Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Mar - 08 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane

EC Number:

286-075-2

EC Name:

Fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane

Cas Number:

85186-89-6

IUPAC Name:

85186-89-6

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 supplemented with 5% (v/v) heat-inactivated horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from rats pretreated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment I:
- 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (8%, v/v))
Experiment II:
- 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (12%, v/v))

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

+ S9: cyclophosphamide, 15 and 5 µg/mL for 3 and 24 h treatment, respectively; - S9: methylmethanesulfonate, 7.5 µg/mL

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Pre-incubation period: No
- Exposure duration: cells were exposed to the test material for 3 h and 24 h in the presence and absence of S9-mix, respectively.
- Expression time (cells in growth medium): For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. For determination of the mutation frequency cells were plated and incubated for 11-12 days. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Several criteria including a concentration-related, or a reproducible increase in mutation frequencies determined a positive result.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 333 µg/mL

RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h respectively with a number of increasing test substance concentrations. The highest concentration tested was 750 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h or 24 h incubation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls

Any other information on results incl. tables

Table 1: Results of experiment 1

Dose (µg/ml)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	mutation frequency x 10E6
					total
Without metabolic activation, 3 h treatment
SC1	100	104	100	100	74
SC2		85			97
0.3	99	98	104	102	74
1	101	102	108	109	71
3	100	101	107	107	94
10	93	98	104	97	67
33	120	94	100	120	63
100	113	101	107	121	61
333*	104	113	120	124	64
750*	405	101	107	112	74
MMS	71	68	72	51	835
With 8% (v/v) metabolic activation, 3 h treatment
SC1	100	70	100	100	65
SC2		69			64
0.3	96	60	86	83	74
1	115	68	98	113	60
3	109	40	57	62	84
10	127	72	104	132	52
33	114	46	66	75	84
100	122	76	108	133	63
333*	115	62	89	102	72
750*	104	58	84	87	53
CP	50	32	45	22	1617

 

Table 2: Results of experiment 2

Dose (µg/ml)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	mutation frequency x 10E6
					total
Without metabolic activation, 24 h treatment
SC1	100	66	100	100	90
SC2		79			75
0.3	112	77	106	119	88
1	116	80	110	128	82
3	117	72	100	117	79
10	120	85	117	140	66
33	114	74	101	116	83
100	121	69	95	115	83
333*	116	70	97	112	70
750*	116	66	91	106	71
MMS	101	49	67	68	1502
With 12% (v/v) metabolic activation, 3 h treatment
SC1	100	93	100	100	80
SC2		93			76
0.3	103	84	90	93	74
1	113	83	89	101	81
3	107	97	104	112	60
10	105	94	101	107	80
33	103	93	100	103	67
100	102	105	114	116	57
333*	106	91	99	104	74
750*	103	93	100	103	73
CP	72	75	81	58	1082

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Applicant's summary and conclusion

Conclusions:

negative