[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-11-13 to 2017-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Given the slight solubility of the test item it was diluted at 8 mM (4X) in treatment medium, 4% DMSO instead of 200 mM.

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

1

Reference substance name:

Bis and tris and tetra{N-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino]-N’-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino] methaniminium} [phthalocyanine(bis and tris and tetra)sulfonato-κ4N29,N30,N31,N32]cuprate(II)

Cas Number:

265115-84-2

Molecular formula:

C32H16-τN8Cu(SO3C17H22N3)τ

IUPAC Name:

Bis and tris and tetra{N-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino]-N’-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino] methaniminium} [phthalocyanine(bis and tris and tetra)sulfonato-κ4N29,N30,N31,N32]cuprate(II)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Test system:
* Type of cells : Keratinosens TM
* Origine : Givaudan
* Culture medium : DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin - stored at 5°C ± 3°C
* Culture conditions : 37°C, 5% CO2.
* Mycoplasma statut: : no mycoplasma

* Passage numbers:
- repetition 1 : 24
- repetition 2 : 16
- repetition 3: 18

* Number of replicates per repetition
- for induction : 3 replicates
- for cytotoxicity : 2 replicates



Media
- Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin - stored at 5°C ± 3°C
- Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum - stored at 5°C ± 3°C
- Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum - stored at 5°C ± 3°C

Controls
* positive control : Cinnamaldehyde:
* Negative solvent control: DMSO


Study course

- Cell seeding (first day)
*Cell seeding: 80%
* Cell density of the cell suspension : 8.10^4 cells/ml in seeding medium
* Seeding : 125 µl of the cell suspension at 8.10^4 cells/ml (i.e. 10^4 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity.
*Incubation : 24 hours ± 1 hour at 37°C, 5% CO2.
* Number of plates : 5


- Preparation of the test item dilution (second day)
* Preparation of the test item stock solution: Given the slight solubility of the test item it was diluted at 8 mM, in treatment medium, 4% DMSO instead of 200 mM.


* Preparation of the positive control stock solution: concentration at 200 mM of positive control in DMSO. The solution is diluted to a concentration of 6.4 mM.

*Preparation of the 100 X plate (positive and negative control): 100-fold concentrated dilutions prepared in 96-well plate.

*Preparation of the 4 X dilution plate :
Test item: series dilutions prepared from the stock solution
positive and negative control: 100 X plate diluted 25 times in the 4X plate


- Contact between the cells and the test and reference items (second day)
Culture medium replaced with 150µL of fresh treatment medium
50 µl from the 4 X plate are placed in the different plates, then the plates are incubated.

Conditions of incubation
Time : 48 hours +/- 1 hour
Temperature: 37°C
CO2: 5%
Plates : covered with foil

- Luciferase activity (day 4)
Preparation of the plates:
Medium is removed after 48 hours and cells are washed with phosphate buffer saline
100µL of Luciferase substrate is added to each well
Incubation of the plates for 15 minutes at room temperature

Measurement in the luminometer:
* integration time : 2 seconds

- Cell viability assessment with MTT method (day 4)
Preparation of the plates:
Medium is removed after 48 hours and cells are washed with phosphate buffer saline
225 µL of MTT solution (0.6 mg/ml in treatment medium) is added to each well, then the plates are incubated.

Conditions of incubation
Time : 4 hours ± 30 min
Temperature: 37°C
CO2: 5%
Plates : covered with foil


After 4 hours, the MTT solution is removed and cells are lysed by adding 10% SDS overnight, in the dark.
After homogeneisation, the absorbances are measured at 540 nm.

Results and discussion

Positive control results:

Positive control : Cinnamaldehyde
Geometric mean EC1.5 = 16.19
Mean Imax = 3.55

Remark :A third assay was performed as in repetition 2, EC.1.5 was higher than the historical data.
All other validation criteria are met and validates the test.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Run 1

Parameter:

other: IC 50 µM

Value:

16.81

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Run 1

Parameter:

other: Imax

Value:

0.47

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: Run 2

Parameter:

other: IC 50 µM

Value:

35.64

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Run / experiment:

other: Run 2

Parameter:

other: Imax

Value:

0.51

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 5

Run / experiment:

other: Run 3

Parameter:

other: IC 50 µM

Value:

10.67

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 6

Key result

Run / experiment:

other: Run 3

Parameter:

other: Imax

Value:

0.89

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 7

Run / experiment:

other: Run 1

Parameter:

other: IC 30 µM

Value:

11.84

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 8

Run / experiment:

other: Run 2

Parameter:

other: IC 30 µM

Value:

16.11

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 9

Run / experiment:

other: Run 3

Parameter:

other: IC 30 µM

Value:

0.98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: For negative (solvent) control, the coefficient of variation was below 20% for all replicates
- Acceptance criteria met for positive control: For repetition 2, EC1.5 was higher the historical data value. A third assay was performed. EC1.5 was on the range of the historical values dataset for replicates 1 and 3.

Any other information on results incl. tables

Positive control

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1,29	1,60	2,24	2,37	5,84	6,70	5,84
Rep 2	1,14	1,10	1,29	1,49	2,26	32,53	2,26
Rep 3	0,96	1,13	1,36	2,00	2,54	19,48	2,54
Mean	1,13	1,28	1,63	1,95	3,55	16,19*	3,55

* geometric mean

Negative control

Control solvent	CV %  control solvent
Rep 1	18,4
Rep 2	8,6
Rep 3	8,1

Test item

	VIABILITY		INDUCTION
	IC30  µM	IC50  µM	Imax	Linear EC1.5  µM	EC1.5 Lin/Log  µM
Rep 1	11.84	16.81	0.47	-	-
Rep 2	16.11	35.64	0.51	-	-
Rep 3	< 0.98	10.67	0.89	-	-
Mean	-	-	0.62	-	-
Geometric mean	-	18.56	-	-	-

Imax < 1.5: no EC1.5 is determined

Mean Viability Percentage

Concentration µM	0,98	1,95	3,91	7,81	15,6	31,3	63	125	250	500	1000	2000
Rep 1	89,80	97,72	89,93	87,47	53,60	9,60	0,65	0,52	0,84	1,23	2,66	4,80
Rep 2	81,87	94,00	81,01	82,73	70,40	57,32	5,06	0,00	0,38	0,86	2,20	10,03
Rep 3	55,71	57,09	56,36	59,27	32,47	37,26	1,16	0,00	0,00	0,00	1,89	16,05
Viability	75,8	82,9	75,8	76,5	52,2	34,7	2,3	0,0	0,1	0,4	2,2	10,3

Mean Induction

Concentration µM	0,98	1,95	3,91	7,81	15,63	31,25	62,50	125	250	500	1000	2000
Rep 1	0,46	0,44	0,46	0,38	0,47	0,47	0,05	0,00	0,00	0,00	0,00	0,00
Rep 2	0,51	0,47	0,35	0,33	0,37	0,29	0,13	0,00	0,00	0,00	0,00	0,00
Rep 3	0,89	0,73	0,61	0,55	0,52	0,46	0,33	0,01	0,01	0,00	0,00	0,00
Induction	0,62	0,55	0,47	0,42	0,45	0,41	0,17	0,01	0,00	0,00	0,00	0,00
SD	0,23	0,16	0,13	0,12	0,08	0,10	0,14	0,01	0,00	0,00	0,00	0,00

Student-t test

Rep 1	0,001	0,001	0,004	0,001	0,002	0,062	0,000	0,000	0,000	0,000	0,000	0,000
Rep 2	0,001	0,000	0,000	0,000	0,000	0,000	0,000	0,000	0,000	0,000	0,000	0,000
Rep 3	0,388	0,002	0,001	0,002	0,000	0,000	0,000	0,000	0,000	0,000	0,000	0,000

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the retained experimental conditions the test item may be classified as not sensitizer.

The test method KeratinoSensTM is considered scientifically valid to be used as part of an integrated approaches to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.

Executive summary:

The test was performed to assess the skin sensitization potential of the test item in KeratinoSens TM cells.

Cells were treated with the test item at 12 concentrations from 0.98 to 2000 µM in DMSO, according to a geometric progression of ratio 2. 

Another group of cells were treated with the positive control (cinnamaldehyde) at 5 concentrations from 4 to 64 µM according to a geometric progression of ratio 2.

A further group of cells were treated with DMSO (1% in treatment medium).

The study was composed of three different repetitions, with 3 replicates for the measurement of induction and 2 replicates for the measurement of cytotoxicity for each repetition. Induction and cytotoxicity were determined after a cell treatment period of 48 hours with the test item, positive and negative controls.

The experimental protocol was established according to the O.E.C.D GuidelineNo 442-D, dated February, 04th, 2015 and the ECVAM DB-ALM protocol 155:KeratinoSens^TM.

For viability the geometric mean of the IC 50 is 18.56.

In both repetitions, Imax are lower than 1.5, and no EC1.5 is determined.

Under the retained experimental conditions the test item may be classified as not sensitizer.

The test method KeratinoSens^TM is considered scientifically valid to be used as part of an integrated approaches to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.