Amides, C16-18 (even numbered) saturated and C18 unsaturated, N,N'-[[(2-hydroxyethyl)imino]di-2,1-ethanediyl]bis-, ethoxylated (1-2.5 mol)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-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:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: GB/T21794-2008 Chemicals-Test method of in vitro mammalian chromosome aberration

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C16-18 and C18-unsatd., N,N'-[[(2-hydroxyethyl)imino]di-2,1-ethanediyl]bis-,ethoxylated, lactates (salts)

Cas Number:

1434133-00-2

Molecular formula:

C47H96N3O5+ C3H5O3-

IUPAC Name:

Amides, C16-18 and C18-unsatd., N,N'-[[(2-hydroxyethyl)imino]di-2,1-ethanediyl]bis-,ethoxylated, lactates (salts)

Test material form:

solid: bulk

Details on test material:

Batch ST02697671

Specific details on test material used for the study:

purity 86.8%

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

other: peripheral human lymphocytes (isolated from the blood of a healthy adult, non-smoking, male volunteers (26-31 years old))

Details on mammalian cell type (if applicable):

- Type and identity of media: culture medium consisted of RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated foetal bovine serum, heparine, Colchicine, NADP and G-6-P, Mitomycin and Cyclophosphamide

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Without and with S9-mix: 62.5, 31.2 and 15.6 µg/mL culture medium (4 h exposure time, 24 h fixation time)

Vehicle / solvent:

- Culture medium: RPMI 1640 (85%); FBS (15%), PHA (200 µg/L), heparin (10U/mL), penicillin (100U/mL), strptomycin (100 U/mL).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

mitomycin C (-S9): 0.25 µg/mL (4 h exposure period), 0.2 and 0.3 µg/mL (24 h exposure period) and 0.1 and 0.15 µg/mL (48 h exposure period ), cyclophosphamide (+S9): 9.4 µg/mL for the 4 h exposure period (24 and 48 h fixation time)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h; 24h
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (1 µg/mL medium, 4h)
STAIN (for cytogenetic assays): Giemsa (5% (v/v))

NUMBER OF REPLICATIONS: duplicate in two independent experiments

NUMBER OF CELLS EVALUATED: 200 per concentration and solvent control group, 100 for positive control group.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

Evaluation criteria:

The chromosome aberration test was considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the range of the laboratory historical control data.
b) 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.
c) A homogeneous response between the replicate cultures was observed.
d) 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:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) 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.

Results and discussion

Test results

Species / strain:

other: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the continuous experiment at the highest dose of 300 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test.
The cytotoxicity result showed that no cell survived and no mitotic phase occured at the concentrations tested over 62.5 µg/mL, and the mitotic index showed a significant reduction at the concentration of 62.5 µg/mL (>50%); There were no cytotoxicity at the concentration of 15.6 µg/mL.
According to the above results of cytotoxicity test, three doses of 62.5, 31.2, 15.6 µg/ml were used.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1: Test results of the experiment

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 4h, fixation time 24h, without S9 mix
dist.water	10	14.8	0	1
MMC	0.25	8.9	1	19
Test substance	15.6	14.8	0.5	0.5
	31.2	12.4	0.5	0.5
	62.5	9.2	0.5	0
Exposure period 4h, fixation time 24h, with S9 mix
dist.water	10	15.5	0	1.5
CP	4	8.5	0	21
Test substance	15.6	14.4	1	0.5
	31.2	11.6	0	0.5
	62.5	7.7	0	1.5
Exposure period 24h, fixation time 24h, without S9 mix
dist.water	10	16.1	0	1
MMC	0.25	7.8	2	23
Test substance	15.6	13.3	0.5	0
	31.2	10.8	0.5	0
	62.5	5.7	0.5	0.5

- MMC: Mitomycin C; CP: Cyclophosphamide

- *: CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Applicant's summary and conclusion

Conclusions:

negative