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

Endpoint summary

Description of key information

First key event (molecular initiating event): Key study. Test method according to the OECD 442C Guideline with GLP. Under the experimental conditions the test item showed a co-elution for both cysteine and lysine which is considered a “non conclusive” result in the DPRA skin sensitization test.

Second key event (activation of keratinocytes): Key study. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test item may be classified as not skin sensitizer using the KeratinoSensTM test method.

Third key event (activation of dendritic cells): Key study. Test method according to the OECD 442E Guideline with GLP. Under the experimental conditions the test item may be classified as skin sensitizer using the h-CLAT test method.

Conclusion: Based on a positive result obtained from the experimental studies in one of the key events of the skin sensitisation Adverse Outcome Pathway (AOP) and also considering the observational and practical experience, e.g. in manufacture or handling the substance at test facility, it was concluded to classify the substance for skin sentitisation as a precautionary measure.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 June 2020 - 18 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Details on study design:

TEST ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was acetonitrile.

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, LLC; Ref. Ac RFAACAA-COOH; batch no 112119; purity 94.96%)
Lysine peptide (supplier RS Synthesis, LLC; Ref. Ac-RFAAKAA-COOH; batch no 112119; purity 94.96%)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKCG8499; purity 99.5%)
Co-elution control: test item in phosphate buffer (100 mM; pH 7.5 ± 0.05) for cysteine peptide and ammonium acetate buffer (100 mM; pH 10.2 ± 0.05) for lysine peptide.
Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy.
Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time.
Reference control C: prepared with acetonitrile, the test item and the positive control solvent, in order to check its influence on the peptide stability.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: 81.9 mg was pre-weighted in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution with acetonitrile.
Positive control solution: 38 µL of the positive control were distributed in a 5 ml glass vial in order to prepare, right before use, 3 ml of a limpid 100 mM solution with acetonitrile.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: 12.6 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 25.2 mL of phosphate buffer (100 mM; pH 7.5 ± 0.05).
Lysine solution: 11.8 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 22.8 mL of ammonium acetate buffer (100 mM; pH 10.2 ± 0.05).

TEST SOLUTIONS
Samples were dissolved immediately before use (100 mM positive control solution was kept for the 2 runs).
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
All the replicates were prepared with the same peptide stock solutions.

1 mL of each solution was prepared according to the following quantities:
Cysteine test solution (0.5 mM Peptide, 5 mM Sample): 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µL of sample or solvent for Reference controls + 200 µL of acetonitrile.
Lysine test solution (0.5 mM Peptide, 25 mM Sample): 750 µL of lysine peptide solution (buffer only to check co-elution) + 250 µL of sample or solvent for Reference controls.

The vials were capped and mixed carefully avoiding bubbling, then placed in the HPLC system sampler at 25°C ± 2.5°C. HPLC analysis started 24 hours ± 2 hours after addition of peptides.
Immediately upon addition of the test item solution to the peptide solution, just prior the beginning of the HPLC analysis and at the end of the analysis, samples were checked.

Replicates: Each sample was tested 3 times from 3 independent solutions

HPLC ANALYSIS
-Apparatus: Waters e2695 HPLC; Waters 2489 UV detector; Xbridge Peptide BEH C18 3.5 µm; dimensions 2.1 x 100 mm
-Calibration curve: Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
-Equilibration of the column: at 30°C with 50% phase A (0.1% (v/v) trifluoroacetic acid in water) and 50% phase B (0.085% (v/v) trifluoroacetic acid in acetonitrile) for at least 20 minutes before running.
-Volume injected: 7 µL of each sample
-Flow rate: 0.40 mL/min
-Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
-Duration of re-equilibration to the initial conditions between injections: at least 4 min.
-Analysis sequence:
The analysis was programmed according to the following principles:
The standards of the calibration curve and the reference controls A were placed at the beginning of the sequence.
The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
The reference controls C were placed at the beginning of each repetition.
Lysine and cysteine analysis were conducted on separate day and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours.

DEVIATIONS FROM OECD GUIDELINE: No.

Positive control results:

The depletion mean rate was 76.82% for cysteine peptide and 50.67% for lysine peptide.

Key result

Run / experiment:

other: 3 runs

Parameter:

other: %depletion in lysine peptide

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test item presented co-elution with the lysine peptide.

Key result

Run / experiment:

other: 3 runs

Parameter:

other: %depletion in cysteine peptide

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The test item presented co-elution with the cysteine peptide.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.503 mM for lysine and 0.491 mM for cysteine which are equal to 0.50± 0.05 mM
- Acceptance criteria met for reference control B/C: yes, the CV (coefficient of variation) of the controls B/C was 0.62% for lysine and 0.97% for cysteine which is lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.504 mM for lysine and 0.497 mM for cysteine which are equal to 0.50± 0.05 mM.
- Acceptance criteria met for positive control: Yes, SD of the 3 repetitions for each peptide was 0.44% for cysteine and 0.80% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 76.82% for cysteine peptide and 50.67% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.
- Acceptance criteria met for variability between replicate measurements: co-elution of the test item with the two peptides occurred then no depletion measurements were obtained.

Table 3: Positive control

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	50.11	76.43
Repetition 2	51.59	76.72
Repetition 3	50.32	77.30
SD (Standard Deviation)	0.80	0.44
Mean	50.67	76.82
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100
CV	1.58%	0.58%

Interpretation of results:

other: DPRA test result is considered as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.

Conclusions:

The test item presents a co-elution for both cysteine and lysine which is considered a “non conclusive” result in the DPRA skin sensitization test.

Executive summary:

A DPRA skin sensitization test was performed for the test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in acetonitrile as solvent. Reference controls A, B and C were prepared with acetonitrile in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C ± 2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested 3 times from 3 independent solutions. All validity criteria were fulfilled. The test item showed a co-elution for both cysteine and lysine, thus the analysis is considered “non conclusive”.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 June 2020 - 03 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Details on study design:

REAGENTS AND MEDIA
-DMSO (Supplier ref. 41640, Sigma Aldrich; Batch no I3540; purity 99.9%)
-DMEM 1 g/l glucose (Supplier ref. 21885025, Fisher Bioblock; Batch no 2095746)
-Non-heat inactivated foetal calf serum (Supplier ref. 11563397, Fisher Bioblock; Batch no 42A0001K)
-Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin.
-Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum.
-Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum.
-Staining solution: 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS (phosphate buffered saline). Prepared extemporaneously and used within the day.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in maintenance medium.
-Passage number: cells were used at passage 19 in repetition 1 and passage 25 in repetition 2.

CONTROLS
-Positive control: cinnamaldehyde (CAS 104-55-2; Sigma Aldrich Ref W228613, batch no MKCG8499; purity 99.5%)
-Negative (solvent) control: DMEM 1 g/l glucose, 1% DMSO, 1% non-heat inactivated foetal calf serum.

CELLS SEEDING.
-Culture plates: 5; 3 white cell culture plates (96 wells) for luminescence reading (induction measurement) + 2 transparent cell culture plates (96 wells) for absorbance reading (cytotoxicity).
-Cells suspension: 125 µl at 8E4 cells/ml in seeding medium were distributed in the culture plates.
-Cell density: 1E4 cells per well.
-Incubation: the seeded plates were incubated 24 hours ± 1 hour at 37ºC, 5% CO2

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Test item stock solution: 200 mM in DMSO
Positive control stock solution: The positive control was prepared at 200 mM in DMSO then diluted to the final concentration of 6.4 mM.

1) 100 X plate: A 100-fold concentrated dilutions series was prepared in 96-well plate:
-Test item: The test item was placed in one of the rows B to F. 100 µl of DMSO were distributed from columns 1 to 11. 200 µl of the 200 mM stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl from column 12 to column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
-Positive control: 100 µl of DMSO were distributed in row G from columns 7 to 10. 200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl of the column 11 in the column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
-Negative control: 100 µl of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12.

2) 4X dilution plate: The 100 X plate was diluted 25 fold in a new plate (4 X) in treatment medium.

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37ºC, 5% CO2).

CONCENTRATIONS TESTED
-Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
-Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 µM.

REPLICATES: The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

LUCIFERASE ACTIVITY.
-Apparatus: Luminometer: GloMax™ (Promega)
-Validity of luminometer: a quality control analysis was performed according to the procedure described in Annex 3 of the OECD guideline 442D .
-Luciferase substrate: luciferine + ATP + lysing agent. Bright Glo™ Luciferase Assay System (Promega).
-Procedure: After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

CITOTOXICITY ASSESSMENT.
-Apparatus: MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -linearity range 0 - 2.200 units of Absorbance.
-Procedure: After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37ºC, 5% CO2). After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS (sodium dodecyl sulfate) one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbance was measured at 540 nm.

DEVIATIONS FROM OECD GUIDELINE: No.

Positive control results:

Repetition 1: The maximal average induction of luciferase activity (Imax) was 4.77 at a concentration of 64 µM. The mean value EC1.5 was 7.76 µM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 3.62 at a concentration of 64 µM. The mean value EC1.5 was 8.82 µM.

Key result

Run / experiment:

other: Repetition 1

Parameter:

other: Imax

Value:

1.21

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax is lower than 1.5, thus the EC1.5 was not determined.

Key result

Run / experiment:

other: Repetition 2

Parameter:

other: Imax

Value:

1.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax = 1.80 at the concentration 2000 µM but the corresponding viability is lower than 70% (i.e. 60.18%). The induction factor is <1.5 for all concentrations given more 70% viability. The EC1.5 is not determined in agreement with the acceptance criteria.

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442D were performed, obtaining values that fall within the respective reference range for at least 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition 1 was 12.7% and for repetition 2 was 4.6% which are not higher than 20%.
- Acceptance criteria met for positive control: Yes, the luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The average induction values at 64 µM were 4.77 and 3.62 in each repetition which are between 2 and 8. The EC1.5 values were 7.76 and 8.82 µM for each repetition which are within 2 SD of the historical mean of the testing facility and between 7 µM and 30 µM based on the OECD validation dataset.

Table 1: Reference items

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.19	1.52	1.93	2.77	4.77	7.76	4.77
Rep 2	1.31	1.45	1.96	2.38	3.62	8.82	3.62
Mean	1.25	1.48	1.95	2.58	4.19	8.28*	4.19

*geometric mean

Control solvent	CV %  control solvent
Rep 1	12.7
Rep 2	4.6

Table 2: Test item summary results

	VIABILITY		INDUCTION
ID-20/03794	IC70  (µM)	IC50  (µM)	Imax	Linear EC1.5  (µM)	EC1.5 Lin/Log  (µM)
Rep 1	312.96	-	1.21	-	-
Rep 2	1362.22	-	1.80	-	-
Mean	-	-	1.51	-	-
Geometric mean	652.94	-	-	-	-

Table 3: Test item mean viability percentage

Concentration µM	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Rep 1	95.21	112.31	102.27	107.36	90.0	84.67	81.94	71.28	71.40	65.83	63.60	56.28
Rep 2	104.83	106,24	102.65	90.72	85.71	84.43	79.68	83.40	80.71	76.09	75.58	60.18
Viability	100	109.3	102.5	99.0	87.9	84.6	80.8	77.3	76.1	71.0	69.6	58.2

Table 4: Test item mean induction

Concentration µM	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Rep 1	0.95	1.03	1.19	1.19	1.21	0.97	0.96	0.72	0.63	0.61	0.62	0.89
Rep 2	1.14	1.18	1.09	0.96	1.22	0.91	0.86	0.81	0.78	0.85	0.99	1.80
Induction	1.05	1.11	1.14	1.07	1.22	0.94	0.91	0.76	0.70	0.73	0.80	1.35
SD	0.13	0.10	0.07	0.17	0.01	0.05	0.07	0.06	0.10	0.17	0.27	0.65

Interpretation of results:

other: KeratinoSensTM test result is considered as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442D.

Conclusions:

Under the experimental conditions the test item may be classified as not skin sensitizer using the KeratinoSensTM test method.

Executive summary:

The KeratinoSensTM test method was performed for the test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours ± 1 hour at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 2 plates (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 1 hour at 37ºC, 5% CO2. 2 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. In the first repetition the induction was lower than 1.5 and thus the EC1.5 was not determined. In the second repetition the induction was above 1.5 at the concentration 2000 µM but the corresponding viability was lower than 70% (i.e. 60.18%). The induction factor was less than 1.5 for all concentrations given more 70% viability and thus the EC1.5 was not determined either in agreement with the acceptance criteria. Bases on these findings, a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Reference 3

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

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

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.

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%.

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%.

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%.

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%.

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).

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is classified for skin sensitization (cat.1) according to CLP Regulation no. 1272/2008.