2,2,6,6-Tetramethylpiperidin-4-yl dodecanoate

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 and OECD guideline compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21 Jul 1997)

Deviations:

yes

Remarks:

(2-aminoanthracene is the only indicator for the examination of the S9-capability)

GLP compliance:

yes (incl. QA statement)

Remarks:

(BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,6,6-tetramethylpiperidin-4-yl dodecanoate

EC Number:

700-503-1

Cas Number:

101238-01-1

Molecular formula:

C21 H41 N O2

IUPAC Name:

2,2,6,6-tetramethylpiperidin-4-yl dodecanoate

Details on test material:

- Analytical purity: 94%

Method

Target gene:

his+ / trp+

Species / strain

Species / strain / cell type:

other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction

Test concentrations with justification for top dose:

1st Experiment (Standard plate test with and without S9 mix): 0, 33, 100, 333, 1000, 2500 and 5400 μg/plate (strong bacteriotoxicity was observed in the 1st standard plate test, therefore a second standard plate test with lower concentrations was conducted)
2nd Experiment (Standard plate test with and without S9 mix): 0, 0.1, 0.33, 1, 3.3, 10 and 33 μg/plate
3rd Experiment (Preincubation test with and without S9 mix): 0, 0.033, 0.1, 0.33, 1, 3.3 and 10 μg/plate (without S9 mix); 0, 0.1, 0.33, 1, 3.3, 10 and 33 μg/plate (with S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

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

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA

Evaluation criteria:

Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(SPT: 10 and 33 µg/plate (-S9), 33 µg/plate (+S9); PIT: 3.3 and 10 µg/plate (-S9), 10 and 33 µg/plate (+S9))

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found at 5400 μg/plate with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A strong 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 standard plate test and in the preincubation assay depending on the strain and test conditions from about 1 μg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Strong bacteriotoxicity was observed in the 1st standard plate test, therefore a second standard plate test with lower concentrations was conducted. The results of the second standard plate test and of the preincubation test are showen in the following tables:

Table 1. Test results of experiment 2 (standard plate test).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		TA 100	TA1535	E.coli WP2 uvrA	TA98	TA1537
–	0	53 ± 3	10 ± 1	64 ± 5	18 ± 3	8 ± 1
–	0.1	46 ± 9	9 ± 2	62 ± 6	17 ± 3	8 ± 0
–	0.33	47 ± 5	10 ± 1	57 ± 8	17 ± 3	7 ± 1
–	1	55 ± 14	10 ± 4	58 ± 3	16 ± 1	6 ± 1
–	3.3	39 ± 2	9 ± 1	63 ± 12	16 ± 1	5 ± 2
–	10	12 ± 1B	4 ± 2B	58 ± 5B	10 ± 3B	0B
–	33	0B	0B	22 ± 3B	0B	0B
Positive controls, –S9	Name	MNNG	MNNG	4-NQO	NOPD	AAC
	Concentrations (μg/plate)	5	5	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	1595 ± 30	1018 ± 55	757 ± 84	940 ± 9	1019 ± 133
+	0	57 ± 6	11 ± 2	68 ± 4	23 ± 4	10 ± 1
+	0.1	56 ± 5	10 ± 2	63 ± 3	17 ± 3	10 ± 2
+	0.33	58 ± 9	12 ± 2	75 ± 4	25 ± 5	9 ± 2
+	1	55 ± 9	12 ± 2	69 ± 5	25 ± 5	8 ± 1
+	3.3	53 ± 3	13 ± 3	71 ± 5	24 ± 1	7 ± 0
+	10	53 ± 10	11 ± 1	67 ± 7	20 ± 5	9 ± 2
+	33	45 ± 7B	11 ± 1B	55 ± 5B	23 ± 5B	5 ± 3B
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	1535 ± 41	228 ± 21	292 ± 10	1624 ± 60	204 ± 26

 

B = reduced background growth

 

Table 2. Test results of experiment 3 (preincubation test).

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		TA 100	TA1535	E.coli WP2 uvrA	TA98	TA1537
–	0	31 ± 2	11 ± 1	52 ± 2	16 ± 1	6 ± 1
–	0.033	30 ± 2	10 ± 1	54 ± 2	15 ± 1	4 ± 1
–	0.1	31 ± 2	10 ± 3	51 ± 6	19 ± 8	6 ± 1
–	0.33	32 ± 3	8 ± 1	50 ± 6	17 ± 3	6 ± 1
–	1	32 ± 3	11 ± 2	57 ± 6	10 ± 2	5 ± 1
–	3.3	19 ± 3B	7 ± 1B	30 ± 3B	11 ± 3B	0B
–	10	0B	0B	15 ± 3B	0B	0B
Positive controls, –S9	Name	MNNG	MNNG	4-NQO	NOPD	AAC
	Concentrations (μg/plate)	5	5	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	1009 ± 42	1113 ± 108	1305 ± 41	772 ± 73	669 ± 228
+	0	35 ± 4	11 ± 2	44 ± 6	21 ± 2	8 ± 2
+	0.1	38 ± 1	11 ± 2	39 ± 4	21 ± 1	9 ± 1
+	0.33	39 ± 9	11 ± 2	37 ± 5	18 ± 2	6 ± 1
+	1	37 ± 2	8 ± 2	40 ± 4	17 ± 3	7 ± 2
+	3.3	38 ± 1	9 ± 2	40 ± 3	19 ± 2	5 ± 1
+	10	37 ± 4B	8 ± 3B	40 ± 6B	19 ± 4B	5 ± 1B
+	33	39 ± 1B	11 ± 3B	38 ± 6B	18 ± 3B	6 ± 3B
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	925 ± 32	148 ± 12	161 ± 13	1015 ± 15	169 ± 13

 

B = reduced background growth

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.