Fatty acids, C18-unsatd., dimers, polymers with 2-ethylhexanol and neopentyl glycol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Apr - 11 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., dimers, hydrogenated, 2-ethylhexyl esters

EC Number:

500-214-9

EC Name:

Fatty acids, C18-unsatd., dimers, hydrogenated, 2-ethylhexyl esters

Cas Number:

68440-06-2

Molecular formula:

C44H84O4

IUPAC Name:

Fatty acids, C18-unsatd., dimers, hydrogenated, 2-ethylhexyl esters

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: yellow liquid
- Storage condition of test material: room temperature in the dark
- Expiration date of the lot/batch: 05 May 2016

Method

Target gene:

his/trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: All TA strains carry a mutation of the uvr B gene coding for the DNA excision repair system (uvr B) and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: The E.coli tester strain contains an uvr A DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats induced with phenobarbitone/β-naphthoflavone

Test concentrations with justification for top dose:

Experiment I:
- 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (with and without metabolic activation)
Experiment II:
- 15, 50, 150, 500, 1500 and 5000 μg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and DMSO at 50 mg/mL, but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

- S9: ENNG: 2 µg/plate (WP2uvr A), 3 µg/plate (TA100), 5 µg/plate (TA1535); 9AA: 80 µg/plate (TA1537); 4NQO: 0.2 µg/plate (TA98); +S9: 2AA: 1 µg/plate (TA100), 2 µg/plate (TA1535, TA1537), 10 µg/plate (WP2uvr A); BP: 5 µg/plate (TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation, pre-incubation

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity (relative total growth) and precipitate by using a dissecting microscope.

Evaluation criteria:

A test system is considered as mutagenic if there is
- a dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979)
- a reproducible increase at one or more concentrations
- biological relevance against in-house historical control ranges
- statistical analysis of data as determined by UKEMS (Mahon et. al., 1989)
- a fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: S. typhimurium TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TESTSPECIFIC CONFOUNDING FACTORS: Precipitation of the test material was recorded at concentrations equal or greater than 1500 μg/plate in both independent experiments (with and without metabolic activation).

COMPARISON WITH HISTORICAL CONTROL DATA: The results were within range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material did not induce cytotoxicity in any of the tested strains at any tested concentration.

Any other information on results incl. tables

Table 1: Test results: Experiment 1 – Plate incorporation

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
–	0	124 ± 5.5	17 ± 2.1	32 ± 6.1	21 ± 2.5	11 ± 2.6
–	1.5	126 ± 13.1	17 ± 0.6	24 ± 5.8	21 ± 4.7	12 ± 3.0
–	5	130 ± 12.1	14 ± 2.3	24 ± 11.5	24 ± 6.1	14 ± 1.2
–	15	115 ± 3.1	17 ± 3.8	25 ± 1.7	21 ± 9.5	14 ± 2.6
–	50	126 ± 13.1	15 ± 3.5	23 ± 2.5	23 ± 2.5	16 ± 0.0
–	150	123 ± 4.0	13 ± 5.6	24 ± 5.0	23 ± 9.5	16 ± 0.6
–	500	125 ± 9.5	15 ± 6.0	20 ± 3.5	22 ± 1.2	13 ± 2.0
–	1500 P	121 ± 11.0	17 ± 4.9	24 ± 3.5	20 ± 0.6	13 ± 5.3
–	5000 P	121 ± 9.5	11 ± 1.7	24 ± 5.1	23 ± 7.6	12 ± 4.0
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	983 ± 56.6	271 ± 9.5	1220 ± 41.7	192 ± 14.6	1719 ± 200.1
+	0	119 ± 8.5	15 ± 3.5	24 ± 4.6	24 ± 7.5	15 ± 3.5
+	1.5	115 ± 3.6	17 ± 6.7	32 ± 5.9	21 ± 1.7	15 ± 5.1
+	5	122 ± 7.9	9 ± 1.5	34 ± 6.0	29 ± 5.3	12 ± 1.0
+	15	140 ± 7.2	11 ± 1.5	24 ± 2.3	23 ± 2.1	15 ± 3.5
+	50	126 ± 8.0	10 ± 3.1	29 ± 10.5	23 ± 8.2	13 ± 3.5
+	150	133 ± 8.7	16 ± 4.2	24 ± 3.6	24 ± 8.1	9 ± 3.8
+	500	133 ± 2.1	11 ± 3.5	20 ± 1.0	27 ± 4.7	15 ± 3.5
+	1500 P	121 ± 14.9	12 ± 4.5	28 ± 6.1	24 ± 7.0	15 ± 2.1
+	5000 P	118 ± 20.4	10 ± 2.0	27 ± 5.0	26 ± 7.4	13 ± 6.1
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1878 ± 130.8	330 ± 12.2	331 ± 24.2	239 ± 14.4	406 ± 10.4

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4NQO: 4-nitroquinoline-N-oxide

9AA: 9-aminoacridine

2AA: 2-aminoanthracene

BP: Benzo(a)pyrene

P: Precipitate

Table 2: Test results: Experiment 2 – Pre-incubation

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
–	0	138 ± 28.6	18 ± 4.2	29 ± 2.5	30 ± 7.0	20 ± 3.5
–	15	137 ± 24.9	15 ± 5.3	29 ± 3.2	29 ± 5.3	28 ± 2.3
–	50	119 ± 3.6	22 ± 4.2	33 ± 5.5	29 ± 4.0	24 ± 4.0
–	150	138 ± 24.9	16 ± 3.5	24 ± 6.4	29 ± 7.6	21 ± 5.5
–	500	136 ± 9.3	21 ± 6.4	27 ± 5.9	28 ± 4.2	20 ± 8.0
–	1500 P	131 ± 17.9	22 ± 6.1	26 ± 4.9	27 ± 4.5	22 ± 9.5
–	5000 P	135 ± 12.5	17 ± 4.0	33 ± 4.9	29 ± 6.4	20 ± 2.3
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	1094 ± 52.9	1087 ± 103.1	1277 ± 159.0	258 ± 24.7	1318 ± 75.5
+	0	146 ± 15.8	17 ± 2.3	42 ± 6.6	29 ± 5.0	27 ± 3.5
+	15	134 ± 13.5	16 ± 5.3	28 ± 6.1	33 ± 2.9	25 ± 1.5
+	50	132 ± 1.5	13 ± 2.1	34 ± 8.3	29 ± 9.8	18 ± 2.1
+	150	126 ± 10.1	14 ± 2.5	35 ± 5.7	33 ± 3.8	18 ± 4.0
+	500	118 ± 12.7	16 ± 3.1	31 ± 9.2	30 ± 7.5	19 ± 5.0
+	1500 P	117 ± 21.7	15 ± 6.4	33 ± 0.6	34 ± 6.2	19 ± 6.4
+	5000 P	117 ± 23.9	12 ± 5.0	36 ± 8.3	36 ± 3.8	21 ± 4.4
Positive controls, +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	921 ± 86.1	297 ± 6.7	210 ± 54.3	462 ± 58.0	556 ± 28.2

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4NQO: 4-nitroquinoline-N-oxide

9AA: 9-aminoacridine

2AA: 2-aminoanthracene

BP: Benzo(a)pyrene

P: Precipitate