Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16/06/2015 - 13/07/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Japanese Regulatory Authorities including METI, MHLW and MAFF
GLP compliance:
yes (incl. certificate)
Remarks:
analysis for concentration, homogeneity and stability f the test item is not a requirement of the test guidelines and is not determined.
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: E15-01025
- Expiration date of the lot/batch: Not supplied
- Source: University of California, Berkeley, on culture discs on 4 August 1995 + British Industrial Biological Research Association on nutrient agar plate on 17 august 1987.
- Storage conditions: -196 °C in Statebourne loquid nitrogen freezer, model SXR34
- Purity test date: 100%
- Physical state/appearance: black powder
- Storage conditions: room temperature in the dark

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
frame shift mutations + base pair substitutions
Additional strain characteristics:
other: rfa-, uvrB-. for TA98 and TA100: R-factor
Metabolic activation:
with and without
Metabolic activation system:
S9
Species / strain:
E. coli WP2 uvr A
Remarks:
base-pair substitutions
Additional strain characteristics:
other: trp-, uvrA-
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1: 8 concentrations between 1,5 - 5000 µg/plate: 1,5 - 5 - 15 - 50 - 150 - 500 - 1500 - 5000 µg/plate
Experiment 2: 6 concentrations between 1,5 - 5000 µg/plate: 15 - 50 - 150 - 500 - 1500 - 5000 µg/plate
5000 µg/plate is the maximum recommended dose level.
Vehicle:
- Vehicle(s)/solvent(s) used: dimethyl formamide at max concentration of 50 mg/ml
- half-log dilutions of the test item are prepared in the vehicle by mixing on a vortex mixer and sonication for 5 minutes at room temperature on the day of each experiment.
- All formulations are used within 4 hours of preparation and were assumed to be stable for this period.
- The test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes.
Controlsopen allclose all
Negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
series without S9-mix
Negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
series with S9-mix
Details on test system and conditions:
NUMBER OF REPLICATIONS: 3

METHODS :
- Experiment 1: direct plate incorporation method (test for mutagenicity) with + without metabolic activation
- Experiment 2: pre incubation method with + without metabolic activation
- The S9 mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

Evaluation criteria:
- Sensitivity of the assay and the efficacy of the S9-mix are validated in the (positive) controls

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not determined
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
- There was no visible reduction in the growth of the bacterial background lwan at any dose level, either in the presence or absence of metabolic activation S9 mix, in the first mutation test and in the second mutation test.
- Small, statistically significant increases in TA100 reveratnt colony frequency observed in the absence of S9-mix at 15 and 150 µg/plate in the first mutation test. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the indidivudal revertant colony counts at 15 and 150 µg/plate were within the control range
- Acceptance criteria are met

Applicant's summary and conclusion

Conclusions:
GASIR 5 was considered to be non-mutagenic under te 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 TestingofChemicals No.471Bacterial Reverse Mutation Test,,Method B13/14ofCommission Regulation (EC) number440/2008of30 May 2008 and the USA, EPA OCSPP harmonized guideline-Bacterial Reverse MutationTest.

Methods

Salmonella typhimurium strains TA1535,Ta 1^j7?TA98 and TA100 and Escherichia coli strain WFluvr Awere treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standardco-factors).The dose range for Experiment1 was predetermined and was 1.5to 5000ig/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh culturesofthe bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment1 and was15to 5000jug/plate.

Six test item concentrations were selected in Experiment2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the testitem following the change in test methodology.

Results

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All ofthe 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000jug/plate.There was no visible reduction in the growth of the bacterial background lawn at any dose level,either in the presence or absence of metabolic activation (S9-mix),in the first mutationtest(plate incorporation method) and consequently the same maximum dose level was used m the second mutationtest.Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix),in the second mutationtest(pre-incubation method).A test item precipitate (black and powdery in appearance) was initially observed under an inverted microscope at 1500|iig/plate and by eye at 5000|ug/plate?this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequencyofrevertant colonies recorded for anyofthe bacterial strains,with any doseofthetestitem, either with or without metabolic activation (S9-mix) in Experiment1(plate incorporation method). Similarly, 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 (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA100 revertant colony frequency were observed in the absence of S9-mix at15and150 |iig/plate in the first mutation test.These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 15and150 |ng/plate were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only1.2 times the concurrent vehicle control.

Conclusion
GASIR5was considered to be non-mutagenic under the conditionsofthistest.