Docosyl docosanoate

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:

30 Nov - 13 Dec 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Docosyl docosanoate

EC Number:

241-646-5

EC Name:

Docosyl docosanoate

Cas Number:

17671-27-1

Molecular formula:

C44H88O2

IUPAC Name:

docosyl docosanoate

Test material form:

solid

Method

Target gene:

his operon (for S. typhimurium strains) and 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

Details on mammalian cell type (if applicable):

CELLS USED
- Source: Trinova Biochem GmbH, Giessen, Germany

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source: Trinova Biochem GmbH, Giessen, Germany

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Type: Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 80 mg/kg bw phenobarbital i.p. (Desitin, Hamburg, Germany) and 13-naphthoflavone p.o. (Aldrich, Steinheim, Germany) each on three consecutive days
- source of S9 : male Wistar Hanlbm rats, 8 - 12 weeks old, approx. 220 - 320 g
- quality controls of S9: checked regularly

Test concentrations with justification for top dose:

Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate

Concentrations in Experiment II were chosen as no toxic effects were observed at 5000 µg/plate in Experiment I.

Vehicle / solvent:

- Vehicle used: acetone (MERCK, Darmstadt, Germany, purity > 99%)

- Justification for choice of solvent/vehicle: Chosen based on solubility properties and its relative non-toxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation, Experiment I) and preincubation (Experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 1 h at 37 °C (Experiment II)
- Exposure duration/duration of treatment: 48 h at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met.

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Mean values and standard deviation were calculated.

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:

Precipitation at and above 333 µg/plate in experiment I and at and above 33 µg/plate in experiment II, each +/- S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

cytotoxicity

Remarks:

In experiment II at and above 1000 µg/plate, with and without S9 mix; precipitation at and above 333 µg/plate in experiment I and at and above 33 µg/plate in experiment II, each +/- S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

cytotoxicity

Remarks:

In experiment II at and above 2500 µg/plate, without S9 mix; precipitation at and above 333 µg/plate in experiment I and at and above 33 µg/plate in experiment II, each +/- S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

Precipitation at and above 333 µg/plate in experiment I and at and above 33 µg/plate in experiment II, each +/- S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

Precipitation at and above 333 µg/plate in experiment I and at and above 33 µg/plate in experiment II, each +/- S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item is poorly soluble in water; hence, DMSO was chosen to prepare the dosing suspensions.
- Precipitation and time of the determination: The test item precipitated in the overlay agar in the test tubes at and above 1000 µg/plate in Experiment I and at and above 2500 µg/plate in Experiment II. Precipitation of the test item was observed on the incubated agar plates at and above 333 µg/plate in Experiment I and at and above 33 µg/plate in experiment II with and without metabolic activation. The undissolved particles had no effect on the data recording.

RANGE-FINDING/SCREENING STUDIES:
In the range finding test, the substance was tested up to 5000 µg/plate in the absence and presence of S9 mix in all S. typhimurium and E. coli strains. No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed. The highest concentration analysed was selected based on the solubility of the test substance in the cell culture medium. Since the range finding experiment was conducted using the acutal experimental conditions and fulfilled all acceptability and evaluation criteria, it was considered as Experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in a separate pdf document attached under 'Attached background material'.

Ames test:
- Individual plate counts and mean number of revertant colonies per plate and standard deviations are reported in a separate pdf document attached under 'Attached background material'.

HISTORICAL CONTROL DATA
- Positive and negative (solvent/vehicle) historical control data are reported in Table 1 under 'Any other information on results incl. tables'.

Any other information on results incl. tables

Table 1: Historical control data

Strain	Control	without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	17	4.51	7	40	21	4.95	8	41
	Negative control	17	4.57	9	34	21	5.55	9	42
	Positive control	1878	203.83	852	2347	270	83.95	98	636
TA1537	Solvent control	11	2.91	6	23	16	4.19	6	35
	Negative control	11	2.97	6	24	17	4.97	7	37
	Positive control	125	41.01	75	424	180	64.61	73	475
TA98	Solvent control	32	6.78	18	66	40	6.24	24	64
	Negative control	35	6.67	17	62	41	6.56	23	67
	Positive control	S-34	163.95	172	1916	1193	491.42	184	2759
TA 100	Solvent control	138	25.59	84	213	157	27.89	94	254
	Negative control	145	21.66	97	210	161	25.76	94	217
	Positive control	1953	492.35	572	2943	1763	713.99	542	3886
WP2uvrA	Solvent control	50	8.32	32	82	63	8.86	39	90
	Negative control	50	7 .89	34	76	63	8.53	44	89
	Positive control	913	460.35	191	2023	327	128.21	161	1345

Mean: mean value of revertants/plate

SD: standard deviation

Min: minimal value

Max: maximal value

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

A reliable study conducted in accordance with OECD guideline 471 and GLP found the test material not to induce gene mutation in bacteria. No base substitution or frame shift mutations were detected in S. typhimurium and E. coli strains.