Tin dioxide

Endpoint summary

Description of key information

Skin irritation (human epidermis test method, OECD 439): non-irritant
Skin corrosion (human skin test method, OECD 431): non-corrosive
Eye irritation (bovine corneal opacity and permeability test method, OECD 437): mild irritant
Eye irritation (rabbit, OECD 405): non-irritant
Respiratory irritation: not tested

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 7th February 2012 to 10th February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD No. 431. In vitro skin corrosion:Human skin model test.13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Species:

other: human skin models: EpiDerm(TM)

Strain:

other: human skin models: EpiDerm(TM)

Details on test animals or test system and environmental conditions:

not applicable

Type of coverage:

open

Preparation of test site:

other: not applicable

Vehicle:

water

Controls:

other: not applicable

Amount / concentration applied:

25 mg of the test item was applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistured with 25 µl of water. The test item was spead to match size of the tissue.

Duration of treatment / exposure:

3 and 60 minutes.

Observation period:

one single application.

Number of animals:

not applicable

Details on study design:

Upon receipt of the EpiDermTM - kit, the tissues were transferred into 6-well plates containing 900 µL prewarmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37 ± I °C, 5.0% CO2 / 95% air for at least 1 h. Then the medium was replaced by 900 µL fresh assay medium. The 6-well plate used for the 3 min. experiment was placed back into the incubator. The other plate was used for the 60 min treatment. About 1 h before the end of the first treatment period the MTT solution was prepared by mixing the MT’ concentrate with the MTT diluent and pre-warmed in the incubator.

60 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the 6-well plate was incubated at 37 ± 1 °C, 5.0% CO2 / 95% air.

3 min experiment: the tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 sec. was kept between dosing.
After 3 min of application, with foreceps, the first insert was removed from the 6-well plate. Using a wash bottle the tissue was gently rinsed about 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well “holding plate” containing 300 µL prewarmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were transferred into a prepared 24-well “MTT assay plate” containing 300 µL prewarmed MTT solution. The plate was incubated for 3 h at 37 ± I °C, 5.0% CO2 / 95% air.

60 min experiment: after 60 min application, with foreceps, the first insert was removed from the 6-well plate. Using a wash bottle the tissue was gently rinsed about 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. All inserts were treated in the same manner.
Then the inserts were transferred into a prepared 24-well “MIT assay plate” containing 300 µL prewarmed MTT solution. The plate was incubated for 3 h at 37 ± 1 DC, 5.0% CO2 / 95% air.

3 min and 60 min experiment: after the 3 h MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. Then the inserts were transferred into new 24-well “extraction plates”. 2 mL of isopropanol were pipetted into each insert, thus the insert was covered from both sides. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out over night without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. Then the inserts were discarded and the extraction plates were placed on a shaker for 15 mm.

Per each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

ca. 99

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control

Value:

ca. 100

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

positive control

Value:

ca. 15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no corrosive effects. The test item is classified as "non corrosive".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 28th may 2012 to 3rd June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in compliance with international recognized guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, 97633 Sulzfeld, Germany
Sex: female
Body weight at the beginning of the study: > 2 kg
Age at the beginning of the study: approximately 17 - 18 weeks old
Number of animals: 3
The animals were derived from a controlled full -barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare [9] the animals were bred for experimental purposes.
- Semi barrier in an air-conditioned room
- Temperature: 18± 3 °C
- Relative humidity: 55±10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits (lot no. 1530), rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE - Housed in ABS - plastic rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

single dose of 0.1 g was appliedin the conjuntival sac of one eye.

Duration of treatment / exposure:

single dose

Observation period (in vivo):

72 hours after dosing.

Number of animals or in vitro replicates:

The in vivo test was performed initially using one animal then two additional animals were treated to confirm the first response.

Details on study design:

24 hours before the test an health inspection was performed to ensure the good state of health of the animals.
One hour before the application of the test item, 0.01 mg/Kg of byprenorphine was administareted subcutaneously in order to achieve a therapeutic level of systemic analgesia.
5 minutes prior to the application of the test item, 2-3 drops of an ocular anaesthetic ( proparacaine hydrochloride ophtalmic 0.5% solution) were administrated in both the treated and the control eye of each animal.
The test item was applied at a single dose in the conjunctival of one eye of each test animal after pulling the lower lid away from the eyeball. The lids were gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control.
The animals were observed for 72 hours after dosing.
The eye irritation was scored and recorded according to the grades reported in the field "attached background material".
For the calculation only the 24, 48 and 72 hours were used.

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

other: 1 hour

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 1 hour

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 1h

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 1h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: 1 hour

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: 1 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: 1, 24, 48, 72 hours

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

other: 1, 24, 48, 72 hours

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: 1 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

other: 1, 24, 48, 72 hours

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

other: 1, 24, 48, 72 hours

Score:

0

Max. score:

2

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

other: 1, 24, 48, 72 hours

Score:

0

Max. score:

4

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

The test item produced irritant but not corrosive effects in all animals.
Conjuntival redness was observed in all animals one hour post-application.
Conjuntival chemosis and discharge were observed only in animal n.2 and 3 one hour after application. These changes were fully reversible within 24 hours.

Other effects:

Neither mortalities nor significant clinical signs of toxicity were observed.
Upon fluoreshein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, a single ocular application of the test item Tin dioxide to rabbits at a dose of 0.1 g produced slight initial irritant effects. Neither mortalities nor significant clinical signs of toxicity were observed.
According to the CLP criteria for classification and labelling requirements for dangerous substances the test item Tin dioxide is not to be classified as eye irritant.

Reference 2

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:

other: GLP study in compliance with international recognized guidelines

Qualifier:

according to guideline

Guideline:

other: OECD 437 " Bovine Corneal Opacity and Permeability Test Method for Identifying ocular corrosives and severe irritants".

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
2012-000203
- Expiration date of the lot/batch:
07/02/2013, W0167
- Purity test date:
> 99.9%, 23/01/2011

INFORMATION ON NANOMATERIALS
- Chemical Composition:
Tin Dioxide
- Density:
6.936 g/cm³
- Particle size & distribution:
10-100 nm; 78
- Specific surface area: 7.4293 ± 0.0132 m²/g

Species:

other: isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals, e.g. from Attenberger Fleish GmbH & Co. KG.

Strain:

not specified

Vehicle:

physiological saline

Controls:

other: 3 corneas as negative control and 3 corneas as positive controls were used.

Duration of treatment / exposure:

750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mE sodium fluorescein solution was added to the anterior chamber and the comeas were incubated for 90 minutes at 32 ± 1 °C, Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

Observation period (in vivo):

After 90 minutues, the optical density at 490 nm was determined, using a spectrophotometer

Number of animals or in vitro replicates:

3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCI
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl

Irritation parameter:

in vitro irritation score

Run / experiment:

Bovine corneal opacity and permeability assay

Value:

15.01

Table 1: Evaluation of the BCOP Assay

mean score: 0 -3: non irritant

mean score: 3.1 -25: mild irritant mean score: 25.1 - 55: moderate irritant mean score: 55.1 - 80: severe irritant mean score: >80.1: very severe irritant

Interpretation of results:

other: mild irritant

Remarks:

Criteria used for interpretation of results: expert judgment

Conclusions:

As reported in the ECHA Guideline: "Guidance on the application of CLP criteria", only positive results in the BCOP, ICE, IRE and HET-CAM in vitro assays can be used for classification as severe eye irritants. Negative results are not conclusive for a non classification. There are currently no validated in vitro eye irritation test methods available. However, two reconstituted human tissue models (the EpiOcularTM and SkinEthicTM HCE models) are undergoing formal validation."

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

According to Regulation 440/2008/EC and its amendments registrants are encouraged to conduct in-vitro tests wherever is possible. Therefore in vivo study can be waived since in vitro study is available.

Both an in-vitro skin irritation test and an in-vitro skin corrosion test have been carried out on the substance tin dioxide in certified GLP labs. These studies can be ranked as Klimisch code 1: reliability without restrictions. The results of them showed that no toxic effect was found for Tin Dioxide NP. So the irritative effect of tin dioxide can be concluded as non irritating/non corrosive.

Eye irritation:

An in vitro test of the substance was performed on eye irritation complying with OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method for identifying ocular corrosives and severe irritants.

This study is regarded as key study as it can be ranked as Klimisch code 1: reliability without restrictions. The result of the test showed that tin oxide is a mild irritant.

As reported in the ECHA Guideline: "Guidance on the application of CLP criteria", only positive results in the BCOP, ICE, IRE and HET-CAM in vitro assays can be used for classification as severe eye irritants. Negative results are not conclusive for a non classification. There are currently no validated in vitro eye irritation test methods available. However, two reconstituted human tissue models (the EpiOcularTM and SkinEthicTM HCE models) are undergoing formal validation."

An in vivo test on rabbits was performed complying with OECD Guideline 405: acute eye irritation/corrosion (Klimisch code: 1). The results of the test showed that tin dioxide is not irritant.

Based on the result of this in-vivo study, Tin dioxide NP is considered to be non irritating to eyes.

Justification for selection of skin irritation / corrosion endpoint:
GLP study in compliance with international recognized guidelines.

Justification for selection of eye irritation endpoint:
in vivo test

Justification for classification or non-classification

The available data for eye and skin irritation indicate that the substance is not irritating to the eyes or skin. Thus, the data are conclusive but not sufficient for classification for eye and skin irritation/corrosion according the CLP Regulation.