alpha-Cyclodextrin (synonyms, cyclohexaamylose, cyclomaltohexaose, alpha-Schar-dinger dextrin) is a non-reducing cyclic saccharide comprised of six glucose units linked by alpha-1,4 bonds. It is produced by the action of cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) on hydrolysed starch syrups at neutral pH (6.0–7.0) and moderate temperature (35–40 ^oC). The annular (or doughnut-shaped) structure of alpha-cyclodextrin provides a hydrophobic cavity that allows formation of inclusion complexes with a variety of non-polar organic molecules of appropriate size. The hydrophilic nature of the outer surface of the cyclic structure makes alpha-cyclodextrin water-soluble.

The hydrophobic cavity and the hydrophilic outer surface of alpha-cyclodextrin form the basis for its use in the food industry. alpha-Cyclodextrin, like its homologues beta- and gamma-cyclodextrin, can function as a carrier and stabilizer for flavours, colours, and sweeteners; as an absorbent for suppression of undesirable flavours and odours in foods; as an absorbent for suppression of halitosis (in breath-freshening preparations); and as a water-solubilizer for fatty acids and vitamins.

The principal method for the isolation and purification of alpha-cyclodextrin takes advantage of its complex-forming ability. At completion of the reaction, 1-decanol is added to the reaction mixture to form an insoluble 1:1 alpha-cyclodextrin:1-decanol inclusion complex. The complex is continuously mixed with water and separated from the reaction mixture by centrifugation. The recovered complex is re-suspended in water and dissolved by heating. Subsequent cooling leads to re-precipitation of the complex. The precipitate is recovered by centrifugation, and 1-decanol is removed by steam distillation. Upon cooling, alpha-cyclodextrin crystallizes from solution. The crystals are removed by filtration and dried, yielding a white crystalline powder with a water content under 11%. The purity on a dried basis is at least 98%.

alpha-Cyclodextrin had not been evaluated previously by the Committee, but structurally related compound beta-cyclodextrin was evaluated at the forty-first and forty-fourth meetings (Annex 1, references 107 and 116) and gamma-cyclodextrin was evaluated at the fifty-first and fifty-third meetings (Annex 1, references 137 and 143). The Committee noted the close structural similarity between alpha-cyclodextrin and beta-cyclodextrin (seven glucose units) and gamma-cyclodextrin (eight glucose units), which permitted comparisons between the metabolism and toxicity of these compounds.

While alpha-cyclodextrin is hydrolysed by alpha-amylases of fungal and bacterial origin (Jodál et al., 1984; Saha & Zeikus, 1992), human salivary and pancreatic amylases cannot hydrolyse alpha-cyclodextrin to a significant extent (Marshall & Miwa, 1981; Kondo et al., 1990).

In an early experiment, the metabolism of alpha- and beta-cyclodextrin was examined in groups of Wistar rats given ¹⁴C-labelled cyclodextrins or starch. The respired ¹⁴CO₂ was collected for 16–23 h, and radiolabel was measured in excreted urine and faeces and in the carcass at the end of the experiment. After administration of radiolabelled starch, ¹⁴CO₂ appeared rapidly in the breath and reached a maximum at 1 h. After administration of the radiolabelled cyclodextrins, ¹⁴CO₂ appeared in the breath only after about 3.5 h and reached a maximum at about 8 h. The cumulative amount of ¹⁴CO₂ was similar with the three compounds. More radiolabel was recovered from the gut after administration of the cyclodextrins than starch. Thus, alpha- and beta-cyclodextrin are not metabolized to any significant extent in the small intestine but are fermented by the intestinal microflora (Andersen et al., 1963).

[¹⁴C]alpha-Cyclodextrin (radiochemical purity, 99%) was administered to groups of four male and four female conventional Wistar rats (Crl:WI(WU)BR) and germ-free (Wistar/Wag/Rij strain) rats to examine the absorption, distribution, metabolism, and excretion profiles. The time course of ¹⁴CO₂ production was measured after oral administration and the occurrence and kinetics of [¹⁴C]alpha-cyclodextrin and its metabolites were examined in blood after oral and intravenous administration. The role of gut microflora in the metabolism of alpha-cyclodextrin was also examined in these studies.

In the first experiment, most (60%) of an oral dose of 200 mg/kg bw (25 µCi/kg bw) was excreted as ¹⁴CO₂ in expired air by animals of each sex within 24 h. The highest rate of excretion was at 7 h after 200 mg/kg bw and at 8.5–11 h after 100 mg/kg bw, but the cumulative 24-h excretion was identical at the two doses. The faeces and the contents of the gastrointestinal tract contained 18–19% of the radiolabel at 48 h, while the urine contained 2.6–4.1%. The amount of radiolabel retained was 6.4–6.7% after 24 h and 2.4–3.5% after 48 h at both doses. The retained radiolabel was found predominantly in the liver. Profiling of the urine by high-perfomance liquid chromatography (HPLC) revealed a peak corresponding to alpha-cyclodextrin at 0–4 h; this decreased with time, and an early eluting peak was observed, which peaked at 8–24 h.

In the second experiment, the kinetics of excretion and the time course in blood over 24 h were examined after a single oral dose of 200 mg/kg bw (100 µCi/kg bw) alpha-cyclodextrin. The blood concentration was low for the first 4 h (< 0.2% of the dose), and the maximum concentration was reached by 12 h. The blood concentration had decreased by 50% by 48 h. The half-time was approximately 36 h. HPLC analysis showed a small peak that co-eluted with alpha-cyclodextrin at 8 h and a second peak that co-eluted with glucose. At 12 h, only the peak co-eluting with glucose was found.

In the third experiment, the kinetics of excretion and the time course in blood over 24 h were examined after a single intravenous dose of 50 mg/kg bw (100 µCi/kg bw) [¹⁴C]alpha-cyclodextrin. The main route of excretion was the urine, with small amounts in the faeces (0.50% and 0.37% for males and females, respectively). Excretion of CO₂ was also significant, with inter-individual variation (1.6–9.9% in males and 2.4–12% in females). The total amount of radiolabel retained in the carcass was 4.0% in males and 7.4% in females, most being found in the liver, kidneys, and gastrointestinal tract. The time course of removal of radiolabel from the blood was rapid, with calculated half-times of 26 min for males and 21 min for females. HPLC profiling revealed that the predominant radioactive species was alpha-cyclodextrin.

In the fourth experiment, the excretion kinetics over 24 h was examined in germ-free rats given a single oral dose of 200 mg/kg bw (50 µCi/kg bw) [¹⁴C]alpha-cyclodextrin. During this period, 27% of the administered dose was excreted by males and 27% by females, almost exclusively (85–90%) via the gastrointestinal tract. Only 0.7–1.8% was excreted in urine and 1.2–1.4% as exhaled ¹⁴CO₂. At 24 h, most of the retained radiolabel was in the gastrointestinal tract, mainly in the caecum. Little radiolabel was found in other organs. HPLC profiling revealed only alpha-cyclodextrin in urine and faeces.

Overall, the studies indicate that alpha-cyclodextrin can be absorbed intact at a level of approximately 2% from the small intestine, but most absorption takes place after metabolism by the microflora in the caecum. Intact alpha-cyclodextrin that is absorbed is excreted rapidly in the urine. Absorption of the metabolites of alpha-cyclodextrin leads to slow removal, mainly via exhaled CO₂ or urine. The metabolites may be partially stored in the liver (Van Ommen & de Bie, 1995).

2.2.1 Acute toxicity

The results of studies of the acute toxicity of alpha-cyclodextrin in rats and mice treated intraperitonerally or intravenously are shown in Table 1.

Administration of a single intravenous dose to Sprague-Dawley rats produced alterations in the vacuolar organelles of the proximal convoluted tubule, characterized by an increase in the number of apical vacuoles and the appearance of giant lysosomes distorted by enclosed microcrystals. Other cell organelles, notably the mitochondia, were degenerated, with manifestation of giant vacuoles. The nephrosis caused by cyclodextrin is considered to be an exaggerated form of osmotic nephrosis, probably due to lack of degradation of cyclodextrin by lysosomal amylases, leading to renal failure (Frank et al., 1976).

Administration of a single intravenous dose to Swiss-outbred mice (Crl:CD) produced signs of toxicity including sluggishness and piloerection within 1 h to a few days after treatment at all doses. Deaths occurred in all groups, and the survivors had a decreased growth rate; however, the surviving animals recovered and appeared healthy at the end of the observation period. Macroscopic examination of animals found dead and of survivors at the end of the observation period did not reveal gross treatment-related alterations (Riebeek, 1990a).

Administration of a single intravenous dose to Wistar outbred rats (Crl:WI(WU)BR) produced severe signs of sluggishness and piloerection after 4 h in all animals at a dose of 1000 mg/kg bw and in one male at 750 mg/kg bw. Moderate to severe signs of sluggishness, piloerection, or pallor were generally observed in males and in one female at 750 mg/kg bw by 24 h. Death occurred within a few days of treatment. The surviving animals recovered from an initially decreased growth rate and appeared healthy at the end of the observation period. Macroscopic examination revealed no gross treatment-related alterations (Riebeek, 1990b).

The toxicity of single intraperitoneal doses of 4000 mg/kg bw alpha-cyclodextrin, 2000 or 4600 mg/kg bw gamma-cyclodextrin, and 4000 mg/kg bw glucose given as solutions in sterile saline was compared in groups of three male Wistar outbred rats (Crl:WI(WU)BR). All animals given alpha-cyclodextrin showed moderate signs of sluggishness within 1 h and died within a few hours after treatment. Animals given gamma-cyclodextrin or glucose showed no ill effects. All rats started to gain weight after 3 days, and no deaths occurred. Macroscopic examination of the survivors at the end of the observation period of 14 days did not reveal gross treatment-related alterations (Riebeek, 1990c).

Administration of a single intravenous dose to Wistar outbred rats (Hsd/Cpb:WU) caused the death of nearly 100% of animals at doses ž 1000 mg/kg bw, of one male and no females at 750 mg/kg bw dose group, and no deaths at 500 mg/kg bw. Sluggishness and piloerection were observed at the two higher doses. All surviving rats gained weight during the 14-day observation period. Macroscopic examination at the end of the observation period of animals surviving after receiving 500–1000 mg/kg bw revealed a pale renal cortex (Prinsen, 1991a).

alpha-Cyclodextrin was examined for its ability to induce ocular irritation in albino rabbits in two separate studies. In the first study, a dose of 0.062 g instilled directly into the conjunctival cul-de-sac of the right eye of three rabbits was irritating but not corrosive (Prinsen, 1990). In the second experiment, in which two groups of three rabbits were given alpha-cyclodextrin as a 14.5% or a 50% dilution in demineralized water, no or minimal irritation was found in the eyes, and there was no corrosion (Prinsen, 1991b).

In chicken enucleated eyes ex vivo, a 14.5% or 50% solution of alpha-cyclodextrin was not irritating or corrosive (Prinsen, 1991b).

A sample of 0.5 g of alpha-cyclodextrin moistened with tap water was applied to the shaven backs and flanks of three albino rabbits for 4 h under a semi-occlusive dressing. There were no signs of skin irritation up to 72 h (Prinsen, 1991c).

Similarly, in guinea-pigs, a 10% or 30% solution of alpha-cyclodextrin induced no signs of sensitization in intradermally or topically induced animals (Prinsen, 1992).

2.2.2 Short-term studies of toxicity

Groups of mature male mice were given alpha- or beta-cyclodextrin by gavage for 15 days at a dose of 0 or 60 mg/kg bw per day. There was no treatment-related effect on growth rate or on relative liver weight (Miyazaki et al., 1979).

Groups of five Wistar rats of each sex were fed diets containing alpha-cyclodextrin at a concentration of 0, 1, 5, or 15%, equivalent to 0, 500, 2500, or 7500 mg/kg bw per day, for 4 weeks. For comparison, an additional group of five rats of each sex was fed a diet containing 5% beta-cyclodextrin, equivalent to 2500 mg/kg bw per day, for the same period.

There were no deaths. Animals given 15% alpha-cyclodextrin had severe diarrhoea from day 6 until the end of the study, while animals given 5% had soft stools only occasionally. The animals fed 5% beta-cyclodextrin had slight to severe diarrhoea from the start of the study until day 7. Animals given 15% alpha-cyclodextrin group also showed emaciation, humpback behaviour, and soiled and rough fur during week 2; these signs had generally disappeared in weeks 3 or 4. The mean body weights were significantly decreased in males fed 15% alpha-cyclodextrin or 5% beta-cyclodextrin. Food intake and food conversion efficiency were decreased in animals fed 5 or 15% alpha-cyclodextrin, while the water intake of animals given 15% alpha-cyclodextrin increased from day 6.

In females fed 15% alpha-cyclodextrin, the haemoglobin concentration, erythrocyte count, packed cell volume, and monocyte and leukocyte counts were all increased. In males fed 15% alpha-cyclodextrin or 5% beta-cyclodextrin, the erythrocyte count was slightly decreased, while the mean corpuscular volume and mean corpuscular haemoglobin were slightly increased. Males and females at 15% alpha-cyclodextrin had increased alkaline phosphatase activity, increased concentrations of albumin and chloride, a decreased concentration of bilirubin, and decreased gamma-glutamyl transpeptidase activity and urea concentrations. Urinary density and urinary pH were decreased in females, while the urine volume was increased at the higher concentration. In animals fed beta-cyclodextrin, the activity of gamma-glutamyl transpeptidase was decreased in males and those of alanine and aspartate aminotransferases were increased in females.

The absolute and relative weights of the liver were decreased in males and females fed 15% alpha-cyclodextrin. The weights of the filled and empty caeca were increased significantly in animals fed 15% alpha-cyclodextrin and to a lesser extent in those fed 5% alpha-cyclodextrin. The absolute weights, but not the relative weights, of the heart and kidneys were also decreased in males fed 15% alpha-cyclodextrin. Gross examination at autopsy revealed signs of emaciation and enlarged caeca in rats fed 15% alpha-cyclodextrin. Microscopic examination revealed changes to the caecum consistent with enlargement, and changes in the liver related to depletion of glycogen in the periportal area. The NOEL was 2500 mg/kg bw per day (Lina & Bruyntjes, 1987).

Groups of 20 Wistar rats of each sex were fed diets containing alpha- or gamma-cyclodextrin at a concentration of 0, 1.5, 5, or 20%, equivalent to 0, 750, 2500, and 10 000 mg/kg bw per day, for 13 weeks. A control group of 20 animals per sex was fed a diet containing 20% lactose (equivalent to 10 000 mg/kg bw per day) for the same period. In order to examine the reversibility of any effects seen, groups of 10 animals of each sex were fed either control diet or a diet containing 20% alpha-cyclodextrin, 20% gamma-cyclodextrin, or 20% lactose for 13 weeks and were then fed control diet for 1 month. The animals were examined for clinical signs of toxicity, and food and water consumption were monitored throughout the study. Ophthalmic examinations were conducted before and at the end of treatment period on controls and animals at the high dose. Body weight and food and water consumption were monitored throughout the study. Clinical chemical, haematological, and urinary parameters were measured at the beginning and end of treatment and after the recovery period. At the end of the study, the animals were killed, organs were examined grossly, and tissues were prepared for histopathological examination.

There were no treatment-related deaths during the study. Soft stools were observed in the early weeks in rats given 20% alpha-cyclodextrin, 5% or 20% gamma-cyclodextrin, or lactose. Ophthalmoscopic examination revealed no treatment-related effects. Slight growth retardation was observed in males at 20% alpha-cyclodextrin, gamma-cyclodextrin, or lactose, which was accompanied by decreased food efficiency in rats given 20% alpha-cyclodextrin or lactose and increased food intake in those given 20% gamma-cyclodextrin. Water intake was increased in rats at the high dose of alpha-cyclodextrin or lactose but decreased in those given 20% gamma-cyclodextrin.

Erythrocyte parameters changed sporadically, with no relation to treatment. The total leukocyte count was increased in males given 20% lactose or alpha-cyclodextrin, but the differential counts were unchanged. No other changes were observed. Decreased plasma gamma-glutamyltransferase activity and phospholipid, triglyceride, and total protein concentrations were found in rats given 20% alpha-cyclodextrin, and the phospholipid, triglyceride, and total protein concentrations were also decreased in males given lactose. The urinary calcium concentration was increased in rats given 20% alpha-cyclodextrin, gamma-cyclodextrin, or lactose. The faecal dry weight and excretion of faecal nitrogen were increased, and faecal pH was decreased in rats given 20% alpha-cyclodextrin or lactose. At the end of recovery period, no changes were found that could be attributed to treatment with alpha-cyclodextrin. The only treatment-related change in urinary parameters was an increase in the urinary calcium concentration in the groups treated with 20% alpha-cyclodextrin, gamma-cyclodextrin, or lactose, which may have been associated with the increased load of osmotically active substances in the large intestine.

Significant increases were found in both the absolute and relative weights of the full and empty caeca in males and females at 5 or 20% alpha-cyclodextrin or 20% lactose when compared with the control group; smaller changes were seen in rats given 20%gamma-cyclodextrin. Differences were still present at the end of the recovery period but were significantly reduced. The relative weights of the spleen and the liver were increased in males given 20% alpha-cyclodextrin and in females given 20% lactose. At the end of the recovery period, only changes in caecal weight were observed. The only gross pathological change observed was caecal enlargement in rats given 20% alpha-cyclodextrin or 20% lactose. Microscopic examination revealed increased corticomedullary mineralization in the kidneys of some rats, but this effect was considered to be relatively common in rats and not related to treatment. There were no treatment-related histopathological changes.

The effects in rats receiving 20% alpha-cyclodextrin in the diet thus appear to be related largely to the presence of high concentrations of osmotically active substances in the large intestine and to be of no toxicological significance. Similar and generally more pronounced effects were observed when the diet contained 20% lactose, while similar but less pronounced effects were observed when the diet contained 20%gamma-cyclodextrin. The NOEL for alpha-cyclodextrin was 20% in the diet, equivalent to 10 000 mg/kg bw per day (Lina, 1992).

Groups of four beagle dogs of each sex were fed diets containing alpha-cyclodextrin at a concentration of 0, 5, 10, or 20%, equivalent to 0, 1250, 2500, or 5000 mg/kg bw per day, for 13 weeks. Body weights and food consumption were recorded weekly throughout the study. Ophthalmoscopic observations were made at the beginning and end of the study. Blood was collected before the start of the study and during weeks 6 and 12 for routine haematological and clinical chemical investigations. Urinary analyses were carried out during week 13. All animals were killed in week 14, their organs were examined, and tissues were examined microscopically.

There were no deaths during the study. Diarrhoea occurred in all treated groups, increasing in incidence and severity with increasing concentration of alpha-cyclodextrin. No other treatment-related toxic effects were observed. Ophthalmoscopy revealed no treatment-related effects.

The weight gain was comparable in all groups except for females given 20% alpha-cyclodextrin, which gained slightly less weight than the control group, although the difference was not statistically significant. The food intake of animals fed 20% alpha-cyclodextrin was slightly higher than that of the control group. No treatment-related changes were seen in haematological parameters. The total plasma bilirubin concentration was increased, but the effect was not dose-related and considered to be unrelated to treatment. The urinary pH was lower in animals given 20% alpha-cyclodextrin than in controls, and the difference was statistically significant in females.

The only changes in organ weights were in the absolute and relative weights of the caecum, either filled or empty, at 10 or 20% alpha-cyclodextrin. Gross examination at autopsy revealed no treatment-related changes apart from the caecal enlargement. Histopathological findings were unremarkable.

The effects observed appeared to be related to the presence of an osmotically active substance in the large intestine. The NOEL was 20% alpha-cyclodextrin, equivalent to 5000 mg/kg bw per day (Til & van Nesselrooij, 1993).

2.2.3 Genotoxicity

alpha-Cyclodextrin was tested for its ability to induce reverse mutation in the histidine-requiring Salmonella typhimurium mutants TA1535, TA1537, TA1538, TA98, and TA100, with or without a liver microsomal fraction, at concentrations of 0.25–20 mg per plate. Under the conditions of this assay, alpha-cyclodextrin was not mutagenic (Blijleven, 1991).

alpha-Cyclodextrin was tested for its potential to induce micronuclei in bone marrow of Charles River CD-1 mice given a dose of 10 000 mg/kg bw. Under the conditions of this assay, alpha-cyclodextrin did not increase the incidence of micronuclei in bone-marrow cells (Immel, 1991).

2.2.4 Developmental toxicity

In a study of embryotoxicity and teratogenicity, groups of presumed pregnant Swiss albino mice were fed diets containing alpha-cyclodextrin at a concentration of 0, (control), 5, 10, or 20%, equal to 0, 14, 23, and 49 mg/kg bw per day, on days 6–16 of gestation. The mice were examined for clinical signs, body weight, and food and water consumption at regular intervals. They were killed on day 17 and examined for reproductive performance. Fetuses were examined for signs of toxicity, external malformations, and soft-tissue defects and were stained for detection of skeletal anomalies.

The dams showed no signs of toxicity, but those at higher doses had increased relative food consumption and increased weight gain at the end of treatment. The absolute and relative weights of the liver and uterus were comparable to those of controls. The numbers of viable litters and corpora lutea and the mean number of implantation sites were similar in all groups. Fetal body weight, the average number of live fetuses, and the sex ratio per litter were similar in all groups. Examination of the fetuses revealed no treatment-related increase in gross, skeletal, or visceral abnormalities. Under the conditions of this assay, alpha-cyclodextrin at 20% in the diet of mice was not teratogenic (National Toxicology Program, 1994a).

In a study of embryotoxicity and teratogenicity, groups of 25 presumed pregnant Wistar WU albino rats were fed diets containing alpha-cyclodextrin (purity, > 98%) at a concentration of 0 (control), 1.5, 5, 10, or 20%, equivalent to 0, 750, 2500, 5000, and 10 000 mg/kg bw per day, on days 0–21 of gestation. A separate group received a diet containing 20% lactose instead of pre-gelatinized potato starch. The rats were killed on day 21 and examined for reproductive performance. The fetuses were examined for signs of toxicity, external malformations, and soft-tissue defects and were stained for detection of skeletal anomalies.

No deaths occurred during the study, and there were no signs of toxicity in the dams, even at the highest dose. Maternal body weight and body-weight gain during gestation were comparable in all groups. Food consumption was slightly but significantly increased in the groups at 10 and 20% alpha-cyclodextrin on days 16–21 of gestation. Necropsy of the dams showed no adverse effects that could be attributed to treatment. The numbers of viable litters and corpora lutea and the mean number of implantation sites were similar in all groups. Fetal length and body weight were also similar in all groups. Examination of the fetuses revealed no treatment-related increase in the incidence of gross, skeletal, or visceral abnormalities. Under the conditions of this assay, alpha-cyclodextrin at 20% in the diet was not teratogenic (Verhagen & Waalkens-Berendsen, 1991).

In a study of embryotoxicity and teratogenicity, groups of 25 presumed pregnant Sprague-Dawley rats were fed diets containing alpha-cyclodextrin at a concentration of 0 (control), 5, 10, or 20%, equal to 0, 4.2, 9.9, or 20 g/kg bw per day, on days 0–16 of gestation. The rats were killed on day 20 and examined for reproductive performance. The fetuses were examined for signs of toxicity, external malformations, and soft-tissue defects and were stained for detection of skeletal anomalies.

The relative food consumption was increased among dams at 10% and 20% during treatment but had returned to normal on days 18–20. The average maternal body weight and body-weight gain did not increase during or after treatment. The weights of the maternal liver and uterus were also not affected by treatment. The numbers of viable litters and corpora lutea and the mean number of implantation sites were similar in all groups. Fetal body weight, the average number of live fetuses, and the sex ratio per litter were similar in all groups. Examination of the fetuses revealed no treatment-related increase in the incidence of gross, skeletal, or visceral abnormalities. Under the conditions of this assay, alpha-cyclodextrin at 20% in the diet of rats was not teratogenic (National Toxicology Program, 1994b).

In a study of embryotoxicity and teratogenicity, groups of 16 presumed pregnant New Zealand white rabbits were fed diets containing alpha-cyclodextrin (purity, > 98%) at a concentration of 0, 5, 10, or 20%, equivalent to 0, 1500, 3000, and 6000 mg/kg bw per day, on days 0–29 of gestation. A separate group of 16 animals received a diet containing 20% lactose instead of wheat starch. The animals were examined throughout the study, and body weights and food consumption were recorded regularly.

No deaths occurred during the study, and there were no treatment-related effects on maternal body weight. Food intake was significantly decreased among does at 20% during the first week of gestation but not thereafter. Necropsy of the does showed no adverse effects that could be related to treatment. The numbers of viable litters and corpora lutea and the mean number of implantation sites were similar in all groups. Examination of the fetuses revealed no treatment-related increase in the incidence of gross, skeletal, or visceral abnormalities. Under the conditions of this assay, alpha-cyclodextrin at concentrations up to 20% in the diet was not teratogenic (Waalkens-Berendsen & Smits-van Prooije, 1992).

2.2.5 Special studies

The interactions between alpha-, beta-, and gamma-cyclodextrin and membrane phospholipids, liposomes, and human erythrocytes were studied in vitro. alpha-Cyclodextrin and other cyclodextrins did not increase the permeability of dipalmityol-phosphatidylcholine liposomes, nor did they affect the active transport of ⁴²K⁺ into erythrocytes at a concentration of 10^–2 mol/L. beta-Cyclodextrin but not gamma-cyclodextrin or alpha-cyclodextrin increased the release of ⁴²K⁺, ⁸⁶Rb⁺, and ¹³⁷Cs⁺ from erythrocytes by passive transport at a concentration of 1.7 x 10^–2 mol/L (Szejtli et al., 1986).

alpha-Cyclodextrin induced haemolysis of human erythrocytes in vitro in isotonic saline after 30 min, with a threshold concentration of 6 mmol/L (approximately 7.5 mg/L). A similar effect was seen with gamma-cyclodextrin at 16 mmol/L and with beta-cyclodextrin at 3.5 mmol/L. The haemolytic effect of the cyclodextrins was ascribed to cyclodextrin-mediated extraction of cholesterol and other lipids from the erythrocyte membrane (Irie et al., 1982).

Other studies have shown that human erythrocytes tolerate alpha-cyclodextrin better than beta-cyclodextrin. Concentrations of 5–10 mml/Lalpha-cyclodextrin were required for the induction of haemolysis (Okada et al., 1988; Skiba, 1990; Leroy-Lechat et al., 1994).

beta-cyclodextrin forms stable inclusion complexes with cholesterol, while alpha-cyclodextrin forms complexes with phospholipids (Irie et al., 1982; Ohtani et al., 1989; Debouzy et al., 1998; Nishijo et al., 2000).

In a study on the differential effects of cyclodextrins on human erythrocytes in vitro, alpha-, beta-, and gamma-cyclodextrin were incubated at increasing concentrations with erythrocytes for 30 min. The cyclodextrins induced a change in the shape, from discocyte to spherocyte, but with beta-cyclodextrin haemolysis occurred before the change was complete. beta-Cyclodextrin, at a concentration of 1 mmol/L led to a significant release of cholesterol and protein from the erythrocytes, whereas alpha-cyclodextrin at this dose led to release of phospholipid and not of cholesterol or protein (Ohtani et al., 1989).

The effects of the cyclodextrins on P388 murine leukaemia cells were examined by exposing the cells to increasing concentrations of cyclodextrins for 48 h in a medium containing 10% fetal calf serum. Initial cytotoxicity was elicited at 11 mmol/L alpha-cyclodextrin, 2.5 mmol/L beta-cyclodextrin, and 55 mmol/L gamma-cyclodextrin (Leroy-Lechat et al., 1994).

In a study of the effects of cyclodextrins on the luminescence of an Escherichia coli suspension, the concentrations of natural and chemically modified cyclodextrins required to reduce the luminescence by 20% and 50% were determined as an indication of their cytotoxicity. gamma- and alpha-Cyclodextrin interfered minimally with the bacterial luminescence and consequently were essentially non-toxic (Bär & Ulitzur, 1994).

In a human cell suspension culture system that exhibits ciliogenesis, the ciliary beat frequency remained stable to gamma-cyclodextrin at 10% (w/v) and alpha-cyclodextrin at 2% (w/v) after 30 min of exposure. At 5–10% (w/v), however, alpha-cyclodextrin caused mild to severe inhibition after 45 min. The effect was partially reversible (Agu et al., 2000).

In a study to examine whether cyclodextrins act as carriers for peptides, the digestion and absorption of two model peptides, glycosylated calcitonin and octreotide, was examined in vitro in a human colorectal carcinoma cell line (Caco-2) and in rats in situ. Some evidence of enhancement of absorption was seen in vitro but not in situ with alpha-cyclodextrin at 1% (w/v). In contrast, beta-cyclodextrin enhanced absorption in both systems. To examine the effect of cyclodextrins on the integrity of tight junctions between cell monolayers, similar tests were performed with PEG-4000. alpha-Cyclodextrin had a greater effect on permeation than beta-cyclodextrin. The results suggest that the absorption-enhancing effect of cyclodextrins depends on the compounds and the cyclodextrin tested (Haeberlin et al., 1996).

In another study with the Caco-2 cell culture system, the effect of alpha-cyclodextrin was examined on the permeability of the membrane to mannitol. Enhancement was observed only at a concentration of 5%, and no effect was seen at 0.1 or 1% (McAllister & Taylor, 1999).

In a further study with the Caco-2 cell culture system, the effect of cyclodextrins on absorption of PEG-4000 was examined. No effect was seen at concentrations up to 5% (w/v), in contrast to the previous study. It was concluded that alpha-cyclodextrin does not open tight junctions of Caco-2 monolayers (Hovgaard & Brondsted, 1995).

In order to examine the effects of alpha- and beta-cyclodextrin on intestinal absorption, the absorption of sulfanilic acid, a non-absorbed drug, was studied in ligated loops of rat small intestine in situ. The experiments were performed in the presence or absence of N-acetyl-L-cysteine which removes the mucus that covers the intestinal mucosa.

After perfusion with 10 mmol/l of alpha- and beta-cyclodextrin, absorption of sulfanilic acid was not enhanced. However, when beta-cyclodextrin was given in combination with a N-acetyl-L-cysteine, there was a significant increase in the absorption of sulfanilic acid. alpha-Cyclodextrin in combination with N-acetyl-L-cysteine had no effect. Analysis of the perfusate after removal of the mucous layer indicated that alpha-cyclodextrin promoted the release of phospholipids, while beta-cyclodextrin promoted the release of cholesterol. The increased permeability was considered to occur via transcellular rather than paracellular pathways (Nakanishi et al., 1992).

The gene coding for cyclodextrin-glycosyl transferase (CGTase) is derived from a strain of the Klebsiella oxytoca M5a1 (formally K. pneumoniae M5a1). K. oxytoca is a gram-negative rod, faculative anaerobe which belongs to the family Enterobacteriaceae and is found in the faecal microflora of 30–40% of persons. K. oxytoca M5a1 is considered to be non-pathogenic; it has a history of safe use and a negligible capacity to persist in humans. After the gene coding for CGTase had been isolated from this strain and characterized, it was cloned in pHE3, isolated again, and inserted in the expression vector pJF118EH. PJF11EH is derived from PBR322, a widely used vector which is considered to be safe. The cloned DNA fragment of K. oxytoca contained only the CGTase gene, as demonstrated by sequence analysis (Bender, 1977; Henneke et al., 1982; Fürste et al., 1986). A strain derived from E. coli K12 was used as the host for the CGTase expression and secretion vector. E. coli K12 is considered to be a non-pathogenic strain and is used for production of enzymes for food use. For the production of CGTase, the recombinant E. coli strain was cultured in a standard medium. The pH of the culture broth was adjusted by the addition of food-grade ammonia or phosphoric acid. The E. coli was grown to a certain density, after which enzyme production was initiated by addition of isopropyl thiogalactoside and the supernatant filtered and concentrated to the crude CGTase preparation.

A CGTase preparation derived from E. coli K12 but differing in the source organism of the gene coding for CGTase (Bacillus firmus/lintus) was considered previously by the Committee (Annex 1, reference 137). In a 3-month study in rats receiving the enzyme in the diet, there was no evidence of treatment-related effects. In a study of mutagenicity in S. typhimurium strains TA1535, TA1537, TA98, and TA100, CGTase had no mutagenic activity.

alpha-Cyclodextrin is used as a carrier for flavours, colours, and sweeteners in foods such as dry mixes, baked goods, and instant teas and coffee, as a stabilizer for flavours, colours, vitamins, and polyunsaturated fatty acids in dry mixes and dietary supplements (< 1% of the final product), as a flavour modifier in soya milk (< 1%), and as an absorbent (breath freshener) in confectionery (10–15% of the final product).

No national assessments of the intake of alpha-cyclodextrin were submitted. An assessment for the USA formed part of the submission from the sponsor (Bär, 2001). Data for the national population were based in individual dietary records from a continuing survey of food consumption in 1994–98 and on the predicted use of alpha-cyclodextrin in a wide variety of foods at maximum levels (Table 2). The mean intake of alpha-cyclodextrin by consumers was 1.7 g/day or 32 mg/kg bw per day, while that at the 90th percentile of consumption was 3 g/day or 67 mg/kg bw per day. The intake of alpha-cyclodextrin by 13–19-year-olds was higher than that for the whole population (mean, 2 g/day; high percentile, 3.6 g/day), but on a body weight basis the intake was highest for children aged 2–6 years (mean, 87 mg/kg bw per day; high percentile, 140 mg/kg bw per day) (Table 3). In most age groups, the main contribution to the predicted intake appeared to be from soya milk and sweets.

alpha-Cyclodextrin, like beta-cyclodextrin, is not digested in the gastrointestinal tract but is fermented by the intestinal microflora. In germ-free rats, alpha-cyclodextrin is almost completely excreted in the faeces, whereas gamma-cyclodextrin is readily digested to glucose by the luminal and/or epithelial enzymes of the gastrointestinal tract. alpha-Cyclodextrin is absorbed intact at low levels (approximately 2%) from the small intestine. Absorbed alpha-cyclodextrin is then excreted rapidly in the urine. The majority of the absorption takes place after metabolism by the microflora in the caecum. Although no studies of metabolism in humans in vivo were available, alpha-cyclodextrin and beta-cyclodextrin, unlike gamma-cyclodextrin, cannot be hydrolysed by human salivary and pancreatic amylases in vitro.

The acute toxicity of alpha-cyclodextrin when given by the intraperitoneal or intravenous route indicates that it can cause osmotic nephrosis, probably because it is not degraded by lysosomal amylases. At high doses, this leads to renal failure.

The results of short-term (28- and 90-day) studies of toxicity indicated that alpha-cyclodextrin has little effect when given orally to rats or dogs. After administration of a very high dietary concentration (20%), caecal enlargement and associated changes were seen in both species. This effect is likely to result from the presence of a high concentration of an osmotically active substance in the large intestine. No studies of intravenous administration were available to permit a comparison of the systemic toxicity of this compound with that of beta- and gamma-cyclodextrin.

Studies conducted in mice, rats, and rabbits with alpha-cyclodextrin at concentrations in the diet of up to 20% did not indicate any teratogenic effects. Similarly, the results of assays for genotoxicity were negative. No long-term studies of toxicity, carcinogenicity, or reproductive toxicity have been conducted with alpha-cyclodextrin, but the Committee concluded that, given the known fate of this compound in the gastrointestinal tract, such studies were not required for an evaluation.

In vitro, alpha-cyclodextrin, like beta-cyclodextrin, sequestered components of the membranes of erythrocytes, causing haemolysis. The threshold concentration for this effect was, however, higher than that observed with beta-cyclodextrin.

While the potential interaction of alpha-cyclodextrin with lipophilic vitamins that might impair their bioavailability has not been studied directly, such an effect was considered unlikely by analogy with the results of studies with beta-cyclodextrin. Complexes between fat-soluble vitamins and beta-cyclodextrin have been shown to have a greater bioavailability than uncomplexed forms.

The enzyme cyclodextrin-glycosyl transferase, which is used in the production of alpha-cyclodextrin, is derived from a non-genotoxic, non-toxinogenic source and is completely removed from alpha-cyclodextrin during purification.

The predicted mean intake of alpha-cyclodextrin by consumers, based on individual dietary records for 1994–98 for the USA and proposed maximum levels of use in a variety of foods, would be 1.7 g/day (32 mg/kg bw per day) for the whole population and 1.6 g/day (87 mg/kg bw per day) for children aged 2–6 years. The main contributors to the total intake of alpha-cyclodextrin are likely to be soya milk and sweets. For consumers at the 90th percentile of intake, the predicted intake of alpha-cyclodextrin would be 3 g/day (67 mg/kg bw per day) for the whole population and 2.6 g/day (140 mg/kg bw per day) for children aged 2–6 years.

No studies of human tolerance to alpha-cyclodextrin were submitted to the Committee, despite its potentially high dietary intake. Nevertheless, the Committee was reassured by the relatively low toxicity of this compound in animals and the fact that it was less toxic than beta-cyclodextrin, for which studies of human tolerance were available. Furthermore, the fact that it is fermented in the gastrointestinal tract in an analogous manner to beta-cyclodextrin supported the conclusion that, as in laboratory animals, it would be fermented to innocuous metabolites before its absorption by humans.

The Committee concluded that, on the basis of the available studies on alpha-cyclodextrin and studies on the related compounds beta-cyclodextrin and gamma-cyclodextrin, for which ADIs had been allocated, there was sufficient information to allocate an ADI "not specified"¹. This ADI was based on the known current uses of alpha-cyclodextrin under good manufacturing practices as a carrier and stablizer for flavours, colours, and sweeteners; as a water-solubilizer for fatty acids and certain vitamins; as a flavour modifier in soya milk; and as an absorbent in confectionery.

Agu, R.U., Jorissen, M., Willems, T., Van den Mooter, G., Kinget, R., Verbeke, N. & Augustijns, P. (2000) Safety assessment of selected cyclodextrins—Effect on ciliary activity using a human cell suspension culture model exhibiting in vitro ciliogenesis. Int. J. Pharmacol., 193, 219–226.

Andersen, G.H., Robbins, F.M., Domingues, F.J., Moores, R.G. & Long, C.L. (1963) The utilization of Schardinger dextrins by the rat. Toxicol. Appl. Pharmacol., 5, 257–266.

Bär, A. (2001) alpha-Cyclodextrin. Unpublished report submitted to WHO by Wacker Chemie GmbH, Munich, Germany.

Bär, R. & Ulitzur, S. (1994) Bacterial toxicity of cyclodextrins: Luminous E. coli as a model. Appl. Microbiol. Biotechnol., 41, 574–577.

Bender, H. (1977) [Cyclodextrin-glucanotransferase from Klebsiella pneumoniae, 1. Synthesis, Reinigung und Eigenschaften des Enzymes von K. pneumoniae M5al.] Arch. Microbiol., 111, 271–282 (in German).

Blijleven, W.G.H. (1991) Examination of alpha-cyclodextrin for mutagenic activity in the Ames test. Unpublished report No. V91.023 from TNO-CIVO Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Debouzy, J.C., Fauvelle, F., Crouzy, S., Girault, L., Chapron, Y., Göschl, M. & Gadelle, A. (1998) Mechanism of alpha-cyclodextrin induced hemolysis. 2. A study of the factors controlling the association and serine-, ethanolamine-, and choline-phospholipids. J. Pharm. Sci., 87, 59–66.

Frank, D.W., Gray, J.E. & Weaver, R.N. (1976) Cyclodextrin nephrosis in the rat. Am. J. Pathol., 35, 367–373.

Fürste, J.P., Pansegrau, W., Frank, R., Blöcker, H., Scholz, P., Bagdasarian, M. & Lanka, E. (1986) Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene, 48, 119–131.

Haeberlin, B., Gengenbacher, T., Meinzer, A. & Fricker, G. (1996) Cyclodextrins—Useful excipients for oral peptide administration? Int. J. Pharmacol., 137, 103–110.

Henneke, H., Günther, I. & Binder, F. (1982) A novel cloning vector for the direct selection of recombinant DNA in E. coli. Gene, 19, 231–234.

Hovgaard, L. & Brondsted, H. (1995) Drug delivery studies in caco-2 monolayers. IV. Absorption enhancer effects of cyclodextrins. Pharmacol. Res., 12, 1328–1332.

Immel, H.R. (1991) Examination of alpha-cyclodextrin in the micronucleus test. Unpublished report No. V90.435 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Irie, T. Otagiri, M. Sunada, M. Uekama, K., Ohtani, Y. Yamada, Y. & Sugiyama, Y. (1982) Cyclodextrin-induced hemolysis and shape changes of human erythrocytes in vitro. J. Pharmacol. Dyn., 5, 741–744.

Jodál, I., Kandra, L., Harangi, J., Nánási, P. & Szejtli, J. (1984) Hydrolysis of cyclodextrins by Aspergillus oryzae alpha-amylase. Starch/Stärke, 36, 140–143.

Kondo, H., Nakatani, H. & Hiromi, K. (1990) In vitro action of human and porcine alpha-amylases on cyclomaltooligosaccharides. Carbohydr. Res., 204, 207–213.

Leroy-Lechat, F., Wouessidjewe, D., Andreux, J.P., Puisieux, F. & Duchêne, D. (1994) Evaluation of the cytotoxicity of cyclodextrins and hydroxypropylated derivatives. Int. J. Pharmacol., 101, 97–103.

Lina, B.A.R. (1992) Sub-chronic (90-day) oral toxicity study, including a recovery study, with alpha-cyclodextrin and gamma-cyclodextrin in rats. Unpublished report No. V90.401 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Lina, B.A.R. & Bruyntjes, J.P. (1987) Sub-acute (28-day) oral toxicity study with alpha-cyclodextrin in rats. Unpublished report No. V87.154/261153 from TNO-CIVO Institutes, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Marshall, J.J. & Miwa, I. (1981) Kinetic differences between hydrolyses of gamma-cyclodextrin by human salivary and pancreatic alpha-amylases. Biochem. Biophys. Acta, 661, 142–147.

McAllister, S.M. & Taylor, I.H. (1999) Effect of cyclodextrin complexation on the permeability of testosterone across caco-2 cell monolayers. Proc. Int. Symp. Control. Rel. Bio-act. Mater., 26, 202–203.

Miyazaki, Y., Kawahara, K. & Nozoe, M. (1979) Influence of cyclodextrin on mice. Ann. Rep. Coll. Agric., 47, 17–22.

Nakanishi, K., Nadai, T., Masnada, M. & Miyajima, K. (1992) Effect of cyclodextrins on biological membrane. II. Mechanism of enhancement on the intestinal absorption of non-absorbable drug by cyclodextrins. Chem. Pharm. Bull., 40, 1252–1256.

Nishijo, J., Shiota, S., Mazima, K., Inoue, Y., Mizuno, H. & Yoshida, J. (2000) Interactions of cyclodextrins with dipalmitoyl, distearoyl, and dimyristoyl phosphatidyl choline liposomes. A study by leakage of carboxyfluorescein in inner aqueous phase of unilamellar liposomes. Chem. Pharm. Bull., 48, 48–52.

National Toxicology Program (1994a) Final report on the developmental toxicity of alpha-cyclodextrin (CAS No. 10016-20-3) in Sprague-Dawley (CD) rats (NTIS No. PB 94-139953). National Technical Information Service, Springfield, VA, USA.

National Toxicology Program (1994b) Final report on the developmental toxicity of alpha-cyclodextrin (CAS No. 10016-20-3) in Swiss (CD-1) mice (NTIS No. PB94-180791). National Technical Information Service, Springfield, VA, USA.

Ohtani, Y., Irie, T., Uekama, K., Fukunaga, K. & Pitha, J. (1989) Differential effects of alpha-, beta-, gamma-cyclodextrins on human erythrocytes. Eur. J. Biochem., 186, 17–22.

Okada, Y., Kubota, Y., Koizumi, K., Hizukuri, S., Ohfuji, T. & Ogata, K. (1988) Some properties and the inclusion behavior of branched cyclodextrins. Chem. Pharm. Bull., 36, 2176–2185.

Prinsen, M.K. (1990) Acute eye irritation/corrosion study with alpha-cyclodextrin in albino rats. Unpublished report No. V90.439 from TNO-CIVO Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Prinsen, M.K. (1991a) Acute intraperitoneal toxicity study with alpha-cyclodextrin in rats. Unpublished report No. V91.084/200060 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Prinsen, M.K. (1991b) Acute eye irritation/corrosion studies with alpha-cyclodextrin in albino rats and ex vivo testing of alpha-cyclodextrin in enucleated-eye-test with chicken eyes. Unpublished report No. V91.553/210069 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Prinsen, M.K. (1991c) Acute dermal irritation /corrosion study with alpha-cyclodextrin in albino rats. Unpublished report No. V91.552/210061 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Prinsen, M.K. (1992) Sensitization study with alpha-cyclodextrin in guinea pigs (maximization test). Unpublished report No. V92.574/352063 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Riebeek, W.M. (1990a) Determination of the acute intravenous toxicity of alpha-cyclodextrin in mice. Unpublished report No. V90.221 from TNO-CIVO Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Riebeek, W.M. (1990b) Determination of the acute intravenous toxicity of alpha-cyclodextrin in rats. Unpublished report No. V90.219 from TNO-CIVO Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Riebeek, W.M. (1990c) Determination of the acute intraperitoneal toxicity of alpha-cyclodextrin and y-cyclodextrin in rats. Unpublished Report No. V90.233 from TNO-CIVO Institute, Zeist, The Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Saha, B.C. & Zeikus, J.G. (1992) Cyclodextrin degrading enzymes. Starch/Stärke, 44, 312–315.

Skiba, M. (1990) [Study on the haemolytic effects of natural cyclodextrins and their derivatives on human red blood cells. Thesis, University of Paris XI.

Szejtli, J., Csherháti, T. & Szögyi, M. (1986) Interactions between cyclodextrins and cell membrane phospholipids. Carbohydr. Polymers, 6, 35–49.

Til, H.P. & van Nesselrooij, J.H.J. (1993) Sub-chronic (90-day) feeding study with alpha-cyclodextrin in dogs. Unpublished report No. V92.016 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Van Ommen, B. & de Bie, A.T.H.J. (1995) Oral and intravenous disposition study with [¹⁴C]-alpha-cyclodextrin in rats. Unpublished report No. V94.389 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Verhagen, F.J.J. & Waalkens-Berendsen, D.H. (1991) Oral embryotoxicity/ teratogenicity study with alpha-cyclodextrin and gamma-cyclodextrin in rats. Unpublished report No. V91.210 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

Waalkens-Berendsen, D.H. & Smits-van Prooije, A.E. (1992) Final report of an oral embryotoxicity/teratogenicity study with alpha-cyclodextrin and gamma-cyclodextrin in New Zealand white rabbits. Unpublished report No. V91.564 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Wacker-Chemie GmbH, München, Germany.

¹ ADI ‘not specified’ is used to refer to a food substance of very low toxicity, which, on the basis of the available data (chemical, biochemical, toxicological, and other) and the total dietary intake of the substance arising from its use at the levels necessary to achieve the desired effect and from its acceptable background levels in food, does not, in the opinion of the Committee, represent a hazard to health. For that reason, and for reasons stated in individual evaluations, the establishment of an ADI expressed in numerical form is not deemed necessary. An additive meeting this criterion must be used within the bounds of good manufacturing practice, i.e., it should be technologically efficacious and should be used at the lowest level necessary to achieve this effect, it should not conceal food of inferior quality or adulterated food, and it should not create a nutritional imbalance.

    See Also:
       Toxicological Abbreviations
       alpha-CYCLODEXTRIN (JECFA Evaluation)