Elsevier

Toxicology

Volume 225, Issue 1, 1 August 2006, Pages 48-54
Toxicology

Preclinical studies on safety of fullerene upon acute oral administration and evaluation for no mutagenesis

https://doi.org/10.1016/j.tox.2006.05.001Get rights and content

Abstract

Fullerenes characterized as an antioxidant are believed to reduce various reactive chemical species, such as free radicals, and their characteristic features have been disclosed to furnish many useful medical technologies. Despite the numerous applications for the biological efficacy of fullerenes, less is known about the toxicity of fullerenes in mammals. Hence, the protocol was designed to determine the acute oral median lethal dose and evaluate the acute toxicity of fullerenes when administrated as a single dose to Sprague–Dawley rats. In an acute toxicity test, fullerenes were administered once orally to a single group of male and female at a dose level of 2000 mg/kg. No deaths were observed and the body weights in both sexes of 2000 mg/kg group increased in a similar pattern to the control group. Genotoxicity of fullerenes was also assessed in a bacterial reverse mutation assay (Ames test) and the chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells. Although structural chromosomal aberrations were induced at up to 5000 μg/mL, there was no significant increase in the frequency of chromosomal aberrations at any dose level regardless of presence of S9. Fullerenes did not cause genetic damage in Salmonella typhimurium TA100, TA1535, TA98 and TA1537 and Escherichia coli WP2uvrA/pKM101. These results indicate that fullerenes are not of high toxicological significance.

Introduction

Despite a wide range of attempts on the biological potency of fullerenes, less is known about the toxicity in mammals. Fullerene arranging the 60 carbon atoms with 30 carbon–carbon (C–C) double bonds and 60 C–C single bonds as shown in Fig. 1 was discovered in 1985 (Kroto et al., 1985). Intense interest has been focused on this unique molecule having spherical symmetry in the last two decades for the characterization with chemical and biological properties of fullerene (Krusic et al., 1991, Arbogaast et al., 1991, McEwen et al., 1992). Various water-soluble fullerenes were prepared by the use of chemical modification of hydrophobic fullerene to widely explore biological activities. It is reported that several water-soluble fullerenes indicated low cytotoxicity while displaying their biological efficacy both in vitro (Tsuchiya et al., 1995, Baierl et al., 1996, Dugan et al., 1997) and in vivo (Yamago et al., 1995, Dugan et al., 1996, Satoh et al., 1997). Fullerene was recognized as a free radical scavenger and water-soluble fullerenes were proved to reduce the level of intracellular peroxidation (Xiao et al., 2005). Attempts for pharmaceutical technologies with a series of water-soluble fullerenes have been explored for their potential as anti-HIV (Schinazi et al., 1993, Toniolo et al., 1994, Rajagopalan et al., 1996) and anticancer (Chiang et al., 1995, Miyata and Yamakoshi, 1997) agents. Quite recently, Moussa have reported that pristine C60 is a powerful liver-protective agent against free-radical-mediated liver injury (Gharbi et al., 2005).

For the promising pharmacotherapeutic application, sufficient data regarding in vitro and in vivo toxicological studies of fullerenes should be accumulated. Thus, we focused our attention on the mixture of C60 and C70, a more practical industrial composition of fullerenes (or fullerite), and we have designed a protocol to determine the acute oral median lethal dose and to investigate the acute toxicity of fullerenes.

Section snippets

A single dose oral toxicity study of fullerenes in rats

Fullerenes (the mixture of C60 and C70, fullerite), sublimed technical grade, purity: 99.5%, were supplied from Vitamin C60 BioResearch Corp. (Tokyo, Japan).

Acute toxicity

Compound-colored stools and mean body weights in Sprague–Dawley rats for the duration from 1- to 15-day after administration at the level of 2000 mg/kg of fullerenes were measured (Fig. 2(A) and (B)). As for absolute body weights of the 2000 mg/kg, there were no differences as compared with the control group (0 mg/kg). Compound-colored stool was found at 6 h after administration in four males and one female of the 2000 mg/kg group, as well as in one male of the 2000 mg/kg group on day 2. No changes

Discussion

Fullerenes were administered once orally to both sexes at a dose level of 2000 mg/kg. The control group that received a vehicle only was also arranged. During the observation study period, no deaths were recorded in either sex of the control or 2000 mg/kg group. All groups gained weight in a similar pattern with the control group. Administration of fullerenes did not induce abnormalities in the necropsy. In the observation of clinical signs, compound-colored stool was noted on day 1 in both sexes

Acknowledgments

The authors are grateful to Mitsubishi Chemical Safety Institute Ltd., for technical assistance with analysis and metabolism. The mammal treatment, and measurement were completed by Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd., Japan (GLP, Standards for Conduct of Non-clinical Studies on the Safety of Drugs, MHW, Ordinance no. 21, March 26, 1997).

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