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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:
10 July 2009 to 07 August 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19th August 2008 Date of signature: 4th March 2009
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :DVS005u (aka Weston 705)
Description : Clear colourless viscous liquid
Batch number : T167-20090615
Date received : 25 June 2009
Storage conditions :Approximately 4°C in the dark, under nitrogen

Method

Target gene:
Histidine operon for Salmonella
Tryptophan operon for Escherichia
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):
Not applicable
Additional strain / cell type characteristics:
other: Deletion through the excision repair gene which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeablity of the cell wall.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Deletion in an excision repair gene (uvrA-).
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ beta-naphthalflavone
Test concentrations with justification for top dose:
Preliminary toxity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation test - Experiment 1 (Range-finding test): 50, 150, 500, 1500 and 5000 µg/plate

Mutation test - Experiment 2 (Main test): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone

- Justification for choice of solvent/vehicle: The test material was miscible in acetone at 50 mg/ml in solubility checks performed in house. Acetone was therefore selected as the vehicle. The miscibility of dimethyl sulphoxide and water were not investigated after information from the Sponsor indicated that these solvents would be poorly miscible with the test material.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: (ENNG)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix

Migrated to IUCLID6: (9AA)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix

Migrated to IUCLID6: (4NQO)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: (BP)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
With S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 to 72 hours

SELECTION AGENT (mutation assays): not stated in report
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: triplicate plating

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction

OTHER EXAMINATIONS:
- sterility and solubility
Evaluation criteria:
Where possible the first 100 consecutive sell-spread metaphases from each culture were counted. where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) was reported. If a weak or equivocal polyploidy effect was observed then an additional independent evaluation of the frequency f polyploidy cells was considered.
Statistics:
Statistical methods as recommended by the UKEMS can be used as an aid to evaluation.

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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to the maximum recommended dose level of 5000 µg per plate
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to the maximum recommended dose level of 5000 µg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not stated in report
- Effects of osmolality: not stated in report
- Evaporation from medium: not stated in report
- Water solubility: poorly miscible
- Precipitation: An oily precipitate was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
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.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. 

No toxicologically 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.

A small but statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 500 µg/plate in the range-finding test only. However, this increase exhibited no dose-response relationship, exhibited counts within the in-house historical control range for the strain and was non-reproducible in two separate experiments. The increase was, therefore, considered to be biologically irrelevant. 

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

Introduction.Thethod was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It alsoets the requirents 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 both the Ames plate incorporation and pre-incubation methodsat five 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 was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

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 no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate 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 material, either with or without metabolic activation.

A small but statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 500 µg/plate in the range-finding test only. However, this increase exhibited no dose-response relationship, exhibited counts within the in-house historical control range for the strain and was non-reproducible in two separate experiments. The increase was, therefore, considered to be biologically irrelevant. 

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