Registration Dossier

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:
2018
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

1
Chemical structure
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

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
Conc./plate

 

4-NOPD
10 µg

NaN3
10 µg

NaN3
10 µg

4-NOPD
40 µg

MMS
1 µL

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
Conc./plate

 

2-AA
2.5 µg

2-AA
2.5 µg

2-AA
2.5 µg

2-AA
2.5 µg

2-AA
10 µg

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.