 2-amino-4-thiazole acetic acid in absence of metabolic activation (trial I). (Revertants/plate).
| Conc. | TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | E.coli WP2pKM 101 |
|---|---|---|---|---|---|---|
| (mg/plate) | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD |
| 50000 | 15.00±2.65 | 13.67±2.08 | 23.00±4.58 | 114.33±39.31 | 252.00±31.18 | 233.33±14.19 |
| 15000 | 12.67±4.04 | 10,33±1.15 | 24.33±6.11 | 137.00±10.54 | 262.33±11.68 | 231.67±6.51 |
| 0.4500 | 13.33±1.53 | 15.00±3.00 | 23.33±4.04 | 126.00±35.59 | 273.67±25.32 | 232.33±5.69 |
| 0.1350 | 13.33±4.04 | 15.00±4.58 | 23.00±3.46 | 123.67±35.30 | 250.33±40.51 | 218.67±12.34 |
| 0.0405 | 16.67±1.53 | 17.33±2.89 | 26.67±3.21 | 116.00±36.66 | 259.67±33.86 | 227.33±13.65 |
| 0.0122 | 15.33±2.08 | 15.33±2.89 | 24.67±6.51 | 120.33±3.79 | 247.33±30.99 | 220.33±18.50 |
| 0.0037 | 15.00±1.73 | 13.00±1.73 | 24.67±6.03 | 130.67±32.50 | 267.00±14.73 | 229.33±11.59 |
| 0.0011 | 14.00±3.00 | 13.00±2.65 | 22.33±6.66 | 104.67±24.58 | 233.67±24.11 | 227.00±15.62 |
| NC | 13.37±3.51 | 12.67±1.53 | 26.33±4.73 | 112.67±14.57 | 274.67±22.19 | 231.33±19.40 |
| PC | 599.00±69.20 | 591.67±127.25 | 620.33±84.01 | 1034.33±81.00 | 1082.67±187.70 | 1276.33±167.54 |
NC=controls; PC=positive controls (see Table 1); SD=standard deviation
Table 3. Bacterial reverse-mutation test of 2-amino-4-thiazole acetic acid in absence of metabolic activation (trial II). (Revertants/plate).
| Conc. | TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | E.coli WP2pKM 101 |
|---|---|---|---|---|---|---|
| (mg/plate) | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD |
| 50000 | 13.33±1.53 | 14.00±1.00 | 24.00±5.57 | 102.67±5.03 | 268.00±20.07 | 233.67±6.43 |
| 15000 | 14.33±2.08 | 14.67±1.15 | 28.67±1.53 | 123.67±9.07 | 241.00±44.24 | 224.00±15.59 |
| 0.4500 | 15.00±1.00 | 11.00±3.61 | 21.00±6.24 | 113.00±33.41 | 249.67±9.50 | 227.33±6.51 |
| 0.1350 | 11.67±1.53 | 16.00±3.46 | 24.00±8.19 | 142.33±28.02 | 271.67±18.15 | 213.33±10.12 |
| 0.0405 | 14.33±4.04 | 15.67±2.31 | 23.00±6.56 | 107.00±30.41 | 257.67±14.47 | 239.00±2.65 |
| 0.0122 | 13.00±2.00 | 13.33±4.16 | 25.00±5.57 | 121.67±10.26 | 271.33±24.54 | 224.33±15.50 |
| 0.0037 | 10.67±1.15 | 13.00±1.73 | 23.67±6.43 | 115.00±10.15 | 236.33±22.94 | 238.00±7.81 |
| 0.0011 | 13.67±4.04 | 15.67±3.06 | 26.33±4.51 | 129.00±34.00 | 242.33±16.65 | 223.00±2.00 |
| NC | 15.67±4.16 | 13.67±1.53 | 25.67±7.57 | 120.00±11.53 | 251.33±40.80 | 219.00±14.53 |
| PC | 683.00±75.94 | 650.67±83.03 | 568.67±65.58 | 945.67±91.27 | 1280.67±147.84 | 1253.67±224.30 |
NC=controls; PC=positive controls (see Table 1); SD=standard deviation
Table 4. Bacterial reverse-mutation test of 2-amino-4-thiazole acetic acid in presence of metabolic activation (trial I). (Revertants/plate).
| Conc. | TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | E.coli WP2pKM 101 |
| (mg/plate) | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD |
| 50000 | 13.67±2.52 | 14.33±4.16 | 23.33±2.52 | 127.67±29.48 | 247.67±37.74 | 233.33±9.71 |
| 15000 | 11.00±1.73 | 16.67±2.31 | 22.33±6.03 | 133.00±13.53 | 250.00±36.35 | 217.67±17.47 |
| 0.4500 | 13.33±3.79 | 15.00±4.36 | 23.67±4.93 | 122.33±18.45 | 276.00±9.54 | 220.33±12.01 |
| 0.1350 | 14.00±4.00 | 11.33±3.06 | 24.33±2.52 | 145.00±17.09 | 251.00±31.19 | 217.33±15.14 |
| 0.0405 | 15.67±2.89 | 14.67±4.04 | 27.00±4.36 | 127.67±16.92 | 258.00±9.00 | 220.33±4.73 |
| 0.0122 | 11.00±2.65 | 11.67±2.52 | 22.67±5.86 | 114.33±13.32 | 244.33±15.57 | 219.00±21.70 |
| 0.0037 | 14.67±3.79 | 16.33±2.08 | 19.33±2.08 | 129.00±30.41 | 224.33±37.00 | 216.00±5.57 |
| 0.0011 | 16.00±1.73 | 14.67±2.52 | 20.67±4.73 | 129.67±39.50 | 272.33±30.66 | 229.00±21.66 |
| NC | 14.67±3.06 | 15.67±3.51 | 20.00±4.36 | 135.00±6.00 | 254.00±47.62 | 230.00±10.54 |
| PC | 601.33±24.44 | 531.67±45.28 | 657.67±31.01 | 911.67±53.46 | 1229.67±128.70 | 1257.67±196.46 |
NC=controls; PC=positive controls (see Table 1); SD=standard deviation
Table 5. Bacterial reverse-mutation test of 2-amino-thiazole acetic acid in presence of metabolic activation (trial II). (Revertants/plate).
| Conc. | TA 1537 | TA 1535 | TA 98 | TA 100 | TA 102 | E.coli WP2pKM 101 |
|---|---|---|---|---|---|---|
| (mg/plate) | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD |
| 50000 | 13.33±1.15 | 13.00±4.00 | 21.67±2.08 | 117.67±30.66 | 272.33±20.11 | 224.00±24.52 |
| 15000 | 15.33±4.73 | 14.00±4.58 | 25.67±4.51 | 144.67±23.16 | 239.67±17.79 | 235.33±9.87 |
| 0.4500 | 14.33±3.21 | 14.33±0.58 | 23.33±2.89 | 141.00±20.42 | 244.67±43.19 | 222.67±15.37 |
| 0.1350 | 16.00±1.73 | 12.33±0.58 | 22.33±3.06 | 139.00±31.51 | 230.67±21.78 | 219.33±7.77 |
| 0.0405 | 14.00±4.58 | 15.33±5.51 | 26.33±3.21 | 118.33±33.71 | 226.67±5.86 | 236.33±9.07 |
| 0.0122 | 13.00±2.00 | 14.00±4.00 | 22.33±2.52 | 124.33±39.80 | 244.33±35.44 | 230.33±0.58 |
| 0.0037 | 14.00±4.36 | 13.33±3.51 | 25.33±0.58 | 108.00±9.17 | 244.33±40.55 | 219.67±18.61 |
| 0.0011 | 13.67±1.15 | 14.67±3.51 | 25.33±4.62 | 139.00±27.51 | 246.00±26.23 | 216.33±10.12 |
| NC | 12.67±3.51 | 13.67±4.04 | 25.33±2.31 | 140.33±20.03 | 236.67±47.65 | 221.00±15.87 |
| PC | 583.00±61.02 | 626.33±72.57 | 578.67±42.34 | 928.67±137.78 | 1245.33±188.21 | 1130.33±81.82 |
NC=controls; PC=positive controls (see Table 1); SD=standard deviation
Based on the results obtained with different concentrations of 2-amino-4-thiazole acetic acid in the mutagenicity assay, using TA 1537, TA 1535, TA 98, TA 100, TA 102 strains of S. typhymurium, and E. coli KMWP2p, it was observed that 2-amino-4-thiazole acetic acid did not show mutagenic effects , both with and without metabolic activation.
Discussion
We investigated some structural analogue compounds starting from aminothiazole (CAS 96-50-4); the compounds taken into consideration are: 2-amino-4-methylthiazole (CAS 1603-91-4), 2-amino-5-methylthiazole (CAS 7305-71-7), 3-methylisothiazole-5-amine (CAS 24340-76-9) and our compound 2-aminothiazole-4-acetic acid (CAS 29676-71-9).
Aminothiazole was tested in Salmonella typhimurium TA 100 and K.pneumoniae (5). It was not mutagen in S. thyphymurium TA 100; whereas it was mutagen in K. pneumonia. Another set of tests shows that aminothiazole was mutagen only in S.typhimurium TA 98 and TA 1538 (with metabolic activation only), while it was not mutagen in the other S.typhimurium strains (both with and without metabolic activation) (6). Its mutagenic activity was also confirmed by a test in mammalian cells (i.e. L5178Y TK +/- cells); in particular, this test was positive both with and without metabolic activation (6). From these results the mutagenicity of this compound is not clear.
2-Amino-4-methylthiazole was tested in a bacterial reverse mutation assay in accordance with OECD guideline 471 and under GLP conditions. The test was performed on S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102 strains and on E,coli WP2pKM101, both with and without metabolic activation. This compound was clearly non-mutagen in E. coli and S.thyphimurium strains, both with and without metabolic activation (7). Old results show that this compound is mutagen in K. pneumonia and not in S. thyphimurium TA 100 (5). This compound may be considered non mutagen.
2-Amino-5-methylthiazole is a substance which was notified in accordance with the Directive 67/546/CEE and data on its genotoxicity are shown in the database of the registered substances in ECHA website (8). According to these data, the substance was tested in a bacterial reverse mutation assay in accordance with OECD Guideline 471 and under GLP conditions. The test was performed on S. thyphimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, both with and without metabolic activation. 2-Amino-5-methylthiazole was clearly negative in 3 strains of S. thyphimurium, whereas, according to authors, a weak mutagenic response in 2 other genotypically different strains could not be ruled out definitely. No data by in vitro test on mammalian cells or by vivo test were found. These results indicated some mutagenicity for this compound.
3-Methylisothiazol-5-amine was tested in S. thyphimurium and mammalian cells (L5178Y TK +/-). With regard to bacterial assay, the substance was mutagen only in S. thyphimurium TA 98 and TA 1538 (with metabolic activation only), whereas it was not mutagen in the other S. thyphimurium strains (both with and without metabolic activation) (6). Its mutagenic activity was also confirmed by the test in mammalian cells (i.e. L5178Y TK +/- cells), that was positive with and without metabolic activation (9). This compound is considered as mutagen.
Our compound 2-aminothiazol-4-acetic acid, as shown in this paper, is not mutagen in bacterial reverse mutation assay. For a preliminary observation, we can say that the substituent in position 2 of aminothiazole allow to obtain compounds with no mutagenic activity as it is shown by this compound and by 2-amino-4 methylthiazole.
References
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Authors
Radice M., Fochi C., Industriale Chimica S.r.l., 21047 Saronno (VA), Italy
Faccioli F., Eurofins Biolab S.r.l., 20090 Vimodrone (MI), Italy
Talamini G., Università Cattolica Sacro Cuore, Istituto di chimica agraria e ambientale (PC) Italy
Coppi G., Consultant AFI, Assoc. Farmaceut. Indust., (MI), Italy