Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Mar 2018 to 02 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Physical appearance (with color): Colourless to yellow liquid
Storage Conditions: Ambient (21 to 29°C)
Date of Expiry: 20-12-2020

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate
Test concentrations with justification for top dose:
Pre-test: 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 μL/plate.
Mutation assay: 0.05, 0.16, 0.5, 1.6 and 5 μL/plate (Based on the results of the initial cytotoxicity test)
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 30 minutes (preincubation)
- Exposure duration: 66 hours and 45 minutes (plate incorporation); 65 hours (preincubation)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn
Evaluation criteria:
EVALUATION AND INTERPRETATION OF RESULTS
The conditions necessary for determining a positive result are: there should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item, either in the presence or absence of the metabolic activation system. The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited. A response is evaluated as negative, as it is neither positive nor equivocal.

CRITERIA FOR ACCEPTABILITY OF THE TEST
The mutation test is considered acceptable as it meets the following criteria: All tester strains confirmed to their genetic characteristics. The positive controls showed increase in revertant colony numbers of at least twice or thrice the concurrent vehicle control levels with the appropriate bacterial strain.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Solubility Test: The test item was miscible in dimethyl sulphoxide at a concentration of 50 μL/mL.
- Precipitation Test: The test item showed minimal precipitation at 5 μL/plate, slight precipitation at 0.8, 0.9, 1, 2, 3 and 4 μL/plate and no precipitation at 0.7 and 0.6 μL/plate.
- Initial Cytotoxicity Test: Based on the results of the solubility and precipitation tests, the concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 μL/plate were selected for the initial cytotoxicity test. No cytotoxicity was observed up to 5 μL/plate. On the basis of cytotoxicity test, 5 μL/plate was considered as the highest test concentration for the mutation assay.
- Plate Incorporation Method: Trial I: All the tester strains treated with the test item, at the concentrations tested of 0.05, 0.16, 0.5, 1.6 and 5 µL/plate showed very close resemblance to the vehicle control when tested with and without metabolic activation. There was no appreciable increase in the number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of the vehicle control. The specific positive controls tested simultaneously produced approximately 2.3 to 14.7-fold increase in the mean number of revertants as compared to the vehicle control.
- Preincubation Method: Trial II: All the tester strains treated with the test item, at the concentrations tested of 0.05, 0.16, 0.5, 1.6 and 5 µL/plate showed very close resemblance to the vehicle control when tested with and without metabolic activation. There was no appreciable increase in the number of revertant colonies and no change in bacterial background lawn when compared to that of the vehicle control, among the tester strains. The mean number of revertant colonies/plate and bacterial background lawn in the treatment groups for the tested strains were comparable to that of the vehicle control. The specific positive controls tested simultaneously produced approximately a 2.3 to 13.9-fold increase in the mean number of revertants as compared to the vehicle control.
- Viable Count: Each tester strain was serially diluted to 1E-7 and plated on nutrient agar. Post-incubation of the plate incorporation and for pre-incubation method, the numbers of colonies were counted manually and results were expressed as Colony Forming Units (CFU). Each tester strains resulted in an acceptable range of 1 to 2E9 CFU/mL.
- Quality Control Plates: Control plates with S9 mix, phosphate buffer saline, soft agar, 5 µL/plate of test item, dimethyl sulphoxide and minimal glucose agar plates incubated at 37±1°C for 66 hrs and 45 mins for the plate incorporation method and pre-incubation method were checked for contamination. No microbial contamination was observed.

Applicant's summary and conclusion