Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 11th, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD series on testing and assessment n. 113- supplement to test guidelines 437 and 438.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

EC Number:

290-844-8

EC Name:

Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

Cas Number:

90268-43-2

Molecular formula:

The substance is too complex, no molecular formula is available.

IUPAC Name:

Reaction product of lanolin with maleic anhydride and Sodium sulphate.

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

Test animals / tissue source

Species:

other: bovine cornea

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes were supplied by a butcher service.
- Source: Butcher Service s.r.l- Mattatoio n. 2067M, Viterbo, IT;
- Age of animals: 6-12 months;
- Killing time: from 10:45 to 12:00 in the morning;
- Transport and condition: at 4 °C in eye transport solution

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

MEDIA AND REAGENT
Eyes transport solution (HBSS);
Complete EMEM (with and without phenol red);
Sodium fluorescein solution

Duration of treatment / exposure:

exposition of the corneas in horizontal position for 10 ± 1 minutes, incubated in a liquid bath at 32 ± 1°C. After exposure, corneas were rinsed thoroughly with complete EMEM with phenol red (3 washings with 3 mL each). A final wash with prewarmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled with pre-warmed complete EMEM without phenol red.
POST-EXPOSURE: Corneas were maintained in horizontal position, for further 2 hours ± 10 minutes, incubated in a liquid bath at 32 ± 1°C.

Observation period (in vivo):

Visual observation of corneas through chamber glasses and opacity determination;
Permeability determination after incubation.

Details on study design:

PREPARATION OF CORNEAS
1) Eyes were examined for the presence of any defects (opacity, scratches or pitting of the corneal surface, vascularisation or pigmentation);
2) Cornea excision: each cornea with 2-3 mm of surrounding sclera was dissected from the eye using a scalpel, scissors and forceps and placed into a Petri dish containing HBSS (Hanks balanced salt solution);
3) Mounting in the chamber with the endothelial surface of the cornea placed in contact with the O-ring of the posterior part of the chamber. In the correct position; the chamber was then filled with complete EMEM without phenol red maintained at 32 ± 1°C (posterior part of the chamber first to maintain convexity).
4) Equilibration: the corneas in their holders were incubated in a liquid bath at 32 ± 1°C at least for 1 hour to permit metabolic stabilisation.
At the end of the pre-dose incubation period, the two chambers were drained (anterior first) and re-filled with complete EMEM without phenol red maintained at approximately 32°C (posterior first).
5) Selection: basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (opacitometer).
Cornea s with a basal value >= 7 arbitrary units were excluded from testing.
6) Determination of the mean opacity of the cornea;

METHOD
Test item: open chamber for a viscous substance;
Positive and negative controls: closed chamber method for free-flowing substances.
The medium was completely removed from the anterior chamber. Corneas were treated with 0.75 mL of treatment volumes. After that, in the chambers the treatment was removed and corneas were exposed in horizontal position for 10 ± 1 minutes, incubated in a liquid bath at 32 ± 1°C. After a first washing with complete EMEM with phenol red (3 washings with 3 mL each) a final wash with pre-warmed complete EMEM without phenol red was carried out. Finally, the anterior chamber was re-filled with pre-warmed complete EMEM without phenol red. Corneas were maintained in horizontal position, for further 2 hours ± 10 minutes, incubated in a liquid bath at 32 ± 1°C.

OBSERVATIONS
Visual observation through chamber glasses and pertinent observation recorded (e.g. tissue peeling/exfoliation, residual test substance, non-uniform opacity pattern etc.);

Opacity determination of all corneas: basal opacity of corneas was determined by means of a calibrated meter, measuring difference in light transmission between the corneas placed into the beam paths versus air (Opacitometer);

Permeability by treatment with fluorescein: 1 mL aliquot of 4 mg/mL sodium fluorescein solution in DPBS. The corneas were horizontally incubated for approximately 90 minutes in a liquid batch at 32 ± 1 °C. The quality controls of the fluorescein solutions were assayed.

Permeability determination: measurement of the optical density of each sample using a spectrophotometer set at 490 nm.

Tissue retention.
At the end of the procedure, corneas were retained and fixed in 10% neutral buffered formalin.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

in vitro irritancy score (IVIS)

Run / experiment:

For each test point, the mean value of opacity obtained after exposure will be calculated. As appropriate negative or vehicle control will be subtracted from the opacity of the test item or positive control.

Value:

> 55.1

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Other effects / acceptance of results:

PREPARATION OF CORNEAS
A total of 9 corneas were processed (out of 14 delivered) for a final selection of the 9 required corneas according to basal opacity.
One cornea out of the nine was selected although it showed a very small lesion (no. 1 pre-test).
cornea no. 1 pre-test was assigned to the negative control (code no. n1).


OBSERVATIONS
Negative control samples: did not induce corrosion as the mean opacity values were homogeneous and in line with the expected values for this kind of control.
however, a single cornea (code n1 – pre-test 1) showed a high od value in the permeability assay. This was attributed to the small lesion recorded before the test and the value was then excluded from mean permeability and opacity value.
The positive control induced opacity of the whole cornea surface with a mean increase of the opacity value equal to 187.3. Opacity and exfoliation value were noted in three replicates at the end of the 2-hour post-incubation period.
The corneal permeability was also increased. The calculated mean permeability OD490 value was 3.3493.

Any other information on results incl. tables

Classification of the test item was performed according to the in vitro irritancy score:

> 55.1 Corrosive or severely irritant;

In addition, the test item was evaluated also for the eye irritancy potential according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438:

0-3 Non eye irritant

3.1-25 Mild eye irritant

25.1-55 Moderate eye irritant

Table.1 Cornea selection

Treatment Code	Pre-test cornea number	Basal opacity	Mean group basal opacity
N1	1 ^	0	0.3
N2	4	1
N3	7	0
P1	2	0	0.0
P2	5	0
P3	8	0
A1	3	0	0.0
A2	6	0
A3	9	0
		Mean: 0.11 °
^ small lesion on the surface
° mean of the total number of selected pre-test corneas

Table.2 assessment of BCOP assay. Observation

Treatment		OPACITY					MACROSCOPIC OBSERVATION OF THE CORNEA		PERMEABILITY
		Basal Opacity	Assay Opacity	Value corrected (basal value)		Value Corrected (negative control)			OD490	Dilution factor	Value Corrected (dilution factor)	Value Corrected (negative control)
		Value	Value
N1		0	-2	0	* °	N/A	No macroscopic changes		0,064	1	0,064	N/A	°
N2		0	-1	0	*	N/A	No macroscopic changes		0,008	1	0,008	N/A
N3		0	-1	0	*	N/A	No macroscopic changes		0,01	1	0,01	N/A
	Mean			0				Mean			0,009
	SD			0				SD			0,0014
	CV %			N/A				CV %			15,56
P1		0	185	185		185	Opaque and exfoliated		0,747	5	3,735	3,726
P2		0	183	183		183	Opaque and exfoliated		0,637	5	3,185	3,176
P3		0	194	194		194	Opaque and exfoliated		0,631	5	3,155	3,146
	Mean					187,3		Mean				3,3493
	SD					5,9		SD				0,3265
	CV %					3,2		CV %				9,75
A1		0	1	1		1	No macroscopic changes		0,01	1	0,01	0,001
A2		0	2	2		2	No macroscopic changes		0,009	1	0,009	0
A3		0	7	7		7	No macroscopic changes		0,012	1	0,012	0,003
	Mean					3,3		Mean				0,0013
	SD					3,2		SD				0,0015
	CV %					97		CV %				115,38

° excluded from the mean                          N/A: not applicable                        * Assumed 0 for calculation                       

N1-3: Negative control                 P1-3: Positive control           A1-3: Test item        

Applicant's summary and conclusion

Interpretation of results:

other: Criteria of the OECD Guideline no. 437: not corrosive or severely irritant to the eye. Criteria of OECD Supplement to Test Guidelines nos. 437 and 438: mild irritant effect to the eye.

Conclusions:

Slight alterations of cornea opacity but not of permeability were recorded after treatment with the test item, so according to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye. However, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is an indication of a mild irritant effect to the eye.

Executive summary:

The potential of the test item, Sulfosuccinate of lanolin Alcohol solid, to cause corrosion/severe irritation by using the Bovine Corneal Opacity and Permeability (BCOP) assay, was examined in agreement with OECD Guideline no. 437 (adopted on September 2009) and OECD Supplement to Test Guidelines nos. 437 and 438.

The test item was used as supplied (being a cream) on the epithelial surface of three idoneous bovine corneas, for an exposure period of 10 minutes plus a post incubation period of 4 hours.

Positive and negative controls [a 1% (w/v) sodium hydroxide solution in water and physiological saline alone, respectively] were concurrently tested in the same number of replicates.

The mean opacity detected with an opacitometer at the end of the test item exposure period was 3.3, slightly higher than that of the negative control. At the macroscopic observation the three corneas did not show any abnormality. 

After the determination of opacity, the epithelial surface was treated with a 0.4% solution of sodium fluorescein in DPBS for 90 minutes, to investigate alteration in cornea permeability.

Mean OD490 value of the corneas treated with the test item was essentially unaffected when compared to that of negative control, thus indicating no alteration of corneal barrier.

Negative and positive controls gave the expected results.

The results obtained indicate that the test item induces slight changes in cornea opacity under the reported experimental conditions.

The calculated in vitro irritancy score (IVIS) for the test item is 3.3.

According to the OECD Guideline no. 437, the test item is not to be classified as corrosive or severely irritant to the eye.

However, according to the criteria stated in the OECD Supplement to Test Guidelines nos. 437 and 438, there is an indication of a mild irritant effect to the eye.