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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:
2018-11-19 - 2018-11-30
Reliability:
1 (reliable without restriction)

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)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Reaction product of guerbet alcohols, C24-26, branched and cyclic with 1,2,4-benzenetricarboxylic acid SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 05347/MA
- Expiration date of the lot/batch: retest date 2022-06-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature and darkness

In vitro test system

Details on study design:
The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
Reference items:
Negative control: each culture plate contains 6 wells of solvent control (treatment medium with 1% DMSO final)
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.
Positive control: for each culture plate, the cinnamaldehyde is tested at 5 concentrations from 4 to 64 µM according to a geometric progression of ratio 2.
Blanks: 1 well by culture plate is left without cell and filled with negative control to assess background value.

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.

Test system:
Cells: KeratinoSens™ (Givaudan) maintained according to the current instruction of the test lab.
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 instruction of the test lab.
Cells were used at passage 17 in repetition 1 and passage 19 in repetition 2.

Media and reagents:
Stored at room temperature 20°C ± 5°C until opening
Dulbecco’s PBS Ca2+ and Mg2+ free*
DMSO CAS N° 67-68-5
Desorption solution: 10% SDS in water
EDTA CAS N°6381-92-6

Stored at 5°C ± 3°C
DMEM 1 g/l glucose
MTT powder CAS N°298-93-1
Geneticin 50 mg/ml
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
Dulbecco’s PBS Ca2+ and Mg2+ free complemented with 0.05% EDTA

Stored at - 20°C ± 5°C until opening
Non-heat inactivated foetal calf serum*
Trypsine (0.5 g/l) - EDTA (0.2 g/l)*

Others storage conditions
Luciferase substrate (Promega):
- Lyophilized Bright-Glo™ Substrate: - 20°C ± 5°C
- Bright-Glo™ Buffer: Room temperature 20°C ± 5°C
- Reconstituted reagent: - 80°C ± 10°C
Staining solution: 5 mg/ml MTT in solution in PBS - extemporaneously prepared and used within the day

* Stored at 5°C ± 3°C after opening.

The expiry after opening of the media and reagents used in the study is defined in the form FL REAC 05.

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.

The equipment used is recorded in the study notebook.

Test protocol:
Cells seeding (first day)
The cells were harvested according to the current working instruction of the test lab. 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.
The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2.
Note: the H12 wells were left without cells and will enable the measurement of blanks.

Preparation of test item and positive control dilutions (second day)
Preparation of the test item stock solution
The stock solution was prepared at 200 mM in DMSO.

A volume of DMSO was calculated according to the following formula:

V = 5 x (p ÷100 ) x w /MW - w /1000

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 in DMSO.

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 from column 12 to 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 from column 11 to 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 row G from columns 1 to 6 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) with treatment medium.

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 (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 discussion

Positive control results:
Test validation/historical data
• the gene induction must be statistically significant above the threshold of 1.5 in at least one of the tested concentration,
• the EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Follow-up of positive control: Cinnamaldehyde (CAS N°104-55-2):
Updated on 09/04/2018 - N = 233

Imax EC1.5 IC70
Mean value 4.94 12.38 µM > 64 µM
Standard deviation 2.91 4.71 -

Mean EC1.5 value ± 2 SD: 3 µM ≤ EC1.5 ≤ 22 µM

OECD validation dataset: between 7 µM and 30 µM.

Test results:
Reference item

Cinnamaldehyde 4 µM 8 µM 16 µM 32 µM 64 µM EC1.5 Imax
Rep 1 1.44 1.55 2.09 3.13 5.11 6.15 5.11
Rep 2 1.33 1.47 2.00 2.96 4.52 8.49 4.52
Mean 1.38 1.51 2.04 3.05 4.82 7.23* 4.82
*geometric mean



In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Imax
Remarks:
IC50 > 2000 µM; IC30 > 2000 µM
Run / experiment:
Rep 1
Value:
1.23
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Imax is lower than 1.5, no EC1.5 is determined
Key result
Parameter:
other: Imax
Remarks:
IC50 > 2000 µM: IC30 > 2000 µM
Run / experiment:
Rep 2
Value:
1.16
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Imax is lower than 1.5, no EC1.5 is determined
Key result
Parameter:
other: Imax
Run / experiment:
Mean
Value:
1.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Since all validity criteria are met, study is considered as valid.

No deviation or amendment to the Study Plan has been observed during this study.

Interpretation of the results:
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 equal to or higher than 1.5 times and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s t-test on the raw RLU values),

• the EC1.5 value is strictly below 1000 µM,

• at the lowest concentration with a gene induction equal to or higher than 1.5, the cell viability must be strictly higher than 70%,

• there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.


Negative Control
The average coefficient of variation of the luminescence reading for the solvent controls (3 x 6 wells) should be below 20% in each repetition.

If for one repetition the validity criteria are not met, or in case of equivocal result additional repetitions should be considered.

The validation of the results is carried out by the Study Director according to the current working plan of the test lab.

Any other information on results incl. tables

Control solvent       CV %

control solvent

Rep 1                     10.5

Rep 2                     11.2

      

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the retained experimental conditions the test item REACTION PRODUCT OF GUERBET ALCOHOLS, C24-26, BRANCHED AND CYCLIC WITH 1,2,4-BENZENETRICARBOXYLIC ACID may be classified as not skin sensitizer.
Executive summary:

Under the retained experimental conditions the test item REACTION PRODUCT OF GUERBET ALCOHOLS, C24-26, BRANCHED AND CYCLIC WITH 1,2,4-BENZENETRICARBOXYLIC ACID 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.