Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate

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:

26 Sept 2012 - 6 Nov 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 21 July 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 30 May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate

EC Number:

250-151-3

EC Name:

Sodium N-methyl-N-(1-oxotetradecyl)aminoacetate

Cas Number:

30364-51-3

Molecular formula:

C17H33NO3.Na

IUPAC Name:

sodium N-methyl-N-(1-oxotetradecyl)aminoacetate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

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

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA98, and TA100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbitone/beta-Naphthoflavone

Test concentrations with justification for top dose:

- Preliminary Toxicity Test:
TA100 or WP2uvrA: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
- Mutation Test - Experiment 1
All Salmonella strains (with and without S9-mix): 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate
WP2uvrA (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 μg/plate
- Mutation Test - Experiment 2
All Salmonella strains (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate
WP2uvrA (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: sterile, distilled water
- Justification for choice of vehicle: based on solubility checks performed in-house

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

- S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate (WP2uvrA), 3 μg/plate (TA100), 5 μg/plate (TA1535); 9-Aminoacridine (9AA): 80 μg/plate (TA1537), 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate (TA98)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

+ S9: 2-Aminoanthracene (2AA): 1 μg/plate (TA100), 2 μg/plate (TA1535 and TA1537), 10 μg/plate (WP2uvrA); Benzo(a)pyrene (BP): 5 μg/plate (TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (Experiment 1) and pre-incubation (Experiment 2)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies, a clearing or diminution of the background lawn (Preliminary Toxicity Test)

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preliminary Toxicity Test: TA100 at 500 μg/plate and above (+ and - S9); plate incorporation Experiment 1: all strains at 150 μg/plate and above (+ and - S9); pre incubation Experiment 2: all strains at 50 μg/plate and above (+ and - S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES: RANGE-FINDING/SCREENING STUDIES: Range finding test: in order to select the appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test substance. Ten concentrations of the test item formulation (0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) and vehicle control (sterile distilled water) were tested.
The test item was toxic to TA 100 from 500 µg/plate and non-toxic to WP2 uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA: The results of the vehicle and positive controls (+ and - S9) are within the historical control data of the last two years.

Any other information on results incl. tables

Table 1 Test Results: Experiment 1 (plate incorporation) – Without Metabolic Activation

Test group		Number of revertants (mean) ± SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA1535	WP2uvrA	TA98	TA1537
Solvent control		105±7.6	18±3.8	34±9.8	21±6.8	15±3.5
Test item	1.5 µg/plate	96±3.1	16±2.6	N/T	19±7.4	9±1.5
	5 µg/plate	97±11.7	21±2.6	N/T	22±5.6	16±2.1
	15 µg/plate	94±4.7	16±3.5	29±2.0	16±3.2	9±2.5
	50 µg/plate	107±16.5	13±3.2	28±2.9	22±8.5	10±2.3
	150 µg/plate	79±6.0	11±4.0	35±7.6	13±2.6	9±5.5
	500 µg/plate	58±4.7	7±4.2	32±8.1	12±3..1	2±1.2
	1500 µg/plate	0±0	2±1.0	25±7.0	0±0	0±0
	5000 µg/plate	N/T	N/T	29±5.5	N/T	N/T
Positive controls S9 mix (-)	Name Dose Level No. of revertants	ENNG	ENNG	ENNG	4NQO	9AA
		3 µg	5 µg	2 µg	0.2 µg	80 µg
		514±45.2	453±17.9	859±31.6	159±12.7	1122±74.5

Table 2 Test Results: Experiment 1 (plate incorporation) – With Metabolic Activation

Test group		Number of revertants (mean) ± SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA1535	WP2uvrA	TA98	TA1537
Solvent control		125±10.2	11±1.0	36±6.0	26±4.2	17±7.0
Test item	1.5 µg/plate	122±12.5	11±1.2	N/T	26±7.1	16±7.6
	5 µg/plate	119±7.8	11±2.6	N/T	19±5.0	15±6.1
	15 µg/plate	124±26.0	12±3.5	40±8.1	21±4.2	14±1.2
	50 µg/plate	126±12.8	11±5.5	33±7.1	22±8.7	16±3.8
	150 µg/plate	103±15.5	11±0.6	39±6.7	24±7.1	10±0.6
	500 µg/plate	68±5.2	5±0.6	34±3.8	11±2.6	3±1.2
	1500 µg/plate	0±0	0±0	35±3.5	3±2.6	0±0
	5000 µg/plate	N/T	N/T	30±2.6	N/T	N/T
Positive controls S9 mix (+)	Name Dose Level No. of revertants	2AA	2AA	2AA	BP	2AA
		1 µg	2 µ	10 µg	5 µg	2 µg
		1939±74.5	314±31.6	362±29.3	169±4.2	384±9.3

Table 3 Test Results: Experiment 2 (pre-incubation) – Without Metabolic Activation

Test group		Number of revertants (mean) ± SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA1535	WP2uvrA	TA98	TA1537
Solvent control		109±7.6	21±2.3	29±3.0	19±3.1	12±2.6
Test item	0.5 µg/plate	116±2.5	23±4.2	N/T	19±2.6	15±3.5
	1.5 µg/plate	103±8.5	22±4.0	N/T	19±5.1	12±2.0
	5 µg/plate	87±13.2	19±4.0	N/T	17±3.8	12±4.7
	15 µg/plate	98±8.5	17±3.1	26±8.2	18±4.5	6±1.0
	50 µg/plate	100±11.6	18±3.5	27±13.2	15±1.2	5±2.1(S)
	150 µg/plate	82±2.9	17±1.5	23±2.6	22±5.8	0±0 (V)
	500 µg/plate	0±0	0±0	21±6.5	10±2.1	0±0 (V)
	1500 µg/plate	N/T	N/T	21±5.0	N/T	N/T
	5000 µg/plate	N/T	N/T	27±1.5	N/T	N/T
Positive controls S9 mix (-)	Name Dose Level No. of revertants	ENNG	ENNG	ENNG	4NQO	9AA
		3 µg	5 µg	2 µg	0.2 µg	80 µg
		635±35.8	335±87.5	775±38.9	121±30.0	953±53.4

 

Table 4 Test Results: Experiment 2 (pre-incubation) – With Metabolic Activation

Test group		Number of revertants (mean) ± SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA1535	WP2uvrA	TA98	TA1537
Solvent control		105±15.0	11±1.2	33±7.6	28±4.0	13±5.9
Test item	0.5 µg/plate	106±7.6	9±0.6	N/T	22±7.6	14±2.6
	1.5 µg/plate	101±4.7	13±3.0	N/T	25±3.2	13±3.8
	5 µg/plate	94±6.2	10±0.6	N/T	21±1.5	12±1.7
	15 µg/plate	91±11.9	11±1.2	33±2.6	28±4.2	11±6.7
	50 µg/plate	89ׅ10.6	12±1.7	31±12.4	18±3.2	10±5.2
	150 µg/plate	85±7.1	10±3.6	34±2.0	21±6.0	6±2.3
	500 µg/plate	54±8.3	0±0	29±5.8	18±3.5	0±0
	1500 µg/plate	N/T	N/T	30±5.5	N/T	N/T
	5000 µg/plate	N/T	N/T	32±1.5	N/T	N/T
Positive controls S9 mix (+)	Name Dose Level No. of revertants	2AA	2AA	2AA	BP	2AA
		1 µg	2 µg	10 µg	5 µg	2 µg
		1161±93.3	162±16.7	292±14.0	138±20.6	169±38.7

 

N/T Not tested at this dose level

(S) = Sparse bacterial background lawn

(V) = Very weak bacterial background lawn

solvent control:sterile, destilled water

positive controls (- S9):

-N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate (WP2uvrA), 3 μg/plate (TA100), 5 μg/plate (TA1537)

- 9-Aminoacridine (9AA): 80 μg/plate (TA1537), 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate (TA98)

positive controls (+ S9):

- 2-Aminoanthracene (2AA): 1 μg/plate (TA100), 2 μg/plate (TA1535 and TA1537), 10 μg/plate (WP2uvrA)

- Benzo(a)pyrene (BP): 5 μg/plate (TA98)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative