Methyl 3-pyridyl ketone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: - Gene mutation (Bacterial reverse mutation assay / Ames test), Salmonella typhimurium LT 2 (TA 97a, TA 98, TA 100, TA 102, TA 1535): negative with and without metabolic activation [OECD 471, EU Method B.13/14, GLP]

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: SOP 118 008 03 edition 11, valid from 01. Oct. 2014 "Bestimmung des erbgutverändernden Potentials mit dem Bacterial-Reverse-Mutation-Test (Ames-Test)"

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The strains used are mutants derived from Salmonella typhimurium LT2, mutations of the strains are listed below:
hisD6610 (category: frame shift, effect: histidine deficiency) present in strain TA97a
hisD3052 (category: frame shift, effect: histidine deficiency) present in strain TA98
hisG46 (category: base pair substitution, effect: histidine deficiency) present in strains TA100 and TA1535
hisG428 (category: base pair substitution, effect: histidine deficiency) present in strain TA102
uvrB (category: deletion, effect: UV sensitivity, biotine deficiency) present in strains TA97a, TA98, TA100 and TA1535
rfa (category: deletion, effect: lipopolysaccharide side chain deficiency) present in strains TA97a, TA98, TA100, TA 102 and TA1535
pKM101 (category: plasmide, effect: ampicillin resistance) present in strains TA97a, TA98, TA100 and TA 102
pAQ1 (category: plasmide, effect: tetracyclin resistance) present in strain TA 102

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

First experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Second experiment: 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
In a non-GLP pre-test the solubility of the test item was determined: The test item is sufficiently soluble in DMSO (no precipitates were visible). A solution containing 50 ± 5 g/L will be prepared for the tests. In a non-GLP pre-test, possible turbidity of the resulting test item solutions in combination with phosphate buffer was tested, too. The highest test item concentration (5 mg/plate) was not turbid.
On the base of these results the preparation of the test item was the same for both experiments. On the day of the start of the respective experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared. DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants. The stock solution was used to prepare the geometric series of the concentrations to be tested. Each solution was membrane filtrated to accomplish sterility.

Untreated negative controls:

other: solvent control (DMSO)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylenediamine (TA97a, TA98 & TA102); sodium azide (TA100 & TA1535); 2-amino-anthracene (TA97a, TA100, TA102 & TA1535); benzo-a-pyrene (TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: Only in the 2nd experiment the materials were gently vortexed in a test tube and incubated at 37 °C for 20 minutes (in the 1st experiment they were directly poured onto the selective agar plates).
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: Per strain and dose, four plates with and four plates without S9 mix were used; 2 experiments were performed.

DETERMINATION OF CYTOTOXICITY
- Method: Assessment for numbers of revertant colonies and examination for effects on the growth of the bacterial background lawn.

OTHER:
- Origin and Culture
Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem and were stored as lyophilisates in the fridge at 2-8 °C. The lyophilisates were used to prepare permanent cultures which were filled into vials and stored at < -75°C. One day before the start of each experiment, one vial per strain to be used was taken from the deep freezer. The surface was scraped with an inoculation loop and the aliquot was put into a culture vessel containing nutrient broth. After incubation over night at 37°C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: see vehicle control

Positive controls validity:

valid

Additional information on results:

The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was not detected. The bacterial background lawn was visible and the number of revertants was not significantly decreased. Therefore it can be stated, that under the test conditions, the test item 3-Acetylpyridine is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.
The confirmation tests of the genotype didn't show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (see Attachment 1) and were definitely increased in comparison with the negative controls, as well as showing mutagenic potential of the diagnostic mutagenes.
Spontaneous revertants were not all within the normal range in comparison with the historical data of the laboratory. The values, which lay outside of the historical data do not affect the validity of the study and the deviations are marginal. For these reasons, the result of the test is considered valid.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

First Experiment

- Confirmation of the Criteria and Validity: The treatments for the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see Attachment 1). All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

- Solubility and Toxicity: The test item was dissolved in DMSO. A stock solution containing 50 g/L was prepared. In this experiment, the test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.

- Mutagenicity: No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the test conditions.

The mean revertant values of the four replicates are presented in the following table.

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	125	112	16	15	103	105	345	347	13	14
	sd	6.0	6.0	1.5	1.5	3.5	12.5	14.0	56.0	1.0	1.5
DMSO	Mean	117	114	14	14	103	105	316	340	12	14
	sd	1.5	1.5	1.5	4.0	6.1	12.8	42.3	73.0	1.5	0.6
Positive Controls*	Mean	465	393	484	102	531	508	959	1212	108	108
	sd	24.4	34.9	34.9	10.1	79.4	50.0	135.4	92.3	1.5	6.5
	f(I)	3.97	3.45	34.57	7.29	5.16	4.84	3.03	3.56	8.31	7.71
5000 µg/pl.	Mean	125	137	11	12	103	102	347	349	14	12
	sd	11.5	6.7	1.5	3.2	6.1	9.3	18.0	12.9	4.0	1.5
	f(I)	1.00	1.22	0.69	0.80	1.00	0.97	1.01	1.01	1.08	0.86
1500 µg/pl.	Mean	120	137	13	15	92	101	327	339	12	14
	sd	4.9	5.6	2.1	2.6	7.1	15.1	2.3	60.1	1.0	3.8
	f(I)	0.96	1.22	0.81	1.00	0.89	0.96	0.95	0.98	0.92	1.00
500 µg/pl.	Mean	122	124	17	12	93	95	293	317	11	9
	sd	1.5	3.2	0.6	2.3	2.0	1.5	71.8	26.6	2.5	1.2
	f(I)	0.98	1.11	1.06	0.80	0.90	0.90	0.85	0.91	0.85	0.64
150 µg/pl.	Mean	125	131	15	14	94	107	337	344	14	12
	sd	18.6	10.6	2.1	1.5	2.1	9.5	14.0	25.0	1.2	2.9
	f(I)	1.00	1.17	0.94	0.93	0.91	1.02	0.98	0.99	1.08	0.86
50 µg/pl.	Mean	132	155	18	14	94	97	323	333	11	12
	sd	8.5	9.7	0.6	3.2	1.2	1.5	40.9	10.1	1.0	2.0
	f(I)	1.06	1.38	1.13	0.93	0.91	0.92	0.94	0.96	0.85	0.86

f(I) = increase factor

* Different positive controls were used

To verify this result, a second experiment was performed using the pre-incubation method.

Second Experiment

- Confirmation of the Criteria and Validity: The treatments for the sterility control and the determination of the titre didn't show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see Attachment 1). All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

- Solubility and Toxicity: The test item was dissolved in DMSO. A stock solution containing 50 g/L was prepared. In this experiment, the test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.

- Mutagenicity: No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the test conditions.

The mean revertant values of the four replicates are presented in the following table.

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	129	124	14	13	135	133	365	370	14	10
	sd	8.5	16.9	1.5	0.0	6.1	13.1	17.0	23.6	4.0	0.6
DMSO	Mean	109	109	12	10	132	139	325	397	14	14
	sd	7.8	6.5	2.6	0.0	5.9	8.1	54.3	19.7	3.1	3.2
Positive Controls*	Mean	499	456	109	99	555	717	1076	1071	165	134
	sd	44.1	73.0	1.2	10.1	36.1	184.0	133.1	63.5	39.3	6.7
	f(I)	4.58	4.18	9.08	9.90	4.11	5.16	3.31	2.70	11.79	9.57
5000 µg/pl.	Mean	111	117	10	12	104	101	196	202	9	12
	sd	2.3	4.5	1.7	2.5	5.5	10.7	14.0	44.1	1.0	4.0
	f(I)	1.02	1.07	0.83	1.20	0.79	0.73	0.60	0.51	0.64	0.86
2500 µg/pl.	Mean	123	116	9	12	107	118	135	211	9	10
	sd	13.6	7.0	3.2	4.4	7.5	23.6	1.2	76.4	3.2	2.5
	f(I)	1.13	1.06	0.75	1.20	0.81	0.85	0.42	0.53	0.64	0.71
1250 µg/pl.	Mean	110	120	15	13	102	101	206	261	15	12
	sd	6.4	2.0	2.3	2.6	8.7	8.1	50.0	33.8	4.5	3.2
	f(I)	1.01	1.10	1.25	1.30	0.77	0.73	0.63	0.66	1.07	0.86
625 µg/pl.	Mean	119	126	11	10	112	122	267	253	10	11
	sd	6.4	2.1	3.5	1.5	10.4	24.2	27.7	41.6	0.6	2.0
	f(I)	1.09	1.16	0.92	1.00	0.85	0.88	0.82	0.64	0.71	0.79
313 µg/pl.	Mean	117	122	7	11	105	75	265	246	8	11
	sd	4.2	2.0	1.5	0.6	4.6	6.2	14.0	52.9	1.5	1.5
	f(I)	1.07	1.12	0.58	1.10	0.80	0.54	0.82	0.62	0.57	0.79

f(I) = increase factor

* Different positive controls were used

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD TG471 and EU Method B.13/14 (GLP) without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. In both experiments no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was therefore considered to be non-mutagenic under the conditions of this test.

Executive summary:

Two valid experiments were performed according to the OECD 471 resp. EU Method B.13/14 (GLP):

First Experiment

Five concentrations of the test item, dissolved in DMSO (ranging from 50 to 5000 µg/plate) were used. Five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 hours, using the plate incorporation method. None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item did not show any mutagenic effects in the first experiment. The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment

To verify the results of the first experiment, a second experiment was performed, using five concentrations of the test item (ranging from 313 to 5000 µg/plate) and a modification in study performance (pre-incubation method). The test item did not show mutagenic effects in the second experiment, either. The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined and the test item 3 -Acetylpyridine is considered as "not mutagenic under the conditions of the test".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There is one guideline study (OECD 471 and EU Method B.13/14) available on in vitro genetic toxicity available, assessed with Klimisch 1 and hence considered as sufficiently reliable to cover this endpoint:

The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was not detected. The bacterial background lawn was visible and the number of revertants was not significantly decreased. Therefore it can be stated, that under the test conditions, the test item 3-Acetylpyridine is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

The confirmation tests of the genotype didn't show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were definitely increased in comparison with the negative controls, as well as showing mutagenic potential of the diagnostic mutagenes.

Spontaneous revertants were not all within the normal range in comparison with the historical data of the laboratory. The values, which lay outside of the historical data do not affect the validity of the study and the deviations are marginal. For these reasons, the result of the test is considered valid.

With regard to the data requirements as set out in Annex VII of REACH, the available GLP guideline study, which provides consistent results, is sufficient, no data gaps were identified and there are also no indications that the negative results are not suitable for human risk assessment. Hence, no further studies need to be conducted.

Justification for classification or non-classification

The test material does not meet the criteria for classification and will not require labelling as a mutagen in accordance with European Regulation (EC) No. 1272/2008.