Apple, Malus sylvestris, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: the ECVAM DB-ALM protocol 155: KeratinoSensTM

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Apple, Malus sylvestris, ext.

EC Number:

286-475-7

EC Name:

Apple, Malus sylvestris, ext.

Cas Number:

85251-63-4

IUPAC Name:

Apple, Malus sylvestris, ext.

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Cells seeding (first day)

The cells were trypsinized according to the current working instruction IL 09. Cells suspension were adjusted to a density of 8.104 cells/ml in seeding medium. 125 µl of the cell suspension at 8.104 cells/ml (i.e. 104 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2.
Note: the H12 wells were left without cells and allowed the measurement of blanks.

Preparation of test item dilutions (second day)
Preparation of the test item stock solution:The test item was diluted in sterile water.The stock solution was prepared at 40 mg/ml (i.e. 4%).

Preparation of the positive control stock solution: The positive control was prepared at 200 mM in DMSO then diluted to the final concentration of 6.4 mM.

Preparation of the 100 X plate:A 100-fold concentrated dilutions serie was prepared in 96-well plate.

Test item
The test item was placed in one of the rows B to F.
100 µl of sterile water. were distributed from columns 1 to 11. 200 µl of the 40 mg/ml stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl of the column 12 in the 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.
 
Preparation of the 4 X dilution plate
The 100 X DMSO plate was diluted 25 fold in a new plate (4 X). For the test item, the level of the DMSO is adjusted to 1% final.

Contact between the cells and the test and reference items (second day)
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 (37°C, 5% CO2).

Luciferase activity (day 4)
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 (luciferine + ATP + lysing agent) 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.

Cell viability assessment with MTT method (day 4)
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 (37°C, 5% CO2).
After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS one night (repetition 1) or a weekend (repetition 2) in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.

Results and discussion

Positive control results:

EC1.5 = 15.14
Imax = 3.94

In vitro / in chemico

Results

Key result

Parameter:

other: Luciferase induction

Value:

1.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

other: not sensitizer

Conclusions:

Under the retained experimental conditions, the test item may be classified as not sensitizer.
The test method KeratinoSens 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:

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

The test method KeratinoSens 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.