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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23-Feb-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Also complies with OECD GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
September 2009
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): S-10793
- IUPAC nomenclature - Sodium diisobutyldithiophosphinate
- Lot S-20227-170B
- Appearance - White powder with lumps
- CAS No. 13360-78-6
- Molecular Formula - C8H18PS2.Na
- Molecular Weight - 232 g/mole
- Purity 93-94%
- Expiration date of the lot/batch: 16 December 2013
- Storage condition of test material: At room temperature in the dark

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Vitelco, 's Hertogenbosch, The Netherlands
- Time interval prior to initiating testing: < 24 hours
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 300 mg per cornea

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole


Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored at 32 °C in a petri dish with cMEM (Eagle‟s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 °C. The corneas were incubated for the minimum of 1 hour at 32 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Physiological saline solution

POSITIVE CONTROL USED
20% (w/v) Imidazole solution

APPLICATION DOSE AND EXPOSURE TIME
301 to 314 mg of the test substance applied directly on the corneo
750 µl of positive and negative controls
240 minutes at 32 °C

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3 times with MEM with phenol red, and then once with fresh cMEM

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post -treatment reading.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

DECISION CRITERIA: decision criteria as indicated in the TG used
In vitro score range / Ìn vitro classification
0 - 3 / Non irritant
3.1 - 25 / Mild irritant
25.1 - 55 / Moderate irritant
≥ 55.1 / Severe irritant

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
190
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 115 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

In Vitro irritancy score

Eye

Negative control correctedFinal Opacity

Negative control correctedFinal OD490

In vitroIrritancy Score1

Negative control

1

0

0.004

0.1

2

0

-0.001

0.0

3

-1

-0.002

-1.0

Positive control

4

69

2.310

103.7

5

72

4.494

139.4

6

68

2.622

103.3

S-10793

10

116

4.818

188.3

11

117

4.278

181.2

12

122

5.280

201.2

 

1        In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).

 

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The test material is a severe irritant in the Bovine Corneal Opacity and Permeability test.

Executive summary:

Screening for eye irritancy potential of the test material was investigated using the Bovine corneal opacity and permeability test (BCOP test) following OECD TG 437 and under GLP conditions. The test was done by topical application of the pure material (ca. 300 mg) for approximately 240 minutes.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score (IVIS) of the positive control (20% (w/v) Imidazole) was 115 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test material induced severe ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 190 after 240 minutes of treatment.

Since it induced an IVIS ≥ 55.1, it is concluded that the test material is a severe irritant in the Bovine Corneal Opacity and Permeability test.