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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:
2016-12-08 to 2016-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): JNJ-42398681-AAA (T001589)
- Physical state: liquid
- Appearence: light brown
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16FB3011
- Expiration date of the lot/batch: 2018-03-14 (retest date)
- Date of manufacture: 2016-06-15
- Purity: 87%


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 1.15
During the performance of the study, the test item was handled with a correction factor of 1.14 (as initially given by the sponsor). In the course of the development it has been identified that the test item should be corrected for the purity/composition with a factor of 1.15. Therefore, the actual concentrations were 0.99% lower than stated in the report.

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver microsomal enzymes (S9-mix)
Test concentrations with justification for top dose:
Dose range finding test: 1.7, 5.4, 17, 52, 163, 508, 1586 and 4957 μg/plate with and without 5% S9-mix;
Mutation experiment 1: 17, 52, 163, 508, 991 and 1586 μg/plate with and without 5% S9-mix;
In DMSO, the test item was soluble at 49.6 mg/ml (= 4957 μg/plate). Based on these solubility findings, DMSO was selected as vehicle and 4957 μg/plate was selected as the maximum final concentration for the dose range finding test.
A top dose of 1586 µg/plate was selected for the mutation experiment based on the toxicity observed in the dose range finding test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 49.6 mg/ml. In DMSO, the test item was soluble at 49.6 mg/ml (= 4957 μg/plate). Based on these solubility findings, DMSO was selected as vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9: 5 μg/plate for TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without S9: 2.5 μg/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9: 10.0 μg/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9: 650 μg/plate for TA100
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: 10 μg/plate for WP2uvrA
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: 1 μg/plate for TA98 (5% S9) and for TA100 (5% S9), 2.5 μg/plate for TA1535 (5% S9) and TA1537 (5% S9); 15 µg/plate for WP2uvrA (5% 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); tryptophan (E. coli tryptophan-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 fully soluble in DMSO, the highest tested concentration for the Mutation assay was 4957 μg/plate.
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 WP2uvrA 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 WP2uvrA 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 existing criteria, 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 resultsopen allclose all
Species / strain:
other: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Without S9-mix up to 24-fold increase in TA1535, up to 7.0-fold in TA100 and up to 11-fold in WP2uvrA. With S9-mix, increases up to 35-fold in TA1535 and up to 12-fold in TA100
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all tester strains with and without S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
increases in the number of revertants up to 11-fold (absence of S9-mix) and up to 10 fold (presence of S9-mix)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, in the absence and presence of S9-mix
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 item was observed to be insoluble in water at 49.6 mg/ml.
- Precipitation:
Dose range finding test: at 4957 μg/plate with and without S9-mix in WP2uvrA at the start and at the end of the incubation period. No precipitation was observed in any other tester strain.
Mutation experiment: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.

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, 163, 508, 1586 and 4957 μg/plate in the absence and presence of 5% (v/v) S9-mix. The dose range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the mutation experiment. In compliance with the OECD guideline No. 471, there is no requirement for verification of a clear positive response. Since the test results of the mutation experiment showed clear positive responses, the follow-up experiment was not performed.

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.
- Negative (solvent/vehicle) historical control data: The vehicle control values were within the laboratory historical control data ranges.

Any other information on results incl. tables

In the absence of S9-mix, the test item induced dose related increases in the number of revertant colonies in various tester strains. The increase observed in tester strain TA1535 was above the laboratory historical control data range, and was up to 24-fold the concurrent control. The increase observed in tester strain TA100 was above the laboratory historical control data range and was up to 7.0-fold the concurrent control. The increase observed in tester strain WP2uvrA was above the laboratory historical control data range and was up to 11-fold the concurrent control.

In the presence of S9-mix, the test item induced dose related increases in the number of revertant colonies. The increase observed in tester strain TA1535 was above the laboratory historical control data range, and was up to 35-fold the concurrent control. The increase observed in tester strain TA100 was above the laboratory historical control data range and was up to 12-fold the concurrent control. The increase observed in tester strain WP2uvrA was above the laboratory historical control data range and was up to 10-fold the concurrent control.

The test item did not induce a significant dose-related increase in the number of revertant colonies in tester strains TA1537 and TA98.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: positive with and without metabolic activation in strains TA100, TA1535 and WP2uvrA
The test item did not induce a significant dose-related increase in the number of revertant colonies in tester strains TA1537 and TA98.
Based on the results of the study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.