Didocosyl sebacate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jul - 17 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Didocosyl sebacate

EC Number:

255-730-4

EC Name:

Didocosyl sebacate

Cas Number:

42233-75-0

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Decanedioic acid, diesters with Fatty alcohols C20-22 (even numbered)

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Physical state: white solid
- Storage condition of test material: room temperature in the dark
- Expiration date of the lot/batch: 30 Jun 2013

Method

Target gene:

S. typhimurium strains: his operon
E. coli: 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 postmitochondrial fraction (S9-mix), prepared from the livers of rats induced with phenobarbitone/β-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test:
- 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Experiment I:
- 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)
Experiment II:
- 50, 150, 500, 1500 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: Tetrahydrofuran was selected as the solvent of choice based on insolubility in water and limited solubility in DMSO, dimethyl formamide, acetonitrile and acetone.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N´-nitro-N-nitrosoguanidine (ENNG), 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA), Benzo(a)pyrene (BP)

Remarks:

other:-S9-mix: ENNG: 2, 3, 5 µg/plate (E. coli, TA 100, TA 1535); 9AA: 80 µg/plate (TA 1537); 4NQO: 0.2 µg/plate (TA 98); +S9-mix: 2AA: 1, 2, 10 µg/plate (TA 100, TA 1535 + TA 1537, E.coli); BP: 5 µg/plate (TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- A cytotoxicity pre-experiment was carried out with the tester strains TA 100 and E. coli WP2 uvr A to determine the non-toxic concentrations for the main genotoxicity experiments.
- 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 clear and dose-related increase in the number of revertants and/or a
- a reproducible increase at one or more concentrations
- biological relevance against in-house historical control ranges
- statistical analysis of data as determined by UKEMS
- fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)
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

Species / strain:

E. coli WP2 uvr A

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

Test results 2

Species / strain:

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

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:

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

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: Precipitation of the test material was recorded at concentrations equal or greater than 500 µg/plate.

RANGEFINDING/SCREENING STUDIES: In the preliminary toxicity assay, the maximum dose tested was 5000 µg/plate. The test item was non-toxic to the strains of bacteria used (TA 100 and E.coli WP2 uvr A). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test results of experiment I (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
		TA 100	TA 1535	WP2 uvr A	TA 98	TA 1537
–	0	116 ±9.9	22 ± 3.1	40 ± 5.0	29 ± 3.2	14 ± 3.5
–	50	122 ± 3.8	21 ± 1.2	35 ± 3.7	25 ± 4.0	13 ± 1.5
–	150	115 ± 4.5	24 ± 3.2	28 ± 4.7	26 ± 2.0	15 ± 1.5
–	500 P	114 ± 7.0	18 ± 4.4	42 ± 3.6	25 ± 5.9	14 ± 2.1
–	1500 P	117 ± 4.9	22 ± 1.5	37 ± 3.6	28 ± 6.1	17 ± 2.0
–	5000 P	112 ± 7.6	21 ± 3.5	42 ± 3.0	30 ± 2.0	15 ± 4.2
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)	710 ± 45.4	557 ± 20.3	1056 ± 164.5	201 ± 15.7	1207 ± 507.6
+	0	110 ± 2.1	23 ± 3.0	49 ± 5.3	32 ± 6.1	15 ± 2.5
+	50	116 ± 5.0	21 ± 2.5	48 ± 8.0	31 ± 4.4	12 ± 3.5
+	150	103 ± 3.8	22 ± 5.9	41 ± 6.0	30 ± 4.0	15 ± 4.4
+	500 P	112 ± 14.8	21 ± 2.6	47 ± 6.0	33 ± 0.6	14 ± 1.5
+	1500 P	112 ± 11.0	19 ± 1.5	45 ± 3.8	33 ± 1.0	13 ± 3.1
+	5000 P	113 ± 6.1	21 ± 0.6	50 ± 2.6	33 ± 1.7	14 ± 2.3
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)	1171 ± 45.9	207 ± 28.0	377 ± 46.3	300 ± 48.3	151 ± 9.0

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 of experiment II (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
		TA 100	TA 1535	WP2 uvr A	TA 98	TA 1537
–	0	126 ± 7.0	13 ± 6.1	20 ± 5.1	18 ± 5.5	14 ± 5.6
–	50	120 ± 12.2	11 ± 2.1	19 ± 3.5	20 ± 5.7	12 ± 6.6
–	150	117 ± 14.4	13 ± 3.1	22 ± 7.5	19 ± 6.5	17 ± 1.2
–	500 P	117 ± 5.2	13 v 0.0	21 ± 3.2	18 ± 2.1	16 ± 3.5
–	1500 P	110 ± 21.2	17 ± 5.0	24 ± 3.2	21 ± 1.2	15 ± 4.9
–	5000 P	104 ± 14.8	12 ± 0.6	23 ± 3.2	17 ± 1.2	12 ± 2.1
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)	710 ± 49.7	415 ± 28.6	506 ± 8.3	147 ± 21.6	457 ± 166.5
+	0	113 ± 13.5	14 ± 1.0	24 ± 4.2	25 ± 2.1	12 ± 1.5
+	50	109 ± 8.9	12 ± 2.3	21 ± 4.6	23 ± 5.9	13 ± 2.9
+	150	111 ± 9.3	13 ± 2.0	25 ± 3.5	17 ± 6.7	10 ± 0.6
+	500 P	111 ± 9.1	14 ± 1.7	23 ± 4.4	23 ± 5.3	11 ± 2.6
+	1500 P	104 ± 12.4	11 ± 0.6	27 ± 2.0	25 ± 1.0	13 ± 1.5
+	5000 P	98 ± 2.0	13 ± 1.2	20 ± 2.0	23 ± 5.1	14 ± 1.5
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)	1109 ± 68.7	458 ± 17.0	254 ± 16.1	275 ± 15.3	187 ± 13.7

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = Benzo(a)pyrene

P = Precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of the study the test material is considered not to be mutagenic in this bacterial mutagenicity test in vitro.