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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:
between 21 May 2009 and 18 June 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of fatty acids, C18-unsatd., dimers, partly hydrogenated with N,N-dimethyl-1,3-propanediamine
EC Number:
937-688-5
Cas Number:
1391530-05-4
Molecular formula:
C46H82N4O2 to C46H94N4O2
IUPAC Name:
Reaction products of fatty acids, C18-unsatd., dimers, partly hydrogenated with N,N-dimethyl-1,3-propanediamine
Details on test material:
- Substance type: UVCB
- Physical state: amber coloured viscous liquid
- Analytical purity: > 97.5%
- Lot/batch No.: CM71117612
- Expiration date of the lot/batch: 14 April 201 0
- Storage condition of test material: room temperature in the dark

Method

Target gene:
Not required
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):
Non-mammalian study
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Non-mammalian study
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: Range-finding test
Salmonella strains: 5, 15, 50, 150, 500, 1500, 5000 µg/plate
E.coli strain: 50, 150, 500, 1500 µg/plate

Experiment 2: Main test
All Salmonella strains (with and without S9) except TA100 (without S9): 15, 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strain TA100 (without S9): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
E.coli strain WP2uvrA- : 50, 150, 500, 1500, 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Remarks:
Concurrent
Positive controls:
yes
Remarks:
Concurrent
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Remarks:
Concurrent
Positive controls:
yes
Remarks:
Concurrent
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Remarks:
Concurrent
Positive controls:
yes
Remarks:
Concurrent
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

The assay was performed by mixing 0.1 ml of bacterial culture (TAI00 or WP2uvrA-), 0.1 ml of the substance formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). In total ten concentrations of the test material and a vehicle control (acetone) were tested. In addition, 0.1 ml of the maximum concentration of the test material 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 material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
DURATION
- Preincubation period: N/A
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9


NUMBER OF CELLS EVALUATED:
Cell viability at the end of pre-culture
RANGE FINDING TEST
S. typhimurium TA 100 = 1.3 x 10^9/ml
S. typhimurium TA1535 = 1.4 x 10^9/ml
S. typhimurium TA98 = 1.2 x 10^9/ml
S. typhimurium TA1537 = 1.8 x 10^9/ml
E. coli WP2uvrA- = 3.0 x 10^9/ml

MAIN TEST
S. typhimurium TA 100 = 2.0 x 10^9/ml
S. typhimurium TA1535 = 1.4 x 10^9/ml
S. typhimurium TA98 = 2.2 x 10^9/ml
S. typhimurium TA1537 = 1.1 x 10^9/ml
E. coli WP2uvrA- = 1.1 x 10^9/ml



DETERMINATION OF CYTOTOXICITY
- Method: N/A


OTHER EXAMINATIONS:
N/A


OTHER:
Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland D J (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

The substance would be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
NDA

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was toxic at and above 500 μg/plate to TA100 and was non-toxic to WP2uvrA-.

 

Mutation Test

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The test material caused a visible reduction in the growth of the bacterial background lawns of all of theSalmonellastrains both with and without metabolic activation, initially from 1500 μg/plate. The sensitivity of theSalmonellatester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. No toxicity was noted forEscherichia colistrain WP2uvrA- at any test material dose level either in the presence or absence of S9. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 μg/plate. A precipitate (particulate in appearance) was observed at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

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.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 μg/plate, depending on bacterial strain type. The experiment was repeated on a separate day using a similar dose range to the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

Results.The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawns of all of theSalmonellastrains both with and without metabolic activation, initially from 1500 μg/plate. The sensitivity of theSalmonellatester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. No toxicity was noted forEscherichia colistrain WP2uvrA- at any test material dose level either in the presence or absence of S9. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 μg/plate. A precipitate (particulate in appearance) was observed at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.