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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

812-520-2

Cas Number:

1259279-57-6

Molecular formula:

C20H22BFN2O4S

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-63757044-AAA (T003686)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 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

Method

Target gene:

not applicable

Species / strain

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.

Controls

Controls 1

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)

Controls 2

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)

Controls 3

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.