Tangled Multi-Walled Carbon Nanotubes

Endpoint summary

Description of key information

Graphistrength C100 was investigated in a local lymph node assay according to OECD TG 429 and in a Buehler assay according to OECD TG 406. No sensitizing potential was observed.

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 delayed contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA) (Sire, 2008). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice Regulations. A preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, twenty-eight female CBA/J mice were allocated to seven groups:
• five treated groups of four animals receiving the test item Graphistrength® C100 micronized at the concentration of 0.1, 0.25, 0.5, 1 or 2.5%,
• one negative control group of four animals receiving the vehicle (propylene glycol),
• one positive control group of four animals receiving the reference item, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.
The test item was not soluble in the recommended vehicles, consequently, a homogenous suspension, obtained in propylene glycol, was used for the study. The maximum practicable concentration in this vehicle was 2.5%. Therefore, the concentrations selected for the preliminary test were 0.25, 0.5, 1 and 2.5%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (2.5%). No mortality and no clinical signs were observed during the study. A black coloration of the skin of the ears was noted on days 2 and 3 in all animals treated at the concentrations of 1 and 2.5% and on day 6 in 2/4 animals treated at the concentration of 2.5%. No noteworthy increase in ear thickness was observed in the animals of the treated groups. A significant lymphoproliferation was noted in the positive control group given HCA at 25%, the study was therefore considered valid. No noteworthy lymphoproliferation was noted at any of the tested concentrations. Graphistrength® C100 micronized did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

A skin sensitization potential test in Guinea pigs was conducted according to OCED 406 guidelines and the Buehler method (Murthy et al., 2011). The Dunkin Hartley guinea pigs, 300 to 400 grams body weight ranges were used in the experiments. The treatment group consists of 20 males whereas control group consist 10 males. A cotton pad about 4 - 6 cm² in size was loaded with Graphistrength® C100 at a dose of 400 mg to all the animals of treatment group. The cotton pad was applied to the clipped area of animals of treatment group and held in contact with the skin by an occlusive patch and bandage dressing for a period of 6 h. The same treatment was repeated on day 7 and 14. Similarly, the animals in control group were treated with distilled water. On day 28, to the closely clipped posterior flank of both treatment and control groups, the nanomaterials were applied in cotton pad at a dose of 200 mg. As in induction exposure, the cotton pads were held in contact with the skin for 6 h by an occlusive patch and bandage dressing. The skin reactions were observed and scored at 30 h after application of the challenge Patch and 54 h after application of the challenge patch. The skin reactions were graded as per the Buehler sensitization scoring scale. On termination, the experiment animals were euthanized using carbon dioxide. None of the animals treated with Graphistrength® C100 exhibited dermal reactions. Hence Graphistrength® C100 could be considered as non sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

february 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 429)

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Janvier, Le Genest-Saint-Isle, France
- Age at study initiation:the animals of the preliminary test were approximately 10 weeks old and the animals of the main test were approximately 9 weeks old
- Weight at study initiation: 21.2 ± 1.1 g.
- Housing:The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm)
- Diet: free access to SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: free access to tap water (filtered using a 0.22 micron filter) contained in bottles
- 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

Vehicle:

propylene glycol

Remarks:

batch No. S35142-236

Concentration:

0.1, 0.25, 0.5, 1 or 2.5%.

No. of animals per dose:

4 females/dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:insoluble
- Concentrations: the concentrations selected for the preliminary test were 0.25, 0.5, 1 and 2.5%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (2.5%).
- Irritation:
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed on a small number of animals, as follows:
• for 3 consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear),
• measurement of the ear thickness (using a micrometer) was performed each day before treatment and 72 hours after the last application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:LLNA
- Criteria used to consider a positive response:
The test item was considered as a skin sensitizer when the SI for a dose group is = 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was not soluble in the recommended vehicles, consequently, a homogenous suspension, obtained in propylene glycol, was used for the study. The maximum practicable concentration in this vehicle was 2.5%.
For the dosage form preparation at 2.5%, the test item was ground to a fine powder using a mortar and pestle, before to add the vehicle. The lower concentrations were obtained by dilution in the vehicle.
All dosage form preparations were made freshly on the morning of administration and any unused material was discarded that same day.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).
Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein.
Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
The results were expressed as disintegration's/mn (dpm) per group and per node.
Stimulation Indices (SI) were calculated according to the following formula:
SI =dpm of treated group/dpm of control group

Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
On days 1, 2 and 3 (before each cutaneous application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer.
No measurement of ear thickness was performed for the animals of the positive control group.
Any irritation reaction (erythema and edema) was recorded in parallel. Any other observation (coloration, presence of residual test item, …) was noted. The irritation level of the test item was determined according to the following table:
% increase in ear thickness between day 1 and day 3 or 6:
< 10% I Non-irritant (I)
10 - 30% II Slightly irritant (II)
> 30% III Irritant (III)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

no

Positive control results:

In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 8.59) were noted.
The study was therefore considered valid.

Parameter:

SI

Value:

1.13

Test group / Remarks:

0.1%

Parameter:

SI

Value:

0.72

Test group / Remarks:

0.25%

Parameter:

SI

Value:

0.84

Test group / Remarks:

0.5%

Parameter:

SI

Value:

0.97

Test group / Remarks:

1%

Parameter:

SI

Value:

0.76

Test group / Remarks:

2.5%

Table 1:

Groups	Treatment and concentrations	Viability (%)	Cellularity index	Number of nodes per group	dpm per group	dmp per node	Stimulation index (SI)	Increase in ear thickness (% between day 1 and day 6)	Irritation level
1	Vehicule	92.79		8	576.81	72.10		-2.91
2	0.1%	99.46	1.78	8	654.34	81.79	1.13	-0.98	I
3	0.25%	96.43	0.79	8	416.37	52.05	0.72	-3.85	I
4	0.5%	92.13	0.80	8	484.86	60.61	0.84	1.00	I
5	1%	79.31	0.67	8	558.54	69.82	0.97	-1.94	I
6	2.5%	93.62	0.85	8	436.90	54.61	0.76	1.00	I
7	25%	92.24	4.16	8	4956.76	619.60	8.59

Interpretation of results:

GHS criteria not met

Conclusions:

GRAPHISTRENGTH C100 micronised did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

The potential of the test item GRAPHISTRENGTH C100 micronised to induce delayed contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). Evaluation of local irritation was also carried out in parallel. This study was conducted in compliance with the principles of Good Laboratory Practice Regulations. A preliminary test was first performed in order to define the concentrations of test item to be used in the main test. In the main test, twenty-eight female CBA/J mice were allocated to seven groups:

• five treated groups of four animals receiving the test item GRAPHISTRENGTH C100 micronised at the concentration of 0.1, 0.25, 0.5, 1 or 2.5%,

• one negative control group of four animals receiving the vehicle (propylene glycol),

• one positive control group of four animals receiving the reference item,a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%. The test item was not soluble in the recommended vehicles, consequently, a homogenous suspension, obtained in propylene glycol, was used for the study. The maximum practicable concentration in this vehicle was 2.5%. Therefore, the concentrations selected for the preliminary test were 0.25, 0.5, 1 and 2.5%. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (2.5%).

Systemic clinical signs and mortality: No mortality and no clinical signs were observed during the study.

Local irritation: A black coloration of the skin of the ears was noted on days 2 and 3 in all animals treated at the concentrations of 1 and 2.5% and on day 6 in 2/4 animals treated at the concentration of 2.5%. No noteworthy increase in ear thickness was observed in the animals of the treated groups.

Proliferation assay: a significant lymphoproliferation was noted in the positive control group given HCA at 25%, the study was therefore considered valid. No noteworthy lymphoproliferation was noted at any of the tested concentrations.

GRAPHISTRENGTH C100 micronised did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

not specified

Type of study:

Buehler test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: no data
- Weight at study initiation: 300 to 400 grams
- Housing: individually in standard stainless steel cages
- Diet: standard rabbit pellet food supplemented with ascorbic acidad libitum
- Water: UV treated water ad libitum
- Acclimation period: five days

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

Route:

intradermal and epicutaneous

Vehicle:

unchanged (no vehicle)

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

No. of animals per dose:

The treatment group consists of 20 males whereas control group consist 10 males.

Details on study design:

A cotton pad about 4 - 6 cm² in size was loaded with MWCNT 2 at a dose of 400 mg to all the animals of treatment group. The cotton pad was applied to the clipped area of animals of treatment group and held in contact with the skin by an occlusive patch and bandage dressing for a period of 6 h. The same treatment was repeated on day 7 and 14. Similarly, the animals in control group were treated with distilled water. On day 28, to the closely clipped posterior flank of both treatment and control groups, the nanomaterials were applied in cotton pad at a dose of 200 mg. As in induction exposure, the cotton pads were held in contact with the skin for 6 h by an occlusive patch and bandage dressing. The skin reactions were observed and scored at 30 h after application of the challenge Patch and 54 h after application of the challenge patch. The skin reactions were graded as per the Buehler sensitization scoring scale. On termination, the experiment animals were euthanized using carbon dioxide.

Positive control substance(s):

not specified

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

200 mg

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 200 mg. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

200 mg

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 200 mg. No with. + reactions: 0.0. Total no. in groups: 20.0.

Interpretation of results:

GHS criteria not met

Conclusions:

None of the animals treated with MWCNT 2 did not exhibited any dermal reactions. Hence MWCNT 2 could be considered as non sensitizer.

Executive summary:

A skin sensitization potential test in Guinea pigs was conducted according to OCED 406 guidelines and the Buehler method (Murthy et al., 2011). The Dunkin Hartley guinea pigs, 300 to 400 grams body weight ranges were used in the experiments. The treatment group consists of 20 males whereas control group consist 10 males. A cotton pad about 4 - 6 cm² in size was loaded with MWCNT 2 (Graphistrength C100) at a dose of 400 mg to all the animals of treatment group. The cotton pad was applied to the clipped area of animals of treatment group and held in contact with the skin by an occlusive patch and bandage dressing for a period of 6 h. The same treatment was repeated on day 7 and 14. Similarly, the animals in control group were treated with distilled water. On day 28, to the closely clipped posterior flank of both treatment and control groups, the nanomaterials were applied in cotton pad at a dose of 200 mg. As in induction exposure, the cotton pads were held in contact with the skin for 6 h by an occlusive patch and bandage dressing. The skin reactions were observed and scored at 30 h after application of the challenge Patch and 54 h after application of the challenge patch. The skin reactions were graded as per the Buehler sensitization scoring scale. On termination, the experiment animals were euthanized using carbon dioxide. None of the animals treated with MWCNT 2 did not exhibite any dermal reactions. Hence MWCNT 2 (Graphistrength C100) could be considered as non sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Graphistrength C100 is not a respiratory sensitiser by it-self but it increase in dose-dependent manner systemic immune response and airway inflammation, mucus production, and fibrotic response induced by house dust mite in the mouse. These effects are associated with an increased production of the epithelium-derived innate cytokines TSLP, IL-33, and IL-25.

The effect of Graphistrength®C100 on systemic immune response and airway inflammation and remodeling induced by the most frequent allergen so far associated with asthma, house dust mite (HDM) were investigated in the mouse (Ronzani et al., 2013). The production of the innate cytokines thymic stromal lymphopoietin (TSLP), IL-25, IL-33, and GM-CSF was evaluated. Mice exposed to HDM exhibited specific IgG1 in serum and inflammatory cell infiltration, and increased Th2 cytokine production, mucus hyperproduction, and collagen deposition in the airways when compared to naive animals. Levels of total IgG 1 and HDM-specific IgG I, influx of macrophages, eosinophils and neutrophils, production of collagen, total tumor growth factor TGF-b1, and mucus, as well as levels of IL-13, eotaxin, and TARC, were dose-dependently increased in mice exposed to HDM and MWCNT compared to HDM alone. These effects were associated with an increased production of TSLP, IL-25, IL-33, and GM-CSF in the airways. MWCNT increase in a dose-dependent manner systemic immune response, as well as airway allergic inflammation and remodeling induced by HDM in the mouse. The data suggest also a role for airway epithelium and innate cytokines in these effects. However, Graphistrength C100 is not a respiratory sensitiser by it-self.

Justification for classification or non-classification

According to the available data and Regulation (EC) No 1272/2008, no respiratory and skin sensitization classification is warranted for Graphistrength C100.