N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Jan - 06 Feb 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

EC Number:

263-218-7

EC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Cas Number:

61792-31-2

Molecular formula:

C17H36N2O2

IUPAC Name:

N-[3-(dimethylamino)propyl]dodecanamide N-oxide

Method

Target gene:

his operon (S. typhimurium strains) and trp operon (E. coli strains)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix).
- source of S9: Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix: S9 mix was prepared from the livers of rats treated with Aroclor 1254.
- quality controls of S9 mix: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene in S. typhimurium strain TA100 at concentrations of 5 and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

First experiment: 5.4, 17, 52, 164, 512 and 1600 µg/plate with and without metabolic activation in strains TA 98, TA 1535 and TA 1537 and 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in strains TA 100 and WP2 uvrA.
Second experiment: 5.4, 17, 52, 164, 512 and 1600 µg/plate with and without metabolic activation in strains TA 98, TA 100, TA 1535 and TA 1537 and 17, 52, 164, 512, 1600 and 5000 µg/plate in strain WP2 uvrA

The highest concentration was selected based on a preliminary toxicity test in strains TA 100 and WP2 uvrA, in which cytotoxicity was observed at 1600 µg/ plate in strain TA 100 and at 5000 µg/plate in strain WP2 uvrA in the presence and absence of S9 mix. The preliminary toxicity test was reported as part of the direct plate assay (first experiment).

Vehicle / solvent:

- Vehicle/solvent used: Milli-Q water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2.5 µg/plate in DMSO (- S9) for TA 1537 (plate incorporation assay); 2-aminoanthracene (2AA), 2.5 µg/plate in DMSO (+ S9) for TA 1535 and TA 1537, 1 µg/plate in DMSO (+ S9) for TA 98 and TA 100, 15 µg/plate in DMSO (+ S9) for WP2 uvrA

Remarks:

Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, preliminary toxicity test/Experiment 1); preincubation (Experiment 2)

DURATION
- Preincubation period: 30 ± 2 min (Experiment 2)
- Exposure duration: 48 ± 4 h (Experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicates each in 2 independant experiments

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 1600 µg/plate with and without S9 mix in the first experiment and at ≥ 512 µg/plate with and without S9 mix in the second experiment

Vehicle controls validity:

not valid

Remarks:

in the first experiment, please refer to "Additional information on results"

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 1600 µg/plate with and without S9 mix in the first experiment and at ≥ 512 µg/plate with and without S9 mix in the second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 1600 µg/plate with and without S9 mix in the first and in the second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 1600 µg/plate with and without S9 mix in the first experiment and at ≥ 512 µg/plate with and without S9 mix in the second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix in the first and in the second experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: There was no precipitation at any test item concentration in any experiment, neither with or without S9 mix.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed in strains TA 100 and WP2 uvrA at concentrations in the range of 1.7 to 5000 µg/plate with and without metabolic activation. The preliminary toxicity test was reported as part of the first experiment (plate-incorporation assay).

CYTOTOXICITY:
Cytotoxicity, evident as a reduced or absent bacterial background lawn was observed:
- at ≥ 1600 µg/plate with and without S9 mix for strains TA 98, TA 100, TA 1535 and TA 1537 and at 5000 µg/plate with and without S9 mix for strain WP2 uvrA in the first experiment.
- at ≥ 512 µg/plate with and without S9 mix for strains TA 98, TA 100 and TA 1537, at 1600 µg/plate for strain TA 1535, and at 5000 µg/plate for WP2 uvrA in the second experiment.

STUDY RESULTS
- All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

HISTORICAL CONTROL DATA: please refer to Tables 3 and 4 under "Any other information on results incl. tables".
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- Negative (solvent/vehicle) historical control data: The negative control values were within the laboratory historical control data ranges, except the response for TA98 in the absence of S9-mix in the first experiment. However, since the mean number of revertant colonies showed a characteristic number of revertant colonies (5 revertant colonies) when compared against relevant historical control data (6 revertant colonies), the validity of the test was considered not to be affected.

Any other information on results incl. tables

Table 1: Results of the preliminary toxicity test (plate incorporation test with strains TA 100 and WP2 uvrA) and the first experiment (plate incorporation test with strains TA 98, TA 1535 and TA 1537)

Experiment 1: Plate incorporation test
Strain	TA 1535		TA 1537		TA 98		TA 100^#		WP2uvrA^#
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
Water mean	3	5	2	4	5	12	116	80	18	25
± SD	± 1	± 4	± 1	± 1	± 1	± 2	± 2	± 9	± 6	± 8
Test item [µg/plate]
1.7 mean	-	-	-	-	-	-	110	95	15	17
± SD	-	-	-	-	-	-	± 15	± 5	± 0	± 10
5.4 mean	5	9	3	1	11	13	107	93	15	13
± SD	± 1	± 1	± 1	± 1	± 4	± 3	± 2	± 8	± 1	± 4
17 mean	7	5	4	2	9	12	104	101	22	19
± SD	± 2	± 3	± 4	± 2	± 2	± 4	± 6	± 12	± 7	± 6
52 mean	7	5	4	0	5	11	104	119	18	15
± SD	± 1	± 3	± 1	± 1	± 2	± 2	± 10	± 10	± 9	± 8
164 mean	5	5	3	2	9	12	106	86	20	23
± SD	± 3	± 1	± 1	± 1	± 5	± 4	± 13	± 6	± 11	± 7
512 mean	4^s	3	4	3	5	10	62^s	71	21	24
± SD	± 2	± 2	± 2	± 2	± 3	± 2	± 10	± 6	± 4	± 4
1600 mean	^eMC	^eMC	^eMC	^eMC	^eMC	1^m	^eMC	^eMC	11	11
± SD	-	-	-	-	-	± 1	-	-	± 7	± 5
5000 mean	-	-	-	-	-	-	0^a	0^a	^eMC	^eMC
± SD	-	-	-	-	-	-	± 0	± 0	-	-
Positive control
^§mean	676	243	1092	254	1102	1117	788	1616	1397	349
± SD	± 35	± 18	± 328	± 62	± 128	± 92	± 59	± 422	± 57	± 19
^#: results of the preliminary toxicity test
MC: Microcolonies; a: bacterial background lawn absent, e: bacterial background lawn extremely reduced, s: bacterial background lawn slighly reduced, m: bacterial background lawn moderately reduced

Table 2: Results of the second experiment (pre-incubation test with all strains)

Experiment 2: Pre-incubation Test
Strain	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
Water mean	6	23	5	5	9	17	107	88	13	17
± SD	± 3	± 18	± 2	± 2	± 6	± 2	± 17	± 2	± 2	± 4
Test item
5.4 mean	8	9	1	5	15	13	92	90	-	-
± SD	± 2	± 1	± 0	± 3	± 6	± 5	± 6	± 13	-	-
17 mean	7	6	5	5	16	13	104	76	19	21
± SD	± 3	± 2	± 2	± 3	± 6	± 1	± 8	± 10	± 4	± 9
52 mean	4	6	3	6	14	22	83	68	14	25
± SD	± 1	± 1	± 0	± 3	± 2	± 3	± 8	± 9	± 2	± 5
164 mean	5^s	6^s	1^m	4^s	5^m	14^s	39^m	59^s	13	16
± SD	± 3	± 2	± 2	± 0	± 3	± 3	± 6	± 7	± 2	± 5
512 mean	4^m	8^m	^eMC	^eMC	^eMC	^eMC	^eMC	^eMC	18	17
± SD	± 3	± 4	-	-	-	-	-	-	± 6	± 5
1600 mean	^eMC	^eMC	^eMC	^eMC	^eMC	^eMC	^eMC	^eMC	12	15
± SD	-	-	-	-	-	-	-	-	± 1	± 4
5000 mean	-	-	-	-	-	-	-	-	^a	^a
± SD	-	-	-	-	-	-	-	-	-	-
Positive control
^§mean	956	227	150	119	1422	418	654	1395	1446	497
± SD	± 58	± 34	± 17	± 3	± 12	± 19	± 68	± 161	± 139	± 19
MC: Microcolonies; a: bacterial background lawn absent, e: bacterial background lawn extremely reduced, s: bacterial background lawn slighly reduced

Table 3: Historical control data for the solvent control generated in the testing laboratory from Nov 2016 - Nov 2019

	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 – 27	2 – 15	2 – 19	6 – 61	8 – 60	61 – 176	60 - 176	11 – 61	13 - 68
Mean	10	11	5	6	15	21	114	108	27	33
SD	3	3	2	2	5	7	18	21	8	9
n	2992	2960	2965	2960	2958	3025	3037	2946	2790	2753

Table 4: Historical control data for the positive control generated in the testing laboratory from Nov 2016 - Nov 2019

	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	107 – 1530	73 – 1481	58 – 1422	48 – 1239	365 – 1978	250 – 2033	439 – 1993	424 – 2666	93 – 1999	109 - 1968
Mean	932	269	812	314	1259	910	900	1354	1117	431
SD	177	116	360	148	228	353	167	401	529	158
n	2879	2810	2497	2893	2920	2895	2917	2864	2689	2729

Applicant's summary and conclusion

Conclusions:

Under the tested conditions, the test compound was not mutagenic in any of the S. typhimurium strains (TA 98, TA 100, TA 1535 and TA1537) and in E. coli strain WP2 uvrA with and without metabolic activation.