Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-12 to 2016-07-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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-17298957-AAA (T001592)
- 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: I15HB2929
- Expiration date of the lot/batch: 2017-08-14 (retest date)
- Date of manufacture: 2015-08-15
- Purity test date: 2015-11-09

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: not indicated

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains)
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
Dose-range finding test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without S9 (TA100) (Top dose selected based on the solubility findings)

Mutation experiment I: 0, 5.4, 17, 52, 164, 512 and 1600 μg/plate with and without S9 (TA1535, TA1537, TA98, TA102) (Top dose based on the dose-range finding test results: the highest concentration of the test item used in the first mutation experiment was 1600 µg/plate, the level at which the test item inhibited bacterial growth.)
Mutation experiment II: 0, 86, 154, 275, 492, 878 and 1568 μg/plate with and without S9 (TA1535, TA1537, TA8, TA100, TA102)
Mutation experiment III: 0, 26, 59, 134, 304, 690 and 1568 μg/plate without S9 (TA1537, TA98, TA102)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml (the test item floated on the water). In DMSO, the test item was dissolved at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9-mix; 5 µg/plate (TA1535)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without S9-mix; 2.5 µg/plate (TA1537)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9-mix; 10 µg/plate (TA98)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9-mix; 650 µg/plate (TA100)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: tert-butyl hydroperoxide (TBH)
Remarks:
Without S9-mix; 250 µg/plate (TA102)
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
With S9-mix; 2.5 µg/plate (TA1535 with 5 and 10% S9-mix; TA1537 with 5% S9-mix), 5 µg/plate (TA1537 with 10% S9), 1 µg/plate (TA98 with 5 and 10% S9-mix; TA100 with 5% S9-mix, 2 µg/plate (TA100 with 10% S9-mix), 10 µg/plate (TA102 with 5 and 10% S9-mix)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains,
- 0.1 ml of a dilution of the test item in DMSO and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time (if incubation with a selection agent): 48 ± 4 h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. Typhimurium strains)

NUMBER OF REPLICATIONS: All concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or TA102 is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or TA102 is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
All experiments, at the highest dose level(s) tested.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml (the test item floated on the water)
- Precipitation:
Dose-range finding test / Mutation experiment I: at 5000 µg/plate (TA100) at the start of the incubation period; no precipitate at the end of incubation period
Mutation experiment II: no precipitation was observed at the start or end of the incubation period
Mutation experiment III: no precipitation was observed at the start or end of the incubation period

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the tester strain TA100 at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Based on the results, the results of the dose-range finding test are reported as a part of the mutation experiment I.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the responses in TA102 (third experiment, absence of S9-mix). The positive control is included as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was 2.2-times greater than the concurrent solvent control value, this deviation in the mean plate count of the positive control had no effect on the validity of the results.
- Negative (solvent/vehicle) historical control data: The vehicle control values were within the laboratory historical control data ranges, except the response for TA102 and TA98 in the second experiment (absence of S9-mix). In the repeat experiment these vehicle control values were within the laboratory historical control data ranges. Therefore this deviation in the mean plate count of the vehicle control had no effect on the validity of the results.

Any other information on results incl. tables

In the second experiment in tester strain TA1537, in the absence of S9-mix only three analysable dose levels were present. Furthermore the test item was cytotoxic at significant lower dose levels than observed in the first mutation experiment. Furthermore, in TA98 and TA102, the solvent control values without S9 were significantly below the lower limit of the historical control range. Therefore, mutation experiment III was conducted.

Applicant's summary and conclusion

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

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) in the absence and presence of S9 metabolic activation in any of the experiments. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay.