Isonicotinaldehyde

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

Study period:

17 February - 28 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isonicotinaldehyde

EC Number:

212-832-3

EC Name:

Isonicotinaldehyde

Cas Number:

872-85-5

Molecular formula:

C6H5NO

IUPAC Name:

pyridine-4-carbaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report) : Pyridine-4-aldehyde
- Physical state : clear yellowish liquid
- Analytical purity :98,7% (GC)
- Lot/batch No. : 1120126/001
- Stability under test conditions : not analysed
- Storage condition of test material : Refrigerator at ca. 4°C , in the dark , under nitrogen

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

see at "any other information on materials and methods incl. tables"

Test concentrations with justification for top dose:

The concentrations for the first experiment were set according to a preliminary toxicity test .
In the preliminary experiment the test substance prevented the growth of the bacterial background lawn at concentrations of 556 µg/plate and higher . It was therefore decided to use 900 µg/plate as the highest concentration which would be toxic and to use 6 instead of the required 5 concentrations . Each of the other 5 concentrations was 1/3 of the preceding one .
In the first experiment clear toxicity was only seen in the highest concentrations of strains TA98 and TA1535 . According to these results a concentration of 2700 µg/plate was added in the second experiment .
The test substance was tested without as well as with an external metabolising system (S9-mix) . The results were verified by a second , independent experiment . Triplicate repetitions were run for each dose group in each of the two separate experiments that were conducted , for the control groups six-fold repetitions were run .
see also at "any other information on materials and methods incl. tables"

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (for test substance and for the negative control group)

Controls

Untreated negative controls:

yes

Remarks:

The solvent , DMSO was used for the negative control group

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

2-nitrofluorene

sodium azide

other: 4-Nitro-o-phenylene-diamine ; t-Butyl-hydroperoxide ; 2-Aminoanthracene ; 1,8-Dihydro-anthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION : in agar (plate incorporation)

CONDITIONS OF CULTIVATION : One day before the Ames test was performed , a small amount from each of the frozen bacterial cultures (stored in small portions in a solution of 6% DMSO in phosphate buffered saline in liquid nitrogen) was transferred to nutrient broth . The liquid cultures were incubated overnight at 37°C and then used for the exposure .

EXPOSURE TECHNIQUE : The exposure was performed according to the ´Plate Incorporation Assay´, in which bacteria test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state . The number of viable cells in the overnight-cultures is in the range of 2 x 10E8 cells per ml .
For each sample the following solutions were combined :
- 0,1 ml of the overnight culture of the bacteria
- 0,5 ml of S9-mix (or phosphate buffered saline for samples without metabolic activation)
- 0,1 ml of the appropriate test- or reference substance solution and
- 2 ml of top agar
The combined solutions were mixed and spread over a plate with minimal agar (9cm diameter) . After the top agar had solidified , the plates were incubated at 37°C until the colonies were visible (2 days) .

COUNTING OF COLONIES :
The plates with less than about 50 revertant colonies , i.e. the plates of TA98 and TA1535 with the exception of the positive controls , were counted visually by marking the colonies with a felt tipped pen . The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program .

Evaluation criteria:

The criteria for a positive result are :
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations :
- For the strains with a low spontaneous revertant rate , i.e. TA98 and TA1535 : The 2 1/2 fold of the amount of the spontaneous revertants .
- For the strains with a high spontaneous revertant rate , i.e. TA97a, TA100 and TA102 : The 1 2/3 fold of the amount of the spontaneous revertants .

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Microcolonies instead of a bacterial background lawn was observed at concentration of 2700 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Microcolonies instead of a bacterial background lawn were observed at 900 µg/plate with and without metabolisation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Microcolonies instead of a bacterial background lawn was observed at concentration of 2700 µg/plate

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

Remarks:

Microcolonies instead of a bacterial background lawn was observed at concentration of 2700 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Microcolonies instead of a bacterial background lawn were observed at concentrations of 2700 µg/plate with and without metabolisation and at 900 µg/plate without metabolisation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation : No precipitation of the test substance was seen in any concentration groups .

RANGE-FINDING/SCREENING STUDIES : The concentrations for the first experiment were set according to a preliminary toxicity test .
In the preliminary experiment the test substance prevented the growth of the bacterial background lawn at concentrations of 556 µg/plate and higher . It was therefore decided to use 900 µg/plate as the highest concentration which would be toxic and to use 6 instead of the required 5 concentrations . Each of the other 5 concentrations was 1/3 of the preceding one .

COMPARISON WITH HISTORICAL CONTROL DATA : see at "any other information on results incl. tables"

Any other information on results incl. tables

COMPARISON WITH HISTORICAL CONTROL DATA :

Genetic properties : (The tests were performed in September 2000 . The strains were stored in liquid nitrogen since that time)

strain:	TA97a	TA98	TA100	TA102	TA1535
Sensitive against crystal violet	yes	yes	yes	yes	yes
Sensitive against ampicillin	no	no	no	no	yes
Sensitive against ampicillin/tetracycline	--	yes	--	no	--
Sensitive against UV	yes	yes	yes	no	yes

-- : not tested

Mutation frequency : (historic data of mean revertants per petri dish , standard SD and number of tests N)

	without metabolisation				with metabolisation (S9 -mix)
strain :	substance, concentration (µg/plate)	revertants/plate			substance, concentration (µg/plate)	revertants/plate
		mean	SD	N		mean	SD	N
TA97a	Control	80	14	92	Control	111	17	92
	4-NoPD, 10 µg	488	113	92	DMBA, 10 µg	388	157	92
TA98	Control	8,4	2,0	92	Control	11,6	2,5	92
	2-Nitro-fluorene, 2 µg	197	54	92	2-Aminoanthracene, 1 µg	481	171	92
TA100	Control	68,7	18,5	92	Control	79,9	15,2	92
	Na-azide, 2 µg	494	131	92	2-Aminoanthracene, 2 µg	1624	425	92
TA102	Control	175	31	92	Control	238	33	92
	tBHPO, 50 µg	542	154	92	DHA, 50 µg	692	189	92
TA1535	Control	10,3	5,7	92	Control	11,0	4,6	92
	Na-azide, 1 µg	250	88	92	2-Aminoanthracene, 2 µg	157	79	92

DHA = 1,8-Dihydroxy-anthraquinone

4-NoPD = 4-Nitro-o-phenylene-diamine

tBHPO = tert.-Butyl-hydroperoxide

DMBA = 7,12-Dimethylbenz[a]anthracene

Table 1 : Mean number of revertants per plate for strain TA97a (Mean, standard deviation SD and number of plates N)

Without metabolisation

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	177,7	22,5	3	900	57,0	7,5	3
300	171,7	5,5	3	300	67,7	11,9	3
100	166,0	23,6	3	100	87,0	20,5	3
33	182,3	27,3	3	33	83,0	1,7	3
11	177,3	4,6	3	11	100,0	27,5	3
3,7	162,0	6,9	3	3,7	71,7	13,3	3
solvent	178,8	15,5	6	solvent	74,7	15,9	6
positive	406,3	30,9	3	positive	334,7	63,4	3

Metabolisation with S9 -mix

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	178,3	4,2	3	900	90,0	4,6	3
300	159,0	6,1	3	300	109,0	1,7	3
100	183,3	13,2	3	100	106,0	39,9	3
33	164,7	19,7	3	33	114,3	15,5	3
11	189,0	19,9	3	11	105,3	6,7	3
3,7	170,3	32,3	3	3,7	106,3	17,0	3
solvent	192,0	10,1	6	solvent	101,5	20,9	6
positive	421,7	7,4	3	positive	255,3	19,6	3

solvent : DMSO

positive : without metabolisation : 4 -Nitro-o-phenylene-diamine (10 µg/plate) ; with metabolisation : 7,12-Dimethylbenz[a]anthracene (10 µg/plate)

toxic : only microcolonies instead of a bacterial background lawn

Table 2 :Mean number of revertants per plate for strain TA98(Mean, standard deviation SD and number of plates N)

Without metabolisation

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	toxic	-	3	900	toxic	-	3
300	6,0	2,6	3	300	4,0	1,7	3
100	11,3	0,6	3	100	8,0	3,6	3
33	9,0	3,0	3	33	6,3	1,5	3
11	7,7	1,5	3	11	8,7	1,2	3
3,7	6,0	3,6	3	3,7	8,7	3,1	3
solvent	8,7	1,0	6	solvent	8,5	3,4	6
positive	156,7	29,4	3	positive	251,3	31,2	3

Metabolisation with S9 -mix

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	toxic	-	3	900	toxic	-	3
300	11,7	1,5	3	300	12,7	5,5	3
100	11,7	2,9	3	100	11,0	1,7	3
33	10,0	2,6	3	33	12,0	5,6	3
11	14,3	2,5	3	11	12,3	4,7	3
3,7	11,3	4,2	3	3,7	11,7	4,5	3
solvent	13,7	2,5	6	solvent	11,3	2,7	6
positive	353,3	11,6	3	positive	330,7	36,5	3

solvent : DMSO

positive : without metabolisation : 2-Nitrofluorene (2 µg/plate) ; with metabolisation : 2-Amino-anthracene (1 µg/plate)

toxic : only microcolonies instead of a bacterial background lawn

Table 3 :Mean number of revertants per plate for strain TA100(Mean, standard deviation SD and number of plates N)

Without metabolisation

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	116,7	11,5	3	900	29,0	7,9	3
300	88,7	3,2	3	300	48,3	9,1	3
100	63,7	7,6	3	100	69,0	16,5	3
33	66,0	13,9	3	33	78,3	25,1	3
11	73,3	11,5	3	11	69,7	4,9	3
3,7	75,7	7,6	3	3,7	85,0	14,0	3
solvent	70,3	10,9	6	solvent	66,3	10,0	6
positive	647,7	105,3	3	positive	408,0	21,7	3

Metabolisation with S9 -mix

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	105,0	8,5	3	900	72,0	3,5	3
300	102,0	12,0	3	300	87,3	8,5	3
100	71,3	2,1	3	100	92,7	13,3	3
33	89,0	16,1	3	33	79,7	14,2	3
11	87,3	5,5	3	11	90,3	12,7	3
3,7	86,0	13,0	3	3,7	92,0	10,4	3
solvent	87,8	8,4	6	solvent	90,3	12,6	6
positive	1786,3	67,4	3	positive	1572,0	31,4	3

solvent : DMSO

positive : without metabolisation : Sodium azide (2 µg/plate) ; with metabolisation : 2-Amino-anthracene (2 µg/plate)

toxic : only microcolonies instead of a bacterial background lawn

Table 4 :Mean number of revertants per plate for strain TA102(Mean, standard deviation SD and number of plates N)

Without metabolisation

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	91,0	20,8	3	900	58,7	18,1	3
300	139,3	8,5	3	300	111,0	12,1	3
100	147,3	11,2	3	100	129,0	17,5	3
33	172,0	9,6	3	33	146,0	20,8	3
11	163,0	13,7	3	11	161,0	19,3	3
3,7	183,3	4,9	3	3,7	133,7	31,2	3
solvent	167,8	22,3	6	solvent	146,7	23,3	6
positive	783,7	104,7	3	positive	340,3	75,1	3

Metabolisation with S9 -mix

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	159,3	30,9	3	900	114,0	6,2	3
300	188,7	18,7	3	300	190,3	26,2	3
100	243,7	18,7	3	100	196,7	25,4	3
33	258,3	35,2	3	33	211,7	14,6	3
11	250,7	10,3	3	11	231,7	30,9	3
3,7	264,0	36,8	3	3,7	200,7	27,2	3
solvent	253,5	21,7	6	solvent	234,4	18,5	6
positive	821,3	21,1	3	positive	753,7	41,9	3

solvent : DMSO

positive : without metabolisation : t-Butyl-hydroperoxide (50 µg/plate) ; with metabolisation : 1,8-Dihydroxy-anthraquinone (50 µg/plate)

toxic : only microcolonies instead of a bacterial background lawn

Table 5:Mean number of revertants per plate for strain TA1535(Mean, standard deviation SD and number of plates N)

Without metabolisation

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	toxic	-	3	900	toxic	-	3
300	7,3	2,1	3	300	7,7	3,2	3
100	9,3	4,7	3	100	15,3	6,4	3
33	9,7	3,2	3	33	13,3	3,2	3
11	7,0	1,0	3	11	8,7	1,5	3
3,7	9,7	5,8	3	3,7	10,0	2,0	3
solvent	10,5	2,3	6	solvent	8,3	2,6	6
positive	307,3	31,7	3	positive	265,3	33,6	3

Metabolisation with S9 -mix

first experiment				second experiment
conc. µg/plate	revertants/plate			conc. µg/plate	revertants/plate
	mean	SD	N		mean	SD	N
-	-	-	-	2700	toxic	-	3
900	7,0	0,0	3	900	7,0	2,6	3
300	9,7	2,3	3	300	10,3	5,8	3
100	7,0	4,4	3	100	6,0	1,0	3
33	9,7	2,5	3	33	8,7	2,5	3
11	7,0	1,0	3	11	9,0	1,0	3
3,7	8,0	1,0	3	3,7	8,3	4,0	3
solvent	8,7	2,6	6	solvent	7,3	3,8	6
positive	114,0	17,7	3	positive	106,3	9,1	3

solvent : DMSO

positive : without metabolisation : Sodium azide (1 µg/plate) ; with metabolisation : 2-Amino-anthracene (2 µg/plate)

toxic : only microcolonies instead of a bacterial background lawn

Applicant's summary and conclusion

Conclusions:

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

The study was performed according to the OECD Guideline 471 without deviations and according to the principles of the good laboratory practice and therefore considered to be of the highest quality (reliability Klimisch 1). All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system. According to the results obtained in this study, "PYRIDINE-4-ALDEHYDE" is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to the limit of toxicity.

Executive summary:

Pyridine-4-aldehyde was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14. The test substance, dissolved in DMSO, was tested at concentrations ranging from 3.7 µg to 2700 µg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9 -mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system. Microcolonies instead of a bacterial background lawn were observed in all strains at concentrations of 2700 µg/plate, at 900 µg/plate in strain TA98 with and without metabolisation and in strain TA1535 without metabolisation. No precipitation of the test substance was seen in any of the concentration groups. In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

According to the results obtained in this study, Pyridine-4 -aldehyde is non-mutagenic in the Ames testwith the strains TA97a, TA98, TA100, TA102 and TA1535 up to the limit of toxicity.