Registration Dossier
Registration Dossier
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EC number: 922-435-3 | CAS number: -
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14 - 21 September 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
- Report date:
- 2010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21st July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine
- EC Number:
- 945-075-9
- Molecular formula:
- Mixture of C47H74N8O4 and C34H58N4O2
- IUPAC Name:
- Reaction product of m-tolylidene diisocyanate and cyclohexylamine and cyclohex-1,2-ylenediamine and (Z)-octadec-9-enylamine
- Test material form:
- solid
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction from Aroclor induced rats
- Test concentrations with justification for top dose:
- 5000 μg, 1667 μg, 556 μg, 185 μg and 62 μg. No justification for the top dose provided.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Water was used for the suspension. The solvent THF was tested, but resulted in a polymer-like precipitation on mixing with the bacterial suspension.
Controls
- Untreated negative controls:
- yes
- Remarks:
- Control cultures were treated with solvent
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- mitomycin C
- other:
- Remarks:
- Concentrations of the positive control susbtances detailed in Table 1
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: In the confirmation test only, for 20 minutes
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 per concentration per test
DETERMINATION OF CYTOTOXICITY
- Method: The number of colonies per plate was counted
- Any supplementary information relevant to cytotoxicity: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.
- OTHER:
Sterility test: The sterility of the test item and the metabolic activation system (S9) were tested. The highest concentration of test item and a sample of the S9 mix were added respectively to top agar preheated at about 45°C and poured over minimal agar medium plates. The plates were incubated for about 72 hours at about 37°C. Presence or absence of colonies was observed. Bacterial growth would be an indication of microbiological contamination of the test item or S9 mix respectively.
Solubility test: Solubility was assessed as precipitation in the final mixture under the actual test conditions. Observation of precipitation by naked eye indicates insolubility. - Rationale for test conditions:
- According to guideline
- Evaluation criteria:
- Several criteria are used for determining a positive result: a dose-response in the range tested and / or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result was considered positive whenever the number of revertants is increased compared to the vehicle-control to at least 2 fold of the vehicle control for TA 98, 100, and 102 strains, and 3-fold of the solvent control for TA 1535 and 1537 strains. However biological relevance of the results was considered first.
Positive results from the bacterial reverse mutation test indicate that a test item induces point mutations or frame-shifts in the genome of the tested bacterial strains. Negative results from the test indicate that under the test conditions, the test item neither mutagenic nor-pro-mutagenic in the tested experimental system. - Statistics:
- Mean and standard deviation results were calculated. Additionally, the R ratio was calculated as follows:
R = Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
Results and discussion
Test resultsopen allclose all
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None reported
- Effects of osmolality: None reported
- Evaporation from medium: None reported
- Water solubility: Solubility was assessed assessed as precipitation in the final mixture under test conditions. No observations of precipitation were reported.
- Precipitation: None reported
- Other confounding effects: No appropriate solvent was found for the test item thus, a suspension in water was used.
RANGE-FINDING/SCREENING STUDIES: None reported
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) :
Strain TA 98 without metabolic activation, CDI+: Mean = 284, Standard Deviation = 62; Range = 150 - 373 (n = 36)
Strain TA 98 without metabolic activation, CDI-: Mean = 12, Standard Deviation = 5; Range = 5 - 30 (n = 36)
Strain TA 98 without metabolic activation, R = 23.4
Strain TA 98 without metabolic activation, CDI-: Mean = 927, Standard Deviation = 218; Range = 250 - 1212 (n = 36)
Strain TA 98 without metabolic activation, CDI+: Mean = 9, Standard Deviation = 3; Range = 4 - 15 (n = 36)
Strain TA 98 without metabolic activation, R = 100.5
Strain TA 98 with metabolic activation, CDI+: Mean = 507, Standard Deviation = 166; Range = 291 - 1025 (n = 36)
Strain TA 98 with metabolic activation, CDI-: Mean = 15, Standard Deviation = 3; Range = 9 - 21 (n = 36)
Strain TA 98 with metabolic activation, R = 34.7
Strain TA 98 with metabolic activation, CDI-: Mean = 951, Standard Deviation = 148; Range = 656 - 1257 (n = 36)
Strain TA 98 with metabolic activation, CDI+: Mean = 22, Standard Deviation = 7; Range = 7 - 35 (n = 36)
Strain TA 98 with metabolic activation, R = 43.4
Strain TA 100 without metabolic activation, CDI+: Mean = 444, Standard Deviation = 59; Range = 308 - 583(n = 36)
Strain TA 100 without metabolic activation, CDI-: Mean = 65, Standard Deviation = 9; Range = 46 - 85(n = 36)
Strain TA 100 without metabolic activation, R = 6.8
Strain TA 100 without metabolic activation, CDI-: Mean = 396, Standard Deviation = 81; Range = 267 - 587 (n = 36)
Strain TA 100 without metabolic activation, CDI+: Mean = 71, Standard Deviation = 21; Range = 125 - 43 (n = 36)
Strain TA 100 without metabolic activation, R = 5.6
Strain TA 100 with metabolic activation, CDI+: Mean = 501, Standard Deviation = 92; Range = 286 - 742 (n = 36)
Strain TA 100 with metabolic activation, CDI-: Mean = 102, Standard Deviation = 23; Range = 46 - 138 (n = 36)
Strain TA 100 with metabolic activation, R = 4.9
Strain TA 100 with metabolic activation, CDI-: Mean = 676, Standard Deviation = 166; Range = 425 - 1121 (n = 36)
Strain TA 100 with metabolic activation, CDI+: Mean = 112, Standard Deviation = 31; Range = 42 - 149 (n = 36)
Strain TA 100 with metabolic activation, R = 6.0
Strain TA 102 without metabolic activation, CDI+: Mean = 1094, Standard Deviation = 133; Range = 865 - 1297 (n = 36)
Strain TA 102 without metabolic activation, CDI-: Mean = 198, Standard Deviation = 33; Range = 114 - 244(n = 36)
Strain TA 102 without metabolic activation, R = 5.5
Strain TA 102 without metabolic activation, CDI-: Mean = 490, Standard Deviation = 208; Range = 218 - 1077(n = 36)
Strain TA 102 without metabolic activation, CDI+: Mean = 199, Standard Deviation = 38; Range = 144 - 309 (n = 36)
Strain TA 102 without metabolic activation, R = 2.5
Strain TA 102 with metabolic activation, CDI+: Mean = 1112, Standard Deviation = 184; Range = 527 - 1369 (n = 36)
Strain TA 102 with metabolic activation, CDI-: Mean = 271, Standard Deviation = 33; Range = 199 - 328 (n = 36)
Strain TA 102 with metabolic activation, R = 4.1
Strain TA 102 with metabolic activation, CDI-: Mean = 591, Standard Deviation = 114; Range = 412 - 841 (n = 36)
Strain TA 102 with metabolic activation, CDI+: Mean = 271, Standard Deviation = 42; Range = 202 - 390 (n = 36)
Strain TA 102 with metabolic activation, R = 2.2
Strain TA 1535 without metabolic activation, CDI+: Mean = 364, Standard Deviation = 74; Range = 135 - 448 (n = 36)
Strain TA 1535 without metabolic activation, CDI-: Mean = 13, Standard Deviation = 3; Range = 6 - 18 (n = 36)
Strain TA 1535 without metabolic activation, R = 27.9
Strain TA 1535 without metabolic activation, CDI-: Mean = 186, Standard Deviation = 62; Range = 112 - 421(n = 36)
Strain TA 1535 without metabolic activation, CDI+: Mean = 11, Standard Deviation = 4; Range = 4 - 23 (n = 36)
Strain TA 1535 without metabolic activation, R = 17.2
Strain TA 1535 with metabolic activation, CDI+: Mean = 558, Standard Deviation = 142; Range = 144 - 729 (n = 36)
Strain TA 1535 with metabolic activation, CDI-: Mean = 27, Standard Deviation = 5; Range = 15 - 35 (n = 36)
Strain TA 1535 with metabolic activation, R = 20.3
Strain TA 1535 with metabolic activation, CDI-: Mean = 483, Standard Deviation = 162; Range = 194 - 922 (n = 36)
Strain TA 1535 with metabolic activation, CDI+: Mean = 25, Standard Deviation = 7; Range = 11 - 39 (n = 36)
Strain TA 1535 with metabolic activation, R = 19.5
Strain TA 1537 with metabolic activation, CDI+: Mean = 15, Standard Deviation = 4; Range = 7 - 25 (n = 36)
Strain TA 1537 with metabolic activation, CDI-: Mean = 6, Standard Deviation = 2; Range = 2 - 9 (n = 36)
Strain TA 1537 with metabolic activation, R = 2.5
Strain TA 1537 with metabolic activation, CDI-: Mean = 459, Standard Deviation = 122; Range = 213 - 753 (n = 36)
Strain TA 1537 with metabolic activation, CDI+: Mean = 8, Standard Deviation = 4; Range = 2 - 18 (n = 36)
Strain TA 1537 with metabolic activation, R = 56.1
Strain TA 1537 without metabolic activation, CDI+: Mean = 73, Standard Deviation = 29; Range = 22 - 131 (n = 36)
Strain TA 1537 without metabolic activation, CDI-: Mean = 9, Standard Deviation = 3; Range = 2 - 18 (n = 36)
Strain TA 1537 without metabolic activation, R = 8.6
Strain TA 1537 without metabolic activation, CDI-: Mean = 26, Standard Deviation = 14; Range = 11 - 74(n = 36)
Strain TA 1537 without metabolic activation, CDI+: Mean = 7, Standard Deviation = 3; Range = 3 - 15 (n = 36)
Strain TA 1537 without metabolic activation, R = 3.5
Where; CDI+ = Positive control direct incorporation method; CPI+ = Positive Control preincubation method; CDI- = Negative control direct incorporation method; CPI- = Negative control preincubation method; n = number of replicates; R = ratio of the number revertant colonies in the positive control divided by the number revertant colonies in the negative control. Note; 95 % confidence intervals for historic data not reported in the study report.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: A reduction in the number of colonies in a dose-dependent manner compared to negative control for any strain and condition might indicate cytotoxicity.
- Other observations when applicable: None reported
Any other information on results incl. tables
Table 4: -S9 direct incorporation (main test); Strain Salmonella typhimurium TA98
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 17 | 19 | 15 | 17.0 | 2.0 | - |
| Positive Control; 2-Nitrofluorene | 5 | 295 | 366 | 309 | 323.3 | 37.6 | 19.0 |
| Test item | 5000 | 13 | 20 | 22 | 18.3 | 4.7 | 1.1 |
| 1667 | 14 | 29 | 18 | 20.3 | 7.8 | 1.2 |
|
556 |
16 |
19 |
17 |
17.3 |
1.5 |
1.0 |
|
185 |
20 |
22 |
17 |
19.7 |
2.5 |
1.2 |
|
62 |
25 |
16 |
15 |
18.7 |
5.5 |
1.1 |
|
Table 5:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA100
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 69 | 78 | 90 | 79.0 |
10.5 |
- |
Positive Control; Sodium Azide |
2.5 |
459 |
545 |
559 |
521.0 |
54.1 |
6.6 |
Test item |
5000 |
78 |
87 |
78 |
81.0 |
5.2 |
1.0 |
1667 |
64 |
67 |
98 |
76.3 |
18.8 |
1.0 |
|
556 |
87 |
88 |
88 |
87.7 |
0.6 |
1.1 |
|
185 |
108 |
90 |
86 |
94.7 |
11.7 |
1.2 |
|
62 |
78 |
98 |
78 |
84.7 |
11.5 |
1.1 |
|
Table 6:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA102
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 247 | 257 | 276 | 260.0 |
14.7 |
- |
Positive Control; Mitomycin C |
0.75 |
947 |
920 |
1026 |
964.3 |
55.1 |
3.7 |
Test item |
5000 |
301 |
272 |
298 |
290.3 |
15.9 |
1.1 |
1667 |
284 |
306 |
303 |
297.7 |
11.9 |
1.1 |
|
556 |
253 |
262 |
330 |
281.7 |
42.1 |
1.1 |
|
185 |
282 |
306 |
262 |
283.3 |
22.0 |
1.1 |
|
62 |
295 |
346 |
237 |
292.7 |
54.5 |
1.1 |
|
Table 7:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA1535
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 13 | 16 | 21 | 16.7 |
4.0 |
- |
Positive Control; Sodium Azide |
2.5 |
465 |
493 |
767 |
575.0 |
166.9 |
34.5 |
Test item |
5000 |
16 |
23 |
14 |
17.7 |
4.7 |
1.1 |
1667 |
15 |
23 |
17 |
18.3 |
4.2 |
1.1 |
|
556 |
18 |
22 |
19 |
19.7 |
2.1 |
1.2 |
|
185 |
25 |
20 |
18 |
21.0 |
3.6 |
1.3 |
|
62 |
24 |
15 |
17 |
18.7 |
4.7 |
1.1 |
|
Table 8:-S9 direct incorporation (main test); Strain Salmonella typhimurium TA1537
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 5 | 7 | 9 | 7.0 |
2.0 |
- |
Positive Control; 9 -Aminoacridine |
30 |
101 |
140 |
138 |
126.3 |
22.0 |
18.0 |
Test item |
5000 |
7 |
3 |
8 |
6.0 |
2.6 |
0.9 |
1667 |
8 |
8 |
10 |
8.7 |
1.2 |
1.2 |
|
556 |
4 |
7 |
7 |
6.0 |
1.7 |
0.9 |
|
185 |
9 |
8 |
2 |
6.3 |
3.8 |
0.9 |
|
62 |
6 |
11 |
8 |
8.3 |
2.5 |
1.2 |
|
Table 9: -S9 preincubation (confirmation test); Strain Salmonella typhimurium TA98
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 12 | 15 | 18 | 15.0 |
3.0 |
- |
Positive Control; 2 -Nitrofluorene |
5 |
650 |
388 |
612 |
550.0 |
141.6 |
36.7 |
Test item |
5000 |
19 |
15 |
21 |
18.3 |
3.1 |
1.2 |
1667 |
15 |
18 |
13 |
15.3 |
2.5 |
1.0 |
|
556 |
21 |
13 |
21 |
18.3 |
4.6 |
1.2 |
|
185 |
16 |
14 |
20 |
16.7 |
3.1 |
1.1 |
|
62 |
12 |
15 |
26 |
17.7 |
7.4 |
1.2 |
|
Table 10:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA100
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 79 | 99 | 84 | 87.3 |
10.4 |
- |
Positive Control; Sodium Azide |
2.5 |
348 |
365 |
589 |
434.0 |
134.5 |
5.0 |
Test item |
5000 |
119 |
91 |
108 |
106.0 |
14.1 |
1.2 |
1667 |
75 |
93 |
70 |
79.3 |
12.1 |
0.9 |
|
556 |
101 |
106 |
99 |
102.0 |
3.6 |
1.2 |
|
185 |
113 |
118 |
106 |
112.3 |
6.0 |
1.3 |
|
62 |
115 |
115 |
94 |
108.0 |
12.1 |
1.2 |
|
Table 11:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA102
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 185 | 266 | 221 | 224.0 |
40.6 |
- |
Positive Control; Mitomycin C |
0.75 |
1172 |
985 |
1136 |
1097.7 |
99.2 |
4.9 |
Test item |
5000 |
249 |
293 |
259 |
267.0 |
23.1 |
1.2 |
1667 |
268 |
250 |
273 |
263.7 |
12.1 |
1.2 |
|
556 |
220 |
266 |
256 |
247.3 |
24.2 |
1.1 |
|
185 |
221 |
251 |
289 |
253.7 |
34.1 |
1.1 |
|
62 |
248 |
254 |
257 |
253.0 |
4.6 |
1.1 |
|
Table 12:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA1535
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 11 | 12 | 16 | 13.0 |
2.6 |
- |
Positive Control; Socium Azide |
2.5 |
201 |
265 |
312 |
259.3 |
55.7 |
19.9 |
Test item |
5000 |
13 |
13 |
12 |
12.7 |
0.6 |
1.0 |
1667 |
19 |
16 |
13 |
16.0 |
3.0 |
1.2 |
|
556 |
12 |
14 |
16 |
14.0 |
2.0 |
1.1 |
|
185 |
13 |
15 |
15 |
14.3 |
1.2 |
1.1 |
|
62 |
15 |
14 |
18 |
15.7 |
2.1 |
1.2 |
|
Table 13:-S9 preincubation (confirmation test); Strain Salmonella typhimurium TA1537
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 10 | 5 | 8 | 7.7 |
2.5 |
- |
Positive Control; 9 -Aminoacridine |
30 |
84 |
98 |
135 |
105.7 |
26.4 |
13.8 |
Test item |
5000 |
9 |
7 |
9 |
8.3 |
1.2 |
1.1 |
1667 |
7 |
6 |
5 |
6.0 |
1.0 |
0.8 |
|
556 |
6 |
8 |
6 |
6.7 |
1.2 |
0.9 |
|
185 |
7 |
5 |
6 |
6.0 |
1.0 |
0.8 |
|
62 |
11 |
7 |
6 |
8.0 |
2.6 |
1.0 |
|
Table 14:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA98
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 26 | 11 | 22 | 19.7 |
7.8 |
- |
Positive Control; 2 -Aminoanthracene |
1.5 |
465 |
564 |
645 |
558.0 |
90.1 |
28.4 |
Test item |
5000 |
36 |
12 |
17 |
21.7 |
12.7 |
1.1 |
1667 |
12 |
17 |
16 |
15.0 |
2.6 |
0.8 |
|
556 |
16 |
15 |
19 |
16.7 |
2.1 |
0.8 |
|
185 |
11 |
19 |
17 |
15.7 |
4.2 |
0.8 |
|
62 |
17 |
14 |
12 |
14.3 |
2.5 |
0.7 |
|
Table 15:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA100
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 91 | 80 | 69 | 80.0 |
11.0 |
- |
Positive Control; 2 -Aminoanthracene |
2.5 |
599 |
382 |
292 |
424.3 |
157.8 |
5.3 |
Test item |
5000 |
93 |
79 |
68 |
80.0 |
12.5 |
1.0 |
1667 |
72 |
74 |
87 |
77.7 |
8.1 |
1.0 |
|
556 |
85 |
79 |
76 |
80.0 |
4.6 |
1.0 |
|
185 |
80 |
68 |
80 |
76.0 |
6.9 |
1.0 |
|
62 |
87 |
75 |
75 |
79.0 |
6.9 |
1.0 |
|
Table 16:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA102
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 235 | 355 | 255 | 281.7 |
64.3 |
- |
Positive Control; 2 -Aminoanthracene |
30 |
1020 |
897 |
923 |
946.7 |
64.8 |
3.4 |
Test item |
5000 |
327 |
302 |
309 |
312.7 |
12.9 |
1.1 |
1667 |
339 |
314 |
256 |
303.0 |
42.6 |
1.1 |
|
556 |
317 |
299 |
284 |
300.0 |
16.5 |
1.1 |
|
185 |
312 |
347 |
292 |
317.0 |
27.8 |
1.1 |
|
62 |
327 |
324 |
291 |
314.0 |
20.0 |
1.1 |
|
Table 17:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA1535
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 16 | 13 | 25 | 18.0 |
6.2 |
- |
Positive Control; 2 -Aminoanthracene |
30 |
456 |
254 |
357 |
355.7 |
101.0 |
19.8 |
Test item |
5000 |
12 |
16 |
17 |
15.0 |
2.6 |
0.8 |
1667 |
12 |
16 |
18 |
15.3 |
3.1 |
0.9 |
|
556 |
9 |
15 |
15 |
13.0 |
3.5 |
0.7 |
|
185 |
13 |
23 |
13 |
16.3 |
5.8 |
0.9 |
|
62 |
17 |
22 |
17 |
18.7 |
2.9 |
1.0 |
|
Table 18:+S9 direct incorporation (main test); Strain Salmonella typhimurium TA1537
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 9 | 8 | 8 | 8.3 |
0.6 |
- |
Positive Control; 2 -Aminoanthracene |
10 |
95 |
65 |
45 |
68.3 |
25.2 |
8.2 |
Test item |
5000 |
7 |
11 |
13 |
10.3 |
3.1 |
1.2 |
1667 |
7 |
5 |
2 |
4.7 |
2.5 |
0.6 |
|
556 |
2 |
5 |
4 |
3.7 |
1.5 |
0.4 |
|
185 |
9 |
8 |
6 |
7.7 |
1.5 |
0.9 |
|
62 |
14 |
5 |
7 |
8.7 |
4.7 |
1.0 |
|
Table 19:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA98
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 27 | 18 | 22 | 22.3 |
4.5 |
- |
Positive Control; 2 -Aminoanthracene |
1.5 |
976 |
541 |
941 |
819.3 |
241.7 |
36.7 |
Test item |
5000 |
23 |
18 |
23 |
21.3 |
2.9 |
1.0 |
1667 |
18 |
22 |
28 |
22.7 |
5.0 |
1.0 |
|
556 |
22 |
14 |
21 |
19.0 |
4.4 |
0.9 |
|
185 |
17 |
18 |
21 |
18.7 |
2.1 |
0.8 |
|
62 |
21 |
20 |
19 |
20.0 |
1.0 |
0.9 |
|
Table 20:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA100
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 126 | 96 | 102 | 108.0 |
15.9 |
- |
Positive Control; 2 -Aminoanthracene |
2.5 |
690 |
684 |
927 |
767.0 |
138.6 |
7.1 |
Test item |
5000 |
128 |
119 |
99 |
115.3 |
14.8 |
1.1 |
1667 |
112 |
116 |
108 |
112.0 |
4.0 |
1.0 |
|
556 |
100 |
133 |
99 |
110.7 |
19.3 |
1.0 |
|
185 |
114 |
113 |
115 |
114.0 |
1.0 |
1.1 |
|
62 |
116 |
122 |
107 |
115.0 |
7.5 |
1.1 |
|
Table 21:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA102
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 274 | 250 | 284 | 269.3 |
17.5 |
- |
Positive Control; 2 -Aminoanthracene |
30 |
933 |
815 |
875 |
874.3 |
59.0 |
3.2 |
Test item |
5000 |
314 |
334 |
315 |
321.0 |
11.3 |
1.2 |
1667 |
350 |
268 |
303 |
307.0 |
41.1 |
1.1 |
|
556 |
316 |
312 |
311 |
313.0 |
2.6 |
1.2 |
|
185 |
323 |
294 |
339 |
318.7 |
22.8 |
1.2 |
|
62 |
310 |
310 |
306 |
308.7 |
2.3 |
1.1 |
|
Table 22:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA1535
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 25 | 21 | 32 | 26.0 |
5.6 |
- |
Positive Control; 2 -Aminoanthracene |
30 |
457 |
446 |
648 |
517.0 |
113.6 |
19.9 |
Test item |
5000 |
26 |
29 |
26 |
27.0 |
1.7 |
1.0 |
1667 |
25 |
28 |
24 |
25.7 |
2.1 |
1.0 |
|
556 |
26 |
27 |
23 |
25.3 |
2.1 |
1.0 |
|
185 |
29 |
21 |
20 |
23.3 |
4.9 |
0.9 |
|
62 |
26 |
25 |
30 |
27.0 |
2.6 |
1.0 |
|
Table 23:+S9 pre-incubation (confirmation test); Strain Salmonella typhimurium TA1537
| Substance | Dose / plate (µg) | Replicate | Mean | S.D. | R | ||
| 1 | 2 | 3 | |||||
| Negative Control | - | 9 | 7 | 8 | 8.0 |
1.0 |
- |
Positive Control; 2 -Aminoanthracene |
10 |
69 |
91 |
49 |
69.7 |
21.0 |
8.7 |
Test item |
5000 |
8 |
7 |
9 |
8.0 |
1.0 |
1.0 |
1667 |
7 |
7 |
4 |
6.0 |
1.7 |
0.8 |
|
556 |
6 |
8 |
4 |
6.0 |
2.0 |
0.8 |
|
185 |
6 |
2 |
12 |
6.7 |
5.0 |
0.8 |
|
62 |
8 |
4 |
11 |
7.7 |
3.5 |
1.0 |
|
Applicant's summary and conclusion
- Conclusions:
- The mutagenic and cytotoxic potential of the test item was investigated in an Ames test, and was found to non mutagenic and non pro-mutagenic under the test conditions and no evidence of cytoxicity was observed.
- Executive summary:
The present bacterial reverse mutation test (Ames test) was performed in order to evaluate the mutagenic potential of the test item. Suspensions of 5 amino-acid requiring strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535, TA1537) were exposed by the direct plate incorporation method to five doses of the test item ranging from 5000 μg to 62 μg per plate in the presence and in the absence of an exogenous metabolic activation system. Both tests were repeated with the pre-incubation method. Revertant bacteria due to point or frameshift-mutations at specific locus are able to grow, forming colonies. These colonies were counted and compared to the number of spontaneous revertant colonies on the solvent control plate (negative control). Similarly, specific standard mutagens were tested and used as positive controls.
Based on the results obtained in this study, the test item was found to be non-mutagenic and non-pro-mutagenic under the test conditions.
The study is a GLP compliant guideline experimental study and is acceptable without restriction for assessment of this endpoint.
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