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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-06-10 to 2004-09-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Ministry of Health and Welfare (MHW), Japanese Guidelines for Nonclinical Studies of Drugs Manual, Section 5. Reverse Mutation Test in Bacteria (1995).
Deviations:
no
Qualifier:
according to
Guideline:
other: FDA Toxicological Principles for the Safety Assessment of Direct Food Ingredients. Section IV.C1.a: Bacterial Reverse Mutation Test, “Redbook 2000.” U.S. FDA Washington DC, 2000.
Deviations:
no
Qualifier:
according to
Guideline:
other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
Version / remarks:
S2A document recommended for adoption at step 4 of the ICH process on July 19, 1995. Federal Register 61;18198-18202, April 24, 1996.
Deviations:
no
Qualifier:
according to
Guideline:
other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Genotoxicity : A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
Version / remarks:
S2B document recommended for adoption at step 4 of the ICH process on July 16, 1997. Federal Register 61;16026-16030, November 21, 1997.
Deviations:
no
GLP compliance:
no
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-4754724-AAA (T002488)
- Physical state: solid
- Appearance: light brown powder
Specific details on test material used for the study:
Batch-no.: RT002488PFA021 - conversion factor = 1.0000
Purity: 100.3%
Acceptance date: 2004-06-07
Retest date: 2006-05-25

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: See below for additional strain characteristics.
Remarks:
The genotypes and viability of the bacteria were checked when a new set of frozen permanent cultures was prepared. Overnight cultures for the plate incorporation tests were checked for their genotypes and viability.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
Range finding study: 0.64, 1.22, 2.44, 4.88, 9.77, 19.53, 39.07, 78.13, 156.25, and 312.5 μg/plate.
As in the range finding study, no bacteriotoxicity was observed, and the revertants could still be scored at the dose level of 321.5 μg/plate, it was decided to select 625 µg/plate as top dose for the first study.
First, second and third study: 9.77, 19.53, 39.07, 78.13, 156.25, 312.5 and 625 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was found to be not soluble in water at the concentration of 50 mg/mL. In DMSO, the test substance was found soluble at the concentration of 50 mg/mL (5000 µg/plate).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9; at 5 µg/plate for 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; at 1 µg/plate for TA1535 and TA100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9; at 50 µg/plate for TA1537
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; at 1 µg/plate for TA98, TA100 and TA1535; at 2.5 µg/plate for TA1537 and TA102
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 ; at 5 µg/plate for TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation)

EXPERIMENTAL PERFORMANCE:
The following solutions were successively added to 2 ml histidine-biotine supplemented top agar: 0.1 ml of an overnight bacterial culture of the tester strain, 0.1 ml of a dilution of T002488 and either 0.5 ml S9-mix containing 100 μl S9/ml for the activation portion, or 0.5 ml phosphate buffer for the non-activation portion. The content of the tube was then mixed and poured onto minimal glucose agar petri dishes.
The plates were incubated in the dark at 37°C for 48 to 72 hours.

DURATION
- Exposure duration: 48 to 72 hours
- Selection time: 48 to 72 hours (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: All concentration levels of the test substance, vehicle controls and positive controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects of the test substance were detected by a decrease in the number of revertant colonies, thinning of the bacterial background lawn or occurrence of pinpoints.

OTHER EXAMINATIONS:
- Agar, top agar, S9 mix and the highest concentration of the test substance were checked for sterility and were incubated at the same time and for the same length of time as the test plates.
Evaluation criteria:
The mean number of the revertant colonies on the vehicle control plates was taken as the background level of the mutation test. The mean reversion values were considered as positive if:
- the test was valid;
- the test substance produced at least a two-fold increase in the mean number of revertants with one of the strains TA98, TA102 or TA100, or a threefold increase in the mean number of revertants with one of the strains TA1535, TA1537 at one or more concentration levels;
- a dose effect relationship was observed;
- these effects could be reproduced in an additional study.
When equivocal results were obtained, more tests may have been conducted. If the test substance produces a positive response in a single test that cannot be reproduced in additional runs, the initial positive test data will be discounted.
If the test substance produced a positive response in a single test that could not be reproduced in additional runs, the initial positive test data was discounted.
Statistics:
No statistical analysis was used.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA102, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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 substance was found to be not soluble in water at the concentration of 50 mg/mL. In DMSO, the test substance was found soluble at the concentration of 50 µg/mL. However, precipitation was observed upon mixing with water down to the concentration of 156.25 µg/plate. At the concentration of 78.13 µg/plate, a colourless solution was obtained upon mixing the test substance with water. The concentration of 312.5 µg/plate was considered to be the highest applicable concentration for the range finding study
- Precipitation: In study 1, at the concentration of 39.07 or 156.25 µg/plate onwards in the absence of S9 mix and at 78.13 or 156.25 µg/plate onwards in the presence of S9 mix, a dose related increase in precipitation was observed. At the top dose of 625 µg/plate, no bacterial background could be observed due to the heavy precipitation. In study 3, at the concentration of 78.13 µg/plate onwards in the absence and in the presence of S9, a dose related increase in precipitation was observed. At the top dose of 625 µg/plate, no bacterial background could be observed due to the heavy precipitation.

RANGE-FINDING/SCREENING STUDIES:
A range finding study was performed with strain T100 in the absence and presence of S9-mix at concentrations ranging from 0.64-312.5 µg/plate using the plate incorporation assay. At concentrations up to 312.5
µg/plate the test substance did not reveal a biologically significant increase in the number of revertant colonies in the absence and in the presence (50 µL S9 homogenate) of S9-mix. No bacteriotoxic effects visualized by a decrease in the number of revertant colonies, thinning of the background lawn or occurrence of pinpoints were observed.
At concentrations of 78.13 µg/plate onwards in the absence and in the presence of rat liver S9-mix a dose related increase in precipitation was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous and vehicle control revertant colonies for the strains in the absence and in the presence of S9-mix fell within the range of the laboratory's historical data. The positive controls showed a significant increase in the number of revertant colonies indicating their mutagenic activity.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
With all the stains, no bacteriotoxic effects visualized by a decrease in the number of revertant colonies, thinning of the background law or occurrence of pinpoints were observed.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

First study

Sterility checks, genotypes of all the bacterial strains and bacterial titre of all the strains were according to the criteria. The tests with all strains were considered acceptable for the evaluation of the mutagenic potential of the test substance. At the concentrations up to 625 µg/plate, the test substance did not reveal a biologically significant increase in the number of revertant colonies in the absence and in the presence (20 µL S9 homogenate) of S9-mix.

Second study

The same concentrations were applied in the second study as were used in the first study. As a bad lot of prepared bottom agar was used in the second study which did not allow the growth of bacteria, the second study was completely invalidated.

Third Study

Sterility checks, genotypes of all the bacterial strains and bacterial titre of all the strains were according to the criteria. The tests with all strains were considered acceptable for the evaluation of the mutagenic potential of the test substance. At concentrations up to 625 µg/plate, the test substance did not reveal a biologically significant increase in the number of revertant colonies in the absence of and in the presence (50 µL S9 homogenate) of S9-mix.

Applicant's summary and conclusion

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

The tests were performed at maximum concentrations of 625 µg of the test substance/plate, which was not bacteriotoxic to the strains but which strongly precipitated. Decreasing concentration levels from the highest acceptable concentration level provided adequate number of data points to ensure that any possible dose-response would have been detected. All the tests satisfied the criteria for a valid test.
Based on the lack of a biologically significant increase in the reversion rate, it can be concluded that the test substance in the presence and in the absence of a rat liver metabolic activation system, has no mutagenic properties towards the Salmonella typhimurium strains under the test conditions described in the report.