2-hydroxy-3-propyl-2H-furan-5-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Aug 2018

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-hydroxy-4-propyl-2,5-dihydrofuran-2-one

Cas Number:

78920-10-2

Molecular formula:

C7H10O3

IUPAC Name:

5-hydroxy-4-propyl-2,5-dihydrofuran-2-one

Test material form:

liquid: viscous

Details on test material:

- Appearance: Brown viscous liquid
- Storage condition of test material: At room temperature

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL: 750 µL per cornea
POSITIVE CONTROL: 750 µL Ethanol per cornea
NEGATIVE CONTROL: 750 µL of physiological saline per cornea

Duration of treatment / exposure:

10 minutes (at 32°C)

Duration of post- treatment incubation (in vitro):

120 minutes (at 32°C)

Number of animals or in vitro replicates:

3 corneas for each treatment group

Details on study design:

SELECTION, PREPARATION AND QUALITY CHECK OF CORNEAS
- Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Corneas with those exhibiting defects were discarded.
- The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea.

REMOVAL OF TEST SUBSTANCE
The corneas were rinsed until no color change of the medium was observed anymore, as a pH effect of the test substance was observed on the rinsing medium.

POST-TREATMENT
The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

OPACITY
The opacity of a cornea was measured by the diminution of light passing through the cornea. The change in opacity for each individual cornea was calculated, by subtracting opacity before treatment from opacity after post-treament. Subsequently negative control corrected. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

PERMEABILITY
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
- The mean OD490 for each treatment was calculated using cMEM corrected OD490 values.

SCORING SYSTEM
In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA
- A test substance that induces a mean IVIS >55 is classified with Eye Irr. Cat. 1 in accordance with the CLP Regulation
- A test substance that induces a mean IVIS ≤ 3 is not classified for Eye irritation/corrosion in accordance with the CLP Regulation
- For a test substance that induces a mean IVIS of >3 - ≤55, no prediction on the classification can be made. More information is needed.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3

Value:

134

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Historical Control Data (period: May 2015 to May 2018):
Negative control Positive control
Opacity Permeability In vitro Irritancy Score In vitro Irritancy Score
Range -2.9 – 3.0 -0.034 – 0.100 -2.8 – 3.0 28.0 – 110.9
Mean 0.25 0.00 0.31 55.03
SD 1.13 0.01 1.19 15.08
n 118 118 118 94

Any other information on results incl. tables

Summary of results:

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score
Negative control	2.5	0.012	2.7
Positive control	20	3.377	71
Test substance	66	4.525	134

Individual in vitro irritancy scores:

Treatment	In vitro Irritancy Score
Negative control	1.9
	3.5
	2.7
Positive control	66
	78
	68
Test substance	131
	144
	127

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 (irreversible effects on the eye) based on GHS and CLP criteria

Conclusions:

Based on a mean in vitro irritancy score of 134 in a Bovine Corneal Opacity and Permeability test, it is concluded that the substance needs to be classified with Category 1 (irreversible effects on the eye) based on GHS and CLP criteria.

Executive summary:

Using the Bovine Corneal Opacity and Permeability test (BCOP test) the substance was screened for its eye irritancy potential in accordance with OECD 437 and according to GLP principles. The liquid substance was applied as such on the corneas. Adequate negative and positive controls were included. The mean in vitro irritancy score was 134 and the acceptance criteria were met. Since the substance induced a mean IVIS >55, the substance needs to be classified for eye irritation/corrosion with Eye Irr. Cat. 1 in accordance with the CLP Regulation.