Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion

WoE: In vivo skin irritation (rabbit, Ema 2011, OECD Guideline 404, N-MWCNT): not skin irritating

WoE: In vivo skin irritation (rabbit, Ema 2011, OECD Guideline 404, MWCNT-7): not skin irritating

WoE: In vivo skin irritation (rabbit, Murthy 2011, OECD Guideline 404, MWCNT1): not skin irritating

WoE: In vivo skin irritation (rabbit, Murthy 2011, OECD Guideline 404, MWCNT2): not skin irritating

Eye irritation/corrosion

WoE: In vivo eye irritation (rabbit, Ema 2011, OECD Guideline 405, N-MWCNT): not eye irritating

WoE: In vivo eye irritation (rabbit, Ema 2011, OECD Guideline 405, MWCNT-7): not eye irritating

WoE: In vivo eye irritation (rabbit, Ema 2011, OECD Guideline 405, SWCNT): not eye irritating

WoE: In vivo eye irritation (rabbit, Murthy 2011, OECD Guideline 405, MWCNT1): potential eye irritant

WoE: In vivo eye irritation (rabbit, Murthy 2011, OECD Guideline 405, MWCNT2): potential eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Species:
rabbit
Strain:
other: Kbl:NZW
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kitayama Labes Co. Ltd. (Ina, Japan)
- Age at study initiation: 17 weeks
- Weight at study initiation: 2.7 - 3.6 kg
- Housing: individually in standard aluminum cages
- Diet: LRC4; Oriental Yeast Co. Ltd., Tokyo, Japan, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days prior to the start of the experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 °C
- Humidity (%): 40-70%
- Air changes (per hr): ventilation of 15-21 air changes/h
- Photoperiod (hrs dark / hrs light): 12-h light (7:00-19:00)/dark (19:00-7:00) cycle
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: olive oil
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): MWCNT-7 was moistened with a minimum amount of olive oil to prepare a paste for dermal application. The paste contained 2% MWCNT-7. This amount of MWCNT-7 was the maximum not leading to overlow when patched and the sample fully coated the skin. Olive oil was used to prepare the paste, because the olive oil was highly refined (neutral, denatured, and free of antioxidants) and widely used as the negative control in these expermiments.
An area of about 10 x 15 cm on the back of each rabbit was made free of fur using electric clippers and an electric shaver 24 h prior to testing. The MWCNT-7 paste (0.5 g) was evenly spread on a lint cloth (2.5 x 2.5 cm), applied to the skin, and covered with a gauze patch, which was held in place with non-irritating elastic bandage (Silkytex, Alcare Co. Ltd., Tokyo Japan).
Duration of treatment / exposure:
4 hours
Observation period:
Test sites were evaluated for signs of dermal irritation approximately 60 min, 24, 48, and 72 h after test substance removal.
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 6.25 cm2 (2.5 x 2.5 cm) clipped area of skin
- Type of wrap if used: gauze patch which was held in place with non-irritating elastic bandage

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
60 min, 24 h, 48 h, and 72 h

SCORING SYSTEM: Test sites were evaluated according to OECD TG 404.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0.6
Reversibility:
fully reversible within: 72 h
Irritant / corrosive response data:
No clinical signs or changes in body weight gain were observed in any groups treated with MWCNT-7. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with MWNT-7. The PII was calculated to be 0.
Other effects:
None
Interpretation of results:
GHS criteria not met
Conclusions:
No clinical signs or changes in body weight gain were observed. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with MWNT-7. The PII was calculated to be 0.
Executive summary:

Acute dermal irritation was analyzed in a study performed according to method described in OECD guideline 404. New Zealand white rabbits received doses of 0.5 g of MWCNT-7-pastes containing 2% MWCNT-7 by application to a clipped area of the skin.

No clinical signs or changes in body weight gain were observed in any groups treated with MWCNT-7. No dermal responses, including erythema/eschar or edema, were found in rabbits treated with MWNT-7. The PII was calculated to be 0 in these group.

Hence based on the above findings MWCNT-7 test item was considered as non irritant to the skin of rabbits.

According to the criteria of Classification, Labelling and Packaging of Substances and Mixtures Regulation (EC) No 1272/2008 (CLP Regulation), classification as skin irritant is not warranted.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Species:
rabbit
Strain:
other: Kbl:NZW
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kitayama Labes Co. Ltd. (Ina, Japan)
- Age at study initiation: 17 weeks
- Weight at study initiation: 2.7 - 3.6 kg
- Housing: individually in standard aluminum cages
- Diet: LRC4; Oriental Yeast Co. Ltd., Tokyo, Japan, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days prior to the start of the experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 °C
- Humidity (%): 40-70%
- Air changes (per hr): ventilation of 15-21 air changes/h
- Photoperiod (hrs dark / hrs light): 12-h light (7:00-19:00)/dark (19:00-7:00) cycle
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: olive oil
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): N-MWCNT was moistened with a minimum amount of olive oil to prepare a paste for dermal application. The paste contained 1% N-MWCNTs. This amount of N-MWCNT was the maximum not leading to overlow when patched and the sample fully coated the skin. Olive oil was used to prepare the paste, because the olive oil was highly refined (neutral, denatured, and free of antioxidants) and widely used as the negative control in these expermiments.
An area of about 10 x 15 cm on the back of each rabbit was made free of fur using electric clippers and an electric shaver 24 h prior to testing. The N-MWCNT paste (0.5 g) was evenly spread on a lint cloth (2.5 x 2.5 cm), applied to the skin, and covered with a gauze patch, which was held in place with non-irritating elastic bandage (Silkytex, Alcare Co. Ltd., Tokyo Japan).
Duration of treatment / exposure:
4 hours
Observation period:
Test sites were evaluated for signs of dermal irritation approximately 60 min, 24, 48, and 72 h after test substance removal.
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 6.25 cm2 (2.5 x 2.5 cm) clipped area of skin
- Type of wrap if used: gauze patch which was held in place with non-irritating elastic bandage

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
60 min, 24 h, 48 h, and 72 h

SCORING SYSTEM: Test sites were evaluated according to OECD TG 404.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0.6
Reversibility:
fully reversible within: 72 h
Irritant / corrosive response data:
In the group treated with N-MWCNTs, three rabbits exhibited very slight erythema, which was barely perceptible, 24 h after removal of the patches. In one rabbit, the erythema disappeared 48 h after the patch was removed. In two rabbits, the erythema disappeared 72 h after removal of the patches. The PII was calculated to be 0.6 in this group.
Other effects:
None
Interpretation of results:
GHS criteria not met
Conclusions:
In the group treated with N-MWCNTs, three rabbits exhibited very slight erythema, which was barely perceptible, 24 h after removal of the patches. The erythema disappeared after up to 72 h after removal of the patches. The PII was calculated to be 0.6 in this group.
Executive summary:

Acute dermal irritation was analyzed in a study performed according to method described in OECD guideline 404. New Zealand white rabbits received doses of 0.5 g of N-MWCNT-pastes containing 1% N-MWCNT by application to a clipped area of the skin. In the group treated with N-MWCNTs, three rabbits exhibited very slight erythema, which was barely perceptible, 24 h after removal of the patches. The erythema disappeared after up to 72 h after removal of the patches. The PII was calculated to be 0.6 in this group.

Hence based on the above findings N-MWCNT test item was considered as non irritant to the skin of rabbits.

According to the criteria of Classification, Labelling and Packaging of Substances and Mixtures Regulation (EC) No 1272/2008 (CLP Regulation), classification as skin irritant is not warranted.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Specific details on test material used for the study:
MWCNT1: 5-8 microns in length with 3-8 nm inside diameter and outside diameter of 140 ± 30 nm; Product No. 659258 purchased from Sigma-Aldrich, USA.
The multi-walled carbon nanotubes used in the study were produced by catalytic chemical vapor deposition (CCVD). MWCNT1 were composed of 99.9 % carbon with small amount (<0.1% Fe) of iron. The manufacturer specification for nanomaterial characterization was confirmed by the following techniques. MWCNT1 characteristics were assessed in the as-synthesized form prior to use for experiments or after dispersion in the vehicle (distilled water) for dosing. Solution characteristics were measured with dynamic light scattering (DLS).
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.0 - 3.0 kg
- Housing: individually in standard stainless steel cages and sterilized paddy husk as bedding. Bedding material was changed daily, whereas water bottles were changed on alternate days.
- Diet (e.g. ad libitum): adlibitum supply of standard rabbit pellet food (supplier: M/s. Amrut Laboratory Animal Feed, Pune)
- Water (e.g. ad libitum): adlibitum supply of UV treated water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a single dermal dose of 0.5 gram of the test material (MWCNT1) moistened with minimum volume (0.25 ml) of distilled water was applied to a 6 cm2 clipped area of skin.
Duration of treatment / exposure:
4 hours
Observation period:
Test sites were evaluated by Draize score for signs of dermal irritation approximately 60 min, 24, 48, and 72 h after test substance removal
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 6 cm2 clipped area of skin
- Type of wrap if used: 2-ply gauze square which was held in place with non-irritating tape and covered with porous tape for a semi-occlusive dressing

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
60 min, 24 h, 48 h, and 72 h

SCORING SYSTEM: Test sites were evaluated by Draize score
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
not specified
Irritant / corrosive response data:
None of the rabbits treated with MWCNT1 showed any dermal reactions.
Other effects:
None
Interpretation of results:
not irritating
Conclusions:
None of the rabbits treated with MWCNT1 showed any dermal reactions at 60 min, 24, 48 and 72 h after patch removal. Thus the Primary Irritation Index (PII) of MWCNT1 is zero.
Executive summary:

Acute dermal irritation was analyzed in a study performed according to method described in OECD guideline 404. New Zealand white rabbits received 0.5 g of MWCNT1 by application to a clipped area of the skin.

None of the rabbits treated with test material showed any dermal reactions at 60 min, 24, 48 and 72 h after patch removal. None of the animals showed clinical signs of toxicity. Body weight gain of the individual animal was normal during the study period. Thus the Primary Irritation Index (PII) of MWCNT1 is zero. Hence based on the above findings MWCNT1 was considered as non irritant to the skin of the New Zealand white rabbits.

According to the criteria of Classification, Labelling and Packaging of Substances and Mixtures Regulation (EC) No 1272/2008 (CLP Regulation), classification as skin irritant is not warranted.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Specific details on test material used for the study:
MWCNT 2: 1-10 microns in length with 2-6 nm inside diameter and outside diameter of 10-15 nm; Product No. 677248, purchased from Sigma-Aldrich, USA.
The multi-walled carbon nanotubes2 used in the study were produced by catalytic chemical vapor deposition (CCVD). The MWCNT2 were composed of 99.9 % carbon with small amount (<0.1% Fe) of iron. The manufacturer specification for nanomaterial characterization was confirmed by the following techniques. MWCNT2 characteristics were assessed in the as-synthesized form prior to use for experiments or after dispersion in the vehicle (distilled water) for dosing. Solution characteristics were measured with dynamic light scattering (DLS).
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.0 - 3.0 kg
- Housing: individually in standard stainless steel cages and sterilized paddy husk as bedding. Bedding material was changed daily, whereas water bottles were changed on alternate days.
- Diet (e.g. ad libitum): adlibitum supply of standard rabbit pellet food (supplier: M/s. Amrut Laboratory Animal Feed, Pune)
- Water (e.g. ad libitum): adlibitum supply of UV treated water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a single dermal dose of 0.5 gram of the test material (MWCNT2) moistened with minimum volume (0.25 ml) of distilled water was applied to a 6 cm2 clipped area of skin.
Duration of treatment / exposure:
4 hours
Observation period:
Test sites were evaluated by Draize score for signs of dermal irritation approximately 60 min, 24, 48, and 72 h after test substance removal
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: 6 cm2 clipped area of skin
- Type of wrap if used: 2-ply gauze square which was held in place with non-irritating tape and covered with porous tape for a semi-occlusive dressing

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
60 min, 24 h, 48 h, and 72 h

SCORING SYSTEM: Test sites were evaluated by Draize score
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
not specified
Irritant / corrosive response data:
None of the rabbits treated with MWCNT2 showed any dermal reactions.
Other effects:
None
Interpretation of results:
not irritating
Conclusions:
None of the rabbits treated with MWCNT2 showed any dermal reactions at 60 min, 24, 48 and 72 h after patch removal. Thus the Primary Irritation Index (PII) of MWCNT2 is zero.
Executive summary:

Acute dermal irritation was analyzed in a study performed according to method described in OECD guideline 404. New Zealand white rabbits received 0.5 g of MWCNT2 by application to a clipped area of the skin.

None of the rabbits treated with test material showed any dermal reactions at 60 min, 24, 48 and 72 h after patch removal. None of the animals showed clinical signs of toxicity. Body weight gain of the individual animal was normal during the study period. Thus the Primary Irritation Index (PII) of MWCNT2 is zero. Hence based on the above findings MWCNT1 was considered as non irritant to the skin of the New Zealand white rabbits.

According to the criteria of Classification, Labelling and Packaging of Substances and Mixtures Regulation (EC) No 1272/2008 (CLP Regulation), classification as skin irritant is not warranted.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
no details given
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Species:
rabbit
Strain:
other: Kbl:NZW
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Kitayama Labes Co. Ltd. (Ina, Japan)
- Age at study initiation: 11 weeks
- Weight at study initiation: 2.2 - 2.5 kg
- Housing: individually in standard aluminum cages
- Diet: LRC4; Oriental Yeast Co. Ltd., Tokyo, Japan, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days prior to the start of the experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 °C
- Humidity (%): 40-70%
- Air changes (per hr): ventilation of 15-21 air changes/h
- Photoperiod (hrs dark / hrs light): 12-h light (7:00-19:00)/dark (19:00-7:00) cycle
Vehicle:
other: see remarks
Remarks:
The test substance was mixed with a minimum amount of olive oil to prepare a test material available in liquid form for ocular instillation.
Controls:
yes, concurrent no treatment
Amount / concentration applied:
1% MWNT-7; this amount was the maximum resulting in good contact and uniform distribution to the eye, but no overlow, when instilled into the conjunctival sac. The concentration of the MWCNT-7 was the maximum allowable for administration.
Duration of treatment / exposure:
no details given
Observation period (in vivo):
The responses, including corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were evaluated at 1, 24, 48 and 72 h
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
3
Details on study design:
The eye irritation experiment was conducted according to the OECD Guideline 405 ''Acute Eye Irritation/Corrosion''. Only rabbits with no abnormalities in the anterior eye parts were used. After gently pulling the lower lid away from the eyeball, 0.1 mL of CNT in liquid form was instilled into the conjunctival sac of the left eye of each rabbit. The lids were gently held together for about 1 s in order to prevent loss of the material. The right eye, which remained untreated, served as a control. The initial test was carried using one rabbit. No corrosive effects were observed at 1 h after the ocular instillation of any CNT. The test was then repeated with two additional rabbits to confirm the initial indings. This strategy was used for the test CNTs. The responses, including corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were evaluated at 1, 24, 48 and 72 h according to OECD Guideline 405 (2002b). At 72 h after the ocular instillation, the corneal epithelium was stained using sodium luorescein. Damage to the corneal epithelium was determined according to McDonald and Shadduck (1977) method.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
3
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
not applicable
Other effects:
The corneal epithelium damage was scored according to McDonald and Shadduck (1977): score = 0
Conclusions:
The test item MWCNT-7 was not irritant to eyes.
Executive summary:

The eye irritation potential of MWCNT-7 test material was conducted in accordance to the OECD Guideline 405 ''Acute Eye Irritation/Corrosion'' with Kbl:NZW rabbits. The concentration of the MWCNT-7 was the maximum allowable for administration. No clinical signs or changes in body weight gain were observed in any groups after the instillation of MWCNT-7. Ocular responses, such as corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were not detected in rabbits instilled with MWCNT-7. The score of the corneal epithelium damage was calculated to be 0. The test item MWCNT-7 was not irritant to eyes.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
no details given
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Species:
rabbit
Strain:
other: Kbl:NZW
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Kitayama Labes Co. Ltd. (Ina, Japan)
- Age at study initiation: 11 weeks
- Weight at study initiation: 2.2 - 2.5 kg
- Housing: individually in standard aluminum cages
- Diet: LRC4; Oriental Yeast Co. Ltd., Tokyo, Japan, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days prior to the start of the experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 °C
- Humidity (%): 40-70%
- Air changes (per hr): ventilation of 15-21 air changes/h
- Photoperiod (hrs dark / hrs light): 12-h light (7:00-19:00)/dark (19:00-7:00) cycle
Vehicle:
other: see remarks
Remarks:
The test substance was mixed with a minimum amount of olive oil to prepare a test material available in liquid form for ocular instillation.
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.25% N-MWNT; the amount of CNT was the maximum resulting in good contact and uniform distribution to the eye, but no overlow, when instilled into the conjunctival sac. The concentration of the N-MWCNT was the maximum allowable for administration.
Duration of treatment / exposure:
no details given
Observation period (in vivo):
The responses, including corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were evaluated at 1, 24, 48 and 72 h
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
3
Details on study design:
The eye irritation experiment was conducted according to the OECD Guideline 405 ''Acute Eye Irritation/Corrosion''. Only rabbits with no abnormalities in the anterior eye parts were used. After gently pulling the lower lid away from the eyeball, 0.1 mL of CNT in liquid form was instilled into the conjunctival sac of the left eye of each rabbit. The lids were gently held together for about 1 s in order to prevent loss of the material. The right eye, which remained untreated, served as a control. The initial test was carried using one rabbit. No corrosive effects were observed at 1 h after the ocular instillation of any CNT. The test was then repeated with two additional rabbits to confirm the initial indings. This strategy was used for the test CNTs. The responses, including corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were evaluated at 1, 24, 48 and 72 h according to OECD Guideline 405 (2002b). At 72 h after the ocular instillation, the corneal epithelium was stained using sodium luorescein. Damage to the corneal epithelium was determined according to McDonald and Shadduck (1977) method.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 1h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
48 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: 1h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
48 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: 1h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
48 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 1h
Score:
1
Max. score:
3
Reversibility:
fully reversible
Remarks on result:
probability of weak irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
48 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
48 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
48 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
72 h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: 1h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
48 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
not applicable
Other effects:
The corneal epithelium damage was scored according to McDonald and Shadduck (1977): score = 0
Conclusions:
MWCNT (test item in this study designated as N-MWCNT) is not considered as irritating to rabbit eyes.
Executive summary:

The eye irritation potential of N-MWCNT test material was conducted in accordance to the OECD Guideline 405 ''Acute Eye Irritation/Corrosion'' with Kbl:NZW rabbits. The concentration of N-MWCNT was the maximum allowable for administration. No clinical signs or changes in body weight gain were observed in any groups after the instillation of N-MWCNT. No rabbits exhibited corneal opacity, abnormality of the iris, or chemosis eye at any time point after the instillation of N-MWCNTs. Although hyperanemia of some blood vessels in the conjunctiva was observed in three rabbits at one timepoint after the instillation of N-MWCNTs, the observed slight effects were fully reversible within 48 h. The score of the corneal epithelium damage was calculated to be 0. The test item N-MWCNT was not irritant to eyes.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
no details given
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Species:
rabbit
Strain:
other: Kbl:NZW
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Kitayama Labes Co. Ltd. (Ina, Japan)
- Age at study initiation: 11 weeks
- Weight at study initiation: 2.2 - 2.5 kg
- Housing: individually in standard aluminum cages
- Diet: LRC4; Oriental Yeast Co. Ltd., Tokyo, Japan, ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 days prior to the start of the experiment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0-25.0 °C
- Humidity (%): 40-70%
- Air changes (per hr): ventilation of 15-21 air changes/h
- Photoperiod (hrs dark / hrs light): 12-h light (7:00-19:00)/dark (19:00-7:00) cycle
Vehicle:
other: olive oil
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1% N-SWCNTs, the amount of CNT was the maximum resulting in good contact and uniform distribution to the eye, but no overlow, when instilled into the conjunctival sac. The concentration of the N-MWCNT was the maximum allowable for administration.
Duration of treatment / exposure:
no details given
Observation period (in vivo):
The responses, including corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were evaluated at 1, 24, 48 and 72 h
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
3
Details on study design:
The eye irritation experiment was conducted according to the OECD Guideline 405 ''Acute Eye Irritation/Corrosion''. Only rabbits with no abnormalities in the anterior eye parts were used. After gently pulling the lower lid away from the eyeball, 0.1 mL of CNT in liquid form was instilled into the conjunctival sac of the left eye of each rabbit. The lids were gently held together for about 1 s in order to prevent loss of the material. The right eye, which remained untreated, served as a control. The initial test was carried using one rabbit. No corrosive effects were observed at 1 h after the ocular instillation of any CNT. The test was then repeated with two additional rabbits to confirm the initial indings. This strategy was used for the test CNTs. The responses, including corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were evaluated at 1, 24, 48 and 72 h according to OECD Guideline 405 (2002b). At 72 h after the ocular instillation, the corneal epithelium was stained using sodium luorescein. Damage to the corneal epithelium was determined according to McDonald and Shadduck (1977) method.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
3
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
other: 1h, 24h, 48h, 72h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
not applicable
Other effects:
The corneal epithelium damage was scored according to McDonald and Shadduck (1977): score = 0
Conclusions:
SWCNTs were not irritants to eyes.
Executive summary:

The eye irritation potential of SWCNT test material was conducted in accordance to the OECD Guideline 405 ''Acute Eye Irritation/Corrosion'' with Kbl:NZW rabbits. The concentration of the SWCNT was the maximum allowable for administration. No clinical signs or changes in body weight gain were observed in any groups after the instillation of SWCNT. Ocular responses, such as corneal opacity, conjunctival redness, abnormality of the iris, and chemosis, were not detected in rabbits instilled with N-SWCNTs. The score of the corneal epithelium damage was calculated to be 0. SWCNT were considered not irritant to rabbit eyes.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Specific details on test material used for the study:
MWCNT1 (5-8 microns in length with 3-8 nm inside diameter and outside diameter of 140 ± 30 nm; Product No. 659258) was produced by catalytic chemical vapor deposition (CCVD). The MWCNT1 was composed of 99.9 % carbon with small amount (<0.1% Fe) of iron. The manufacturer specification for nanomaterial characterization was confirmed by the following techniques. MWCNT1 characteristics were assessed in the as-synthesized form prior to use for experiments or after dispersion in the vehicle (distilled water) for dosing. Solution characteristics were measured with dynamic light scattering (DLS).
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.0 - 3.0 kg
- Housing: individually in standard stainless steel cages and sterilized paddy husk as bedding. Bedding material was changed daily, whereas water bottles were changed on alternate days.
- Diet (e.g. ad libitum): ad libitum supply of standard rabbit pellet food (supplier: M/s. Amrut Laboratory Animal Feed, Pune)
- Water (e.g. ad libitum): ad libitum supply of UV treated water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle
Vehicle:
not specified
Controls:
yes
Amount / concentration applied:
0.1 g of MWCNT1
Duration of treatment / exposure:
A single dose of MWCNT1 was applied into the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The eyes of the test animals were washed with distilled water at 24 hours following the application of test materials
Observation period (in vivo):
1, 24, 48, 72 h and 5 days following application of the test material.
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
3
Details on study design:
The eyes of the test animals were washed with distilled water at 24 hours following the application of test material. The conjunctiva, iris, and cornea of both treated and control eyes were evaluated and scored according to Draize method at the end of 1, 24, 48, 72 h and 5 days following application of the test materials.
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 1h
Score:
4
Reversibility:
fully reversible within: 5 d
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
24 h
Score:
8
Reversibility:
fully reversible within: 5 d
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
48 h
Score:
ca. 4.7
Reversibility:
fully reversible within: 5 d
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
72 h
Score:
ca. 2.7
Reversibility:
fully reversible
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 96 h
Score:
ca. 1.3
Reversibility:
fully reversible within:
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 5 d
Score:
0
Reversibility:
fully reversible
Irritant / corrosive response data:
Animals exhibited conjunctival redness, chemosis and discharge (score 1 or 2) from 1 hour onwards. All the animals recovered from above ocular lesions on 5th day. Hence MWCNT1 was considered as minimally irritating to the eye of the New Zealand white rabbits. Data in detail see table 2 for MWCNT 1.
Other effects:
no details given
Interpretation of results:
GHS criteria not met
Conclusions:
Animals exhibited conjunctival redness, chemosis and discharge (score 1 or 2) from 1hour onwards. All the animals recovered from above ocular lesions on 5th day. Hence the MWCNT1 was considered as minimally irritating to the eye of the New Zealand white rabbits.
Executive summary:

Acute eye irritation was analyzed in a study performed according to OECD guideline 405. New Zealand white rabbits received doses of 0.1 g of MWCNT1 by application into the conjunctival sac of one eye.

The eyes of the rabbits were examined at 1, 24, 48, 72, 96 hours and 5 days after treating with test material. The maximum mean score for ocular lesions observed was 8.0 for MWCNT1 (Table 2). Animals exhibited conjunctival redness, chemosis and discharge (score 1 or 2) from 1 hour onwards. All the animals recovered from above ocular lesions on 5th day. Hence the MWCNT1 was considered as minimally irritating to the eye of the New Zealand white rabbits.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no details given
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
not specified
Remarks:
no information on GLP compliance available in this publication
Specific details on test material used for the study:
MWCNT 2 (1-10 microns in length with 2-6 nm inside diameter and outside diameter of 10-15 nm; Product No. 677248, purchased from Sigma-Aldrich, USA) was produced by catalytic chemical vapor deposition (CCVD). The MWCNT2 was composed of 99.9 % carbon with small amount (<0.1% Fe) of iron. The manufacturer specification for nanomaterial characterization was confirmed by the following techniques. MWCNT2 characteristics were assessed in the as-synthesized form prior to use for experiments or after dispersion in the vehicle (distilled water) for dosing. Solution characteristics were measured with dynamic light scattering (DLS).
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.0 - 3.0 kg
- Housing: individually in standard stainless steel cages and sterilized paddy husk as bedding. Bedding material was changed daily, whereas water bottles were changed on alternate days.
- Diet (e.g. ad libitum): ad libitum supply of standard rabbit pellet food (supplier: M/s. Amrut Laboratory Animal Feed, Pune)
- Water (e.g. ad libitum): ad libitum supply of UV treated water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle
Vehicle:
not specified
Controls:
yes
Amount / concentration applied:
0.1 g of MWCNT2
Duration of treatment / exposure:
A single dose of MWCNT2 was applied into the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material. The eyes of the test animals were washed with distilled water at 24 hours following the application of test material
Observation period (in vivo):
1, 24, 48, 72 h and 5 days following application of the test material.
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
3
Details on study design:
The eyes of the test animals were washed with distilled water at 24 hours following the application of test material. The conjunctiva, iris, and cornea of both treated and control eyes were evaluated and scored according to Draize method at the end of 1, 24, 48, 72 h and 5 days following application of the test material.
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 1 h
Score:
4.7
Reversibility:
fully reversible within: 5 d
Remarks on result:
other: test material: MWCNT 2
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
24 h
Score:
10
Reversibility:
fully reversible within: 5 d
Remarks on result:
other: test material: MWCNT 2
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
48 h
Score:
6
Reversibility:
fully reversible within: 5 d
Remarks on result:
other: test material: MWCNT 2
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
72 h
Score:
3.3
Reversibility:
fully reversible within: 5 d
Remarks on result:
other: test material: MWCNT 2
Irritation parameter:
maximum mean total score (MMTS)
Basis:
mean
Time point:
other: 5 d
Score:
0
Reversibility:
fully reversible
Remarks on result:
other: test material: MWCNT 2
Irritant / corrosive response data:
Animals exhibited conjunctival redness, chemosis and discharge (score 1 or 2) from 1 hour onwards. All the animals recovered from above ocular lesions on 5th day. Hence MWCNT2 was considered as minimally irritating to the eye of the New Zealand white rabbits. Data in detail see table 2 for MWCNT 2.
Other effects:
no details given
Interpretation of results:
GHS criteria not met
Conclusions:
Animals exhibited conjunctival redness, chemosis and discharge (score 1 or 2) from 1hour onwards. All the animals recovered from above ocular lesions on 5th day. Hence MWCNT2 was considered as minimally irritating to the eye of the New Zealand white rabbits.
Executive summary:

Acute eye irritation was analyzed in a study performed according to OECD guideline 405. New Zealand white rabbits received doses of 0.1 g of MWCNT2 by application into the conjunctival sac of one eye.

The eyes of the rabbits were examined at 1, 24, 48, 72, 96 hours and 5 days after treating with test material. The maximum mean score for ocular lesions observed was 10.0 for MWCNT2 (Table 2). Animals exhibited conjunctival redness, chemosis and discharge (score 1 or 2) from 1 hour onwards. All the animals recovered from above ocular lesions on 5th day. Hence MWCNT2 was considered as minimally irritating to the eye of the New Zealand white rabbits.

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

Additional information

Justification for classification or non-classification

Skin irritation / corrosion:

No skin irritation potential is expected for the test item graphene. Nanomaterials usually do not penetrate the skin as recently summarized e.g. by the ECETOC Nano Task Force (Landsiedel et al., 2017). The assumption of neglectable skin irritation potential of graphene is further supported by in vivo studies on related carbon-based nanomaterials: Multiwalled carbon nanotubes (MWCNT) were found not skin irritating in rabbits (OECD TG 404, Murthy et al., 2011 and Ema et al., 2011; see cross-references), singlewalled carbon nanotubes (SWCNT) were found not skin irritating in rabbits (OECD TG 404, Ema et al., 2011), and fullerene C60 were also not skin irritation in rabbits (OECD TG 404, Ema et al., 2013). In conclusion, classification of graphene as skin irritant according to Regulation (EC) No 1272/2008 is not warranted.

Eye irritation:

No eye irritation potential is expected for the test item graphene based on findings for related carbon-based nanomaterials in several in vivo eye irritation studies conducted according to OECD 405.

Multiwalled-carbon nanotubes (MWCNT) were evaluated with respect to their eye irritation potential in rabbits in two published studies, whereof one compared two MWCNT test items with different specifications (Ema et al., 2011 and Murthy et al., 2011). Overall, Murthy et al. concluded that the MWCNT were minimally irritating to the rabbit eyes, and effects were fully reversible within 5 days. In contrast to that, Ema et al. did not find an eye irritating effect of both specifications of MWCNT. The carbon-based singlewalled-carbon nanotubes (two specifications) were also not eye irritating in rabbits in a study conducted according to OECD 405 (Ema et al., 2011). In addition, C60 fullerence, another related carbon-based nanomaterial, was not eye irritating in a study conducted according to OECD 405 (Ema et al., 2013). This is additionally supported by a negative eye irritation study (OECD 405) on graphite, consisting of multiple layers of graphene, reported in the disseminated ECHA dossier. In conclusion, classification of graphene as eye irritant according to Regulation (EC) No 1272/2008 is not warranted.