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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:
from 2011-10-11 to 2011-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted in the spirit of compliance with 21 CFR Part 588, OECD principles of Good Laboratory Practice (1998)9, OECD guideline 471 (1997)10, and in accordance with the appropriate J&JPRD SOPs (unless specifically stated in the protocol).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: US FDA Good laboratory Practices 21 CFR Part 58
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: J&JPRD SOPs
Deviations:
no
GLP compliance:
yes
Remarks:
The study was conducted in the spirit of compliance with OECD principles of GLP (1998)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid
EC Number:
689-958-4
Cas Number:
1173807-85-6
Molecular formula:
C8H13NO2.0.5H2SO4
IUPAC Name:
bis(ethyl (1R,2S)-1-amino-2-ethenylcyclopropane-1-carboxylate); sulfuric acid
Details on test material:
- Name of test material (as cited in study report): JNJ-31052047-ABI
- Analytical purity: 100.1 %
- Purity test date: 6 July 2011
- Lot/batch No.: Lot Number : 549620 / Batch ID: 39046749
- Storage condition of test material: Room temperature

Method

Target gene:
His- and Trp-Operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S9-mix from male Sprague-Dawley rat liver
Test concentrations with justification for top dose:
Range finding : 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/plate
Mutation Assay : 250, 500, 1000, 2000, 3000, 4000, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
- Lot number : 98072023
- Description : clear liquid
- Supplier : Omni pur
- Storage : Room temperature (15-30°)
- Justification for choice of solvent/vehicle: Water was selected as the vehicle for this study based on stability information.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: 2-Aminoanthracene (TA98, TA100, TA1535 and TA1537: 2 µg/plate ; WP2uvrA: 25 µg/plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: Fenaminosulf (200 µg/plate for TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide (2 µg/plate for TA100 and TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: ICR-191 (1 µg/plate for TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: 4-Nitroquinoline-N-Oxide (1 µg/plate for WP2uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation).

DOSE-RANGE FINDING EXPERIMENT:
A toxicity experiment via the plate incorporation methodology was performed with all tester strains to determine the maximum concentration of the test article to be used in the mutation experiment. Test article doses tested were: 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 μg/plate. All dose levels of the test article, vehicle, and positive controls were evaluated in duplicate plates, with or without S9, as appropriate. The appropriate quantity of test or control article (0.05 mL), 2 mL of supplemented molten top agar (containing 0.1 mL tester strain), and 0.5 mL phosphate buffer or S9 were combined and overlaid onto minimal agar plates.

MUTATION ESSAY: The appropriate quantity of test or control article (0.05 mL), 2 mL of supplemented molten top agar (containing 0.1 mL tester strain), and 0.5 mL phosphate buffer or S9 were combined and overlaid onto minimal agar plates.

DURATION (for both dose range finding experiment and mutation essay): 46-72 hours at 37 ± 2 °C

NUMBER OF REPLICATIONS: Range-finding experiment: 2plates/dose/experiment ; mutation essay: 3 plates/dose/experiment

NUMBER OF CELLS EVALUATED: The density of tester strain cultures must be ≥ 0.5 x 10E09 bacteria/mL to demonstrate that appropriate numbers of bacteria are plated.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is detectable as a decrease in the number of revertant colonies/plate and/or by a thinning or disappearance of the bacterial background lawn.
Evaluation criteria:
Criteria for a Positive Response:
A test article is considered to be positive (mutagenic), if it induces a dose-dependent increase in revertant frequency to at least 2-fold (3-fold for
TA1535 and TA1537) that observed in the appropriate concurrent vehicle control. In addition, the response should be reproducible.

Criteria for a Negative Response:
A test article is considered to be negative (non-mutagenic), if no reproducible dose-dependent or 2-fold (3-fold for TA1535 and TA1537) increases in revertant frequency are observed.

Criteria for an Equivocal Response:
Occasionally, a test article cannot be judged to be positive or negative (e.g., dose-dependent increases that fail to reach 2-fold control values, or  2-fold increases that do not appear to be dose dependent). In these rare instances, the results may be classified as equivocal.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at ≥ 1000 μg/plate.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium Strains TA98, TA100 and TA1537
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
Species / strain:
E. coli WP2 uvr A
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:
Testing of JNJ-31052047-ABI in the Range-Finding Assay
The mean values and standard deviations were calculated on the basis of individual plate readings for all test, vehicle and positive control plates . All doses of all test and control articles were evaluated in duplicate plates with and without S9. Indications of toxicity [reduced bacterial colony counts, sparse lawns (reduced lawns), or absent bacterial lawns] were not observed. Precipitation was not observed in top agar or on plates. Revertant frequencies in TA1535 and TA1537 without S9 at 5000 μg/plate were 3.5 and 3.3 times higher, respectively, than those observed in the concurrent vehicle control cultures. Revertant frequencies in TA1535 with S9 at 2500 and 5000 μg/plate were 4.5 and 5.5 times higher, respectively, than those observed in the concurrent vehicle control cultures. All positive and vehicle control values were within acceptable ranges, as were tester strain characterization results. The results of the range-finding assay were consistent with the results observed in the mutation assay. All criteria for a valid assay were met . Therefore this range-finding experiment is considered as the initial mutation experiment.

Testing of JNJ-31052047-ABI in the Mutation Assay
The mean values and standard deviations were calculated on the basis of individual plate readings. All doses of all test and control articles were evaluated in triplicate plates with and without S9. Indications of toxicity were not observed. Precipitation was not observed in top agar or on plates. A dose-dependent increase (2.3 to 5.7 fold) in revertant colonies above those observed in the concurrent vehicle control cultures was observed in TA1535 with S9 at ≥ 1000 μg/plate. All positive and vehicle control values were within acceptable ranges, as were tester strain characterization results.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Control Data

Acceptable negative control data for all four S. typhimurium strains and E. coli strain WP2uvrA were obtained. The positive indicators Fenaminosulf (tested in TA98), ICR-191 (tested in TA1537), sodium azide (tested in TA100 and TA1535), 4-NQO (tested in WP2uvrA), and 2-aminoanthracene (tested in all strains), a promutagen in this test system, were mutagenic. These results and the strain characterization data confirmed the responsiveness and identity of the test organisms, as well as the functionality of the liver microsomal preparations used in this study.

Applicant's summary and conclusion

Conclusions:
These results indicate JNJ-31052047-ABI was positive in the in vitro bacterial/microsomal activation reverse mutation assay under the conditions, and according to the criteria, of the test protocol.