Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Nov 2012 - 10 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Remarks:

The Department of Health of the Government of the United Kingdom

Test material

Test material information

Constituent 1

Reference substance name:

Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts

EC Number:

263-174-9

EC Name:

Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts

Cas Number:

61791-42-2

Molecular formula:

UVCB

IUPAC Name:

Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts

Details on test material:

- Name of test material (as cited in study report): trade name given; Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
- Physical state: white powder
- Analytical purity: >90%
- Lot/batch No.: 524038
- Expiration date of the lot/batch: 31 May 2013
- Storage condition of test material: at room temperature in the dark

Test animals / tissue source

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used to classify substances as “ocular corrosives and severe irritants”. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in isolated bovine corneas. The opacity and permeability assessments of the corneas are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: local abattoir as by-product from freshly slaughtered animals
- Transport medium and temperature conditions: Hank’s balanced salt solution (HBSS) supplemented with penicillin/streptomycin, transported on ice paks

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Test medium and temperature conditions used in the cornea holder: complete Minimum Essential Medium (MEM); holders were incubated at 32 ± 1 °C for 60 min
- Quality check of the equilibrated corneas: free of macroscopic defects

DETERMINATION OF THE INITIAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using an opacitometer
- Specification of the device: Opacitometer TX307

Test system

Vehicle:

other: 0.9% w/v sodium chloride solution

Controls:

other: negative control: sodium chloride solution; positive control: imidazole

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20%

NEGATIVE CONTROL
0.9% w/v sodium chloride solution

POSITIVE SUBSTANCE
- Substance: imidazole
- Concentration (if solution): 20% in 0.9% sodium chloride solution

Duration of treatment / exposure:

240 min at 32 ± 1 °C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

number of corneas for each the test item and controls: 3

Details on study design:

TEST CONDITIONS
- Short description of the method used: closed-chamber method
The MEM was removed from the anterior chamber of the BCOP holder and the test item preparation or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 240 minutes.

POST-EXPOSURE TREATMENT
- Removal of the test substance: at the end of the exposure period, corneas were rinsed three times with fresh complete MEM containing phenol red followed by a final rinsing with complete MEM without phenol red. Afterwards, the anterior and posterior chamber were filled with fresh complete MEM.
- Medium for washing the corneas: complete MEM containing phenol red
- Medium for final rinsing: complete MEM

DETERMINATION OF THE FINAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using an opacitometer
- Time of determination: after the post-exposure treatment and visual inspection of each cornea

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced wit 1 mL sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. After incubation, the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm was measured. If values greater than 1.5 OD492 were obtained, a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.
The corneas were retained after testing for possible conduct of histopathology.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

<= 55

Any other information on results incl. tables

Table 1: Individual and mean corneal opacity and permeability measurements

Treatment	Cornea number	Opacity				Permeability		In vitro irritancy score
		Pre-treatment	Post-treatment	Post-treatment - Pre-treatment	Corrected value		Corrected value
Negative control	1	4	6	2		0.048
	2	4	7	3		0.067
	3	3	9	6		0.065
				3.7*		0.060§		4.6
Positive control	4	2	67	65	61.3	1.449	1.389
	5	3	65	62	58.3	3.420	3.360
	6	3	64	61	57.3	1.570	1.510
					59.0#		2.086#	90.3
Test item	10	2	64	62	58.3	0.112	0.052
	11	1	58	57	53.3	0.052	0.000
	12	1	54	53	49.3	0.048	0.000
					53.7#		0.017#	53.9

§: Mean permeability

*: Mean of the post incubation - pre-treatment values

#: Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered as severe eye irritant (Serious eye damage Category 1) based on a positive result in the Bovine Corneal Opacity and Permeability (BCOP) test. Negative in-vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant (Category 2) and shall therefore be subject to further evaluation.