4-methylpyridine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacteria reverse mutation: Weight of evidence: A couple of experimental studies with the substance are available for in vitro genetic toxicity following a method similar to guideline OECD 471. The substance was tested with S. typhimurium strains TA 97, TA 98, TA 100 and TA 1535 with and without S9 mix in one study and with  S. typhimurium strains TA 1538, TA 1535, TA 1537, TA 98 and TA 100 with S9 mix in the other. All results were negative. Thus, the substance can be considered as not mutagenic in all strains tested with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable with restrictions; basic data given, comparable to guidelines/standards.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

precedes development of guideline

Deviations:

yes

Remarks:

Not tested in the absence of metabolic activation

Principles of method if other than guideline:

Plate incorporation assay

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

standard test strains

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with

Metabolic activation system:

Liver fraction (S9) from Aroclor-induced rats.

Test concentrations with justification for top dose:

0.01, 0.05, 0.10, 0.50 and 1.0 mg/plate

Vehicle / solvent:

Dimethylsulfoxide was used as the solvent and negative control.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

for strain TA 100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

For strain TA 98

Details on test system and experimental conditions:

The plate incorporation assay was employed. The bacterial cultures (approximately 2 x 108 cells/plate) were treated with the test substance in the presence of S9 mix, , in two ml of molten top agar and poured over previously prepared Vogel-Bonner minimal agar medium. Plates were incubated at 37 degrees C for 48 hours before counting the reverted colonies. General toxicity was determined over a 1000 fold concentration range, up to a maximum of 5 mg/plate. The experiments were repeated independently at least two times in the nontoxic and effect dose range using three plates per dose.

Evaluation criteria:

No specific data, other than critical slope values of the linear dose-response curves.

Statistics:

The specific mutagenic activities were determined from the slope values of linear dose-response curves and were given as revertants per mg of the test substance.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Although all the tester strains were used, TA98 was the most sensitive strain and was the strain for which most data were reported in this article.

Conclusions:

Mutagenic activity was not observed with this test substance in the presence of S9 mix.

Executive summary:

The genetic toxicity in vitro of the test item has been tested following a method similar to guideline OECD 471. It has been tested in S. typhimurium strains: TA 1538, TA 1535, TA 1537, TA 98 and TA 100. It has been used DMSO as vehicle and for positive controls 2-acetylaminofluorene and benzo(a)pyrene. 

Mutagenic activity was not observed with this test substance in the presence of S9 mix.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Remarks:

no data on the test report

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurim strains: Strain TA100, strain TA1535, strain TA97, Strain TA98

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

Extracts of rat or hamster liver enzymes (S9 mix).

Test concentrations with justification for top dose:

Initial test concentrations: 100, 333, 1000, 3333 and 10000 µg/plate.

Details on test system and experimental conditions:

PRINCIPLE OF ASSAY
Each of the bacterial strains has a mutation that prevents it from making an amino acid (a protein building block) required for normal cell growth. The S. typhimurium strains are unable to manufacture histidine; the E. coli strain cannot manufacture tryptophan. The tester strains will not be able to grow without amino acid supplementation, unless the mutated gene is changed back to the correct DNA sequence (reversion mutation). Being exposed to a mutagenic chemical increases the chance that the mutant gene will be restored to the correct sequence, which allows the bacteria to grow and form colonies. Each substance under study is tested in a variety of bacterial strains with different types of altered DNA sequences in order to provide a comprehensive assessment of the mutagenic potential of the substance.

PREPARING CULTURES
Multiple sets of cultures are prepared using a range of doses and different amounts of liver enzymes for each bacterial strain. Each culture is prepared in a test tube containing a suspension of one bacterial strain and either an S9 mix or plain buffer solution. Then the test substance is added. Negative control cultures are established using the same ingredients, but without the test substance. Positive control cultures are established using known mutagens active in particular bacterial strains and metabolic conditions (for example, with or without S9). Once prepared, each culture is incubated for 20 minutes at 37° C. Agar is then added to the cultures, and the contents of each tube are poured onto the surface of a Petri dish that was prepared with standard bacterial culture medium. The plates are then incubated, usually for two days.

Evaluation criteria:

To validate the test, a trained investigator compares the number of colonies on the chemical-treated plates to the number of colonies on the negative control plates. If the substance under test is mutagenic, the chemical-treated plates will have a much greater number of colonies than the negative control plates.

- A positive response is a reproducible, dose-related increase in mutant colonies in any single strain, with or without the addition of S9 metabolic enzymes. While there is no minimum percentage of increase required for a result to be considered positive, a twofold increase in mutant colonies in a treated plate is usually considered to be a positive (mutagenic) response.

- An equivocal response is any increase that is not reproducible, not dose-related, or not high enough in magnitude to be considered positive.

- A negative response occurs when no increases in mutant colonies are seen in the cultures treated with the test chemical, compared with the control.

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Strain TA100
Dose (µg/Plate)	Without S9	Without S9	With 10% Rat S9	With 30% Rat S9	With 10% Hamster S9	With 30% Hamster S9
Vehicle control	109 +/- 7.5		142 +/- 7.2	119 +/- 17.6	110 +/- 3.5	95 +/- 4.3
100	100 +/- 3.3	121 +/- 14.5	132 +/- 6.8	142 +/- 2.3	130 +/- 6.5	111 +/- 2.0
333	98 +/- 5.8	128 +/- 13.0	125 +/- 11.0	125 +/- 8.5	114 +/- 8.4	101 +/- 5.2
1000	106 +/- 1.5	131 +/- 2.0	142 +/- 0.7	116 +/- 3.2	124 +/- 11.7	102 +/- 4.7
3333	117 +/- 0.9	146 +/- 9.5	137 +/- 4.7	101 +/- 10.4	121 +/- 6.1	96 +/- 6.1
10000	Toxic	106 +/- 3.5	101 +/- 1.3	80+/- 3.4	90 +/- 6.4	83 102 +/- 4.7 8.7
Trial Summary	Negative	Negative	Negative	Negative	Negative	Negative
Positive control	818 +/- 29.6 (4)	947 +/- 19.1 (4)	480 +/- 34.5 (3)	427 +/- 9.8 (5)	617 +/- 12.9 (1)	554 102 +/- 22.4 (3)

 

Strain TA1535
Dose (µg/Plate)	Without S9	Without S9	With 10% Rat S9	With 30% Rat S9	With 10% Hamster S9	With 30% Hamster S9
Vehicle control	9 +/- 1.5	12 +/- 3.0	14 +/- 1.9	10 +/- 1.5	7 +/- 0.6	7 +/- 0.3
100	11 +/- 1.3	13 +/- 1.0	12 +/- 1.5	15 +/- 1.5	5 +/- 1.0	10 +/- 1.9
333	10 +/- 2.1	11 +/- 1.8	8 +/- 1.8	10 +/- 1.3	9 +/- 2.8	10 +/- 1.2
1000	13 +/- 2.3	11 +/- 3.5	10 +/- 1.2	11 +/- 1.8	5 +/- 1.9	9 +/- 1.9
3333	12 +/- 2.7	13 +/- 1.5	10 +/- 1.7	12 +/- 2.0	13 +/- 3.5	9 +/- 0.6
10000	3 +/- 1.8	8 +/- 0.7	6 +/- 0.6	10 +/- 2.0	4 +/- 1.8	5 +/- 0.9
Trial Summary	Negative	Negative	Negative	Negative	Negative	Negative
Positive control	818 +/- 29.6 (4)	947 +/- 19.1 (4)	58 +/- 2.0 (5)	82 +/- 7.3 (6)	82 +/- 7.3 (3)	147 +/- 123 (5)

 

Strain TA97
Dose (µg/Plate)	Without S9	Without S9	With 10% Rat S9	With 30% Rat S9	With 10% Hamster S9	With 30% Hamster S9
Vehicle control	157 +/- 5.0	117 +/- 3.4	147 +/- 4.3	191 +/- 7.3	116 +/- 9.9	183 +/- 2.0
100	152 +/- 5.4	121 +/- 4.5	145 +/- 5.7	190 +/- 2.7	120 +/- 7.9	167 +/- 2.5
333	124 +/- 11.0	129 +/- 2.6	159 +/- 9.3	214 +/- 3.0	130 +/- 16.4	145 +/- 14.2
1000	161 +/- 10.3	120 +/- 14.0	187 +/- 7.5	210 +/- 7.3	146 +/- 2.6	166 +/- 6.7
3333	162 +/- 1.5	130+/- 3.5	181 +/- 7.8	188 +/- 10.0	143 +/- 7.0	155 +/- 7.0
10000	70 +/- 7.0	104 +/- 14.3	138 +/- 8.0	140+/- 9.2	123 +/- 9.8	122 +/- 3.7
Trial Summary	Negative	Negative	Negative	Negative	Negative	Negative
Positive control	479  +/- 12.5 (8)	479 +/- 12.5 (8)	450 +/- 15.7 (3)	560 +/- 3.5 (5)	577 +/- 32.9 (7)	605 +/- 1.5 (3)

 

Strain TA98
Dose (µg/Plate)	Without S9	Without S9	With 10% Rat S9	With 30% Rat S9	With 10% Hamster S9	With 30% Hamster S9
Vehicle control	17 +/- 2.5	18 +/- 1.2	26 +/- 1.9	16 +/- 3.8	22 +/- 3.5	18 +/- 3.2
100	16 +/- 1.5	15 +/- 0.6	21 +/- 2.6	20 +/- 2.3	25 +/- 2.1	18 +/- 3.5
333	17 +/- 2.6	19 +/- 4.0	24 +/- 4.7	17 +/- 2.0	15 +/- 5.8	19 +/- 4.3
1000	16 +/- 2.9	21 +/- 4.6	21 +/- 3.3	12 +/- 1.2	27 +/- 5.5	19 +/- 2.5
3333	10 +/- 1.5	23 +/- 3.3	25 +/- 3.6	11 +/- 1.5	26 +/- 1.9	16 +/- 4.4
10000	Toxic	11 +/- 2.0	16 +/- 1.9	11 +/- 2.0	21 +/- 3.7	9 +/- 1.0
Trial Summary	Negative	Negative	Negative	Negative	Negative	Negative
Positive control	600 +/- 50.9 (9)	472 +/- 41.4 (9)	338 +/- 11.2 (3)	215 +/- 11.2 (5)	215 +/- 10.1 (7)	242 +/- 18.7 (3)

1: Vehicle control: water 

2: 1.0 µg/plate Sodium Azide 

3: 2.0 µg/plate 2-Aminoanthracene 

4: 5.0 µg/plate sodium azide 

5: 5.0 µg/plate 2-Aminoanthracene 

6: 10.0 µg/plate 2-Aminoanthracene  

7: 1.0 µg/plate 2-Aminoanthracene  

8: 50.0 µg/plate 9-Aminoacridine 

9:2.5 µg/plate 4-Nitro-O-Phenylenediamine 

Conclusions:

Mutagenic activity was not observed with this test substance in the presence or absence of S9 mix.

Executive summary:

The genetic toxicity in vitro of the test item has been tested following a method similar to guideline OECD 471. It has been tested in S. typhimurium strains: Ta 97, TA 98, TA 100 and TA 1535. Mutagenic activity was not observed with this test substance with and without metabolic activity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.