Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Mar 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

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)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

EC Number:

265-723-8

EC Name:

Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

Cas Number:

65380-84-9

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

2-methyl-4,4-bis(propan-2-yloxy)-3,5-dioxa-8-aza-4-titanadecan-10-amine; 7,7-bis(propan-2-yloxy)-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; 7,7-bis({2-[(2-aminoethyl)amino]ethoxy})-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; 7-{2-[(2-aminoethyl)amino]ethoxy}-7-(propan-2-yloxy)-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; tetrakis(propan-2-yloxy)titanium

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: greater than 35 weeks of age
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in a refrigerated container containing Hank's Balanced Salt Solution (HBSS) with penicillin/streptomycin.
- Time interval prior to initiating testing: The bovine eyes were transported to the testing facility laboratory within 24 h of harvest and the test was performed the same day.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were examined after receipt from the abattoir. Any cornea with visible evidence of neovascularization, pigmentation, opacity or scratches was discarded.
- Indication of any antibiotics used: Penicillin/streptomycin containing HBSS was used for transport.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL

NEGATIVE CONTROL:
- Amount applied: 750 µL
- Lot No. SLBV3954

POSITIVE CONTROL:
- Amount applied: 750 µL
- Concentration: 100%
- Lot No. SHBH4983V

Duration of treatment / exposure:

10 ± 1 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

triplicates for each treatment and control groups

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas free of visible defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The excised corneas were then placed in a container of fresh Hank's Balanced Salt Solution (HBSS). A specifically designed corneal holder was used to mount each cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied by the opacitometer. Fresh MEM was added before taking these baseline opacity readings. Any cornea with a value greater than 7 units was discarded.

TREATMENT METHOD: Closed chamber method
The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. The entire holder was incubated at 32 ± 1 °C and allowed to equilibrate for at least one hour, but not longer than two hours. Then the corneas were exposed to the appropriate test or control substances for 10 ± 1 min.

REMOVAL OF TEST SUBSTANCE
After the incubation, either the test item or the control substance was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP-KIT, Electro-Design Corporation, RIOM, France). Opacity measurements were made following the 10 min exposure and MEM solution refill after the 2 h post-treatment incubation.
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a spectrophotometer (Spectronic 20-D Colorimeter Spectrophotometer Milton/Roy, model: 333175) at 490 nm (OD 490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA
Test substance with an IVIS > 55 was considered as severe irritant/corrosive and labelled Category 1 according to CLP/EPA/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneas

Run / experiment:

10 min exposure

Value:

36.45

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: The IVIS value of the positive control did not fall within the acceptance range which was two standard deviations of the current historical mean. Although the IVIS of the positive control was slightly higher, the assay was considered acceptable since the IVIS still demonstrated the anticipated outcome of bovine corneas exposed to 100% ethanol for a 10 min exposure: a moderately irritating, non-corrosive response. Additionally, the IVIS of the negative control group was within the range of the historical mean.

Any other information on results incl. tables

Table 1: Results of the test item on optical density/permeability and opacity

Cornea ID	Pretest Opacity Score	10 min Opacity Score	2 h Opacity Score	Corrected Opacity Scores		OD490 nm (Permeability)	Corrected Optical Density
				10 min	2 h
10	0	10	27	10	27	1.08	1.063
11	0	13	24	13	24	0.875	0.858
12	1	11s	22	10	21	0.586	0.569
Corrected Mean Optical Density =							0.83
2 h Corrected Mean Opacity Score =							24

Table 2: Results of the negative control on optical density/permeability and opacity

Cornea ID	Pretest Opacity Score	10 min Opacity Score	2 h Opacity Score	Corrected Opacity Scores		OD490 nm (Permeability)
				10 min	2 h
1	0	0	0	0	0	0.017
2	0	0	0	0	0	0.016
3	0	0	0	0	0	0.019
Mean of Individual Optical Density =						0.017
2 h Corrected Mean Opacity Score =						0

Table 3: Results of the positive control on optical density/permeability and opacity

Cornea ID	Pretest Opacity Score	10 min Opacity Score	2 h Opacity Score	Corrected Opacity Scores		OD490 nm (Permeability)	Corrected Optical Density
				10 min	2 h
4	0	37	35	37	35	0.512	0.495
5	1	39	36	38	35	0.985	0.968
6	0	42	39	42	39	0.88	0.863
Corrected Mean Optical Density =							0.775
2 h Corrected Mean Opacity Score =							36.33

Table 4: Calculated in vitro irritancy scores

Test Article	Negative Control (MEM)	Positive Control (100% Ethanol)
24.00 + (15 x 0.830)	0.00 + (15 x 0.017)	36.33 + (15 x 0.775)
24.00 + 12.45	0.00 + 0.255	36.33 + 11.625
IVIS = 36.45	IVIS = 0.26	IVIS = 47.96*

* Although the positive control had an IVIS that was slightly greater than two standard deviations of the historical mean, the Study Director, in consultation with the Sponsor, considered the assay to be acceptable since the IVIS still demonstrated the anticipated outcome of bovine corneas exposed to 100% ethanol for a 10-minute exposure: a moderately irritating, non-corrosive response. Additionally, the IVIS of the negative control group was within the range of the historical mean.

Table 5: Historical control data

Historical Data	Positive Control	Negative Control
Mean IVIS	27.72	0.51
Standard Deviation	5.54	0.89
n	61	61

Applicant's summary and conclusion

Interpretation of results:

other: Eye Irrit. 2, (H319) according to Regulation (EC) No. 1272/2008

Conclusions:

The irritation potential of the test item was assessed in the in vitro Bovine Corneal Opacity and Permeability Test (BCOP). The test is capable of identifying substances that can induce serious eye damage (Category 1) and substances not requiring classification for eye irritation. Under the conditions of the current test, no prediction could be made for the registered substance (IVIS > 3 to ≤ 55). However, the test item caused an increase of the corneal opacity and of the permeability above the values characteristic for substances not requiring classification as eye irritants. Based on a worst-case assumption, the test item is classified as Eye Irrit. 2 (H319) according to Regulation (EC) No. 1272/2008.
CLP: Eye Irrit. 2 (H319)