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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 2018 - 18 January 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:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
dated 4 February 2015
GLP compliance:
yes (incl. certificate)
Remarks:
dated 27 April 2017
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
OECD 442D is one of the in-vitro skin sensitization test method now recommended for REACh registration.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vitro test system

Details on study design:
STUDY OBJECTIVE:
A skin sensitizer refers to a substance that will lead to an allergic response following skin contact.
One of the biological key events takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling pathways such as the antioxidant/electrophile response element (ARE) dependent pathways.
The genes under the ARE control, including AKR1C2 gene identified as a target gene for detecting skin sensitizers in keratinocytes, are induced by the protein Nrf2 via Keap1.
The test consists of evaluating the activation of AKR1C2 in transformed keratinocytes (KeratinoSens™), by monitoring the induction of the luciferase gene fused to AKR1C2. The luciferase produced by the cells complexes with luciferin which, in the presence of ATP, produces light measured in relative light units (RLU).
After contact between a potentially sensitizing element with a KeratinoSens™ monolayer, the induction of the luciferase is quantified. In parallel, the cytotoxicity is measured, in order to exclude a false positive generated by skin irritation.
Since activation of the Keap1-Nrf2-ARE pathway addresses only the second key event of the skin sensitization AOP, information from test methods based on the activation of this pathway is unlikely to be sufficient when used on its own to conclude on the skin sensitization potential of chemicals.
Therefore data generated should be considered in the context of integrated approaches.
This study was carried out according to the OECD Guideline 442D dated February, 04th, 2015 and the ECVAM DB-ALM protocol 155: KeratinoSensTM.

SERIES DEFINITION:
- Test item: The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
- Reference item:
Negative control: 6 wells of solvent control (1% DMSO in treatment medium) on each culture plate.
Positive control: 5 concentrations of cinnamaldehyde on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2.
- Blanks: 1 well by culture plate is left without cell and was filled with negative control.
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.

REFERENCE ITEMS:
- Positive control: Cinnamaldehyde (SIGMA ALDRICH Ref W228613):
- Negative control: Treatment culture medium, 1% DMSO, 1% Non-heat inactivated foetal calf serum. The negative control is prepared just prior the test and used within the day.

TEST SYSTEM:
Cells: KeratinoSens™ (Givaudan) maintained according to the current working instruction.
Cells are cultured in maintenance medium at 37°C, 5% CO2.
Cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current working instruction.
Cells were used at passage 17 in repetition 1 and passage 19 in repetition 2.

MEDIA AND REAGEANTS:
- 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
- Trypsin (0.5 g/l) - EDTA (0.2 g/l) - stored at -20°C ± 5°C
- Diluent for the test item: Sterile water - stored at room temperature 20°C ± 5°C
- Diluent for the positive control: DMSO (1% maximum final concentration) - stored at room temperature 20°C ± 5°C
- Luciferase substrate: Bright Glo™ Luciferase Assay System (Promega) - stored at -80°C after reconstitution
- Cell washing solution: Dulbecco’s PBS Ca2+ and Mg2+ free with 0.05% EDTA - stored at 5°C ± 3°C
- Dulbecco’s PBS Ca2+ and Mg2+ free - stored at room temperature 20°C ± 5°C
- Staining solution: 5 mg/ml MTT* (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS - prepared extemporaneously and used within the day
- Desorption solution: 10% SDS in water - stored at room temperature 20°C ± 5°C
* Note: MTT powder is stored at 5°C ± 3°C

EQUIPMENT AND CONSUMABLES:
- Luminometer: GloMax™ (Promega)
- MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -linearity range 0 - 2.200 units of Absorbance
- White cell culture 96-well plates for luminescence reading
- Transparent cell culture 96-well plates for absorbance reading
- Plastic adhesive foils
- Conventional cell culture laboratory equipment.

TEST PROTOCOL:
- Cells seeding (first day)
The cells were trypsinized according to the current working instruction IL 09.
After removal the culture medium from the culture flask, the cell layer was rinsed with PBS 0.05% EDTA, Ca2 + and Mg² + free to remove all traces of serum. The solution of trypsin-EDTA was added and left for a few minutes at 37°C, 5% CO2 until the detachment of cells. The action of trypsin was stopped by addition of maintenance medium.
Cell concentration was determined on Malassez cell. Cells suspension was 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 cell culture plates (96 wells) for the induction measurement and two transparent cell culture plates (96 wells) to assess the cytotoxicity. In order to ensure a good homogeneity of seeding, cells suspension was regularly mixed all along the seeding.
Note: the H12 wells were left without cells for the measurement of blanks.

- Preparation of test item and positive control dilutions (second day)
Given the high MW of the test item, the stock solution was prepared at 79.2 mM* (4X) in treatment medium, 4% DMSO instead of 100X.
*During the experimentation, the purity of the test item was considered at 10.1 %; taking into account the 100% purity, the concentration of the stock solution was 79.2 mM.
The positive control stock solution was prepared at 200 mM in DMSO, then diluted to the concentration of 6.4 mM in DMSO.

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 from the column 11 to 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
Test item: The test item was placed in one of the rows B to F.
100 µl of treatment culture medium 4% DMSO were distributed from columns 1 to 11. 200 µl of the 79.2 mM stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl from the column 12 to the column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Positive and negative control
The 100 X DMSO plate was diluted 25 fold with treatment medium in the 4X plate.

- 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 homogeneity of the test item dilutions are checked, no precipitation was observed. The medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferin + 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 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 one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.

RESULTS AND INTERPRETATION:
Two parameters are measured, the luciferase induction and the cytotoxicity.
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. Otherwise the KeratinosensTM prediction is considered as negative:
• the Imax is strictly 1.5 fold higher of the basal luciferase activity* statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student’s t-test on the raw RLU values),
* If the Imax is exactly equal to 1.5, the test item is rated as negative and no EC1.5 value is calculated
• the EC1.5 value is strictly below 1000 µM,
• at the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e. EC1.5 < IC70),
• there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.
In rare cases, the test items which induce the gene activity at a concentration very close to the cytotoxic levels, are positive in some repetitions at non-cytotoxic levels, and in other repetitions only at cytotoxic levels. In this case, the test item must be tested again with a narrower range using a dilution factor of 4/3 instead of 2.
Test items that only induce the gene activity at cytotoxic levels are not rated as positive, as it is the case for some non-sensitizing skin irritants.
If, in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, in case of a test item with poor solubility tested at a concentration lower than 1000 µM, a negative result obtained should also be considered as inconclusive.
Negative results should be interpreted with caution as substances with an exclusive reactivity towards lysine-residues can be detected as negative by the test method. Furthermore, because of the limited metabolic capability of the cell line used and because of the experimental conditions, pro-haptens (i.e.chemicals requiring enzymatic activation for example via P450 enzymes) and pre-haptens (i.e. chemicals activated by auto-oxidation) in particular with a slow oxidation rate may also provide negative results.
Test chemicals that do not act as a sensitizer but are nevertheless chemical stressors may lead on the other hand to false positive results. Furthermore, highly cytotoxic test chemicals cannot always be reliably assessed.
Finally, test chemicals that interfere with the luciferase enzyme can confound the activity of luciferase in cell-based assays causing either apparent inhibition or increased luminescence.

Results and discussion

Positive control results:
Rep 1:
4 µM: 1.26 - 8 µM: 1.46 - 16 µM: 1.94 - 32 µM: 2.40 - 64 µM: 4.13 - EC1.5 = 8.74 and Imax = 4.13
Rep 2:
4 µM: 1.14 - 8 µM: 1.24 - 16 µM: 1.43 - 32 µM: 1.67 - 64µM: 3.95 - EC1.5=20.67 and Imax = 3.95
Mean:
4 µM: 1.20 - 8 µM: 1.35 - 16 µM: 1.69 - 32 µM: 2.03 - 64µM: 4.04 - EC1.5=13.44 (geometric mean) and Imax = 4.0

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: EC1.5
Remarks:
expressed in mM
Run / experiment:
induction
Value:
< 0.01
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Imax
Remarks:
mean value
Run / experiment:
viability
Value:
145.79
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: IC70
Remarks:
geometric mean expressed in mM
Run / experiment:
induction
Value:
0.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
All validity criteria are met, study is considered valid.

Any other information on results incl. tables

The test item dilutions were checked by visual inspection after 48 hours at 37°C, no undissolved residues or precipitate or phase separation remained.

Interference of the test item with the detection of luminescence was not checked.

   VIABILITY  INDUCTION      
   IC70 (mM)  Imax  Linear EC 1.5 (mM)  EC 1.5 Lin/lLog (mM)
 Rep 1  0.07 56.36  < 0.010   < 0.010
 Rep 2  0.19  235.22  < 0.010  < 0.010
 Mean    145.79    
 Geometric mean  0.11      

      

Applicant's summary and conclusion

Interpretation of results:
other: May be classified as a potential skin sensitizer
Remarks:
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.
Conclusions:
Under the retained experimental conditions of this OECD 442D test, test item may be classified as potential skin sensitizer.