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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Remarks:
WoE

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
Adopted: 25. June 2018
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD. Performance standards for assessment of proposed similar or modified in vitro skin sensitisation ARE-Nrf2 Luciferase test methods. ENV/JM/MONO(2015)6
Version / remarks:
22. May 2015
Deviations:
no
Guideline:
other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method
Version / remarks:
Adopted 14. Feb. 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In vitro study should be performed first. An in vivo study shall be conducted only if in vitro/in chemico test methods described under point 8.3.1 of REACH are not applicable.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Storage conditions: Room Temperature (20 ± 5 °C)
- Expiry date: 16. Dec. 2019

In vitro test system

Details on study design:
This in vitro study was performed to assess the potential of the test item to activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” (Bauch et al. 2012).
It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signalling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010). In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two, but a maximum of three independent and valid experiments are performed.
The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible. For a confirmation of a negative result another in vitro skin sensitisation test has to be performed.

Results and discussion

Positive control results:
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

Resultsopen allclose all
Parameter:
other: Relative Viability %
Run / experiment:
Experiment I; test item concentrations 0.526 µM to 1.309 µM
Value:
>= 74
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Relative Viability %
Run / experiment:
Experiment II; test item concentrations 0.526 µM to 1.571 µM
Value:
>= 74
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Applicant's summary and conclusion

Interpretation of results:
other: negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential)
Conclusions:
The study was performed according to OECD TG 442D under GLP on the registered substance itself. Positive and negative controls were valid.
The ARE-Nrf2 Luciferase Test addresses the second key event of the skin sensitisation adverse outcome pathway (AOP) that was defined by the OECD in 2012 and is a part of the AOP-based “two out of three” skin sensitisation integrated testing strategy for hazard identification (Bauch et al., 2012).
Under the experimental conditions of this study, the test item, 3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential). The substance will be further tested according to OECD TG 442C.
Executive summary:

This in vitro study according to OECD 442D (GLP) was performed to investigate the potential of 3,3-bis(4-hydroxy-5-isopropyl-o-tolyl)phthalide to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II:

0.526 µM, 0.631 µM, 0.758 µM, 0.909 µM, 1.091 µM, 1.309 µM, 1.571 µM, 1.886 µM, 2.263 µM, 2.715 µM, 3.258 µM, 3.91 µM

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold. 

Since all acceptability criteria of the assay were met the study is valid.

 

In experiment I a cytotoxic effect was observed at 1.571 µM up to 3.91 µM. In experiment II, a cytotoxic effect was observed at 1.886 µM up to 3.91 µM.

Finally the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction:

Experiment I: 0.526 µM, 0.631 µM, 0.758 µM, 0.909 µM, 1.091 µM, 1.309 µM

Experiment II: 0.526 µM, 0.631 µM, 0.758 µM, 0.909 µM, 1.091 µM, 1.309 µM, 1.571 µM

 

In experiment I only one non-cytotoxic test item concentration induced an increase in luciferase induction above 1.5 fold in comparison to the solvent control. This concentration (1.309 µM) showed an increase in luciferase induction of 1.6 fold.

 In none of the other tested non cytotoxic concentrations the luciferase induction was equal or above 1.5 fold in comparison to the solvent control.

The test item concentration 1.571 µM induced a 1.7 fold increase, was however already in the cytotoxic range and therefore the value is not used for the final evaluation of luciferase induction.

In experiment II, none of the tested non cytotoxic concentrations induced a luciferase induction above or equal 1.5 fold in comparison to the solvent control.

The test item concentration 1.886 µM induced a 1.3 fold increase of luciferase induction but showed a viability of 62.1 %. This test item concentration did not induce an increase in luciferase induction equal or above 1.5 fold in comparison to the solvent control and because of low viability the value is not used for the final evaluation of luciferase induction.

According to the classification criteria the result of both experiments is negative.

In conclusion, it can be stated that under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).