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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

950-123-7

Cas Number:

97849-65-5

Molecular formula:

C44H44N6O6S

IUPAC Name:

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

Test material form:

solid: particulate/powder

In vitro test system

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).

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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.

Results 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.