Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 23 Jan 2018. Experimental completion date: 20 Feb 2018
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
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
Draft version July 2017 including LuSens test
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-oxydiethanol
EC Number:
203-872-2
EC Name:
2,2'-oxydiethanol
Cas Number:
111-46-6
Molecular formula:
C4H10O3
IUPAC Name:
(2-hydroxyethoxy) ethan-2-ol
Constituent 2
Chemical structure
Reference substance name:
Diethylene glycol 6-hydroxyhexanoate
Molecular formula:
C10H20O5
IUPAC Name:
Diethylene glycol 6-hydroxyhexanoate
Constituent 3
Chemical structure
Reference substance name:
Diethylene glycol di-6-hydroxyhexanoate
Molecular formula:
C16H30O7
IUPAC Name:
Diethylene glycol di-6-hydroxyhexanoate
Constituent 4
Chemical structure
Reference substance name:
Diethylene glycol tri-6-hydroxyhexanoate
Molecular formula:
C22H40O9
IUPAC Name:
Diethylene glycol tri-6-hydroxyhexanoate
Constituent 5
Chemical structure
Reference substance name:
Diethylene glycol tetra-6-hydroxyhexanoate
Molecular formula:
C28H50O11
IUPAC Name:
Diethylene glycol tetra-6-hydroxyhexanoate
Constituent 6
Chemical structure
Reference substance name:
Diethylene glycol penta-6-hydroxyhexanoat
Molecular formula:
C34H60O13
IUPAC Name:
Diethylene glycol penta-6-hydroxyhexanoat
Constituent 7
Chemical structure
Reference substance name:
Diethylene glycol hexa-6-hydroxyhexanoate
Molecular formula:
C40H70O15
IUPAC Name:
Diethylene glycol hexa-6-hydroxyhexanoate
Test material form:
liquid: viscous
Details on test material:
Appearance viscous, colorless liquid
CAS No. 36890-68-3
EINECS-No. 500-092-7

In vitro test system

Details on the study design:
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) up-stream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck). The test system employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity.
The assay included a cytotoxicity range finder test (CRFT) and three independent experiments (experiment I, II and III) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the three experiments were deter-mined.
In the experiments, the highest nominal applied concentration (2000 µg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.
Three experiments (experiment I, II and III) were performed in the same way. Experiment III serves only to confirm the results of experiment I and II.

Results and discussion

Positive control results:
All control substances indicated the expected effect. No considerable reduction of the viability was de-tected (all values > 92 %). The positive control induced a clear effect with an induction value of 4.9 fold in comparison to the solvent control.

In vitro / in chemico

Results
Key result
Parameter:
other: Fold induction
Value:
1.5
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:
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 SEN-SITISATION ARE-NRF2 LUCIFERASE 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.

The following criteria for acceptability were met:

- The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.
- 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%.
- The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.
- At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.

Any other information on results incl. tables

The following test item concentrations showed a viability ≥ 70 %, were declared as valid and could therefore be evaluated for luciferase induction:

Experiment I: 269µg/mL, 323 µg/mL, 338 µg/mL, 465 µg/mL, 558 µg/mL.

Experiment II: 269µg/mL, 323 µg/mL, 338 µg/mL, 465 µg/mL.

Experiment III: 269µg/mL, 323 µg/mL, 338 µg/mL.

A substantial and reproducible dose-dependent and a statistically significant increase in luciferase induction was measured in the test item concentrations 323 µg/mL to 558 µg/mL in experiment I, 269 µg/mL to 465 µg/mL in experiment II and 269 to 388 µg/mL in experiment III. Here, the induction was equal or above threshold of 1.5 fold in comparison to the solvent control.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The test item ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol is considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential) under the conditions of the LuSens test.
Executive summary:

An in vitro study (conducted according to OECD Test Guideline 422D) has been carried out to evaluate the potential of the test item ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization 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.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). Since July 2017 a reviewed version of the OECD 442D is available, which includes the LuSens test. Therefore this study is performed in accordance to this draft OECD 442D with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (2000 µg/mL) was chosen based on the results obtained in the CRFT.A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

A substantial and reproducible dose-dependent increase in luciferase induction above 1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in both experiments.

Under the experimental conditions of this study, the test item,ε-Caprolactone, oligomeric reaction products with 2,2'-oxydiethanol, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).