Ethyl 3,4-dihydroxybenzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2016 to 03 May 2016

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 3,4-dihydroxybenzoate

EC Number:

223-529-0

EC Name:

Ethyl 3,4-dihydroxybenzoate

Cas Number:

3943-89-3

Molecular formula:

C9H10O4

IUPAC Name:

ethyl 3,4-dihydroxybenzoate

Test material form:

solid: particulate/powder

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): cooled in physiological saline
- Time interval prior to initiating testing: not indicated
- indication of any existing defects or lesions in ocular tissue samples: free of lesions, eyes were checked for opacity, scratches, pigmentation and neovascularization in light

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 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 303-308 mg (applied as such)

CONTROLS:
- negative: physiological saline (750 µL)
- positive: 20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline (750 µL)

Duration of treatment / exposure:

240 ± 10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

90 ± 5 min at 32 ± 1 °C.

Number of animals or in vitro replicates:

3

Details on study design:

Initial opacity was measured immediately after the incubation period.
After treatment the solutions and the test substance were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed. Thereafter corneas were treated with Na-fluorescien and incubated for 90 ± 5 minutes at 32 ± 1 °C. 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).
All values were corrected for negative controls. The overall irritation score was calculated according to:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range 0.4-1.4

Results 2

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

mean

Value:

4.827

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: range 3.870 to 6.064

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

range 60-92

Run / experiment:

mean

Value:

73.3

Negative controls validity:

valid

Remarks:

range 0.5-1.9

Positive controls validity:

valid

Remarks:

range 87-159

Remarks on result:

other: range 60-92

Other effects / acceptance of results:

no effects on pH

In vivo

Irritant / corrosive response data:

irritant

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores

 

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	0.9	0.022	1.2
Positive control	114.2	1.333	134.2
CH02700	0.9	4.827	73.3

 

¹         Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

²          In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to CLP the substance is considered a severe eye-irritant

Executive summary:

The substance was tested in a BCOP assay. After exposure of the corneas of young cattle during 240 minutes, the substance showed an overall irritation score > 55. Mean effects were on the permeability of the tissue. Based on these findings the substance is considered severely irritant to the eye.