Benzamide, N-(5-bromo-3-methyl-2-pyridinyl)-N-methyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2020 - 17 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h CLAT)"

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Boronic acid, [6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-

Cas Number:

446299-81-6

Molecular formula:

C14H15BN2O3

IUPAC Name:

Boronic acid, [6-(benzoylmethylamino)-5-methyl-3-pyridinyl]-

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Details on study design:

TEST SYSTEM
- Cell line used, storage conditions and source: THP-1 (ATCC, #TIB-202), stored in liquid nitrogen and sub-cultured twice weekly.
- Culture medium and conditions: RPMI 1640 medium, GlutaMAX™ supplement including 25 mM HEPES, supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin).
The cells were grown using general culture procedures and maintained in a humidified incubator set at 37± 1.5ºC, 5± 0.5% CO2 and were not allowed to exceed a cell density of 1 E06 cells/mL or more than 30 passages. Reactivity checks were performed to qualify the cells before testing.
The passage numbers of the used THP-1 cells were 22 K1 and 22 K2 in the cytotoxicity tests and 23 and 25 in the h CLAT for runs 1 and 2, respectively.
- Cell culture for testing: On the day of the cytotoxicity or main experiment, directly before the treatment of the cells, a volume of 500 µL with a cell density of 1.8 - 2 E06 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.

CONTROLS
- Medium Control: culture medium.
- Solvent Control for the Test Item: DMSO (final concentration for cytotoxicity and for h-CLAT 0.2%).
- Positive control: 2,4-Dinitrochlorobenzene (DNCB, purity ≥ 99%) at 3 and 4 µg/mL in DMSO (final concentration 0.2%).
- Solvent Control for the Positive Control: dimethylsulfoxide (DMSO, purity ≥ 99%), dissolved at 0.2% in culture medium.

STUDY DESIGN
- Solubility assessment: As tested by a solubility test, 1000 µg/mL in 0.2% (v/v) DMSO in culture medium was used as highest test item concentration in the cytotoxicity test, as recommended in the OECD 442E.

- Dose Finding assay (PI Assay):
The test item concentrations investigated in the main experiments (h-CLAT) were determined with two cytotoxicity tests. The tests were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run. The test item dilutions were prepared freshly before each experiment. Each volume (500 µL) of the dilutions of the test item, culture medium and solvent control (0.2% (v/v) DMSO in culture medium) were added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.
Each test item-treated and not test item-treated cells were collected in sample tubes, centrifuged (approx. 250 x g, 5 min), washed twice (2 - 8 °C) with 2 mL FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added in each sample tube.
The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal.

- Main test (CD86/CD54 expression measurement):
The test item was tested in two independent runs. The tests were performed with independent cell cultures (cells are collected from different culture flasks) and on different days. The test item was prepared separately for each run. Since the CV75 could not be determined, the OECD 442E guideline recommended maximal to be tested test item concentration (1000 µg/mL) prepared in DMSO was used as highest concentrations for the h-CLAT. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) DMSO in the medium. Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures. The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

INTERPRETATION OF RESULTS
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs:
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
− Otherwise, the h-CLAT prediction is considered NEGATIVE.

ACCEPTANCE CRITERIA
- Cell viability of medium control and DMSO control should be more than 90%.
- In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
- For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
- Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. If 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90%.

DEVIATIONS FROM OECD GUIDELINE: No.

Results and discussion

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result obtained in one tested concentration (1000 µg/mL), at which also cell viability was >50%.

Results 2

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result obtained in one tested concentration (1000 µg/mL), at which also cell viability was >50%.

Results 3

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result obtained in one tested concentration (1000 µg/mL), at which also cell viability was >50%.

Results 4

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Result obtained in one tested concentration (1000 µg/mL), at which also cell viability was >50%.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated.

ACCEPTANCE OF RESULTS: All acceptance criteria were met in each run (see table below).

Any other information on results incl. tables

Table 1. Results of the Dose Finding Assay (Cytotoxicity Test) no. 1.

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	97.18
Solvent Control	-	no	97.78
Test Item	7.83	no	97.63
	15.7	no	97.35
	31.3	no	96.64
	62.5	no	97.21
	125	no	97.24
	250	no	97.25
	500	no	96.40
	1000	no	95.97

Due to the lack of cytotoxicity in the Flow Cytometric Evaluation of the Cytotoxicity Test, a CV75 value could not be calculated.

Table 2. Results of the Dose Finding Assay (Cytotoxicity Test) no. 2.

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	97.20
Solvent Control	-	no	97.99
Test Item	7.83	no	97.63
	15.7	no	97.62
	31.3	no	97.51
	62.5	no	97.57
	125	no	97.70
	250	no	97.41
	500	no	96.24
	1000	no	96.06

Due to the lack of cytotoxicity in the Flow Cytometric Evaluation of the Cytotoxicity Test, a CV75 value could not be calculated.

Table 3. Results of the h-CLAT Test. First run.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	98.12
DMSO Control	-	100.0	100.0	97.99
Positive Control (DNCB)	3.0	371.2*	649.1*	89.80
	4.0	537.0*	644.7*	86.16
Test Item	279	104.1	106.5	97.04
	335	98.6	98.2	97.14
	402	106.8	116.7	97.22
	482	124.7	121.5	96.03
	579	143.8	105.1	95.47
	694	152.1	134.2	96.50
	833	143.8	105.1	94.32
	1000	221.9*	279.3*	85.40

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Table 4. Results of the h-CLAT Test. Second run.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	97.00
DMSO Control	-	100.0	100.0	97.30
Positive Control (DNCB)	3.0	333.3*	147.7^#	89.16
	4.0	470.2*	768.8*	79.64
Test Item	279	109.5	83.9	96.93
	335	119.0	112.8	97.40
	402	150.0	126.1	96.31
	482	116.7	114.7	96.71
	579	131.0	103.2	96.09
	694	139.3	118.8	95.68
	833	131.0	125.7	94.82
	1000	211.9*	242.7*	84.97

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

# CD86 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criteria (CD86 ≥ 150%) due to a technical error. This value was not evaluated.

Table 5. Acceptance criteria of the h-CLAT Test. First run.

Cell viability of medium control and DMSO control should be more than 90%.            Medium:        98.12%            DMSO:          97.99%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       371.2%            3 µg/mL DNCB (CD 86):       649.1%            4 µg/mL DNCB (CD 54):       537.0%            4 µg/mL DNCB (CD 86):       644.7%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   112.3%            CD86:    91.1%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         124.6%            Medium CD 86:         214.4%            DMSO CD 54:           127.2%            DMSO CD 86:           202.6%

Table 6. Acceptance criteria of the h-CLAT Test. Second run.

Cell viability of medium control and DMSO control should be more than 90%.            Medium:        97.00%            DMSO:          97.30%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.            3 µg/mL DNCB (CD 54):       333.3%            3 µg/mL DNCB (CD 86):       147.7%^#            4 µg/mL DNCB (CD 54):       470.2%            4 µg/mL DNCB (CD 86):       768.8%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).            CD54:   129.2%            CD86:   103.3%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%.            Medium CD 54:         123.7%            Medium CD 86:         177.0%            DMSO CD 54:           131.3%            DMSO CD 86:           181.3%

# CD86 RFI value of the positive control (3.0 µg/mL DNCB) did not fulfil the positive criteria (CD86 ≥ 150%) due to a technical error. This value was not evaluated.

Table 7. Historical control data.

	Positive Control				(MFI - MFI(ISO)DMSO)/ (MFI - MFI(ISO)Medium)
	DNCB (3 µg/mL)		DNCB (4 µg/mL)
Antibody	CD 54	CD 86	CD 54	CD 86	CD 54	CD 86
Value should be	≥ 200%	≥ 150%	≥ 200%	≥ 150%	< 200%	< 150%
Mean Value [%]	320.4	476.2	416.0	508.5	114.1	108.4
Standard Deviation	81.2	324.2	120.7	278.0	18.0	12.7
Max Value [%]	551.0	1952.4	697.1	1672.8	172.5	140.7
Low Value [%]	168.0	183.0	127.6	191.2	76.1	77.6
Total number of runs	45

These values represent historical control data conducted in 2019.

Applicant's summary and conclusion

Interpretation of results:

other: The h-CLAT test result is considered as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442E.

Conclusions:

The test item activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive in the h-CLAT Test.

Executive summary:

An in vitro Human Cell Line Activation Test (h-CLAT) was performed as part of an integrated approach to support the identification of the sensitization potential of the test item in accordance with OECD Guideline 442E, under GLP conditions. Based on the results from a solubility assay, two Dose-Range Finding (cytotoxicity) assays were conducted with the test item dissolved in DMSO and further diluted in culture medium (final concentration of 0.2% (v/v) DMSO in culture medium). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore, maximal test item concentration of 1000 µg/mL was used as recommended in the Guideline. The concentrations 279, 335, 402, 482, 579, 694, 833, 1000 µg/mL of the test item were tested in 2 independent main experiments (h-CLAT). In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%, with one exception. The CD86 RFI value of the positive control (3.0 µg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (CD86 ≥ 150%). However, this one exception is considered to be acceptable since the CD86 RFI value of the positive control (4.0 µg/mL DNCB) in the second h-CLAT run exceeded the positive criteria. The RFI of CD86 and/or CD54 for the test item was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.