Registration Dossier
Registration Dossier
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EC number: 202-124-2 | CAS number: 92-09-1
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:
- 11 - 28 Sep 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Adopted: 21st July 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- N-[2-[(4-amino-m-tolyl)ethylamino]ethyl]methanesulphonamide
- EC Number:
- 202-124-2
- EC Name:
- N-[2-[(4-amino-m-tolyl)ethylamino]ethyl]methanesulphonamide
- Cas Number:
- 92-09-1
- Molecular formula:
- C12H21N3O2S
- IUPAC Name:
- N-{2-[(4-amino-3-methylphenyl)(ethyl)amino]ethyl}methanesulfonamide
1
Method
- Target gene:
- his operon for S. typhimurium strains
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:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared at Eurofins Munich from the livers of rats treated with phenobarbital and β-naphthoflavone.
- Test concentrations with justification for top dose:
- Pre-experiment (TA98 and TA100):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment 1 (plate incorporation test):
3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate (top dose selceted based on toxicity observed in the pre-experiment)
Experiment 2 (pre-incubation test):
1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate (top dose selceted based on toxicity observed in the pre-experiment) - Vehicle / solvent:
- - Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene (2-AA), 4-nitro-o-phenylene-diamine (4-NOPD)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate-incorporation test, pre-incubation test
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS:
- triplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: observation of bacterial growth inhibition or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control. - Rationale for test conditions:
- Test concentration were based on toxicity observed in the pre-experiment.
- Evaluation criteria:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system. - Statistics:
- Mean numbers of the revertant colonies of each concentration of each test system was calculated and compared to that of the solvent control.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Plate-incorporation test: Background lawn reduced at 1000 and 2500 µg/plate (- S9), 2500 µg/plate (+ S9). Pre-incubation test: Background lawn reduced at 316, 1000 and 2500 µg/plate (- S9), 1000 and 2500 µg/plate (+ S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Plate-incorporation test: Background lawn reduced at 1000 and 2500 µg/plate (- S9), 2500 µg/plate (+ S9). Pre-incubation test: Background lawn reduced at 316, 1000 and 2500 µg/plate (- S9), 1000 and 2500 µg/plate (+ S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Plate-incorporation test: Background lawn reduced at 1000 and 2500 µg/plate (- S9), 1000 (reduced to mean: 4) and 2500 µg/plate (+ S9). Pre-incubation test: Background lawn reduced at 316, 1000 and 2500 µg/plate (- S9), 1000 and 2500 µg/plate (+ S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Plate-incorporation test: Background lawn reduced at 1000 and 2500 µg/plate (- S9), 2500 µg/plate (+ S9). Pre-incubation test: Background lawn reduced concentrations of 316, 1000 and 2500 µg/plate (- S9), 1000 and 2500 µg/plate (+ S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Plate-incorporation test: Background lawn reduced at 1000 and 2500 µg/plate (- S9), 2500 µg/plate (+ S9). Pre-incubation test: Background lawn reduced at 316 µg (reduced to mean: 88) 1000 and 2500 µg/plate (- S9), 1000 and 2500 µg/plate (+ S9).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
- Other observations when applicable: Toxic effects of the test item were noted in all tester strains evaluated in experiment 1 and 2.
In experiment 1 toxic effects of the test item were observed in tester strains TA98, TA100, TA1537 and TA102 at concentrations of 1000 µg/plate and higher (without metabolic activation) and at a concentration of 2500 µg/plate (with metabolic activation). In tester strain TA1535 toxic effects of the test item werenoted at concentrations of 1000 µg/plate and higher (with and without metabolic activation).
In experiment 2 toxic effects of the test item were noted in all tester strains used at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation).
Any other information on results incl. tables
Table 1: Historical Laboratory Control Data of the Negative Control (in 2015 - 2017) without S9 (-S9)
|
|
TA98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
Mean |
25.5 |
94.1 |
16.7 |
10.5 |
283.3 |
|
SD |
6.8 |
16.6 |
6.6 |
5.5 |
53.5 |
|
Min |
11 |
49 |
4 |
3 |
142 |
|
Max |
58 |
155 |
41 |
35 |
472 |
|
RSD [%] |
26.5 |
17.6 |
39.4 |
51.9 |
18.9 |
|
n |
|
1071 |
1258 |
1040 |
1035 |
819 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertants/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 2: Historical Laboratory Control Data of the Positive Control (in 2015 - 2017) without S9 (-S9)
|
|
TA98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
Substance |
|
4-NOPD |
NaN3 |
NaN3 |
4-NOPD |
MMS |
Mean |
440.0 |
650.2 |
875.3 |
107.3 |
1610.3 |
|
SD |
162.5 |
219.1 |
289.3 |
37.3 |
546.3 |
|
Min |
89 |
146 |
56 |
36 |
412 |
|
Max |
1895 |
2493 |
1854 |
570 |
3437 |
|
RSD [%] |
36.9 |
33.7 |
33.1 |
34.7 |
33.9 |
|
n |
|
1077 |
1264 |
1049 |
1039 |
824 |
S9: metabolic activation
Conc.: concentration
Mean: mean of revertants/plate
Min.: minimum of revertants/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 3: Historical Laboratory Control Data of the Negative Control (in 2015 - 2017) with S9 (+S9)
|
|
TA98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
Mean |
29.3 |
95.5 |
13.3 |
10.7 |
345.7 |
|
SD |
7.0 |
14.6 |
5.4 |
5.2 |
69.3 |
|
Min |
15 |
60 |
3 |
3 |
157 |
|
Max |
59 |
155 |
38 |
36 |
586 |
|
RSD [%] |
23.8 |
15.3 |
40.4 |
48.1 |
20.0 |
|
n |
|
1064 |
1252 |
1033 |
1028 |
812 |
S9: metabolic activation
Mean: mean of revertants/plate
Min.: minimum of revertants/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 4: Historical Laboratory Control Data of the Positive Control (in 2015 - 2017) with S9 (+S9)
|
|
TA98 |
TA100 |
TA1535 |
TA1537 |
TA102 |
Substance |
|
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
Mean |
1615.9 |
1533.5 |
167.7 |
221.6 |
841.2 |
|
SD |
687.6 |
563.7 |
158.2 |
110.5 |
215.0 |
|
Min |
70 |
169 |
23 |
23 |
310 |
|
Max |
3287 |
3092 |
1954 |
888 |
3588 |
|
RSD [%] |
42.5 |
36.8 |
94.3 |
49.8 |
25.6 |
|
n |
|
1067 |
1254 |
1039 |
1031 |
815 |
S9: metabolic activation
Conc.: concentration
Mean: mean of revertants/plate
Min.: minimum of revertants/plate
Max.: maximum of revertants/plate
SD: Standard Deviation
RSD: Relative Standard Deviation
n: Number of control values
Table 5: Results of the pre-Experiment
Substance |
Dose (µg/plate) |
TA 98 Mutation factor (toxicity*) |
TA 100 Mutation factor (toxicity*) |
||
Metabolic activation |
|
without S9 |
with S9 |
without S9 |
with S9 |
Solvent Control (DMSO) |
|
1.0 |
1.0 |
1.0 |
1.0 |
4-NOPD |
10.0 |
12.5 |
- |
- |
- |
NaN3 |
10.0 |
- |
- |
5.6 |
- |
2-AA |
2.50 |
- |
13.0 |
- |
6.4 |
TestItem |
3.16 |
0.8 |
1.0 |
1.0 |
0.9 |
10.0 |
0.8 |
1.0 |
1.0 |
1.0 |
|
31.6 |
0.9 |
1.1 |
1.1 |
1.0 |
|
100 |
0.8 |
1.0 |
1.2 |
1.1 |
|
316 |
0.9 |
1.0 |
1.2 |
1.1 |
|
1000 |
0.0B |
1.0 |
0.0B |
1.1 |
|
2500 |
0.0B |
0.0 B |
0.0B |
0.0B |
|
5000 |
0.0B |
0.0 B |
0.0B |
0.0B |
|
* [toxicity parameter]: B = Background lawn reduced; N = No background lawn
Table 6: Results of experiment 1 (Plate-incorporation test)
Substance |
Dose (µg/plate) |
TA98 |
TA100 |
TA 1535 |
TA 1537 |
TA 102 |
|||||||||||||||
without S9 |
with S9 |
without S9 |
with S9 |
without S9 |
with S9 |
without S9 |
with S9 |
without S9 |
with S9 |
||||||||||||
|
|
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Aqua dest. |
|
24 |
4.9 |
21 |
3.2 |
82 |
4.4 |
77 |
5.5 |
9 |
1.5 |
9 |
0.6 |
11 |
3.1 |
10 |
1.2 |
237 |
6.0 |
298 |
9.3 |
DMSO |
|
26 |
3.5 |
24 |
2.1 |
73 |
2.5 |
75 |
9.8 |
8 |
2.1 |
10 |
2.0 |
10 |
1.2 |
12 |
2.0 |
247 |
9.5 |
300 |
8.6 |
TestItem |
3.16 |
22 |
6.1 |
24 |
4.2 |
71 |
9.5 |
64 |
4.6 |
9 |
1.2 |
9 |
1.2 |
11 |
2.3 |
10 |
3.0 |
258 |
4.7 |
303 |
10.8 |
10.0 |
20 |
5.6 |
23 |
3.8 |
70 |
10.1 |
74 |
4.2 |
10 |
1.5 |
9 |
3.6 |
12 |
0.6 |
9 |
2.1 |
255 |
6.2 |
300 |
11.9 |
|
31.6 |
24 |
3.2 |
27 |
1.0 |
79 |
3.2 |
74 |
8.9 |
8 |
1.5 |
9 |
1.7 |
11 |
3.0 |
12 |
0.6 |
259 |
7.0 |
302 |
10.5 |
|
100 |
22 |
6.7 |
24 |
7.8 |
87 |
4.0 |
82 |
5.6 |
10 |
0.6 |
9 |
2.1 |
12 |
2.6 |
12 |
1.5 |
261 |
7.0 |
310 |
15.7 |
|
316 |
24 |
2.9 |
24 |
4.7 |
87 |
9.1 |
82 |
3.1 |
10 |
2.3 |
10 |
2.5 |
10 |
2.0 |
10 |
1.0 |
261 |
10.2 |
308 |
11.7 |
|
1000 |
0B |
0B |
23 |
4.2 |
0 B |
0 |
84 |
9.0 |
0B |
0 |
4 |
2.3 |
0B |
0 |
8 |
2.6 |
0B |
0 |
291 |
39.3 |
|
2500 |
0B |
0B |
0B |
0 |
0 B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
|
4-NOPD |
10 |
320 |
70.9 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
4-NOPD |
40 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
179 |
59.0 |
/ |
/ |
/ |
/ |
/ |
/ |
NaN3 |
10.0 |
/ |
/ |
/ |
/ |
407 |
19.0 |
/ |
/ |
194 |
20.0 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
MMS |
1 µL |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
1146 |
176.9 |
/ |
/ |
2-AA |
2.50 |
/ |
/ |
307 |
65.0 |
/ |
/ |
477 |
72.9 |
/ |
/ |
207 |
14.2 |
/ |
/ |
193 |
25.9 |
/ |
/ |
/ |
/ |
2-AA |
10 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
923 |
83.0 |
SD: Standard-deviation P: Precipitation
B: Background lawn
reduced C: Contamination
N: No background lawn
Table 7: Results of experiment 2 (Pre-incubation test)
Substance |
Dose (µg/plate) |
TA98 |
TA100 |
TA 1535 |
TA 1537 |
TA 102 |
|||||||||||||||
without S9 |
with S9 |
without S9 |
with S9 |
without S9 |
with S9 |
without S9 |
with S9 |
without S9 |
with S9 |
||||||||||||
|
|
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Aqua dest. |
|
20 |
2.5 |
21 |
0.6 |
79 |
1.5 |
78 |
5.1 |
8 |
2.1 |
9 |
1.7 |
17 |
3.5 |
22 |
2.6 |
242 |
6.8 |
304 |
2.1 |
DMSO |
|
21 |
2.1 |
21 |
2.6 |
74 |
1.5 |
73 |
2.9 |
8 |
0.6 |
9 |
0.5 |
10 |
3.5 |
19 |
3.1 |
243 |
4.6 |
301 |
8.2 |
TestItem |
1.00 |
20 |
5.2 |
18 |
1.0 |
64 |
8.2 |
71 |
1.5 |
9 |
1.5 |
8 |
1.5 |
12 |
1.5 |
18 |
3.1 |
237 |
2.1 |
303 |
2.9 |
3.16 |
23 |
2.9 |
24 |
1.2 |
73 |
2.5 |
78 |
7.0 |
8 |
0.6 |
8 |
0.6 |
13 |
1.5 |
15 |
4.0 |
242 |
5.7 |
302 |
4.2 |
|
10.0 |
19 |
6.5 |
19 |
5.1 |
78 |
8.5 |
71 |
11.5 |
8 |
2.6 |
8 |
2.0 |
10 |
2.9 |
11 |
3.1 |
238 |
13.8 |
302 |
10.8 |
|
31.6 |
22 |
1.5 |
22 |
5.3 |
72 |
9.1 |
76 |
5.6 |
8 |
1.2 |
8 |
1.7 |
6 |
1.5 |
12 |
1.0 |
234 |
5.9 |
300 |
6.7 |
|
100 |
20 |
4.6 |
23 |
2.1 |
61 |
4.7 |
77 |
3.8 |
7 |
1.5 |
9 |
1.5 |
7 |
2.3 |
13 |
5.6 |
210 |
33.2 |
305 |
9.3 |
|
316 |
13 |
3.0 |
23 |
2.6 |
0B |
0 |
84 |
2.3 |
0B |
0 |
9 |
1.0 |
2 |
2.5 |
14 |
4.0 |
88 |
2.1 |
309 |
5.9 |
|
1000 |
0B |
0 |
2 |
0.6 |
0B |
0 |
3 |
5.8 |
0B |
0 |
1 |
1.7 |
0B |
0 |
2 |
1.5 |
0B |
0 |
207B |
51.4 |
|
2500 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
0B |
0 |
6B |
10.4 |
|
4-NOPD |
10 |
314 |
114.2 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
4-NOPD |
40 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
155 |
49.3 |
/ |
/ |
/ |
/ |
/ |
/ |
NaN3 |
10.0 |
/ |
/ |
/ |
/ |
465 |
42.9 |
/ |
/ |
267 |
26.4 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
MMS |
1 µL |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
1116 |
174.2 |
/ |
/ |
2-AA |
2.50 |
/ |
/ |
357 |
74.3 |
/ |
/ |
478 |
50.2 |
/ |
/ |
170 |
33.9 |
/ |
/ |
200 |
11.6 |
/ |
/ |
/ |
/ |
2-AA |
10 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
1042 |
8.7 |
SD: Standard-deviation P: Precipitation
B: Background lawn
reduced C: Contamination
N: No background lawn
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of the conducted test the substance was not mutagenic in any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and TA 102) tested with and without metabolic activation test up to toxic concentrations of the test item.
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