Registration Dossier

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Description of key information

The acute oral and dermal toxicity of Graphistrength® C100 was low with LD0 values exceeding 2000 mg/kg

 

Oral route

The acute oral toxicity of Graphistrength® C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) was evaluated in rats according to OECD n° 423 and Good Laboratory Practice Regulations (Pelcot, 2008a). The test item was prepared in 0.5% methylcellulose and was administrated by oral route (gavage), to groups of three fasted females Sprague-Dawley rats. Clinical signs, mortality and body weight gain were checked for a period of up to 14 days following the single administration of the test item. All animals were subjected to necropsy. No deaths and no clinical signs were noted at 300 mg/kg. No deaths occurred but hypoactivity, piloerection and dyspnea were noted in 3/6 animals on day 1, at 2000 mg/kg. The oral LD0 of Graphistrength® C100 (micronized) was equal to or higher than 2000 mg/kg in rats.

 

Acute oral toxicity –up and down procedure was conducted as per OECD 425 guidelines with slight modifications in terms of usage of sexes and animal number (Murthy et al. 2011). A single dose of Graphistrength® C100 suspended in distilled water was administered by oral gavage to group of rats at a dose of 2000 mg/ kg b.w. The test solution was prepared shortly prior to the administration. The dose volume was maximum 10 ml/kg b. w. Similarly, control group of animals (5 males and 5 females) were dosed with distilled water alone. Animals were observed for mortality/morbidity, clinical signs of toxicity and weekly body weight during the experimental period. Gross pathology was performed at the end of experimental period (day 14). There was no mortality evident in the study. Animals of the treated group showed normal and consistent gain in body weight when compared to the control group of animals. No gross lesions were observed. Based on the above findings, the oral LD0 for Graphistrength® C100 was greater than 2000 mg/kg b. w for Wistar rats.

 

Inhalation route

No study is available to evaluate the acute inhalation toxicity of Graphistrength® C100.

 

Dermal route

The acute dermal toxicity of Graphistrength® C100 (micronized, the mean diameter of the agglomerates was reduced to 30 µm instead 400 µm to increase the bioavailability) was evaluated in rats according to OECD 402 and Good Laboratory Practice Regulations (Pelcot, 2008b). The test item was applied to the skin of one group of ten Sprague-Dawley rats (five males and five females). The application was performed with the test item in its original form at the dose-level of 2000 mg/kg. The test site was then covered by a semi-occlusive dressing for 24 hours. Clinical signs, mortality and body weight gain were checked for a period of 14 days following the single application of the test item. All animals were subjected to necropsy. No deaths and no clinical signs were observed during the study. A black coloration of the skin was noted in all the animals from day 2 until day 10, in all the males and in one female between day 11 and day 15. This coloration masked the evaluation of cutaneous reactions in all the animals from day 2 until day 6. Crusts were observed in 1/5 males and 2/5 females between day 11 and day 15. No apparent abnormalities were observed at necropsy in any animal. The dermal LD0 of the test item Graphistrength® C100 micronized was equal to or higher than 2000 mg/kg in rats.

The acute dermal toxicity test was conducted according to OECD 402 guidelines (Murthy et al., 2011). A limit dose of 2000 mg/ kg b. w. of Graphistrength® C100 mixed with minimum volume (0.25ml) of distilled water was applied uniformly to a clipped 10% body surface area of a group of rats comprising of 5 males and 5 females. The test substance was held in contact with skin with a porous gauze dressing and bandaged with non-irritating adhesive tape up to 24 h. Following this, the residual test substance was wiped off gently from the skin using cotton soaked in water. Restrainer was used to prevent the ingestion of the test substance from the application site. Control group of animals (5 males and 5 females) were handled similarly without any treatment. Animals were observed for mortality/morbidity, clinical signs of toxicity and weekly body weight during the experimental period. Gross pathology was performed at the end of experimental period (day 14). No mortality was evident in the study. Animals of treated groups showed normal and consistent gain in body weight when compared to the control group of animals. No gross lesions were observed. Based on the above findings, the dermal LD0 for Graphistrength® C100 was greater than 2000 mg/kg b. w for Wistar rats.

 

Intratracheal or intranasal routes

Intratracheal instillation and pharyngeal aspiration are not physiological routes of exposure for humans but have nevertheless been used in mice and rats to investigate potential pulmonary and systemic toxicity of high doses of MWCNTs.

 

The pulmonary response of mice was evaluated after exposure to Graphistrength® C100 (Tabet et al., 2011). The MWCNTs 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 MWCNTs. 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-a and 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 MWCNTs. 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 MWCNTs 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 MWCNTs. No modification of GPX-1, SOD-1 or TGF-ß mRNA expression was observed.

 

A synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol, cholesterol and bovine serum albumin (BSA), was elaborated as a vehicle to study the lung toxicity of Graphistrength® C100 in mice (6.25 µg/mouse) (Ronzani et al., 2012). Deposition of surfactant-dispersed MWCNT in the lung of BALB/c mice after a single administrations was analyzed by histology and TEM. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF) -ß1 and collagen, and by histology. Surfactant dispersed Graphistrength® C100 distributed all throughout the mouse airways and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. Mice showed neutrophil infiltrate and greater concentrations of tumor necrosis factor (TNF) -a, keratinocyte-derived chemokine (KC) and interleukin (IL) -17 in BALF when compared to controls.

Male C57BL/6J mice were dosed by oropharyngeal aspiration with vehicle, Graphistrength C100 as produced (AP-MW), or polymer coated Graphistrength C100 (PC-MW) at 4 µg or 40 µg. Mice were sacrificed at 4 h, 1, 7, 28 and 84 d post-exposure with collection of lavage fluid, lung, and tracheobronchial lymph node (TBL) (Bishop et al., 2017). AP-MW induced dose- and time-dependent measures of pulmonary cytotoxicity, inflammatory cell influx, and inflammatory proteins. Histopathology identified small granulomas at terminal bronchioles at 84 d but no significant alveolar fibrosis at the 40 µg dose. In neither case did PC-MW enhance the effects of AP-MW. With mass as a dose metric, inflammation was less from exposure to PC-MW and, at 84 d, remaining material was localized to large extracellular aggregates with minimal or no inflammation. All MW were mostly cleared by 84 d. Translocation to the TBL was observed but unlike larger diameter MWCNT, no translocation to systemic organs was detected for any material.

C57BL/6 mice were pharyngeally aspirated with Graphistrength® C100 and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF) (Vietty et al., 2013). Graphistrength® C100 induced a dose-dependent collagen accumulation in the lungs at the highest doses (50–100µg/mouse), similar in extent to 2.5 mg Min-U-Sil (SiO2 positive control). Graphistrength® C100 enhanced the cytokines in a dose-dependent manner in BALF.

 

The systemic levels of protein serum amyloid A, SAA1/2 and SAA3 proteins, after pulmonary exposure to NM-402 (Graphistrength C100) were assessed in female C57BL/6J mice by intratracheal instillation to 3 doses (6, 18 or 54 µg/mouse) and 3 time points (1, 28 and 92 days post-exposure) (Poulsen et al., 2017). Expression of hepatic Saa1 and pulmonary Saa3 genes on days 1 and 28 were assessed to address the origin of the circulating SAA proteins. NM-402 did not induce an increase of SAA1/2 levels on day 1, 28 and 92 but induced a significant increase of SAA3 levels compared to controls 1 day after exposure (protein levels had returned to control levels 28 and 92 days after exposure). The hepatic Saa1 expression was highest on day 1 and dose-dependent. On day 28, all expression levels had returned to control levels. The mRNA expression levels of Saa3 in the lungs was dose-dependent on day 1 and increased expression of Saa3 was still observed 28 days after exposure to the 54 µg/mouse dose. At this dose, NM-402 induced a significantly high transcription level. In addition, NM-402 exposure also induced significantly increased Saa3 expression at the lower doses (6 and 18 µg/mouse).

 

Intravenous injection

The leukocyte influx into the liver was evaluated following IV exposure of mice to NM 402 (Graphistrength®C100) (Kermanizadeh et al., 2013). The changes in the expression of complement factor C (C3), interleukin 6 (IL6), chemokine C-X-C motif Ligand 2 (CXCL2), IL10, Tumour Necrosis Factor-a(TNF-a), Fas ligand, albumin, Intracellular Adhesion Molecule 1 (ICAM-1) and the total glutathione were also evaluated in the liver. The findings suggest that intravenous exposure of mice to Graphistrength® C100 results in a weak neutrophil governed acute effects in the liver.

 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: oral
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 423)
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Janvier, Le genest-Saint-Isle, France
- Age at study initiation: approximately 8 weeks old
- Weight at study initiation: 189 +/- 6g
- Fasting period before study:18 hours before dosing
- Housing: polycarbonate cages with stainless steel lid (48cmx27cmx20cm)
Each cage contained one to seven animals during the acclimatation period and three rats of the same group during the treatment period. Each cage contained autoclaved sawdust; Sawdust is analysed by the supplier for the composition and contaminant levels.
- Diet :SsniffR/M-H pelleted diet, ad libitum
- Water :filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period:at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2
- Humidity (%):30 to 70
- Air changes (per hr):approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light):12h/12h

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methycellulose
Details on oral exposure:
The administration was performed once (300mg/Kg) or twice (2000mg/Kg) at a 2 hours interval by oral route using a metal gavage fitted to a 2 or 5 mL plastic syringe. The volume administered to each animal was adjusted according to body weight determined on the day of treatment.

VEHICLE
- Concentration in vehicle:0.5% methycellulose
- Amount of vehicle (if gavage):no data
- Justification for choice of vehicle:The test item was not soluble at the concentration of 100mg/mL in purifies water, 0.5% methycellulose and corn oil. At the maximal concentration of 70 mg/mL, a suspension was obtained in purified water or in 0.5% methycellulose. As the suspension was more homogeneous in 0.5% methycellulose, this vehicule was retained.
- Lot/batch no. (if required):no data
- Purity:no data

MAXIMUM DOSE VOLUME APPLIED: 10mL/Kg (for a dose-level of 300mg/Kg)
14.3mL/Kg (for a dose-level of 2000mg/Kg)

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:yes
The dose-level used as the starting dose-level was selected from one of four fixed levels, 5, 50, 300 or 2000mg/Kg body weight.
As no information on the toxic potential of the test item was available, for animal welfare reasons, the starting dose-level of 300mg/Kg was chosen.
Doses:
starting dose-level = 300mg/Kg
After the first assay, as no deaths occured, another assay was carried out on three animals at 2000mg/Kg.
After the second assay, as no deaths occured,another assay was carried out on three animals at 2000mg/Kg.

No. of animals per sex per dose:
3 females/dose
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations :frequently during the hours following administration.Thereafter at least once a day.
- Frequency of weighing:individually just before administration on day 1, 8 and 15.
- Necropsy of survivors performed: yes
Statistics:
no
Key result
Sex:
female
Dose descriptor:
LD0
Effect level:
>= 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality
Mortality:
no
Clinical signs:
at 300mg/Kg: no clinical signs
at 2000mg/Kg: hypoactivity, piloerection, dyspnea, we were noted in 3/6 animals on day 1.
Body weight:
The body weight gain of the treated animals was compared to that of CIT control animals with the same initial body weight.
Gross pathology:
Sacrifice on day 15 of all animals by carbon dioxide asphyxiation.
Macroscopic examination as soon as possible after death of the main organs (digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any organs with ovious abnormalities).
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: other: Regulation n°1272/2008
Conclusions:
The oral LD0 of the test item GRAPHISTRENGTH C100 micronised was equal to or higher than 2000mg/Kg in rats.
Executive summary:

The acute oral toxicity of GRAPHISTENGTH C100 micronised was evaluated in rats according to OECD n°423 and Good Laboratory Pratice Regulations. The test item was prepared in 0.5% methylcellulose and was administrated by oral route (gavage), to groups of three fasted females Sprague-Dawley rats. Clinical signs, mortality and body weight gain were checked for a period of up to 14 days following the single administration of the test item. All animals were subjected to necropsy.

No deaths and no clinical signs were noted at 300mg/Kg. No deaths occured but hypoactivity, piloerection and dyspnea were noted in 3/6 animals on day 1, at 2000mg/Kg. The oral LD0 of GRAPHISTRENGTH C100 micronised was equal to or higher than 2000mg/Kg in rats.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
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 425 (Acute Oral Toxicity: Up-and-Down Procedure)
Deviations:
yes
Remarks:
slight modifications in terms of usage of sexes and animal number
GLP compliance:
not specified
Test type:
up-and-down procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
No data
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
A single dose of MWCNT 2 suspended in distilled water was administered by oral gavage to group of rats at a dose of 2000 mg/ kg b.w.. The test solution was prepared shortly prior to the administration. The dose volume was maximum 10 ml/kg b.w. Similarly, control group of animals (5 males and 5 females) were dosed with distilled water alone.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
Animals were observed for mortality/morbidity, clinical signs of toxicity and weekly body weight during the experimental period. Gross pathology was performed at the end of experimental period (day 14).
Sex:
male/female
Dose descriptor:
LD0
Effect level:
>= 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality
Mortality:
There was no mortality evident in the study.
Body weight:
Animals of the treated group showed normal and consistent gain in body weight when compared to the control group of animals.
Gross pathology:
No gross lesions were observed.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The oral LD0 of MWCNT 2 (Graphistrength® C100) was greater than 2000 mg/kg b.w for Wistar rats.
Executive summary:

Acute oral toxicity –up and down procedure was conducted as per OECD 425 guidelines with slight modifications in terms of usage of sexes and animal number (Murthy et al. 2011). A single dose MWCNT 2 ( Graphistrength® C100) suspended in distilled water was administered by oral gavage to group of rats at a dose of 2000 mg/ kg b.w.. The test solution was prepared shortly prior to the administration. The dose volume was maximum 10 ml/kg b.w. Similarly, control group of animals (5 males and 5 females) were dosed with distilled water alone. Animals were observed for mortality/morbidity, clinical signs of toxicity and weekly body weight during the experimental period. Gross pathology was performed at the end of experimental period (day 14).There was no mortality evident in the study. Animals of the treated group showed normal and consistent gain in body weight when compared to the control group of animals. No gross lesions were observed. Based on the above findings, the oral LD0 for MWCNT 2 ( Graphistrength® C100 ) was greater than 2000 mg/kg b.w for Wistar rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: dermal
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 402)
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
acclimatation: 4 days
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Janvier, Le Genest-Saint-Isle, France
- Age at study initiation:approximately 8 weeks old
- Weight at study initiation:287+/- 6g for the males and 214+/- 5g for the females
- Fasting period before study:no
- Housing:During the acclimatation period, 1 to 7 animals of the same sex were housed in polycarbonate cages with stainless steel lid (48cm x 27cm x 20cm). During the treatment period, the animals were housed individually in polycarbonate cages with stainless steel lid (35.5cm x 23.5cm x 19.3cm). Each cage contained autoclaved sawdust, sawdust is analyzed by the supplier for composition and contaminant levels.
- Diet:SsniffR/M-H pelleted diet, ad libitum
- Water:filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period:4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2
- 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
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
Purified water was used in order to to moisten the test item and ensure a good contact with a skin.
TEST SITE
- Area of exposure:dorsal area (5cm x 7cm for the males and 5cm x 6cm for the females)
- % coverage:approximately 10%
- Type of wrap if used:a hydrophilic gauze pad (pre-moistened with 2mL of purified water) held in contact with the skin for 24 hours by means of an adhesive hypoallergenic aerated semi-occlusive dressing and a restraining bandage. The dressing prevented from ingestion of the test item by the animal.

REMOVAL OF TEST SUBSTANCE
- Washing (if done):no
- Time after start of exposure:24 hours
- For solids, paste formed: yes
Duration of exposure:
24 hours
Doses:
2000 mg/Kg
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: frequently during the hours following administration of the test item; thereafter at least once a day until day 15.
-Frequency of weighing:individually, just before administration of the test item on day 1 and then on days 8 and 15.
- Necropsy of survivors performed: yes
Statistics:
no
Preliminary study:
no
Key result
Sex:
male/female
Dose descriptor:
LD0
Effect level:
>= 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality
Mortality:
no deaths occured during the study
Clinical signs:
no clinical signs were observed during the study.
A black coloration of the skin was noted in all the animals from day 2 until day 10, in all the males and in one female between day 11 and day 15.
This coloration masked the evaluation of cutaneous reactions in all the animals from day 2 until day 6.
Crusts were observed in 1/5 males and 2/5 females between day 11 and day 15.
Body weight:
not affected by the treatment
Gross pathology:
macroscopic examination of the main organs of the animals (digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any other organs with obvious abnormalities) revealed no apparent abnormalities.
Other findings:
no
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: other: Regulation n°1272/2008
Conclusions:
Under the experimental conditions of this study, the dermal LD0 of the test item GRAPHISTRENGTH C100 micronised was equal to or higher than 2000mg/Kg in rats.
Executive summary:

The acute dermal toxicity of the test item GRAPHISTRENGTH C100 micronised was evaluated in rats according to OECD 402 and Good Laboratory Practice Regulations. The test item was applied to the skin of one group of ten Sprague-Dawley rats (five males and five females). The application was performed with the test item in its original form at the dose-level of 2000 mg/Kg.

The test site was then covered by a semi-occlusive dressing for 24 hours. Clinical signs, mortality and body weight gain were checked for a period of 14 days following the single application of the test item. All animals were subjected to necropsy.

No deaths and no clinical signs were observed during the study. A black coloration of the skin was noted in all the animals from day 2 until day 10, in all the males and in one female between day 11 and day 15. This coloration masked the evaluation of cutaneous reactionsin all the animals from day 2 until day 6. Crusts were observed in 1/5 males and 2/5 females between day 11 and day 15.

No apparent abnormalities were observed at necropsy in any animal.

The dermal LD0 of the test item GRAPHISTRENGTH C100 micronised was equal to or higher than 2000 mg/Kg in rats.

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
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 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
not specified
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
No data
Type of coverage:
semiocclusive
Vehicle:
water
Details on dermal exposure:
A limit dose of 2000 mg/ kg b.w. of MWCNT 2 mixed with minimum volume (0.25ml) of distilled water was applied uniformly to a clipped 10% body surface area of a group of rats comprising of 5 males and 5 females. The test substance was held in contact with skin with a porous gauze dressing and bandaged with non-irritating adhesive tape up to 24 h. Following this, the residual test substance was wiped off gently from the skin using cotton soaked in water. Restrainer was used to prevent the ingestion of the test substance from the application site. Control group of animals (5 males and 5 females) were handled similarly without any treatment.
Duration of exposure:
24h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were observed for mortality/morbidity, clinical signs of toxicity and weekly body weight during the experimental period. Gross pathology was performed at the end of experimental period (day 14).
Sex:
male/female
Dose descriptor:
LD0
Effect level:
>= 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality
Mortality:
No mortality was evident in the study.
Body weight:
Animals of treated groups showed normal and consistent gain in body weight when compared to the control group of animals
Gross pathology:
No gross lesions were observed.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The dermal LD0 of MWCNT 2 was greater then 2000 mg/kg b.w for Wistar rats.
Executive summary:

The acute dermal toxicity test was conducted according to OECD 402 guidelines (Murthy et al., 2011). A limit dose of 2000 mg/ kg b.w. of MWCNT 2 (Graphistrength C100) mixed with minimum volume (0.25ml) of distilled water was applied uniformly to a clipped 10% body surface area of a group of rats comprising of 5 males and 5 females. The test substance was held in contact with skin with a porous gauze dressing and bandaged with non-irritating adhesive tape up to 24 h. Following this, the residual test substance was wiped off gently from the skin using cotton soaked in water. Restrainer was used to prevent the ingestion of the test substance from the application site. Control group of animals (5 males and 5 females) were handled similarly without any treatment. Animals were observed for mortality/morbidity, clinical signs of toxicity and weekly body weight during the experimental period. Gross pathology was performed at the end of experimental period (day 14). No mortality was evident in the study. Animals of treated groups showed normal and consistent gain in body weight when compared to the control group of animals. No gross lesions were observed. Based on the above findings, the dermal LD0 for MWCNT 2 (Graphistrength C100) was greater then 2000 mg/kg b.w for Wistar rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw

Additional information

Justification for classification or non-classification

According to the available data on Graphistrenght C100 and Regulation (EC) No 1272/2008, no classification is required for acute oral and dermal toxicity.