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EC number: 240-354-5 | CAS number: 16230-35-6
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:
- December 8, 2016 - January 5, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]
- EC Number:
- 240-354-5
- EC Name:
- N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]
- Cas Number:
- 16230-35-6
- Molecular formula:
- C19H24N2O3Si
- IUPAC Name:
- N,N'-[ethoxy(methyl)silanediyl]bis(N-methylbenzamide)
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.
Method
- Target gene:
- his D (Salmonella typhimurium TA 98)
his C (Salmonella typhimurium TA 1537)
his G (Salmonella typhimurium TA 100 and TA1535)
tryp E (Escherichia coli WP2 uvrA pKM101)
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa, pKM 101
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: Δuvr B, rfa, pKM 101
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Additional strain / cell type characteristics:
- other: Δuvr A, pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 50, 150, 500, 1500 and 5000 μg/plate. The preliminary study showed no toxicity of the test item up to 5000 μg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: the item is completely soluble in the vehicle
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- NaCl 0.9%, Acetone, DMSO, Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-Anthramine; cis-Platinum (II) Diammine Dichloride
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Assay 1:in agar (plate incorporation) with and without metabolic activation
A stock solution of the test item was prepared at 100 mg / mL. In a test tube, 0.1 mL of the bacterial suspension containing 1-9x109 bacteria/mL and 0.1 mL of each dilution of the original solution and 0.5 mL of sterile phosphate buffer are successively added to 2 mL of overlay agar maintained super cooled in 45ºC containing 5% (v/v) of a L-Histidine-D-Biotine solution (0.5 mM) for Salmonella Typhimurium strains or containing 5% (v/v) of nutrient broth nº2 to which are added 5 μL of a L-Tryptophane solution at 2 mg/mL for Escherichia coli strain.
In the assay with metabolic activation, either a standard plate incorporation method where the protocol is similar to the described above, except that, 500 μL of S9-mix fraction is quickly added, before pouring the mixture onto the plates.
After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate. Data are presented as the number of revertant colonies (mean ± standard deviation) per plate. The following ratio is calculated:
R= Number of revertant colonies in the presence of the test item / Number of revertant colonies in the absence of the test item
Assay 2: preincubation with and without metabolic activation
A stock solution of the test item was prepared at 100 mg / mL. The assay without metabolic activation was performed as mendioned in assay 1.
In the assay with metabolic activation, the solution of the test item solution with the test strain and 500 μL of S9-mix fraction are preincubated with shaking for 30 min, at 37ºC prior to mixing with the overlay agar and pouring onto the minimal agar plate.
After a 48-72 hour incubation period at 37ºC, revertant colonies are counted in each plate. Data are presented as the number of revertant colonies (mean ± standard deviation) per plate, and the ratio is calculate as mentioned before.
If the first assay is positive, the second one is performed in the same manner.
If the first assay, in presence of the test item, is negative, the pre-incubation test is performed for the second assay.
- Cell density at seeding (if applicable): 1:9x109 bacteria/mL
DURATION
- Preincubation period: 30 min at 37ºC (Assay 2)
- Exposure duration: 48-72 hours at 37ºC
SELECTION AGENT (mutation assays):
Salmonella Thyphimuriun strains: lack of Histidine in the media
Escherichia coli: lack of Tryptophane in the media
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: Preliminary cytotoxicity test (Strain TA100): In a test tube 0.1 mL of the bacterial suspension (1-9 x103 bacteria /mL) and 0.1 mL of the stock solution of L-Histidine-D-Biotine (2.5 mM). After homogenization, the content of the tube is poured onto a Petri plate (90 mm in diameter) containing minimal agar (20 mL). 3 plates per concentration are incubated for 48-72 h at 37ºC, and the colonies counted. A negative control containing the blank alone is run in parallel. In case of bacteriostatic activity
is detected, the highest concentration to be retined is that exhibiting a bacteriostatic activity of 75% or less. The precipitate, if present, should not interfere with the scoring. The following four dilutions studied are distributed according to a semi-logarithmic progression.
- OTHER:
STERILITY TESTS: Test item and the corresponding dilutions are added to 2 mL of top agar maintained at 45ºC, and poured after homogenization on the bottom agar (20 mL) onto a Petri plate (90 mm in diameter) (n=3). Plates are incubated for 48-72 hours at 37ºC and then examined. There should be no bacterial growth on any plate. S9-mix sterility is checked using the same protocol.
PREPARATION OF THE METABOLIC ACTIVATION SYSTEM:
Obtention of S9 fraction: S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOX (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11101-5-3607 validated on 04.2016 - expiry date: 24.03.2018).
Preparation of S9-mix 10% (v/v): The final concentration of co-factors and salts is as follows: S9 fraction 10%; MgCl2-6H2O 8 mM; KCl 33 mM; Glucose-6-Phophate Na2 5 mM; NADP Na2 4mM; Phosphate buffer pH 7.4 0.1 M.
CONTROL OF STRAINS
- Histidine and tryptophane requirements with cultrues in presence and in absence of L-histidine and L-triptophane for Samonella typhimurium and Escherichia coli strains respectively.
- Loss of cell wall LPS (rfa mutation) measuring crystal violet inhibition for Salmonella typhimurium strains.
- Ampicillin resistance for the strains which have the pKM 101 plasmide.
- Δuvr B mutation i.e. U.V.B. sensitivity for Salmonella typhimurium and Δuvr A mutation i.e. U.V.A. sensitivity for Escherichia coli.
- Spontaneous revertant rate.
- Sensitivity to reference mutagens. - Rationale for test conditions:
- Results of sterility controls show the absence of any bacterial growth in the presence of the various concentrations of the test item and in the presence of S9-mix.
Results of the bacteriostatic activity control show toxicity consistent with the maximum tolerated 75% in presence of 5000 µg/plate.
Values and frequency ranges from the laboratory's historical control - Evaluation criteria:
- The result of the test is considered as negative if the revertant number is below 3-fold the number of spontaneous reversions for TA 1535 and TA 1537 strains, and below 2-fold the number of spontaneous reversions for TA 98, TA 100 and E. coli WP2 (uvrA-) (pKM 101) strains with and without metabolic activation.
The result of the test is considered as positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least 2-fold that of spontaneous revertant colonies for TA 98, TA 100 and E. coli WP2 (uvrA-) (pKM 101), and 3-fold for TA 1535 and TA 1537.
Results and discussion
Test resultsopen allclose all
- Key result
- 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
- Key result
- 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
- Key result
- 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
- Key result
- 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
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- 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
- Precipitation: No precipitation of the test item was observed.
RANGE-FINDING/SCREENING STUDIES:
Neither original solution nor dilutions have bacteriostatic effect. The test item is tested at these doses: 5000, 1500, 500, 150 and 50 μg/plate.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: (2007-2016)
S. typhimurium TA 1535: without metab. activation (Sodium Azide): n= 975; 702.3 ± 203.4 (mean ± SD); 190 / 1481 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n= 525; 104.0 ± 53.0 (mean ± SD); 26 / 269 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n= 519; 73.2 ± 34.9 (mean ± SD); 25 / 185 (min / max)
S. typhimurium TA 1537: without metab. activation (9-Aminoacridine): n=975; 851.2 ± 428.1 (mean ± SD); 219 / 1967 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n= 525; 55.8 ± 23.4 (mean ± SD); 24 / 170 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n= 519; 49.5 ± 22.5 (mean ± SD); 21 / 182 (min / max)
S. typhimurium TA 98: without metab. activation (2-Nitrofluorene): n= 975; 512.6 ± 219.5 (mean ± SD); 187 / 1667 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n=525; 574.5 ± 209.5 (mean ± SD); 219 / 1499.0 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n= 519; 474.6 ± 196.5 (mean ± SD); 174 / 1370 (min / max)
S. typhimurium TA 100: without metab. activation (Sodium Azide): n=975; 934.4 ± 325.2 (mean ± SD); 381 / 1690 (min / max); with metab. activation (without pre-incubation) (2-Anthramine): n=522; 862.1 ± 359.1 (mean ± SD); 361 / 2163.0 (min / max); with metab. activation (with pre-incubation) (2-Anthramine): n=522; 682.4 ± 290.0 (mean ± SD); 309 / 1889 (min / max)
E. coli WP2 (pKM 101) (uvr A-): without metab. activation (cis-Platinium (II) Diamine Dichloride): n= 717; 502.8 ± 168.1 (mean ± SD); 248 / 1089 (min / max); with metab. activation (without pre-incubation) (Dimethyl Benzanthracene): n= 372; 707.3 ± 248.0 (mean ± SD); 378 / 1680 (min / max); with metab. activation (with pre-incubation) (Dimethyl Benzanthracene): n=372; 701.8 ± 229.7 (mean ± SD); 397 / 1680 (min / max)
- Negative (solvent/vehicle) historical control data: (2007-2016)
S. typhimurium TA 1535: without metab. activation: n= 975; 10.9 ± 3.6 (mean ± SD); 4 / 23 (min / max); with metab. activation (without pre-incubation): n= 525; 12.3 ± 4 (mean ± SD); 3 / 23 (min / max); with metab. activation (with pre-incubation): n= 519; 12.7 ± 4.2 (mean ± SD); 5 / 25 (min / max)
S. typhimurium TA 1537: without metab. activation: n= 975; 6.0 ± 2.4 (mean ± SD); 1 / 14 (min / max); with metab. activation (without pre-incubation): n= 525, 8.0 ± 3.1 (mean ± SD); 1 / 24 (min / max); with metab. activation (with pre-incubation): n= 519; 8.3 ± 3.2 (mean ± SD); 1 / 19 (min / max)
S. typhimurium TA 98: without metab. activation: n= 975; 16.0 ± 3.8 (mean ± SD); 6 / 29 (min / max); with metab. activation (without pre-incubation): n= 525; 23.2 ± 4.8 (mean ± SD); 12 / 38 (min / max); with metab. activation (with pre-incubation): n= 519; 23.3 ± 5.2 (mean ± SD); 11 / 36 (min / max)
S. typhimurium TA 100: without metab. activation: n= 975; 62.2 ± 14.3 (mean ± SD); 41 / 126 (min / max); with metab. activation (without pre-incubation): n= 522; 101.7 ± 22.9 (mean ± SD); 58 / 189 (min / max); with metab. activation (with pre-incubation): n=519; 101.3 ± 24.8 (mean ± SD); 51 / 189 (min / max)
E. coli WP2 (pKM 101) (uvr A-): without metab. activation: n= 717; 75.0 ± 29.2 (mean ± SD); 41 / 188 (min / max); with metab. activation (without pre-incubation): n= 372; 154.8 ± 33.6 (mean ± SD); 80 / 264 (min / max); with metab. activation (with pre-incubation): n= 372; 157.5 ± 35.4 (mean ± SD); 69 / 250 (min / max)
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects of the test item were observed in any of the five tested strains used up to the highest dose (with and without metabolic activation) in assay 1 and 2.
Any other information on results incl. tables
Table 1. Sterility control
Serie |
Doses |
Colony number/plate |
||
Control nº 1 |
1 |
2 |
3 |
|
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg / plate |
0 |
0 |
0 |
1500 µg / plate |
0 |
0 |
0 |
|
500 µg / plate |
0 |
0 |
0 |
|
150 µg / plate |
0 |
0 |
0 |
|
50 µg / plate |
0 |
0 |
0 |
|
S9-mix |
500µL / plate |
0 |
0 |
0 |
Control nº 2 |
1 |
2 |
3 |
|
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg / plate |
0 |
0 |
0 |
1500 µg / plate |
0 |
0 |
0 |
|
500 µg / plate |
0 |
0 |
0 |
|
150 µg / plate |
0 |
0 |
0 |
|
50 µg /plate |
0 |
0 |
0 |
|
S9-mix |
500 µL / plate |
0 |
0 |
0 |
Table 2. Bacteriostatic activity control nº2
|
|
Doses (/plate) |
|||||||
|
|
0 (negative control) |
DMSO |
50 µg |
150 µg |
500 µg |
1500 µg |
2500 µg |
5000 µg |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA081216-S4) |
N1 |
259 |
249 |
289 |
334 |
304 |
238 |
303 |
311 |
N2 |
288 |
293 |
302 |
320 |
301 |
297 |
312 |
287 |
|
N3 |
304 |
308 |
302 |
302 |
218 |
312 |
287 |
260 |
|
N |
284±23 |
283± 31 |
298±8 |
319± 16 |
274± 49 |
282± 39 |
301±13 |
286± 26 |
|
% |
- |
100% |
105% |
112% |
97% |
100% |
106% |
101% |
|
N1 Number of colonies in plate 1
N2 Number of colonies in plate 2
N3 Number of colonies in plate 3
N Mean per plate
% Percent of survival compared to negative control
Table 3. Bacteriostatic activity control nº3
|
|
Doses (/plate) |
||||||
|
|
0 (negative control) |
DMSO |
50 µg |
150 µg |
500 µg |
1500 µg |
5000 µg |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
N1 |
221 |
239 |
235 |
239 |
241 |
250 |
239 |
N2 |
241 |
237 |
234 |
235 |
237 |
241 |
230 |
|
N3 |
223 |
264 |
216 |
185 |
227 |
215 |
237 |
|
N |
228± 11 |
247± 15 |
228± 11 |
220± 30 |
235±7 |
235±18 |
235± 5 |
|
% |
- |
108% |
100% |
96% |
103% |
103% |
103% |
|
N1 Number of colonies in plate 1
N2 Number of colonies in plate 2
N3 Number of colonies in plate 3
N Mean per plate
% Percent of survival compared to negative control
Table 4. TA 1535 - Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
7 |
6 |
7 |
6.67 |
0.58 |
- |
Positive control solvent |
5 µL |
6 |
8 |
6 |
6.67 |
1.15 |
- |
Positive control: Sodium azide |
5 µg in 5 µL |
513 |
616 |
583 |
570.67 |
52.60 |
85.60 |
Vehicle |
50 µL |
8 |
7 |
8 |
7.67 |
0.58 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
4 |
5 |
6 |
5.00 |
1.00 |
0.65 |
1500 µg |
8 |
17 |
8 |
11.00 |
5.20 |
1.43 |
|
500 µg |
11 |
9 |
5 |
8.33 |
3.06 |
1.09 |
|
150 µg |
5 |
7 |
8 |
6.67 |
1.53 |
0.87 |
|
50 µg |
7 |
5 |
8 |
6.67 |
1.53 |
0.87 |
|
Table 5. TA 1535 - Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
14 |
8 |
5 |
9.00 |
4.58 |
- |
Positive control solvent |
20 µL |
7 |
6 |
5 |
6.00 |
1.00 |
- |
Positive control: 2-Anthramine |
2 µg in 20 µL |
90 |
71 |
61 |
74.00 |
14.73 |
12.33 |
Vehicle |
50 µL |
10 |
8 |
7 |
8.33 |
1.53 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
9 |
9 |
10 |
9.33 |
0.58 |
1.12 |
1500 µg |
7 |
14 |
14 |
11.67 |
4.04 |
1.40 |
|
500 µg |
4 |
5 |
8 |
5.67 |
2.08 |
0.68 |
|
150 µg |
9 |
6 |
5 |
6.67 |
2.08 |
0.80 |
|
50 µg |
7 |
10 |
13 |
10.00 |
3.00 |
1.20 |
|
Table 6. TA 1535 - Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
14 |
16 |
7 |
12.33 |
4.73 |
- |
Positive control solvent |
5 µL |
11 |
12 |
12 |
11.67 |
0.58 |
- |
Positive control: Sodium azide |
5 µg in 5 µL |
735 |
642 |
692 |
689.67 |
46.54 |
59.11 |
Vehicle |
50 µL |
18 |
10 |
14 |
14.00 |
4.00 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
14 |
10 |
14 |
12.67 |
2.31 |
0.90 |
1500 µg |
15 |
12 |
18 |
15.00 |
3.00 |
1.07 |
|
500 µg |
16 |
14 |
13 |
14.33 |
1.53 |
1.02 |
|
150 µg |
12 |
20 |
17 |
16.33 |
4.04 |
1.17 |
|
50 µg |
7 |
16 |
14 |
12.33 |
4.73 |
0.88 |
|
Table 7. TA 1535 - Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
13 |
17 |
11 |
13.67 |
3.06 |
- |
Positive control solvent |
10 µL |
12 |
12 |
10 |
11.33 |
1.15 |
- |
Positive control: 2-Anthramine |
1 µg in 10 µL |
64 |
49 |
67 |
60.00 |
9.64 |
5.29 |
Vehicle |
50 µL |
11 |
19 |
19 |
16.33 |
4.62 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
10 |
17 |
12 |
13.00 |
3.61 |
0.80 |
1500 µg |
12 |
15 |
13 |
13.33 |
1.53 |
0.82 |
|
500 µg |
14 |
15 |
13 |
14.00 |
1.00 |
0.86 |
|
150 µg |
14 |
15 |
20 |
16.33 |
3.21 |
1.00 |
|
50 µg |
17 |
15 |
16 |
16.00 |
1.00 |
0.98 |
|
Table 8. TA 1537 - Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
5 |
5 |
7 |
5.67 |
1.15 |
- |
Positive control solvent |
20 µL |
4 |
3 |
6 |
4.33 |
1.53 |
- |
Positive control: 9-Aminoacridine |
50 µg in 20 µL |
1055 |
1110 |
933 |
1032.67 |
90.59 |
238.31 |
Vehicle |
50 µL |
7 |
2 |
9 |
6.00 |
3.61 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
7 |
4 |
4 |
5.00 |
1.73 |
0.83 |
1500 µg |
8 |
13 |
12 |
11.00 |
2.65 |
1.83 |
|
500 µg |
8 |
7 |
8 |
7.67 |
0.58 |
1.28 |
|
150 µg |
13 |
7 |
11 |
10.33 |
3.06 |
1.72 |
|
50 µg |
2 |
4 |
7 |
4.33 |
2.52 |
0.72 |
|
Table 9. TA 1537 - Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
7 |
4 |
6 |
5.67 |
1.53 |
- |
Positive control solvent |
20 µL |
12 |
8 |
10 |
10.00 |
2.00 |
- |
Positive control: 2-Anthramine |
2 µg in 20 µL |
25 |
39 |
30 |
31.33 |
7.09 |
3.13 |
Vehicle |
50 µL |
7 |
10 |
6 |
7.67 |
2.08 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
14 |
7 |
14 |
11.67 |
4.04 |
1.52 |
1500 µg |
10 |
8 |
16 |
11.33 |
4.16 |
1.48 |
|
500 µg |
12 |
10 |
10 |
10.67 |
1.15 |
1.39 |
|
150 µg |
12 |
7 |
9 |
9.33 |
2.52 |
1.22 |
|
50 µg |
3 |
12 |
7 |
7.33 |
4.51 |
0.96 |
|
Table 10. TA 1537 - Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
7 |
4 |
4 |
5.00 |
1.73 |
- |
Positive control solvent |
20 µL |
3 |
2 |
4 |
3.00 |
1.00 |
- |
Positive control: 9-Aminoacridine |
50 µg in 20 µL |
1716 |
1860 |
1415 |
1663.67 |
227.07 |
554.56 |
Vehicle |
50 µL |
1 |
4 |
5 |
3.33 |
2.08 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
2 |
6 |
3 |
3.67 |
2.08 |
1.10 |
1500 µg |
3 |
3 |
2 |
2.67 |
0.58 |
0.80 |
|
500 µg |
2 |
3 |
3 |
2.67 |
0.58 |
0.80 |
|
150 µg |
4 |
4 |
3 |
3.67 |
0.58 |
1.10 |
|
50 µg |
4 |
2 |
3 |
3.00 |
1.00 |
0.90 |
|
Table 11. TA 1537 - Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
8 |
5 |
9 |
7.33 |
2.08 |
- |
Positive control solvent |
20 µL |
2 |
3 |
5 |
3.33 |
1.53 |
- |
Positive control: 2-Anthramine |
1 µg in 10 µL |
28 |
25 |
22 |
25.00 |
3.00 |
7.50 |
Vehicle |
50 µL |
6 |
10 |
4 |
6.67 |
3.06 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
5 |
3 |
4 |
4.00 |
1.00 |
0.60 |
1500 µg |
3 |
4 |
4 |
3.67 |
0.58 |
0.55 |
|
500 µg |
5 |
6 |
5 |
5.33 |
0.58 |
0.80 |
|
150 µg |
6 |
7 |
6 |
6.33 |
0.58 |
0.95 |
|
50 µg |
5 |
4 |
5 |
4.67 |
0.58 |
0.70 |
|
Table 12. TA 98 - Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
22 |
11 |
22 |
18.33 |
6.35 |
- |
Positive control solvent |
20 µL |
22 |
25 |
18 |
21.67 |
3.51 |
- |
Positive control: 2-Nitrofluorene |
2 µg in 20 µL |
191 |
229 |
217 |
212.33 |
19.43 |
9.80 |
Vehicle |
50 µL |
19 |
29 |
17 |
21.67 |
6.43 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
14 |
15 |
23 |
17.33 |
4.93 |
0.80 |
1500 µg |
26 |
22 |
23 |
23.67 |
2.08 |
1.09 |
|
500 µg |
22 |
21 |
22 |
21.67 |
0.58 |
1.00 |
|
150 µg |
24 |
27 |
28 |
26.33 |
2.08 |
1.22 |
|
50 µg |
28 |
25 |
21 |
24.67 |
3.51 |
1.14 |
|
Table 13. TA 98 - Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
18 |
22 |
23 |
21.00 |
2.65 |
- |
Positive control solvent |
20 µL |
30 |
23 |
34 |
29.00 |
5.57 |
- |
Positive control: 2-Anthramine |
2 µg in 20 µL |
253 |
233 |
239 |
241.67 |
10.26 |
8.33 |
Vehicle |
50 µL |
32 |
24 |
30 |
28.67 |
4.16 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
21 |
16 |
20 |
19.00 |
2.65 |
0.66 |
1500 µg |
32 |
36 |
22 |
30.00 |
7.21 |
1.05 |
|
500 µg |
35 |
32 |
28 |
31.67 |
3.51 |
1.10 |
|
150 µg |
32 |
31 |
29 |
30.67 |
1.53 |
1.07 |
|
50 µg |
33 |
33 |
30 |
32.00 |
1.73 |
1.12 |
|
Table 14. TA 98 - Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
27 |
13 |
22 |
20.67 |
7.09 |
- |
Positive control solvent |
20 µL |
17 |
17 |
16 |
16.67 |
0.58 |
- |
Positive control: 2-Nitrofluorene |
2 µg in 20 µL |
252 |
266 |
312 |
276.67 |
31.39 |
16.60 |
Vehicle |
50 µL |
21 |
20 |
18 |
19.67 |
1.53 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
10 |
14 |
10 |
11.33 |
2.31 |
0.58 |
1500 µg |
16 |
17 |
10 |
14.33 |
3.79 |
0.73 |
|
500 µg |
21 |
18 |
16 |
18.33 |
2.52 |
0.93 |
|
150 µg |
23 |
22 |
18 |
21.00 |
2.65 |
1.07 |
|
50 µg |
18 |
25 |
21 |
21.33 |
3.51 |
1.08 |
|
Table 15. TA 98 - Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
28 |
26 |
26 |
26.67 |
1.15 |
- |
Positive control solvent |
20 µL |
19 |
16 |
32 |
22.33 |
8.50 |
- |
Positive control: 2-Anthramine |
1 µg in 10 µL |
254 |
259 |
237 |
250.00 |
11.53 |
11.19 |
Vehicle |
50 µL |
32 |
25 |
29 |
28.67 |
3.51 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
25 |
31 |
32 |
29.33 |
3.79 |
1.02 |
1500 µg |
25 |
35 |
32 |
30.67 |
5.13 |
1.07 |
|
500 µg |
37 |
13 |
27 |
25.67 |
12.06 |
0.90 |
|
150 µg |
24 |
33 |
34 |
30.33 |
5.51 |
1.06 |
|
50 µg |
20 |
35 |
27 |
27.33 |
7.51 |
0.95 |
|
Table 16. TA 100 - Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
54 |
53 |
51 |
52.67 |
1.53 |
- |
Positive control solvent |
20 µL |
57 |
52 |
50 |
53.00 |
3.61 |
- |
Positive control: Sodium azide |
20 µg in 20 µL |
1334 |
1134 |
1256 |
1241.33 |
100.80 |
23.42 |
Vehicle |
50 µL |
73 |
63 |
54 |
63.33 |
9.50 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
56 |
52 |
58 |
53.33 |
3.06 |
0.87 |
1500 µg |
57 |
53 |
60 |
56.67 |
3.51 |
0.89 |
|
500 µg |
55 |
57 |
52 |
54.67 |
2.52 |
0.86 |
|
150 µg |
58 |
57 |
52 |
55.67 |
3.21 |
0.88 |
|
50 µg |
55 |
53 |
48 |
52.00 |
3.61 |
0.82 |
|
Table 17. TA 100 - Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
71 |
72 |
91 |
78.00 |
11.27 |
- |
Positive control solvent |
20 µL |
72 |
73 |
72 |
72.33 |
0.58 |
- |
Positive control: 2-Anthramine |
2 µg in 20 µL |
392 |
373 |
395 |
386.67 |
11.93 |
5.35 |
Vehicle |
50 µL |
81 |
73 |
71 |
75.00 |
5.29 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
85 |
88 |
82 |
85.00 |
3.00 |
1.13 |
1500 µg |
74 |
79 |
90 |
81.00 |
8.19 |
1.08 |
|
500 µg |
78 |
77 |
76 |
77.00 |
1.00 |
1.03 |
|
150 µg |
71 |
74 |
93 |
79.33 |
11.93 |
1.06 |
|
50 µg |
79 |
72 |
71 |
74.00 |
4.36 |
0.99 |
|
Table 18. TA 100 - Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
54 |
67 |
60 |
60.33 |
6.51 |
- |
Positive control solvent |
20 µL |
60 |
51 |
60 |
57.00 |
5.20 |
- |
Positive control: Sodium azide |
20 µg in 20 µL |
1531 |
1505 |
1569 |
1535.00 |
32.19 |
26.93 |
Vehicle |
50 µL |
64 |
74 |
62 |
66.67 |
6.43 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
48 |
53 |
44 |
48.33 |
4.51 |
0.73 |
1500 µg |
47 |
52 |
47 |
48.67 |
2.89 |
0.73 |
|
500 µg |
48 |
52 |
56 |
52.00 |
4.00 |
0.78 |
|
150 µg |
49 |
47 |
46 |
47.33 |
1.53 |
0.71 |
|
50 µg |
57 |
52 |
46 |
51.67 |
5.51 |
0.78 |
|
Table 19. TA 100 - Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
120 |
95 |
88 |
101.00 |
16.82 |
- |
Positive control solvent |
20 µL |
76 |
78 |
75 |
76.33 |
1.53 |
- |
Positive control: 2-Anthramine |
1 µg in 10 µL |
356 |
390 |
370 |
372.00 |
17.09 |
4.87 |
Vehicle |
50 µL |
71 |
77 |
69 |
72.33 |
4.16 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
58 |
71 |
69 |
66.00 |
7.00 |
0.91 |
1500 µg |
61 |
73 |
75 |
69.67 |
7.57 |
0.96 |
|
500 µg |
76 |
72 |
77 |
75.00 |
2.65 |
1.04 |
|
150 µg |
73 |
64 |
78 |
71.67 |
7.09 |
0.99 |
|
50 µg |
74 |
68 |
75 |
72.33 |
3.79 |
1.00 |
|
Table 20. E. COLI - Assay nº1 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
107 |
109 |
105 |
107.00 |
2.00 |
- |
Positive control solvent |
10 µL |
131 |
109 |
99 |
113.00 |
16.37 |
- |
Positive control: cis-Platinum (II) |
1 µg in 10 µL |
310 |
324 |
341 |
325.00 |
15.52 |
2.88 |
Vehicle |
50 µL |
94 |
96 |
96 |
95.33 |
1.15 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
108 |
131 |
114 |
117.67 |
11.93 |
1.23 |
1500 µg |
101 |
117 |
128 |
115.33 |
13.58 |
1.21 |
|
500 µg |
136 |
125 |
119 |
126.67 |
8.62 |
1.33 |
|
150 µg |
102 |
113 |
120 |
111.67 |
9.07 |
1.17 |
|
50 µg |
137 |
104 |
131 |
124.00 |
17.58 |
1.30 |
|
Table 21. E. COLI - Assay nº1 – with metabolic activation (10% S9-mix) – without pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
182 |
158 |
171 |
170.33 |
12.01 |
- |
Positive control solvent |
5 µL |
152 |
124 |
140 |
138.67 |
14.05 |
- |
Positive control: Dimethylbenzanthracene |
5 µg in 5 µL |
424 |
503 |
436 |
454.33 |
42.57 |
3.28 |
Vehicle |
50 µL |
175 |
182 |
143 |
166.67 |
20.79 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA191216-S1) |
5000 µg |
171 |
145 |
155 |
157.00 |
13.11 |
0.94 |
1500 µg |
141 |
157 |
168 |
155.33 |
13.58 |
0.93 |
|
500 µg |
173 |
136 |
149 |
152.67 |
18.77 |
0.92 |
|
150 µg |
155 |
162 |
171 |
162.67 |
8.02 |
0.98 |
|
50 µg |
154 |
162 |
170 |
162.00 |
8.00 |
0.97 |
|
Table 22. E. COLI - Assay nº2 – without metabolic activation (-S9-mix)
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
137 |
144 |
122 |
134.33 |
11.24 |
- |
Positive control solvent |
10 µL |
125 |
156 |
128 |
136.33 |
17.10 |
- |
Positive control: cis-Platinum (II) |
1 µg in 10 µL |
345 |
332 |
328 |
335.00 |
8.89 |
2.46 |
Vehicle |
50 µL |
142 |
145 |
125 |
137.33 |
10.79 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
110 |
127 |
130 |
122.33 |
10.79 |
0.89 |
1500 µg |
118 |
138 |
111 |
122.33 |
14.01 |
0.89 |
|
500 µg |
132 |
151 |
147 |
143.33 |
10.02 |
1.04 |
|
150 µg |
130 |
125 |
111 |
122.00 |
9.85 |
0.89 |
|
50 µg |
136 |
123 |
134 |
131.00 |
7.00 |
0.95 |
|
Table 23. E. COLI - Assay nº2 – with metabolic activation (10% S9-mix) – with pre-incubation
Serie |
Dose/plate |
Plate |
Mean |
Standard deviation |
R |
||
nº1 |
nº2 |
nº3 |
|||||
Negative control |
100 µL |
215 |
198 |
186 |
199.67 |
14.57 |
- |
Positive control solvent |
5 µL |
211 |
192 |
204 |
202.33 |
9.61 |
- |
Positive control: Dimethylbenzanthracene |
2.5 µg in 5 µL |
447 |
438 |
506 |
463.67 |
36.94 |
2.29 |
Vehicle |
50 µL |
160 |
188 |
201 |
183.00 |
20.95 |
- |
Solution of N,N’-(ethoxymethylsilylene)bis[N-methylbenzamide] (CAS No. 16230-35-6) BATCH: 1000106346 (LEMI code: GGA020117-S1) |
5000 µg |
167 |
173 |
182 |
174.00 |
7.55 |
0.95 |
1500 µg |
159 |
168 |
185 |
170.67 |
13.20 |
0.93 |
|
500 µg |
171 |
182 |
188 |
180.33 |
8.62 |
0.99 |
|
150 µg |
181 |
186 |
193 |
186.67 |
6.03 |
1.02 |
|
50 µg |
178 |
188 |
180 |
182.00 |
5.29 |
0.99 |
|
Applicant's summary and conclusion
- Conclusions:
- The test item do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2.
- Executive summary:
The determination of the mutagenic potential of the test item has been assessed by the bacterial reverse mutation test, performed according to OECD 471 method and under GLP conditions. A preliminary study showed no toxicity up to 5000 μg/plate. A stock solution of the test item was prepared at 100 mg/mL and various concentrations of the test item (5000, 1500, 500, 150 and 50 μg/plate) were put in contact with the strains TA 1535, TA 1537, TA98, TA100 of Salmonela typhimurium and Escherichia coli WP2 (uvrA-) (pKM 101), with and without metabolic activation. Two independent assays were performed. For assay nº1, the different concetrations of the test item were put in contact with the strains in the absence and presence of a metabolic activation system S9-mix 10% (v/v) for 48 -72 h at 37ºC . For assay nº2, the different concentrations of the test item were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metablic activation system. For both assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item, with and without metabolic activation in the strains tested. The different doses prepared from solutions of test item, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA-) (pKM 101) with or without metabolic activation.
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