Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

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)
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The LuSens cell line was specially designed for this test system by the BASF SE (Lud-wigshafen, Germany). It employs the use of a luciferase reporter gene placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
In accordance with the OECD guideline 442D (draft version 21. Dec. 2017), the maximum final test item concentration should be 2000 µM. For a test chemical which has no de-fined molecular weight, the final test item concentration 2000 µg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).
In the case of a cytotoxic result, the concentrations for experiment III and IV should be determined so that at least one of them is in the cytotoxic range.
Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentra-tions were chosen for experiment III and VI:
7.81 µM, 9.37 µM, 11.25 µM, 13.50 µM, 16.19 µM, 19.43 µM, 23.32 µM, 27.98 µM, 33.58 µM, 40.30 µM, 48.36 µM, 58.03 µM

Results and discussion

Positive control results:
Name: EGDMA (Ethylene glycol dimethylacrylate)
CAS no.: 97-90-5
Solvent: DMSO
Supplier: Sigma-Aldrich
Purity: 98 %
Lot no.: SHBG0572V
Expiry Date: 27. Jan. 2021
Final concentration: 120 µM
Storage: Fridge
Stability: Stable under specified storage conditions
The solution was freshly prepared on the day of the experiment.

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 86 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.3 fold, negative control: 1.0 fold). However, the posi-tive control induced a clear effect with an induction value of 4.8 fold in comparison to the solvent control.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Relative Viability
Run / experiment:
2000 µM
Value:
ca. 65.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Relative Viability
Run / experiment:
500 µM
Value:
ca. 73.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Relative Viability
Run / experiment:
125 µM
Value:
ca. 58.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Relative Viability
Run / experiment:
31.25 µM
Value:
ca. 55.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Relative Viability
Run / experiment:
1.95 µM
Value:
ca. 83
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use, the validity of the LuSens test at LAUS GmbH was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LU-CIFERASE TEST METHODS) were tested. As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70%.

- Acceptance criteria met for positive control: The average induction for the positive control should be ≥ 2.5 fold and it should have a rela-tive viability of at least 70 %.

- Acceptance criteria met for variability between replicate measurements: The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, it can be stated that under the experimental conditions of this study, the test item, CARMINE LAKE, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing po-tential).