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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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 / strainopen allclose all
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 / 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 resultsopen allclose all
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
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
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
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
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.