1-methylpiperidine

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 1983)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

(adopted 1983)

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 1997)

Deviations:

yes

Remarks:

2-Aminoanthracene was the sole positive control in the presence of S9-Mix.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-methylpiperidine

EC Number:

210-959-9

EC Name:

1-methylpiperidine

Cas Number:

626-67-5

Molecular formula:

C6H13N

IUPAC Name:

1-methylpiperidine

Details on test material:

- Name of test material (as cited in study report): 1-Methylpiperidin ca. 80% b. 100%
- Physical state: colorless liquid
- Analytical purity: 80.2%
- Lot/batch No.: Tank 73
- Stability: The stability of the test substance throughout the study period has been verified by reanalysis.
- Storage condition of test material: room temperature (under nitrogen conditions)

Method

Target gene:

Salmonella: HIS
E. coli: TRP

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA1535, TA100, TA1537, TA98, E. coli wp2 uvra

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 induced rat liver.

Test concentrations with justification for top dose:

First experiment, standard plate test:
TA1535, TA1537, TA100, TA98, E.coli: 25, 125, 625, 3125, 6250 µg/plate
Second experiment, standard plate test:
TA98, E. coli: 1000, 2000, 3000, 4000, 5000 µg/plate
Third experiment, preincubation test:
TA1535, TA1537, TA100, TA98, E.coli: 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

Water

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9-mix: TA 100, TA 98, TA 1537 and TA 1535: 2.5 µg 2-AA; E. coli WP2 uvrA: 60 µg 2-AA; Without S9-mix: TA100, TA1535: 5 µg MNNG; TA98: 10 µg NOPD; TA1537: 100 µg AAC; E. coli WP2 uvrA: 10 µg ENNG

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: background growth


Positive control:
with S-9 mix: 2.5 µg 2-aminoanthracene (2-AA, dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535.
60 µg 2-aminoanthracene (2-AA, dissolved in DMSO) for the strain E. coli WP2 uvrA.
without S-9 mix: 5 µg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, dissolved in DMSO) for the strains TA 100 and TA 1535.
10 µg 4-nitro-o-phenylendiamine (NOPD, dissolved in DMSO) for the strain TA 98.
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537
10 µg N-ethyl-N'-nitro-N-nitrosoguanidin (ENNG, dissolved in DMSO) for the strain E. coli WP2 uvrA

S9-fraction:
At least 5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 per kg body weight 5 days before sacrifice. Five days after administration the rats are sacrificed, and the livers are prepared. The livers are weighed and washed in an equivalent volumne of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (so-called S-9 fraction) are quickly deep-frozen in dry ice and stored at -700C° to -800C°.
S9-mix:
One volume of S-9 fraction is mixed with 9 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice until used. The concentrations of the cofactors in the S-9 mix are: MgCl2 8 mM, KCl 33 mM, Glucose-6-phosphate 5 mM, NADP 4 mM, Phosphate buffer (pH 7.4) 15 mM

Standard plate test, Salmonella typhimurium:
Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination af mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order: 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. Composition of the minimal glucose agar: 980 mL aqua dest., 20 mL Vogel-Bonner E medium, 15 g Difco bacto agar, 20 g D-glucose, monohydrate. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his revertants) are counted.

Standard plate test, E. coli:
Test tubes containing 2 mL portions of soft agar which consists of 100 mL agar (0.6% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45°C, and the remaining components are added in the following order: 0.1 mL test solution or vehicle 0.1 mL bacterial suspension, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48-72 hours in the dark, the bacterial colonies are counted.

Titer determination:
In general, the titer is determined only in the experiments with S-9 mix both without test substance (vehicle only) and after adding the two highest amounts of substance. For this purpose, in the standard plate test 0.1 mL of the overnight cultures is diluted to 10xE-6 in each case. Test tubes containing 2 mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order: 0.1 mL vehicle (without and with test substance), 0.1 mL bacterial suspension (dilution: 10E-6), 0.5 mL S-9 mix.
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37°C for 20 minutes. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium TA1535, TA100, TA1537, TA98,E. coli wp2 uvra

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the preincubation assay at doses > 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitation was found.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his or trp background growth, decrease in the number of his or trp revertants, reduction in the titer) was observed in the preincubation assay at doses > 2500 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number (arithmetic mean) of colonies of histidine and tryptophan-prototrophic back-mutants in experiments without microsomal activation in the standard plate test.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	E.coli WP2 uvrA
Test substance
0	27	137	18	12	29
25	24	134	19	10	30
125	30	133	19	9	28
625	27	121	17	7	34
3125	41	107	18	6	33
6250	29	114	16	7	32
MNNG
5		1414	1842
AAC
100				415
NOPD
10	1038
ENNG
10					565

 

 

Table 2: Number (arithmetic mean) of colonies of histidine and tryptophane-prototrophic back-mutants in experiments with microsomal activation in the standard plate test.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	E.coli WP2 uvrA
Test substance
0	33	125	16	13	32
25	35	137	17	7	36
125	37	128	15	8	42
625	32	142	12	5	42
3125	62	164	21	10	50
6250	49	132	19	12	40
2-AA
60					225
2.5	621	1073	122	171

Table 3: Number (arithmetic mean) of colonies of histidine and tryptophan-prototrophic back-mutants in experiments without microsomal activation in the second standard plate test.

Concentration (µg/plate)	TA98	E.coli WP2 uvrA
Test substance
0	26	32
1000	27	27
2000	31	31
3000	28	30
4000	25	27
5000	23	29
NOPD
10	1138
ENNG
10		504

 

Table 4: Number (arithmetic mean) of colonies of histidine and tryptophane-prototrophic back-mutants in experiments with microsomal activation in the second standard plate test.

Concentration (µg/plate)	TA98	E.coli WP2 uvrA
Test substance
0	43	43
1000	33	42
2000	35	44
3000	38	42
4000	30	40
5000	28	42
2-AA
60		248
2.5	798

 

 

Table 5: Number (arithmetic mean) of colonies of histidine and tryptophan-prototrophic back-mutants in experiments without microsomal activation in the preincubation test.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	E.coli WP2 uvrA
Test substance
0	30	132	24	11	32
20	29	130	21	10	32
100	29	135	20	8	29
500	35	136	19	9	25
2500	10	62	11	1	10
5000	0 (B)	0 (B)	0 (B)	0 (B)	0 (B)
MNNG
5		1122	1385
AAC
100				412
NOPD
10	1556
ENNG
10					515

  (B): reduced background growth

 

 

Table 6: Number (arithmetic mean) of colonies of histidine and tryptophane-prototrophic back-mutants in experiments with microsomal activation in the preincubation test.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	E.coli WP2 uvrA
Test substance
0	47	145	23	12	35
20	47	145	20	11	31
100	44	136	13	10	27
500	44	128	11	7	33
2500	38	146	8	8	24
5000	17	0 (B)	0 (B)	0 (B)	10
2-AA
60					202
2.5	759	1024	131	115

  (B): reduced background growth

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative