6.1. General
Fungal beta-1,3/1,6-glucans have a 3000-year long history as a medical treatise in traditional Eastern medicine, a practice also adapted in clinical cancer therapy in Japan today (Noda et al. 1992). The research on beta-1,3/1,6-glucans in the Western Hemisphere has mainly focused on beta-1,3/1,6-glucans from baker’s yeast (Saccharomyces cerevisiae). The compound is regarded as very safe in use when administered orally or topically. It is isolated from, and a major part of, a GRAS organism and it is composed solely of glucose units. For years, beta-1,3/1,6-glucans isolated from yeast have been used as dietary supplements for humans (Bell et al. 1999) and for topical applications (Zülli et al. 1997) with no reports of adverse or toxic effects. The few toxicological events observed after administration of yeast beta-1,3/1,6-glucans are restricted to intravenous or intraperitoneal injection of beta-1,3/1,6- glucans (Di Luzio et al. 1980; Takahashi et al. 2001; Williams et al. 1996).
6.2. Experience from practical use
More than 30 different scientific papers investigating primarily the efficacy, but to a certain extent also the safety, of yeast beta-1,3/1,6-glucan have been published in peer reviewed journals. These papers describe several hundreds of experiments where the yeast beta-1,3/1,6-glucan had been administered to the animals by injection, in feed, by intubation, by immersion, in the water supply etc. So far, no reports have indicated any toxic effect of the beta-1,3/1,6-glucan preparations. In addition to published papers, feed companies have carried out more than 100 feeding trials to establish the optimal feeding regimes for implementing the yeast beta-1,3/1,6-glucan in feed to animals like pigs, calves, broiler, dogs, cats, rabbits, several fish species, and larvae and juveniles of many aquatic species. In some trials, the beta-1,3/1,6-glucan was fed throughout the whole life cycle of the animal. In addition, yeast beta- 1,3/1,6-glucan has been used as a dietary supplement for several years. On this background it seems safe to assume that the yeast beta-1,3/1,6- glucan is not encumbered with any toxicological side effects.
6.3. No immunogenicity
In purified form beta-1,3/1,6-glucans are not immunogenic—no antibody can be raised against this molecular structure. Immunogenicity is very important for safety evaluations of pure products, in particular with regard to allergy. Antibodies that have been raised against fungal and yeast preparations of beta glucan are therefore always directed against other constituents in the preparations, such as mannose-proteins in yeast and other glycoproteins in mushrooms.
6.4. Formal safety studies: Basis for clinical trials
6.4.1. Micro-particulate yeast beta-1,3/1,6-glucan
A parallel, double blind, placebo controlled study with 140 individuals with mild to moderate hypercholesterolemia, but otherwise healthy, showed no adverse reactions or signs which indicate any safety concerns related to a daily intake for 8 weeks of 0.7 grams of a yeast beta-1,3/1,6-glucan preparation (Ref. Biotec Pharmacon ASA, Tromsø, Norway). The same product has been used for years as supplement for humans, and no reports on adverse effects have been reported.
6.4.2. Soluble native beta-1,3/1,6-glucan from yeast
The following independent toxicity tests (Covance Laboratories Ltd. England) of soluble native yeast beta-1,3/1,6-glucan have been completed: 1) “Reverse mutation in five histidine-requiring strains of Salmonella typhimurum” (52 pages); 2) Induction of chromosome aberrations in cultured Chinese hamster ovary (CHO) cells (46 pages); 3) Single dose intravenous toxicity study in the mouse—estimation of approximate lethal dose (22 pages); 4) Single dose intravenous toxicity study in the rat—estimation of approximate lethal dose (23 pages); 5) 28 day oral (Gavage) administration toxicity study in the rat (188 pages). The results showed no toxicity in any of these tests which were carried out according to Good Laboratory Practice. Even at intravenous dosages as high as 400 mg/kg in mice or rats, there was no mortality and no clinical signs of reaction to the product (Ref. Biotec Pharmacon ASA, Tromsø, Norway).
Subsequently, a human safety test has been carried out at the National Hospital in Oslo with healthy volunteers. No toxicity or any side-effects was reported with healthy volunteers receiving 100, 200, and 400 mg/day orally for 4 consecutive days (Ref. Biotec Pharmacon ASA, Tromsø, Norway). Based on completed and satisfactory safety documentation of this product, it is accepted for clinical trials in humans.
Prior to these safety studies, several clinical safety studies have been performed with the poly(1-6)-B-D-glucopyranosyl-(1-3)-B-D-glucopyranose (PGG-glucan) named Betafectin provided by Alpha-Beta Technology, Inc., Worcester, MA, USA. The investigational drug PGGglucan was derived from a proprietary, non-recombinant yeast strain of Saccharomyces cerevisiae. Initial in vitro characterizations of the product had revealed a high affinity for receptors of human monocytes and neutrophils. Clinical trials were conducted with the PGG-glucan in healthy volunteers and the results showed that a single intravenous dose of 0.05 to 2.25 mg/kg was safe and well tolerated. Clinical assessments of physical conditions, vital signs and electrocardiograms showed no clinically significant abnormalities, and the product did not produce persistent fever, nausea, myalgia or bone pain. There were transient increases in total white blood cell counts, and in the monocyte and neutrophil counts, indicating a possible clinically useful response. Furthermore, at doses of 0.5 mg/kg and 2.25 mg/kg, an increased neutrophil and monocyte microbial killing activity against S. aureus was observed.
A: A soluble beta-1,3/1,6-glucan has been subjected to pre-clinical safety evaluation in mice, rats, guinea pigs and rabbits (Williams et al.1998). ICR/HSD mice and Harlan Sprague-Dawley rats received a single i.v. injection of soluble glucan in doses ranging from 40 to 1000 mg/kg. Soluble glucan administration did not induce mortality, appearance or behavioural changes in mice or rats. In subsequent studies, mice and guinea pigs were injected i.p. with glucan (250 mg/kg) for 7 consecutive days. ICR/HSD mice gained weight at the same rate as the saline-treated controls. In contrast, guinea pigs receiving i.p. injections of soluble glucan showed a significant (P less than 0.05) 10-13% decrease in weight gain over the 7-day period. No other toxicological, behavioural or appearance changes were noted. To examine chronic toxicity, soluble glucan was administered twice weekly for a period of 30 or 60 days to ICR/HSD mice in the dose of 40, 200 or 1000 mg/kg. No deaths were observed in any group. Chronic glucan administration did not alter body weight, liver, lung or kidney weight. However, a significant splenomegaly was observed in both the 30 and 60-day study. Histopathologic examination showed no tissue alterations at 40 or 200 mg/kg. However, at 1000 mg/kg a mononuclear infiltrate was observed in the liver. Pyrogenicity testing, employing New Zealand white rabbits, revealed that parenteral glucan administration (5 mg/kg) did not significantly alter body temperature. These data indicate that the systemic administration of soluble glucan over a wide dose range does not induce mortality or significant toxicity, an important consideration in preparing soluble glucan for parenteral administration to human populations.
B: Lentinan (beta-1,3-glucan) was studied on the acute toxicities using both sexes of mice (ICR) and rats (CD) treated intravenously (i.v.) intraperitoneally (i.p.), subcutaneously (s.c.) and orally (Moriyuki and Ichimura 1980). LD50 values in mg/kg body weight were essentially the same regardless of species as well as sexes and estimated as follows: 250-500 (i.v.) greater than 2500 (i.p., s.c., and p.o.). Cyanosis, convulsion and death were observed in both species of animals administered (i.v.) with only higher dosages of lentinan. No remarkable toxic signs being specific from lentinan were observed in any cases of treatment, i.p., s.c., and p.o. Gross findings: enlargement of the spleen (i.v., i.p., s.c.) and coarse nodular surface of the kidney (i.v.) in the both species of animals, erythema of the ears (i.v., i.p., s.c.) in mice, mesenteric petechial haemorrhage of the lung and abdomen (i.v.), enlargement of the mesenteric lymph nodes (i.v.) and oedema of the diaphragm and intestine (i.p.) in rats were observed. In parallel, another sample of lentinan for clinical use prepared by freeze-dried procedure was tested in both sexes of mice and rats treated by i.v. alone, comparing with an original sample mentioned above. So far as the acute toxicities of lentinans concerned, no significant differences between the two preparations were observed.
C: Mice, rats and rabbits of both sexes were used for pharmacological an acute toxicological study with antitumor polysaccharide, schizophyllan (Matsuo et al. 1982). The physiologically tolerable maximum doses SPG (schizophyllan) were administered by i.m., s.c., i.p. and i.v. routes, respectively. No dead case was found in any species and any administration route during 14 days after a single administration of up to 2000 mg/kg i.p, 300 mg/kg i.v.; 100 mg/kg i.m., and 2000 mg/kg s.c. Sub-acute toxicology was evaluated after s.c. administration on alternate days for 3 months with doses up to 40 mg/kg. No dead cases were found, no significant alteration on body weight, food consumption of water intake was observed.