Indium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Version / remarks:

version 7th September 2009

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc chloride

EC Number:

231-592-0

EC Name:

Zinc chloride

Cas Number:

7646-85-7

Molecular formula:

ZnCl2

IUPAC Name:

Zinc dichloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Product name: Indium powder
Molecular formula: In
Batch number: In-05252012A1
CAS number: 7440-74-6
Appearance: silver grey, powder
Manufacture Date: 06 June 2012
Expiry date: 06 June 2014
Storage conditions: Controlled Room Temperature (15-25°C, below 70 RH%)
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

up to 240 minutes (at 30, 75, 120, 180 and 240 min) after post-treatment rinse for the cornea thickness and cornea opacity
Fluorescein retention was measured at base line (t=0) and at 30 minutes after rinse.

Number of animals or in vitro replicates:

3 test item treated eyes, 3 positive control treated eyes and 1 negative control eye

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Eyes selection:
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (v/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 ml isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
Preparation of eyes:
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period (45-60min), a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not increase by more than 5-7 % between the -45 and the zero time. Slight changes in thickness (-1% to 1%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
3 test item treated eyes, 3 positive control treated eyes and 1 negative control eye

NEGATIVE CONTROL USED
treated with 30µL isotonic saline

SOLVENT CONTROL USED (if applicable): not applicable

POSITIVE CONTROL USED
treated with 30 mg imidazole

APPLICATION DOSE AND EXPOSURE TIME
test substance treated chicken eye: treated with 30 mg during 10 seconds

OBSERVATION PERIOD
30, 75, 120, 180 and 240 min after post-treatment. The cornea thickness and cornea opacity were measured at all time points.
Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: cornea surface was rinsed thoroughly with 20ml isotonic saline

METHODS FOR MEASURED ENDPOINTS:

- Corneal opacity: examined with slit lamp microscope
- Damage to epithelium based on fluorescein retention: examined with hand-held slit lamp or slit lamp microscope
- Swelling: examined with slit lamp microscope

SCORING SYSTEM: see below other info on mat and meth
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment


DECISION CRITERIA: as indicated in the TG

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 75min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 240min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

cornea opacity score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

fluorescein retention score

Value:

0.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity and fluorescein retention are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.

Test Item: Indium powder

Observation	Value	ICE Class*
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.67	II
Other Observations	The Test item was stuck on the cornea surface after the post-treatment rinse. The cornea surface was not cleared 240 min after the post-treatment rinse.
Overall ICE Class*	2xI 1xII

In this in vitro eye irritation in the isolated chicken eyes test with Indium powder,the results suggest that the test item was not irritating.No conclusion of in vivo significance can be made from the adherence of the test item to the cornea, since in vivo eye lids will probably clear the surface, but abrasion may occur. An in vivo study is required for classification.

Positive Control: Imidazole

Observation	Value	ICE Class*
Mean maximum corneal swelling at up to 75 min	1 %	I
Mean maximum corneal swelling at up to 240 min	6 %	II
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	2.83	IV
Other Observations	The Imidazole was stuck on the cornea surface after the post-treatment rinse. The cornea surface was not cleared 240 minutes after the post-treatment rinse.
Overall ICE Class*	1xII 2xIV

The positive control Imidazole was classed as severely irritating,GHS Classification: Category 1.

Negative Control: Sodium chloride

Observation	Value	ICE Class*
Mean maximum corneal swelling at up to 75 min	0 %	I
Mean maximum corneal swelling at up to 240 min	0 %	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class*	3xI

The negative control Sodium chloride 0.9% had no significant effects on the chicken eye in this study.

Table:Assessment of the general IN VITRO eye irritancy and regulatory GHS classification.

 

The following table is used to identify the probably eye irritancy potential of test items. In the case where the result indicates Corrosive/Severely Irritating, then the test item can be classified as Severe. In all other cases the probable level of irritancy can be reported, but a regulatory in vivo rabbit eye irritation test is required for regulatory classification and labelling purposes.

EC and GHS Classification	Combinations of the three ICE Classes
A=Not irritating	3×I 2×I, 1×II 2xII, 1xI^4
B= Slightly irritating (GHS^3category 2B: Mild irritant / causes eye irritation)	3×II 2×II, 1×III 1×I, 1×II, 1×III^1
C= Moderately irritating (GHS^3category 2A: Irritant / causes eye irritation)	3×III 2×III, 1×II 2xI, 1xIV^1 2×III, 1×IV^2 2×III, 1×I 2×II, 1×IV^1 1×II, 1×III, 1×IV^1
D= Corrosive/severely irritating (GHS^3category 1: Irreversible effects on the eye / serious damage to the eye)	3×IV 2×IV, 1×III 2×IV, 1×II^1 2×IV, 1×I^1 Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) Corneal opacity = 4 at any time point (in at least 2 eyes) Severe loosening of epithelium (in at least 1 eye)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro eye irritation study in the Isolated Chicken Eyes model with Indium powder, the results suggest that the test item is not irritating. According to the guideline OECD 438, Indium powder does not require a classification as a severe eye irritant. Indium powder remained adhered to the cornea surface after the post-treatment rinse.

Executive summary:

An in vitro eye irritation study of the test item Indium powder was performed in chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (07^thSeptember 2009).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg of Indium powder was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 mg Imidazole. The negative control eye was treated with 30 µL of isotonic saline.

In this in vitro eye irritation study in the Isolated Chicken Eyes model with Indium powder, the results suggest that the test item is not irritating. According to the guideline OECD 438,Indium powder does not require a classification as a severe eye irritant. Indium powder remained adhered to the cornea surface after the post-treatment rinse.