2-hydroxy-N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]benzamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-02 to 2012-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

31 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]stearamide

EC Number:

231-609-1

EC Name:

N-[3-(dimethylamino)propyl]stearamide

Cas Number:

7651-02-7

Molecular formula:

C23H48N2O

IUPAC Name:

N-[3-(dimethylamino)propyl]octadecanamide

Test material form:

solid: pellets

Method

Target gene:

not applicable, chromosome aberration assay

Species / strain

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated foetal calf serum, 2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin and 30 U/mL heparin

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
0, 1, 3, 10, 33, 100, 333 and 500 μg/mL

First experiment:
without and with S9-mix (3 h exposure time, 24 h fixation time): 0, 1, 3 and 10 μg/mL

Second experiment:
without S9-mix (24 h and 48 h exposure time, 24 h and 48 h fixation time): 0, 3, 6, 10, 15, 20 and 25 μg/mL
with S9-mix (3 h exposure time, 48 h fixation time): 0, 1, 3 and 10 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (final concentration in culture medium: 0.5% (v/v))

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other: (without metabolic activation)

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: (with metabolic activation)

Details on test system and experimental conditions:

TEST SYSTEM
METHOD OF APPLICATION: in medium

DURATION
first experiment, without and with S9-mix: 3 h exposure time, 24 h fixation time
second experiment, without S9-mix: 24 h and 48 h exposure time, 24 h and 48 h fixation time
second experiment, with S9-mix: 3 h exposure time, 48 h fixation time

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates; 2 independent experiment

NUMBER OF CELLS EVALUATED: 1000 for mitotic index; 100 for chromosome aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
- The number of chromosome aberrations found in the solvent control cultures should be within the laboratory historical control data range.
- The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
- A homogeneous response between the replicate cultures is observed.
- A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
- It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
- A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

Chi-square test, one-sided

Results and discussion

Test results

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: a concentration of 10 µg/mL already precipitated in the culture medium

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test a concentration of 33 µg/mL reduced the mitotic index to 0-10% of the control. Higher concentrations lead to cell lysis.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations, number of polyploid cells and cells with endoreduplicated chromosomes were within the laboratory historical control data range.

Remarks on result:

other: strain/cell type: peripheral human lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

	mitotic index	number of cell with aberrations (-gaps)
first experiment, without metabolic activation, 3 h exposure, 24 h fixation time
control	100	1
1	111	0
3	114	1
10	186	0
MMC 0.5 µg/mL	43	50
first experiment, with metabolic activation, 3 h exposure, 24 h fixation time
control	100	2
1	135	3
3	115	2
10	90	3
CP 10 µg/mL	30	52
second experiment, without metabolic activation, 24 h exposure, 24 h fixation time
control	100	2
3	87	4
6	73	1
10	57	3
15	26	not scored
20	7	not scored
25	4	not scored
MMC 0.2 µg/mL	30	35
second experiment, without metabolic activation, 48 h exposure, 48 h fixation time
control	100	2
3	92	2
6	79	1
10	55	2
15	13	not scored
20	4	not scored
25	3	not scored
MMC 0.1 µg/mL	54	49
second experiment, with metabolic activation, 3 h exposure, 48 h fixation time
control	100	0
1	87	2
3	64	0
10	59	2
CP, 10 µg/mL	**	38
** CP was fixed after 24 h, mitotic index was not calculated as % of control

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it is concluded that Stearic acid 3-(dimethylaminopropyl)amide is not clastogenic in the in vitro mammalian chromosome aberration test.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473, adopted 21 July 1997 and EU Method B.10, May 2008, peripheral human lymphocyte cultures were exposed to Stearic acid 3-(dimethylaminopropyl)amide in Ethanol at the following concentrations:

 

First experiment:

without and with S9-mix (3 h exposure time, 24 h fixation time): 0, 1, 3 and 10 μg/mL

 

Second experiment:

without S9-mix (24 h and 48 h exposure time, 24 h and 48 h fixation time): 0, 3, 6, 10, 15, 20 and 25 μg/mL

with S9-mix (3 h exposure time, 48 h fixation time): 0, 1, 3 and 10 μg/mL

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Stearic acid 3-(dimethylaminopropyl)amide was tested up to precipitating concentrations (10 µg/mL).

Both in the absence and presence of S9-mix Stearic acid 3-(dimethylaminopropyl)amide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of Stearic acid 3-(dimethylaminopropyl)amide on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Stearic acid 3-(dimethylaminopropyl)amide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

There was no evidence of chromosome aberrations induced over background.

This study is classified as acceptable and satisfies the requirement for OECD Test Guideline 473 for in vitro cytogenetic mutagenicity data.