Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-02 to 2016-06-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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
Chemical structure
Reference substance name:
METHYL (1R,2R,4S)-2-[HEX-5-EN-1-YL(METHYL)CARBAMOYL]-4-({7-METHOXY-8-METHYL-2-[4-(PROPAN-2-YL)-1,3-THIAZOL-2-YL]QUINOLIN-4-YL}OXY)CYCLOPENTANECARBOXYLATE
EC Number:
922-147-8
Cas Number:
1042695-87-3
Molecular formula:
C32H41N3O5S
IUPAC Name:
METHYL (1R,2R,4S)-2-[HEX-5-EN-1-YL(METHYL)CARBAMOYL]-4-({7-METHOXY-8-METHYL-2-[4-(PROPAN-2-YL)-1,3-THIAZOL-2-YL]QUINOLIN-4-YL}OXY)CYCLOPENTANECARBOXYLATE
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-39125593-AAA (T003019)
- Physical state: solid
- Appearance: white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15FB2925
- Expiration date of the lot/batch: 2017-05-31 (retest date)
- Date of manufacture: 2016-06-01
- Purity test date: 2015-11-09

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was dissolved in DMSO at 50 mg/ml (= 5000 μg/plate) applying treatment with ultrasonic waves resulting in a clear light yellow solution

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains)
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:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
Dose-range finding test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without S9 (TA100) (Top dose selected based on the solubility findings)
Mutation experiment I: ,0 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without S9 (TA1535, TA1537, TA98, TA102) (Top dose based on the dose-range finding test results)
Mutation experiment II: 0, 87, 154, 275, 492, 878 and 1600 μg/plate with and without S9 (TA1535, TA1537, TA8, TA100, TA102) (Top dose based on the first experiment test results)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml (the test item precipitated and floated on the water). In DMSO, the test item was dissolved at 50 mg/ml (= 5000 μg/plate) applying treatment with ultrasonic waves resulting in a clear light yellow solution. Based on these solubility findings, DMSO was selected as vehicle.
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-mix; 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
Remarks:
Without S9-mix; 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-mix; 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-mix; 650 μg/plate (TA100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: tert-butyl hydroperoxide (TBH)
Remarks:
Without S9-mix; 250 µg/plate (TA102)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
With S9-mix; 2.5μg/plate (TA1535 with 5 and 10% S9-mix; TA1537 with 5% S9-mix), 5μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix; TA100 with 5% S9-mix), 2μg/plate (TA100 with 10% S9-mix), 10μg/ plate (TA102 with 5 and 10% S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: 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 non-activation 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): 48 ± 4 h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium 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 TA102 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 TA102 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 below, 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 results
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102
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
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml (the test item precipitated and floated on the water).
- Precipitation:
Dose-range finding test / mutation experiment I: at 512 µg/plate and upwards at the start and end of the incubation period (TA100, TA102); at 1600 µg/plate and upwards at the end of the incubation period (TA1535, TA1537, TA98)
Mutation experiment II: at 878 and 1600 µg/plate at the beginning and end of the incubation period in all tester strains

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the tester strain TA100 at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Based on the results, the results of the dose-range finding test are reported as a part of the mutation experiment I.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: strain-specific positive control values were within the laboratory historical control data ranges, except the response for TA102 (absence of S9-mix, mutation experiment I). Since the value was 1.8 times greater in the absence of S9-mix than the concurrent solvent control value, this deviation in the mean plate count of the positive control had no effect on the validity of the test results.
- Negative (solvent/vehicle) historical control data: the vehicle control values were within the laboratory historical control data ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies

Any other information on results incl. tables

First mutation experiment

The test item induced an up to 4.2-fold increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix at the mid dose of 164 μg/plate in tester strain TA1537 and an up to 3.5-fold increase in the absence of S9-mix at the mid dose of 512 μg/plate in tester strain TA98. However, since the increases were observed at a mid-dose level only and were caused by one outlier (TA1537: 5, 1, 54 and TA98: 159, 27 and 23 revertant colonies), these increases are considered to be not biologically relevant.

Second mutation experiment:

Initially the results with tester strain TA98 were rejected, due to infection of the highest dose levels tested. This part of the study was repeated. In this repeat experiment the mid dose of 275 μg/plate was not tested, due to a technical error.

Applicant's summary and conclusion

Conclusions:
Interpretation of 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.