Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-21 to 2015-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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 the test material (as cited in study reports): JNJ-63757109-AAA (T003690)
- Physical state: solid (powder)
- Appearance: white to slightly yellow power
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15HD2977
- Purity: 99.0%
- Expiration date of the lot/batch: 2017-08-10 (retest date)

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

OTHER SPECIFICS: correction factor is 1

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver metabolic activation system (S9-mix)
Test concentrations with justification for top dose:
Dose range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (the highest concentration of the test item used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item exhibited limited solubility).
Mutation experiment 1 A: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix;
Mutation experiment 1 B: 52, 164, 512, 1600 and 5000 μg/plate (with and without 5% (v/v) S9-mix)
Mutation experiment 2: 275, 492, 878, 1568 and 2800 μg/plate (with and without 10% (v/v) S9-mix)
In DMSO, the test item was soluble at 28 mg/ml (= 5000 μg/plate). Based on these solubilityfindings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was not soluble in Milli-Q water and in dimethyl sulfoxide (DMSO) a slightly translucent solution was obtained at a concentration of 50 mg/mL. Therefore, DMSO was selected as a vehicle and 5000 µg/plate was selected as the maximum final concentration for the dose range finding test.
At concentrations of 28 mg/mL and lower the test item was fully soluble in dimethyl sulfoxide (DMSO).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9-mix, 5 µg/plate for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without S9-mix, 2.5 µg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9-mix 10 μg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9-mix, 650 μg/plate for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: tert-butyl hydroperoxide
Remarks:
Without S9-mix, 250 μg/plate for TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9: 1 μg/plate for TA98 (5% and 10 % S9) and for TA100 (5% S9), 2.5 μg/plate for TA1535 (5% and 10% S9) and TA1537 (5% S9); 5 μg/plate for TA1537 (10 % S9); 2 μg/plate for TA100 (10 % S9); 10 μg/plate for TA102 (5 % and 10 % S9);
Details on test system and experimental 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 histidine-dependent strains);

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, reduction of the bacterial background lawn or presence of microcolonies
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD and EC).
Solubility limitations: Since the test item was not soluble in Milli-Q water and in dimethyl sulfoxide a slightly translucent solution was obtained at a concentration of 50 mg/ml, 5000 µg/plate was selected as the maximum final concentration for the dose range finding test and dimethyl sulfoxide was selected as vehicle.
Evaluation criteria:

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 control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent 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.

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 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.

Statistics:
No formal hypothesis testing was done.

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml.
- Precipitation: Dose range finding test: at the start of the incubation period at the concentration of 5000 µg/plate and at 1600 and 5000 µg/plate at the end of the incubation period.
Mutation experiment 1: at the start of the incubation period at the concentration of 5000 µg/plate in the absence of S9-mix and at 512 µg/plate and above in the presence of S9-mix. Precipitation of the test item on the plates at the end of the incubation period was observed at 1600 and 5000 µg/plate.
Mutation experiment 2: at the start of the incubation period at the concentration of 2800 µg/plate in the absence of S9-mix and at 492 µg/plate and above in the presence of S9-mix. Except in the tester strains TA1537, TA102 in the absence and presence of S9-mix and TA100 in the absence of S9-mix where precipitate was observed at 1568 µg/plate and 2800 µg/plate.
Precipitation of the test item on the plates was observed at the end of the incubation period at concentrations of 1568 and 2800 µg/plate in the absence and presence of S9-mix. Except in the tester strains TA1537, TA102 in the absence and presence of S9-mix and TA100 in the absence of S9-mix where precipitate was observed at 2800 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
In a dose range finding test, the test item was tested at a 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.
Mutation experiment 1: 52, 164, 512, 1600, 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.
Mutation experiment 2: 275, 492, 878, 1568 and 2800 μg/plate in the absence and presence of 5% (v/v) S9-mix.


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 response for TA102 in the absence of S9-mix (first experiment) and absence and presence S9-mix in the second experiment.
- Negative historical control data: The negative control values were within the laboratory historical control data ranges

Since the mean number of revertant colonies showed in the first experiment a 4.0-fold increase and in the second experiment 1.8- and 2.0 fold increases compared to the concurrent vehicle controls and were just outside the historical control data, the validity of the test was considered to be not affected.

Applicant's summary and conclusion

Conclusions:
Interpretation of results
negative

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-mix 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.