Reaction mass of 2,6-dimethylpyridinium chloride and 3-methylpyridinium chloride and 4-methylpyridinium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, 4 instead of 5 strains tested (old OECD protocol)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Pyridine, alkyl derivs., hydrochlorides

EC Number:

271-753-2

EC Name:

Pyridine, alkyl derivs., hydrochlorides

Cas Number:

68607-19-2

IUPAC Name:

68607-19-2

Test material form:

other: liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: TA 98: hisD3052, rfa, uvrB, pKM101; TA 100: hisG46, rfa, uvrB, pKM101; TA 1535: hisG46, rfa, uvrB; TA 1537: hisC3076, rfa, uvrB

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat)

Test concentrations with justification for top dose:

50 - 5000 µg/plate

Vehicle / solvent:

Water: test item and positive control sodium azide
DMSO: positive control 2-nitrofluorene and 9-aminoacridine

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

water

Controls 2

Positive controls:

yes

Remarks:

2.5 µg/plate

Positive control substance:

2-nitrofluorene

Remarks:

TA98 with and without metabolic activation

Controls 3

Positive controls:

yes

Remarks:

2.5 µg/plate

Positive control substance:

sodium azide

Remarks:

TA100 with and without metabolic activation

Controls 4

Positive controls:

yes

Remarks:

2.5 µg/plate

Positive control substance:

sodium azide

Remarks:

TA1535 with and without metabolic activation

Controls 5

Positive controls:

yes

Remarks:

25 µg/plate

Positive control substance:

9-aminoacridine

Remarks:

TA1537 with and without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st main experiment: in agar (plate incorporation); 2nd main experiment: preincubation

DURATION
- 1st main experiment: 72 hours incubation (37°C)
- 2nd main experiment: 30 min preincubation (30°C), 72 hours incubation (37°C)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; determination of background lawn

Evaluation criteria:

The following criteria must be met for the mutagenicity assay to be considered valid:
- In the solvent control, each tester strain culture must exhibit a characteristic mean number of spontaneaus revertants.
- To ensure that appropriate numbers of bacteria are plated, overnight culture titers must be in excess of 10^8 bacteria/ml
- The mean of each positive control must exhibit a significant increase in the number of revertants over the mean value of the respective vehicle control
- Normally, at least four non-toxic dose Ievels are required to evaluate assay data, deviations from this requirement must be justified.

For a test compound to be considered positive, it must (in two independent experiments) cause at least a doubling in the mean revertants per plate of at least
one tester strain. This increase must be accompanied by a dose response to increasing concentrations of the test article. A test article that does not meet these
criteria will be called non-motagenie in bacteria. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independent tests are considered non-relevant. lf however these increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A reduced growth of the bacterial background lawn, indicative of test compound induced cytotoxicity was detectable only with TA 1537 in the preincubation test -S9 mix (at the highest test compound concentration of 5000 µg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Plate lncorporation test, with metabolic activation

Dose	Tester strains
	TA 98		TA 100		TA 1535		TA 1537
	R/P	MF	R/P	MF	R/P	MF	R/P	MF
Negative control (water)	37± 6	1.0	165±19	1.0	12±2	1.0	17±4	1.0
Positiv control  2-Aminoanthracene 2.5 µg/plate	1570 ± 283	42.4	1729±506	10.5	202±12	16.4	341±24	19.7
50 µg/plate	49±11	1.3	153± 8	0.9	11±4	0.9	16±3	0.9
160 µg/plate	50±5	1.4	181±14	1.1	11±3	0.9	21±1	1.2
500 µg/plate	51±16	1.4	158±26	1.0	10±1	0.8	16±2	0.9
1600 µg/plate	41± 3	1.1	153± 6	0.9	14±3	1.2	19±4	1.1
5000 µg/plate	41± 5	1.1	163± 6	1.0	10±2	0.8	14±5	0.8

Plate lncorporation test, without metabolic activation

Dose	Tester strains
	TA 98		TA 100		TA 1535		TA 1537
	R/P	MF	R/P	MF	R/P	MF	R/P	MF
Negative control (water)	40 ± 8	1.0	159±27	1.0	11±2	1.0	9±3	1.0
50 µg/plate	167±19	1.0	167±19	1.0	9±2	0.9	8±2	0.9
160 µg/plate	159±19	1.0	159±19	1.0	10±3	1.0	6±1	0.7
500 µg/plate	155±13	1.0	155±13	1.0	13±6	1.2	7±2	0.8
1600 µg/plate	158±18	1.0	158±18	1.0	16±2	1.5	7±3	0.8
5000 µg/plate	148±13	0.9	148±13	0.9	11±5	1.1	7±3	0.8
Positive control	146 ± 10  (2-Nitrofluorene)  2.5µg/plate	0.8	520± 2 Sodiumazide  2.5µg/plate	3.3	478±40 Sodiumazide  2.5µg/plate	44.8	50±12 9-Aminoacridine 25µg/plate	5.8

R/P: number of revertants/plate (mean values)

MF: mutation factor

Preincubation test, with metabolic activation

Dose	Tester strains
	TA 98		TA 100		TA 1535		TA 1537
	R/P	MF	R/P	MF	R/P	MF	R/P	MF
Negative control (water)	39± 4	1.0	139±13	1.0	11±6	1.0	13±4	1.0
50 µg/plate	45±14	1.2	149±21	1.1	9±4	0.9	12±1	0.9
160 µg/plate	40±1	1.0	118±12	0.8	14±2	1.3	14±5	1.1
500 µg/plate	38±6	1.0	123±13	0.9	11±2	1.0	14±0	1.1
1600 µg/plate	33± 9	0.8	143±14	1.0	10±3	0.9	15±6	1.2
5000 µg/plate	40±4	1.20	135±19	1.0	11±2	1.0	12±3	0.9
Positive control  (2-Aminoanthracene)  2.5µg/plate	1081±328	27.7	1453±59	10.4	169±16	15.8	188±33	14.4

Preincubation test,

without metabolic activation

Dose	Tester strains
	TA 98		TA 100		TA 1535		TA 1537
	R/P	MF	R/P	MF	R/P	MF	R/P	MF
Negative control (water)	30±8	1.0	140±13	1.0	7±2	1.0	9±4	1.0
50 µg/plate	34±8	1.1	168±13	1.2	6±2	1.2	10±1	0.9
160 µg/plate	34±3	1.1	146± 7	1.0	8±3	1.2	11±3	0.7
500 µg/plate	31±2	1.0	137±17	1.0	6±2	1.5	13±4	0.8
1600 µg/plate	30±9	1.0	140± 6	1.0	6±2	0.6	5±1	0.8
5000 µg/plate	28±7	0.9	172±11	1.2	6±1	0.3	3±1	0.8
Positive control	193±26 (2-Nitrofluorene)  2.5µg/plate	6.4	388±31 Sodiumazide  2.5µg/plate	2.8	287±47 Sodiumazide  2.5µg/plate	43.1	51±9 9-Aminoacridine 25µg/plate	5.8

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the experimental conditions of this test the test item did not induce a mutagenic effect in S. typhimurium.
lt is therefore not considered to be a bacterial mutagen.