1,2-Pyrrolidinedicarboxylic acid, 4-oxo-, 1-(1,1-dimethylethyl) ester, (2S)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylic acid

EC Number:

617-556-0

Cas Number:

84348-37-8

Molecular formula:

C10H15NO5

IUPAC Name:

(2S)-1-(tert-butoxycarbonyl)-4-oxopyrrolidine-2-carboxylic acid

Test material form:

solid

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% suspension (w/v) in saline

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin). The corneae were isolated on the same day after delivery of the eyes.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

NEGATIVE CONTROL USED
Saline (0.9% (w/v) NaCl in deionised water)

POSITIVE CONTROL USED
10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline using sonication

APPLICATION DOSE AND EXPOSURE TIME
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted 240 minutes.

REMOVAL OF TEST SUBSTANCE
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: microplate reader

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG

Results and discussion

In vitro

Results

Irritation parameter:

cornea opacity score

Run / experiment:

main

Value:

27.83

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For the negative control (saline), neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.94).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 106.87) corresponding to a classification as serious eye damaging (EU CLP/UN GHS (Category 1).
Relative to the negative control, the test item N-tert.-Butoxycarbonyl-4-oxo-L-proline caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 27.83 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

Applicant's summary and conclusion

Interpretation of results:

other: prediction cannot be made

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, a prediction for the damage hazard cannot be made (UN GHS) for N-tert.-Butoxycarbonyl-4-oxo-L-proline.