Tangled Multi-Walled Carbon Nanotubes

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3 October 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

GRAPHISTRENGTH C100

IUPAC Name:

GRAPHISTRENGTH C100

Constituent 2

Reference substance name:

Tangled Multi-Walled Carbon Nanotubes

EC Number:

701-160-0

Cas Number:

7782-42-5

Molecular formula:

Hollow tubular carbon, 1-dimensional nano structures with hexagonal arrangement of carbon atoms

IUPAC Name:

Tangled Multi-Walled Carbon Nanotubes

Test material form:

solid: nanoform

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca 6 weeks
- Weight at study initiation: males 233-259 g, females: 167-197g
- Fasting period before study: none
- Housing: up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages
- Diet (ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l.)
- Water: ad libitum
- Acclimation period: approximately 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C± 2
- Humidity (%): 55%±15
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): The test item Graphistrength C100 will be formulated, using powdered diet, by initial preparation of a pre-mix followed by dilution with further quantities of diet and mixing. The formulations will be prepared separately for each group. Fresh diets will be prepared at weekly intervals, unless specified otherwise, at fixed concentrations of 100, 1000 and 10000 ppm. Concentrations will be calculated and expressed in terms of test item as supplied.
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analysis has been performed by ICP-OES (inductively coupled plasma optical emission spectrometry) of Al and Fe concentrations resulting from the residual amount of catalist. Samples have been mineralized beforehand by alkali fusion or micro-wave digestion. Some tests have also been conducted by XRF (X-ray fluorescence). However, the analysis couldn’t confirm the Graphistrength® C100 concentrations in the diet formulations containing nominally 100, 1000 and 10000 ppm of Graphistrength® C100. The main issue comes from the low Graphistrength® C100 concentrations to measure (and so the very low Al and Fe concentrations) in rat diet formulations that contains already non negligible Al and Fe amounts.
Due to the uncertainty of the measurements (with Alkali fusion + ICP-OES), concentrations of 100 and 1000 ppm of Graphistrength® C100 have no chance to be detected. The 10000 ppm Graphistrength® C100 concentration is the only one that could be quantified but the recovery (ca. 72%) was lower than the expected values.

Duration of treatment / exposure:

28 days

Frequency of treatment:

ad libitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg diet

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg diet

Doses / concentrations 3

Dose / conc.:

10 000 mg/kg diet

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was followed except that the final check was carried out at approximately mid-day

DETAILED CLINICAL OBSERVATIONS: Yes
Observations were performed at the same time interval each day. Observation of the cage was also performed. The data are not presented in this report but retained as study raw data. Once before commencement of treatment and once per week from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.
Once during Week 4 of treatment, an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed.

BODY WEIGHT: Yes
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food consumed by each cage of rat was recorded daily, starting from the allocation. Due allowance was made for any spilled food in each cage. The group mean daily intake per rat was calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of Week 4 of treatment
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters examined:
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Heinz bodies
– Differential leucocyte count
· Neutrophils
· Lymphocytes
· Eosinophils
· Basophils
· Monocytes
· Large unstained cells
– Platelets
– Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of Week 4 of treatment
- Animals fasted: Yes
- How many animals: all
- Parameters examined:
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Bile acids
– Inorganic phosphorus
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: end of Week 4 of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:
– Appearance
– Volume
– Specific gravity
– pH
– Protein
– Glucose
– Ketones
– Bilirubin
– Urobilinogen
– Blood
Part of the sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined, microscopically for:
– Epithelial cells
– Leucocytes
– Erythrocytes
– Crystals
– Spermatozoa and precursors
– Other abnormal components (e.g. the presence of Multiwalled carbon nanotubes, MWCNT)

NEUROBEHAVIOURAL EXAMINATION: Yes
The motor activity (MA) of all animals was measured once during Week 4 of treatment by an automated activity recording. Measurements were performed using a computer generated random order.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).

ORGAN WEIGHT: Yes
See Table TISSUE PROCESSING

HISTOPATHOLOGY: Yes
See Table TISSUE PROCESSING

Other examinations:

CytoViva hyperspectral microscopy was employed to spectrally identify Graphistrength C100 multiwalled carbon nanotubes (MWCNT) in H&E stained kidney and urinary bladder tissues of Sprague-Dawley rats exposed to 10,000 ppm of Graphistrength C100 MWCNT via diet for 4 weeks. The samples were imaged with transmitted brightfield. Each tissue sample was surveyed at 10x magnification under brightfield illumination to identify any dark particles that appeared to be Graphistrength C100 MWCNT. As no dark deposits were found in any samples, 60x magnification hyperspectral and optical images were acquired at random areas in the tissue. No mapping was required because no dark deposits were found.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No signs of toxicological relevance were recorded. Animals of the mid- and high dose groups showed black appearance of the general body surface including tail (see attached pictures).
Additionally, at observation of the cage tray, black staining was also recorded in the mid- and high dose groups. One low dose animal (no. 20) showed moderate decreased activity, piloerection and hunched posture during the last days of study. Mild ulceration in the stomach was recorded at histological examination that might account for the presence of such clinical signs. No other relevant signs were recorded in the low dose group.
Weekly observation of animals at removal from the cage and in an open arena (open-field assessment) did not reveal changes attributable to the test item.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean values of the achieved dosage, calculated over the first two weeks, were 11, 104 and 1003 mg/kg/day for males and 11, 111 and 1152 mg/kg/day for females, respectively for the low, mid- and high dose level. During the last two weeks, the mean values of the achieved dosage were 9, 86 and 900 mg/kg/day for males and 10, 99 and 995 for females, for the low, mid- and high dose level respectively. The overall achieved dosages for the 4 weeks of study were 10, 95 and 951 mg/kg/day for males and 11, 105 and 1073 mg/kg/day for females.

Haematological findings:

no effects observed

Description (incidence and severity):

Some statistically significant changes were recorded in treated animals, such as: increase of erythrocytes, haemoglobin and haematocrit in males dosed at 100 ppm, increase of haemoglobin and haematocrit in those receiving 10000 ppm and decrease of mean corpuscular haemoglobin concentration in females treated at 100 ppm. Due to the minimal severity (3% to 8%) and/or the absence of dose-relation, the above findings were considered incidental.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Alkaline phosphatase was reduced in some females dosed at 100 ppm and 10000 ppm. Mean group values were 25% and 21% below controls, respectively. In addition, male no. A2321030 (1000 ppm) showed high aspartate aminotransferase value (69% above mean control data). Due to the minimal incidence and/or the absence of dose-relation, the above findings were considered incidental.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination of fresh samples of urine sediment revealed the presence of few dark amorphous aggregates for three males and two females dosed at 10000 ppm. As additional analysis, the urine sediments were further investigated by the Sponsor (Godillot, 2017), these formations were identified by scanning electronic microscopy as MWCNT agglomerates of several tens of micrometers (Godillot, 2017).

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No relevant changes were noted in treated animals at post mortem, when compared with control animals. The changes observed in treated groups of both sexes, such as brown staining of the head (muzzle) or dark colour of skin and/or tail are suggested to be likely due to the colour of the test item. The remaining sporadic findings observed in few treated animals could be considered incidental.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No changes or traces related to the treatment with Graphistrength C100 were noted in the organs/tissues. All observed lesions are known to occur spontaneously in Sprague Dawley SD rats of the same age and/or have a comparable incidence in the control group.
As additional analysis, kidneys and urinary bladder tissue samples were further investigated by the Sponsor using brightfield hyperspectral microscopy (Jonhson, 2017). No trace of Graphinstrength C100 MWCNT was observed which could account for the MWCNT agglomerates observed in the urine sediments of some rats exposed to the top concentration.

Other effects:

no effects observed

Description (incidence and severity):

Oestrous cycle
Evaluation of the oestrous cycle at the end of study (Day 29) did not indicate particular differences between groups.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 10 000 mg/kg diet

Based on:

test mat.

Remarks:

ca. 1000 mg/kg bw/d

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

A 4 week toxicity study on Graphistrength C100 was conducted in Spraque Dawley rats. The test item was given orally, via diet, at constant concentrations of 100, 1000 and 10000 ppm, corresponding to mean achieved dose levels over 4 weeks of 10, 95 and 951mg/kg body weight/day for males and 11, 105 and 1073mg/kg body weight/day for females, respectively.
The in-life phase did not show changes of toxicological relevance. The most remarkable clinical signs observed in treated animals of the mid- and high dose groups was dark staining of the general body surface including tail resulting from the accumulation of MWCNT in the fur after the contamination of the litter by spillage of diet and/or excretion of MWCNT in the feces. In addition, data collected from detailed multiple functional observation battery tests and specific functional tests including motor activity, landing foot splay and sensory reactivity to stimuli, did not show evidence of neurotoxic effects of the test compound.
Body weight and food consumption were unaffected by treatment.
At clinical pathology investigations, no important changes were recorded in hematological, biochemistry or urinalysis parameters that could be considered as treatment-related. The presence of large dark aggregates was observed in the urine sediment of many high dose animals, which were identified as MWCNT by scanning electron microscopy (Godillot, 2017).
At necropsy examination, the macroscopic findings were limited to dark soft content of caecum and stomach or dark colour of skin and/or tail in treated animals. The remaining sporadic findings observed in few treated animals could be considered incidental.
The histological analysis did not reveal changes related to inflammatory reactions or cell injury in any organ/tissues. The remaining sporadic findings observed in few treated animals could be considered incidental.
A specific histological investigation was performed by CytoViva© Inc. (Johnson, 2017) using hyperspectral microscopy to spectrally identify any dark particles that could appear to be Graphistrength C100 in H&E stained kidney and urinary bladder tissues of rats exposed to 10000 ppm. No dark deposits were found in ant tissue samples. The results support the conclusion that the MWCNT observed in some urinary sediments was due to an external contamination (via the fur and/or feces) during the urine collection.
In conclusion, no signs of toxicity were observed in male or female animals at any of the dose levels investigated.

Executive summary:

The toxicity of Graphinstrength C100 MWCNT was investigated in Spraque Dawley rats after 4 weeks when given orally, via diet. The animals were assigned to four groups of 5 animals/sex and received the test item, mixed in the rodent diet, at fixed concentrations of 100, 1000 and 10000 ppm, corresponding to mean achieved dose levels over 4 weeks of 10, 95 and 951 mg/kg body weight/day for males and 11, 105 and 1073 mg/kg body weight/day for females, respectively. Routine in vivo analyses included daily clinical observations and food consumption. At weekly interval, body weight and assessment of detailed clinical observations (removal from the home cage and observation in open arena) were performed. In addition, towards the end of treatment specific functional tests (hindlimb landing foot splay, sensory reactivity to stimuli including grip strength, and motor activity) were performed for neurotoxicity assessment. Oestrous cycle was also evaluated on Day 29, before dispatch to necropsy. Bleeding for clinical pathology investigation including urinalysis was carried out. At term, the animals were subjected to a detailed macroscopic examination along with organ weights and tissue retention.

No mortality occurred in the study. The main clinical signs were limited to a general black discolouration of the body surface due to the black colour of the test item. Additionally, at observation of the cage tray, black staining was also recorded in the mid- and high dose groups. Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item. No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups. Comparable body weight values were recorded in the control and treated groups through the study. Food consumption did not show relevant changes through the study. No treatment-related effects were seen at hematology or clinical chemistry analyses. The presence of black staining in the urine sediment was detected and associated with the presence of large MWCNT aggregates due to an external contamination of the urine by the test item retained in the fur and/or excreted in feces. The dark discolouration of skin, muzzle and/or tail was likely due to the colour of the test item. No changes were recorded at histopathological examination of tissues that could be related to the test item. In conclusion, no treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated.