branched and cross-linked multi-walled carbon nanotubes

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date (animal arrival): 23 February 2021
Experimental Completion Date: 16 March 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 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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:

Identification: Athlos ™ 200 Carbon Nano Structures
EC Number: 951-407-3
Batch: V20169
Purity: 98.2%
Physical state/Appearance: Black pellets
Expiry Date: 17 June 2025
Storage Conditions: stored at 15-25°C protected from light.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

Species Selection
The sex and strain of mouse was selected for this study in conformance with the design of the validated test method (OECD Guidelines for Testing of Chemicals Method 429 adopted 22 July 2010).
Animal Specifications and Acclimatisation
Nulliparous, non-pregnant female CBA/CaCrl strain mice were obtained from Charles River (UK) Ltd., Margate. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. The condition of the animals was assessed daily throughout the acclimatisation period of 8 to 15 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 on the day prior to commencement of treatment.
Animals in the main study were in a body weight range of 18 to 21 g on Day 1 of dosing. Individual body weights were within ±20% of the mean body weight for mice on the study. Based on information from the supplier the mice were approximately 9 to 10 weeks old on Day 1.

Environmental Conditions, Diet and Water
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 during acclimatisation. From Day 1 the preliminary animal was singly housed while the Main Study animals were housed in pairs 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 softwood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Covance. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study

Water
Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been 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 Covance.

Diet
5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. 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 Covance.

Environment
The animal rooms were designed to permit a minimum of 15 air changes per hour. The target 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 nesting materials. The nesting materials were removed from the cages prior to dosing on Day 1.

Animal Identification and Assignment to Study
A unique number inscribed onto the tail with indelible ink prior to dose on Day 1 individually identified the mice. A colour-coded card on each cage gave information including study number, animal number and sex.
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. Overtly healthy animals were arbitrarily allocated to the study groups on the day of commencement of treatment.

Study design: in vivo (LLNA)

Vehicle:

other: Corn oil

Concentration:

Low concentration: 1% w/v
Intermediate concentration 2.5% w/v
High concentration 5% w/v

No. of animals per dose:

4 mice per group

Details on study design:

Preliminary Screening test
In the absence of toxicological information regarding irritation and/or systemic toxicity, a preliminary screening test was conducted with one animal. The mouse was treated by daily application of 25 mg of the test article at the maximum suitable concentration (5% w/v in corn oil) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day 1 and prior to termination on Day 6. Both ears were observed for erythema and scored using the following scale:

Observation: Score
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema: 4

Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.

Main Study
Doses were selected from the concentration series 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected on the basis of the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation.

Inlife Procedures
Test Article Administration
Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate negative control or vehicle dispensed from an automatic pipette (0.025 mL/pinna) or test formulation using a metal spatula (nominal 25 mg/pinna).

Treatment Regimen
The five groups of four female mice were subjected to application of the vehicle control, positive control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3.
On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment, the mice were returned to their cages. Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exsanguination under a deep plane of inhalation anasthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.
Clinical Signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article.
Routine Health Checks
All animals were examined at the beginning and end of the working day throughout
the acclimatisation and study periods to ensure they were in good health.
Body Weights
Mice were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of 3HTdR.

Terminal Procedures
Recovery of Lymph Nodes
Once death had been confirmed each mouse was placed on a bench lined with paper with its ventral aspect uppermost. A mid-line incision from the lower jaw to the sternum was made and the cervical structures were exposed by reflecting the skin. The auricular lymph nodes were located and removed using curved end forceps. Any connective tissue was removed from the capsule of the nodes. The auricular lymph nodes of all mice from each dose group were placed into a petri dish containing 5 mL phosphate buffered saline.
Preparation for Scintillation Count
The lymph nodes collected into each petri dish were cut open and disaggregated by squashing the fragments with a sharp blade. The resultant liquor was transferred into code-identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer, debris such as fragments of capsule, were retained in the petri dish wherever possible.
After 5 minutes the pooled liquor was filtered into a second conical tube by transferring the liquor into a 10 mL syringe and passing it through a stainless steel gauze containing a fabric filter, cut to size (Clarcor UK, Lockertex Filtration Products, Warrington, UK). Any visible sediment remaining prior to filtering was left in the conical tube. The liquor was centrifuged at 200 g for 10 minutes. Following centrifugation, the supernatant was discarded and the pellet was resuspended in 5 mL
phosphate buffered saline. This was centrifuged at 200 g for 10 minutes. The supernatant was discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8°C (nominal 4°C).
On the following day the suspension was re-centrifuged at 200 g for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid then subjected to ultrasonic dispersion for 25 minutes to ensure a homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added.
Scintillation Counting
Once prepared the scintillation vials were placed in the appropriate carrier racks. Two background vials were prepared, one containing ca 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and ca 10 mL scintillation fluid. The carrier rack was passed into the scintillation counter. All vials, including the background samples, were submitted for liquid scintillation counting for 10 minutes, using a 3H quench curve.
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

α-Hexylcinnamaldehyde at concentrations of 25%, 25 % and 35 % in acetone in olive oil resulted in stimulation indices of 5.17, 5.54 and 5.42 respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.46

Test group / Remarks:

5 % of Test Article in Applied Formulation (% w/v)

Results 2

Parameter:

SI

Value:

0.98

Test group / Remarks:

2.5 % of Test Article in Applied Formulation (% w/v)

Results 3

Parameter:

SI

Value:

1.09

Test group / Remarks:

1% of Test Article in Applied Formulation (% w/v)

Cellular proliferation data / Observations:

Preliminary Screening Test
Death or signs of systemic toxicity/excessive irritation were not noted.
Based on this information the dose levels selected for the main test were 1%, 2.5% and 5% w/v in corn oil.

Main Test
Mortality
All animals survived treatment with Athlos™ 200 Carbon Nano Structures.
Clinical Signs
There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 1, 2.5 or 5% w/v formulations of the test article. Greasy fur behind the ears and to the back of the neck were observed in all animals.
Body Weights
There was no indication of a treatment related effect on body weight.

Applicant's summary and conclusion

Conclusions:

The Local Lymph Node Assay demonstrated that Athlos™ 200 Carbon Nano structures does not have the potential to cause skin sensitisation under the conditions of this assay.

Executive summary:

SUMMARY
This study was conducted to assess the potential of the test article, Athlos™ 200 Carbon Nano Structures, to cause skin sensitisation in the mouse.

Following a preliminary screening test using a 5% w/v formulation, the test article was prepared for administration at 1, 2.5 and 5% w/v in corn oil.
Groups of four female CBA / CaCrl mice were subjected to topical applications of vehicle control, positive control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.
Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the vehicle control and test groups relative to the corresponding mean scintillation count obtained from the negative control. The
threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

	Concentration of Test Article in Applied Formulation (% w/v)
	Vehicle (Corn Oil)	1%	2.50%	5%
Stimulation Index	1.71	1.09	0.98	1.46

The Local Lymph Node Assay demonstrated that Athlos™ 200 Carbon Nano Structures does not have the potential to cause skin sensitisation.