N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

The study was conducted under GLP following the most up to date OECD guideline

Adequacy of study:

key study

Study period:

06 June 2017 - 13 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C34H68N2O3

IUPAC Name:

Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 2

Reference substance name:

Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C35H70N2O3

IUPAC Name:

Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 3

Reference substance name:

Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C36H72N2O3

IUPAC Name:

Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 4

Reference substance name:

Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C37H74N2O3

IUPAC Name:

Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 5

Reference substance name:

Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C38H76N2O3

IUPAC Name:

Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 6

Reference substance name:

Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C39H78N2O3

IUPAC Name:

Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 7

Reference substance name:

Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C40H80N2O3

IUPAC Name:

Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 8

Reference substance name:

Stearic acid

EC Number:

200-313-4

EC Name:

Stearic acid

Cas Number:

57-11-4

Molecular formula:

C18H36O2

IUPAC Name:

stearic acid

Constituent 9

Reference substance name:

Palmitic acid

EC Number:

200-312-9

EC Name:

Palmitic acid

Cas Number:

57-10-3

Molecular formula:

C16H32O2

IUPAC Name:

palmitic acid

Constituent 10

Reference substance name:

Myristic acid

EC Number:

208-875-2

EC Name:

Myristic acid

Cas Number:

544-63-8

Molecular formula:

C14H28O2

IUPAC Name:

myristic acid

Constituent 11

Reference substance name:

2-(2-aminoethylamino)ethanol

EC Number:

203-867-5

EC Name:

2-(2-aminoethylamino)ethanol

Cas Number:

111-41-1

Molecular formula:

C4H12N2O

IUPAC Name:

2-(2-aminoethylamino)ethanol

Test material form:

solid

Details on test material:

Identification: N-(2-{[C16-18 (EVEN NUMBERED) ALKANOYL]AMINO}ETHYL)-N-(2-HYDROXYETHYL)[C16-18 (EVEN NUMBERED) ALKYLAMIDE
Storage Conditions: Room temperature

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in THF (tetrahydrofuran) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilutions prepared in tetrahydrofuran by mixing on a vortex mixer on the day of each experiment. Formulated concentrations purity correction was not required.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of stock solution = 200 mg/mL.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 micro g/plate in triplicate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: the test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but fully soluble in tetrahydrofuran at 200 mg/mL.

Controls

Untreated negative controls:

yes

Remarks:

untreated control

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation; in suspension (main test)
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: presence of bacterial lawn
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.

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 (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation: none
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the range-finding test (plate incorporation method) the test item did not cause any visible reduction in the growth of the bacterial background lawns of all of the tester strains, at all dose levels in the absence and presence of S9-mix. Therefore, the maximum recommended dose level of 5000 µg/plate was selected for main experiment.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: Yes (2009 - 2010 data)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: presence of bacterial lawn
- Other observations when applicable: no

Any other information on results incl. tables

Table 1:        Test Results; Experiment 1 – With and Without Metabolic Activation (Plate Incorporation)

Concentration (µg/plate)		Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)
Solvent Control (THF)		79	81	22	18	29	35	19	20	14	12
1.5 µg		75	75	18	23	24	37	13	24	13	16
5 µg		76	87	14	20	23	31	15	23	16	14
15 µg		78	92	22	21	25	30	13	23	16	15
50 µg		91	85	19	23	34	32	19	21	17	16
150 µg		76	89	18	21	27	33	18	14	18	15
500 µg		73	94P	21P	23P	28P	36P	19P	18P	17P	14P
1500 µg		86	83P	26P	22P	25P	30P	21P	22P	15P	14P
5000 µg		66	62P	16P	20P	22P	26P	16P	19P	10P	9P
Positive controls	Name	ENNG	2AA	ENNG	2AA	ENNG	2AA	4NQO	BP	9AA	2AA
	Dose Level	3 µg	1 µg	5 µg	2 µg	2 µg	10 µg	0.2 µg	5 µg	80 µg	2 µg
	No. of Revertants	430	1797	313	217	742	153	170	144	397	422

ENNG - N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO - 4-Nitroquinoline-1-oxide

9AA - 9-Aminoacridine

2AA - 2-Aminoanthracene

BP -  Benzo(a)pyrene

P  -  Test item precipitate

Table 2:       Test Results; Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Concentration (µg/plate)		Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
		S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)	S9-Mix (-)	S9-Mix (+)
Solvent Control (THF)		76	72	10	17	16	24	13	25	10	9
15 µg		66	67	10	21	17	19	17	23	8	8
50 µg		66	65	10	14	14	19	15	17	8	12
150 µg		62	72	9	13	18	17	14	17	12	8
500 µg		61P	72P	10P	18P	18P	18P	15P	28P	10P	7P
1500 µg		69P	69P	9P	20P	14P	20P	14P	27P	10P	9P
5000 µg		63P	75P	11P	21P	18P	21P	12P	19P	9P	8P
Positive controls	Name	ENNG	2AA	ENNG	2AA	ENNG	2AA	4NQO	BP	9AA	2AA
	Dose Level	3 µg	1 µg	5 µg	2 µg	2 µg	10 µg	0.2 µg	5 µg	80 µg	2 µg
	No. of Revertants	623	977	1434	169	534	120	262	165	225	292

ENNG - N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO - 4-Nitroquinoline-1-oxide

9AA - 9-Aminoacridine

2AA - 2-Aminoanthracene

BP -  Benzo(a)pyrene

P  -  Test item precipitate

Applicant's summary and conclusion

Conclusions:

It was concluded that the test itme was non-mutagenic under the conditions of the test.

Executive summary:

OECD 471 (2011) - In a reverse gene mutation assay in bacteria strains: TA100, TA1535, TA98 and TA1537 of S. Typhimurium and WP2uvrA of E. Coli were exposed to

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

in tetrahydrofuran at concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate with and without metabolic activation in plate incorporation and pre-incubation methods test system.

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was tested up to cytotoxic concentrations. The positive controls induced the appropriate responses in the corresponding strains. The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range.

 

There was no evidence of induced mutant colonies over background in the test item treated colonies.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.