2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-20 to 2017-05-24

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adpted July 26, 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)

Version / remarks:

December 09, 2010

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

EC Number:

815-462-6

Cas Number:

124451-79-2

Molecular formula:

C17H26N2O5

IUPAC Name:

2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

Test material form:

liquid

Details on test material:

2-({[3-(isocyanatomethyl)-3, 5, 5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate of Evonik Degussa GmbH Batch SB 4663 of 24 November 2016

Specific details on test material used for the study:

The test item was used as supplied.

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial
contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution2 (HBSS) containing 1% Penicillin/Streptomycin3.
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation.
Only corneas from eyes free of defects were used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl test item
750 µl negative control item, 0.9% sodium chloride solution
750 µl positive control item, 1% NaOH solution in aqua ad iniectabilia

Duration of treatment / exposure:

Exposure period: 10 minutes

Duration of post- treatment incubation (in vitro):

After rinsing the corneas were incubated at 32°C ± 1°C for two hours. After this post incubation period, the corneas were examined.

Number of animals or in vitro replicates:

Three corneas were used for each treatment group (test item, negative control and positive control).

Details on study design:

PREPARATION OF BOVINE EYES
- Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse .
- To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks' Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL .
- Corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium)
chambers
- The chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM)
- Corneal holder was equilibrated at 32 ± 1°C for at least one hour
- After equilibration period, fresh pre-warmed EMEM was added to both chambers
- Baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation,
neovascularisation) or an opacity >7 opacity units were discarded
- Mean opacity of all equilibrated corneas was calculated by use of an opacitometer
- A minimum of three corneas with opacity values close to the median value for all corneas were selected as solvent control corneas
- The remaining corneas were then distributed into treatment, solvent and positive control groups
ADMINISTRATION
- Three corneas were used for each treatment group
Negative control item: 0.9% sodium chloride solution
Positive control item: 1% NaOH solution in aqua ad iniectabilia
Test item: The test item was used undiluted
- Exposure period: 10 minutes
- After the exposure period of 10 minutes the exposure solution was removed from each chamber and the epithelium was washed with EMEM containing phenol red at least three times.
- The open-chamber method was used.
- The window-locking ring and glass window from the anterior chamber were removed prior to treatment.
- The control or test chemical was applied directly to the epithelial surface of the cornea using a micro-pipet.
- Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible.
- The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber.
- The chamber was then filled with EMEM without phenol red.
- After rinsing, the glass window was replaced on the anterior chamber to recreate a closed system and the corneas were incubated at 32°C±1°C for two hours.
- After this post-exposure incubation period, the corneas were examined.
EXAMINATION
- Corneal injury was assessed by evaluating the opacity and permeability of the cornea
- Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer
resulting in opacity values measured on a continuous scale
- To determine the corneal permeability 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior
chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM
- The holder was incubated in a horizontal position at 32 ± 1°C for 90 ± 5 minutes
- Amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader
(Tecan Sunrise Magellan Version 7.2).
- Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values
based upon a visible light spectrophotometer using a standard 1 cm path length

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

BOVINE CORNEAL OPACITY AND PERMEABILITY TEST

Value:

0.171

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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

Any other information on results incl. tables

Opacity Values

	Cornea No.	Opacity [Opacity Units]	Corrected Opacity
				Mean of group	Standard deviation
0.9% NaCl	1	0.916	-	0.438	0.414
	2	0.199	-
	3	0.199	-
1% NaOH	4	59.443	59.005	58.049	1.831
	5	59.642	59.204
	6	56.375	55.937
Test item	7	0.637	0.199	0.186	0.379
	8	0.997	0.559
	9	0.239	-0.199

Permeability OD Values (490 nm)

	Cornea no.	Permeability [OD]	Mean of Triplicates	Corrected Permeability [OD]
					Per Cornea		Per Group
					Mean	SD	Mean	SD
0.9% NaCl	1	0.008	0.006	-			0.010	0.006
		0.004		-
		0.007		-	0.006	0.002
	2	0.017	0.017	-	0.017	0.001
		0.018		-
		0.016		-
	3	0.005	0.006	-	0.006	0.001
		0.005		-
		0.007		-
1% NaOH	4	1.436	1.459	1.426	1.449	0.021	1.577	0.369
		1.476		1.466
		1.466		1.456
	5	1.288	1.298	1.278	1.288	0.009
		1.300		1.290
		1.306		1.296
	6	1.988	2.003	1.978	1.993	0.031
		1.982		1.972
		2.038		2.028
Test item	7	0.021	0.020	0.011	0.010	0.001	-0.001	0.010
		0.020		0.010
		0.020		0.010
	8	0.002	0.003	-0.008	-0.007	0.002
		0.002		-0.008
		0.005		-0.005
	9	0.003	0.004	-0.007	-0.006	0.002
		0.003		-0.007
		0.006		-0.004

SD : standard deviation

OD : optical density

In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
0.9% NaCl	1	0.916	0.006	1.006	0.583	0.376
	2	0.199	0.017	0.454
	3	0.199	0.006	0.289
1% NaOH	4	59.005	1.449	80.740	81.699	3.747
	5	59.204	1.288	78.524
	6	55.937	1.993	85.832
Test item	7	0.199	0.010	0.349	0.171	0.402
	8	0.559	-0.007	0.454
	9	-0.199	-0.006	-0.289

Applicant's summary and conclusion

Conclusions:

Under the present test conditions test item tested in the in vitro BCOP test method, had an IVIS value of 0.171, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification

Executive summary:

The purpose of this study was to determine the ocular irritancy potential of test item in an in vitro system. The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability in isolated corneas from bovine eyes.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, negative control and positive control). The liquid test item was used undiluted as recommended in the test guideline OECD 437. 0.9% NaCl solution was used as the negative control and 1% NaOH in water (highly purified water) as the positive control item.

The test and control items were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 10 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.438 ± 0.414 and a mean permeability value of 0.010 ± 0.006. The calculated IVIS value of 0.583 ± 0.376 was well below the cut-off value of 3 (UN GHS no category).

The corneas treated with the positive control item 1% NaOH in highly purified water revealed a mean opacity value of 58.049 ± 1.831 and a mean permeability value of 1.577 ± 0.369 compared to the solvent control. The calculated IVIS value of 81.699 ± 3.747 was within two standard deviations of the current historical mean and well above the cut-off value of 55.

The acceptance criteria of validity were fulfilled in this test.

Following treatment with test item a mean opacity of 0.186 ± 0.379 and a mean permeability value of <0.01 compared to the negative control were determined. The calculated IVIS of 0.171 ± 0.402 is below the cut-off value of 3 (UN GHS no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

 

Conclusion

Under the present test conditions test item tested in the in vitro BCOP test method, had an IVIS value of 0.171, which is below the cut-off value of 3 (UN GHS no category) and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.