[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 - 25 Sep 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dimethylbutyl (E)-but-2-enoate

Molecular formula:

C10H18O2

IUPAC Name:

1,3-dimethylbutyl (E)-but-2-enoate

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™

Details on test animals or tissues and environmental conditions:

TEST MODEL (EpiOcular™ Kit)
- Source: MatTek Corporation, Ashland, USA
- Lot No.: 21572

TEST METHOD
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. Irritant materials are identified by their ability to damage the underlying cell layers which is determined through a decrease in cell viability as determined by MTT reduction.

ADAPTATION TO CELL CULTURE CONDITIONS
1.0 mL assay medium (37 °C) was aliquoted into 6-well plates. The inserts with EpiOcular™ tissues were transferred aseptically into the plates and pre-incubated at standard culture conditions for 1 h. Afterwards, the medium was replaced by 1 mL fresh assay medium and the EpiOcular™ tissues were incubated at standard culture conditions overnight (18 h). After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca²+ Mg²+ free DPBS. The tissues were incubated at standard culture conditions for 30 min.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1.5
- CO2 gas concentration (%): 5 ± 0.5
- Humidity (%): 95

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Applied volume: 50 µL

POSITIVE SUBSTANCE
- Substance: methyl acetate
- Applied volume: 50 µL

NEGATIVE CONTROL
- Substance: deionised water
- Applied volume: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

The test was performed in duplicate for each treatment and control group.

Details on study design:

TEST SITE
- Area of exposure: 0.6 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the treatment time, the test substance was removed by extensively rinsing the tissues with Ca²+Mg²+ free DPBS in clean beakers.
- Post-treatment incubation period: 2 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, a volume of 300 µL MTT solution was added to each well for 3 h at standard culture conditions. After removal of the MTT solution, wells were rinsed three times with Ca²+Mg²+ free DPBS. Extraction of the formazan product was carried out in 2 mL isopropanol. At the end of the extraction period the optical density (OD) was measured.

EVALUATION OF RESULTS
1) The mean OD value of the blank control wells (ODBlk) for each experiment were calculated.
2) The ODBlkfrom each OD value of the same experiment (blank corrected values) were subtracted.
3) The mean value of the two aliquots for each tissue (= corrected test substance OD) were calculated.
4) The mean value of the two relating tissues for each control and test substance (= corrected mean OD) were calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability.
6) The percent viability of each of the two relating tissues for each control and test substance relative to the negative control (100% control) were calculated.
Viability (%) = [(corrected test substance OD) / (corrected mean negative control OD)] x 100

EVALUATION CRITERIA
A test substance leading to a tissue viabilty > 60% (relative to the negative control tissue viability) is considered as non-irritating.
A test substance leading to a tissue viabilty < 60% (relative to the negative control tissue viability) is considered as irritating.

ACCEPTABILITY OF THE ASSAY
1. The negative control OD is > 0.8 and < 2.5
2. The mean relative viability of the positive control is below 60% of the negative control viability.
3. The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test substance). This applies also to the killed controls (substance and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Results

Irritation parameter:

other: cell viability (%)

Remarks:

mean values of 2 tissues

Run / experiment:

30 min exposure

Value:

100.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The optical pre-experiment (colour interference pre-experiment) to investigate the test substance’s colour change potential in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.

ACCEPTANCE OF RESULTS:
All acceptance criteria were met.

Any other information on results incl. tables

Table 1. Results after 30 min incubation time

Test group	Absorbance*		Mean absorbance of 2 tissues*	Rel. absorbance (%)**		Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2	Rel. absorbance (% of negative control)**
	Tissue 1	Tissue 2		Tissue 1	Tissue 2
Negative control	1.568	1.653	1.611	97.4	102.6	5.3	100.0
Positive control	0.107	0.114	0.110	6.6	7.1	0.5	6.9
Test substance	1.675	1.563	1.619	104.0	97.0	7.0	100.5

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no calssification according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance did not exhibit irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model.

Executive summary:

An in vitro eye irritation test using a human cornea model according to OECD 492 was conducted in compliance with GLP. Since the viability value of the test substance exposed tissues did not decrease below 60% (100.5%), the test substance is not considered to possess an eye irritating potential.