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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:
2005-10-17 to 2005-10-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP study with an expert report performed according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay) with the following deviations: only three strains are tested. However, an expert statement is added in the field "any other remarks" to justify the fact that no further testing is necessary.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
3 bacterial strains used instead of 5
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D (dd 2000-05-19)
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: IRA091
- Expiration date of the lot/batch: not specified
- Purity: 100%
- Purity test date: not specified

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:
Water: 19 g/L
Methanol: 8.5 g/L
Dichloromethane: 0.02 g/L
Acetone: 0.03 g/L
Ethanol: 1.2 g/L
2-propanol: 0.25 g/L
N,N-Dimethylforamide: 1.5 g/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

Method

Target gene:
Histidine-locus
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 98
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / bèta-Naphthoflavone induced rat liver S9
Species / strain:
S. typhimurium TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / bèta-Naphthoflavone induced rat liver S9
Species / strain:
S. typhimurium TA 102
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / bèta-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

A top dose of 5000 µg/plate was chosen since no toxic effects or precipitation were observed at this concentration.
Vehicle:
- Vehicle(s)/solvent(s) used: Methanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9, 10 µg/plate (TA100)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without S9, 10 µg/plate (TA 98)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9, 4 µL/plate (TA102)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9, 2.5 µg/plate (TA98, TA100) and 10 µg/plate (TA102)
Details on test system and conditions:
METHOD OF APPLICATION: plate incorporation test (experiment I) and pre-incubation test (experiment II)
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 μL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 μL Overlay agar
After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

DURATION
- Preincubation period (Experiment II only): not indicated
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 48h, simultaneous with exposure period

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: triplicate, for each strain and dose level (including the controls)

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not required.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
minor toxic effects at 5000 µg/plate without metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
minor toxic effects at 5000 µg/plate with metabolic activation
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 19 g/L
- Precipitation: No precipitation of the test item in the overlay agar was observed.

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). The pre-experiment is reported as main experiment I.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: positive control values were within historical control data range, except the positive control for TA98 without metabolic activation which was above in the second experiment. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
- Negative (solvent/vehicle) historical control data: vehicle control values were within historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The plates incubated with the test item showed normal background growth up to the highest concentration with the exception of strain TA 100 where a reduction in the background growth was observed at 2500 and 5000 μg/plate without metabolic activation in experiment I. No toxic effects, evident as a reduction in the number of revertants, were observed in experiment I. In experiment II, minor toxic effects were observed at 5000 μg/plate in strain TA 98 without metabolic activation and in strain TA 102 with metabolic activation.

Applicant's summary and conclusion

Conclusions:
Expert conclusion: Based on the results described in the draft study report of the bacterial reverse mutation assay (Ames test), it is concluded that JNJ-1782820-AAC has no mutagenic properties towards the various S. typhimurium strains under the experimental conditions reported.