Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genotoxicity of the target substance was assessed in an in vitro bacterial reverse mutation assay under GLP (OECD 471). There was no evidence of induced mutant colonies over background and therefore 1-Benzyl-3-carbomoyl-pyridinium, chloride can be considered as not genotoxic.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-12 to 2016-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
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 mix
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as
the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in the pre-experiment as well as experiment I & II.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (A. dest.)
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
A. dest.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-NOPD (4-nitro-o-phenylene-diamine) without metabolic activation; 2-AA (2-aminoanthracene) with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, experiment I); pre-incubation (experiment II)

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Evaluation criteria:
Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
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 TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 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:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
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 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 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:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. The reduction in the number of revertants down to a mutation factor of smaller than 0.5 found in experiment II in tester strain TA 1535 at a concentration of 100 µg/plate (without metabolic activation), at a concentration of 100, 316 and 2500 !µg/plate (with metabolic activation) and in in tester strain TA 1537 at a concentration of 1000 µg/plate (without metabolic activation) and 31.6 µg/plate (with metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 1-Benzyl-3-carbamoyl-pyridinium, chloride at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.
Remarks on result:
other: Experiment I & II

Results Pre-Experiment

Substance

Dose [µg/plate]

TA 98

Mutation Factor [toxicity]*

TA 100

Mutation Factor [toxicity]*

without S9

with S9

without S9

with S9

Solvent Control (A. dest.)

 

1.0

1.0

1.0

1.0

4-NOPD

10.0

28.4

-

-

-

NaN3

10.0

-

-

11.0

-

2-AA

2.50

-

98.2

-

30.8

Test item

3.16

1.2

0.9

1.2

1.5

10.0

1.4

0.9

1.2

1.6

31.6

1.3

1.2

1.2

1.4

100

1.5

1.0

1.1

1.5

316

1.4

1.1

1.2

1.5

1000

1.8

1.2

1.5

1.4

2500

0.9

1.3

1.4

1.4

5000

1.3

1.2

1.2

1.4

*[toxicity parameter]: B =Background lawn reduced; N =No background lawn

Results Tables Experiment 1 (Plate-Incorporation Test) 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 98 - Experiment 1

A. dest.

 

19

13

24

19

5.5

18

25

30

24

6.0

1.0

1.0

Test item

31.6 µg

21

27

27

25

3.5

23

32

34

30

5.9

1.3

1.2

100 µg

28

25

33

29

4.0

23

22

29

25

3.8

1.5

1.0

316 µg

21

30

28

26

4.7

30

30

22

27

4.6

1.4

1.1

1000 µg

27

38

37

34

6.1

21

24

42

29

11.4

1.8

1.2

2500 µg

11

19

18

16

 

4.4

29

33

34

32

2.6

0.9

1.3

5000 µg

20

25

29

25

4.5

28

32

28

29

2.3

1.3

1.2

4-NOPD

10 µg

463

436

590

530

63.7

/

/

/

/

/

28.4

/

2-AA

2.5 µg

/

/

/

/

/

2347

2238

2582

2389

175.8

/

98.2

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 100 - Experiment 1

A. dest.

 

70

93

93

85

13.3

85

62

75

74

11.5

1.0

1.0

Test item

31.6 µg

91

106

105

101

8.4

106

107

105

106

1.0

1.2

1.4

100 µg

104

79

96

93

12.8

106

110

114

110

4.0

1.1

1.5

316 µg

96

104

109

103

6.6

102

128

99

110

15.9

1.2

1.5

1000 µg

127

119

135

127

8.0

108

101

102

104

3.8

1.5

1.4

2500 µg

115

126

121

121

5.5

105

93

102

100

6.2

1.4

1.4

5000 µg

118

99

86

101

16.1

95

101

107

101

6.0

1.2

1.4

NaN3

10 µg

885

1102

940

942

58.2

/

/

/

/

/

11.0

/

2-AA

2.5 µg

/

/

/

/

/

2155

2353

2337

2282

110.0

/

30.8

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 1535 - Experiment 1

A. dest.

 

15

18

19

17

2.1

18

14

16

16

2.0

1.0

1.0

Test item

31.6 µg

13

14

15

14

1.0

9

9

10

9

0.6

0.8

0.6

100 µg

20

16

18

18

2.0

16

13

18

16

2.5

1.0

1.0

316 µg

16

25

20

20

4.5

17

15

12

15

2.5

1.2

0.9

1000 µg

14

18

18

17

2.3

14

12

14

13

1.2

1.0

0.8

2500 µg

23

24

23

23

0.6

9

13

11

11

 

20

1.3

0.7

5000 µg

10

17

15

14

3.6

8

11

13

11

2.5

0.8

0.7

NaN3

10 µg

573

624

698

632

65.9

/

/

/

/

/

36.4

/

2-AA

2.5 µg

/

/

/

/

/

181

212

193

195

15.6

/

12.2

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 1537 - Experiment 1

A. dest.

 

18

15

14

16

2.1

11

10

10

10

0.6

1.0

1.0

Test item

31.6 µg

14

19

19

17

2.9

9

7

8

8

1.0

1.1

0.8

100 µg

12

15

14

14

1.5

12

8

8

9

2.3

0.9

0.9

316 µg

10

10

19

13

5.2

12

8

8

8

1.2

0.8

0.7

1000 µg

14

11

15

13

2.1

10

8

13

10

2.5

0.9

1.0

2500 µg

9

9

18

12

5.2

7

9

8

8

1.0

0.8

0.8

5000 µg

11

10

12

11

1.0

10

9

10

10

0.6

0.7

0.9

4-NOPD

40 µg

120

104

116

113

8.3

/

/

/

/

/

7.2

/

2-AA

2.5 µg

/

/

/

/

/

361

378

433

391

37.6

/

37.8

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 102 - Experiment 1

A. dest.

 

142

141

143

142

1.0

158

160

172

163

7.6

1.0

1.0

Test item

31.6 µg

145

153

149

149

4.0

181

216

164

187

26.5

1.0

1.1

100 µg

155

160

116

144

24.1

200

167

144

170

28.1

1.0

1.0

316 µg

145

155

142

147

6.8

149

188

182

173

21.0

1.0

1.1

1000 µg

215

176

220

204

24.1

153

191

206

183

27.3

1.4

1.1

2500 µg

143

145

141

143

2.0

189

169

170

176

11.3

1.0

1.1

5000 µg

171

157

187

172

15.0

178

152

173

168

13.8

1.2

1.0

MMS

1 µL

827

978

994

933

92.1

/

/

/

/

/

6.6

/

2-AA

10 µg

/

/

/

/

/

308

321

395

341

46.9

/

2.1

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

Results Tables Experiment 2 (Pre-Incubation Test):

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 98 -Experiment 2

A. dest.

 

21

25

18

21

3.5

30

31

24

28

3.8

1.0

1.0

Test item

31.6 µg

15

20

16

17

2.6

18

24

22

21

3.1

0.8

0.8

100 µg

15

21

15

17

3.5

23

14

20

19

4.6

0.8

0.7

316 µg

23

22

18

21

2.6

15

13

25

18

6.4

1.0

0.6

1000 µg

16

14

23

18

4.7

23

18

17

19

3.2

0.8

0.7

2500 µg

17

26

22

22

4.5

16

21

19

19

2.5

1.0

0.7

5000 µg

13

21

27

20

7.0

18

21

29

23

5.7

1.0

0.8

4-NOPD

10 µg

190

229

217

212

20.0

/

/

/

/

/

9.9

/

2-AA

2.5 µg

/

/

/

/

/

1090

1397

1470

1319

201.7

/

46.6

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 100 -Experiment 2

A. dest.

 

74

73

75

75

1.0

82

72

77

77

5.0

1.0

1.0

Test item

31.6 µg

71

69

63

68

4.2

89

81

87

86

4.2

0.9

1.1

100 µg

57

79

73

70

11.4

82

72

83

79

6.1

0.9

1.0

316 µg

73

71

80

75

4.7

84

82

79

82

2,5

1.0

1.1

1000 µg

72

59

74

68

8.1

78

75

76

76

1.5

0.9

1.0

2500 µg

79

68

65

71

7.4

73

75

86

78

7.0

1.0

1.0

5000 µg

78

69

66

71

6.2

72

76

74

74

2.0

1.0

1.0

NaN3

10 µg

424

402

376

401

24.0

/

/

/

/

/

5.4

/

2-AA

2.5 µg

/

/

/

/

/

812

789

1247

949

258.0

/

12.3

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 1535 -Experiment 2

A. dest.

 

20

21

17

19

2.1

18

18

21

19

1.7

1.0

1.0

Test item

31.6 µg

13

15

12

13

1.5

9

12

13

11

2.1

0.7

0.6

100 µg

7

12

11

10

2.6

8

10

7

8

 

1.5

0.5

0.4

316 µg

10

8

4

11

3.1

9

11

10

10

1.0

0.6

0.5

1000 µg

14

12

10

12

2.0

7

15

11

11

4.0

0.6

0.6

2500 µg

9

12

15

12

3.0

10

8

8

9

1.2

0.6

0.5

5000 µg

10

10

17

12

4.0

13

7

15

12

 

4.2

0.6

0.6

NaN3

10 µg

588

643

679

637

45.8

/

/

/

/

/

32.9

/

2-AA

2.5 µg

/

/

/

/

/

393

366

414

391

24.1

/

20.6

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

 

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 1537 -Experiment 2

A. dest.

 

18

7

10

12

5.7

10

12

15

12

2.5

1.0

1.0

Test item

31.6 µg

6

10

8

8

2.0

5

5

7

6

1.2

0.7

0.5

100 µg

6

10

6

7

2.3

10

12

10

11

1.2

0.6

0.9

316 µg

10

8

5

8

2.5

6

7

11

8

2.6

0.7

0.6

1000 µg

8

5

5

6

1.7

6

8

8

7

1.2

0.5

0.6

2500 µg

5

8

11

8

3.0

12

7

12

10

2.9

0.7

0.8

5000 µg

6

10

6

7

2.3

6

6

11

8

2.9

0.6

0.6

4-NOPD

40 µg

49

57

44

50

6.6

/

/

/

/

/

4.3

/

2-AA

2.5 µg

/

/

/

/

/

118

120

113

117

3.6

/

9.5

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

Substance

Dose plate

REVERTANT COLONIES PER PLATE

Mutation Factor

without activation (-S9)

without activation (-S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

Tester Strain: TA 102 -Experiment 2

A. dest.

 

166

206

159

177

25.4

296

233

299

276

37.3

1.0

1.0

Test item

31.6 µg

174

189

181

18

7.5

280

247

240

256

21.4

1.0

0.9

100 µg

186

195

146

176

26.1

259

240

246

248

9.7

1.0

0.9

316 µg

174

204

155

177

24.8

219

282

266

256

32.7

1.0

0.9

1000 µg

162

176

180

173

9.5

254

290

234

259

28.4

1.0

0.9

2500 µg

191

178

201

190

11.5

239

240

251

243

6.7

1.1

0.9

5000 µg

168

182

170

173

7.6

249

219

270

246

25.6

1.0

0.9

MMS

1 µL

1198

1665

2125

1663

463.5

/

/

/

/

/

9.4

/

2-AA

10 µg

/

/

/

/

/

862

814

925

867

55.7

/

3.1

SD: Standard-deviation                    P:Precipitation
B: Background lawn reduced           C:Contamination
N: No background lawn
Mutation factor=mean revertants (test item) / mean revertants (vehicle control)

 

 

Conclusions:
1-Benzyl-3-carbamoyl-pyridinium, chloride is not genotoxic in the bacterial reverse gene mutation assay (OECD 471) in the presence and absence of mammalian metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA 102 were exposed to 1-Benzyl-3-carbamoyl-pyridinium, chloride (~96% purity) at concentrations of 31.6, 100, 316, 1000, 2.500 and 5.000 µg/plate in the presence and absence of mammalian metabolic activation.

The test item was tested up to the limit concentration of 5.000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation assay).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The mutagenic potential of 1-Benzyl-3-carbamoyl-pyridinium, chloride was investigated in a study conducted in accordance with OECD Guideline 471. Two independent experiments (experiment I: plate incorporation test; experiment II: pre-incubation test) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation (S9). Test substance concentrations were 0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Neither precipitation nor toxic effects were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II. Biologically relevant increases in revertant colony numbers of any of the five tester strains were not observed following treatment with 1-Benzyl-3-carbamoyl-pyridinium, chloride at any concentration level (with and without meatbolic activation). Thus, it can be concluded that 1-Benzyl-3-carbamoyl-pyridinium, chloride can be considered non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Based on available testing data (OECD 471), the substance 1-Benzyl-3-carbamoyl-pyridinium, chloride does not have to be classified for mutagenicity.