(ethyl acetoacetato-O1',O3)(pentane-2,4-dionato-O,O')[propane-1,3-diolato(2-)-O,O']titanium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-31 to 2018-05-31

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

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)

Version / remarks:

26 July 2013

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(ethyl acetoacetato-O1',O3)(pentane-2,4-dionato-O,O')[propane-1,3-diolato(2-)-O,O']titanium

EC Number:

279-899-9

EC Name:

(ethyl acetoacetato-O1',O3)(pentane-2,4-dionato-O,O')[propane-1,3-diolato(2-)-O,O']titanium

Cas Number:

82089-64-3

Molecular formula:

C14H22O7Ti

IUPAC Name:

13-ethoxy-8,10,15-trimethyl-1λ³,5λ³-dioxa-7λ³,11λ³,12,16-tetraoxa-6-titanaspiro[5.5⁶.5⁶]hexadeca-7,10-diene-6,6,6,6-tetrakis(ylium)-1,5,9,14-tetraide

Test material form:

liquid: viscous

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- source: abattoir
- Characteristics of donor animals (e.g. age, sex, weight): adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- indication of any existing defects or lesions in ocular tissue samples: no; those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

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): 0.75mL same amount for test item, positive and negative control

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

two hours

Number of animals or in vitro replicates:

3 per treatment

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60minutes.

At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES
3 per treatment

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

POSITIVE CONTROL USED
Ethanol (purity: >99.8% )

APPLICATION DOSE AND EXPOSURE TIME
0.75mL for 10 minutes

POST-INCUBATION PERIOD: yes, 2 hours

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
3 times washing with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein:
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability Determinations:
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. A dilution was performed on the positive controls and the optical density was re-measured.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability:
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
negative control: IVIS = opacity value + (15 x OD492 value)
positive control and the test item: IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD492 value)

DECISION CRITERIA:
IVIS <= 3: No category. Not requiring classification to UN GHS or EU CLP
IVIS > 3; <= 55: No prediction of eye irritation can be made
IVIS > 55: classified as Category 1 UN GHS or EU CLP Causes serious eye damage

HISTOPATHOLOGY: Yes

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

45.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Treatment	Cornea Number		Opacity					Permeability (OD492)		In Vitro Irritancy Score
			Pre-Treatment	Post-Treatment	Post- Incubation	Post-Incubation - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1		5	5	6	1	-	0.033	-	-
	2		2	1	2	0	-	0.004	-	-
	3		2	2	2	0	-	0.007	-	-
	-		-	-	-	0.3*	-	0.015¿	-	0.6
Positive Control	4		4	27	32	28	27.7	2.435	2.420	-
	5		3	26	28	25	24.7	1.765	1.750	-
	6		2	31	32	30	29.7	1.790	1.775	-
	-		-	-	-	-	27.3•	-	1.982•	57.1
Test Item	7		3	32	48	45	44.7	0.116	0.101	-
	9		6	32	48	42	41.7	0.230	0.215	-
	10		4	37	48	44	43.7	0.100	0.085	-
	-		-	-	-	-	43.3•	-	0.134•	45.3

OD = Optical density * = Mean of the post-incubation  - pre-treatment values ¿ = Mean permeability   • = Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

With an IVIS Score of 45.3 no prediction of eye irritation can be made. The histopathology evaluation of corneas from eyes exposed to the test item gave a score exceeding that of the positive control corneas.