Table 1. Positive control for the various strains with and without S9 mix.
| Compound | (µg/plate) | Strain | S9 |
| Sodium azide (NaN3) | 1.5 | S.typhimuriumTA 1535S. typhumurium TA 100 | without |
| 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. typhimuriumTA 1537S. typhimuriumTA 98S. typhimuriumTA 102E. coli WP2pKM101 | with |
| Ciclophosphamide CP) | 100 | S. typhimuriumTA 1535S. typhimurium TA 100 | with |
In the Table 2 we report the genetic characteristics of the utilized strains.
Table 2. Genetic characteristics of the utilized strains.
| Strain | rfa | His / Trp | UVrA/UVrB | R. amp | R. tetra |
| S. typhimurium TA 1535 | rfa | His | UVrB | – | – |
| S. typhimurium TA 1537 | rfa | His | UVrB | – | – |
| S. typhimurium TA 98 | rfa | His | UVrB | R. amp | – |
| S. typhimurium TA 100 | rfa | His | UVrB | R. amp | – |
| S.typhimurium TA 102 | rfa | His | – | R. amp | R.tetra |
| E. coli WP2pMK101 | – | Trp | UVrA | R.amp | – |
Rfa: sensitivity to cristal violet
His: need for histidine as a growth factor
Trp: need for triptophan as a growth factor
UVrA: sensitivity to A ultraviolet radiation
UVrB: sensitivity to B ultraviolet radiation
R. amp: resistance to ampicillin
R. tetra: resistance to tetracicline
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-methylthiazole in the Ames test are shown in Tables 3-6, whereas the results with sulfathiazole are shown in Tables 7-10.
Table 3. Bacterial reverse-mutation test of 2-amino-4-methylthiazole in absence of metabolic activation (trial I). (Revertants/plate).
| Conc.(µg/plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.coliWP2pKM 101Mean + SD |
| 5,0000 | 26.00+4.6 | 27.00+5.6 | 17.67+4.5 | 158.33+35.2 | 204.00+43.5 | 225.33+19.0 |
| 1,5000 | 19.33+7.1 | 30.33+4.6 | 26.33+1.1 | 159.00+6.1 | 209.33+22.9 | 224.00+19.0 |
| 0,4500 | 15.67+2.1 | 32.33+2.1 | 21.67+3.1 | 157.67+14.6 | 189.00+45.7 | 223.67+21.7 |
| 0,1350 | 21.33+2.1 | 25.67+4.7 | 22.67+3.1 | 156.67+23.8 | 209.00+45.0 | 236.67+9.5 |
| 0,0405 | 21.67+5.7 | 26.00+2.6 | 20.67+2.1 | 141.67+31.0 | 196.00+17.0 | 215.33+17.2 |
| 0,0122 | 19.33+5.5 | 32.67+10.1 | 19.33+5.0 | 145.67+11.2 | 173.33+31.8 | 220.33+2.5 |
| 0,0037 | 17.00+5.6 | 33.00+9.6 | 25.33+5.5 | 134.00+19.7 | 219.00+12.8 | 236.33+9.1 |
| 0,0011 | 25.33+2.3 | 32.67+2.9 | 20.33+2.5 | 154.00+12.5 | 209.33+10.2 | 227.00+14.0 |
| NC | 19.00+8.7 | 27.33+5.5 | 24.00+2.0 | 147.33+26.8 | 193.00+34.0 | 222.00+11.4 |
| PC | 240.67+24.9 | 239.00+23.4 | 226.33+40.2 | 412.67+15.4 | 610.67+36.9 | 733.00+72.7 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation
Table 4. Bacterial reverse-mutation test of 2-amino-4-methylthiazole in absence of metabolic activation (trial II). (Revertants/plate).
| Conc.(µg/plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.coliWP2pKM 101Mean + SD |
| 5,0000 | 27.00+1.0 | 37.00+2.6 | 22.67+9.4 | 136.67+12.1 | 201.67+43.5 | 222.00+14.8 |
| 1,5000 | 16.00+5.3 | 26.00+2.8 | 18.33+2.3 | 167.67+17.2 | 173.33+7.5 | 234.00+3.5 |
| 0,4500 | 19.33+7.6 | 24.33+4.2 | 21.67+6.8 | 140.33+13.3 | 204.00+25.2 | 221.67+12.1 |
| 0,1350 | 24.33+1.1 | 32.00+1.7 | 19.33+6.0 | 149.33+16.3 | 184.67+34.0 | 217.67+14.2 |
| 0,0405 | 21.00+5.2 | 31.33+4.9 | 15.00+2.0 | 151.00+25.2 | 234.67+1.5 | 230.67+26.6 |
| 0,0122 | 20.67+5.5 | 30.33+5.9 | 23.33+5.1 | 155.67+16.3 | 215.67+30.0 | 229.00+7.8 |
| 0,0037 | 19.00+7.5 | 31.33+6.0 | 18.67+5.5 | 163.33+18.2 | 230.67+6.5 | 231.33+5.9 |
| 0,0011 | 15.00+3.6 | 27.33+4.7 | 18.33+1.2 | 151.33+23.4 | 207.00+37.4 | 225.00+17.4 |
| NC | 21.67+9.7 | 32.67+4.0 | 19.00+5.3 | 135.67+28.0 | 213.67+46.5 | 237.33+3.1 |
| PC | 207.33+30.2 | 248.33+14.3 | 250.00+16.5 | 377.67+1.2 | 663.33+86.4 | 779.00+46.2 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation
Table 5. Bacterial reverse-mutation test of 2-amino-4-methylthiazole in presence of metabolic activation (trial I). (Revertants/plate).
| Conc.(µg/plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.coliWP2pKM 101Mean + SD |
| 5,0000 | 19.00+6.1 | 27.67+5.0 | 25.00+6.9 | 165.33+14.6 | 213.33+50.9 | 222.33+11.7 |
| 1,5000 | 15.00+3.0 | 31.00+7.0 | 19.33+7.5 | 142.33+5.1 | 185.00+40.0 | 234.67+12.7 |
| 0,4500 | 16.33+6.1 | 31.33+8.6 | 20.00+8.9 | 146.67+14.8 | 232.00+8.9 | 238.67+5.5 |
| 0,1350 | 21.00+1.7 | 32.00+7.2 | 23.67+5.0 | 132.33+8.5 | 200.67+41.5 | 229.67+4.0 |
| 0,0405 | 23.00+7.2 | 28.00+5.0 | 20.00+6.1 | 155.33+18.5 | 206.33+39.6 | 233.67+11.1 |
| 0,0122 | 20.00+4.6 | 28.67+1.5 | 16.67+4.0 | 140.33+4.6 | 173.00+14.0 | 214.67+10.1 |
| 0,0037 | 23.00+2.6 | 26.00+7.8 | 23.00+7.9 | 155.33+32.3 | 216.33+17.6 | 231.67+9.3 |
| 0,0011 | 17.67+6.4 | 30.33+7.6 | 21.00+3.6 | 142.00+16.6 | 198.00+ 31.4 | 227.33+5.7 |
| NC | 16.00+4.4 | 33.00+5.3 | 19.33+6.1 | 150.67+9.5 | 211.67+33.0 | 229.33+22.0 |
| PC | 229.33+14.0 | 231.67+24.4 | 218.67+32.0 | 394.33+23.6 | 669.00+51.0 | 731.00+91.4 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation
Table 6. Bacterial reverse-mutation test of 2-amino-4-methylthiazole in presence of metabolic activation (trial II). (Revertants/plate).
| Conc.(µg/Plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.coliWP2pKM 101Mean + SD |
| 5,0000 | 15.67+4.6 | 31.33+5.0 | 20.67+6.1 | 138.33+28.4 | 191.00+47.3 | 222.00+10.4 |
| 1,5000 | 21.00+7.2 | 30.00+2.0 | 24.33+3.8 | 161,00+34.6 | 212.33+36.1 | 226.33+10.6 |
| 0,4500 | 23.67+10.1 | 27.00+1.7 | 24.00+6.1 | 167.67+7.1 | 192.67+8.0 | 221.00+15.7 |
| 0,1350 | 22.00+4.6 | 26.33+5.8 | 22.67+5.9 | 145.00+20.0 | 183.67+36.8 | 208.00+7.6 |
| 0,0405 | 20.00+3.6 | 32.67+1.5 | 17.67+2.3 | 154.33+18.6 | 192.33+39.7 | 217.33+12.5 |
| 0,0122 | 23.33+6.4 | 30.33+3.2 | 19.00+6.2 | 151.67+19.6 | 220.33+12.7 | 217.00+10.4 |
| 0,0037 | 17.33+8.4 | 28.00+4.4 | 17.00+4.0 | 148.00+16.4 | 205.67+16.8 | 219.67+19.4 |
| 0,0011 | 18.33+1.5 | 30.00+6.6 | 20.33+7.8 | 157.00+4.0 | 200.67+27.6 | 222.33+16.6 |
| NC | 23.33+3.8 | 26.67+3.5 | 21.67+2.1 | 138.33+17.2 | 226.33+24.8 | 212.67+7.4 |
| PC | 218.33+34.5 | 249.33+15.5 | 203.00+11.8 | 409.00+17.3 | 581.33+50.7 | 757.00+43.2 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation
Table 7. Bacterial reverse-mutation test of sulfamethylthiazole in absence of metabolic activation (trial I). (Revertants/plate).
| Conc.(µg/plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.coliWP2pKM 101Mean + SD |
| 5,0000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 1,5000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,4500 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,1350 | 15.00+1.0 | 22.00+2.6 | 16.33+2.1 | 126.00+6.6 | 208.33+8.0 | 207.00+5.3 |
| 0,0405 | 24.33+4.0 | 32.33+5.8 | 13.67+1.5 | 142.00+13.8 | 217.00+25.5 | 211.67+6.7 |
| 0,0122 | 26.67+4.2 | 32.67+4.7 | 17.67+6.0 | 144.00+25.4 | 187.67+38.1 | 220.33+24.0 |
| 0,0037 | 22.67+2.1 | 36.33+4.7 | 22.00+9.6 | 157.33+33.5 | 207.33+30.6 | 232.33+10.2 |
| 0,0011 | 16.67+0.6 | 32.33+5.7 | 19.33+7.8 | 140.67+3.2 | 186.33+29.9 | 227.67+12.7 |
| NC | 19.33+2.5 | 31.00+5.6 | 21.33+5.9 | 136.67+4.7 | 200.33+38.7 | 222.67+20.8 |
| PC | 226.67+10.0 | 243.33+26.2 | 237.67+25.7 | 395.00+9.9 | 691.33+29.1 | 644.33+88.3 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation
Table 8. Bacterial reverse-mutation test of sulfamethylthiazole in absence of metabolic activation (trial II). (Revertants/plate).
| Conc.(µg/plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.Coli WP2pKM 101Mean + SD |
| 5,0000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 1,5000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,4500 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,1350 | 14.00+1.0 | 32.00+5.2 | 20.67+8.7 | 132.33+13.3 | 201.00+18.5 | 208.33+9.1 |
| 0,0405 | 27.00+3.6 | 23.67+2.5 | 19.00+5.0 | 142.00+21.1 | 216.00+40.6 | 237.00+14.9 |
| 0,0122 | 17.00+3.6 | 37.00+2.7 | 25,67+4.2 | 149.67+23.9 | 181.00+20.4 | 220.00+16.6 |
| 0,0037 | 27.67+2.5 | 30.83+8.1 | 20.33+9.1 | 154.00+20.4 | 212.33+10.8 | 219.00+17.3 |
| 0,0011 | 22.00+4.4 | 31.33+7.4 | 21.00+8.5 | 158.67+26.4 | 188.67+34.3 | 219.33+7.1 |
| NC | 20.00+4.6 | 31.00+7.9 | 18.67+6.1 | 141.33+35.2 | 188.33+11.2 | 216.67+12.5 |
| PC | 212.33+5.1 | 258.67+19.6 | 222.67+39.6 | 402.33+18.6 | 613.00+89.6 | 729.33+92.5 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation
Table 9. Bacterial reverse-mutation test of sulfamethylthiazole in presence of metabolic activation (trial I). (Revertants/plate).
| Conc.(µg/plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.coli WP2pKM 101Mean + SD |
| 5,0000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 1,5000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,4500 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,1350 | 21.00+2.0 | 26.00+2.7 | 17.67+2.1 | 153.00+7.9 | 190.67+8.0 | 210.33+8.1 |
| 0,0405 | 18.33+10.1 | 27.33+10.1 | 20.33+6.1 | 164.67+8.7 | 212.00+33.0 | 224.33+20.0 |
| 0,0122 | 22.33+4.5 | 32.33+8.0 | 13.00+1.7 | 138.33+22.2 | 199.67+25.6 | 228.00+14.1 |
| 0,0037 | 19.00+4.4 | 30.67+4.7 | 19.67+3.2 | 157.67+31.6 | 210.00+30.6 | 226.33+15.5 |
| 0,0011 | 25.67+3.2 | 34.33+2.1 | 20.00+8.9 | 137.00+21.4 | 195.00+33.6 | 218.00+3.0 |
| NC | 25.67+3.2 | 35.67+5.8 | 21.33+8.1 | 163.00+6.2 | 203.00+27.2 | 221.33+18.9 |
| PC | 238.00+7.9 | 242.67+24.2 | 221.33+11.1 | 390.00+18.7 | 528.33+14.4 | 697.00+16.1 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation
Table 10. Bacterial reverse-mutation test of sulfamethylthiazole in presence of metabolic activation (trial II). (Revertants/plate).
| Conc.(µg/plate) | TA 1537Mean + SD | TA 1535Mean + SD | TA 98Mean + SD | TA 100Mean + SD | TA 102Mean + SD | E.coli KMWp2p 101Mean + SD |
| 5,0000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 1,5000 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,4500 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 | 0.00+0.0 |
| 0,1350 | 20.00+2.0 | 30.00+3.0 | 22.67+2.5 | 122.33+6.1 | 195.33+19.4 | 206.67+4.9 |
| 0,0405 | 17.67+7.2 | 26.00+1.0 | 22.67+2.1 | 151.00+7.8 | 214.67+26.7 | 222.67+14.3 |
| 0,0122 | 24.00+5.0 | 28.33+10.1 | 21.33+6.7 | 152.00+10.4 | 210.67+37.2 | 232.00+14.1 |
| 0,0037 | 22.33+3.1 | 30.00+4.6 | 22.33+5.5 | 149.00+29.6 | 217.33+5.0 | 218.67+7.8 |
| 0,0011 | 23.67+5.5 | 31.67+6.1 | 24.00+7.0 | 137.00+7.0 | 226.33+10.6 | 228.33+5.0 |
| NC | 24.00+2.0 | 24.67+2.5 | 26.67+2.9 | 144.00+17.6 | 199.67+31.5 | 211.00+9.9 |
| PC | 217.67+36.8 | 236.00+27.6 | 232.33+11.6 | 401.33+17.2 | 664.67+93.5 | 749.33+68.5 |
NC=controls ; PC=positive controls (see Table 1) ; SD=standard deviation