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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:
27th November - December 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
EC Number:
248-227-6
EC Name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate
Cas Number:
27107-89-7
Molecular formula:
C38H74O6S3Sn
IUPAC Name:
2-ethylhexyl 10-ethyl-4-({2-[(2-ethylhexyl)oxy]-2-oxoethyl}sulfanyl)-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecan-1-oate
Constituent 2
Reference substance name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate>97.9%
IUPAC Name:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate>97.9%
Details on test material:
2-ethylhexyl 10-ethyl-4-[[2-[(2-ethylhexyl)oxy]-2-oxoethyl]thio]-4-octyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate>97.9%

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Heads of the animals were obtained from poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, the Netherlands.
- Age at study initiation: in vitro study, eyes from 7 week old chicks were used.
- Weight at study initiation: 1.5 - 2.5 kg


ENVIRONMENTAL CONDITIONS
not applicable.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): N/A


VEHICLE
not applicable.
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
Approximately 30 minutes after treatment observations were recorded
Number of animals or in vitro replicates:
in vitro study:
1 negative control eye.
3 positive control eyes
3 test eyes
Details on study design:
EXPERIMENTAL DESIGN
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus (see Figure 1) using the following procedure: First the eye lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2.0% w/v (Minims, Smith & Nephew Ltd., Romford, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein treated cornea were examined with a slit lamp microscope (Slit lamp 900 BP, Haag Streit AG, Liebefeld Bern, Switzerland) to ensure that the cornea is not damaged. If undamaged, the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye ball from the orbit without cutting off the optical nerve too short.

The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of ca 0.10 0.15 mL/min (peristaltic pump set at 5.00, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32ºC (water pump set at 34ºC; Lauda 103, Germany).

After placing in the superfusion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they are not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag Streit slit lamp microscope. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.

Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that show opacity (score higher than 0.5), or are unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that show any other signs of damage, were rejected as test eyes and replaced.

In accordance with the proposed OECD guideline no. 438, three test eyes, one negative control eye and three positive control eyes were selected for testing. Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45 60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.
At time t = 0 (i.e. immediately after the zero reference measurement), the following procedure was applied for each test eye: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards. Next, the eyes (corneas) were treated with the study substances 30µl for 10 seconds, followed by rinsing in 20ml saline.After rinsing, each eye in the holder was returned to its chamber.

The eyes were examined at ca 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system given in Annex 1. Fluorescein retention was only scored at ca 30 minutes after treatment. All examinations were carried out with the slit lamp microscope.
After the final examination the test substance treated eyes and the negative and positive control eyes were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 µM and stained with PAS (Periodic Acid-Schiff). The microscopic slides were filed in the archives and kept available for histopathological examination if considered relevant. In the case of Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE), histopathological examination was considered not relevant, because no significant corneal effects were observed.

CONTROLS
- Positive control: 5% (w/w) aqueous dilution of benzalkonium chloride
- Negative control: Saline.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed with 20 mL of saline solution
- Time after start of exposure: 10 seconds


SCORING SYSTEM:
In the ICE test, the eyes were examined at several time intervals after treatment to determine ocular effects using the parameters of corneal thickness (swelling), corneal opacity and fluorescein retention. Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV). Four classes of eye irritancy (not irritating; slightly irritating; moderately irritating; severely irritating) were identified in the ICE test by combination of the categories defined for each of the evaluation parameters using the Prediction Model described in the box below.
Regulatory classification of eye irritants was based on the standard in vivo Draize Eye test. Various international regulatory classification systems are in use. Some systems recognize four classes, viz. non irritant, mild irritant (Category 2B), irritant (Category 2A), and severe irritant (Category 1) such as in the Globally Harmonized System (UN/GHS). The new EC Regulation 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP) entered into force on the 20 January 2009. CLP implements the Globally harmonised System (GHS). The EC/GHS-classification system (CLP) recognizes three classes, i.e. non-classified/non-irritant (NC/NI), irritant (Category 2) and severe irritant (Category 1).
Table 1 below shows the criteria used in the EC/GHS-regulatory classification system to identify the different classes of eye irritants

In order to translate the eye irritancy scores from the ICE test to an EC/GHS-regulatory classification, it was necessary to reconcile four irritancy classes from the ICE study into three classes within the EC/GHS-regulatory classification scheme. This was achieved through application of the prediction model defined below, which was based on scientific judgement and which was supported by several years of experience with conduct of the ICE test in parallel to the in vivo OECD 405 test. As a result of the reconciliation of the four ICE classes of irritancy into the 3 classes of the EC/GHS-regulatory classification system, it is possible that materials defined as slightly irritant using the ICE test Prediction Model may be identified in the EC-regulatory classification system as not classified/non-irritant.

TOOL USED TO ASSESS SCORE: hand-slit lamp and fluorescein

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test material
Value:
22
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
no effects
Positive controls validity:
valid
Remarks:
Irritation Index = 149

Any other information on results incl. tables

Table 2: Summary of Results

Test material

 

Maximum mean score for:

Irritation

categories1

Irritation

Index2

Classification

(General, GHS and EC/GHS)

Swelling %

Opacity

Fluorescein

retention

MOTE

2

0.5

0.5

I;I;I

22

NI/NI/NI

BAC 5%

295

3.0

3.0

III;IV;IV

149

severe/I/I

Saline (negative control)6

0

0.0

0.0

n.a.7

n.a.

n.a.

1        I = no effect; II = slight effect; III = moderate effect; IV = severe effect.

2        Irritation Index = maximum mean corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20).

3        2B = mild irritant; 2A = irritant; 1 = serious eye damage. United Nations-Economic Commission for(UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). UN,and, 2007 (http://www.unece.org/trans/danger/publi/ghs/ghs_rev02/02files_e.html)

4        Blistering of epithelium.

5        Individual values based on one eye.

6 n.a. = not applicable

EC/GHS‑CLASSIFICATION (CLP) OF EYE IRRITANTS; EXTRAPOLATION FROM IN VITRO RESULTS TO IN VIVO CLASSIFICATION.

 

Extrapolation from thein vitroirritancy results to the EC/GHS classification will be carried out using scientific judgement, re-dividing the original four irritancy classes to three. The combinations of the three categories that are allowed for each of the three classifications are mentioned in the scheme below.

 

Regulatory Classification Combinations of the three categories.

NC/NI = not classified/not irritating            3 x I

                                                                          2 x I, 1 x II

                                                                          2 x II, 1 x I3

 

EC/GHS Category 24:                                    3 x II

Irritating to eyes                                             2 x II, 1 x III

                                                                          1 x I, 1 x II, 1 x III¹

                                                                          3 x III

                                                                          2 x III, 1 x II

                                                                          2 x III, 1 x I

                                                                          2 x I, 1 x IV¹

                                                                          2 x II, 1 x IV¹

                                                                          2 x III, 1 x IV2

                                                                          1 x II, 1 x III, 1 x IV¹

 

EC/GHS Category 14:                                     3 x IV

Irreversible effects on the eye/                     2 x IV, 1 x III

serious damage to eyes                                 2 x IV, 1 x II¹

                                                                          2 x IV, 1 x I¹

                                                                          immediate corneal opacity score 3

                                                                          corneal opacity score 4

                                                                          severe loosening of epithelium

¹ Combinations of categories less likely to occur.

 2The combination of 2 x III, and 1 x IV can be considered as a borderline case between irritating and severely irritating.

 3The combination of 2 x II, and 1 x I can be considered as a borderline case between not irritating and Category 2.

 4Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006.

 

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to EU criteria
Conclusions:
The negative control eye showed no corneal effects. The positive control caused moderate swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 149. Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) caused very slight swelling, very slight opacity and very slight fluorescein retention. The calculated Irritation Index was 22.

On the basis of the results obtained in the ICE test and according to its classification schemes, Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) was considered not irritating to eyes.
Executive summary:

Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) was evaluated neat in the Isolated Chicken Eye (ICE) test, according to OECD guideline no. 438. Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of the test sample for 10 seconds using an application volume of 30 µL. Benzalkonium chloride (BAC) 5% was used as the positive control and physiological saline as the negative control. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells.

The negative control eye showed no corneal effects. The positive control caused moderate swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 149. Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) caused very slight swelling, very slight opacity and very slight fluorescein retention. The calculated Irritation Index was 22.

On the basis of the results obtained in the ICE test and according to its classification schemes, Octyltin tris(2-ethylhexylmercaptoacetate) (MOTE) was considered not irritating to eyes.