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All relevant and available information on intrinsic properties of MWCNT are cited in chapter 7 of the IUCLID. Although a large number of studies on the toxicity of MWCNTs have recently been published, clear characterization of the test materials including sample preparation which are quite essential for ensuring reproducibility and reliability in the toxicity test using suspension, in vivo and in vitro methods, is often missing. Importantly, modifications of some specific characteristics of the MWCNT tested such as variations in particle size distribution or length might give rise to differences in the toxicological profile. 

Toxic effects of different MWCNTs seem to depend on the form (length) and physico-chemical properties (metal content, aggregation/agglomeration, surface chemistry, and functionalisation). Thus for the time being, a case-by-case approach would be appropriate and the hazard assessment for MWCNT meeting the form described in section 4.5 of the IUCLID dossier is based solely on the information collected with this MWCNT. That is the reason why a comprehensive documentation in the IUCLID dossier and the chemical safety report involved automatically is restricted to all available and relevant data on the MWCNT meeting the form described in section 4.5 of the IUCLID dossier. That means on the other hand that the values used for the risk assessment exercise should not be used in general for all MWCNTs or CNTs, as the results obtained with one particular type of MWCNTs may not necessarily be relevant for other CNTs with other dimensions and properties

In addition the information on MWCNT not meeting the form described in section 4.5 of the IUCLID dossier (the differences can be tracked back to the original papers or because in others the characterization of the materials was unclear or inclomplete) is also included in the IUCLID dossier but the entries are restricted to the information on data source to give a quick overview of all available data.

 

The assessment of the test material meeting the form described in Section 4.5 of the IUCLID relies on data from the guideline studies.

An Ames test with theS. typhimuriumstrains TA 98, TA 100, TTA 102 , A 1535, and TA 1537 with the substance meeting the form described in Section 4.5 of the IUCLID dossier revealed no mutagenic activity in the absence and in the presence of a metabolic activation system (OECD TG 471; Herbold, 2006). A second Ames test according to OECD TG 471 showed also no mutagenic activity for the test substance meeting the form decribed in Section 4.5 of the IUCLID dossier in the absence and in the presence of a metabolic activation system with theS. typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 as well asE. colistrain WP2uvrA (Verbaan, 2010).

In further tests on gene mutation, the test substance meeting the form decribed in Section 4.5 of the IUCLID dossier was investigated at the hypoxanthine-guanine phosphoribosyl transferase locus (HPRT test) in Chinese hamster V79 cells (Entian, 2010) according to OECD TG 476. There was no evidence for induction of gene mutation in mammalian cells.

The clastogenic potential of test substances meeting the form described in section 4.5 of the IUCLID dossier was evaluated in a chromosome aberration test on Chinese hamster V79 cells (Thum, 2006, 2007), in the presence and absence of S9 mix according to OECD TG 473. Based on these test results, the test substance meeting the form described in Section 4.5 of the IUCLID dossier is considered to be not clastogenic for mammalian cells in vitro.  

Whereas the MNvit is a validated assay by now (OECD TG 487, July 2010) and the assay appear to have usefulness in hazard identification the published positive results from cytokinesis-block micronucleus assays conducted with thetest material meeting the form described in Section 4.5 of the IUCLID dossier are disregarded, as the studies show several methodological deficiencies :

 

  • There are no data available to support the validity of a rat lung epithelial (RLE) cell line and human breast carcinome cell line (MCF-7). It is recommended that cell types with a low, stable background frequency of micronucleus formation be used.

 

  • The adequacy of the concurrent positive control (tungsten carbide-cobalt mixture) is questionable. The positive controls should employ known inducers of micronucleus formation at concentrations expected to give small, but reproducible increases over background, and demonstrate the sensitivity of the test system. Positive control substances used should be appropriate for the cell type

 

  • The test substance-induced cytotoxicity was separately determined in several cytotoxicity tests (MTT and LDH release assay) and showed nonuniform results. In addition the suitability of the tests is questionable (e.g. interaction with the dy). Therefore the absence of dose -response relationship may be based on the choice of inappropriate concentrations.

 

  • In addition the authors could not replicate the findings with MCF-7 cells in a second independent experiment (Muller et al., 2008a, b).

 

In an ex vivo micronucleus assay on type II pneumocytes (AT-II cells) conducted by Muller et al. (2008b) female Wistar rats were exposed to the test substance meeting the form described in section 4.5 of the IUCLID dossier by a single intratracheal and then left for 3 days before being killed and isolated and cultured AT-II cells being examined for micronuclei. The authors reported an increase in micronuclei in the test substance treated animals as compared to controls. Positive results were generally confined to the high dose group that was accompanied with signs of marked inflammation in the lungs. In addition there are several methodological problems with this study including: the test was not carried out according to standard protocol, lack of historical data in AT-II cells, lack of data about the viability of the isolated AT-II cells and lack of a valid positive control group. As a result of these flaws in the study the data cannot be interpreted and hence are disregarded.

The micronucleus test from the oral subacute study (0.05 and 0.5 mg/kg) with rats (Rolin et al., 2008a) is not sufficiently documentated and therefore these data are not used for assessment.

Although an in vitro micronucleus (MNvit) assay(Thurnherr et al., 2011)confirms that there is no evidence for in vitro the induction of chromosomal aberration, in vitro and also no induction of DNA damage in A549 cells was observed in a Comet assay(Thurnherr et al., 2011), both assays are considered to be inappropriate for assessing the mutagenic potential of the test material described in Section 4.5 of the IUCLID Dossier, due to several methodological deficiencies .

Additional supportive information are stored under section 13.2 (Additional genetic toxicity information related to IU section 7.6)

 

Justification for classification or non-classification

No classification required according to EU-Directive 67/548/EEC, Annex VI.

No classification required according to Regulation (EC) No 1272/2008, Annex I.