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Description of key information

Skin irritation
The skin irritation potential of charcoal was evaluated in an in vitro study using the human skin model EpiDermTM as well as in an in vivo skin irritation test in rabbits.
While the in vitro human skin model test was conducted with all three charcoal test samples Probe 1 (C-Fix=73.3%), Probe 2 (C-Fix=80.5%), and Probe 3 (88.7%), the in vivo test was conducted with Probe 2 only.
Eye irritation
The skin irritation potential of charcoal was evaluated in an in vitro study in incubated chicken eggs, the HET-CAM test, as well as in an in vivo eye irritation test in rabbits (Draize test).
While the HET-CAM test was conducted with all three charcoal test samples Probe 1 (C-Fix=73.3%), Probe 2 (C-Fix=80.5%), and Probe 3 (88.7%), the in vivo test was conducted with Probe 2 only.
Respiratory irritation
Respiratory irritation caused by charcoal was not studied.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation

In vitro human skin model test

This in vitro human skin model test was performed according to EU method B.46. For conducting the test, 25 mg of the respective test sample was spread on the surface of three sections of an EpiDermTMtissue and incubated for 60 min. The negative control consisting of Dulbecco’s phosphate buffered saline was applied to another three tissue sections. As positive control a SDS-solution (50 g/L) was used for three other tissue sections. After rinsing and two incubation steps of altogether 42 h an enzyme assay to determine cell viability was performed. The tissues were incubated with MTT-reagent for 3 h. The tissues were washed with PBS, dried and transferred to a 24-well-plate. After incubating with isopropanol at room temperature over night, the concentration of the formazan reaction product was measured in a spectral photometer at 570 nm. The mean percentage values of formazan production were calculated in comparison to the negative control.

After treatment with charcoal samples Probe 1, Probe 2, and Probe 3, the relative absorbance was reduced to 70.4%, 82.9%, and 89.4% of the negative control values. These values were well above the threshold for irritation (50%) used in this test. A concurrently performed positive control showed a reduction of formazan production to 7.8%.

Accordingly, the test item charcoal was considered as not irritant to skin under the experimental conditions used in this study.

In vivo skin irritation test

The potential skin irritant properties of charcoal Probe 2 were investigated in an in vivo skin irritation study in 3 male New Zealand White rabbits. The study was performed according to OECD guideline no. 404.The test item (particle size: ≤ 500μm) was applied occlusively to the intact skin of rabbits in a single dose of 0.5 g. The irritation symptoms were examined at 1, 24, 48, and 72 h after patch removal.

No primary irritation symptoms, such as erythema and oedema, or other signs occurred during the observation period. 72 h after treatment, the study was terminated, since there were no primary irritation symptoms.

During the study, the general state and behaviour of animals were normal. There were no notable body weight changes during the contact and observation period.

Accordingly, the test item charcoal was considered to be non-irritant to the skin of rabbits under the conditions of this present study.

 

Eye irritation

In vitro HET-CAM test

This in vitro study (HET-CAM) was performed to assess the irritating potential of charcoal (Probe 1: C-Fix = 73.3%). The respective test item (46-63 mg) was applied to the surface of the chorioallantoic membrane (CAM) of 6 freshly laid hen’s eggs which had been incubated at 38.5 °C and 58-60% humidity for 9 days. The occurrence of vascular injection, hemorrhage, and coagulation was observed and recorded over a period of 5 min after application of the test item. Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the blood vessels of the(mean irritation score, IS = 0.00). Solutions of SDS (1%) and NaOH (0.1 N) were used as positive controls. The positive controls induced a severe irritation on the blood vessels with IS of 11.25 and 19.99, respectively.

None of the three charcoal test samples showed any effects on the blood vessels of the. In each case, the calculated mean IS was 0.00.

In conclusion, it can be stated that the test charcoal did not show any eye irritation potential under the conditions used in these HET-CAM tests.

In vivo eye irritation test

The eye irritation properties of charcoal Probe 2 were evaluated in an in vivo eye irritation study in 3 male New Zealand White rabbits. The study was conducted according to OECD guideline no. 405.

The test item (particle size: ≤ 500μm) was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. 0.1 g of the test item was used for the study in pure state, as a single dose. The eyes of the test animals were not washed out after the application of test item.

The eyes were examined at 1, 24, 48, 72 h, and then 1 week after the application.

1 h after the single application of the test item, slight redness, moderate discharge and slight chemosis occurred. 48 h after treatment, slight redness, slight chemosis and severely increased discharge were detected in animals. One animal became symptom-free by this observation time. 72 h after treatment two animals were symptom-free. One animal had slight redness and chemosis, as well as moderately increased discharge. At 1 week after treatment the study was terminated, as all animals were free of symptoms of irritation. Iris and cornea irritation was not found during the study.

During the study, the control eyes of animals were symptom-free.

General state and the behaviour of animals were normal throughout the study period. There were no notable body weight changes during the contact and observation period.

In conclusion, the test item charcoal, applied to the rabbits' eye mucosa, caused slight to severe conjunctival irritant effects, fully reversible within one week. Cornea and iris showed no irritant reaction.


Effects on eye irritation: slightly irritating

Justification for classification or non-classification

Based on the in results of the skin and eye irritation studies, charcoal is not classified as a skin or eye irritant as defined in the current EU-CLP regulation.

A classification of charcoal for respiratory irritation is not possible, as no studies with charcoal were conducted for this endpoint.

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