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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:
2017-08-10 to 2018-03-26
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)
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-39722280-AAA (4-Nitrobenzene-1-sulfonyl chloride)
- Physical state: solid (powder)
- Appearance: Slightly yellow to yellowish brown crystalline powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16LB4523
- Expiration date of the lot/batch: 2018-06-23
- Purity test date: 2018-04-17

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: A correction factor of 1.00 for the purity/composition of the test item was applied in this study.

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
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:
The maximum final concentration for the dose range finding test was based on thesolubility of the test item in DMSO (maximum recommended dose = 5000 µg/plate).
Dose-range finding test: 1.7, 5.4, 17, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5% (v/v) S9-mix

The top doses for the mutation experiments were selected based on the toxicity observed in the dose range finding test.
Mutation experiment 1: 17, 52, 164, 512, 1600 and and 5000 μg/plate in TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix
Mutation experiment 2: 188, 375, 750, 1250, 2500 and and 5000 μg/plate with and without 10% (v/v) S9- mix

Mutation experiment 3: To verify the mutagenic response observed in tester strain TA98 with and without 10% (v/v) S9-mix, this part of the experiment was repeated with the following doses: 1000, 1500, 2000, 2500, 3000, 3500 and 4000 μg/plate

Since the test item is not stable in DMSO, the mutation experiments were repeated with anhydrous THF as solvent in additional fourth and fifth mutation assays.
The maximum final concentration was based on the stability and solubility of the test item in THF (maximum recommended dose = 5000 µg/plate) and toxicity observed in previous experiments.
Mutation experiment 4: 17, 52, 164, 512, 1600 and 5000 μg/plate; with and without 5% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Mutation experiment 5: 5.12, 12.8, 32, 80, 200, 500 and 750 μg/plate; with and without 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Mutation experiment 6: To verify the mutagenic response observed in the tester strains TA98 and TA1537 in the fifth mutation experiment, this part of the experiment was repeated.
without S9-mix in tester strain TA98: 9.4, 18.8, 37.6, 75, 100, 150 and 200 μg/plate,
with 5% (v/v) S9-mix in tester strain TA1537: 5.12, 12.8, 32, 80, 200 and 500 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: anhydrous tetra hydrofuran (THF)
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item was soluble at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test. However, due to the instability of the test item in DMSO, the mutation experiments were repeated with anhydrous tetra hydrofuran (THF) as vehicle. The test item was soluble at 200 mg/ml in THF and 5000 μg/plate was selected as the maximum final concentration for the fourth mutation experiment.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9-mix; 5 μg/plate (TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: OCR-191
Remarks:
Without S9-mix; 2.5 μg/plate (TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9-mix; 10 μg/plate (TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9-mix; 650 μg/plate (TA100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9-mix; 10 μg/plate (WP2uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2.5 μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5 μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2 μg/plate (TA100 with 10% S9-mix), 15 μg/ plate (WP2uvrA with 5 and 10% S9-mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
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 or 0.025 ml of a dilution of the test item in DMSO or THF, respectively and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation 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): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent 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 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.

In addition to the criteria stated above, 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.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
Mutation experiment 1, 2 and 3: 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.
Mutation experiment 4: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 164 μg/plate and above at the end of the incubation period in all tester strains in the absence and presence of S9-mix.
Mutation experiment 5: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 500 μg/plate and upwards and at 80 μg/plate and above at the end of the incubation period in all tester strains in the absence and presence of S9-mix
Mutation experiment 6: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 500 μg/plate and at 200 μg/plate and above at the end of the incubation period in both tester strains.

RANGE-FINDING/SCREENING STUDIES:In the dose range finding test, the test item was tested at concentrations of 1.7, 5.4, 17, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. It was reported as part of the first mutation experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The vehicle control values were within the laboratory historical control data ranges, except the response for TA1537 in the fifth experiment (presence of S9-mix). Since the mean number of revertant colonies showed a slightly lower number of revertant colonies (2 revertant colonies) when compared against relevant historical control data (3 revertant colonies), the validity of the test was considered not to be affected. In addition, this tester strain was repeated in the sixth experiment.
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 TA1535 (fifth 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 above the historical control data range, this deviation in the mean plate count of the positive control had no effect on the validity of the results.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutation Expeirment 1: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix
Mutation Expeirment 2: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains, except in tester strain WP2uvrA
Mutation Expeirment 3: Cytotoxicity, as evidenced by a reduction of the background lawn and/or the presence of microcolonies, was observed in both the absence and presence of S9-mix at concentrations of 2500 μg/plate and upwards
Mutation Expeirment 4: Cytotoxicity, as evidenced by a reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Mutation Expeirment 5: Cytotoxicity, as evidenced by a decrease in the number of revertants, a reduction of the background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Mutation Expeirment 6: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Any other information on results incl. tables

Additional information on mutation frequencies

Mutation experiment 1, 2 and 3 (DMSO):

- In the absence of S9-mix, the test item induced increases in tester strain TA98. Although the increases were above the historical control data range, the increases were not three-fold and therefore the increases are considered equivocal. In the presence of 10% (v/v) S9-mix, the test item induced increases in tester strain TA98. The increases observed were up to 3.1-fold compared to the concurrent vehicle control. However, the increases were within the historical control data range and therefore the increases are considered equivocal.

- All other bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant and/or significant dose-related increase (three-fold for TA1535 and TA1537 or two-fold for TA100 and WP2uvrA) in the number of revertants in any of the experiments.

- Since the test item is not stable in DMSO the first, second and third mutation experiment are considered invalid for the

evaluation of the mutagenic activity of the test item. Therefore, the experiments were repeated in the fourth, fifth and sixth experiment in the absence and presence of S9-mix in all five tester strains with anhydrous THF as solvent.

Mutation experiment 4, 5 and 6 (anhydrous THF):

- In the tester strains TA98 and TA1537, the test item induced up to 2.7- and 4.0-fold increases in the number of revertant colonies compared to the solvent control in the absence and presence of 10% (v/v) S9-mix, respectively. The increases observed were within the historical control data range and were only observed in one experiment. Therefore, these increases are not considered biologically relevant. In the other tester strains, no increase in the number of revertants was observed.

- The test item did not induce a biologically relevant and/or dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 metabolic activation in the fourth, fifth and sixth experiment.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay when dissolved in a suitable vehicle (i.e. anhydrous THF).