Octadecanoic acid, C16-18-alkyl esters

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:

26 Sep - 18 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

National Institute of Pharmacy, Budapest, Hungary

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Docosyl stearate

EC Number:

244-971-0

EC Name:

Docosyl stearate

Cas Number:

22413-03-2

Molecular formula:

C40H80O2

IUPAC Name:

docosyl octadecanoate

Method

Target gene:

his operon (for S. typhimurium strains), trp operon (for E. coli strains)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment for toxicity:
10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA98 and TA100

Based on the results of the pre-experiment the following concentrations were chosen for the main experiment for all strains:
First experiment: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate with and without metabolic activation
Second experiment: 1.581, 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Solvents used: DMF

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine: -S9: 4 µg/plate for TA98; 2-aminoanthracene: +S9: 2 µg/plate for all S. typhimurium strains and 50 µg/plate for WP2uvrA

Remarks:

The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, 1st experiment); preincubation (2nd experiment)

DURATION
- Preincubation period: 20 min (preincubation method)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: number of revertant colonies

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor (mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate) values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than two times higher than the reversion rate of the negative (solvent) control.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Reduced background lawn was observed at 1581 µg/plate without metabolic activation in the pre-incubation method.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Slightly reduced and reduced background lawn was observed at 1581 and 5000 µg/plate without metabolic activation in the pre-incubation method, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Reduced background lawn was observed at 5000 µg/plate without metabolic activation in the pre-incubation method.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Slightly reduced background lawn was observed at 158.1 µg/plate and higher without metabolic activation in the pre-incubation method.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate or slight precipitate was observed in all tester strains at 1581 and 5000 µg/plate with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: Lower or slightly reduced numbers of revertant colonies (compared to the solvent control plates) were observed in Salmonella typhimurium TA98 bacterial strain with metabolic activation, although no effect on the background lawn was observed. However, they had no biological significance, and this fact did not affect the proper dose selection.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control values were within the range of the historical control data and therefore considered to be valid (please refer to table 1 under "any other information on materials and methods incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data and therefore considered to be valid (please refer to table 1 under "any other information on materials and methods incl. tables").

Any other information on results incl. tables

Table 2: Results of the pre-experiment for toxicity

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	17.0	20.3	121.0	125.3
	MF	0.98	0.51	1.08	0.87
Distilled water control	Mean	-	-	114.7	-
	MF	-	-	1.02	-
DMSO control	Mean	19.7	19.7	-	153.0
	MF	1.13	0.49	-	1.06
DMF control	Mean	17.3	40.0	112.3	143.7
	MF	1.00	1.00	1.00	1.00
5000	Mean	15.0	25.7	96.0	103.3
	MF	0.87	0.64	0.85	0.72
2500	Mean	17.3	18.3	122.0	117.7
	MF	1.00	0.46	1.09	0.82
1000	Mean	15.0	17.7	115.0	124.7
	MF	0.87	0.44	1.02	0.87
316	Mean	15.0	18.3	123.0	141.0
	MF	0.87	0.46	1.09	0.98
100	Mean	15.7	17.0	134.0	149.3
	MF	0.90	0.43	1.19	1.04
31.6	Mean	14.0	21.7	121.0	130.7
	MF	0.81	0.54	1.08	0.91
10	Mean	17.0	24.0	134.3	123.7
	MF	0.98	0.60	1.20	0.86
NPD (4 µg)	Mean	235.3	-	-	-
	MF	11.97	-	-	-
2AA (2 µg)	Mean	-	2444.0	-	2794.7
	MF	-	124.27	-	18.27
SA (2 µg)	Mean	-	-	1273.3	-
	MF	-	-	11.10	-

NPD: 4-nitro-1,2-phenylene-diamine

2AA: 2-aminoanthracene

SA: sodium azide

MF: mutation factor

Table 3: Summary of Experiment I (plate incorporation)

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	25.7	29.3	111.0	127.3	9.0	10.0	8.0	9.7	25.7	39.0
	MF	1.60	1.06	0.89	0.91	0.87	1.20	0.75	1.21	1.40	1.27
Distilled water control	Mean	-	-	124.0	-	7.3	-	-	-	25.3	-
	MF	-	-	0.99	-	0.71	-	-	-	1.38	-
DMSO control	Mean	21.0	36.3	-	210.7	-	7.7	8.0	8.3	-	46.7
	MF	1.31	1.31	-	1.50	-	0.92	0.75	1.04	-	1.52
DMF control	Mean	16.0	27.7	125.3	140.3	10.3	8.3	10.7	8.0	18.3	30.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	21.0	26.3	104.7	123.3	8.3	11.3	9.7	7.7	23.0	41.3
	MF	1.31	0.95	0.84	0.88	0.81	1.36	0.91	0.96	1.25	1.35
1581	Mean	29.3	26.3	115.3	126.0	11.7	11.0	7.0	8.7	27.0	33.7
	MF	1.83	0.95	0.92	0.90	1.13	1.32	0.66	1.08	1.47	1.10
500	Mean	22.3	26.0	123.0	151.7	7.7	6.0	11.7	9.0	27.7	39.7
	MF	1.40	0.94	0.98	1.08	0.74	0.72	1.09	1.13	1.51	1.29
158.1	Mean	25.3	30.0	133.0	151.0	7.3	7.0	7.7	8.3	24.3	36.3
	MF	1.58	1.08	1.06	1.08	0.71	0.84	0.72	1.04	1.33	1.18
50	Mean	23.7	26.0	130.0	151.7	6.7	8.3	8.0	8.0	29.3	33.0
	MF	1.48	0.94	1.04	1.08	0.65	1.00	0.75	1.00	1.60	1.08
15.81	Mean	21.0	22.0	122.7	134.7	5.3	10.0	7.3	10.3	30.7	39.3
	MF	1.31	0.80	0.98	0.96	0.52	1.20	0.69	1.29	1.67	1.28
5	Mean	19.3	25.3	119.3	139.0	4.7	9.3	8.3	5.3	28.7	32.7
	MF	1.21	0.92	0.95	0.99	0.45	1.12	0.78	0.67	1.56	1.07
NPD (4 µg)	Mean	289.3	-	-	-	-	-	-	-	-	-
	MF	13.78	-	-	-	-	-	-	-	-	-
2AA (2 µg)	Mean	-	2400.0	-	2273.3	-	214.0	-	125.3	-	-
	MF	-	66.06	-	10.79	-	27.91	-	15.04	-	-
2AA (50 µg)	Mean	-	-	-	-	-	-	-	-	-	253.3
	MF	-	-	-	-	-	-	-	-	-	5.43
SA (2 µg)	Mean	-	-	1244.0	-	1101.3	-	-	-	-	-
	MF	-	-	10.03	-	150.18	-	-	-	-	-
9AA (50 µg)	Mean	-	-	-	-	-	-	509.3	-	-	-
	MF	-	-	-	-	-	-	63.67	-	-	-
MMS (2 µL)	Mean	-	-	-	-	-	-	-	-	1037.3	-
	MF	-	-	-	-	-	-	-	-	40.95	-

NPD: 4-nitro-1,2-phenylene-diamine

9AA: 9-aminoacridine

2AA: 2-aminoanthracene

SA: sodium azide

MMS: methylmethanesulfonate

MF: mutation factor

Table 4: Summary of Experiment II (pre-incubation method)

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	22.0	26.7	110.0	122.3	7.0	11.0	5.7	12.0	34.0	38.3
	MF	1.10	1.10	1.47	1.22	0.72	1.10	0.81	1.24	1.00	1.02
Distilled water control	Mean	-	-	119.0	-	7.0	-	-	-	44.3	-
	MF	-	-	1.59	-	0.72	-	-	-	1.30	-
DMSO control	Mean	21.7	29.3	-	132.0	-	12.7	8.0	8.0	-	42.3
	MF	1.08	1.21	-	1.31	-	1.27	1.14	0.83	-	1.12
DMF control	Mean	20.0	24.3	75.0	100.7	9.7	10.0	7.0	9.7	34.0	37.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	12.3	21.0	57.0	94.3	7.7	9.3	5.3	11.3	29.7	28.7
	MF	0.62	0.86	0.76	0.94	0.79	0.93	0.76	1.17	0.87	0.76
1581	Mean	18.0	21.3	81.0	99.7	6.7	6.7	5.3	8.7	20.3	32.0
	MF	0.90	0.88	1.08	0.99	0.69	0.67	0.76	0.90	0.60	0.85
500	Mean	14.3	20.3	76.3	99.7	7.7	9.0	8.0	8.0	30.0	30.3
	MF	0.72	0.84	1.02	0.99	0.79	0.90	1.14	0.83	0.88	0.81
158.1	Mean	16.7	18.0	52.0	91.3	6.0	8.0	2.0	6.3	31.0	35.0
	MF	0.83	0.74	0.69	0.91	0.62	0.80	0.29	0.66	0.91	0.93
50	Mean	10.7	24.3	83.0	93.0	4.3	7.7	2.7	9.3	22.3	36.3
	MF	0.53	1.00	1.11	0.92	0.45	0.77	0.38	0.97	0.66	0.96
15.81	Mean	16.0	23.7	58.3	101.3	7.0	10.3	5.7	7.3	27.0	36.7
	MF	0.80	0.97	0.78	1.01	0.72	1.03	0.81	0.76	0.79	0.97
5	Mean	14.7	17.7	80.7	91.0	5.3	7.3	6.3	7.7	24.3	32.3
	MF	0.73	0.73	1.08	0.90	0.55	0.73	0.90	0.79	0.72	0.86
1.581	Mean	15.3	23.7	63.3	97.0	5.3	7.3	7.0	5.7	23.0	32.7
	MF	0.77	0.97	0.84	0.96	0.55	0.73	1.00	0.59	0.68	0.87
NPD (4 µg)	Mean	298.7	-	-	-	-	-	-	-	-	-
	MF	13.78	-	-	-	-	-	-	-	-	-
2AA (2 µg)	Mean	-	2374.7	-	2468.0	-	192.3	-	207.3	-	-
	MF	-	80.95	-	18.70	-	15.18	-	25.92	-	-
2AA (50 µg)	Mean	-	-	-	-	-	-	-	-	-	235.3
	MF	-	-	-	-	-	-	-	-	-	5.56
SAZ (2 µg)	Mean	-	-	1202.7	-	1134.7	-	-	-	-	-
	MF	-	-	10.11	-	162.10	-	-	-	-	-
9AA (50 µg)	Mean	-	-	-	-	-	-	364.0	-	-	-
	MF	-	-	-	-	-	-	45.50	-	-	-
MMS (2 µL)	Mean	-	-	-	-	-	-	-	-	1102.7	-
	MF	-	-	-	-	-	-	-	-	24.87	-

NPD: 4-nitro-1,2-phenylene-diamine

9AA: 9-aminoacridine

2AA: 2-aminoanthracene

SA: sodium azide

MMS: methylmethanesulfonate

MF: mutation factor

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study, the test substance did not show mutagenic properties in S. typhimurium TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2 uvrA with and without metabolic activation.