L-Ascorbic acid, 3-O-ethyl-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 - 8 Dec 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

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:

amended 9 Dec 2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(5R)-5-[(1S)-1,2-dihydroxyethyl]-4-ethoxy-3-hydroxy-2,5-dihydrofuran-2-one

EC Number:

617-849-3

Cas Number:

86404-04-8

Molecular formula:

C8H12O6

IUPAC Name:

(5R)-5-[(1S)-1,2-dihydroxyethyl]-4-ethoxy-3-hydroxy-2,5-dihydrofuran-2-one

Details on test material:

- Name of test material (as cited in study report): Ethyl ascorbic acid
- Storage condition of test material: in the freezer (≤ -15 °C)
- Substance type: white to very pale yellow, crystalline, powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Vitelco, 's Hertogenbosch, The Netherlands
- Characteristics of donor animals: Bovine eyes from young cattle
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v)

POSITIVE CONTROL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) in physiological saline

NEGATIVE CONTROL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

240 ± 10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

in triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
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. Those exhibiting defects were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the initial opacity was determined. Each cornea with a value of the initial opacity >7 was discarded.

TREATMENT METHOD:
The isolated corneas were mounted in a corneal holder (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 °C. The corneas were incubated for a minimum of 1 h at 32 ± 1 °C. At the end of the equilibration period, the medium was removed from the anterior compartment and 0.75 ml of either test substance or control was applied to the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE:
After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany)
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite M200 Pro Plate Reader) at 490 nm
- Others: After removal of the test substance, possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as severe irritant/corrosive and labelled Category 1.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55 no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean value of 3 corneae

Value:

13

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: After the incubation period with the test substance the corneas were clear with a spot. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed.

Any other information on results incl. tables

Table 2. Results after 240 min incubation time.

Test group	Opacity value		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative control	-0.7	-0.9	-0.001	0.000	-0.7	-0.9
	0.6		-0.003		0.6
	-2.7		0.004		-2.6
Positive control	107.8		0.907		121.4	113.4
	81.1		1.851		108.9
	87.5		1.501		110.0
Test substance	11.8		0.59		20.7	13.0
	1.0		0.219		4.3
	5.1		0.586		13.9

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

The irritation potential of the test substance was assessed in the BCOP assay. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 13.0. According to OECD Guideline 437 no prediction on the irritation potential can be made but the test substance does not have to be classified as serious eye irritant (Eye Dam. Cat. 1)