2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-04-18 to 2017-04-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

Deviations:

yes

Remarks:

please refer to 'principles of method if other than guideline'

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD. (22. May 2015). 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:

2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: BASF SE: Protocol LuSens Assay, Last update: 16. May 2014

Version / remarks:

16. May 2014

Deviations:

no

Principles of method if other than guideline:

The used cell line as well as the negative and the positive control is different in comparison to the OECD 442D guideline. Also the test performance differs in details (e.g. performance of a CRFT, amount of tested concentrations of controls, incubation period of MTT solution or lysis buffer).
Prior to routine use, the validity of the LuSens test at the test facility was demonstrated in a proficiency study. For all control substances historical data are available, which demonstrates the reliability and the validity of those substances.
For this reason, all deviations of the LuSens test in comparison to the OECD 442D are declared as uncritical. The end result is not affected by those changes.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

appropriate test method for determination of the skin sensitising potential

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

EC Number:

237-655-9

EC Name:

2,2'-[oxybis(2,1-ethanediyloxy)]bisacetic acid

Cas Number:

13887-98-4

Molecular formula:

C8H14O7

IUPAC Name:

2-{2-[2-(carboxymethoxy)ethoxy]ethoxy}acetic acid

Test material form:

other: liquid: clear colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 03/2016_T_R_JM
- Expiration date of the lot/batch: 26. Jun. 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (20 ± 5°C)
- Solubility of the test substance in the solvent/vehicle: The test item was soluble in DMSO at the required concentration of 200 mM.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution containing 200 mM (nominal) of the test item in DMSO was prepared and used for preparatrion of a geometric series of the subsequent concentrations. After slight shaking, the test item was completely solved. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The assay 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 signaling pathway. 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 will be performed. The assay is used for discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the subcategories 1 A and 1 B is not possible.

Results and discussion

Positive control results:

All control substances indicated the expected effect. Regarding the Luciferase induction, the positive control induced a clear effect with an induction value of 6.8 fold in comparison to the solvent control.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: II

Parameter:

other: induction of luciferase

Remarks:

fold induction compared to solvent control

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: II

Parameter:

other: cell viability [%]

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: I

Parameter:

other: induction of luciferase

Remarks:

fold induction compared to solvent control

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Run / experiment:

other: I

Parameter:

other: cell viability [%]

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
Concerning the test item, no cytotoxic effect was observed at all test item concentrations when compared to the solvent control. The viability values were all ≥101%. Therefore all concentrations were analysable for luciferase induction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values > 99 %). 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: 0.9 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 6.8 fold in comparison to the solvent control. Concerning the test item, no cytotoxic effect was observed at all test item concentrations when compared to the solvent control. The viability values were all ≥ 101 %. Therefore all concentrations were analysable for luciferase induction. None of the tested concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal concentration of the test item. Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor.

Executive summary:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor 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). 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 μM) 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 none of the experiments. DMSO (final concentration: 1 %) was used as solvent control. Lactic acid (5000 μM) was used as negative control and EGDMA (120 μM) as positive control. No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal concentration of the test item. Under the experimental conditions of this study, the test item was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor.