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Graphistrength® C100 is slightly irritating to the skin and irritating to the eyes of rabbits. Graphistrength® C100 induced a pulmonary inflammatory in rats after intratracheal and inhalation exposures.

 

Skin irritation

In vitro study

The three-dimensional human epidermis model (EPISKIN) was used to assess the skin irritation potential of Graphistrength® C100 (Kishore et al., 2009). Skin Ethic RHE is histologically similar to in vivo human epidermis and features a functional permeability barrier, which is one of the functions of viable skin (OECD 431). Ten milligrams of Graphistrength® C100were applied as such onto the tissue inserts, as well as positive (10 µL of 5% SLS) and negative controls (sterile distilled water). After application tissues were exposed for 15 min. The cell viability was assessed by a MTT test, measuring optic density (OD). Percentage cell viability of positive control was 9.18% and the negative control was 99.99%. Cell viability of Graphistrength® C100was 99.68%. Since the mean tissue viability is > 50%, Graphistrength® C100was considered to be non-irritant to RHE.

 

In vivo study

The potential of Graphistrength® C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) to induce skin irritation was evaluated in rabbits according to OECD No. 404 and Good Laboratory Practice Regulations (Pelcot, 2008c). The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit. Since the test item was neither severely irritant nor corrosive on this first animal, it was then applied simultaneously for 4 hours to two other animals. A single dose of 500 mg of the test item in its original form was applied to the closely-clipped skin of one flank. The test item was held in contact with the skin by means of a semi-occlusive dressing. Cutaneous reactions were observed approximately 1 hour, 24, 48 and 72 hours after removal of the dressing and then daily until reversibility of cutaneous reactions (day 8). The mean values of the scores for edema were calculated for each animal. After a 3-minute exposure (one animal), no cutaneous reactions were observed. A grey coloration of the skin was noted from day 1 until day 4. After a 1-hour exposure (one animal), a grey coloration of the skin was noted from day 1 until day 4. This coloration could have masked a possible very slight erythema on day 1. No cutaneous reactions were observed from day 2 until day 4. After a 4-hour exposure (three animals), a grey coloration of the skin was noted in all the animals all over the observation period. This coloration could have masked a possible very slight or well-defined erythema from day 1 until day 2, 3 or 4. A very slight erythema was noted in 1/3 animals on days 4 and 5. A dryness of the skin was noted in 2/3 animals between day 4 and day 7. Due to the coloration of the skin, the mean scores over 24, 48 and 72 hours for each animal were not calculable for erythema. For edema, the mean scores over 24, 48 and 72 hours for each animal were 0.0, 0.0 and 0.0. However, taking into account the possible erythema masked by the coloration, the maximal mean values over 24, 48 and 72 hours for erythema could be: 0.3,1.0and 1.3. Graphistrength® C100 (micronized), induced a skin coloration preventing conclusions about its skin irritation potential. However, taking into account the possible maximal mean values for erythema, the test item Graphistrength® C100 micronized could be considered as slightly irritating.

 

The acute dermal irritation potential of Graphistrength® C100 was determined according to OECD 404 test guideline (Kishore et al, 2009; Murthy et al., 2011). Three females were treated with a dose of 0.5 g Graphistrength® C100moistened with minimum volume of distilled water to prepare a paste which was applied to the clipped skin area on the left side of the experimental animals and the areas were covered with a gauze patch, which was held in place with non-irritating tape. The right untreated side was kept as control areas. The patch was loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period. Initially test was carried out using one animal. At the end of the 4 h exposure period, the residual test substance was removed, using water without altering the existing response or the integrity of the epidermis. No dermal reactions were observed at 60 min and 24h after patch removal.Since the animal in the initial test did not exhibit any dermal reaction, the test was repeated with two additional animals to confirm the initial test findings. The dermal responses (erythema and eschar formation, edema) were scored at 60 min, and then at 24, 48 and 72 h after patch removal. Dermal reactions were graded and recorded according to Draize. The results of the acute dermal irritation study indicated that Graphistrength® C100was non-irritant to the skin of rabbits.

 

Eye irritation

The eye irritation potential of Graphistrength® C100 was evaluated in three in vivo assays (OECD TG #405) and four in vitro assays (OECD TG #437 and 492 and HE-CAM test). In one of the in vivo assays, Graphistrength® C100 was tested grounded to reduce the mean size of the agglomerates from c. a. 400 µm to c. a. 30 µm and to increase the bioavailability.

 

In vitro study

The potential of Graphistrength® C100 (mean agglomerate size 473 µm) to cause corrosion/severe irritation by using the Bovine corneal opacity and permeability (BCOP) Assay was examined in agreement with OECD guideline no. 437 (adopted 7 September 2009) and Good Laboratory Practice Regulations (Sire, 2011). Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and preincubated for 1 hour at 32°C. Three corneas were used for each treated series (test item, positive and negative controls). Before the treatment, a first opacity measurement was performed using an opacitometer. For the treatment, the test item was used in its original form (neat). The test item was tested in a single experiment using a treatment time of 4 hours. At the completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at 32°C. At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then the cornea was observed for opaque spots and other irregularities. No notable opaque spots or irregularities were observed on negative control corneas following the treatment. Residual test item was observed on corneas treated with the test item. The test item In Vitro Irritancy Score (IVIS) was: 0.2. As the test item Graphistrength® C100 (mean agglomerate size 473 µm) induced an IVIS < 55.1, it was not identified as ocular corrosive or severe irritant. According to the current guideline, Graphistrength® C100 should not be classified as an eye irritant.

 

In another BCOP test including histopathology of the bovine corneas (Kolle et al., 2016), Graphistrength C100 (NM-402) did not induce corneal effects.

 

The in vitro eye irritation potential of Graphistrength C100 (NM-402) was investigated in a EpiOcular™ Eye Irritation Test (EpiOcular™-EIT) (Kolle et al., 2016). No eye irritation potential was revealed.

 

An in vitro Hen Egg Chorion Allantoic Membrane (HE­CAM) test was performed on Graphistrength® C100 (Kishore et al, 2009). The CAM of each egg was applied directly with 0.3 ml of the positive/negative control (0.1N NaOH and 1% SDS, and distilled water) and 0.3 mg of Graphistrength® C100. Two eggs for controls and three for test substance were used for each assay. The reactions of haemorrhage, coagulation and lysis on the CAM were observed over a period of 5 min. The time for each reaction was recorded in seconds and an irritation score (IS) was calculated. After treating for 5 min the main reaction (haemorrhage, lysis or coagulation) was scored (0 = no reaction; 1 = slight reaction; 2 = moderate reaction; 3 = severe reaction). Graphistrength® C100 showed no reaction and the mean irritation threshold and irritation score was zero. Graphistrength® C100 revealed non-irritant result in HE-CAM test.

 

In vivo study

The potential of Graphistrength® C100 (mean agglomerate size 473 µm), to induce irritation following a single ocular administration to rabbits was evaluated according to OECD (No. 405, 24th April 2002) and Commission Regulation (EC) (No. 440/2008, B.5, 30 May 2008) guidelines (Gerbeix, 2011). The study was conducted in compliance with the principles of Good Laboratory Practice. Graphistrength® C100, was first administered to a single male New Zealand White rabbit. Since the test item was irritant on the first animal, it was then evaluated in sequential manner on two other animals. The quantity of the test item administered was 100 mg. The test item was introduced into the conjunctival sac of the left eye. The right eye, which remained untreated, served as control. Approximately 24 hours after instillation of the test item, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl). Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions (two animals) or until the end of the observation period (one animal). The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal. Body weight was recorded at the beginning and the end of the observation period. On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

First animal: a marked or severe chemosis was noted from day 1 until day 3, followed by a slight or moderate from day 4 until the end of the observation period (except on day 12 where a marked chemosis was observed). A severe redness of the conjunctiva was noted on days 1 and 2, then a slight or moderate redness of conjunvtiva was observed from day 3 until day 21 (except on days 12 and 13, where a severe redness of the conjunctiva was noted). A clear discharge was noted on days 1, 3 and 4 and then from day 11 until day 14. Scoring of iris lesions and corneal opacity was masked on day 1 by residual test item. Iris lesions (grade 1) were noted on days 2 and 11. A marked opacity of cornea, which covered the whole area of the cornea on day 2, was observed from day 2 until day 4. Then, a slight or moderate opacity of the cornea was noted from day 5 until day 21. Neovascularisation was noted from day 4 until day 7 and from day 11 until day 15.

Second animal: a marked chemosis was noted on days 1 and 2, followed by a slight or moderate chemosis from day 3 until day 7. On day 1, a moderate redness of the conjunctiva was observed, followed by a severe redness of the conjunctiva on day 2. Then, a slight or moderate redness of the conjunctiva was noted from day 3 until day 7. A clear discharge was observed on days 1 and 3. Iris lesions were noted on days 2 and 3. A slight or moderate corneal opacity was noted from day 2 until day 4.

Third animal: a marked or severe chemosis was noted on days 1 and 2. Then, a slight or moderate chemosis was observed from day 3 until day 10. A severe redness of the conjunctiva was noted from day 1 until day 3, followed by a slight or moderate redness of the conjunctiva from day 4 until day 9. A clear discharge was recorded on days 1 and 3. Scoring of iris lesions and corneal opacity was masked on day 1 by residual test item. Iris lesions were noted from day 2 until day 4. A marked opacity of cornea, which covered the whole area of the cornea, was observed on day 2. Then, a slight or moderate opacity of the cornea was noted from day 3 until day 7.

In conclusion, mean scores calculated for each animal over 24, 48 and 72 hours were 3.0, 2.0 and 2.7 for chemosis, 2.3, 2.0 and 2.7 for redness of the conjunctiva, 0.3, 0.7 and 1.0 for iris lesions and 3.0, 1.7 and 2.3 for corneal opacity. Graphistrength® C100 (mean agglomerate size 473 µm) was irritating when administered by ocular route to rabbits.

 

The potential of the test item Graphistrength® C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) to induce ocular irritation was evaluated in rabbits according to OECD No. 405 and Good Laboratory Practice Regulations (Pelcot, 2008b). The test item was first administered to a single male New Zealand White rabbit. Since the test item was irritant on this first animal, but without persistence until the end of the observation period, it was then evaluated sequentially in two other animals. A single dose of the test item in its original form was introduced into the left conjunctival sac. The right eye was not treated and served as control. The eyes were not rinsed after administration of the test item. Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until complete reversibility of the ocular reactions (day 9 at the latest). The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal. Mean scores calculated for each animal over 24, 48 and 72 hours were 2.7, 2.3 and 2.7 for chemosis, 2.0, 1.7 and 2.7 for redness of the conjunctiva, 0.3, 0.3 and 1.3 for iris lesions and 2.0, 1.0 and 2.0 for corneal opacity. Under the experimental conditions of the study, Graphistrength® C100 (micronized) was irritating when administered by ocular route to rabbits.

 

The acute ocular irritation potential of Graphistrength® C100 was determined according to OECD 405 test guideline (Kishore et al., 2009; Murthy et al., 2011). The conjunctiva, iris, and cornea of both treated and control eyes were evaluated and scored according to Draize method (expressed as overall irritation score over 110) at the end of 1, 24, 48, and 72 h following application of the test materials. Since animals exhibited ocular lesions the observation period was extended until the lesions disappeared and then euthanized using carbon dioxide. The eyes of the rabbits were examined at 1, 24, 48, 72 and 96h after treating with test material. The maximum mean score for ocular lesions observed was 10 at 48 hours. Animals exhibited conjunctival redness and discharge (score 1or 2) from 1 h onwards. All the animals recovered from above ocular lesions at 96h. Graphistrength® C100was considered as slightly irritating to the eye of the New Zealand white rabbits.

 

In conclusion, slight to moderate eye irritation was observed in three in vivo assays with ground or unground Graphistrength® C100. Corneal opacity was observed in all animals in 2 tests (Pelcot 2008b; Gerbeix, 2011), but in none animals in the Kishore et al. (2009) study. However, no corneal opacification was observed in a Bovine corneal opacity and permeability (BCOP) assay with the ungroung Graphistrength® C100 (Sire, 2011; Kolle et al, 2016) and the HE-CAM test (Kishore et al., 2009) and the EpiOcular™ Eye Irritation Test (Kolle et al., 2016) revealed no irritant effect, both indicating that the corneal opacification, and probably the other lesions (conjunctive and iris), didn’t result from a cytotoxic effect but were secondary to an abrasive (mechanical) action. In one animal tested with the unground Graphistrength® C100, a slight chemosis (grade 1) was still observed at the end of the observation period on day 22, whereas the corneal lesion was fully reversible. Considering that in the five other rabbits tested the lesions were fully reversible between 8 and 11 days, it can be expected that a full reversibility will be observed in this animal a few days after the end of the observation period. Therefore, Graphistrength® C100 will be classified as an eye irritant according to CLP (category 2, H319) criteria.

 

Respiratory tract irritation

According to the Guidance on the Application of Regulation (EC) No 1272/2008, the assessment for classification STOT SE is based on the respective criteria together with available adequate and robust test data/information. Generally, information relevant to STOT-SE respiratory tract irritation (RTI) can be obtained from human experience or acute toxicity studies in animals. As no human data is available, the standard animal studies that provide this information are acute and repeated inhalation toxicity (Annex 1: 3.8.2.2.1 of the Guidance) studies which can include clinical observations and detailed macroscopic and microscopic examination to enable the toxic effects on target tissues/organs to be identified. The generic term RTI covers two different effects: “sensory irritation” and “local cytotoxic effects”. Classification in STOT-SE Category 3 for respiratory tract irritation is generally limited to local cytotoxic effects.

 

No acute inhalation study is available on Graphistrength™ C100, information on the respiratory tract effects of Graphistrength™ C100 are available from a 5-d (Schuler, 2010) and 90-day (Pothmann, 2015) inhalation toxicity study (see section Repeated dose toxicity). Supporting information is also provided by acute intratracheal/intranasal administration studies (Tabetet al., 2011; Ronzaniet al., 2012; Viettyet al., 2013; see section 2.4.) and in vitro cytotoxicity assays (Tabetet al., 2009; see section 9.).

 

Inhalation exposure

See section Repeated dose toxicity (Schuler, 2010; Broich, 2016).

 

Intratracheal administration

The pulmonary response of mice was evaluated after exposure to Graphistrength® C100 (Tabet et al., 2011). Graphistrength® C100 suspension in sterile Dulbecco’s modified Eagle’s medium (DMEM) was introduced into mice lungs by intratracheal administration. Male Balb/C mice were treated with either 10 or 100 µg of Graphistrength® C100. Mice were sacrificed at 1, 7, 30, 90 or 180 days post-instillation. Bronchoalveolar Lavage (BAL) and lung were collected. The percentage of macrophages having internalized the different CNT was analyzed. Quantification of the mRNA expression of different genes involved in oxidative stress (SOD-2 and HO-1) and inflammation (TNF-aand MIP-2) was performed by quantitative RT-PCR. In addition, expression of collagen-1 and -3 was analyzed, as markers of interstitial fibrosis. Lung histological analysis was performed in a subset of animals different from that in which BAL analysis and lung gene expression were analyzed.

The results showed significant cellular influx by a single exposure to Graphistrength® C100. Yields of total cells and the number of neutrophils in BAL cells were significantly elevated in MWCNTs-treated mice post-treatment day 1. Histological analysis of the lungs at 1 day after instillation demonstrated the presence of micrometric agglomerates of Graphistrength® C100 at the level of the distal bronchi and the alveolar wall. The agglomerates were preferentially located in the distal bronchi. The late lung response resulted in infiltration and encasement by macrophages to form a connective tissue rich granulomatous inflammation which is typical of the lungs response to an insoluble particle. These lesions were observed from 3 months after instillation and persisted at 6 months post-instillation. No fibrosis was observed. mRNA expression of various genes implicated in oxidative stress, inflammation and fibrosis was quantified in lung homogenates. No modification in the expression of these genes was observed in animals exposed to Graphistrength® C100. No modification of GPX-1, SOD-1 or TGF-ß mRNA expression was observed.

In conclusion, intratracheal instillation of Graphistrength® C100 to mice induced an acute cellular and a persistent granulomatous inflammation in lungs.

 

In vitro cytotoxicity assays

The adverse effects of Graphistrength® C100 were evaluated on the human epithelial cell line A549 (Tabet et al., 2009). MWCNTs were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in two other media: ethanol (EtOH) and phosphate-buffered saline (PBS). Effects of MWCNTs were also compared to those of two asbestos fibres (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells but also in mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. Graphistrength® C100 formed agglomerates on top of both cell lines (surface area 15-35 µm²) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 µg/ml Graphistrength® C100 induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither Graphistrength® C100 cellular internalization nor oxidative stress was observed. In contrast, asbestos fibres penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects.

Incubation with 100 µg/ml Graphistrength® C100 induced a decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither Graphistrength® C100 cellular internalization nor oxidative stress was observed. This study demonstrates that Graphistrength® C100 exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines.

 

In conclusion, five- and 90–day inhalation toxicity studies and single intratracheal/intranasal studies have demonstrated that the respiratory tract is the target organ after exposure to Graphistrength™ C100. If the clearance capacity of the lung are not overloaded, the inflammatory effect is reversible. Therefore, Graphistrength™ C100 is classified as a respiratory tract irritant, STOT-SE category 3 (H335) according to CLP.

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:
key study
Study period:
july 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 404)
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Grimaud frères selection S.A.S., La Corbière, Roussay, France
- Age at study initiation:2 to 4 months old
- Weight at study initiation:2.5 +/- 0.1 Kg
- Housing:individually in Techniplast (64 cm x 63 cm x 30 cm) or Pajon (50 cm x 57 cm x 75 cm) cages.
- Diet :110C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum.
Food is analyzed regularly by the supplier for composition and contaminant levels.
- Water :filtered by a FG Millipore membrane (0.22 micron), ad libitum.
Bacteriological and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and
nitrosamines).
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18 +/- 3
- Humidity (%):30 to 70
- Air changes (per hr):approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light):12h/12h

Type of coverage:
semiocclusive
Preparation of test site:
other: clipped using electric clippers
Vehicle:
unchanged (no vehicle)
Controls:
other: The untreated skin served as control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):Doses of 500 mg of the test item in its original form
Duration of treatment / exposure:
The durations of exposure were 3 minutes, 1 hour and 4 hours, for the first single animal.
Since the test item was neither severely irritant nor corrosive on this first animal, it was then applied simultaneously for 4 hours to two other animals.
Observation period:
The skin was examined approximately 1 hour, 24, 48 and 72 hours after removal of the dressing. Since there were persistent irritation reactions at 72 hours, the observation period was extended up to their complete reversibility (day 8).
Number of animals:
The test item was first evaluated on a single animal; since the test item was neither severely irritant nor corrosive on this first animal, it was then applied simultaneously to two other animals.
Details on study design:
TEST SITE
- Area of exposure: the anterior left flank (application for 3 minutes), the anterior right flank (application for 1 hour) or the posterior right flank (application for 4 hours) of the animals.
- coverage:approximately 6 cm2
- Type of wrap if used:The test item in its original form were placed on a gauze pad moistened with purified water.The gauze pad was held in contact with the skin by means of an adhesive hypoallergenic aerated semi-occlusive dressing and a restraining bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done):After removal of the dressing, any residual test item was wiped off by means of a dry cotton pad or a cotton pad moistened with acetone then with water.
- Time after start of exposure:3 minutes, 1 hour, 4 hours

SCORING SYSTEM:Draize scale:
-Erythema and eschar formation
-Edema formation
Irritation parameter:
edema score
Basis:
animal: #1, 2 and 3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: 4 hours exposure
Irritation parameter:
erythema score
Basis:
animal: # 1, 2 and 3
Time point:
24/48/72 h
Remarks on result:
other: Not calculated due to the coloration of the skin
Irritant / corrosive response data:
After a 3-minute exposure (one animal): No cutaneous reactions were observed.
A grey coloration of the skin was noted from day 1 until day 4.

After a 1-hour exposure (one animal):
A grey coloration of the skin was noted from day 1 until day 4. This coloration could have masked a possible very slight erythema (grade 1) on day 1. No cutaneous reactions were observed from day 2 until day 4.

After a 4-hour exposure (three animals):
A grey coloration of the skin was noted in all the animals all over the observation period. This coloration could have masked a possible very slight or well-defined erythema (grade 1 or 2) from day 1 until day 2, 3 or 4.
A very slight erythema (grade 1) was noted in 1/3 animals on days 4 and 5. A dryness of the skin was noted in 2/3 animals between day 4 and day 7.

Due to the coloration of the skin, the mean scores over 24, 48 and 72 hours for each animal were not calculable for erythema. For edema, the mean scores over 24, 48 and 72 hours for each animal were 0.0, 0.0 and 0.0.
However, taking into account the possible erythema masked by the coloration, the maximal mean values over 24, 48 and 72 hours for erythema could be: 0.3, 1.0 and 1.3.

Interpretation of results:
GHS criteria not met
Conclusions:
When applied topically to rabbits, GRAPHISTRENGTH C100 micronised, induced a skin coloration preventing conclusions about its skin irritation potential. However, taking into account the possible maximal mean values for erythema, the test item GRAPHISTRENGTH C100 micronised could be slightly irritant.
However, taking into account the possible maximal mean values for erythema, the test item GRAPHISTRENGTH C100 micronised could be slightly irritant.
Executive summary:

The potential of the test item GRAPHISTRENGTH C100 micronised to induce skin irritation was evaluated in rabbits according to OECD No. 404 and Good Laboratory Practice Regulations. The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit. Since the test item was neither severely irritant nor corrosive on this first animal, it was then applied simultaneously for 4 hours to two other animals. A single dose of 500 mg of the test item in its original form was applied to the closely-clipped skin of one flank. The test item was held in contact with the skin by means of a semi-occlusive dressing. Cutaneous reactions were observed approximately 1 hour, 24, 48 and 72 hours after removal of the dressing and then daily until reversibility of cutaneous reactions (day 8). The mean values of the scores for edema were calculated for each animal.

After a 3-minute exposure (one animal):

No cutaneous reactions were observed. A grey coloration of the skin was noted from day 1 until day 4.

After a 1-hour exposure (one animal):

A grey coloration of the skin was noted from day 1 until day 4. This coloration could have masked a possible very slight erythema on day 1. No cutaneous reactions were observed from day 2 until day 4.

After a 4-hour exposure (three animals)

A grey coloration of the skin was noted in all the animals all over the observation period. This coloration could have masked a possible very slight or well-defined erythema from day 1 until day 2, 3 or 4.

A very slight erythema was noted in 1/3 animals on days 4 and 5. A dryness of the skin was noted in 2/3 animals between day 4 and day 7.

Due to the coloration of the skin, the mean scores over 24, 48 and 72 hours for each animal were not calculable for erythema. For edema, the mean scores over 24, 48 and 72 hours for each animal were 0.0, 0.0 and 0.0. However, taking into account the possible erythema masked by the coloration, the maximal mean values over 24, 48 and 72 hours for erythema could be: 0.3, 1.0 and 1.3.

The test item GRAPHISTRENGTH C100 micronised, induced a skin coloration preventing conclusions about its skin irritation potential. However, taking into account the possible maximal mean values for erythema, the test item GRAPHISTRENGTH C100 micronised could be slightly irritant.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: House IIBAT animal house facility
All procedures using animals were reviewed and approved by the Institution! Animal Ethics Committee.

- nulliparous and non-pregnant
- Age at study initiation: no data
- Weight at study initiation: 2-3 kg
- Housing: housed 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: ad libitum standard rabbis pellet food
- Water: ad libitum supply of UV treated water.
The feed and water were analyzed to ensure that they did not contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: for minimum period of five days in the controlled environment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+/3
- Humidity (%): 50+/-20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:
semiocclusive
Preparation of test site:
other: clipped skin area
Vehicle:
water
Controls:
other: right untreated side was kept as control area
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
0.5 g on area of 6 cm2

VEHICLE
- Amount(s) applied (volume or weight with unit):
distilled water
Duration of treatment / exposure:
4 h exposure period
Observation period:
The dermal responses (erythema and eschar formation, edema) were scored at 60 min, and then at 24,-48 and 72 h after patch removal.
Number of animals:
3 (Since the animal in the initial test did not exhibit any dermal reaction, the test was repeated with two additionl animals to confirm the initial test findings)
Details on study design:
TEST SITE
- Area of exposure: 6 cm2, on both sides of the dorsal surface of the trunk of the animals
- % coverage: no data
- Type of wrap if used: The areas were covered with a gauze patch, which was held in place with non-irritating tape.
The patch was loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period.

Care was taken to avoid abrading the skin, and only animals with healthy, intact skin were used.
Restrainer (neck coller) was used to prevent the ingestion of the test substance from the application site.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, using water without altering the existing response or the integrity of the epidermis.
- Time after start of exposure: 4 h

SCORING SYSTEM: Dermal reactions were graded and recorded according to Draize:
erythema and eschar formation, edema
Irritation parameter:
erythema score
Basis:
other: all animals
Time point:
other: 24-48-72hrs
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
other: all animals
Time point:
other: 24-48-72hrs
Score:
0
Max. score:
4
Irritant / corrosive response data:
None of the rabbits treated showed any dermal reactions at 60 min, 24, 48 and 72 h after patch removal.
Other effects:
None of the animals showed clinical signs of toxicity. Body weight gain of the individual animal was normal during the study period.
Interpretation of results:
GHS criteria not met
Conclusions:
No dermal reactions like erythema and oedema were observed in rabbits. Hence MWCNT 2 is non-irritating to skin of rabbits.
Executive summary:

The acute dermal irritation potential of MWCNT 2 (Graphistrength® C100: 1-10 µm in length, with 2-6 nm inside diameter and outside diameter of 10-15 nm) was determined according to OECD 404 test guideline (Kishore et al, 2009; Murthy etal., 2011). Three females were treated with a dose of 0.5 g MWCNT 2 moistened with minimum volume of distilledwater to prepare a paste which was applied to the clipped skin area on the left side of the experimental animals and the areas were covered with a gauze patch, which was held in place with non-irritating tape. The right untreated side was kept as control areas. The patch was loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period. Initially test was carried out using one animal. At the end of the 4 h exposure period, the residual test substance was removed, using water without altering the existing response or the integrity of the epidermis. No dermal reactions were observed at 60 min and 24h after patch removal.Since the animal in the initial test did not exhibit any dermal reaction, the test was repeated with two additional animals to confirm the initial test findings. The dermal responses (erythema and eschar formation, edema) were scored at 60 min, and then at 24, 48 and 72 h after patch removal. Dermal reactions were graded and recorded according to Draize. The results of the acute dermal irritation study indicated that MWCNT 2 wasnon-irritant to the skin of rabbits.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
september 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
yes
Remarks:
15 min of exposure, positive control: 5 % SLS
GLP compliance:
not specified
Remarks:
data published
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg of the MWCNT 2 were applied as such onto the tissue inserts



Duration of treatment / exposure:
15 min
Observation period:
immediately
Details on study design:
The three-dimensional human epidermis model (EPISKIN-SMtm) used in this study was, procured from EPISKIN SNC Lyon, France. Adult human derived epidermal keratinocytes were seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen.A highly differentiated and stratified epidermis model was obtained after culturing on air-liquid interface for 13-dey period comprising the main basal, supra basal, spinous and granulat layers and a functional stratum corneum.
Skin Ethic RHE is histologically similar to in vivo human epidermis and features a functional permeability barrier, which is one of the functions of viable skin.
The Skin Ethic human tissue models and media were manufactured in accordante to ISO 9001.

Skin Ethic RHE tissues were shipped and soon after the arrival, test was performed. Upon receipt, the tissues were kept at room temperature (18-22°C) on transport medium (agarose) until use. On the day of testing, tissues were removed from transport agarose medium, transferred to growth medium (3 ml/well) and incubated for 24h at 37°C, in a 5 % C02 atmosphere. Ten milligrams of test chemicals were applied as well as positive (10 µL of 5% SLS) and negative controls (sterile distilled water).
After application tissues were exposed for 15 min.
Subsequently, the treatment was stopped by adding D-PBS.
The tissue inserts were washed with DPBS at least 20 Limes.
Tissues were transferred to fresh growth medium (3 mL/well) and incubated for 42h at (37°C, 5% C02, humidified atmosphere). Tissues were transferred to wells (2 mL/well) contalning MTT of 0.3 mg/mL of growth medium and incubated for 3 h at (37°C, 5% CO2, humidified atmosphere).
After incubation, the tissues were separated and placed in tubes (one tissue/tube) containing acidic isopropanol (500 µL/tube). These tubes were incubated for 4 h in dark with periodic vortexing.
At the end of the incubation period the tubes were re-vortexed to get a homogenous color. Then per tissue 2x 200 µL of sample/well (equal to 2 wells per tissue) from each tube were transferred to 96-well flat bottom microtitre plate and absorbante was read at 570 nm with acidified isopropanol as blank without using a reference filter.

Irritation / corrosion parameter:
other: other: % viability (MTT test)
Value:
99.68
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Remarks: MWCNT 2 (Graphistrength® C100). (migrated information)
Irritation / corrosion parameter:
other: other: % viability (MTT test)
Value:
99.99
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Remarks: Negative control. (migrated information)
Irritation / corrosion parameter:
other: other: % viability (MTT test)
Value:
9.18
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Remarks: Positive control: 5% SLS. (migrated information)
Interpretation of results:
GHS criteria not met
Conclusions:
Percentage cell viability of positive control was 9.18% and the negative control was 99.99%. Cell viability of MWCNT2 was 99.68%. Since the mean tissue viability is > 50%, MWCNT 2 was considered to be non-irritant to RHE.WCNT 1 and 2 were considered to be non-corrosive to RHE.
Executive summary:

The three-dimensional human epidermis model (EPISKIN) was used to assess the skin irritation potential of MWCNT 2 (Graphistrength® C100, 1-10 µm in length, with 2-6 nm inside diameter and outside diameter of 10-15 nm ) (Kishore et al., 2009). Skin Ethic RHE is histologically similar to in vivo human epidermis and features a functional permeability barrier, which is one of the functions of viable skin (OECD,431). Ten milligrams of MWCNT 2 were applied as such onto the tissue inserts, as well as positive (10 µL of 5% SLS) and negative controls (sterile distilled water). After application tissues were exposed for 15 min. The cell viability was assessed by a MTT test, measuring optic density (OD). Percentage cell viability of positive control was 9.18% and the negative control was 99.99%. Cell viability of MWCNT2 was 99.68%. Since the mean tissue viability is > 50%, MWCNT 2 (Graphistrength® C100)

was considered to be non-irritant to RHE.

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:
March-June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Adequate coherence between data and conclusions. GLP and guideline compliant.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Remarks:
deviations from agreed study plan but not from the guideline
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand Black
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Hypharm, La Corbière, Roussay, France
- Age at study initiation: 2 to 4 months
- Weight at study initiation: 3.1 kg ± 0.3 kg
- Housing: individually housed in Pajon cages (50 cm x 57 cm x 75 cm)
- Diet (e.g. ad libitum): free access to breeding pelleted diet "type 110C"
- Water (e.g. ad libitum): free access to tap water (filtered using a 0.22 µm filter)
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 15 March 2011 To 10 April 2011
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
single; around 24h before rinsing of the eye
Observation period (in vivo):
1 hour, 24, 48 and 72 hours and up to day 22
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): at 24h

SCORING SYSTEM:

Conjunctival Chemosis (lids and/or nictitating membranes)
. no swelling ...................................................................................................................................0
. any swelling above normal (includes nictitating membranes) .............................................1
. obvious swelling with partial eversion of lids..........................................................................2
. swelling with lids about half-closed..........................................................................................3
. swelling with lids more than half-closed .................................................................................4
Conjunctival Redness (refers to palpebral and bulbar conjunctivae, cornea and iris)
. blood vessels normal ..................................................................................................................0
. a number of blood vessels definitely hyperemic (injected)...................................................1
. diffuse, crimson colour, individual vessels not easily discernible ......................................2
. diffuse, beefy red..........................................................................................................................3
Conjunctival Discharge
. absence of discharge ..................................................................................................................0
. slight discharge (does not include small amounts normally found in
inner canthus) ................................................................................................................................1
. discharge with moistening of lids and hairs adjacent to lids................................................2
. discharge with moistening of lids and hairs on wide area around the eye.........................3
Iris lesions
. normal ...........................................................................................................................................0
. markedly deepened rugae, congestion, swelling, moderate circum-corneal
hyperemia, or injection, any of these or combination of any thereof, iris still
reacting to light (sluggish reaction is positive) ........................................................................1
. no reaction to light, haemorrhage, gross destruction (any or all of these) .......................2
Cornea Opacity (degree of intensity: area most dense taken for reading)
. no ulceration or opacity..............................................................................................................0
. scattered or diffuse areas of opacity (other than slight dulling or normal lustre),
details of iris clearly visible ..........................................................................................................1
. easily discernible translucent area, details of iris slightly obscured ...................................2
. nacreous areas, no details of iris visible, size of pupil barely discernible ..........................3
. opaque cornea, iris not discernible through the opacity.......................................................4
Cornea Area of opacity
. one quarter (or less) but not zero..............................................................................................1
. greater than one quarter but less than a half...........................................................................2
. greater than one half but less than three quarters..................................................................3
. greater than three quarters up to whole area. .........................................................................4

TOOL USED TO ASSESS SCORE: hand-slit lamp + fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
fully reversible within: 22 days
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
fully reversible within: 5 days
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
fully reversible within: 8 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not fully reversible within: 22 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 8 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
fully reversible within: 11 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.3
Max. score:
3
Reversibility:
fully reversible within: 22 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 8 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
fully reversible within: 10 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 5 days
Irritant / corrosive response data:
First animal
A marked or severe chemosis was noted from day 1 until day 3, followed by a slight or moderate from day 4 until the end of the observation period (except on day 12 where a marked chemosis was observed).
A severe redness of the conjunctiva was noted on days 1 and 2, then a slight or moderate redness of conjunvtiva was observed from day 3 until day 21 (except on days 12 and 13, where a severe redness of the conjunctiva was noted).
A clear discharge was noted on days 1, 3 and 4 and then from day 11 until day 14.
Scoring of iris lesions and corneal opacity was masked on day 1 by residual test item.
Iris lesions (grade 1) were noted on days 2 and 11.
A marked opacity of cornea, which covered the whole area of the cornea on day 2, was observed from day 2 until day 4. Then, a slight or moderate opacity of the cornea was noted from day 5 until day 21.
Neovascularisation was noted from day 4 until day 7 and from day 11 until day 15.
On day 22, when the animal was sacrificed, only a slight chemosis (grade 1) was still observed indicating a trend to total reversibility.

Second animal
A marked chemosis was noted on days 1 and 2, followed by a slight or moderate chemosis from day 3 until day 7. On day 1, a moderate redness of the conjunctiva was observed, followed by a severe redness of the conjunctiva on day 2. Then, a slight or moderate redness of the conjunctiva was noted from day 3 until day 7. A clear discharge was observed on days 1 and 3.
Iris lesions were noted on days 2 and 3.
A slight or moderate corneal opacity was noted from day 2 until day 4.

Third animal
A marked or severe chemosis was noted on days 1 and 2. Then, a slight or moderate chemosis was observed from day 3 until day 10. A severe redness of the conjunctiva was noted from day 1 until day 3, followed by a slight or moderate redness of the conjunctiva from day 4 until day 9. A clear discharge was recorded on days 1 and 3.
Scoring of iris lesions and corneal opacity was masked on day 1 by residual test item.
Iris lesions were noted from day 2 until day 4.
A marked opacity of cornea, which covered the whole area of the cornea, was observed on day 2. Then, a slight or moderate opacity of the cornea was noted from day 3 until day 7.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Mean scores calculated for each animal over 24, 48 and 72 hours were 3.0, 2.0 and 2.7 for chemosis, 2.3, 2.0 and 2.7 for redness of the conjunctiva, 0.3, 0.7 and 1.0 for iris lesions and 3.0, 1.7 and 2.3 for corneal opacity.
Executive summary:

The potential of the test item, Graphistrenght C100, to induce irritation following a single ocular administration to rabbits was evaluated according to OECD (No. 405, 24th April 2002) andCommission Regulation (EC) (No. 440/2008, B.5, 30 May 2008)guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice.

 

Graphistrength C100, was first administered to a single male New Zealand White rabbit. Since the test item was irritant on the first animal, it was then evaluated in sequential manner on two other animals. The quantity of the test item administered was 100 mg. The test item was introduced into the conjunctival sac of the left eye. The right eye, which remained untreated, served as control. Approximately 24 hours after instillation of the test item, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl). Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions (two animals) or until the end of the observation period (one animal). The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal. Body weight was recorded at the beginning and the end of the observation period. On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

 

First animal

A marked or severe chemosis was noted from day 1 until day 3, followed by a slight or moderate from day 4 until the end of the observation period (except on day 12 where a marked chemosis was observed). A severe redness of the conjunctiva was noted on days 1 and 2, then a slight or moderate redness of conjunvtiva was observed from day 3 until day 21 (except on days 12 and 13, where a severe redness of the conjunctiva was noted). A clear discharge was noted on days 1, 3 and 4 and then from day 11 until day 14. Scoring of iris lesions and corneal opacity was masked on day 1 by residual test item. Iris lesions (grade 1) were noted on days 2 and 11. A marked opacity of cornea, which covered the whole area of the cornea on day 2, was observed from day 2 until day 4. Then, a slight or moderate opacity of the cornea was noted from day 5 until day 21. Neovascularisation was noted from day 4 until day 7 and from day 11 until day 15.

 

Second animal

A marked chemosis was noted on days 1 and 2, followed by a slight or moderate chemosis from day 3 until day 7. On day 1, a moderate redness of the conjunctiva was observed, followed by a severe redness of the conjunctiva on day 2. Then, a slight or moderate redness of the conjunctiva was noted from day 3 until day 7. A clear discharge was observed on days 1 and 3. Iris lesions were noted on days 2 and 3. A slight or moderate corneal opacity was noted from day 2 until day 4.

 

Third animal

A marked or severe chemosis was noted on days 1 and 2. Then, a slight or moderate chemosis was observed from day 3 until day 10. A severe redness of the conjunctiva was noted from day 1 until day 3, followed by a slight or moderate redness of the conjunctiva from day 4 until day 9. A clear discharge was recorded on days 1 and 3. Scoring of iris lesions and corneal opacity was masked on day 1 by residual test item. Iris lesions were noted from day 2 until day 4. A marked opacity of cornea, which covered the whole area of the cornea, was observed on day 2. Then, a slight or moderate opacity of the cornea was noted from day 3 until day 7.

In conclusion, mean scores calculated for each animal over 24, 48 and 72 hours were 3.0, 2.0 and 2.7 for chemosis, 2.3, 2.0 and 2.7 for redness of the conjunctiva, 0.3, 0.7 and 1.0 for iris lesions and 3.0, 1.7 and 2.3 for corneal opacity. Graphistrength C100 was irritating when administered by ocular route to rabbits.

 

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
july 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 405)
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source:Grimaud frères selection S.A.S., La Corbière, Roussey, France
- Age at study initiation:2 to 4 months old
- Weight at study initiation:2.9+/- 0.1 Kg
- Housing:The animals were housed individually in Techniplast (64 cm x 63 cm x 30 cm) or Pajon (50 cm x 57 cm x 75 cm) cages.
- Diet:110C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum.
Food is analyzed regularly by the supplier for composition and contaminant level.
- Water :filtered by a FG Millipore membrane (0.22 micron), ad libitum.
Bacteriological and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18+/-3
- Humidity (%):30 to 70
- Air changes (per hr):approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light):12h/12h

Vehicle:
unchanged (no vehicle)
Controls:
other: right eye as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):the maximal quantity of the test item administrable into the eye was approximately 1/3 of the 100 mg required quantity


Duration of treatment / exposure:
no data
Observation period (in vivo):
The eyes were examined approximately 1 hour, 24, 48 and 72 hours after administration of the test item.
Since there were persistent ocular reactions at 72 hours, the observation period was extended up to their complete reversibility (day 9 at the latest).
Number of animals or in vitro replicates:
The test item was first administered to a single animal. Since the test item was irritant, but without persistence at the end of the observation period, on this first animal, it was then evaluated sequentially on 2 others animals.

Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done):The eyes were not rinsed after administration of the test item.
- Time after start of exposure:no data

SCORING SYSTEM:Draize scale

TOOL USED TO ASSESS SCORE: For the evaluation of corneal opacification (presence or absence, affected area), the eyes were examined under a UV lamp after instillation of one or two drops of 0.5% sodium fluorescein solution (a clear fluorescence is visible in the areas of opacification). This evaluation was performed on day 2 and repeated thereafter whenever necessary.
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
fully reversible within: 9 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 9 days
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
cornea opacity score
Remarks:
intensity
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritation parameter:
conjunctivae score
Remarks:
chemosis
Basis:
animal #2
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
fully reversible within: 8 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
3
Reversibility:
fully reversible within: 6 days
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
cornea opacity score
Remarks:
intensity
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
chemosis
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
fully reversible within: 9 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
fully reversible within: 9 days
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.3
Max. score:
2
Reversibility:
fully reversible within: 5 days
Irritation parameter:
cornea opacity score
Remarks:
intensity
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 6 days
Irritant / corrosive response data:
A slight to marked chemosis (grades 1 to 3) was noted in all the animals from day 1 until day 7 (1/3 animals) or 8 (2/3 animals). A slight to severe redness of the conjunctiva (grades 1 to 3) was observed in all the animals from day 1 until day 5 (1/3 animals) or 8 (2/3 animals). A clear discharge was observed in all the animals from day 1 until day 2, 5 or 6.
An iritis of grade 1 was noted in 2/3 the animals on day 2. In the third animal, an iritis of grade 2 was noted on day 2; an iritis of grade 1 persisted until day 4.
A slight or moderate corneal opacity (grade 1 or 2) was recorded in all the animals on day 2; it persisted until day 3 (1/3 animals) or 5 (2/3 animals). This corneal opacity covered the whole area of the cornea of 1/3 animals on day 2.

Mean scores calculated for each animal over 24, 48 and 72 hours were 2.7, 2.3 and 2.7 for chemosis, 2.0, 1.7 and 2.7 for redness of the conjunctiva, 0.3, 0.3 and 1.3 for iris lesions and 2.0, 1.0 and 2.0 for corneal opacity.
Other effects:
slight increase of the body weight gain, between the day of the treatment and the end of the observation period.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Mean scores calculated for each animal over 24, 48 and 72 hours were 2.7, 2.3 and 2.7 for chemosis, 2.0, 1.7 and 2.7 for redness of the conjunctiva, 0.3, 0.3 and 1.3 for iris lesions and 2.0, 1.0 and 2.0 for corneal opacity. GRAPHISTRENGTH C100 micronised was irritant when administered by ocular route to rabbits.
Executive summary:

The potential of the test item Graphistrength C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) to induce ocular irritation was evaluated in rabbits according to OECD No. 405 and Good Laboratory Practice Regulations.

The test item was first administered to a single male New Zealand White rabbit. Since the test item was irritant on this first animal, but without persistence until the end of the observation period, it was then evaluated sequentially in two other animals. A single dose of the test item in its original form was introduced into the left conjunctival sac. The right eye was not treated and served as control. The eyes were not rinsed afteradministration of the test item.

Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until complete reversibility of the ocular reactions (day 9 at the latest).

The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal.

Mean scores calculated for each animal over 24, 48 and 72 hours were 2.7, 2.3 and 2.7 for chemosis, 2.0, 1.7 and 2.7 for redness of the conjunctiva, 0.3, 0.3 and 1.3 for iris lesions and 2.0, 1.0 and 2.0 for corneal opacity. Graphistrength C100 micronized was irritating when administered by ocular route to rabbits.

 

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
not specified
Remarks:
data published
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: House IIBAT animal house facility
All procedures using animals were reviewed and approved by the Institution! Animal Ethics Committee.

- nulliparous and non-pregnant
- Age at study initiation: no data
- Weight at study initiation: 2-3 kg
- Housing: housed 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: ad libitum standard rabbis pellet food
- Water: ad libitum supply of UV treated water.
The feed and water were analyzed to ensure that they did not contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: for minimum period of five days in the controlled environment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+/3
- Humidity (%): 50+/-20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
water
Controls:
other: right eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 18 mg (equivalent weight to 0.1 ml) of test material (MWCNT 1 and 2)



Duration of treatment / exposure:
no data
Observation period (in vivo):
at 1, 24, 48, 72 and 96 h after treating with test material
Number of animals or in vitro replicates:
3 (Initially, test was carried out using one animal. The responses obtained in the initial test were confirmed with two more additional animals in the confirmatory test.)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: yes
- Time after start of exposure: The eyes of the test animais were washed with distilled water at 24 h following the application of test materials, to remove the presence of residual test substance if any.

SCORING SYSTEM:
The conjunctiva, iris, and cornea of both treated and control eyes were evaluated and scored according to Draize method

TOOL USED TO ASSESS SCORE:
Both eyes of the experimental animals selected for testing were examined with an Ophthalmoscope 24 h prior to starting experiment. None of the animals selected for the study showed any ocular defects or pre-existing cornai injury.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24 h
Score:
10
Max. score:
110
Remarks on result:
other: individual scores are not available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
48 h
Score:
6
Max. score:
110
Remarks on result:
other: individual scores are not available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
72 h
Score:
3.3
Max. score:
110
Remarks on result:
other: individual scores are not available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 96 h
Score:
1.3
Max. score:
110
Remarks on result:
other: individual scores are not available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 5 days
Score:
0
Max. score:
110
Remarks on result:
other: individual scores are not available
Irritant / corrosive response data:
The maximum mean score for ocular lesions observed was 10.0/110. 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.
Interpretation of results:
GHS criteria not met
Conclusions:
MWCNT 2 was considered as minimally irritating to the eye of the New Zealand white rabbits.
Executive summary:

The acute ocular irritation potential of MWCNT 2 (Graphistrength® C100, 1-10 µm in length, with 2-6 nm inside diameter and outside diameter of 10-15 nm) was determined according to OECD 405 test guideline (Kishore et al., 2009; Murthy et al., 2011). The conjunctiva, iris, and cornea of both treated and control eyes were evaluated and scored according to Draize method (expressed as overall irritation score over 110 ) at the end of 1,24, 48, and 72 h following application of the test materials. Since animals exhibited ocular lesions the observation period was extended until the lesions disappeared and then euthanized using carbon dioxide.The eyes of the rabbits were examined at 1, 24, 48, 72 and 96h after treating with test material. The maximum mean score for ocular lesions observed was 10 at 48 hours. Animals exhibited conjunctival redness and discharge (score 1 or 2) from 1 h onwards. All the animals recovered from above ocular lesions at 96h. MWCNT 2 was considered as slightly irritating to the eye of the New Zealand white rabbits.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
GLP compliance:
not specified
Vehicle:
water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
20 % (w/w) dry suspension was prepared mixing 750 mg MWCNT NM-402 in highly de-ionized water shortly before application by stirring with a spatula.
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
none
Number of animals or in vitro replicates:
3 corneas
Details on study design:
SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS: no data

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (BASF-OP2.0, BASF SE, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)
- Others: histopathological evaluation by light microscopy

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The In Vitro Irritancy Score (IVIS) was calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation (SD) was determined: IVIS = mean opacity value + (15 x mean permeability value). An IVIS >55 indicates a risk of serious damage to the eyes.

DECISION CRITERIA:
Acceptance criteria laid down in OECD TG 437
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
-3.9
Vehicle controls validity:
valid
Remarks:
4.9 ± 5.7
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
123.0 ± 17.4
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
-3.8
Vehicle controls validity:
valid
Remarks:
4.7 ± 5.7
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
78.5 ± 16.4
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein leakage
Run / experiment:
Mean
Value:
-0.01
Vehicle controls validity:
valid
Remarks:
0.01 ± 0.02
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
3.0 ± 0.8
Remarks on result:
no indication of irritation
Irritation parameter:
histopathological observations
Value:
0
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Executive summary:

The in vitro eye irritation potential of Graphistrength C100 (NM-402) was investigated in a BCOP test including histopathology of the bovine corneas. No corneal effects were observed.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25 May 2011 - 17 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Adequate coherence between data and conclusions. GLP and guideline compliant.
Qualifier:
according to guideline
Guideline:
other: OECD 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants
Deviations:
no
Remarks:
deviations from study plan but not from guideline
GLP compliance:
yes (incl. QA statement)
Details on test animals or tissues and environmental conditions:
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir (SOCAVIA, Cany Barville, France or SOCAVIA, Beuvillers, France).

Age: bovine cattle were up to 12 months old.

Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

Transport from Supplier to CIT: the eyes were transported to CIT at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

Upon arrival at CIT, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, neovascularization, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.

Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.

Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used immediately or within a maximum of 24 hours.

Storage of the corneas: if the corneas were used immediately, after washing they were stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

Experimental dates: 26 May 2011 - 17 June 2011
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro controls (see above)
Amount / concentration applied:
An amount of 750 mg ± 75 mg was applied on each cornea using the open-chamber method
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
Immediately after treatment
Number of animals or in vitro replicates:
Three corneas were used for each treated series (test item, positive control and negative control).
Details on study design:
Negative control: 0.9% NaCl
Positive control: 20% imidazole solution in 0.9% NaCl (w/v)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed.

TOOL USED TO ASSESS SCORE: opacitometer, fluorescein and spectrophotomter.

OPACITY MEASUREMENT:
- opacity through the center of each mounted cornea
- two opacity measurements (OPT0 before treatment and OPT2 at 4h),
- change in opacity determined by subtracting the initial base-line opacity measurement (OPT0) from the post treatment opacity reading (OPT2).
- Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.
- In Vitro Irritancy Score: IVIS = corrected opacity + (15 x cOD490 nm)

PERMEABILITY MEASUREMENT:
- done after the second opacity measurement
- The concentration of the fluoresceine solution was 5 mg/mL
- from the fluoresceine application time of the first cornea of the series, the holders were incubated in a water bath at 32 °C for 90 minutes.
- At the end of incubation, the cMEM recoverable from the posterior chamber of each holder was subjected to determination of OD490 nm using a spectrophotometer
Irritation parameter:
in vitro irritation score
Run / experiment:
4 hours
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.5
Positive controls validity:
valid
Remarks:
146.7
Irritant / corrosive response data:
No notable opaque spots or irregularities were observed on negative control corneas following the treatment.
Residual test item was observed on corneas treated with the test item.
The individual and mean opacity and permeability values for the test item, positive and negative controls are given in the attached Table.
The test item In Vitro Irritancy Score (IVIS) was: 0.2.
As the test item induced an IVIS < 55.1, it was not identified as ocular corrosive or severe irritant.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item is not considered to be a corrosive/severe irritant substance to the eye.
Executive summary:

The potential of the test item Graphistrength C100 to cause corrosion/severe irritation by using the Bovine corneal opacity and permeability (BCOP) Assay was examined in agreement with OECD guideline no. 437 (adopted 7 September 2009) and Good Laboratory Practice Regulations.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and preincubated for 1 hour at 32°C. Three corneas were used for each treated series (test item, positive and negative controls). Before the treatment, a first opacity measurement was performed using an opacitometer. For the treatment, the test item was used in its original form (neat). The test item was tested in a single experiment using a treatment time of 4 hours. At the completion of the treatment period, the test item was removed from the front opening of the anterior chamber and the epithelium was rinsed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluoresceine solution. The holders were then incubated vertically for 90 minutes at 32°C. At the end of the incubation, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then the cornea was observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on negative control corneas following the treatment. Residual test item was observed on corneas treated with the test item. The test item In Vitro Irritancy Score (IVIS) was: 0.2. As the test item Graphistrength C100 induced an IVIS < 55.1, it was not identified as ocular corrosive or severe irritant.

 

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In vitro Hen Egg Chorion Allantoic Membrane (HE-CAM) test was performed. The CAM of each egg was applied directly with 0.3 ml of the positive/negative control and 0.3 mg of test chemical. Two eggs for controls and three for test substance were used for each assay. The reactions of haemorrhage, coagulation and lysis on the CAM were observed over a period of 5 min. After treating for 5min the main reaction (haemorrhage, lysis or coagulation) was scored.
GLP compliance:
not specified
Remarks:
data published
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.3 mg of test chemical, 0.3 mL of the positive/negative control


Duration of treatment / exposure:
5 min
Observation period (in vivo):
immediately after treatment
Details on study design:
Day 9 old eggs of White Leghorn chicken were obtained from the Poultry Research centre, Chennai.
Each hen egg (on day 9) was tested with candling light to ensure that all were viable. White Leghorn chicken has been selected because the eggs of this breed with no hereditary defects yield very consistent and reproducible results.
Cold lamp was used to ensure optimal illumination of the chorioallantoic membrane. On day 10, the air cell was marked with a pencil, using a rotating dentist-saw blade removed a section of the shell off.
The membrane was carefully moistened with 0.9% NaCl solution at 37°C. The eggs were replaced in the incubator until ready for assay (maximum of 30 min between opening the eggs and commencement of the assay). The moistening solution (0,9% NaCl) was poured off from the opened egg and using a tapered forceps the membrane was carefully removed (without injuring any underlying blood vessels).

The CAM of each egg was applied directly with 0.3 ml of the positive/negative control and 0.3 mg of test chemical. Two eggs for controls and three for test substance were used for each assay. The reactions of hemorrhage, coagulation and lysis on the CAM were observed over a period of 5 min.
The time for each reaction was recorded in seconds and an irritation score (IS) was calculated according to the formula:

IS = 5 x( 301-Sec H/300) + 7 x (301-Sec L/300) + 9 x (301-Sec C/300)

H=hemorrhage; L=.vessel lysis; C=coagulation; Séc=start second.
After treating for 5min the main reaction (hemorrhage, lysis or coagulation) was scored as follows:

0 = no reaction; 1 = slight reaction; 2 = moderate reaction; 3 = severe reaction

Each test was done in triplicate and the mean score of the three eggs was determined.
At the end of each assay the embryos were killed quickly by placing the eggs into a freezer at -20 °C.

Positive controls: 0.1N NaOH and 1% SDS
The mean irritation score for 0.1N NaOH and 1% SDS was 17.03 and 13.52 respectively

Negative control: distilled water, non-specific changes in the test system was observed (IS=0)



Irritation parameter:
in vitro irritation score
Run / experiment:
5 min
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
0
Positive controls validity:
valid
Remarks:
17.03 (0.1 N NaOH), 13.52 (1% SDS)
Interpretation of results:
GHS criteria not met
Conclusions:
MWCNT 2 showed no reaction and the mean irritation threshold and irritation score was zero.
Executive summary:

An in vitro Hen Egg Chorion Allantoic Membrane (HE­CAM) test was performed on Graphistrength® C100 (1-10 µm in length, with 2-6 nm inside diameter and outside diameter of 10-15 nm) (Kishore et al, 2009). The CAM of each egg was applied directly with 0.3 ml of the positive/negative control (0.1N NaOH and 1% SDS, and distilled water) and 0.3 mg of Graphistrength C100. Two eggs for controls and three for test substance were used for each assay. The reactions of haemorrhage, coagulation and lysis on the CAM were observed over a period of 5 min. The time for each reaction was recorded in seconds and an irritation score (IS) was calculated. After treating for 5 min the main reaction (haemorrhage, lysis or coagulation) was scored as follows: 0 = no reaction; 1 = slight reaction; 2 = moderate reaction; 3 = severe reaction. Graphistrength C100 showed no reaction and the mean irritation threshold and irritation score was zero. Graphistrength C100 revealed non-irritant result in HE-CAM test.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
yes
Remarks:
The test protocol differs in respect to the exposure and post-exposure immersion periods laid down for solid test materials.
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
MatTek Corp. (USA) and MatTek In Vitro Life Science Laboratories (Slovakia) provided the test systems: EpiOcular™ OCL-200 kit (containing 24 OCL-200 reconstructed cornea tissues, 0.6 cm2 surface area, cultured in Millicells® with 1-cm diameter).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
8 mg corresponding to 2x 50 µL bulk volume
Duration of treatment / exposure:
90 min
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
Pre-tests to determine direct MTT reduction
Pre-tests as described in the OECD TG 492 were performed that precluded the test materials’ ability to directly reduce MTT.

Main tests
In the main tests, two tissues were treated with either the test materials, the NC or the PC. On the day of arrival in the laboratory, the EpiOcular™ tissues were transferred to sterile 6-well plates with 1 mL DMEM and pre-conditioned at standard culture conditions (37 °C, 5 % CO2, 90–95 % humidity) in the incubator for 16–24 h. After pre-incubation, the tissues were pre-treated with 20 µL PBS and further incubated at standard culture conditions for 30 min. Using a sharp spoon, the dry-powder were applied to cover the entire tissue surface. Control tissues were concurrently exposed to 50 µL highly de-ionized water (NC) or methyl acetate (PC). After test material application, the tissues were placed into the incubator for 90 min.
To remove the test materials, the tissues were washed with sterile PBS and immediately immersed into 12-well plates, pre-filled with 5 mL pre-warmed medium per well to remove test material residuals. After 12 min, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL pre-warmed medium per well (post-exposure immersion). Subsequently, the tissues were incubated at standard culture conditions (post-exposure incubation) for 18 h. During the post-exposure immersion and incubation periods, weak cytotoxic effects might reverse, and more pronounced effects might increase.
Upon completion of the respective post-exposure periods, the assay medium was replaced by 0.3 mL MTT solution. After incubating the tissues for 3 h, the tissues were washed with PBS to terminate the MTT incubation. The produced formazan was extracted by incubating the tissues in isopropanol at room temperature overnight or on a plate shaker for at least 2 h. The optical density of the formazan extracts was determined spectrophotometrically at a wavelength of 570 nm (OD570). For each microtitre plate, blank values were established from 4 wells filled with isopropanol.

Calculation of mean relative tissue viability
Tissue OD570 values were calculated by subtracting the mean blank value of the respective microtitre plate from the measured tissue OD570 value, and mean OD570 values were calculated for the two tissues of each treatment group. The quotient of the mean OD570 values of the test material-treated tissues and those of the NC (i.e. the mean relative tissue viability) was determined to evaluate whether or not a test material is an irritant:
• Mean relative tissue viabilities =60 % indicated ‘irritancy to the eye’;
• Mean relative tissue viabilities >60 % indicated ‘no irritancy to the eye’.

Irritation parameter:
other: % viability of tissue relative to negative control
Run / experiment:
1
Value:
98
Vehicle controls validity:
valid
Remarks:
Mean OD570 = 1.241
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
18%
Irritation parameter:
other: % viability of tissue relative to negative control
Run / experiment:
2
Value:
100
Other effects / acceptance of results:
Acceptance criteria
In case one of the following acceptance criteria (AC) as described in the OECD TG 492 was not met, repetition of the EpiOcular™-EIT was considered.
• AC for the NC: The OD570 of the NC reflects the laboratory-specific tissue viability under the specific conditions of the assay. It was considered acceptable if the mean OD570 of the NC was =0.8 and =2.5 and the historical in-house mean at the respective time of testing was met (variant 1: OD570 of NC for liquids / solids: 1.490¿±¿0.106 / 1.361¿±¿0.138; variant 2: OD570 of NC for solids: 1.650¿±¿0.159).
• AC for the PC: In-house, the PC methyl acetate usually elicits relative tissue viabilities of approx. 25 % (historical in-house means at the time of testing in accordance with variant 1: OD570 of PC for liquids/solids: 0.388¿±¿0.098/0.318¿±¿0.119; variant 2: OD570 of PC for solids: 0.396¿±¿0.098). In addition to these historical means, all relative tissue viability values <50 % were considered acceptable.
• AC for tissue variability: The relative inter-tissue variability (ITV%) between the two tissues of a treatment group was considered acceptable if it was =20 %.

Irritant / corrosive response data:
After washing, test material residues were observed on the EpiOcular™ tissues. However, since NM-402 was able to reduce MTT directly, it was concluded that these residues do not to interfere with the MTT assay.
Interpretation of results:
GHS criteria not met
Executive summary:
The in vitro eye irritation potential of Graphistrength C100 (NM-402) was investigated in a EpiOcular™ Eye Irritation Test (EpiOcular™-EIT). No eye irritation potential was revealed.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification

According to the available data on the Graphistrength C100 and Regulation (EC) No 1272/2008, Annex I:

- No skin irritation classification is warranted for Graphistrength C100,

- Graphistrength C100 should be classified as eye irritant, category 2 (H319),

- Graphistrength C100 should be classified as a respiratory tract irritant, STOT-SE category 3 (H335).