1-Propanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-, sodium salt (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2012 to 07 March 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

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:

07 September 2009

Deviations:

no

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:

09 December 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium 3-(cyclohexylamino)-2-hydroxypropane-1-sulfonate

EC Number:

700-631-8

Cas Number:

102601-34-3

Molecular formula:

C9 H19 N O4 S . Na

IUPAC Name:

sodium 3-(cyclohexylamino)-2-hydroxypropane-1-sulfonate

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

not applicable

Test system

Vehicle:

other: 0.9% sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL/per eye
- Concentration: 20%

VEHICLE
- Amount applied:750 µL/per eye
- Concentration: 0.9 %

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

No post-treatment incubation

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Fresh bovine eyes were obtained from the slaughterhouse Muller Fleisch GmbH, Enzstr. 2.4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank's balanced salt solution (supplemented with 0.01 % streptomycin and 0.01 % penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32°C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1 % fetal calf serum (= complete MEM) and stored in a water bath at 32 °C ± 1 °C. The same was performed with the MEM with phenol red. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without phenol red was filled. The holders were then incubated for one hour in the incubation chamber at 32 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the arrival of the corneas they were examined and only corneas which were free from defects were used.

NUMBER OF REPLICATES
Two experiments were performed; the first experiment was not valid, because the positive control was not in the range of the validity data. The raw data of this experiment will be archived together with the other raw data, but will not be reported. The second experiment was valid. For each treatment group (negative control, positive control and test item), three replicates were used.

SOLVENT CONTROL USED
0.9 % sodium chloride solution

POSITIVE CONTROL USED
20 % imidazole solution

APPLICATION DOSE AND EXPOSURE TIME : 750 µL, 4 hours at 32°C

TREATMENT METHOD: Open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was calculated from the measured absorption at 570 nm. The difference between opacity before and after exposition is used for further evaluation.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
According to OECD Guideline no. 437 (2009) and EU method B.47, a substance that induces an IVIS > 55.1 is defined as a corrosive or severe irritant. Substances with IVIS < 55.1 may be considered as non-corrosive resp. not severely irritant.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

100.839

Vehicle controls validity:

valid

Remarks:

IVIS 1.458

Positive controls validity:

valid

Remarks:

IVIS 68.763

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, in both experiments
- Acceptance criteria met for positive control: yes, in the second experiment only

Any other information on results incl. tables

Table 1: In vitro irritation score (IVIS)

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative control (0.9 % NaCl)	1.447	1.458	0.7 %
	1.463
	1.464
Test item:	90.021	100.839	9.7 %
	103.615
	108.880
Positive control (20 % Imidazole)	75.187	68.763	8.1 %
	66.091
	65.010

 

Table 2: Absorption and Opacity Values

Parameter	Negative control			Positive control			Test item
Absorption before exposition	0.3707	0.3678	0.3751	0.2007	0.1812	0.1432	0.4041	0.2615	0.3362
Absorption after exposition	0.5714	0.5784	0.5847	1.6052	1.4840	1.4825	1.4320	1.5996	1.6576
Opacity before exposition	2.3480	2.3324	2.3719	1.5874	1.5177	1.3906	2.5357	1.8260	2.1687
Opacity after exposition	3.7273	3.7879	3.8433	40.2903	30.4789	30.3739	27.0396	39.7741	45.4569
Opacity difference	1.3793	1.4555	1.4713	38.7028	28.9612	28.9833	24.5039	37.9481	43.2882

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In an in vitro eye irritation test according to OECD Test Guideline 437 (BCOP), the test item was severely eye irritating/corrosive.

Executive summary:

In an in vitro eye irritation test according to OECD Test Guideline 437 (BCOP), the corneal irritation and damage potential of the test item was investigated by quantitative measurements of changes in opacity and permeability in a bovine cornea. Two experiments were performed; the first experiment was not valid, because the positive control was not in the range of the validity data. The raw data of this experiment will be archived together with the other raw data, but will not be reported. The second experiment was valid. The test item was applied onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes/four hours at 32 ± 1 °C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured. Physiological sodium chloride solution was used as negative control, imidazole (20 % solution in 0.9 % sodium chloride solution) was used as positive control. The positive control induced a severe irritation on the cornea, mean IVIS was 68.763. The negative control showed no irritation, mean IVIS was 1.458. The test item was tested as 20 % solution. A mean IVIS of 100.839 was calculated, corresponding to an ICCVAM classification as "very severely eye irritant". According to OECD Guideline no. 437 (2009) and EU method B.47, a substance that induces an IVIS 55.1 is defined as a corrosive or severe irritant. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.