General properties (Specifications) of C60 and their safety assessment in animals
Fullerenes, first discovered by Kroto et al. (1985), are carbon allotropes similar in structure to graphene but rolled up to form closed-cage, hollow spheres. Diamond and graphite are the other allotropes of carbon. Graphite contains layers of carbon atoms. The layers slide over each other easily because there are only weak forces between them, making graphite slippery. Diamond has a giant molecular structure. Each carbon atom is covalently bonded to four other carbon atoms. A lot of energy is needed to separate the atoms in diamond. This is because covalent bonds are strong, and diamond contains very many covalent bonds. The structures of C60 fullerene, graphite and diamond are shown in Figure 1.
Carbon60 (C60) fullerenes have further gained considerable attention due to their anti-oxidant and radical scavenging properties. Their current applications include targeted drug delivery, energy application, polymer modifications and cosmetic products. The production of fullerenes and their use in consumer products is expected to increase in future [2, 3]. The physical properties of C60 fullerene are given in the Table 1.
C60 fullerenes are found in nature. They are naturally produced by combustion in events such as forest fires and volcanic eruptions. Coal fired power plants are also a source of fullerene exposure. Spectroscopic studies have been able to detect a mixture of C60 and C70 in the soot from hydrocarbon combustion. Coal fired power plants are also a source of fullerene exposure. Naturally-occurring fullerenes were detected in the 1990s in materials affected by high energy events such as lightning strikes, meteors and meteor-impacted or metamorphic materials, and in geologic samples. C60 occurs in soot generated by combustion of hydrocarbons and oxygen, commercially-available charcoal, and soot produced by candle flames [3-9].
Biological applications of C60 is well documented in the literature [10-13]. It is also reported that C60 is 250 times stronger antioxidant than Vitamin C . Lipophilic fullerene C60 has only been reported to be safe in animals even at high dosage levels till to date [10, 11].
In a study by Takahashi group to obtain information on the possible repeated-dose oral toxicity of fullerene C60, rats were administered fullerene C60 by gavage once daily at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days, followed by a 14-day recovery period. It is reported that no deaths occurred in any groups, and there were no changes from controls in detailed clinical observations, body weights, and food consumption in any treatment groups. Moreover, it was observed that no treatment-related histopathological changes were found in any organs examined at the end of the administration period and at the end of the recovery period. However, blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. There were no changes from controls in the liver and spleen weights at the end of the administration period, but those weights in males in the 1,000 mg/kg/day group increased at the end of the recovery period .
To address the inhalation toxicity and lung toxicokinetics of C60 fullerene nanoparticles and microparticles, male rats were exposed to C60 fullerene nanoparticles (2.22 mg/m3, 55 nm diameter) and microparticles (2.35 mg/m3, 0.93 micron diameter) for 3 h a day, for 10 consecutive days using a nose-only exposure system. Direct observation of inflammation, total and neutrophilic cell counts in bronchoalveolar lavage fluid (BALF), and cytokine amounts in BALF have been used as indicators of alveolar inflammation. For the nano-sized C60 fullerenes, there were no significant differences in BALF cytokines in rats. Although a rise in BALF protein concentration was noted, this could not be considered as an adverse effect because a slight increase in protein concentration does not directly indicate a toxic effect. At necropsy, no gross or microscopic lesions were observed in either group of C60 fullerene exposures rats .
Toxicokinetic investigations are considered important to identify possible target organs for fullerenes toxicity following exposure via inhalation, oral or dermal exposure. Published studies as shown in Table 2, indicate that the minimal exposure of C60 fullerene without any modifications is not toxic to the living cells .
“Companion60” is LivePet’s first health product that contains fullerene C60 as one of the components. To evaluate the safety, toxicity and efficacy of Companion60 and thus fullerene C60, a clinical trial was conducted with aged dogs to test the safety and efficacy of Companion60 and compared to over-the-counter anti-inflammatory products. This controlled laboratory study demonstrated the safety and efficacy of a test compound on the quality of life of aged dogs when compared to over the counter (OTC) anti-inflammatory products. The study consisted of 35 dogs (5 per group) randomized to seven treatment groups. Dosing with the test compound was well tolerated at all dose levels (0.5X, 1X, 3X and 5X), 5X being 2.5 mg/Kg, when administered for three consecutive days and then every other day for a total of 19 dose administrations. No adverse effects related to administration of the test compound were noted in any groups. No notable changes in body weights, food consumption, blood oxygen saturation levels, clinical pathology or urinalysis were noted. In conclusion, administration of the test compound at 0.5X, 1X, 3X and 5X therapeutic dose, did not result in any toxicity to the dogs .
Another controlled laboratory study was conducted using “Companion60” to determine an optimal dosing regimen of “Companion60” for a consistent reduction in systemic inflammation as determined by a decrease in pro-inflammatory cytokines. The study consisted of 15 dogs (5 per group) randomized to one of three treatment groups. Dosing with the test compound was well tolerated and palatable at all dose levels when administered daily for 28 consecutive days. The therapeutic dosage level was 0.125 mg/Kg which was 20 times lower than the highest dosage in the first trial . No adverse effects related to administration of the test compound were noted in any groups. No notable changes in body weights or urinalysis were noted in any groups .
Carbon60 (C60) present in the lipofullerene and thus in Companion60 acts as an anti-oxidant and free radical scavenger helping to maintain overall good health. Fullerene C60 is also known to be able to inactivate hydroxyl radicals by attaching to the double bonds. The majority of reactive oxygen species (ROS) are generated in the mitochondrial respiratory chain and the presence of C60 inside the cells helps remove ROS. Water-soluble derivatives of buckminsterfullerene (C60) indicated that they are capable of eliminating both superoxide anion and H2O2, and were effective inhibitors of lipid peroxidation, as well. Syntheses of water soluble fullerene derivatives and their enhancing effect on neurite outgrowth in NGF-treated PC12 cells has been reported in the literature. Fullerene-based antioxidants demonstrated robust neuroprotection against excite-toxic, apoptotic and metabolic insults in cortical cell cultures. They were also capable of rescuing mesencephalic dopaminergic neurons from both MPP+ and 6-hydroxydopamine-induced degeneration. Therefore, the lipofullerene present in Companion60 acts as a free radical sponge, decreases the cellular stress, improves immunity and thus it is safe in animals and improves the overall quality of life of animals [10-19].
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- Clinical Trial 1 by LivePet “Safety and Efficacy of a Test Compound (Companion 60) on the Quality of Life in Dogs Over 3 Years of Age”, LRI Study Number: 14.5155.001, Nov 2014.
- Clinical Trial 2 by LivePet, “Determination of an Optimal Dosing Regimen of a Test Compound (Companion 60) for a Consistent Reduction in Systemic Inflammation”, LRI Study Number: 14.5155.002, May 2015.
Table 1. Physical properties of C60 fullerene
Table 2. Summary of hazard assessment of fullerenes.
Figure 1. Structural comparison of (a) C60 fullerene and (b) graphite.
(a) C60 Fullerene