Bis (3‐(diethylamino)‐7‐hydroxy‐5‐phenylphenazinium) sulphate

Endpoint summary

Description of key information

First key event (molecular initiating event): Data waiving. Preliminary to the study in accordance with OECD Guideline 442C, the solubility at 100 mM was assessed in all required solvents. The test substance was only soluble in water. However, when diluted in peptide buffer, the test item precipitated in all cases. In these conditions, OCDE 442C stipulates that only a positive DPRA prediction could be taken into account and thus no study was performed.

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

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Preliminary to the study in accordance with OECD Guideline 442C, the solubility at 100 mM was assessed in all required solvents. The test substance was only soluble in water. However, when diluted in peptide buffer, the test item precipitated in all cases. In these conditions, OCDE 442C stipulates that only a positive DPRA prediction could be taken into account and thus no study was performed. See attached a summary of this preliminary solubility test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2019 - 04 October 2019

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 STBH8906; purity 99.9%)
-DMEM 1 g/l glucose (Supplier ref. 21885025, Fisher Bioblock; Batch no 2095710)
-Non-heat inactivated foetal calf serum (Supplier ref. 11563397, Fisher Bioblock; Batch no 42F6480K)
-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 16 in repetition 1 and passage 18 in repetition 2.

CONTROLS
-Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
-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 8x10e4 cells/ml in seeding medium were distributed in the culture plates.
-Cell density: 10e4 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 solution: 2000 µM (1X) in treatment medium 1% 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 (positive and negative control): A 100-fold concentrated dilutions series was prepared in 96-well plate:
-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 (in cytotoxicity repetition 2, negative control was distributed in row H).

2) 4X dilution plate (positive and negative control): The 100 X plate was diluted 25 fold in a new plate (4 X).

3) Preparation of the 1X dilution for the test item:
The test item was placed in one of the rows B to F.
1100 µl of treatment medium, 1% DMSO were distributed columns 1 to 10 in a masterblock. 2200 µl of the 2000 µM solution were placed in column 12 then the series dilutions were prepared by transferring 1100 µl of the column 12 in the column 11 and so until the column 1. Dilutions were mixed by repeated pipetting at least 3 times, between each concentration.

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.

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
-Negative and positive control: 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).
-Test item: In the 5 seeded plates, the medium was aspirated. The 1X masterblock was replicated 5 times: 200 µl from the 1X masterblock were placed in each of the three white plates and in the two transparent plates. The plates (1X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37ºC ± 1°C, 5% CO2).

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:
The dilution strategy is different from the OECD 442D TG (paragraph 22). Given the slight solubility of the test item, the dilution was prepared directly 1X in treatment medium-1% DMSO at 2000 µM. This has no impact on the study result because the test item was tested in the expected final condition (i.e. 2000 µM as maximal concentration).

Positive control results:

Repetition 1: The maximal average induction of luciferase activity (Imax) was 6.75 at a concentration of 64 µM. The mean value EC1.5 was 8.15 µM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 5.92 at a concentration of 64 µM. The mean value EC1.5 was 13.23 µM.

Key result

Run / experiment:

other: Repetition 1

Parameter:

other: Imax

Value:

1.96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The induction is greater than 1.5 but at the EC1.5 concentration the viability is less than 70%, thus this repetition is negative.

Key result

Run / experiment:

other: Repetition 2

Parameter:

other: Imax

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The induction is less than 1.5, thus this repetition is negative.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.
- In both repetitions, at the end of the 48 hours incubation, sediments were observed in culture wells from 62.5 µM to 2000 µM which led to an overestimation of the viability at these concentrations. However, a dose dependent decrease of viability from 0.98 µM (99.5%) to 31.25 µM (4.4%) was observed which means that in the wells where sediments were formed, viability would be lower than 4.4% (see tables 3 and 4 below). Inductions are not taken into account if viability is lower than 70%, so the test item sediments did not impact the study outcome.

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 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 20% and for repetition 2 was 16% 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 6.75 and 5.92 in each repetition which are between 2 and 8. The EC1.5 values were 8.15 and 13.23 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	1.34	1.49	2.23	3.28	6.75	8.15	6.75
Rep 2	1.11	1.25	1.63	2.78	5.92	13.23	5.92
Mean	1.23	1.37	1.93	3.03	6.34	10.38*	6.34

*geometric mean

Control solvent	CV %  control solvent
Rep 1	20
Rep 2	16

Table 2: Test item summary results

	VIABILITY		INDUCTION
ID-19-09379	IC30 µM	IC50 µM	Imax	Linear EC1.5 µM	EC1.5 Lin/Log µM
Rep 1	2.50	2.99	1.96	3.38	3.24
Rep 2	2.66	3.04	1.11	-	-
Mean	-		1.53	-	-
Geometric mean	2.58	3.02	-	-	-

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	88,81	92,45	12,40	5,13	4,96	2,32	6,28	10.58	22,33	47,96	151,65	255,18
Rep 2	110,19	106,61	5,15	4,26	2,91	6,49	14,33	24,64	71,00	198,21	434,71	620,83
Viability	99,5	99,5	8,8	4,7	3,9	4,4	10,3	17,6	46,7	123,1	293,2	438,0

* presence of sediments

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	0.89	1,00	1,69	1,89	1,96	0,04	-0,01	-0,01	-0,01	-0,01	-0,01	-0,01
Rep 2	0,92	0,93	1,11	0,98	0,81	0,11	-0,02	-0,02	-0,02	-0,02	-0,02	-0,02
Induction	0,90	0,96	1,40	1,44	1,38	0,08	-0,01	-0,02	-0,02	-0,02	-0,02	-0,02
SD	0,02	0,05	0,41	0,64	0,81	0,05	0,01	0,01	0,01	0,01	0,01	0,01

Table 5: Student t-test

Rep 1	0.299	0.959	0.010	0.012	0.092	0.001	0.001	0.001	0.001	0.001	0.001	0.001
Rep 2	0.281	0.322	0.319	0.777	0.072	0.001	0.000	0.000	0.000	0.000	0.000	0.000

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 greater than 1.5 but at the EC1.5 concentration the viability was less than 70% and in the second repetition the induction was lower than 1.5, thus 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:

18 November 2019 - 28 November 2019

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 16 and 17 in the cytotoxicity tests and 19 and 20 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
- Solvent/vehicle control: dimethylsulfoxide (DMSO, purity ≥ 99%), dissolved at 0.2% in culture medium.
- Medium control: culture medium.
- Positive control: 2,4-Dinitrochlorobenzene (DNCB, purity ≥ 99%) at 3 and 4 µg/mL.

STUDY DESIGN
- Solubility assessment: Medium and DMSO was tested in the solubility test. Since the test item could be resolved in DMSO, it was used as suitable solvent. The highest tested test item concentration for the cytotoxicity test was 150 µg/mL in 0.2% DMSO diluted in culture medium.

- Dose Finding assay (PI Assay): For each of 2 independent cytotoxicity tests, 8 concentrations of test item were prepared by 1:2 serial dilutions from a test item stock solution of 75 mg/mL in DMSO. These dilutions were then further diluted 250-fold into culture medium and finally diluted with a dilution factor of 2 into the THP-1 cell suspension in the plate. The plates were incubated for 24 ± 0.5 h. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Then, cells were transferred into sample tubes and collected by centrifugation, washed twice with 2 mL of 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. Then, the flow cytometer (FACSCalibur, Becton Dickinson GmbH) was calibrated and 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. The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.

- Main test (CD86/CD54 expression measurement). 2 independent validated runs were conducted on different days. The CV75 value of both cytotoxicity tests was not calculated, since precipitation was observed in the four highest test item concentrations (18.75, 37.5, 75 and 150 µg/mL). Therefore, the lowest test item concentration with observed precipitation (18.8 µg/mL, rounded value) was used as highest concentration for the main experiments. Therefore, dilutions were prepared by 1:1.2 serial dilution from a 9.40 mg/mL test item stock solution. These dilutions were further diluted 1:250 into culture medium and finally diluted with a dilution factor of 2 into the THP-1 cell suspension in the plate. Exposure was carried out as in the Dose Finding assay. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Then cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2-8ºC (on ice) for approximately 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 - 8ºC or on ice during the staining and analysis procedures. Those cells were mixed and incubated for 30 ± 5 min. at 2 - 8ºC, protected from light. The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry as in the DRF. Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis.

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%.

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 all tested concentrations

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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 all tested concentrations except for the lowest at 5.3 µg/mL.

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.
- Precipitation:
1) DRF test: The three highest concentrations of both cytotoxicity tests could not be microscopically evaluated due to observed strong precipitation.
2) Main test: Precipitation was observed at the three highest concentrations of both runs.

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 'overall remarks').

Table 3. Results of the first h-CLAT run for the Test Item.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	97.95
DMSO Control	-	100.0	100.0	97.84
Positive Control (DNCB)	3.0	368.8*	468.5*	90.20
	4.0	474.0*	713.8*	81.10
Test Item	5.3	170.1	399.4*	97.29
	6.3	184.4	259.2*	94.93
	7.6	198.7	308.7*	89.83
	9.1	149.4	408.7*	77.93
	10.9	129.9	547.6*	62.14
	13.1^P	63.6	706.8*	59.32
	15.7^P	87.0	651.4*	46.91#
	18.8^P	61.0	987.8*	52.46

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

P: Precipitation, is excluded from the evaluation

#: cell viability below 50%, values excluded from the evaluation.

Table 4. Results of the second h-CLAT run for the Test Item.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	98.14
DMSO Control	-	100.0	100.0	98.28
Positive Control (DNCB)	3.0	311.4*	328.9*	87.87
	4.0	322.7*	345.6*	86.73
Test Item	5.3	109.1	149.7	98.59
	6.3	125.0	150.3*	97.91
	7.6	114.8	193.1*	96.74
	9.1	108.0	241.5*	95.66
	10.9	83.0	336.5*	86.15
	13.1^P	114.8	389.3*	74.28
	15.7^P	46.6	510.4*	67.68
	18.8^P	0.0	806.0*	56.16

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

P: Precipitation, is excluded from the evaluation

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 RFI of CD86 was equal or greater than 150% in at least one concentration of both runs. Therefore, the test item activated THP-1 cells under the test conditions of this study and it is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

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 at highest dose of 150 µg/mL in 0.2% DMSO diluted in culture medium. The CV75 value of both cytotoxicity tests was not calculated since precipitation was observed in the four highest test item concentrations (18.75, 37.5, 75 and 150 µg/mL). Therefore, the lowest test item concentration with observed precipitation (18.8 µg/mL) was used as highest concentration for the main experiments. Two validated successive test runs were performed. In each run, the test item formulations ( 5.25, 6.30, 7.56, 9.07, 10.9, 13.1, 15.7 and 18.8 µg/mL) were applied to THP-1 cells and cultured for 24 hours ± 0.5 hours at 37ºC, 5% CO2. Negative (medium), vehicle (DMSO), and positive (DCNB) controls were run in parallel. After incubation, the expression of CD86 and CD54 was measured by flow cytometry, and the viability of the cells was determined after staining with 7-Aminoactinomycin D. The Mean Fluorescence Intensity (MFI) was obtained for each test sample and then corrected. The corrected MFI values were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. All validity criteria were met. The RFI of CD86 was equal or greater than 150% 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.