Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 March 2018 to 23 July 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
Qualifier:
according to
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:
adopted 9 October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch no. 99671, supplied by the sponsor
- Expiration date of the lot/batch: 9 October 2019
- Purity test date:6 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a closed vessel, at room temperature (20±5°C)
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The item is a liquid substance. It was tested directly, without dilution or preparation of a solution
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Neat, as supplied

Test animals / tissue source

Species:
cattle
Strain:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse Muller Fleish
- Number of animals: 9 eyes were used
- Characteristics of donor animals (e.g. age, sex, weight): The cattle were between 12 and 60 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour
- Time interval prior to initiating testing: Eyes were examined and initiated the day of the arrival
- indication of any existing defects or lesions in ocular tissue samples: no lesions
- Indication of any antibiotics used: not specified

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): 750µL
- Concentration (if solution): pure

VEHICLE
No vehicle was used
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours at 32±1°C
Number of animals or in vitro replicates:
Triplicates were used per condition
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C

QUALITY CHECK OF THE ISOLATED CORNEAS
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES
Triplicates were sued per condition

NEGATIVE CONTROL USED
HBSS

POSITIVE CONTROL USED
Dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME
750 µL of test substance or control substances were exposed for 10 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes 2 hours

REMOVAL OF TEST SUBSTANCE AND POST-EXPOSURE INCUBATION:
- Number of washing steps after exposure period: Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treat-ment opacity reading for each cornea.
The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The IVIS of each replicate of the negative control was calculated from the following equa-tion:
IVIS = opacity difference + (15 x corrected OD492 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used:
IVIS.≤ 3 : No category
IVIS > 3 and ≤ 55 : No prediction can be made
> 55 : Eye damage Category I

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item Triisononylamine (Mean)
Value:
0.55
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No damage

DEMONSTRATION OF TECHNICAL PROFICIENCY: the positive control condition showed technical proficiency of the test system

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: valid
- Acceptance criteria met for positive control: valid
- Range of historical values if different from the ones specified in the test guideline: valid

Any other information on results incl. tables

Table 1          Results : IVIS

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
HBSS

1.20

1.05

12.64%

0.95

0.99

Test Item
Triisononylamine

-1.27

0.55

384.46%

0.07

2.84

Positive Control
DMF undiluted

71.14

72.16

9.35%

79.36

65.97

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental condition of the study, the test item Triisononylamine showed no effects on the cornea of the bovine eye. The calculated IVIS was 0.55.
According to CLP criteria, a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
Executive summary:

This GLP compliant in vitro study was performed to assess the corneal damage potential of Triisononylamine by quantitative measurements of changes in opacity and permeability in a bovine cornea according to OECD 437 Guideline method.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old. The test item Triisononylamine was brought onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) is 1.05.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS was 72.16.

Under the conditions of this study, the test item Triisononylamine showed no effects on the cornea of the bovine eye. The calculated IVIS was 0.55. According to CLP criteria, a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.