Multi-Walled Carbon Nanotubes (MWCNT), synthetic graphite in tubular shape

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

other: 2 (reliable with restriction) because of the dose selection and the number of dose groups it is not possible to draw conclusions with regard to regulatory hazard assessment

Rationale for reliability incl. deficiencies:

other: Study meets the requirements of research purposes but is not suitable for regulatory hazard assessment

Data source

Reference

Reference Type:

other: project report of Nanotoxico Pole of Excellence, RW/FUNDP research convention N° 516252 01/06/2007 to 31/08/2008

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: on the basis of OECD TG 407

Deviations:

yes

Remarks:

dosage: at least 3 test groups and dose level: The highest dose level should be chosen with the aim of inducing toxic effects but not death or severe suffering.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

936-414-1

Molecular formula:

C

IUPAC Name:

carbon

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ico: OFA-SD (IOPS Caw), Charles River
- Age at study initiation: 5 weeks
- Weight at study initiation: 135 to 150 g
- Fasting period before study: No
- Housing:
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): 45-65%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Pluronic F108

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Carbon nanotubes are dispersed in a solution of Pluronic F108 (1%) (1% p/v) at a stock concentration of 1mg/ml. Concentrations of 0.1 mg/ml and
0.01 mg/ml for doses of 0.5 mg/kg and 0.05 mg/kg administered at the animal are prepared daily by dilution of this stock suspension. The stock
suspension (1 mg/ml) is freshly prepared each week and are subjected to ultrasounds with a probe during one hour (VC 50T, 50 watts, 20kHz- Sonics & Materials Inc).


VEHICLE
- Justification for use and choice of vehicle (if other than water): Pluronic F108 is a surfactant largely used and well known for its innocuousness. The physico-chemical studies of the suspensions of CNT prepared with this surfactant have shown by electronic microscopy (TEM) an excellent
dispersion with few aggregates. It will also give the opportunity to compare the results with those obtained in the oral acute test.
- Concentration in vehicle: 1% p/v
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

once per day, 5 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
0.05 mg/kg ; 0.5 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses have been selected based on the results of the oral toxicity tests with carbon nanotubes and due to the fact that small inflammatory granulomatous changes were observed in the hepatic parenchyma from the dose of 0.5 mg/kg for suspensions in HPC and in Pluronic F108.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: daily


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: no data


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:twice per week
- Anaesthetic used for blood collection: No
- Animals fasted: Yes


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last administration
- Animals fasted: No
- How many animals: 18 animals
- Parameters checked in table [No.1] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: each day
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No


OTHER: - Duration of observation period following administration: 28 day- Frequency of observations and weighing:The animals are observed 10, 30 minutes, 18 and 24 hours after administration - Necropsy of survivors performed: yes- Other examinations performed: clinical signs, organ weights, blood analysis, urine analysis, genotoxicity, macroscopic examinations of organs and histopathology, other:- Urines were harvested and quantified each day. Blood samples were taken twice a week in the tails of rats to assess the presence of micronucleus in erythrocytes. 28 days after the first administration, the animals are anaesthetized (nembutal, 60 mg/kg. An incision is done from the cloaca to the chin following the medio-ventralline. The inferior vena cava is isolated in order to take blood in this vessel. Plasma is obtained after centrifugation.- Measurements of: sodium, potassium, glucose, total cholesterol, alkaline phosphatase and albumin.- Measurements of biochemical parameters reflecting the hepatic function such as the aspartate aminotransferase (TGO) and alanine aminotransferase (TGP) and of biochemical parameters reflecting the renal function such as urea and creatinine.-

Genotoxicity: Blood smears were stained during 5 minutes in methanol prior coloration according to the acridine orange method. Briefly, the slides were colored in acridine orange (12mg/100ml phosphate buffer) during 1 minutes prior the rinsing in 2 successive bath containing phosphate buffer. The slides are then analysis with a fluorescent microscope.

The kidneys, the spleen, the heart-lung block, the reproductive organs and the bladder were removed. All the organs were directly preserved in formaline 10%. Three samples of liver were also removed and preserved in glutaraldehyde in order to analyse them by transmission electron microscopy (TEM).

Sacrifice and pathology:

Macroscopic examination and autopsy: organs observed: stomach, small and large intestine, liver, pancreas, kidneys, spleen, the heart-lung block, the reproductive organs and the bladder.- Histological analysis: observed organs: stomach, small and large intestine, liver, pancreas, kidneys, spleen.The samples are wraped up in paraffin blocks which are cut in slices of 6µm thick. The slices are put on microscop plates which are developped by a technique of conventional histology coloration, the hematoxylin and eosine (H&E). The slides are observed by optical microscopy (Olympus Prov is AX70) and pictures are taken with a camera (Zeiss Axiocam).The histological cuts have been done in the stomach, small and large intestine, spleen, pancreas, liver and kidneys. The other organs (the heart-lung block, the reproductive organs and the bladder) are stored in formalin (10%) if further investigations are needed. The histopathological examination has been realised in collaboration with the Institute of Pathology and Genetic (IPG, Gosselies).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

no particular symptoms

Mortality:

no mortality observed

Description (incidence):

no particular symptoms

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals have shown particular sign of discomfort (lethargy, nausea, vomiting, diarrhoea...). One animal treated with CNT (0.05 mg/kg) died the 10th day of exposure. The macroscopic examination haven't revealed anything.

BODY WEIGHT AND WEIGHT GAIN
The animals were weighted daily and no diminution of the weight gain has been observed during the experiment. At the end of the experiment, the animals weigh between 200 and 250 g, so normal values described for Sprague Dawley females of 10 weeks.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption has been quantified and it appears that there is no diminution of the food consumption during the 28 days of treatment. The animals have eaten on average 71 g/kg of food per day.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The animals have drunk in average 102 ml/kg of water per day.


HAEMATOLOGY
A smear test is realised in order to detect the presence of micro-nucleus, a realistic approach of the genotoxic risk. The count of micro-nucleus is recognized as a biological marker of genotoxic effects. Micro-nucleus are nuclear entities independant of the nucleus, present in the cytoplasm, coming from full chromosomes or part of them during the anaphase. The slides, colored by acridine orange, a selective dye for nucleic acids, are read by fluorescent microscopy (x1000). For each subject, 1000 intact reticulocytes are observed. Results showed a slight increase of the number of micronucleaed reticulocytes for the dose of 0.5 mg/kg but which is not statistically significant.

CLINICAL CHEMISTRY
Study of the biochemical parameters in the plasma:
All the measured parameters are within the limit range described for the rat. The absence of significant increase of plasmatic enzymes TGO and TGP suggests that the hepatic function is not affected by the administration of the studied compound.

URINALYSIS: The urinary analysis showed that the excretory capacity of the animals exposed to 0.05 and 0.5 mg/kg of CNT is similar to the values observed in the control group. Urines were also observed after digestion in sulfuric acid, filtration and centrifugation to detect the presence of CNT by TEM analysis. Qualitative analysis showed that the CNT are eliminated via the urinary pathway.

ORGAN WEIGHTS
The liver, the spleen and the kidneys were weighted; the values are in table 2. 24 hours after the administration of the last dose of CNT, no significant difference is observed between the weight of the animals treated with 0.05 mg/kg and 0.5 mg/kg and the one of the control group.

GROSS PATHOLOGY
No change has been observed in the removed organs such as the stomach, the small and large intestine but also incidental glandular organs such as the liver and pancreas.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histological cuts mainly realised first on the liver have revealed the presence of inflammatory granulomatous changes in the hepatic parenchyma as well as in the interlobular space from the dose of 0.05mg/kg, such as for the animals which were subjected to the dose of 5 mg/kg during the acute test (24 hours) and with Pluronic F108 as well as HPC as surfactant. These granulomatous changes are mainly constituted by polynuclear cells (especially macrophages revealed by immunohistochemistry) and lymphocytes. Beside the presence of those granulomatous changes, the cellular structure is well preserved and no steatosis is observed, nor cholestasis, hepatocellular insufficiency or sign of suffering. The set of hepatic granulomatous changes of each rat exposed to the suspension of nanoparticles have been quantified. Considering that the size of the slides of liver is homogeneous, the number of granulomatous changes present on each slide have been counted (minimum 7-10 sections by liver of animals) in order to determined the mean for minimum 7 sections. The surfaces of the slides of liver were quantified with the help of a software of morphometry (Histolab) which allows to digitalised surfaces in order to determined the number of granulomatous changes or the quantified surface for a granulomatous change. The mean surface of the liver evaluated for the counts of granulomatous changes is 2985 ± 227 mm². The number of granulomatous changes per cm2 is higher in the group treated with the CNT (0.05 and 0.5 mg/kg) than in the control group (vehicle). The difference observed is statistically significant.
The histological analysis performed on the others organs (oesophagus, stomach, small and large intestine, mucous membrane of the gastro-intestinal tract, spleen, pancreas, kidneys(cortex and medullar)) did not reveal any changes in comparison of the control group.

Effect levels

Dose descriptor:

dose level:

Effect level:

0.05 - 0.5 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Biochemical parameters in blood plasma (mean ± S.D. (n= 4, * : p<0.05 compared with the control group))

Parameters (n. physiol.)	Control ; Pluronic F108	Group 1 ; 0.05 mg/kg	Group 2 ; 0.5 mg/kg
Sodium 130-150 (mmol/L)	142.50 ± 0.88	144.20 ± 2.28	143.80 ± 2.31
Potassium 4.5-6.0 (mmol/L)	4.48 ± 0.51	4.36 ± 0.83	4.07 ± 0.646
Glucose 0.89-1.833 (g/L)	1.26 ± 0.73	1.50 ± 0.65	1.27 ± 0.18
Urea 0.32-0.54 (g/L)	0.47 ± 0.08	0.50 ± 0.12	0.50 ± 0.15
Creatinine 3.9-22.9 (mg/L)	4.55 ± 0.68	4.14 ± 0.84	4.18 ± 0.99
Total proteins 59.0-84.0 (g/L)	60.00 ± 5.90	57.80 ± 2.39	56.83 ± 2.79
Albumine 32.0-43.0 (g/L)	39.67 ± 3.61	39.20 ± 1.09	37.50 ± 3.08
PA 39-216 (UI/L)	135.50 ± 31.59	130.40 ± 49.30	135.70 ± 18.28
TGO 39-92 (UI/L)	99.68 ± 25.33	79.40 ± 9.23	80.50 ± 12.00
TGP 17-50 (UI/L)	33.83 ± 13.08	31.20 ± 10.76	32.50 ± 12.34
Total cholesterol 0.5-1.0 (g/L)	0.68 ± 0.15	0.75 ± 0.10	0.65 ± 0.15

Table 2: Weights (g) of barious target organs: spleen, liver, kidneys: (mean ± S.D.)

	spleen	liver	left kidney	right kidney
Control	0.54 ± 0.08	6.64 ± 0.74	0.69 ± 0.07	0.7 ± 0.07
0.05 mg/kg	0.52 ± 0.17	6.37 ± 1.66	0.74 ± 0.05	0.74 ± 0.07
0.5 mg/kg	0.54 ± 0.08	6.98 ± 1.7	0.72 ± 0.06	0.74 ± 0.06

Applicant's summary and conclusion

Executive summary:

Test substance meeting the form described in section 4.5 of IUCLID (0.05 and 0.5 mg/kg) dossier was administered to rats by gavage during 28 days. During this 28-days study, none of the animal presented particular abnormal symptoms except one animal died after 10 days. The reason of the death was not clearly established. The follow up of the weight as well as the feed and water consumption did not reveal abnormalities. TEM analysis in urine revealed the presence of CNT demonstrating their urinary excretion..

The analysis of the various biochemical parameters in the blood plasma did not reveal damages or particular dysfunctions. All the measured values were within normal range described for the rat.

The micronucleus test on blood smears taken along all the protocol reveal that even if the number of micronucleated erythrocytes tends to increased during the exposure period for the dose of 0.5 mg/kg, this evolution is not statistically significant.

The macroscopic examination of viscera did not reveal particular morphological change. The liver, the kidneys and the spleen of all animals were weighted and the weight of those organs of the exposed groups was similar to the one of control group.

The histological analysis performed on oesophagus, stomach, small and large intestine, mucous membrane of the gastro-intestinal tract, spleen, pancreas and kidneys (cortex and medullar)) did not reveal any changes in comparison of the control group.The analysis of the liver reveals the presence of inflammatory granulomatous changes in the hepatic parenchyma of the animals exposed to MWCNT. This increase in granulomatous changes is statistically different by comparison of the control group. Beside the presence of those granulomatous changes, the cellular structure is well preserved and no steatosis is observed, nor cholestasis, hepatocellular insufficiency or sign of suffering. For none of the tissues that was examined histopathologically the presence of MWCNT in the slides is documented.