Tetrabutyltin

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrabutyltin

EC Number:

215-960-8

EC Name:

Tetrabutyltin

Cas Number:

1461-25-2

Molecular formula:

C16H36Sn

IUPAC Name:

tetrabutylstannane

Test material form:

liquid

Details on test material:

Sponsor's identification: CAS No 1461-25-2
Description: clear colourless liquid
Date received: 06 April 2010
Expiry date: 28 March 2012
Storage conditions: approximately 4°C in the dark
Purity: 95.7 %

The integrity of supplied data relating to the identity, purity and stability of the test material is the responsibility of the Sponsor.

Test animals / tissue source

Species:

rabbit

Strain:

not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

The test material was applied as evenly as possible to the surface of the cornea. After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C).

Observation period (in vivo):

Assessment of corneal cloudiness was made pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment

Number of animals or in vitro replicates:

Three eyes were treated with test material, two additional eyes remained untreated for control purposes.

Details on study design:

PRE-TEST PROCEDURES
SUPERFUSION CHAMBER
The water heating circulator (Julabo MP5, Jencons (Scientific) Ltd., Leighton Buzzard, Beds, UK), was adjusted so that the temperature of the water flowing through the water jacket of the superfusion apparatus, gave a stable temperature, of 32 ± 1.5 °C, within the chambers of the apparatus. A peristaltic pump (205S/BA, Watson Marlow Ltd, Falmouth, Cornwall; UK) was used to supply saline solution at a flow rate of 0.15 to 0.4 mL/minute (at approximately 30 °C) into the rear of each chamber of the apparatus in order to irrigate the surface of the cornea.

SELECTION OF EYES
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1 % w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope (Keeler Ltd, Windsor, Berks; UK). Corneal thickness values were also recorded using the DGH-1000 Ultrasonic pachymeter (DGH Technology Inc, Solana Beach, CA). Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes.

ENUCLEATION OF EYES
The donor rabbits were sacrificed by intravenous administration of an overdose of sodium pentobarbitone. Immediately afterwards, two to three drops of saline solution (approximately 32 °C) were applied to the cornea to prevent desiccation during excision. The eye was then carefully removed, positioned in a perspex clamp and placed within the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber adjusted so that saline solution was allowed to irrigate the surface of the cornea. The eyes were then allowed to equilibrate for approximately thirty minutes. Following the equilibration period, the eyes were re-examined to ensure they had not been damaged during excision. Corneal thickness was also measured using the ultrasonic pachymeter. Any eyes in which the corneal swelling was greater than 10 % relative to the pre-enucleation measurement, or in which the cornea was stained with fluorescein, were rejected. The post equilibration corneal thickness values for each eye were recorded.

MAIN TEST PROCEDURE
TEST MATERIAL ADMINISTRATION
Three eyes were treated with test material, two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally into a petri dish.
The test material was used undiluted as supplied. A volume of 0.1 mL of the test material was applied as evenly as possible to the surface of the cornea.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): After ten seconds the test material was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32 °C). Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye. The untreated eyes were similarly washed and used for control purposes.
- Time after start of exposure: 10 seconds

SCORING SYSTEM:

CORNEA
The scoring scheme measures the severity of corneal cloudiness and the area of the cornea involved. 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 with diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the 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 the 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.

The surface of the cornea relative to the area of cloudiness is divided into five grades from 0 to 4.
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 to 75 % area of stromal cloudiness
4 = 76 to 100 % area of stromal cloudiness

FLUORESCEIN
The use of fluorescein is a valuable aid in defining epithelial damage for fluorescein staining. 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 confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
2 = Moderate fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
3 = Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination underlying structures are barely visible but are not completely obliterated.
4 = Extreme fluorescein staining. With diffuse illumination the underlying structures cannot be seen.

REFERENCE:
Hackett R B and McDonald T O, Eye Irritation. In: Advances in Modern Toxicology: Dermatoxicology. 4th ed. (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749 815.

TOOL USED TO ASSESS SCORE:
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment. This was carried out using the cobalt blue filter of the slit lamp biomicroscope, following application of Fluorescein Sodium drops.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Time point 240 minutes

Value:

3

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

other: corneal swelling

Run / experiment:

Time point 60 minutes

Value:

13

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

other: corneal swelling

Run / experiment:

Time point 120 minutes

Value:

18.4

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Results 4

Irritation parameter:

other: corneal swelling

Run / experiment:

Time point 240 minutes

Value:

26.9

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Results 5

Irritation parameter:

other: Fluoroscein uptake

Run / experiment:

Time point 240 minuted

Value:

3

Negative controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

CORNEAL OPACITY
Individual scores for corneal opacity are given in Table 1.
Some loss of transparency was noted in all test eyes. No corneal effects were noted in the control eyes during the study period.

CORNEAL THICKNESS
Individual and mean corneal thickness measurements and corneal swelling calculations are given in Table 2 and Table 3.
Corneal swelling of the test eyes during the study period was considerably greater than that observed in the control eyes over the same period.

CORNEAL CONDITION
The condition of the corneal epithelium following treatment is given in Table 4.
Sloughing of the cornea was noted in two test eyes. One test eye and the control eyes appeared normal during the study period.

FLUORESCEIN UPTAKE
Individual scores for fluorescein uptake are given in Table 5.
Slight fluorescein uptake was noted in the test eyes 240 minutes following test material application. No fluorescein uptake was noted in the control eyes 240 minutes following treatment.

Any other information on results incl. tables

Please refer to illustration for tabulated results (Tables 1 -5)

Interpretation of Results

The data for all endpoints was assessed and an estimate of the test material ocular irritancy potential was made based on the following cut-off values:

REET Parameter*	REET Cut‑Off Value
Maximum Corneal Opacity (Corneal Cloudiness x Area)	> or = 4
Maximum Fluorescein Uptake (Intensity x Area)	> or = 4
Mean Corneal Swelling (mins): 60, 120, 240	> or = 25 %
Corneal Epithelium Observations	Any with pitting, mottling or sloughing

*Any parameter that meets or exceeds the cut-off values indicates a severe eye irritant

Endpoints included corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling). For each test and control eye, the percentage change in corneal thickness following treatment (60, 120, 180 and 240 minutes) was calculated based upon the pre‑treatment value as follows:

((mean corneal thickness post-treatment) – (mean corneal thickness post equilibration) /(mean corneal thickness post equilibration)) x 100

- A mean value for corneal swelling was then calculated for the test and control eyes for the 60, 120 and 240 minute post treatment observation periods.

- A negative ocular irritancy potential may require further investigation using an in vivo ocular irritation study.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Following assessment of the data for all endpoints, the test material was considered to have the potential to cause severe ocular irritancy in vivo.

Executive summary:

A study was performed to assess the ocular irritancy potential of the test material in the rabbit following application onto the cornea of the enucleated eye. The results of the study are believed to be of value in predicting the ocular irritation potential of the test material in man. 

0.1 mL of the test material was applied onto the cornea of each of three enucleated eyes which had been maintained at a temperature of 32 ± 1.5 °C within the superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9 % Sodium Chloride).

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

Corneal Opacity	Fluorescein Uptake	Corneal Swelling (%)						Condition of Corneal Epithelium
		Test Eyes*			Control Eyes**
Cloudy x Area	Int x Area	60 mins	120 mins	240 mins	60 mins	120 mins	240 mins
3	3	13.0	18.4	26.9+	5.1	5.1	3.5	Sloughing+

Following assessment of the data for all endpoints the test material was considered to have the potential to cause severe ocular irritancy in vivo.

*For each time point the swelling recorded is the mean of three eyes

**For each time point the swelling recorded is the mean of two eyes

Int = Intensity of fluorescein uptake

+ = Meets or exceeds cut-off value indicating a severe ocular irritant