Tangled Multi-Walled Carbon Nanotubes

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

distribution

Principles of method if other than guideline:

Organ and blood samples were collected and evaluated for the presence of MWCNTs after the i.v. administration of MWCNT radiolabeled with 14C atoms to allow detection in the body.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

see below

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, France
- Weight at study initiation: 226 and 234g for females and 256 and 266g for males

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

intravenous

Vehicle:

other: 0.05 wt% Rat Serum Albumin (RSA) in ultra pure water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A 2.56 mg/ml stock solution was prepared by prewetting the CNT powder in 0.5 vol % ethanol followed by dispersion in 0.05 wt% Rat Serum Albumin (RSA) in ultra pure water. The suspensions were sonicated for 15/16 minutes on ice.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
After preparation dispersed 14Cmultiwall carbon nanotubes (MWCNT) solutions were observed by optical microscopy to control sample homogeneity. According to the observations, depending on the MWCNT, the dispersion solutions were stable from one to several hours and therefore were immediately used after their preparation for IV injection in rats.

Duration and frequency of treatment / exposure:

single (day 1) or repeated (on 5 consecutive days, day 1-5) administrations

Remarks:

Doses / Concentrations:
single dose groups: 9.6 – 10 mg/kg b.w. for male animals, and 10.9 – 11.3 mg/kg b.w. for female.
repeated dose groups: total cumulative dose between 48 – 50 mg/kg b.w after 5 days treatment, for male animals, and 54.5 – 56.5 mg/kg b.w. for female.

No. of animals per sex per dose / concentration:

6 animals per time/group (3 males and 3 females), except for day 14 and day 30 for repeated dosages for which only 3 males were investigated.

Control animals:

other: Vehicle treated animals were investigated as follows; males n=2 at days 2, 6, and 90, and females n=1 at days 2, 6 and 90.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled:
Various organs and blood were collected including liver, lung, spleen, kidney, heart and testes. Organs were immediately removed and immersed in an - 80°C mixture of dry ice and isopentane to prevent MWCNT tissue redistribution.
Blood samples were collected on abdominal aortic vein at days post administration of MWCNT. Quantitative determination of the radioactivity in blood was carried out by 1 µL blood deposition on glass slides and radioactivity counting using the b-imagerTM 2000.
For tissue collection rats were anesthetised with isoflurane 2% and sacrificed by exsanguination (abdominal aorta). Organs were immediately removed and immersed in an -80° C mixture of dry ice and isopentane to prevent MWCNT tissue redistribution. After blocking organs in mounting medium, 50 tissue sections (20 m) were made at -20° C with a slicing microtome (LEICA Microsystems, France), selecting representative organ regions. After lay down on glass slides, these sections were left either in freezer for optical study, or were kept at room temperature for 1 day in the presence of silica gel to ensure complete drying, before radioimaging analysis. The quantitative determination of the radioactivity in dried tissue sections and imaging were carried out using a high performance
autoradiography imager (b-imagerTM 2000, detection threshold of 0.01 cpm/mm2 for 14C Biospace Lab, Paris, France).
For each animal, the percentage (%) of the injected dose for whole organs was calculated by radioactivity integration to the whole organ volume, using the radioactivity level detected in the 50 tissue sections for each organ (integration procedure developed previously in our laboratories). The % of the injected dose for each analysed organ is reported.
- Time and frequency of sampling:
Tissue samples were obtained at various time points after the administration. Samples were collected at day 2 (24 hour after the single administration at day 1), and at day 90 after the single administration. After the five times repeated administrations at days 1-5, samples were collected at day 6 (24 hour after the last of the administrations days 1 to 5), and at day 14, day 30 and day 90. At autopsy at day 2, day 6, day 14, day 30 and day 90 after administration blood was collected.

Design of toxicokinetic study for determination of tissue distribution of NM 402:
Treatment Autopsy
Day 2 Day 6 Day 14 Day 30 Day 90
MN (1x) 3M+3F - - - 3M+3F
MN (5x) - 3M+3F 3M 3M 3M+3F
Vehicle 2M+1F 2M+1F - - 2M+1F

Type:

distribution

Results:

Single IV dose, total recovery in organs on days 1 and 90, 8 and 4% (male) and 24 and 6 % (female). In liver, 7 and 3% and 23 and 5%.

Type:

distribution

Results:

5-day IV dosing, total recovery in organs on days 6 and 90, 5 and 4% (male) and 11 and 4 % (female). In liver, 4 and 3% and 10 and 3%.

In blood obtained at 24 hours after the administration of the 14C-MWCNT very low to almost no radioactivity was detected whatever the protocol of MWCNT administration in either male or female rats. Table 6-2 shows the 14C tissue distribution after a single IV MWCNT administration, and Table 6-3 shows the distribution after five repeated IV administrations.

After a single IV dose for male rats for NM-402 only 8% of the injected dose was observed in organs at day 1, with the liver being the main target organ (7%). In females the recovery was 24% after a single dose (Table 6-2). At day 90 a 50% reduction of the level of NM-402 was observed when compared to day 1 in most organs, while for the lung a similar % of injected dose was observed both on day 1 after a single administration and at day 90 after repeated administration. After the repeated IV dosing for male rats compared to the data of day 6, almost no elimination of NM-402 was observed at day 90 after administration (Table 6-3). However, when comparing the elimination after repeated exposures and single exposure, NM-402 elimination appeared much more reduced. After a single exposure a 50% elimination was observed while almost no elimination was observed after repeated IV doses. A particular feature with this NM-402 was the observation that spleen was more loaded than lung at any time point.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

distribution

Principles of method if other than guideline:

Organ and blood samples were collected and evaluated for the presence of CNTs after the oral administration of MWCNT radiolabeled with 14C atoms to allow detection in the body.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

see below

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, France
- Weight at study initiation: 226 and 234g for females and 256 and 266g for males

ENVIRONMENTAL CONDITIONS
No data

Route of administration:

oral: gavage

Vehicle:

other: 0.05 wt% Rat Serum Albumin (RSA) in ultra pure water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A 2.56 mg/ml stock solution was prepared by prewetting the CNT powder in 0.5 vol % ethanol followed by dispersion in 0.05 wt% Rat Serum Albumin (RSA) in ultra pure water. The suspensions were sonicated for 15/16 minutes on ice.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
After preparation dispersed 14Cmultiwall carbon nanotubes (MWCNT) solutions were observed by optical microscopy to control sample homogeneity. According to the observations, depending on the MWCNT, the dispersion solutions were stable from one to several hours and therefore were immediately used after their preparation for IV injection in rats.

Duration and frequency of treatment / exposure:

single (day 1) or repeated (on 5 consecutive days, day 1-5) administrations

Remarks:

Doses / Concentrations:
single dose groups: 9.6 – 10 mg/kg b.w. for male animals, and 10.9 – 11.3 mg/kg b.w. for female.
repeated dose groups: total cumulative dose between 48 – 50 mg/kg b.w after 5 days treatment, for male animals, and 54.5 – 56.5 mg/kg b.w. for female.

No. of animals per sex per dose / concentration:

6 animals per time/group (3 males and 3 females), except for day 14 and day 30 for repeated dosages for which only 3 males were investigated.

Control animals:

other: Vehicle treated animals were investigated as follows; males n=2 at days 2, 6, and 90, and females n=1 at days 2, 6 and 90.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled:
Various organs and blood were collected including liver, lung, spleen, kidney, heart and testes. Organs were immediately removed and immersed in an - 80°C mixture of dry ice and isopentane to prevent MWCNT tissue redistribution.
Blood samples were collected on abdominal aortic vein at days post administration of MWCNT. Quantitative determination of the radioactivity in blood was carried out by 1 µL blood deposition on glass slides and radioactivity counting using the b-imagerTM 2000.
For tissue collection rats were anesthetised with isoflurane 2% and sacrificed by exsanguination (abdominal aorta). Organs were immediately removed and immersed in an -80° C mixture of dry ice and isopentane to prevent MWCNT tissue redistribution. After blocking organs in mounting medium, 50 tissue sections (20 m) were made at -20° C with a slicing microtome (LEICA Microsystems, France), selecting representative organ regions. After lay down on glass slides, these sections were left either in freezer for optical study, or were kept at room temperature for 1 day in the presence of silica gel to ensure complete drying, before radioimaging analysis. The quantitative determination of the radioactivity in dried tissue sections and imaging were carried out using a high performance
autoradiography imager (b-imagerTM 2000, detection threshold of 0.01 cpm/mm2 for 14C Biospace Lab, Paris, France).
For each animal, the percentage (%) of the injected dose for whole organs was calculated by radioactivity integration to the whole organ volume, using the radioactivity level detected in the 50 tissue sections for each organ (integration procedure developed previously in our laboratories). The % of the injected dose for each analysed organ is reported.
- Time and frequency of sampling:
Tissue samples were obtained at various time points after the administration. Samples were collected at day 2 (24 hour after the single administration at day 1), and at day 90 after the single administration. After the five times repeated administrations at days 1-5, samples were collected at day 6 (24 hour after the last of the administrations days 1 to 5), and at day 14, day 30 and day 90. At autopsy at day 2, day 6, day 14, day 30 and day 90 after administration blood was collected.

Design of toxicokinetic study for determination of tissue distribution of NM 402:
Treatment Autopsy
Day 2 Day 6 Day 14 Day 30 Day 90
MN (1x) 3M+3F - - - 3M+3F
MN (5x) - 3M+3F 3M 3M 3M+3F
Vehicle 2M+1F 2M+1F - - 2M+1F

Type:

distribution

Results:

After oral administration 14C-MWCNT didn't show translocation from the GI-tract into the systemic circulation or any of the organs investigated (including spleen, liver, and lung).

Description of key information

In the frame of the Nanogenotox program, eighteen male and 12 female rats received a single (day 1) or repeated (on 5 consecutive days, day 1-5) oral or intravenous (IV) administrations of 14C-labelled Graphistrength™ C100 (named NM 402 in the report) dispersed in 0.05 wt% Rat Serum Albumin (RSA) in ultra-pure water (Jacobsen et al., 2013). 14C-labelling of carbon nanotubes was carried out using a three steps chemical process. The specific activity was 0.36 MBq/mg (9.7µCi/mg), which correspond to 14C/12C ratios ranging from 2/1000. Six male and three female animals were treated with the vehicle used for the dispersion. The single dose groups received between 9.6 – 10 mg/kg b. w. for male animals, and 10.9 – 11.3 mg/kg b. w. for female animals depending on the actual weight of the animal. The repeated dose groups received a total cumulative dose per animal between 48 – 50 mg/kg b. w after 5 days of treatment, for male animals, and 54.5 – 56.5 mg/kg b. w. for female animals depending on the actual weight of the animal. Tissue samples were obtained at various time points after the administration. After single treatment, radioactivity in tissues was evaluated in 3 male or female rats at days 2 and 90, whereas after five times repeated treatments tissue levels were evaluated for 3 males at days 6, 14, 30 and 90, and for females at days 6 and 90. Vehicle treated animals were investigated as follows; males n=2 at days 2, 6, and 90, and females n=1 at days 2, 6 and 90. Various organs and blood were collected including liver, lung, spleen, kidney, heart and testes. Organs were immediately removed and immersed in an -80°C mixture of dry ice and isopentane to prevent MWCNT tissue redistribution.

After oral administration, 14C-labelled Graphistrength™ C100 didn't show translocation from the GI-tract into the systemic circulation or any of the organs investigated (including spleen, liver, and lung). In blood obtained at 24 hours after the i.v. administration of the 14C-labelled Graphistrength™ C100 very low to almost no radioactivity was detected whatever the protocol of administration in either male or female rats.

After a single i.v. dose, only 8% of the injected dose was observed in organs of males at day 1, with the liver being the main target organ (7% of the dose). In females the recovery was 24% after a single dose. At day 90 a 50% reduction of the level of 14C labelled Graphistrength™ C100 was observed when compared to day 1 in most organs, while for the lung a similar % of injected dose was observed both on day 1 after a single administration and at day 90 after repeated administration. After the repeated i.v. dosing for male rats compared to the data of day 6, almost no elimination of Graphistrength™ C100 was observed at day 90 after administration. However, when comparing the elimination after repeated exposures and single exposure, 14C-labelled Graphistrength™ C100 elimination appeared much more reduced. After a single exposure a 50% elimination was observed while almost no elimination was observed after repeated i.v. doses. A particular feature with this 14C-labelled Graphistrength™ C100 was the observation that spleen was more loaded than lung at any time point (<= 0.5% of the dose).

 

In a subchronic inhalation toxicity study, multiwalled carbon nanotubes Graphistrength C100 were administered 5 days per week by nose-only, flow-past inhalation to groups of 35 Wistar rats of both sexes at aerosol concentrations of 0.05, 0.25 and 5.0 mg/m3 air (groups 2, 3 and 4, respectively) for a period of 13 weeks (Broich, 2016). A control group was treated similarly with air, only (group 1). At the end of the 13-week treatment period, histological examination was performed on all tissues and organs from 10 animals per sex of groups 1 and 4 and on the respiratory tract organs from animals of groups 2 and 3. Deposition of black particles was only observed in the lungs and the tracheobronchial lymph nodes. Using brightfield hyperspectral microscopy, no sign of MWCNT translocation was observed in the other organs indicating the lack of biodisponibility by inhalation exposure (Johnson, 2017).

Key value for chemical safety assessment

Absorption rate - oral (%):

0

Absorption rate - dermal (%):

0

Absorption rate - inhalation (%):

0