Registration Dossier

Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Cytotoxicity of multi-walled carbon nanotubes in three skin cellular models: Effects of sonication, dispersive agents and corneous layer of reconstructed epidermis
Author:
Vankoningsloo, S. et al.
Year:
2010
Bibliographic source:
Nanotoxicology 4, 84-97

Materials and methods

Principles of method if other than guideline:
Test substance stock suspensions were diluted at 100 mg/ml in PBS and applied topically for 24 h on reconstructed human epidermises (RHE), at the interface with air. The impact of test substance on the viability of living cells inside RHE was assessed with several toxicological assays described in section 7.9.3 of the IUCLID dossier. Trans-epithelial electrical resistance (TEER) measurements were performed in addition.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
no

Administration / exposure

Details on study design:
DOSE PREPARATION
RHE were incubated for 24 h with PBS (CTL, control), with 1 mg/ml benzalkonium chloride (BC), with dispersing agents alone (HPC or F108), or with 100 µg/ml MWCNTs suspended in water (H2O + MWCNT), 100 µg/ml MWCNTs sonicated in water (H2O + sonicated MWCNT), 100 µg/ml MWCNTs sonicated in dispersing agents (HPC + sonicated MWCNT or F108 + sonicated MWCNT).

MWCNT stock suspensions were prepared by four different protocols:
. protocol i: strong stirring into pure water, raw MWCNTs formed big agglomerates of various diameters, with a peak centred at 1.4 µm.
. protocol ii: gentle sonication (5 W) for 90 min, a second peak spanning around 0.4 µm, which represents 40% in total weight
. protocol iii: sonicating MWCNTs for 90 min into sterile solutions of 1% HPC.
. protocol iv: sonicating MWCNTs for 90 min into sterile solutions of 1% F108. Indeed, virtually all MWCNTs (99.9% in weight) were found as mono-dispersed particles or very small agglomerates, as indicated by narrow peaks around 40 nm in HPC and 30 nm in F108.
TEM analysis confirmed that nanotubes were well isolated in HPC and F108, while water-suspended samples appeared as bundles containing thousands of interweaved nanotubes

APPLICATION OF DOSE:
These MWCNT stocks suspensions were diluted in cell culture medium in PBS (for RHE) to achieve a final concentration of 100 µg/ml before cell incubations.

Results and discussion

Absorption in different matrices:
Test substance meeting the form described in section 4.5 of the IUCLID dossier (MWCNTs suspension and dry powder) is not able to enter epidermis through the corn barrier within 24-48 h, at least not at a sufficient amounts to generate measurable cytotoxicity.

Applicant's summary and conclusion

Executive summary:

The effects of multi-walled carbon nanotubes meeting the form described in section 4.5 of the IUCLID dossier were investigated in reconstructed human epidermises. (RHE) Carbon nanotubes were subjected to dispersion protocols leading to different agglomeration states. No significanttrans-epithelial electrical resistance (TEER) drop was observed, indicating that nanotubes meeting the form described in section 4.5 of the IUCLID dossier do not disconnect the tight intercellular junctions in epidermis within 24 h of exposure.

RHE were also incubated for 24 h with 2.5 mg/cm² dry multi-walled carbon nanotubes meeting the form described in section 4.5 of IUCLID dossier and then assayed for TEER. MWCNT powder did not alter this parameter.