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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:
2010-05-26 to 2010-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Well documented study report, GLP compliant, and according to Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals. No deviations from the study protocol were recorded.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-39453193-AAA (T003063)
- Physical state: solid (powder)
- Appearance: white to almost white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Mitsubishi Tanabe Pharma Corporation, 00541426
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
- Purity: 100%
- Physical description: Pale yellow-white crystalline powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not indicated
- Stability under test conditions: Stable at room temperature (under the airtight condition)
- Solubility and stability of the test substance in the solvent/vehicle: in DMSO: 50 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

OTHER SPECIFICS: The test substance was handled under the yellow light condition and stored under the light-shielded condition, because information about stability of the test substance under the light condition was not provided from sponsor.

Method

Target gene:
histidine locus (histidine-dependent S. typhimurium strains); tryptophan locus (tryptophan-dependent E.coli strain)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/5,6-benzoflavone induced rat liver (S9) from male Sprague-Dawley rats
Test concentrations with justification for top dose:
Dose-finding assay: 0, 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate (maximum dose recommended by the guideline)
Main assay: 156, 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: According to the information provided by the sponsor, the solubility in DMSO is 50 mg/mL or more, and the test substance is stable in DMSO. DMSO was selected as the solvent for the test substance, because the preliminary test on solvent selection in the test facility revealed that the test substance was soluble at 5% (w/v) in DMSO and the solution was stable. DMSO was dehydrated using Molecular Sieves 4A just before use.
Controlsopen allclose all
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-mix, all strains, 0.5 µg/plate (TA98), 1.0 µg/plate (TA100), 2.0 µg/plate (TA1535, TA1537), 10.0 µg/plate (WP2 uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Without S9-mix, 0.01 µg/plate (TA100, WP2 uvrA), 0.1 µg/plate (TA98)
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, 0.5 µg/plate (TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine dihydrochloride
Remarks:
Without S9-mix, 1.0 µg/plate (TA1537)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation method
The 0.5 mL of 0.1 M Na-phosphate buffer with pH 7.4 (or 0.5 mL of S9 Mix in case of metabolic activation assay) was added to 0.1 mL of the test substance solution, and then 0.1 mL each of bacterial culture was added. The mixture was incubated at 37ºC for 20 minutes with shaking (“pre-incubation”). The 2.0 mL of top agar was added to the mixture, and the resultant mixture was overlaid onto a minimum glucose agar plate. The agar solution [0.6% agar (Bacto-Agar DIFCO), 0.5% NaCl] supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin solution for S. typhimurium or with 0.5 mM L-tryptophan solution for E. coli at a volume ratio of 10:1 was used as a top agar.
The plates were incubated at 37ºC for 48 hours, and stored at 4ºC until colony counting if the incubation finished on a holiday. After the incubation, the growth inhibition of bacterial strain and the precipitation were examined, and the revertant colonies were counted.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48h
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine (S. typhimurium); tryptophan (E. coli)

NUMBER OF REPLICATIONS: duplicate at each dose level; triplicate for negative control

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition (dose-related)

COLONY COUNTING:
Revertant colonies within a circle with about 80 mm diameter on a plate with 86 mm diameter (inner diameter: 84 mm) were counted with an automatic colony counter. The count data was corrected based on the area for uncounted colonies by using a personal computer. The revertant colonies at 1250 μg/plate or more under the conditions of S9 (±) could not be counted by the automatic colony counter due to the test substance precipitation; therefore, the colonies were counted manually. By manual counting,all number of revertant colonies were counted without correction.

OTHER:
- For all plates, the growth inhibition of bacterial strain was observed with a stereoscopic microscope (40-fold magnification) and the precipitation was observed by unaided eyes.
- For each dose, the mean number of revertant colonies on two or more plates was calculated and rounded off to an integral number.
Evaluation criteria:
When the number of revertant colonies on the test substance treatment plate is increased 2-fold or more compared to the negative control value, and the increase is dose-dependent and reproducible between the dose-finding assay and the main assay, the test substance is judged to be positive for mutagenicity. Otherwise, the test substance is judged as negative. When the test substance is judged to be positive, the activity of mutagenicity is calculated.
Statistics:
No statistical analysis is performed for data analysis.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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: Information about solubility and stability of the test substance in water is not provided.
- Precipitation: In the dose-finding assay, white precipitates of the test substance were observed at 20 μg/plate or more (without S9) and at 78 μg/plate or more (with S9); In the main assay, white precipitates of the test substance were observed at 156 μg/plate or more (with and without S9).

RANGE-FINDING/SCREENING STUDIES:
In the dose-finding assay, 5000 μg/plate was set as the maximum dose, which is recommended by the guideline, and a total of 7 doses were set with a common ratio of 4. Based on the results of the dose-finding assay, the highest dose in the main assay was set at 5000 μg/plate, and a total of 6 doses were set with a common ratio of 2, including at lease one dose producing precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the dose-finding assay and the main assay, the negative control values were within the acceptable range (mean± 2.5SD) specified based on the historical data of the test facility. The positive controls induced revertant colonies more than 2-fold of the negative control value in each strain. No bacterial growth due to contamination was observed in the sterility tests. These results indicated the assays were performed properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No growth inhibition of bacteria by the test substance was observed in any of the two experiments.
Remarks on result:
other: Dose-finding assay

Any other information on results incl. tables

Validity of assays

In the dose-finding assay and the main assay, the negative control values were within the acceptable range (mean±2.5SD) specified based on the historical data of the test facility. The positive controls induced revertant colonies more than 2-fold of the negative control value in each strain. No bacterial growth due to contamination was observed in the sterility tests. These results indicated the

assays were performed properly.

Maximum mutagenic activity

The maximum mutagenic activity was 5.64×10 rev./mg (at 5000 μg/plate in TA100 without S9 in the dose-finding assay).

Applicant's summary and conclusion

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

Based on the results, it was concluded that T003063 was negative for mutagenicity under the test conditions of this study.