Octadecanoic acid, 12-hydroxy-, reaction products with decanoic acid and ethylenediamine

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:

From 2009-10-09 to 2009-12-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Octadecanamide, 12-hydroxy-N-[2-[(1-oxodecyl)amino]ethyl]- and N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide) and Decanamide, N,N'-1,2-ethanediylbis-

EC Number:

907-495-0

Cas Number:

198028-14-7

Molecular formula:

C90H180N6O9

IUPAC Name:

Reaction mass of Octadecanamide, 12-hydroxy-N-[2-[(1-oxodecyl)amino]ethyl]- and N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide) and Decanamide, N,N'-1,2-ethanediylbis-

Test material form:

solid

Details on test material:

Chemical name : Reaction mass of N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide) and Octadecanamide, 12-hydroxy-N-[2-[(1-oxodecyl)amino]ethyl]- and Decanamide, N,N'-1,2-ethanediylbis-
Chemical registery number : EC 907-495-0 / CAS : 198028-14-7

Method

Target gene:

Not applicable, chromosome aberration

Species / strain

Species / strain / cell type:

lymphocytes: human lymphocyte cultures

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced animals

Test concentrations with justification for top dose:

First experiment:
3+17 hour -S-9: 15.1, 130 and 200 µg/mL
3+17 hour +S-9: 54.9, 130 and 200 µg/mL

Second experiment:
20+0 hour -S-9: 125, 150, 175 and 200 µg/mL
3+17 hour +S-9: 140, 160, and 180 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:the test substance was soluble in DMSO at concentrations up to approximately 80.10 mg/mL when heated to 80ºC with the aid of vortex mixing and ultrasonication.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4 Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA)

Remarks:

no remarks

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension

DURATION
- Preincubation period: no
- Exposure duration:
First experiment: 3 hours
Second experiment: 20 hours and 3 hours
- Expression time (cells in growth medium): no data
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SELECTION AGENT (mutation assays): no data
SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): the cells were stained for 5 minutes in filtered 4% (v/v) Giemsa in pH 6.8 buffer.

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and percentage of cells in mitosis

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: hyperdiploidy and structural aberrations

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p < 0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered as positive in this assay if all of the above criteria were met.
The test article was considered as negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result [ ]. Biological relevance was taken into account, for example consistency of response within and between concentrations and/or between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.

Statistics:

No data

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human lymphocyte cultures

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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
- Effects of pH: No marked changes in pH compared to the concurrent vehicle controls, were observed
- Effects of osmolality: No marked changes in osmolality compared to the concurrent vehicle controls, were observed
- Evaporation from medium: no data
- Water solubility: not soluble
- Precipitation: yes
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the proportion of cells with structural aberrations in these cultures fell within current historical vehicle control (normal) ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Any other information on results incl. tables

Table 7.6.1/2: Experiment 1 – Results summary

Treatment	Concentration (mg/mL)	Cytotoxicity (%)	% Cells with Chromosome Aberrations (Excluding Gaps)	Historical(%)^#	Statistical significance
3+17 hour -S-9	Vehicle^a	-	2.00	0-3	-
	15.1	10	1.50		NC
	130	30	1.50		NC
	200	39	1.50		NC
	*NQO, 2.50	ND	20.17		p=0.001
3+17 hour +S-9	Vehicle^a	-	0.50	0-3	-
	54.9	23	1.50		NC
	130	4	0.00		NC
	200	17	1.00		NC
	*CPA, 12.5	ND	15.76		p=0.001
^aVehicle control,DMSO * Positive control ^#95^thpercentile of the observed range NC = Not calculated ND = Not determined

Table 7.6.1/3: Experiment 2 – Results summary

Treatment	Concentration (mg/mL)	Cytotoxicity (%)	% Cells with Chromosome Aberrations (Excluding Gaps)	Historical(%)^#	Statistical significance
20+0 hour -S-9	Vehicle^a	-	0.00	0-3	-
	125	9	1.00		NC
	150	17	0.50		NC
	175	47	0.50		NC
	200	34	0.50		NC
	*NQO, 2.50	ND	40.82		p=0.001
3+17 hour +S-9	Vehicle^a	-	1.50	0-3	-
	140	ND	9.00		NC
	160	0	0.50		NC
	180	1	1.50		NC
	*CPA, 12.5	ND	42.55		p=0.001
^aVehicle control,DMSO * Positive control ^#95^thpercentile of the observed range NC = Not calculated ND = Not determined

Applicant's summary and conclusion

Conclusions:

The test substance did not induce chromosome aberration in cultured human peripheral blood lymphocytes in the absence and in the presence of activation system.

Executive summary:

The test substance was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors. This study was performed in accordance with OECD Guideline 473 and Good Laboratory Practices.

A range-finding study was performed to select the test article concentrations for chromosome analysis, by evaluating the effect of the test substance on mitotic index. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals.

In the first experiment, human lymphocyte cultures were treated with the test substance at concentrations of 15.1, 130 and 200 µg/mL in the absence of S9 and 54.9, 130 and 200 µg/mL in the presence of S9. In the second experiment, human lymphocyte cultures were treated with the test substance at concentrations of 125, 150, 175 and 200 µg/mL in the absence of S9 and 140, 160, and 180 µg/mL in the presence of S9.

Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within current historical vehicle control (normal) ranges. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively.

Treatment of cells with the test substance in the absence and presence of S-9 in both experiments resulted in frequencies of cells with structural aberrations that were generally similar to those observed in concurrent vehicle control cultures for all concentrations analysed.

The only exception to this was observed in one culture at the lowest concentration (140 µg/mL) analysed in the presence of S-9 in Experiment 2, where the number of aberrant cells (excluding gaps) was markedly higher than the concurrent control values. There was therefore no evidence of reproducibility of this isolated effect between cultures or between experiments and the observation was not considered biologically relevant.

No increases in the total frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were observed in cultures treated with the test substance in the absence and presence of S-9 in Experiments 1 and 2.

Therefore, the test substance did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of solubility in culture medium both in the absence and the presence of S-9.