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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Remarks:
Keratinosens
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Alternative in vitro methods are recommended in first intention by ECHA.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on study design:
1. Cell seeding
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.

2. Preparation of test item and positive control dilutions
Preparation of the test item stock solution:
The test item was diluted in DMSO. The stock solution was prepared at 200 mM. A volume of DMSO was calculated according to the following formula: V=(5*[(p/100)*w)/MW) - (w/1000)
where V is the volume of DMSO in ml to be added, p is the purity of the test item in %, MW is the molecular weight of the test item in g/mol, w is the exact weight of the test item in mg.

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

Preparation of the 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 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).

3. Contact between the cells and the test and reference items
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).

4. Luciferase activity
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.

5. Cell viability assessment with MTT method
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 5 mg/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 a week-end (repetition 1) or one night (repetition 2) in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.

Results and discussion

In vitro / in chemico

Results
Key result
Parameter:
other: EC1.5 (uM)
Remarks on result:
not measured/tested
Remarks:
Given that Imax was less than 1.5, the EC1.5 could not be determined

Any other information on results incl. tables

Imax = 1.21 (1st assay) and Imax = 1.13 (2nd assay). Therefore, the mean Imax was 1.17, which is lower than 1.5. No EC1.5 was thus determined.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the retained experimental conditions 1,4-DIBROMONAPHTHALENE may be classified as not skin 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.