Diindium trioxide

Endpoint summary

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

Reference

Reference 1

Endpoint:

skin corrosion: 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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

version 13 April 2004

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: EPISKIN-SM (Manufacturer: SkinEthic, France, Catalogue Number: EPISKIN/S/13
- Tissue batch number(s): 11-EKIN-045
- Expiry date: 12 December 2011
- Date of initiation of testing: 7 December 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 18-28°C
- Temperature of post-treatment incubation (if applicable): not applicable

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: 2.2 mL of 0.3 mg/mL MTT
- 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)


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement):

- The test substance is considered to be corrosive to skin, if the mean relative viability after 4 hours of exposure is below 35% of the negative control.

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: NaCI (9 g/l saline)
- Concentration (if solution): 50µl

POSITIVE CONTROL: glacial acetic acid
- Concentration (if solution): 50µl

Duration of treatment / exposure:

4h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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

Substance	Optical Density (OD)		OD after adjustment*	Viability (%)
Negative Control: NaCl (9g/L saline)	1	0.263		100
	2	0.249
	3	0.231
	mean	0.248
Positive Control: Glacial acetic acid	1	0.000		0
	2	0.028		11
	3	0.005		2
	mean	0.011		4.3
Test Item: Dindium trioxide	1	0.223	0.200	81
	2	0.243	0.220	88
	3	0.242	0.219	88
	mean	0.236	0.213	86

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKIN model test with Diindium trioxide, the results indicate that the test item is not a skin corrosive.

Executive summary:

Disks of EPISKIN (three units / chemical) were treated with test item Diindium trioxide and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9 %). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCI (9 g/l saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control.

The test substance is considered to be corrosive to skin, if the mean relative viability after 4 hours of exposure is below 35% of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control. All test item results were far above 35% of the mean negative control value.

In this in vitro EPISKIN model test with Diindium trioxide, the results indicate that the test item is not a skin corrosive.

Reference 2

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: 14 May 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:

101

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	0.820	108
PBS	2	0.659	87
	3	0.788	104
	mean	0.756	100
	standard deviation (SD)		11.15
Positive Control:	1	0.040	5.3
SDS 5%	2	0.079	10
	3	0.041	5.4
	mean	0.053	6.9
	standard deviation (SD)		2.69
Test Item:	1	0.787	104
Diindium trioxide	2	0.763	101
	3	0.751	99
	mean	0.767	101
	standard deviation (SD)		2.52

 

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro skin irritation test in the EPISKIN model with diindium trioxide 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 trioxide 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 diindium trioxide, the mean treated skin value was 101% 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 diindium trioxide the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

 

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:

One eye was treated with isotonic saline, three eyes with the test item and other three ones with Imidazole

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

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: Diindium trioxide

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	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*	3xI

In this in vitro eye irritation in the isolated chicken eyes test with Diindium trioxide,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	2 %	I
Mean maximum corneal swelling at up to 240 min	8 %	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	1 %	I
Mean maximum corneal swelling at up to 240 min	1 %	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class*	3xI

The negative controlSodium 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)

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

An in vitro eye irritation study of the test item Diindium trioxide 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 antimonide 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 Diindium trioxide, the results suggest that the test item is not irritating. According to the guideline OECD 438, Diindium trioxide does not require a classification as a severe eye irritant. Diindium trioxide 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/corrosion test in the EPISKIN model with diindium trioxide (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 diindium trioxide (Kiss, CiToxLAB Hungary 2012) suggest that the test item is not irritating. According to the guideline OECD 438, diindium trioxide does not require a classification as a severe eye irritant.

Justification for classification or non-classification

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

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