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Genetic toxicity: in vitro

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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:
The experimental phase of this study was performed between 30 October 2012 and 28 January 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Esterification products of Fatty acids C18 unsaturated and triethanolamine
EC Number:
939-649-8
Cas Number:
1474044-69-3
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Esterification products of Fatty acids C18 unsaturated and triethanolamine
Test material form:
liquid: viscous

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
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
Experiment 1:
Salmonella strains (with and without S9 mix): 15, 50, 150, 500, 1500 and 5000 µg/plate
E.coli WP2uvrA (with and without S9 mix: 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2 (Main test):
5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 for all bacterial tester strains
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of bacterial strains
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
The positive controls ENNG, 9AA and 4NQO were used in the series of plates without S9-mix.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of bacterial strains
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
The positive controls 2AA and BP were used in the series of plates with S9-mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
-All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
-All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
-All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per ml.
-Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
-There should be a minimum of four non-toxic test item dose levels.
-There should be no evidence of excessive contamination.

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
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 (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical analysis of data as determined by UKEMS.

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
Remarks:
No significant increases in the frequency of revertant colonies
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See discussion of results for details
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (greasy in appearance) was noted at 5000 µg/plate. This did not prevent the scoring of revertant colonies.


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test: The test item induced toxicity was weakended background lawns to TA100 at 5000 µg/plate (presence and absense of S9-mix) and was non-toxic to WP2uvrA. The test item formulation and S9-mix used in this experiment were both shown to be sterile.

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). 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) are presented in Table 1 (attached background material) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4 (see attached background material for Tables and Figures).

The test item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of TA100, TA1535 and TA1537 (absence of S9-mix only), initially from 1500 µg/plate. Weakened lawns were also noted for TA100 (presence of S9-mix) at 5000 µg/plate using the plate incorporation modification and TA98 (absence of S9-mix) at the same dose level employing pre-incubation methodology. No toxicity either as weakened bacterial background lawns or reductions in colony frequency were noted for any of the remaining tester strains. The test item was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (greasy in appearance) was noted at 5000 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 item, either with or without metabolic activation or exposure method.

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

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

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, 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 harmonized guidelines.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods 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 15 and 5000 µg/plate,depending on bacterial strain type. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range (5 to 5000 µg/plate), fresh cultures of the bacterial strains and fresh test item formulations.

 

Additional dose levels were included and an expanded dose range was selected in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Results

The vehicle (dimethyl sulphoxide) 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 item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of TA100, TA1535 and TA1537 (absence of S9-mix only), initially from 1500µg/plate.Weakened lawns were also noted for TA100 (presence of S9-mix) at 5000µg/plateusing the plate incorporation modification and TA98 (absence of S9-mix) at the same dose level employing pre-incubation methodology. No toxicity either as weakened bacterial background lawns or reductions in colony frequency were noted for any of the remaining tester strains. The test item was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (greasy in appearance) was noted at 5000 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 item, either with or without metabolic activation or exposure method.

Conclusion

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

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