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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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,18-dimethyl octadecanedioate
EC Number:
604-582-2
Cas Number:
1472-93-1
Molecular formula:
C20H38O4
IUPAC Name:
1,18-dimethyl octadecanedioate
Details on test material:
- Name of test material (as cited in study report): Octadecanedioic acid, 1,18-dimethyl ester (ODDAME)
- Physical state: white solid
- Analytical purity: 98.8 %
- Lot/batch No.: 1091-4-B
- Storage condition of test material: room temperature in the dark
- Stability under storage conditions: analyses showed stability of the test material over the period it was stored

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal enzyme fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Mutation test - Experiment 1: 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation test - Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
The test item was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully soluble in acetone (100 mg/ml), dimethyl formamide (50 mg/ml) and tetrahydrofuran (200 mg/ml) in solubility checks performed in-house. Acetone was selected as the vehicle.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535 without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537 without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98 without metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
10 µg/plate for WP2uvrA, 1 µg/plate for TA100, 2 µg/plate for TA1535 and 2 µg/plate for TA1537 with metabolic activation
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and preincubation for Experiment 2.

PRELIMINARY TOXICITY TEST
Ten concentrations of the test item formulation and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test item and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 μg/plate because of excessive test item precipitation.

Mutation Test - Experiment 1:
Six concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Weakened TA100 bacterial background lawns were noted in the preliminary toxicity test, therefore an additional dose level and an expanded dose range were selected in order to achieve four non-toxic dose levels and the potential toxic limit of the test item.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the vehicle, test item formulation or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter. Manual counts were performed at 5000 μg/plate because of excessive test item precipitation.

Mutation Test - Experiment 2:
The test item dose range was the same as Experiment 1. An additional dose level and an expanded dose range were again selected in order to achieve four non-toxic dose levels and the potential toxic limit of the test item.

As it is good scientific practice to alter one condition in the replicate assay, the exposure condition was changed from plate incorporation to pre-incubation. The test item formulations and vehicle control were therefore dosed as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test item formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method described in Section 3.5.2.

All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter. Manual counts were again performed at 5000 μg/plate because of excessive test item precipitation.

NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 0.9 to 9 x 10^09 bacteria per mL.



Evaluation criteria:
There were several criteria for determining a positive result:

1). A dose-related increase in mutant frequency over the dose range tested.
2). A reproducible increase at one or more concentrations.
3). Biological relevance against in-house historical control ranges.
4). Statistical analysis of data as determined by UKEMS.
5). Fold increase greater than 2 times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
preliminary test only
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Weakened bacterial background lawns were noted to TA100 at 5000 μg/plate (absence and presence of S9-mix) in the preliminary toxicity test. However, in both Experiments 1 and 2, the test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. The response noted in the preliminary assay was considered spurious and of no biological relevance. The test item was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA100 revertant colony frequency was observed in the absence of S9-mix at 50 μg/plate in Experiment 2. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.1 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test item, octadecanedioic acid, 1,18-dimethyl ester (ODDAME) was considered to be non-mutagenic under the conditions of this test.