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Description of key information

key studies demonstrate a lack of irritation/corrosion potential

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin 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
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
version 22 July 2010
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM (SM) (Manufacturer: SkinEthic, France
- Tissue batch number(s):12-EKIN-028
- Expiry Date: 16 July 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21.7-22.3°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1wahsing step: rinsing thoroughly with PBS 1x solution (0.9%)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3h
- Spectrophotometer: 96-well plate spectrophotometer
- Wavelength: 540nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE (see any other info on mat and meth)



Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): na; no formulation was required

NEGATIVE CONTROL: PBS
- Concentration (if solution): 50µl

POSITIVE CONTROL: SDS 5%
- Concentration (if solution): 50µl
Duration of treatment / exposure:
15 minutes (± 0.5 min)
Duration of post-treatment incubation (if applicable):
42h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects:
none

The results of the optical density (OD) measured at 540 nm of each extract and the calculated % viability of the cells is presented below:

 

 

Table : Optical Density (OD) and the calculated % viability of the cells

Substance

Optical Density (OD)

Viability (%)

Negative Control:

1

0.797

104

PBS

2

0.744

97

 

3

0.753

98

 

mean

0.765

100

 

standard deviation (SD)

3.79

Positive Control:

1

0.246

32

SDS 5%

2

0.207

27

 

3

0.083

11

 

mean

0.179

23

 

standard deviation (SD)

10.97

Test Item:

1

0.718

94

Indium powder

2

0.683

89

3

0.832

109

mean

0.744

97

 

standard deviation (SD)

10.41

 

 


 

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro skin irritation test in the EPISKIN model with indium powder the results indicated that the test item is Non Irritant (NI)
[UN GHS: No Category].
Executive summary:

Disks of EPISKIN (three units / chemical) were treated with Diindium trisulphide and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of the epidermis on each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.

SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a % relative to negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test substance is considered to be irritant to skin.

Following exposure with indium powder, the mean treated skin value was 97% and therefore non irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

In this in vitro skin irritation test in the EPISKIN model with indium powder the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

 

Endpoint:
skin corrosion: in vitro / ex vivo
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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)
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
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
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
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
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

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, 1xI4

B= Slightly irritating

(GHS3category 2B: Mild irritant / causes eye irritation)

3×II

2×II, 1×III

1×I, 1×II, 1×III1

C= Moderately irritating

(GHS3category 2A: Irritant / causes eye irritation)

3×III

2×III, 1×II

2xI, 1xIV1

2×III, 1×IV2

2×III, 1×I

2×II, 1×IV1

1×II, 1×III, 1×IV1

D= Corrosive/severely irritating

(GHS3category 1: Irreversible effects on the eye / serious damage to the eye)

3×IV

2×IV, 1×III

2×IV, 1×II1

2×IV, 1×I1

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)

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 (07thSeptember 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.


 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

The in vitro skin irritation test in the EPISKIN model with indium powder (Kiss, CiToxLAB Hungary 2012) indicate that the test item is Non Irritant (NI).

Eye irritation:

The in vitro eye irritation study in the Isolated Chicken Eyes model with Indium powder (Kiss, CiToxLAB Hungary 2012) 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.


Justification for classification or non-classification

Based upon OECD 439 in vitro skin irritation data, Indium does not require classification as a skin irritant.

Based upon OECD 438 in vitro eye irritation data, Indium does not require classification as an eye irritant.