2-(oxolan-2-ylmethoxy)ethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 - 06 Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Test material information

Constituent 1

Reference substance name:

2-[(oxolan-2-yl)methoxy]ethan-1-ol

EC Number:

608-659-1

Cas Number:

31692-85-0

Molecular formula:

C5H10O2[C2H4O]n, n = 0, 1, 2, 3, 4, ... (data given for max n of 4)

IUPAC Name:

2-[(oxolan-2-yl)methoxy]ethan-1-ol

Test animals / tissue source

Species:

human

Strain:

other: Keratinocyte strain: 4F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcularTM Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe.
This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) without further testing within a tiered testing strategy from those requiring classification and labelling (UN GHS categories 1 and 2). It therefore can be used for regulatory purposes as an initial step in the Bottom-Up approach or as one of the last steps in a Top-Down approach. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo. All biological components of the used tissue and the kit culture medium have been tested and are free of the presence of viruses, bacteria and mycoplasma. Analysis for tissue functionality and quality was performed and passed.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: undiluted

Duration of treatment / exposure:

30 ± 2 min

Duration of post- treatment incubation (in vitro):

12 ± 2 min (Post-soak immersion incubation)
120 ± 15 min (Post-treatment incubation)

Number of animals or in vitro replicates:

2 replicates

Details on study design:

- Details of the test procedure used
Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion by the EpiOcular™ tissue construct after topical exposure to the test substance. The percent reduction of cell viability in comparison to untreated negative controls is used to predict eye irritation potential.

- RhCE tissue construct used, including batch number
The EpiOcularTM human cell construct (e.g. OCL-200 MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, Lot number: 27007)

- Doses of test chemical and control substances used
50 µL test item; 50 μL positive control (methyl acetate) and 50 μL negative control (destilled water)

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods
exposure: 30 ± 2 min at 37 ± 1 °C
post-exposure immersion (post-soak): 12 ± 2 min at room temperature
post-exposure incubation: 120 ± 15 min at 37 ± 1 °C

- Number of tissue replicates used per test chemical and controls
2 replicates each

- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
570 nm; filter band pass of maximum ± 30 nm

- Description of the method used to quantify MTT formazan
Inserts were removed from the 24-well plate after the incubation time in MTT solution (3 h ± 10 min), the bottom of the insert was blotted on absorbent material and transferred to a 24-well plate containing 2 mL isopropanol per well. The plate was sealed to inhibit evaporation. MTT was extracted overnight at 2-8 °C in the dark or by shaking on an orbital plate shaker for 2 - 3 h at room temperature. 200 μL samples (in duplicate) were placed into the wells of a 96-well plate and the absorbance/ optical density was measured in a plate spectrophotometer to determine cell viability.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test substance is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test substance is labeled irritant or corrosive.

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
The results obtained for the negative and positive controls are slightly below and above the historical control data, respectively, but both lie in the range of the acceptance criteria (please refer to "Any other information on materials and methods incl. tables").

- Complete supporting information for the specific RhCE tissue construct used
yes, available in form of the certificate of analysis provided by the manufacturer (keratinocyte strain 4F1188 used, analysis for potential biological contaminants passed)

- Reference to historical data of the RhCE tissue construct
yes, tissue functionality and quality shown: tissue viability: OD = 1.331 ± 0.206; barrier function: ET-50 = 29.61 min, sterility: no contamination

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
yes, but in a separate document

- Positive and negative control means and acceptance ranges based on historical data
please refer to Table 1 in "Any other information on materials and methods incl. tables"

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: mean viability (%)

Run / experiment:

positive control

Value:

45.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

other: mean viability (%)

Run / experiment:

test substance

Value:

48.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, as the mean OD value (1.148) of the two negative control tissues lies between 0.8 and 2.5.
- Acceptance criteria met for positive control: Yes, as the mean percentage viability for the positive control (45.8%) lies below 50% of the control viability.
- Acceptable variability between tissue replicates for positive and negative controls: Yes, as the variability for positive and negative controls was 10% and 7.3%, respectively, and thus < 20%.
- Acceptable variability between tissue replicates for the test chemical: Yes, as the variability for the test chemical was 13.7%, and thus < 20%.

Any other information on results incl. tables

Table 2: MTT results

	Negative control		Positive control		Test substance
Tissue No.	1	2	1	2	1	2
OD570 (blank-corrected)	1.168	1.069	0.495	0.533	0.63	0.472
	1.154	1.032	0.482	0.517	0.603	0.458
Mean OD570 of the duplicates (blank-corrected)	1.161	1.051	0.488	0.525	0.616	0.465
Total mean OD570of 2 replicate tissues (blank-corrected)	1.106		0.507		0.541
SD OD570	0.07		0.02		0.09
Relative tissue viability [%]	105.0	95.0	44.1	47.5	55.7	42.1
Relative tissue viability difference [%]	10.0		3.4		13.7
CV [% viability]	10.0		7.3		27.9
Mean relative tissue viability [%]	100.0		45.8		48.9

Applicant's summary and conclusion

Interpretation of results:

other: irritating potential (Eye Irrit. 2 or Eye Dam. 1 according to Regulation (EC) No 1272/2008)

Conclusions:

Under the conditions of the test, the test substance was shown to have an irritating or corrosive potential towards reconstructed human epidermis tissue in the EpiOcular™ EIT prediction model. The result does not allow for the classification of the test substance as irritant or corrosive and therefore further evaluation and/or data generation is required.