Dihydro-2,2-dioctyl-6H-1,3,2-oxathiastannin-6-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 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:

no

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)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dihydro-2,2-dioctyl-6H-1,3,2-oxathiastannin-6-one

EC Number:

221-218-4

EC Name:

Dihydro-2,2-dioctyl-6H-1,3,2-oxathiastannin-6-one

Cas Number:

3033-29-2

Molecular formula:

C19H38O2SSn

IUPAC Name:

2,2-dioctyl-1,3,2-oxathiastanninan-6-one

Test material form:

solid: particulate/powder

Details on test material:

- Storage conditions: Dry, < 30 °C

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: 0.8144 g

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes with sodium fluorescein

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. 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 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
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. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control material.

NUMBER OF REPLICATES: 3

TREATMENT METHOD
The EMEM was removed from the anterior chamber of the BCOP holder and the test material preparation or control materials were applied to the cornea. Approximately 0.8144 g of the solid test material was found to adequately cover the corneal surface. 0.75mL of each control material was 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 240 minutes.

REMOVAL OF TEST MATERIAL
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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken and each cornea was visually observed.
- Corneal permeability: Following the 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 (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1°C for 90 minutes.
- 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)
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
- Opacity Measurement:
The change in opacity for each cornea (including the negative control) 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.
- Permeability Measurement:
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.
- In Vitro Irritancy Score:
The following formula was used to determine the In Vitro Irritancy Score:
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 item induced a response through only one of the two endpoints.

DECISION CRITERIA:
The test material was classified according to the following prediction model:
- IVIS ≤ 3: No category. Not requiring classification to UN GHS or EU CLP
- IVIS > 3; ≤ 55: No prediction of eye irritation can be made
- IVIS > 55: Category 1. UN GHS or EU CLP Causes serious eye damage

ACCEPTABILITY CRITERIA
- The test was acceptable if the positive control (20% w/v imidazole ) produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2016 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 65.1 to 123.3.
- The test was acceptable if the negative control (0.9% w/v sodium chloride solution) produced an In Vitro Irritancy Score 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 solids the negative control limit for opacity should be ≤ 2.4 and for permeability ≤ 0.072.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Corneal Opacity and Permeability Measurement:
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are shown in Table 1.

- Corneal Epithelium Condition:
The corneas treated with the test material were clear post treatment. The corneas treated with the negative control material were clear post treatment. The corneas treated with the positive control material were cloudy post treatment.

- Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤ 2.4 and permeability ≤ 0.072. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	3	2	0		0.000
	2	5	7	2		0.004
	3	6	7	1		0.001
				1.0*		0.002¨		1.0
Positive Control	4	4	73	69	68.0	1.290	1.288
	5	2	85	83	82.0	1.147	1.145
	6	5	87	82	81.0	1.014	1.012
					77.0·		1.149·	94.2
Test Material	7	2	3	1	0.0	0.031	0.029
	8	2	3	1	0.0	0.004	0.002
	9	4	3	-1	0.0	0.006	0.004
					0.0·		0.012·	0.2

OD = Optical density           

* = Mean of the post-treatment -pre‑treatment values             

¨ = Mean permeability                     

· = Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

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

The eye damage of the test material was tested in isolated bovine corneas through topical application for 240 minutes. The test material was applied undiluted and concurrent negative and positive controls were run using sodium chloride 0.9 % (w/v) and 20 % w/v imidazole solution in sodium chloride 0.9 % (w/v), respectively. 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 test material had a mean IVIS of 0.2 after 240 minutes of treatment.

The positive control IVIS was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤ 2.4 and permeability ≤ 0.072. The negative control acceptance criteria were therefore satisfied.

Under the conditions of this study, as the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.