Registration Dossier

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:
2005-08-30 to 2005-12-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
(only three tester strains were used and limited information was provided on the test substance)

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:
: only three tester strains were used and limited information was provided on the test substance
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
: only three tester strains were used and limited information was provided on the test substance
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 report): JNJ-559728-AAA (T001250)
- Physical state: solid (powder)
- Appearance: White powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00443418 RT001250G4A661
- Expiration date of the lot/batch: not indicated

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

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100 and TA102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
- Pre-experiment/Experiment 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 , for TA100 at 10 ug/plate
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
without S9, for TA98 at 10 ug/plate
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9, MMS for TA102 at 4.0 uL/plate
Untreated negative controls:
yes
Remarks:
: untreated
Negative solvent / vehicle controls:
yes
Remarks:
: DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S9, for TA98 and TA100 at 2.5 ug/plate and TA102 at 10 ug/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- The following materials were mixed in a test tube and poured onto the selective agar plates: 100 uL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 uL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without S9), 100 uL bacteria suspension (cf. test system, pre-culture of the strains), 2000 uL overlay agar. After solidification the plates were incubated upside down for at least 48 hours at 37 deg C in the dark.

DURATION
- Exposure duration: at least 48 hours
- Selection time: at least 48 hours (simultaneous with exposure)

SELECTION AGENT:
- histidine

SPINDLE INHIBITOR:
- not applicable

STAIN:
- not applicable

NUMBER OF REPLICATIONS:
3

NUMBER OF CELLS EVALUATED:
not applicable

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

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable

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

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
: TA100 with and without S9 from 333 up to 5000 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
: TA100 at 2500 and 5000 ug/plate (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: 0.62 g/L
- Precipitation: No precipitation of the test substance in the overlay agar was observed.
- Other confounding effects: not applicable

RANGE-FINDING/SCREENING STUDIES:
- To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations, 3 to 5000 µg/plate, 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 1 (plate incorporation test). The pre-experiment is reported as main experiment 1.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive controls showed a distinct increase in induced revertant colonies and gave revertant values within historical control data ranges except the positive control for TA98 in the absence of S9 mix, which was slightly above.
- Negative (solvent/vehicle) historical control data: negative, and vehicle controls gave revertant values within historical control data ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The plates incubated with the test substance showed normal background growth up to the highest concentration. Only in strain TA100 reduced background growth was observed in the absence of metabolic activation at 2500 ug/plate and 5000 ug/plate.
- No toxic effects, evident as a reduction in the number of revertants, were observed in any of the strains used.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: positive with and without metabolic activation

The test substance was evaluated for mutagenic potential in the bacterial reverse mutation assay in S. typhimurium strains TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation. Under the conditions of the study, the test substance did induce gene mutations by base pair changes in the genome of strain TA100 with and without metabolic activation.