1-(carboxylatomethyl)methylpyridinium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results obtained from an OECD 471 compliant study, it is concluded that the test item is non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test conditions chosen.

 

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:

2017-12-20 to 2018-03-15

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

induced rat liver S9 mix

Test concentrations with justification for top dose:

Both trials (plate incorporation and pre-incubation) were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: recommended by guideline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene: 4 µg/plate, all strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 10^8 viable cells per plate

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 hrs +/- 30 min (plate incorporation);

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: background lawn

Rationale for test conditions:

According to guideline and based on preliminary dose-range finding study.

Evaluation criteria:

Evaluation:
A substance is considered positive if the following criteria apply:
There should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.

The mutation test is considered acceptable as it met the following criteria:
- All tester strains confirmed to their genetic characteristics.
- The positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.

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:

not examined

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:

not examined

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:

not examined

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:

not examined

Positive controls validity:

valid

Key result

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: well soluble
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes

Table 1 SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I: Plate Incorporation Method

 

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium						Salmonella typhimurium
			TA 98	TA 100	TA 102	TA 1535	TA 1537		TA 98	TA 100	TA 102	TA 1535	TA 1537
Vehicle Control	100 µL of Distilled water	Mean	25.3	102.3	255.3	20.3	8.3		23.0	110.0	256.7	20.7	8.7
		±SD	2.5	6.1	7.8	3.5	2.5		3.0	10.6	5.9	2.1	2.3
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Test item	0.05	Mean	19.7	97.0	257.3	18.7	7.3		21.7	98.3	253.3	19.7	6.3
		±SD	3.5	8.9	8.5	2.5	1.5		2.5	6.5	5.7	0.6	3.2
	Fold Increase		0.8	0.9	1.0	0.9	0.9		0.9	0.9	1.0	1.0	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	23.0	97.0	252.7	20.7	9.3		19.0	96.3	254.7	15.7	9.3
		±SD	3.6	6.0	5.0	1.5	2.3		3.6	5.1	4.0	1.5	2.1
	Fold Increase		0.9	0.9	1.0	1.0	1.1		0.8	0.9	1.0	0.8	1.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	20.7	106.0	254.0	20.0	7.7		18.0	97.7	258.7	17.3	6.3
		±SD	5.0	6.1	7.5	2.6	1.5		2.6	5.7	5.5	2.5	2.1
	Fold Increase		0.8	1.0	1.0	1.0	0.9		0.8	0.9	1.0	0.8	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.6	Mean	22.7	106.0	256.0	20.0	9.0		20.3	99.0	255.0	19.7	8.3
		±SD	4.2	7.8	2.0	3.6	3.6		3.1	7.5	4.6	4.0	2.5
	Fold Increase		0.9	1.0	1.0	1.0	1.1		0.9	0.9	1.0	1.0	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	5	Mean	21.3	107.3	253.3	19.7	7.7		19.7	104.7	257.3	18.3	8.3
		±SD	2.5	4.2	6.1	1.5	3.1		2.5	11.6	7.5	2.5	3.2
	Fold Increase		0.8	1.0	1.0	1.0	0.9		0.9	1.0	1.0	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Positive Control	100 µL of respective Positive Control	Mean	375.7	406.0	602.3	146.0	119.3		354.3	392.0	597.0	142.0	115.7
		±SD	14.3	6.6	10.1	8.9	3.5		14.5	9.5	7.0	7.5	6.0
	Fold Increase		14.8	4.0	2.4	7.2	14.3		15.4	3.6	2.3	6.9	13.3
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

 

 

Table 2SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-II: Preincubation Method

 

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium						Salmonella typhimurium
			TA 98	TA 100	TA 102	TA 1535	TA 1537		TA 98	TA 100	TA 102	TA 1535	TA 1537
Vehicle Control	100 µL of Distilled water	Mean	27.0	106.0	269.0	22.7	9.3		22.0	98.0	269.3	19.0	7.3
		±SD	4.4	5.6	12.5	1.5	3.5		4.6	8.7	11.5	3.6	2.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
1-(carboxylatomethyl)methylpyridinium	0.05	Mean	25.3	98.3	260.3	19.3	9.3		20.3	102.3	253.0	19.0	7.3
		±SD	3.1	5.7	9.7	3.8	3.8		3.1	8.5	7.0	3.0	2.1
	Fold Increase		0.9	0.9	1.0	0.9	1.0		0.9	1.0	0.9	1.0	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	22.7	105.3	263.0	20.7	10.7		20.7	104.0	269.7	17.0	6.0
		±SD	3.8	8.6	3.6	2.5	1.5		1.5	3.6	11.7	2.0	1.7
	Fold Increase		0.8	1.0	1.0	0.9	1.1		0.9	1.1	1.0	0.9	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	23.0	97.7	262.0	20.7	9.7		20.0	97.0	259.7	19.0	6.0
		±SD	1.7	4.2	9.8	4.5	1.5		3.6	5.6	5.9	3.0	2.0
	Fold Increase		0.9	0.9	1.0	0.9	1.0		0.9	1.0	1.0	1.0	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.6	Mean	25.7	98.0	266.3	18.7	7.0		20.7	95.3	255.0	16.3	5.7
		±SD	2.5	8.5	11.8	4.5	2.0		1.5	6.8	8.2	0.6	0.6
	Fold Increase		1.0	0.9	1.0	0.8	0.8		0.9	1.0	0.9	0.9	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	5	Mean	23.3	101.7	262.7	18.0	6.0		21.3	99.0	269.7	16.0	5.7
		±SD	2.5	4.0	10.7	2.0	2.0		3.1	4.4	4.5	2.6	1.5
	Fold Increase		0.9	1.0	1.0	0.8	0.6		1.0	1.0	1.0	0.8	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Positive Control	100 µL of respective Positive Control	Mean	373.3	396.0	605.0	144.3	118.3		348.0	395.7	592.0	139.3	118.3
		±SD	7.5	8.2	7.5	12.1	6.7		6.2	7.5	15.9	9.3	5.0
	Fold Increase		13.8	3.7	2.2	6.4	12.7		15.8	4.0	2.2	7.3	16.1
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

 

 

Positive controls:

With S9:     

For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

Without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA102: 0.5 µg/plate of Mitomycin C

For TA1537: 50 µg/plate of 9-Aminoacridine

 

Conclusions:

Based on the results obtained from this study, it is concluded that the test item is non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 mg/plate under the test conditions chosen.

Executive summary:

The test item was evaluated for mutagenicity in a Bacterial Reverse Mutation Test according to OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test concentrations used in the mutation assay were selected based on the results obtained in the pre-tests. The mutagenic potential of the test item was investigated in two independent experiments, in plate incorporation test (trial 1) and pre-incubation test (trial 2). Each assay was conducted with and without metabolic activation (± S9 mix). Both trials were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate. All concentrations, including the controls, were tested in triplicate. Vehicle control (distilled water) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials without metabolic activation and 2-Aminoanthracene for trials with metabolic activation) were tested simultaneously.

The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA 102, TA1535 and TA1537.

In the performed trials all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both trials in the presence and absence of metabolic activation. There was no substantial increase in number of revertant colonies at any of the tested concentrations in both trials. In the performed experiments an inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

The number of revertant colonies in the positive controls resulted in 2.2 to 16.1 fold increase under identical conditions.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

The test item was evaluated for mutagenicity in a Bacterial Reverse Mutation Test according to OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test concentrations used in the mutation assay were selected based on the results obtained in the pre-tests. The mutagenic potential of the test item was investigated in two independent experiments, in plate incorporation test (trial 1) and pre-incubation test (trial 2). Each assay was conducted with and without metabolic activation (± S9 mix). Both trials were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and5 mg/plate. All concentrations, including the controls, were tested in triplicate. Vehicle control (distilled water) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials without metabolic activation and 2-Aminoanthracene for trials with metabolic activation) were tested simultaneously.

The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA 102, TA1535 and TA1537.

In the performed trials all of the validity criteria regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled.Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both trials in the presence and absence of metabolic activation. There was no substantial increase in number of revertant colonies at any of the tested concentrations in both trials.In the performed experiments an inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

The number of revertant colonies in the positive controls resulted in 2.2 to 16.1 fold increase under identical conditions.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item is not classified as genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.