Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Name of test material (as cited in study report): 20231250
- Physical state: white powder
- Storage condition of test material: room temperature (ca. 20°C) in the dark

Test animals / tissue source

Species:

other: bovine cornea

Details on test animals or tissues and environmental conditions:

Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the local slaughterhouse.
Age: not provided.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to laboratory: the eyes were transported to the laboratory at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].
Upon arrival at the laboratory, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were rinced in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature.
The eyes were used 4 hours 16minutes after slaughter.

(Pre)Incubation T°C: 32°C

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20% (w/w)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): Sigma S8776, Lot batch 128K2337

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

90-min for tPermeability measurement

Number of animals or in vitro replicates:

3 cornea per treatment groups

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the corneas washed, at least three times or until the wash medium (MEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on the top of the holder. The test substance required eight washes.
- Time after start of exposure: 4 hours

SCORING SYSTEM / TOOL USED TO ASSESS SCORE:
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values of positive control and test item treated corneas.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Value:

-0.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

133

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Value:

-1

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

90.3

Results 3

Irritation parameter:

fluorescein leakage

Value:

0.004

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

0.018

Positive controls validity:

valid

Remarks:

2.83

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
No notable opaque spots or irregularities were observed on the three test substance and negative control-treated corneas.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (Negative control: opacity = 0.000 +/- 1.00; permeability = 0.018 +/- 0.008.)
- Acceptance criteria met for positive control: yes ( IVIS = 133 +/- 2.60 The positive control IVIS was within the range of historical mean +/- 2 SD: 112.8 - 164.3).

Any other information on results incl. tables

Table 7.3.2/2: Individual and Mean Corneal Opacity and Permeability Measurements

 

Treatment	Cornea Number	Opacity				Permeability (OD)		In vitro Irritancy Score (IVIS)
		Pre-Treatment	Post-Treatment	Change Post -Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	5	6	1		0.022
	14	5	4	-1		0.024
	16	5	5	0		0.009
	Mean			0.000		0.018
Positive Control	2	6	99	93	93.000	2.475	2.457	129.850
	3	6	100	94	94.000	2.635	2.617	133.250
	4	4	88	84	84.000	3.420	3.402	135.025
	Mean				90.333		2.825	132.7
Test Item	8	6	4	-2	-2.000	0.022	0.004	-1.945
	9	6	8	2	2.000	0.033	0.015	2.220
	10	6	3	-3	-3.000	0.013	-0.005	-3.080
	Mean				-1.000		0.004	-0.9

OD= Optical density

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

the test substance is considered as not requiring classification for eye irritation or serious eye damage.

Executive summary:

A study was performed to assess the ocular irritancy potential of the test substance to the isolated bovine cornea. The study was conducted according to the OECD guideline No. 437 and in compliance with the principles of Good Laboratory Practice .Negative (Sodium chloride 0.9%) and positive (imidazole purity 99.7%) control items were tested concurrently. 

The test substance was mixed using a mortar and pestle. The 0.9% sodium chloride solution was added to achieve the desired concentration of 20%. Then 750 µL of the diluted test substance was applied on each cornea (3 corneas/treatment condition). The corneas were incubated for 4 hours at 32°C. Following incubation, the test substance, positive and negative controls were removed and the epithelial surface of the cornea washed.

The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined to generate an In Vitro Irritancy Score (IVIS). 

To consider the study as valid, the positive control should elicit an In Vitro Score that falls within two standard deviations of the historical mean for the laboratory, which was the case; IVIS of imidazole was found to be 132.7 +/- 2.60. Furthermore, the negative control mean opacity change value should be<2.0 and the permeability mean value<0.1, which was the case as opacity of sodium chloride 0.9% was 0.000 +/- 1.00 and the permeability was 0.018 +/- 0.008.

 

 IVIS of the test item was determined to be -0.90 +/- 2.80, which was below the cut-off value of 3 and thus indicating that the test substance was not requiring classification for eye irritation or serious eye damage.