2-({3-(octadecanoyloxy)-2,2-bis[[](octadecanoyloxy)methyl]propoxy}methyl)-2-[[](octadecanoyloxy)methyl]propane-1,3-diyl dioctadecanoate

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:

8 Feb - 15 Apr 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted in 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C5-9, hexaesters with dipentaerythritol

EC Number:

267-021-7

EC Name:

Fatty acids, C5-9, hexaesters with dipentaerythritol

Cas Number:

647028-25-9

Molecular formula:

Not available (UVCB)

IUPAC Name:

647028-25-9

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Eagle’s MEM medium supplemented with:
-sodium bicarbonate
-HEPES buffer
-L-glutamine
-penicillin/streptomycin
-amphotericin B
-15% foetal calf serum (FCS)

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

First experiment:
4(20) h without and with S9: 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL

Second experiment:
20 (20) h without S9: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL
4(20) h with S9: 156.25; 312.5; 625; 1250; 2500 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide, 25 µg/mL in phopsphate buffered saline, +S9; ethyl methanesulphonate , 750 µg/mL and 500 µg/mL (Experiment 1 and 2 respectively), -S9 in dimethyl sulphoxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (experiment 1, with and without S9); 4 and 20 h (experiment 2, with and without S9, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 20h

SPINDLE INHIBITOR (cytogenetic assays): colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): Gurrs Giemsa R66 5%

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 2000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test or Chi-squared test.

Results and discussion

Test results

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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: there was no observable change in pH
- Effects of osmolality: the osmolality did not increase by more than 50 mOSM
- Precipitation: a cloudy appearance of the test material was noted at all dose levels in both treatment groups, after four hours exposure

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: in Experiment 1 and 2 no dose-related toxicity was observed, a 50% mitotic inhibition was not achieved and the precipitate of the test item had no effect on the toxicity response curve. Thus, 1250, 2500 and 5000 µg/mL dose levels were selected for chromosome analysis.

Any other information on results incl. tables

Table 1: Test results of experiment 1

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 4 h, fixation time 20 h, without S9 mix
Acetone	0	100	3.0	2.5
EMS	750	50	20	12.5
Test substance	1250	78	3.0	2.0
	2500	83	5.5	1.0
	5000	70	2.0	1.5
Exposure period 4 h, fixation time 20 h, with S9 mix
Acetone	0	100	3.5	2.0
CP	25	29	14.5	9.0
Test substance	1250	87	3.0	3.0
	2500	125	3.5	0.5
	5000	117	1.5	1.0

EMS: ethyl methanesulphonate;

CP: Cyclophosphamide (positive controls)

 

Table 2: Test results of experiment 2

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 20 h, fixation time 20 h, without S9 mix
Acetone	0	100	1.5	0.5
EMS	750	58	36.7	20.0
Test substance	1250	123	2.0	0.5
	2500	110	0.5	0.0
	5000	99	2.0	0.0
Exposure period 4 h, fixation time 20 h, with S9 mix
Acetone	0	100	2.0	0.5
CP	25	25	19.0	11.5
Test substance	1250	89	1.5	0.0
	2500	89	1.0	0.0
	5000	112	1.0	0.0

EMS: ethyl methanesulphonate;

CP: Cyclophosphamide (positive controls)

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative