Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21st November 2017 - 21st December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
EC Number:
916-328-0
Molecular formula:
C10H18O3
IUPAC Name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
The eyes, once they arrived at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS:
From 24 eyes the 5 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 7, 8, 9, 13, 14 and 15), 4 eyes were superfluous and 6 corneas were used for testing of other substances.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% NaCl

POSITIVE CONTROL USED: 100% DMFA

APPLICATION DOSE AND EXPOSURE TIME: As a liquid, the test item was tested undiluted for 10 minutes.

TREATMENT METHOD: Closed-chamber method was used, because the test substance was applicable by micropipette. The test substance (750 µL of application form) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.

POST-INCUBATION PERIOD: Yes, two hours at 32 ± 1 ºC

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period, the test item was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The corneas were incubated for an additional two hours
at 32 ± 1 ºC with EMEM. At the end of the post-exposure incubation period, the opacity and permeability of each cornea were recorded.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale

- Corneal permeability: 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length

SCORING SYSTEM: IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: A test is considered acceptable if the positive control gives IVIS that falls within one standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.

The IVIS cut-off value for identifying the test item as including serious eye damage (UN GHS Category 1) and the test item not requiring classification for eye irritation or serious damage (UN GHS No Category) is given in Table 1 below.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
AAG
Value:
1.58
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
58.12
Irritation parameter:
fluorescein retention score
Run / experiment:
AAG
Value:
0.022
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
0.171
Irritation parameter:
in vitro irritation score
Run / experiment:
AAG
Value:
1.91
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
0.85
Positive controls validity:
valid
Remarks:
60.68
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Appearance of corneas was observed before and after application of the test item, negative and positive control (see table 2 and 3). No macroscopic damage was observed on corneas before application. Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control and the test item were without macroscopic damage.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The value of opacity for negative control (0.9% NaCl) obtained during the study was 0.73 and value of permeability was 0.008. The values obtained during this study not exceeded upper limits (2.86 and 0.0446, respectively), so the study is considered acceptable.

- Acceptance criteria met for positive control: The value of IVIS for positive control (100% DMFA) obtained during the study was 60.68. This value is within the acceptance limit (one standard deviations of the current historical mean; 73.89±14.95), so the study is considered acceptable.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the Bovine Corneal Opacity and Permeability (BCOP) assay, the IVIS for AAG was 1.91. As the IVIS is ≤ 3, the classification of the test substance according to UN GHS criteria for eye irritation or serious eye damage is: No category.
Executive summary:

In the Bovine Corneal Opacity and Permeability (BCOP) assay (17-759), isolated bovine corneas were exposed to 100%  Allyl Amyl Glycolate  for 10  minutes using the closed chamber method. 0.9% sodium chloride was used for the negative control and 100% Dimethylformamide was used for the positive control. The corneas were rinsed twice and then incubated for an additional two hours at 32 ± 1 ºC in medium. The opacity and permeability (via sodium fluorescein dye) of each cornea were recorded.

There was no macroscopic damage to any corneas treated with the test substance There was no macroscopic damage to any corneas treated with the test substance (Table 3). The mean opacity value for the test substance was 1.58. The mean permeability OD490 for the test substance was 0.022. The IVIS for the test substance was 1.91. The positive control gave the appropriate response. The IVIS for AAG is ≤ 3 therefore the classification of test substance according to UN GHS criteria for eye irritation or serious eye damage is: No category.

This Bovine Corneal Opacity and Permeability (BCOP) is acceptable and satisfies the guideline requirement for an OECD 437 study.