5-fluoro-1-[(4-methylphenyl)sulfonyl]-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in bacteria

In a first, non-GLP, K2 study (A. Schuermans, B.M. van der Leede and J. Van Gompel; 2014) with five Salmonella typhimurium strains (TA98, TA1537, TA100, TA1535 and TA102), the test item has no mutagenic properties in the absence and presence of a rat liver metabolizing system (S9-mix) when tested up to 1250 μg/plate as a solution including particles.

In a second, GLP, K1 (C.M. Verspeek-Rip, 2015) study with five Salmonella typhimurium strains (TA1535, TA1537, TA98, TA100 and TA102) it was concluded that the test item has mutagenic properties only towards strain TA100 in the absence and presence of S9-mix when tested up to 1600 μg/plate as a solution including particles.

In a third, non-GLP, K2 study (A. Schuermans, B.M. van der Leede and J. Van Gompel; 2016) with the Salmonella typhimurium strain TA100, the test item has no mutagenic properties in the absence and presence of S9-mix using the same experimental design (i.e. testing up 1250 and 1600 μg/plate as a solution including particles and testing up to 5000 μg/plate as a suspension including particles) as in the previous conducted studies.

In a fourth, GLP, K1 study (C.M. Verspeek-Rip, 2016) with five Salmonella typhimurium strains (TA98, TA1537, TA100, TA1535 and TA102), the test item has mutagenic properties towards strain TA100 in the absence and presence of S9-mix when tested up to 1600 μg/plate as a solution including particles.

In view of safety, the overall conclusion from these studies is that the test item has mutagenic properties towards strain TA100 both in the absence and presence of S9-mix.

In vitro cytogenicity in mammalian cells

In an in vitro micronucleus assay in cultured peripheral human lymphocytes performed according to OECD Guideline 487, it was concluded that T003686 was not clastogenic or aneugenic in human lymphocytes in the absence and in the presence of S9 -mix under the experimental conditions described in the report.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-08-24 to 2015-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020734592
- Expiration date of the lot/batch: 2016-01-14 (retest date)
- Purity test date: 2015-06-17

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.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: The stock formulation was treated with ultrasonic waves to obtain a homogeneous suspension.

In the dose range finding test, at concentrations of 5.12 mg/ml and higher the test item was suspended in dimethyl sulfoxide. At concentrations of 1.64 mg/ml and lower the test item was dissolved in dimethyl sulfoxide.
In the first mutation experiment, at concentrations of 1.7 mg/ml and higher the test item was suspended in dimethyl sulfoxide. At the concentration of 0.54 mg/ml the test item was dissolved in dimethyl sulfoxide.
In the second and third mutation experiment, at all concentrations (1.57 to 16 mg/ml) the test item was suspended in dimethyl sulfoxide.

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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 the solubility of the test item in DMSO.
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 with and without 5% (v/v) S9-mix

The top doses for the mutation experiments were selected based on the toxicity and precipitation observed in the dose range finding test.
Mutation experiment 1: 5.4, 17, 52, 164 and 512 μg/plate in TA1535, TA1537, TA98 and TA102 with and without 5% (v/v) S9-mix

Since in the first experiment no toxicity and no precipitate on the plates was observed in all four tester strains, an additional mutation experiment was performed.
Mutation experiment 1A: 512 and 1600 μg/plate in TA1535, TA1537, TA98 and TA102 with and without 5% (v/v) S9-mix

To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix.
Mutation experiment 2: 157, 281, 502, 896 and 1600 µg/plate in TA1535, TA1537, TA98, TA100 and TA102 with and without 10% (v/v) S9-mix

To verify the positive response of tester strain TA100 in the second mutation experiment, a third mutation experiment with this tester strain was performed.
Mutation experiment 3: 157, 281, 502, 896 and 1600 µg/plate in TA100 with and without 10% (v/v) S9-mix results

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: No workable suspension of the test item could be obtained in water or in dimethyl sulfoxide at the concentration of 50 mg/ml. Since the test item formed a homogeneous suspension in dimethyl sulfoxide at a concentration of 25 mg/ml, DMSO was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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

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

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9-mix; 2.5 μg/plate (TA1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: tert-butyl hydroperoxide (TBH)

Remarks:

Without S9-mix; 250 μg/plate (TA102)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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), 10 μg/ plate (TA102 with 5 and 10% S9-mix)

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 or 0.2 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): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-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 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.

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.

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.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: No workable suspension of the test item could be obtained in water at the concentration of 50 mg/ml.
- Precipitation:
Mutation experiment 1: at the start of the incubation period at concentrations of 164 μg/plate and upwards. No precipitation of the test item on the plates was observed at the end of the incubation period.
Mutation experiment 1A: at the start and at the end of the incubation period at the concentration of 1600 μg/plate.
Mutation experiment 2: at the start of the incubation period at concentrations of 492 μg/plate and upwards and at the start of the incubation period at concentrations of 896 and 1600 μg/plate.
Mutation experiment 3: at the start and at the end of the incubation period at concentrations of 896 μg/plate and upwards.

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strain TA100. The dose range finding test results are reported as a part of mutation experiment I.
Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 164 μg/plate and upwards. At the end of the incubation period, precipitation of the test item on the plates was observed at concentrations of 512 μg/plate and upwards.
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 up to the dose level of 1600 μg/plate. The number of revertants at the dose level of 5000 μg/plate could not be determined due to precipitate. No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The negative and 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.
Mutation experiment 1: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested
Mutation experiment 1A: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested
Mutation experiment 2:In tester strain TA100, the test item induced dose related increases in the number of revertants up to 2.1 and 1.9-fold compared to the solvent control in the absence and presence of S9-mix, respectively.
Mutation experiment 3: In tester strain TA100, the test item induced dose related increases in the number of revertants up to 2.4 and 2.3-fold compared to the solvent control in the absence and presence of S9-mix, respectively.

In tester strain TA100, 1.9- to 2.4-fold, dose related increases were observed, both in the absence and presence of S9-mix. The results were observed in two out of three experiments and the number of revertants were above the laboratory historical control data range. Therefore these increases are considered biologically relevant.
All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related increase in the number of revertants in two independently repeated experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Mutation experiment 1: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutation experiment 1A: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Mutation experiment 2: There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
Mutation experiment 3: There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Conclusions:

Based on the results of this study it is concluded that the test item at concentrations up to 1600 μg/plate is mutagenic in tester strain TA100 of the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537, TA98 or TA102).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-01-31 to 2016-02-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020734592
- Expiration date of the lot/batch: 2017-01-14
- Purity test date: 2016-02-01
- Purity: 99.5% (GC)

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.

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

Mutation experiment I: 157, 281, 502, 891, 1250 and 1600 μg/plate in TA1535, TA1537, TA98, TA100 and TA102 with and without 5% (v/v) S9-mix
Mutation experiment II: 157, 281, 502, 896, 1250 and 1600 µg/plate in TA1535, TA1537, TA98, TA100 and TA102 with and without 10% (v/v) S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: solubility test performed in WIL Research project TOX11348

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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

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

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9-mix; 2.5 μg/plate (TA1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: tert-butyl hydroperoxide (TBH)

Remarks:

Without S9-mix; 250 μg/plate (TA102)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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), 10 μg/ plate (TA102 with 5 and 10% S9-mix)

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 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)

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 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 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.

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.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: No workable suspension of the test item could be obtained in water at the concentration of 50 mg/ml.
- Precipitation:
Mutation experiment 1:at the start of the incubation period at concentrations of 1250 μg/plate and upwards. At the end of the incubation period, precipitation on the plates was observed at concentrations of 891 μg/plate and above in the absence of S9-mix and 1250 μg/plate and above in the presence of S9-mix.
Mutation experiment 2: No precipitation of the test item on the plates was observed at the start of the incubation period. At the end of the incubation period, precipitation on the plates was observed at concentrations of 1250 μg/plate and upwards.

RANGE-FINDING/SCREENING STUDIES: was performed in WIL Research project TOX11348

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The negative and 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.

- Cytotoxicity:
Mutation experiment 1: Cytotoxicity was only observed in tester strain TA98 in the absence of S9-mix, where a slight reduction in the number of revertant colonies was observed at the test item concentration of 1600 μg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the other tester strains in the absence and presence of S9-mix.
Mutation experiment 2: There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In tester strain TA102, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed in the absence of S9-mix. However, since the reduction was less than 20% compared to the concurrent vehicle control, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.
In tester strain TA1535, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the lowest dose level of 157 μg/plate in the absence of S9-mix. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

- Mutagenicity:
Mutation experiment 1: In tester strain TA100, the test item induced dose related increases in the number of revertant colonies up to 2.3- and 2.2-fold compared to the solvent control in the absence and presence of S9-mix, respectively.
The test item did not induce a significant dose-related increase in the number of revertant colonies in the tester strains TA1535, TA1537, TA98 and TA102 in the absence and presence of S9-mix.
Mutation experiment 2: In tester strain TA100, the test item induced dose related increases in the number of revertant colonies up to 2.4- and 2.3-fold compared to the solvent control in the absence and presence of S9-mix, respectively.
The test item did not induce a significant dose-related increase in the number of revertant colonies in the tester strains TA1535, TA1537, TA98 and TA102 in the absence and presence of S9-mix.

Conclusions:

Based on the results of this study it is concluded that the test item at concentrations up to 1600 μg/plate is mutagenic in tester strain TA100 of the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537, TA98 or TA102).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-11-17 to 2018-11-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: FDA Toxicological Principles for the Safety Assessment of Direct Food Ingredients. Section IV.C1.a:Bacterial Reverse Mutation Test. “Redbook 2000”.

Version / remarks:

U.S. FDA Washington DC 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ministry of Health and Welfare (MHW), Japanese Guidelines for Nonclinical Studies of Drugs Manual (1995), Section 5. Reverse Mutation Test in Bacteria

Version / remarks:

1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. S2 (R1)

Version / remarks:

document recommended for adoption at step 4 of the ICH process on November 9, 2011

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: kclemen-05-00291-1
- Expiration date of the lot/batch: not indicated
- Purity test date: not indicated
- Purity: 99.5 – 100.4 % w/w (depending on the analysis column)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in a closed and labelled container protected from light
- 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. Fresh test formulations were prepared for each day’s work.

Preparation and handling of formulations
Formulations were prepared on a weight/volume basis without correction for the displacement due to the volume occupied by the test and positive control items.
The test item concentrations were prepared immediately before use by subsequent dilutions from the highest stock formulation. Solutions of positive control items were prepared before the first day of plating, after which aliquots of the solutions were stored at approximately –20ºC for the whole duration of the study. The frozen aliquots were thawed immediately prior to use.
The test and control items were handled at all times in a manner designed to reduce the risk of any accidental exposure of staff and environment.

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

50 µg S9-mix/plate (Aroclor 1254 induced rat liver metabolic activation system)

Test concentrations with justification for top dose:

The maximum final concentration for the first mutation experiment was based on the solubility of the test item in DMSO.

Mutation experiment I: 19.53, 39.06, 78.13, 156.25, 312.5, 625 and 1250 μg/plate in TA1535, TA1537, TA98, TA100 and TA102 with and without 50 μl S9/plate

As the plate incorporation test with strain TA100 of the first mutation experiment was invalidated in the absence and in the presence of S9-mix, this test was repeated in the second mutation experiment using the same concentrations.
Mutation experiment II: 19.53, 39.06, 78.13, 156.25, 312.5, 625 and 1250 μg/plate in TA100 with and without 50 μl S9/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- 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 finally soluble at 6.25 mg/ml (= 625 μg/plate) after warming at 37ºC for about 2 hours, but a moderate milky suspension was obtained upon mixing with water. Sequential dilutions from this concentration resulted in moderate to strong milky suspension upon mixing with water, until at 1.56 mg/ml (= 156.25 μg/plate) a clear solution was obtained upon mixing with water. Based on these solubility findings, DMSO was selected as vehicle and 1250 μg/plate (using a workable suspension at 12.5 mg/ml) was selected as the maximum final concentration for the first mutation experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix; 5 μg/plate (TA98)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9-mix; 1 μg/plate (TA1535, TA100)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix; 50 μg/plate (TA1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix; 2.5 μg/plate (TA98, TA100, TA1535, TA1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix; 7.5 μg/plate (TA102)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix; 5 μg/plate (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
The following solutions were added to 2 ml histidine-biotin supplemented top agar:
- 0.1 ml of an overnight bacterial culture of the tester strain
- 0.1 ml of a dilution of the test item, vehicle control or positive control
- 0.5 ml of S9-mix for the activation portion or 0.5 ml phosphate buffer for the non-activation portion.
The content of the tube was then mixed and poured onto minimal glucose agar plates. The plates were incubated in the dark at 37°C for 48 to 72 hours. If observed, precipitation of the test item into top agar at the start of treatment, evident to the unaided eye, was recorded.

DURATION
- Exposure duration: 48 to 72h
- Selection time (if incubation with a selection agent): 48 to 72h (simultaneous with exposure)

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

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

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; reduction of revertant colonies

Evaluation criteria:

Criteria for positive response:
According to Brusick (1980), a test item is considered positive (mutagenic) if all of the following criteria are met:
- the test item produces at least a two-fold increase in the mean number of revertants with one of strains TA98, TA102 or TA100, or a three-fold increase in the mean number of revertants with one of strains TA1535 or TA1537 at one or more concentrations in comparison to the mean concurrent vehicle control value;
- a concentration-related effect is observed.

Criteria for negative response:
If the test item does not produce (1) a concentration-dependent increase in the number of revertant colonies and (2) a two-fold increase in the mean number of revertants with one of strains TA98, TA102 or TA100, or a three-fold increase in the mean number of revertants with one of strains TA1535 or TA1537 in comparison to the mean concurrent vehicle control value, it will be considered as negative (non-mutagenic) in this test system under the current test conditions.

When criteria for a clear positive or a clear negative are not satisfied (e.g., concentrationdependent increase that fails to reach two-fold in the mean number of revertants for strains TA98, TA102 or TA100, or three-fold in the mean number of revertants for strains TA1535 or TA1537; or biological significant increase in the reversion rate that does not appear to be concentration-dependent), more tests may be required, in order to evaluate the mutagenic potential of the test item. If the test item produces a positive response in a single test that cannot be reproduced in additional testing, the initial positive data will be discounted. If still the test item cannot be judged to be positive or negative, the results may be classified as equivocal.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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: From 625 μg/plate onwards, a concentration-related increase in precipitation (a moderate to strong milky suspension) was observed at the start of treatment with the strains TA98, TA1537, TA1535 and TA102 in the absence and in the presence of S9-mix.
Mutation experiment 2: From 625 μg/plate onwards, a concentration-related increase in precipitation (a moderate to strong milky suspension) was observed at the start treatment with the strain TA100 in the absence and in the presence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: no dose range finding test was conducted

STUDY RESULTS
- Concurrent vehicle negative control:
First mutation experiment
The mean number of spontaneous and vehicle control revertant colonies for all strains in the absence and for strains TA98, TA100, TA1535 and TA102 in the presence of S9-mix fell within the range of the laboratory historical data.
For strain TA1537 in the presence of S9-mix, the mean number of vehicle control revertant colonies fell slightly above the range of the laboratory historical data. This observation had no impact on the scientific validity of the results with this strain as the mean number of spontaneous revertant colonies and the mean number of vehicle control revertant colonies in the absence of S9-mix fell within the range of the laboratory historical control data.
For strain TA1535 in the absence of S9-mix, the mean number of spontaneous revertant colonies fell marginally below the range of the laboratory historical data. This observation had no impact on the scientific validity of the results with this strain as the mean number of vehicle control revertant colonies fell within the range of the laboratory historical data.
For strain TA100 in the absence of S9-mix, the mean number of spontaneous revertant colonies fell below the range of the laboratory historical data. This observation had no impact on the scientific validity of the results with this strain as the mean number of vehicle control revertant colonies fell within the range of the laboratory historical data.

Second mutation experiment
The mean number of spontaneous and vehicle control revertant colonies for TA100 in mutation experiment II in the absence and in the presence of S9-mix fell within the range of the laboratory historical data.

- Positive control data:
First mutation experiment:
The positive controls with the strains TA98, TA1537, TA1535 and TA102 induced a biologically significant increase in the mean number of revertant colonies in comparison to the mean concurrent vehicle control value, indicating the capacity of the test system to identify mutagens. Based on the above findings, the plate incorporation tests with the strains TA98, TA1537, TA1535 and TA102 in the absence and in the presence of S9-mix were considered acceptable for the evaluation of the mutagenic potential of JNJ-63757044-AAA.
The plate incorporation test with the strain TA100 in the absence and in the presence of S9-mix was invalidated as the positive controls did not induce a biologically significant increase in the mean numbers number of revertant colonies in comparison to the mean concurrent vehicle control value.

Second mutation experiment: The positive control induced a biologically significant increase in the mean number of revertant colonies in comparison to the mean concurrent vehicle control value, indicating the capacity of the test system to identify mutagens

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No significant decrease in the number of revertants or reduction in bacterial background lawn observed in any of the experiments

Sterility checks and bacterial titre of the strains were according to the criteria.

Conclusions:

It can be concluded that the test item in the absence and in the presence of an Aroclor 1254 induced rat liver metabolic activation system, has no mutagenic properties towards the various Salmonella typhimurium strains TA98, TA1537, TA100, TA1535 and TA102 under the test conditions described in this report, up to the maximum test concentration of 1250 μg/plate.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-01-11 to 2016-01-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

non-GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Remarks:

but performed on strain only

Qualifier:

according to guideline

Guideline:

other: FDA Toxicological Principles for the Safety Assessment of Direct Food Ingredients. Section IV.C1.a:Bacterial Reverse Mutation Test. “Redbook 2000”.

Version / remarks:

U.S. FDA Washington DC 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. S2 (R1)

Version / remarks:

Document recommended for adoption at step 4 of the ICH process on November 9, 2011

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0020734592
- Expiration date of the lot/batch: not known
- Purity test date: not known
- Purity: 99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in a closed and labelled container protected from light
- 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.

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains)

Species / strain / cell type:

S. typhimurium TA 100

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:

Aroclor 1254 induced male rat liver S9-mix

Test concentrations with justification for top dose:

The maximum final concentration for the mutation experiments was based on the solubility of the test item in DMSO.
Mutation experiment I: 19.53, 39.06, 78.13, 156.25, 312.5, 625 and 1250 μg/plate in TA100 with and without 10% (v/v) S9-mix
Mutation experiment II: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 with and without 5% (v/v) S9-mix
Mutation experiment III: 157, 281, 502, 896, 1600 μg/plate in TA100 with and without 10% (v/v) S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: In DMSO, the test item forms a strong milky suspension with particles at 50 mg/ml (= 5000 μg/plate). Sequential dilutions from this maximum concentration resulted in moderate to slight milky suspensions until at 1.56 mg/ml (= 156.25 μg/plate) a clear solution was obtained. Based on these solubility findings, DMSO was selected as vehicle and the maximum final concentration of 12.5 (clear solution with particles), 25 and 16 mg/ml (slight milky suspensions with particles) was selected for the first, second and third mutation experiment, respectively.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9-mix; 1 μg/plate (TA100)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

without S9-mix; 2.5 μg/plate (TA100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
The following solutions were added to 2 ml histidine-biotin supplemented top agar:
- 0.1 ml of an overnight bacterial culture of the tester strain
- 0.1 ml (and 0.2 ml in the second mutation experiment for the top concentration) of a dilution of the test item, 0.1 ml (and 0.2 ml in the second mutation experiment) vehicle control or 0.1 ml positive control
- 0.5 ml of S9-mix for the activation portion or 0.5 ml phosphate buffer for the non-activation portion.
The content of the tube was then mixed and poured onto minimal glucose agar plates. The plates were incubated in the dark at 37°C for 48 to 72 hours. If observed, precipitation of the test item into top agar at the start of treatment, evident to the unaided eye, was recorded.

DURATION
- Exposure duration: 48 to 72h
- Selection time (if incubation with a selection agent): 48 to 72h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains)

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

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; reduction of revertant colonies

Evaluation criteria:

Criteria for positive response:
According to Brusick (1980), a test item is considered positive (mutagenic) if all of the following criteria are met:
- the test item produces at least a two-fold increase in the mean number of revertants with the strain TA100 at one or more concentrations in comparison to the mean concurrent vehicle control value;
- a concentration-related effect is observed.

Criteria for negative response:
If the test item does not produce (1) a concentration-dependent increase in the number of revertant colonies and (2) a two-fold increase in the mean number of revertants with the strain TA100 in comparison to the mean concurrent vehicle control value, it will be considered as negative (non-mutagenic) in this test system under the current test conditions.

When criteria for a clear positive or a clear negative are not satisfied (e.g., concentrationdependent increase that fails to reach two-fold in the mean number of revertants for the strain TA100; or biological significant increase in the reversion rate that does not appear to be concentration-dependent), more tests may be required, in order to evaluate the mutagenic potential of the test item. If the test item produces a positive response in a single test that cannot be reproduced in additional testing, the initial positive data will be discounted. If still the test item cannot be judged to be positive or negative, the results may be classified as equivocal.

Statistics:

No formal hypothesis testing was done.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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: At the start of treatment, a clear solution with small particles was observed with the strain TA100 in the absence of S9-mix at 625 and 1250 μg/plate. At the end of treatment, slight precipitation was observed with the strain TA100 in the absence and in the presence of S9-mix at 1250 μg/plate.
Mutation experiment 2: At the start of treatment, a moderate milky suspension with particles was observed with the strain TA100 in the absence and in the presence of S9-mix at 5000 μg/plate. At 512 and 1600 μg/plate, a clear solution with particles was observed with the strain TA100 in the absence of S9-mix. At the end of treatment, slight or moderate precipitation was observed with the strain TA100 at 1250 μg/plate, respectively in the absence or in the presence of S9-mix.
Mutation experiment 3: At the start of treatment, a slight milky suspension with particles was observed with the strain TA100 in the absence and in the presence of S9-mix. At the end of treatment, a concentrationrelated increase in precipitation (slight to moderate) was observed with the strain TA100 in the absence of S9-mix from 896 μg/plate onwards. In the presence of S9-mix, slight precipitation was observed with strain the TA100 at 1600 μg/plate.

RANGE-FINDING/SCREENING STUDIES: no dose ranging test was performed

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The mean number of spontaneous and vehicle control revertant colonies for all strains in the absence and the presence of S9-mix fell within the range of the laboratory's historical data.

- Cytotoxicity:
Mutation experiment 1: Up to 1250 μg/plate, JNJ-63757044-AAA did not induce a concentration-related and biologically significant increase (>= 2 -fold) in the number of revertant colonies above the concurrent vehicle control with the strain TA100 in the absence and in the presence of S9-mix (50 μl S9/plate)
Mutation experiment 2: Up to 5000 μg/plate, JNJ-63757044-AAA did not induce a concentration-related and biologically significant increase (>= 2 -fold) in the number of revertant colonies above the concurrent vehicle control with the strain TA100 in the absence and in the presence of S9-mix (25 μl S9/plate)
Mutation experiment 3: np to 1600 μg/plate, JNJ-63757044-AAA did not induce a concentration-related and biologically significant increase (>= 2 -fold) in the number of revertant colonies above the concurrent vehicle control with the strain TA100 in the absence and in the presence of S9-mix (50 μl S9/plate)

- Mutagenicity:
Mutation experiment 1: no additional information on cytotoxicity
Mutation experiment 2: Bacteriotoxic effects, visualised by thinning of the bacterial background lawn was observed with the strain TA100 in the absence and in the presence of S9-mix at 5000 μg/plate.
Mutation experiment 3: no additional information on cytotoxicity

Conclusions:

It can be concluded that the test item in the absence and in the presence of an Aroclor 1254 induced rat liver metabolic activation system, has no mutagenic properties towards the Salmonella typhimurium strain TA100 under the test conditions described in this report, up to the maximum test concentration of 5000 μg/plate.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-20 to 2019-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I18ID2574
- Expiration date of the lot/batch: 2020-09-22 (retest date)
- Purity test date: 2019-01-28 (release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

OTHER SPECIFICS: correction factor of 1.00 for the purity/composition of the test item was applied

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
For lymphocytes:
- Sex, age and number of blood donors: ages 24 and 27
- Whether whole blood or separated lymphocytes were used: Blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitab le size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started. Whole blood was used.
- Whether blood from different donors were pooled or not: not specified
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-in activated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 29 - 95 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 - 38.0 °C).

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

The highest tested concentration was determined by the solubility of the test item in the culture medium.
Dose-range finding test: 3.9, 7.8, 15.6, 31.3, 62.5 and 125 μg/mL without S9-mix (24h treatment).
Based on the absence of cytotoxicity observed in the duplicate cultures for the 3 h treatment period, the dose range finding study (short term exposure period) was used as the first cytogenetic assay.

First cytogenetic assay:
Without and with S9-mix: 31.3, 62.5 and 125 μg/ml culture medium (3 hour exposure time, 27 hour harvest time )

Based on the results of the dose range finding test, an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility of the test item in the culture medium.
Second cytogenetic assay: 1, 5, 15, 20, 25, 30, 40 and 50 μg/mL without S9-mix (24 hour exposure period, 24 hour harvest time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: The test item proved insoluble in RPMI 1640 medium, dimethyl sulfoxide, ethanol, acetone and dimethylformamide but was dissolved tetrahydrofuran. The stock solution was treated with ultrasonic waves until the test item had completely dissolved. Test item concentrations were used within 1 hour after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9-mix; at 0.25 and 0.38 µg/mL (3h treatment); at 0.15 and 0.23 µg/mL (24h treatment)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

without S9-mix; at 0.1 µg/mL (3h treatment); at 0.05 µg/mL (24h treatment)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix; at 15 and 17.5 µg/mL (3h treatment)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
First cytogenetic assay: 3 hour exposure period, 27 hour harvest time, with and without metabolic activation
Second cytogenetic assay: 24 hour exposure period, 24 hour harvest time without metabolic activation

FOR MICRONUCLEUS:
- Identity of cytokinesis blocking substance: Cytochalasin B was added at 5 µg/mL for the 24h expression time after the 3h exposure time to the test item, and simultaneously with the test item for the 24h exposure time.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
To harvest the cells, cell cultures were centrifuged and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68. After centrifugation, the cells in the r emaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately ther eafter, ethanol/ acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation and cells in the pellet were fixated carefully with 3 changes of ethanol / acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) etha nol / ether and cleaned with a tissue. The slides were marked with the Test Facility Study number a nd group number. At least two slides were prepared per culture. Slides were allowed to dry and ther eafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene / pertex and mounted with a coverslip in an automated cover slipper.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
Three analysable concentrations were scored for micronuclei. At least 1000 (with a maximum deviatio n of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. The number of micronuclei per cell was not recorded.
Since the lowest concentration of MMC-C and CP resulted in a positive response the highest con centration was not examined for the presence of micronuclei. Due to cytotoxicity the number of exam ined bi- or mononucleated cells in the positive control groups might be <1000. However, when an exp ected statistical significant increase is observed, this has no effect on the study integrity.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).

Rationale for test conditions:

The highest concentration examined for micronuclei should be cultures that produce 55 ± 5% of cytotoxicity. The lowest concentration should have little or no cytotoxicity (approximately the same as vehicle control). Cultures treated with an intermediate concentration should be examined (24h exposure time). For poorly soluble test items, the highest concentration examined should be the lowest insoluble concentration determined at the end of treatment irrespective of toxicity. The extent of precipitation may not interfere with the scoring of micronuclei. If no precipitate or limiting toxicity is observed, the highest concentration examined should correspond to 2 mg/ml or 0.01 M, whichever is the lowest.

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) The increase is dose-related in at least one experimental condition when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical vehicle control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the historical vehicle control data range.

Statistics:

Fisher exact test was used to identify results significantly different from control group.Since the Fisher exact test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Remarks:

first and second cytogenetic experiments primary assays

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Water solubility: No data
- Precipitation : The test item precipitated in culture medium at the concentration of 25 mg/ml (= final concentration of 125 µg/ml) and above.

RANGE-FINDING/SCREENING STUDIES:
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test: blood cultures were treated with 3.9, 7.8, 15.6, 31.3, 62.5 and 125 μg test item/ml culture medium and exposed for 24 hours in the absence of S9-mix.
Dose-related cytotoxicity was observed after 24 hours of exposure at 3.9 µg/ml culture medium and above. The test item precipitated in the culture medium at the concentration of 125 µg/ml.

STUDY RESULTS
- Concurrent vehicle negative and positive control data Positive historical control data: The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated and binucleated cells with micronuclei. In addition, the number of mono- and bi nucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent) historical control data: The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical vehicle control database.

- Results from cytotoxicity measurements:
Dose range finding test / First cytogenetic assay: Dose-related cytotoxicity was observed after 24 hours of exposure at 3.9 µg/ml culture medium and above.
No appropriate levels of cytotoxicity were observed after 3 hours of exposure up to the test item concentration of 125 µg/ml. The test item precipitated in the culture medium at this dose level.
All dose levels were selected for scoring of micronuclei in the first cytogenetic assay.

Second cytogenetic assay: Appropriate toxicity was reached at 40 and 50 µg/ml culture medium as in one of the duplicate cultures, a reduction in the cytokinesis-block proliferation index to 45 ± 5% was observed.

- Genotoxicity results
Dose range finding test / First cytogenetic assay: All dose levels were selected for scoring of micronuclei in the first cytogenetic assay.
In the absence of S9-mix, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest dose. However, no dose response was observed and the number of cells with micronuclei (3 in 1000 and 7 in 1000 for duplicates A and B, respectively) was clearly within the range of the solvent control historical data range (upper 95%control limit: 8.50 per 1000 cells).

In the presence of S9-mix, the test item induced a statistically significant and dose related (Cochran Armitage trend test p = 0.013) increase in the number of binucleated cells with micronuclei at the highest dose. However, the number of cells with micronuclei (4 in 1000 and 3 in 1000 for duplicates A and B, respectively) was clearly within the range of the solvent control historical data range (upper 95%control limit: 9.23 per 1000 cells).

The increases observed both in the presence and in the absence of S9-mix were therefore not considered biologically relevant.

Second cytogenetic assay: The test item did not induce a statistically significant or biologically relevant increase in the number of mono- or binucleated cells with micronuclei.

Conclusions:

Based on the results of the study, it is concluded that this test is valid and that the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation tests

In a first (non-GLP) study with five Salmonella typhimurium strains (TOX11076), JNJ-63757044-AAA has no mutagenic properties in the absence and presence of a rat liver metabolizing system (S9-mix) when tested up to 1250 μg/plate as a solution including particles.

In a second (GLP) study with five Salmonella typhimurium strains (TOX11348), JNJ-63757044-AAA has no mutagenic properties in the absence and presence of S9 -mix when tested up to 5000 μg/plate as a suspension including particles. In a subsequent experiment, JNJ-63757044-AAA induced dose-related increases in the number of revertants in strain TA100 up to 2.4 and 2.3-fold compared to the vehicle control in the absence and presence of S9-mix, respectively. It was concluded that JNJ-63757044-AAA has mutagenic properties towards strain TA100 in the absence and presence of S9-mix when tested up to 1600 μg/plate as a solution including particles.

In a third (non-GLP) study with the Salmonella typhimurium strain TA100 (TOX11568), JNJ-63757044-AAA has no mutagenic properties in the absence and presence of S9-mix using the same experimental design (i.e. testing up 1250 and 1600 μg/plate as a solution including particles and testing up to 5000 μg/plate as a suspension including particles) as in the previous conducted studies.

To further investigate the discripant results obtained in the first 3 studies, a fourth (GLP) study (TOX11575) was conducted by testing JNJ-63757044-AAA in five Salmonella typhimurium strains in the absence and presence of S9-mix up to 1600 μg/plate with special attention to the preparation of the formulations and scoring of revertant colonies. In strain TA100, JNJ-63757044-AAA induced dose-related increases in the number of revertants up to 2.4 and 2.3-fold compared to the vehicle control in the absence and presence of S9-mix, respectively. It was concluded that JNJ-63757044-AAA has mutagenic properties towards strain TA100 in the absence and

presence of S9-mix when tested up to 1600 μg/plate as a solution including particles.

Although no biologically relevant increases in the number of revertants (< 2-fold compared to vehicle control) were observed in strain TA100 in the first and third study,

the increases observed in the second and fourth study were biologically relevant, but were weak (up to 2.4-fold). All studies were conducted at the limit of test item’s

solubility and as a consequence some of the used test item formulations included particles. It can therefore not be excluded that the nature of the formulations played a role in the discripant results obtained in these studies.

In view of safety, the overall conclusion from these studies is that JNJ-63757044-AAA has mutagenic properties towards strain TA100 both in the absence and presence of S9-mix.

See attachment with expert statement on overall conclusion of the mutagenic properties of test item based on the four Ames test described above.

In vitro cytogenicity in mammalian cells

An in vitro micronucleus assay with JNJ-63757044-AAA (T003686) in cultured peripheral human lymphocytes was performed (Eurlings, 2019, K1).

This report describes the effect of the test item on the induction of micronuclei formed in cultured peripheral human lymphocytes in the presence of S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ß-naphthoflavone) with a 3 hour exposure period and in the absence of S9-mix with a 3 and 24 hour exposure period. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments. 

The study procedures described in this report are in compliance with the most recent OECD and EC guidelines. 

Batch I18ID2574 of the test item was a white powder. The test item was dissolved in tetrathydrofuran. 

In the first cytogenetic assay, the test item was tested up to 125 µg/ml for a 3-hour exposure period with a 27 hour harvest time in the absence and presence of S9-mix. The test item precipitated in the culture medium at this dose level. 

In the second cytogenetic assay, the test item was tested up to 50 µg/ml for a 24 hour exposure period with a 24 hour harvest time in the absence of S9-mix. Appropriate toxicity (reduction in the cytokinesis-block proliferation index to 45 ± 5%) was reached at this dose level. 

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical vehicle control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated and binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system
(S9-mix) functioned properly. 

The test item did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. 

It is concluded that this test is valid and that the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Justification for classification or non-classification

Positive results were observed in the tester strain Salmonella typhimurium TA100 in the Ames test and negative results were obtained in the in vitro micronucleus assay.

Based on the positive results obtained in the bacterial reverse mutation test, the criteria laid out in the CLP regulation and the precautionary principle, the test item T003686 should be classified as Muta 2 - H341 Suspected of causing genetic defects.