Single Wall Carbon Nanotubes (SWCNT)

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Apr - 22 Oct 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted in 2009

Deviations:

yes

Remarks:

Intratrachial administration of test material

GLP compliance:

yes

Test type:

traditional method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

943-098-9

Cas Number:

7440-44-0

Molecular formula:

Carbon. Morphological features: - Tube cylindrical surface is formed by 6-membered rings consisting of carbon atoms linked by a double bond. - Carbon nanotube chirality is random. - Tube length: 1 - 10 μm. - Agglomeration: CNTs tend for form bundles

IUPAC Name:

carbon

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:sd

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Barcelona, Spain
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: mean of groups was 402.4 - 415.0 g
- Fasting period before study: no
- Housing: 5 per cage
- Diet: Global Diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 26 days
- Microbiological status: Specific Pathogen Free (SPF)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 25.6
- Humidity (%): 20 - 63
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Apr 2015 To: 1 Jun 2015

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

other: intratracheal instillation

Vehicle:

other: PBS-1% Tween 80

Details on inhalation exposure:

Preparation of test material:
SWCNT were grinded for approximately 68 h using a ball mill with the aim of reducing the size of particles to make them suitable to be administered intra-tracheally. Then, from the grinded SWCNT, the appropriate amounts for dosing group A (sighting test) and E (main test, high dose) were weighed and resuspended in an appropriate volume of the vehicle (i.e 10 mg were weighed for each mL of the solution/suspension for treatment of group A at 1 mg/animal during the sighting test). SWCNT solution for groups D (mid dose) and C (low dose) were prepared by appropriate serial dilutions of the SWCNT solution for group E (high dose).

ADMINISTRATION OF THE TEST MATERIAL
Animals were anaesthetized with isoflurane and placed on their backs against an angled restraining stand. Illumination with a cold light to the ventral part of the neck was provided to assist the administration. A 0.5 mm diameter-guiding, ball tipped device was then inserted into the mouth and placed into the lumen of the trachea. A 16G Teflon cannula was inserted into the trachea through the guiding device. Once the cannula was placed into the trachea, the guiding device was retrieved. Confirmation of cannula placement into the trachea was provided by coupling an ambu-like device into the syringe lumen and confirmation of inflation. Thereafter, the corresponding volume of Test item/vehicle was administered through the lumen of the cannula with the aid of an automatic pipette. Confirmation of syringe placement into the trachea was performed again after administration with the ambu-like device as described before. Finally, a bolus of approximately 400 µL of air was introduced through the cannula to wash out the remaining Test item/vehicle and to restimulate normal breathing of animals.

Analytical verification of test atmosphere concentrations:

no

Remarks on duration:

single instillation

Concentrations:

Sighting test: 1 mg/animal
Main test: 0.1, 0.5, 1 mg/animal

No. of animals per sex per dose:

Sighting test: 5
Main test: 15 (5 per terminal time point)

Control animals:

yes

Details on study design:

Sighting test:
Body weight and clinical observations were recorded during 14 days. Selections of the doses to be administered during the main test were based on toxic effects observed on body weight and clinical observations recorded during sighting test.

Main test:
- Duration of observation period following administration: 3, 7 and 28 days
- Frequency of observations and weighing: Body weight was recorded during group distribution, on the day of administration prior to treatment and then once weekly during the observation period until sacrifice. Additionally, animals were weighed during sighting test on day 4 (3 days after administration) and day 8 (7 days after administration).
- Necropsy of survivors performed: yes
All animals were exsanguinated via abdominal aorta under phentobarbital anaesthesia (75 mg/kg i.p.) and subjected to a gross necropsy consisting in the examination of the abdominal and thoracic cavities and contents.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant. Additionally and only for animals belonging to the main test, selected organs were weighed, collected and preserved in appropriate fixative medium for further histopathological analysis.
Organs weights: heart, liver, brain, kidneys (weighed together) and spleen
Tissues histopathologically examined: lungs (right lobe used for BALF analysis, left lobes), trachea, larynx and esophagus, tracheobronchial lymph nodes, heart, liver, kidneys, spleen, brain (including sections of cerebrum, cerebellum, and pons/medulla), diaphragm, nasal cavity
- Clinical signs including body weight: Detailed clinical observations in response to treatment were performed approximately 1 hour after administration and weekly thereafter.
- Other examinations performed: On the day of sacrifice, blood was collected by retroorbital puncture prior to sacrifice to animals under isoflurane anaesthesia.
Haematology paratmers examined: White blood cell count (WBC), differential leukocyte counts, red blodd cell count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), mean platelet volume (MPV)
Clinical chemistry parameters examined: albumin (ALB), total proteins (TP), globulins (GLOB), cholesterol (CHOL), triglycerides (TRIGL), glucose (GLUC), urea (URE), total bilirubin (BIL), creatinine (CRE), alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), calcium (Ca2+), chloride (Cl-), inorganic phosphorus (PO43-), potassium (K+), sodium (Na+)

Statistics:

Provided that the sample size was considered adequate, the following parameters were subject to statistical analysis:
- Body weight and body weight gain
- Haematological parameters
- Biochemical parameters
- Absolute and relative organ weights
- BALF biochemical parameters
- Differential cell count (percentage of each cell type) on BALF smear (performed at the test site)

For analysis of data from 2 groups, a test of normal distribution was performed. If the test was positive, an un-paired t-test followed, if not, a Mann-Whitney test was performed. When a test of equal variance was negative, an un-paired t-test with Welch’s correction was performed.
For analysis of data from more than two groups, an one-way ANOVA was performed when a test of normal distribution was positive. The ANOVA was followed by a Dunnett’s post test (comparison versus control group) and/or a Tukey post test (comparison of all groups). If the test of normal distribution was negative, a Kruskal-Wallis and a Dunn’s post test was performed. If a test of equal variance was negative, the same procedure was applied.

A value of P<0.05 was considered statistically significant.

Results and discussion

Preliminary study:

No clinical signs were observed after administration of the test material during the observation period (14 days) in the sighting test (group A, 1mg/animal). Animals belonging to the sighting test gained weight normally after administration of 1 mg/animal.

Black spots in lungs were observed in 3/5 animals.

Effect levels

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

> 1 other: mg/animal

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No unscheduled mortality was recorded and all animals survived the scheduled observation period.

Clinical signs:

other: No relevant clinical sign were observed during the main test, with the exception of loud breathing, recorded 1h after vehicle or test material administration. This effect was transient but the number of animals showing loud breathing increased with dose.

Body weight:

- Sighting test group: The animal gained weight normally after administration.
- Main study: No statistically significant change in body weight was observed during the main study in all dose groups compared with animals administered with vehicle. 7 days after treatment, a statistically significant reduced body weight gain was observed in animals of the high dose group. This reduced body weight gain in animals of the high dose group was also observed at later time points (14, 21 and 28 days after treatment), although the differences did not reach statistical significance.

Summarized data can be found in the Attached background material.

Gross pathology:

Necropsy findings:
Black spots in the lung of test animals were recorded at necropsy. The incideces are summarized in Table 1.
Black spots were recorded in both left and right lung in all animals, with the exception of one animal in the 0.5 mg/animal-Group, where black spots were only observed in the left lung. Macroscopic black spots in lungs correlated microscopically almost in all cases with the presence of macrophages phagocytising blackish material (i.e carbon nanotubes). This finding is considered an indication that the test item reached the lung and is not considered as an adverse event. By contrast, black spots in the lungs of 1 animal in the 0.1 mg/animal group (3 days allocation) and 2 animals of the vehicle group (28 days allocation) found no microscopical correlation during histopathological analysis.
Summarized data can be found in the Attached background material.

Organ weights:
No biologically relevant differences were found in any of the absolute or relative weights of the organs. Statistical significant differences were incidental and not accompanied by histopathological findings and were therefore considered incidental.
Summarized data can be found in the Attached background material.

Other findings:

Haematology
No biologically relevant differences were found in any of the treatment groups when compared to the vehicle control group. Statistical significant differences were considered incidental because mean values were within normal range for animals of this strain and age. Furthermore, no correlated adverse findings or dose-dependent effects were observed in any of these parameters.

Summarized data can be found in the Attached background material.

Clinical chemistry
No biologically relevant differences were found in any of the treatment groups when compared to the vehicle control group. Statistical significant differences were considered incidental because mean values were within normal range for animals of this strain and age. Furthermore, no correlated adverse findings or dose-dependent effects were observed in any of these parameters.

Summarized data can be found in the Attached background material.

Histopathology
Lungs: Alveolar macrophages phagocytising blackish material were observed in all animals treated with the test substance. The grade was minimal (0.1 mg/animal), minimum to slight (0.5 mg/animal and 1 mg/animal). The incidence and group mean severity of this finding in each sacrifice point increased with dose. Independently of the experimental group, the incidence and severity decreased or tended to decrease over time.
Increase and group mean severity of granuloma and/or granuloma-like focus at bronchioalveolar area increased over time in all test item treatment groups.
Hypertrophy of bronchial epithelium was recorded at a minimum or slight severity in the all test item treatment groups examined at sacrifice point day 3. This finding was no longer present at sacrifice points days 7 and 28, with the exception of 1 animal from group D from the 7 days allocation.
Minimum to slight hypertrophy of bronchiolar and/or alveolar epithelium was recorded in all test groups and the vehicle group.
Focal or multifocal inflammatory cell infiltration in alveoli was recorded sporadically at a minimum in all groups including the control group, independently of the sacrifice allocation.
All these findings in lungs were considered to be indicative that the physiological reactions for eliminating foreign substance from the lung were progressing. No other degenerative or proliferative changes that could be attributable to the test item were observed. All other findings in lungs were considered incidental.

Trachea
Mucosal and submucosal inflammation of the trachea as well as hypertrophy of tracheal mucosal epithelium were recorded in all groups including group B (vehicle) at the 3 days allocation. This finding was not recorded in any animal belonging to the 7 or 28 days allocation. These changes were considered a consequence of the instillation procedure and therefore, not related with the test item.

Larynx
Mucosal and submucosal inflammation of larynx was found in the vehicle and in the low-dose group at the 3 day allocation. These changes were considered a consequence of the instillation procedure and therefore, not related with the test item.

Summarized data can be found in the Attached background material.

BALF analysis
Biochemical
No statistically differences were observed in GGT, ALP or LDH activity from the bronchoalveolar fluid from animals treated with the test substance at any dose (0.1, 0.5 or 1 mg/animal, groups C, D and E respectively) when compared to animals administered with vehicle (group B).

Differential cell count
Statistical significant changes were observed in the following parameters:
- A dose-dependent increase in macrophages phagocytising blackish material was observed in all SWCNT-treated groups (groups C-E), independently of the allocation. This increase reached statistical significance when animals treated with the test material at 0.5 mg/animal (group D) or 1 mg/animal (group E) were compared to animals treated with vehicle (group B).
- a dose-dependent increase in neutrophils was observed in all SWCNT-treated groups (groups C-E) in all allocations. This increase only reached statistical significance when animals treated with the test material at 1 mg/animal (group E) were compared to animals treated with vehicle (group B).
- A statistically significant increase in lymphocyte %ratio was found in the 3 days allocation when animals treated with SWCNT at 1 mg/animal (group E) were compared to animals treated with vehicle (group B).

The ratio of macrophages phagocytising blackish material and neutrophils showed a trend to decrease over time. This decreasing trend over time reached statistically significance in the high dose group (group E, 1 mg/animal) when animals from the 7 days or 28 days allocations were compared to animals belonging to the 3 days allocation. This fact could be interpreted as if the test material-mediated inflammation is transient and tend to decrease over time. In addition, the increase in the ratios of neutrophils and lymphocytes did not translate in any histopathological change in the lungs, which suggest that changes found in BALF differential cell counts have no toxicological relevance.

Summarized data can be found in the Attached background material.

Any other information on results incl. tables

Table 1: Incidences of black spots in lungs

Group (mg/animal)	Sighting test	Main test (allocation
		3 days	7 days	28 days
A (1)	3/5
B (0, vehicle)		0/5	0/5	2/5
C (0.1)		0/5	1/5	2/5
D (0.5)		2/5	4/5	3/5
E (1)		1/5	4/5	1/5

Applicant's summary and conclusion

Interpretation of results:

other: According to the classification criteria of the CLP Regulation (EU) No. 1272/2008, no classification is required.

Conclusions:

The test was conducted under GLP conditions. No guideline was followed.

The toxicity of the test item was evaluated after one single intra-tracheal instillation to male Sprague Dawley rats in a two-step procedure: In a sighting test, 5 rats were administered a single dose of 1 mg/rat. In the main test, groups of 15 rats were administered 0.1, 0.5 or 1 mg test material/animal. 5 rats of each group were sacrificed at different times after administration (3 days, 7 days and 28 days).
It was concluded that under the assayed conditions, intra-tracheal administration of the test substance did not induce major toxicological effects. No LD50 could be determined.