Bis(dodecylthio)dioctylstannane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

yes

Remarks:

(please refer to "Principles of method if other than guideline" for further information)

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)

Deviations:

yes

Remarks:

(please refer to "Principles of method if other than guideline" for further information)

Principles of method if other than guideline:

The positive control group had an overall IVIS of 60.3, which was marginally higher than the criteria range set for an acceptable test. As the score was only marginally exceeded, it was decided that the result was acceptable as the positive control group still provided its intended function, which was to show the sensitivity of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dodecylthio)dioctylstannane

EC Number:

244-841-3

EC Name:

Bis(dodecylthio)dioctylstannane

Cas Number:

22205-30-7

Molecular formula:

C40H84S2Sn

IUPAC Name:

bis(dodecylsulfanyl)dioctylstannane

Test material form:

liquid

Details on test material:

- Storage Conditions: Room temperature in the dark
- Physical state/Appearance: Colourless liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes followed by 90 minutes with sodium fluorecein

Number of animals or in vitro replicates:

3 replicates for each condition (test material, negative control and positive control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

TREATMENT METHOD
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes.

REMOVAL OF TEST SUBSTANCE
At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

SODIUM FLUORESCEIN
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. After sodium fluorescein incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The corneal opacity readings were taken using a calibrated opacitometer. The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.

DATA INTERPRETATION
The test material was classified according to the following:
- IVIS ≤ 3 = Not classified for irritation
- IVIS > 3; ≤ 55 = No prediction can be made
- IVIS > 55 = Category 1, H318: Causes serious eye damage

ACCEPTABILITY OF THE ASSAY
For an acceptable test the following control criteria should be achieved:
- The test was acceptable if the positive control produced an IVIS which fell within two standard deviations of the historical mean collated during 2016 for the testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 31.6 to 58.7.
- The test was acceptable if the negative control produced an IVIS which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be ≤ 3.0 and for permeability ≤ 0.077.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- A summary of the results of the study can be seen in Table 1.
- The corneas treated with the test material were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF THE RESULTS
- The positive control In Vitro Irritancy Score was above the range of 31.6 to 58.7 (60.3). The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.
- The negative control gave opacity of ≤ 3.0 and permeability ≤ 0.077. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Table 1: Summary of opacity, permeability and in vitro irritancy scores

Treatment	Mean Opacity	Mean Permeability	Mean In Vitro Irritancy Score
Negative control	0.0	0.002	0.0
Positive control	32.3	1.862	60.3
Test material	0.3	0.034	0.8

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU Criteria

Conclusions:

Under the conditions of this study the test material had an IVIS of 0.8, therefore it is not irritating to the eye.

Executive summary:

The potential of the test material to cause eye irritation was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47 using the Bovine Corneal Opacity and Permeability (BCOP) test method, under GLP conditions.

The test was performed to assess damage by the test material using quantitative measurements of changes in corneal opacity and permeability.

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative and positive control materials were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

The positive control In Vitro Irritancy Score was above the range of 31.6 to 58.7 (60.3). The positive control acceptance criterion was therefore not satisfied and this is reported as a deviation. The negative control gave opacity of ≤ 3.0 and permeability ≤ 0.077 and the negative control acceptance criteria were therefore satisfied.

Under the conditions of this study the test material had an IVIS of 0.8, therefore it is not irritating to the eye.