1-benzyl-3-carbamoylpyridinium chloride

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:

2016-07-12 to 2016-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

1-Benzyl-3-carbamoyl-pyridinium, chloride

Cas Number:

5096-13-9

Molecular formula:

C13H13ClN2O

IUPAC Name:

1-Benzyl-3-carbamoyl-pyridinium, chloride

Test material form:

solid

Method

Target gene:

histidine

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 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.

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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)

 

 

Applicant's summary and conclusion

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).