branched and cross-linked multi-walled carbon nanotubes

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date (animal arrival): 13 April 2021
Experimental Completion Date: 06 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

branched and cross-linked multi-walled carbon nanotubes

Molecular formula:

C

IUPAC Name:

branched and cross-linked multi-walled carbon nanotubes

Test material form:

solid: nanoform, no surface treatment

Specific details on test material used for the study:

Substance: AthlosTM 200 Carbon Nano Structures, also known as Athlos200
Lot number: V20169
Appearance: black pellets.
Received: 16 March 2021
Storage conditions: 15-25°C.
Purity: 98.2%
Expiry date: 17 June 2025

Test animals

Species:

rat

Strain:

other: RccHan:WIST

Sex:

female

Details on test animals or test system and environmental conditions:

Animal Specifications and Acclimatisation
Female (nulliparous, non-pregnant) RccHan:WIST strain rats were obtained from Envigo, Blackthorn, UK. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.
Rats were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages using a sequence that reduced selective bias. The condition of the animals was assessed daily throughout the acclimatisation period of 7 to 9 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups at least one day prior to dosing.
The rats were in a body weight range of 145 to 158 g on Day 1. Based on information from the supplier the rats were approximately 8 to 9 weeks old on Day 1. The weight variation did not exceed ±20% of the mean weight.

Environmental Conditions, Diet, Water and Bedding
The animals were kept in the following conditions except for short periods of time where experimental procedures dictated otherwise.

Housing
The animals were housed in groups of up to five in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014).
Bedding was provided on a weekly basis to each cage by use of clean European soft wood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Labcorp.
Each batch of bedding was analysed for specific constituents and contaminants. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Water
Mains water was provided ad libitum via water bottles. The water was periodically analysed for specific contaminants.
No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Diet
Throughout the study the animals had access to 5LF2 EU Rodent Diet 14%, which was freely available to the animals at all times, except for a period of fasting from the evening of the day prior to dosing (Day-1) until approximately 3 hours after dosing. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Environment
The animal rooms were designed to permit a minimum of 15 air changes per hour. The temperature and humidity ranges were 19 to 25°C and 40 to 70% respectively. Daily recordings of maximum and minimum temperature and humidity were made. Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.

Environmental Enrichment
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.
Environmental enrichment materials were removed during the period of fasting.

Animal Identification
A number written on the tail in indelible ink on the day before dosing individually identified the rats. A colour-coded card on each cage gave information including study number, group number, animal numbers and sex.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

Test Article Formulation
Dose levels were expressed gravimetrically and in terms of test article received (without regard to purity or active content). The test article was dispersed in 1% w/v methylcellulose because the test article did not suspend in purified water.
The maximum attainable was found to be 25 mg/mL in 1% w/v methylcellulose.
The formulated concentrations were calculated from the selected dose level and the dose volume of 15 mL/kg. All formulations were used within two hours of preparation.
Dose containers were repeatedly inverted prior to administration to ensure homogeneity.

Administration
Doses were administered orally, by gavage, using plastic syringes and rubber catheters. Each rat was dosed once on Day 1, by passing the tip of a catheter along the oesophagus and instilling the test article into the gastric lumen.
Individual doses (mL) were calculated using the fasted body weights of the rats on the morning of dosing (Day 1) and the dose volume of 15 mL/kg.

Doses:

Dose level: 300 mg/kg
Dose Concentration: 20 mg/mL
Dose volume: 15 mL/kg

No. of animals per sex per dose:

Sighting study 1 rat
Main study 4 rats

Control animals:

no

Details on study design:

Inlife Procedures
Health Monitoring
All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity.
Clinical Signs
Treated rats were observed closely for clinical signs of reaction to treatment. Clinical signs were recorded immediately post-dose, at approximately 15 and 30 minutes postdose, hourly between 1 and 4 hours post-dose (inclusive), twice daily on Days 2, 3 and 4 and once daily from the fifth to last day of the observation period. Individual records of clinical signs were maintained for each treated rat.
Body Weights
Rats were weighed on Day-1 (day before dosing) and on Days 1, 4, 8 and 15.

Terminal Procedures
Necropsy
Rats were killed on Day 15. Each animal was given isoflurane anaesthesia. Once a suitable deep plane of anaesthesia had been established, the animal was exsanguinated by the severing of major blood vessels. After exsanguination a full macroscopic necropsy was performed and all lesions were recorded.
The necropsy procedure included inspection of external surfaces and orifices, all viscera and tissue within the abdominal, thoracic and cranial cavities, free-hand sectioning of the liver and kidneys and examination of representative sections of mucosal surfaces of the stomach, small and large intestines.
No tissue preservation or histopathological assessment of tissues was undertaken

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: Piloerection was noted in four animals 2 hours after dosing and for one animal lasted up to 4 hours after dosing.

Gross pathology:

No abnormalities were noted at necropsy.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The test article, AthlosTM 200 Carbon Nano Structures, was classified as Category 4 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). This is based on the maximum obtainable dose level of 300 mg/kg (limited by solubility).

Executive summary:

This study was conducted to assess the acute toxicity of the test article, AthlosTM 200 Carbon Nano Structures, following a single oral administration to the rat.
In the preliminary study a fasted female rat was given the test article as a single dose by oral gavage at a dose level of 300 mg/kg.
As the preliminary study animal survived, a further four fasted females were given a single oral dose of test article at a dose level of 300 mg/kg body weight (the maximum attainable dose level based on solubility).
The test article was dispersed in 1% w/v methylcellulose and administered at a dose volume of 15 mL/kg. All animals were killed on Day 15 and subsequently underwent a full necropsy.
There were no deaths.
Piloerection was noted in four animals 2 hours after dosing and for one animal lasted up to 4 hours after dosing.
All surviving rats achieved body weight gains during the first and second weeks of the study.
No abnormalities were noted at necropsy.
The test article, AthlosTM 200 Carbon Nano Structures, was classified as Category 4 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). This is based on the maximum obtainable dose level of 300 mg/kg (limited by solubility).