Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-09 to 2017-01-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in the report): JNJ-47961173-AAA
- Physical state: Liquid
- Colour: Colourless
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15IB3251
- Expiration date of the lot/batch: 2017-04-07
- Purity test date: 2015-09-18


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5%(v/v) S9-mix (top dose selected based on the solubility findings)
Mutation experiment I: 52, 164, 512, 1600 and 5000 μg/plate in TA1535, TA1537 and TA98 with and without 5%(v/v) S9-mix (top dose selected based on the dose range finding test results).
Mutation experiment II: 492, 878, 1568, 2800 and 5000 µg/plate with and without 10%(v/v) S9-mix (top dose selected based on the dose range finding test and mutation experiment I results).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item formed a clear solution at 50 mg/ml (5000 μg/plate). DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9; 5 μg/plate (TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191 dissolved in DMSO
Remarks:
Without S9; 2.5 μg/plate (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9; 10 μg/plate (TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; 650 μg/plate (TA100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9; 10 μg/plate (WP2uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9; 2.5 μg/plate (TA1535 with 5 and 10% S9, TA1537 with 5% S9), 5 μg/plate (TA1537 with 10% S9), 1μg/plate (TA98 with 5 and 10% S9, TA100 with 5% S9), 2 μg/plate (TA100 with 10% S9), 15 μ g/ plate (WP2uvrA with 5 and 10% S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2°C.
The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains
- 0.1 ml of a dilution of the test item in DMSO and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: all concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.
In addition to the criteria stated above, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml.
- Precipitation: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate in the tester strains TA1535, TA1537 and TA98. No precipitation was observed at the end of the incubation period in any of the five tester strains.

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. The dose range finding test results are reported as a part of mutation experiment I.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed

Any other information on results incl. tables

Dose range finding test/Mutation experiment 1

Toxicity: In strain TA1537 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid dose level of 512 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. This reduction is more likely caused by an incidental fluctuation in the number of revertant colonies.

Mutation experiment 2

Mutagenicity: In tester strain TA1535, an increase above the historical control data range was observed at the highest tested dose level in the presence of S9-mix. However, since this increase was just above the historical control data range, only observed in the second mutation experiment, and was not dose-related and not three-fold (a maximum of 2.1-fold was reached), this increase was considered to be not biologically relevant.

Applicant's summary and conclusion

Conclusions:
Interpretation of the results:
negative with and without metabolic activation

All bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant and/or significant dose-related increase in the number of revertants in two experiments. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.