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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:
2017-11 - 2018-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study under GLP conditions

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
GLP compliance:
yes (incl. certificate)
Remarks:
2016-11-02
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non sensitizers in the context of an integrated approach to testing and assessment.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Name: 2,2`-Dihydroxy-4-methoxybenzophenone
Synonym: CYASORB® UV-24 Light Absorber
CAS No.: 131-53-3
Batch No.: CM70314A1
Description: Yellow powder
Storage condition: At room temperature
Expiry date: 14 March2019

In vitro test system

Details on study design:
Cell line used:
- KeratinoSens cells
- Supplier: this cell line was provided by Givaudan.
- Batch: D1.
- Storage condition: at -80°C.
- Mycoplasm: absence of mycoplasm was confirmed.

Preparation of test chemicals:
On the basis of solubility results, the test item was dissolved in DMSO at 200 mM.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2, to obtain a total of 12 concentrations in a 96-well plate; this 96 well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96 well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

Media:
- Maintenance medium No. 1: DMEM containing GlutaMAXTM, 1000 mg/L D-Glucose, Sodium Pyruvate and supplemented with 9.1% Fetal Calf Serum (FCS) and 500 µg/mL G-418,
- Maintenance medium No. 2: DMEM with 9.1% FCS without G-418,
- Treatment medium: DMEM with 1% FCS without G-418,
- Freezing medium: DMEM with 20% FCS and 10% DMSO.

Main test:
- The test item was tested in two independent runs using cells from a different passage number
- Cells were grown using general culture procedures up to 80-90% confluence; the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL; cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 µL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding; after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition
- After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL of treatment medium; from the Master plate 4x, a volume of 50 µL was added to each well of the three white assay plates and 50 µL to the transparent plate for the cytotoxicity evaluation; all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells; the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

Controls:
- DMSO was used as the negative control, and was applied to cells at a concentration of 1% in culture medium.
- Cinnamic aldehyde was used as the positive control. For each run, the positive control item was dissoved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of 5 concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 µM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

Cell viability assay MTT:
For the cell viability assay plate, the medium was replaced by 200 µL of treatment medium; a volume of 27 µL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate; the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes); at the end of the incubation period, the medium was removed and a volume of 200 µL of a 10% SDS solution was added to each well; the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells; after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

Luciferase assay:
After incubation, the supernatants from the white assay plates were discarded; the cells were washed once with D-PBS; a volume of 20 µL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking; the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program: 50 µL of the luciferase substrate was added to each well, and 1 second after this addition, the luciferase signal was integrated for 2 seconds

Evaluation criteria:
The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Validity criteria:
Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose response with increasing luciferase activity at increasing concentrations for the positive control,
- the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

Results and discussion

Positive control results:
First run: Imax 6.63; EC1.5 11.65 µM; Viability >= 108 %
Second run: Imax 4.78; EC1.5 8.64 µM; Viability >= 99 %

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: EC1.50 (µM)
Run / experiment:
Run 1
Value:
13.82
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: EC1.50 (µM)
Run / experiment:
Run 2
Value:
11.26
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
First run was performed using the following concentrations 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.
At these tested concentrations:
- precipitates were observed in treated wells for concentrations ≥ 1000 µM,
- a decrease in cell viability (i.e. cell viability < 70%) was noted for concentrations ≥ 250 µM,
- the corresponding IC30 and IC50 were calculated to be 157.34 and 182.29 µM, respectively,
- statistically gene-fold inductions above the threshold of 1.5 were noted at non-cytotoxic concentrations ≥ 15.63 µM with an apparent dose-response,
- the Imax was 2.25 and the calculated EC1.5 was 13.82 µM.

Second run was performed with the same concentrations as in run 1.
At these tested concentrations:
- precipitates were observed in treated wells for concentrations ≥ 1000 µM,
- a decrease in cell viability (i.e. cell viability < 70%) was noted for concentrations ≥ 250 µM,
- the corresponding IC30 and IC50 were calculated to be 148.83 and 177.28 µM, respectively,
- statistically gene-fold inductions above the threshold of 1.5 were noted at non-cytotoxic concentrations ≥ 15.63 µM with an apparent dose-response,
- the Imax was 2.38 and the calculated EC1.5 was 11.26 µM.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental conditions of this study, the test item was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.
Executive summary:

The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the DPRA may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of substances, two congruent results in these two tests give a good prediction of the sensitizer hazard, in particular when comparing against human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results. 

In both repetitions, induction of the luciferase above the threshold of 1.5 was noted. 2,2'-Dihydroxy-4 -methoxybenzophenone is thus rated positive in the KeratinoSens™ assay. Further testing is required.