Phosphorodithioic acid, O,O-bis(2-methylpropyl) ester, compd. with N,N-dimethylmethanamine (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:

26 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according GLP principles and an inter-laboratory validated method that was well described. Positive results from this test are considered acceptable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

only in vitro test performed as part of step-wise procedure

Principles of method if other than guideline:

A Rabbit Enucleated Eye Test was performed: test substance is applied to 3 excised rabbit eyes after enucleation for 10 seconds, after which the eyes were washed. Corneal opacity and thickness are measured before and at 60, 120, 180 and 240 minutes of exposure. Uptake of fluorescein was investigated before and at 240 minutes after exposure.
The assay has undergone inter-laboratory validation and has been shown to reliably detect test items that are negligible, or moderate to sever ocular irritants. An in-house validation (date not known) has shown the strain of rabbit used here to produce satisfactory responses using known ocular-irritants and non-ocular irritants.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

EC Number:

700-910-4

Cas Number:

1354201-99-2

Molecular formula:

C11H28NO2PS2

IUPAC Name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

Test material form:

solid

Remarks:

pale yellow glassy solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): S-10713
- Physical state: solid
- Colour: brown
- Storage condition of test material: at room temperature in the dark
- Lot/batch No.: S20227-179

Test animals / tissue source

Species:

rabbit

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
Harlan
- Number of animals:
5
- Characteristics of donor animals (e.g. age, sex, weight):
not specified
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples:
- Indication of any antibiotics used: no

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: approx. 97 mg (0.1 ml) per cornea

NEGATIVE CONTROL
- Amount applied : similar amount of 0.9% saline per cornea

POSITIVE CONTROL
None used.

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

3 rabbit eyes for test substance, 2 control eyes, a positive control test was not performed periodically

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Isolated rabbit eye
- Prior enucleation, examination for evidence of ocular irritation or defect following application of Fluorescein Sodium drops BP (1% w/v).
- Immediately after sacrifice, saline solution was applied to the cornea to prevent desiccation.

EQUILIBRATION AND BASELINE RECORDINGS
- The eye was put into a superfusion chamber with a saline drip to irrigate the eye before (30 minutes equilibration) and after treatment.
- After equilibration the eyes were re-examined for damange. Corneal thickness was measured using an ultrasonic pachymeter. Any eyes in which the swelling was > 10% relative to pre-enucleation measurement or which were stained with fluorescein were rejected.

NUMBER OF REPLICATES
3 for treatment and 2 for control

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with minimal 20 ml of saline solution (approx. 32 °C)
- Time after start of exposure: 10 seconds

METHODS FOR MEASURED ENDPOINTS:
- Corneal cloudiness: pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes after treatment; use of slit-lamp biomicroscope.
- Cornea thickness: pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes after treatment; use of ultrasonic pachymeter; measured at opical centre and further four locatiosn at the apex of the corean
- Fluorescein uptake: pre-enucleation, post equilibration and approximately 240 minutes following treatmen; cobalt blue filter of a slit-lamp biomicroscope following applicatio of Fluorescein Sodium drops.

SCORING SYSTEM:
Cornea: Severity of corneal cloudiness is graded as follows:
0 = Normal cornea. Appears with the slit-lamp as having a bright grey line on the epithelial surface and a bright grey appearance of the stroma.
1 = Some loss of transparency. Only the anterior half of the stroma is involved as observed with an optical section of the slit-lamp. The underlying structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent wiht diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way tot he endothelium. The stroma has lost its marble-like appearance and is homogeneously white. With diffuse illumination, underlying structures are clearly visible.
3 = Involvement of the entire thickness of the stroma. With optical section, the endothelial surface is still visible. However, with diffuse illumination teh underlying structures are just visible.
4 = Involvement of the entire thickness of the stroma. With the optical section cannot clearly visualise the endothelium. With diffuse illumination, the underlying structures cannot be seen.

Area of cloudiness:
0 = Normal cornea with no area of cloudiness
1 = 1 to 25% area of stromal cloudiness
2 = 26 to 50% area of stromal cloudiness
3 = 51 t0 75% area of stromal cloudiness
4 = 76 to100% area of stromal cloudiness

Fluorescein
The area can be judged as a 0 to 4 scale using the same terminology as for corneal cloudiness. The intensity of fluorescein staining can be divided into a 0 to 4 scale.
0 = absence of fluorescein staining
1 = slight fluorescein staining
2 = moderate fluorescein staining
3 = marked fluorescein staining
4 = exreme fluorescein staining

DECISION CRITERIA:
REET CUT-OFF VALUES
Maximum corneal opacity (corneal cloudiness x area): >=4
Maximum fluorescein uptake (intensity x area): >=4
Mean corneal swelling (min): 60, 120, 240: >=25%
Corneal epithelium observations: any with pitting, mottling or sloughing
For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 min) was calculated based upon the pre-treatment value as follows:
(mean corneal thickness post-treatment - mean corneal thickness pre-treatment) / mean corneal thickness pre-treatment x 100
A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 min post treatment observations periods.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

240min post-treatment

Value:

8

Remarks on result:

other: All three enucleated eyes treated

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

240min post-treatment

Value:

> 108 - < 126

Remarks on result:

other: All three enucleated eyes treated

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

240min post-treatment

Value:

8

Remarks on result:

other: All three enucleated eyes treated

Other effects / acceptance of results:

Moderate loss of transparency was noted in all test eyes. No corneal effects were noted in the control eyes during the study.
Corneal swelling of the test eyes was considerably greater than that observed in the control eyes (0.9 - 4.1%) over the same period. Sloughing of the corneal epithelium was noted in all test eyes. The condition of the corneal epithelium of the control eyes appeared normal during the study.
Moderate fluorescein uptake was noted in all test eyes 240 minutes following test item application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The substance is considered to have the potential to cause severe ocular irritancy in vivo.

Executive summary:

The potential for ocular irritancy of the test material was evaluated based on the OECD TG 405 and according to GLP principles. Because the available information indicated that it had the potential to produce severe effects in a rabbit eye, a Rabbit Enucelated Eye Test (REET) was first performed for an initial assessment. This step-wise procedure is in accordance with OECD TG 405 and animal welfare considerations.

A study was therefore performed to assess the ocular irritancy potential of the test item in the rabbit following application onto the cornea of enucleated eyes. 0.1 ml of the test tiem was applied to the cornea of three enucleated eyes, and two additional eyes were kept untreated as controls.

Maximal ocular irritation observations recorded for the test eyes were as follows:

Corneal opacity (cloudiness score x area): 8 (exceeds cut-off value)

Fluorescein update (intensity x area): 8 (exceeds cut-off value)

Corneal swelling: from 62.3 (60 mins) to 120.1 % (240 mins) in treated eyes and from 3.5 (60 mins) to 1.4 % (240 mins) (exceeds cut-off value)

Conditions of corneal epithelium: sloughing (meets cut-off value)

Based on these results, the test item is considered to have the potential to cause severe ocular irritancy in vivo. An in vivo eye irritation study is therefore not required. The substance is classifed as GHS Category 1 Eye damage.