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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

    WORLD HEALTH ORGANIZATION





    SAFETY EVALUATION OF CERTAIN 
    FOOD ADDITIVES



    WHO FOOD ADDITIVES SERIES: 42





    Prepared by the Fifty-first meeting of the Joint FAO/WHO
    Expert Committee on Food Additives (JECFA)





    World Health Organization, Geneva, 1999
    IPCS - International Programme on Chemical Safety

    gamma-CYCLODEXTRIN

    First draft prepared by 
    Dr P.J. Abbott
    Australia New Zealand Food Authority, Canberra, Australia 

          Explanation 
          Biological data 
              Biochemical aspects
                   Absorption, distribution, biotransformation, 
                        and excretion 
              Toxicological studies 
                   Acute toxicity
                   Short-term studies of toxicity
                   Genotoxicity 
                   Developmental toxicity 
                   Special studies 
                        Ocular irritation and dermal sensitization 
                        Cell membrane effects 
                        Impurities 
          Estimate of dietary intake 
          Comments 
          Evaluation 
          References 


    1.  EXPLANATION

         gamma-Cyclodextrin is a ring-shaped molecule made up of eight
    glucose units linked by alpha-1,4-bonds. It is produced by the action
    of the enzyme, cyclodextrin-glycosyl transferase (EC 2.4.1.19) on
    hydrolysed starch syrups. Cyclodextrin-glycosyl transferases are
    amylolytic enzymes that are produced naturally by various strains of
     Bacillus and other organisms. The gene coding for the cyclodextrin-
    glycosyl transferase used for the manufacture of gamma-cyclo-dextrin
    was isolated from  Bacillus firmus and  Bacillus lentus (both of
    which are considered non-pathogenic) and cloned into a strain of
     Escherichia coli K12. Mixtures of alpha-, -, and gamma-cyclodextrin
    are formed during the reaction, and the formation of
    gamma-cyclodextrin is optimized by the addition of the complexant,
    8-cyclohexadecen-1-one, which causes precipitation of the
    gamma-cyclodextrin-8-cyclohexadecen-1-one complex, which can then be
    further purified. The final product contains > 98%
    gamma-cyclodextrin. Cyclodextrin-glycosyl transferase is inactivated
    by heat and removed from the final gamma-cyclodextrin product. The
    complexant, 8-cyclohexadecen-1-one, is extracted from the
    gamma-cyclodextrin with  n-decane which, in turn, is separated from
    the final dry gamma-cyclodextrin product. 

         The circular structure of gamma-cyclodextrin (Figure 1) provides
    a hydrophobic cavity which enables complexes to be formed with a
    variety of organic molecules, while the hydrophilic outer surface
    makes gamma-cyclodexrin water soluble. Because of these properties,
    gamma-cyclodextrin can be used in a variety of ways in food, with a
    potential intake in the order of grams per person per day. 

    FIGURE 1

         gamma-Cyclodextrin has not been reviewed previously by the
    Committee; however, the structurally related -cyclodextrin (seven
    glucose units) was evaluated at the forty-first and forty-fourth
    meetings of the Committee (Annex 1, references 107 and 116). 


    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution, biotransformation, and excretion

         Early experiments provided some evidence that gamma-cyclodextrin
    (unlike alpha- or -cyclodextrin) is hydrolysed by salivary amylase
    (French, 1957). More recent studies have confirmed that both human
    salivary amylase and human or porcine pancreatic amylase can readily
    hydrolyse gamma-cyclodextrin, yielding mainly maltose, some
    maltotriose, and smaller amounts of glucose (Abdullah et al., 1966;
    Marshall & Miwa, 1981; Kondo et al., 1990).

         The fate of intravenously administered gamma-cyclodextrin was
    examined in three male and two female rabbits and one male dog. The
    rabbits were each given a dose of 7.5 g in saline (30 ml of a 25% w/v
    solution), while the dog was given a dose of 25 g in saline (500 ml of
    a 5% w/v solution  at 8.3 ml/min). Blood and urine samples were
    collected and examined by high-performance liquid chromatography
    (HPLC) for gamma-cyclodextrin over 4 h. The half-life of
    gamma-cyclo-dextrin in blood was about 50 min in rabbits and 30 min in
    the dog. In rabbits, about 77% of the injected dose was recovered as
    unchanged gamma-cyclodextrin in the urine within 24 h, while in the
    dog, nearly 80% of the injected dose was recovered as unchanged
    gamma-cyclodextrin in the urine within the first 4 h (Matsuda et al.,
    1985). 

         Two male Wistar rats were given 14C-gamma-cyclodextrin (94%
    radiochemical purity) as single oral or intravenous doses. After an
    oral dose of 14C-gamma-cyclo-dextrin (25 Ci/kg bw diluted to 200
    mg/kg bw), 50% of the radiolabel was excreted in the exhaled air
    within 24 h, while 2% was excreted in the urine and 5% in the faeces;
    35% of the radiolabel was retained in the body after 24 h. No
    detectable gamma-cyclodextrin was found in blood during 8 h after
    administration, suggesting metabolism in the gastrointestinal tract. 

         After the intravenous dose of 14C-gamma-cyclodextrin (25 Ci/kg
    bw diluted to 100 mg/kg bw), 90% of the radiolabel was excreted in the
    urine within 8 h, mainly as unchanged gamma-cyclodextrin. After 8 h,
    small amounts of radiolabel were excreted in exhaled air (1.1%), while
    0.5% was detected in the faeces; 6% was retained in the body after 24
    h (de Bie & van Ommen, 1994).

         Groups of four male Wistar rats were given single oral or
    intravenous doses of 14C-gamma-cyclodextrin. The oral dose was 1000
    mg/kg bw, and the concentration of radiolabel was either 25 or 100
    Ci/kg bw. In order to examine the role of the gut microflora in the
    metabolism of gamma-cyclodextrin, a separate group of germ-free male
    rats was given 25 Ci/kg bw gamma-cyclodextrin diluted to 1000 mg/kg
    bw. The intravenous dose was 600 mg/kg bw, and the concentration of
    radiolabel was 25 Ci/kg bw. 

         In the first experiment, the time-course and routes of metabolism
    were examined after oral administration. After administration of
    1000 mg/kg bw (25 Ci/kg bw) 14C-gamma-cyclodextrin, 62% of the
    radiolabel in males and 71% in females was excreted within 48 h. The
    majority was excreted in expired air (57% in males and 65% in
    females), with maximum excretion between 1 and 2 h after dosing.
    Little was excreted in the urine (2.1% for males and 2.5% for females)
    or faeces (2.9% for males and females) during the 48-h sampling
    period. Retention of radiolabel after 48 h was 27% in males and 22% in
    females, with the most significant retention in the liver (11% in
    males, 5.2% in females). The amounts in all other organs were low:
    intestine, 0.42% for males, 0.36% for females; kidney, 0.13% for
    males. 

         In the second experiment, the excretion kinetics and time-course
    in blood over 24 h were examined after a single oral dose of
    1000 mg/kg bw (100 Ci/kg bw) 14C-gamma-cyclodextrin. The major route
    of excretion was exhaled air (55% for males; 53% for females) during
    the 24-h sampling period. The carcass retained 35% of the radioactive
    dose in males and 25% in females at 24 h after treatment. The maximum
    concentration in blood was reached 40 min after treatment, but this
    concentration decreased by 50% after 0.5 h in males and after 6 h in
    females. The urine of animals of each sex contained a peak that eluted
    before glucose at all times and a small peak that co-eluted with
    glucose (6% of total radiolabel). A small amount of unchanged
    gamma-cyclo-dextrin was detected in urine after 4 h (< 0.02% of the
    ingested dose). HPLC analysis of the stomach contents 24 h after
    dosing revealed the presence of gamma-cyclodextrin and a minor peak
    that co-eluted with glucose. HPLC analysis of the blood revealed the
    presence of a radioactive peak that co-eluted with glucose. 

         In the third experiment, the excretion kinetics and time-course
    in blood over 24 h were examined after a single intravenous dose of
    600 mg/kg bw (25 Ci/kg bw) 14C-gamma-cyclodextrin. The major route
    of excretion was the urine (87% for males, 91% for females), and the
    faeces contained about 1% and the gastrointestinal tract about 0.5% of
    the radiolabel; the exhaled air contained 7.8% of the dose for males
    and 3.2% for females. The carcass contained 3.9% of the dose in males
    and 2.2% in females, the liver having the most residue (0.41% in
    males, 0.42% in females). Radiolabel was removed rapidly from the
    blood, with calculated initial half-lives of 20 min in males and 15
    min in females. HPLC analysis of blood revealed predominantly
    gamma-cyclodextrin. 

         In the fourth experiment, excretion kinetics over 24 h was
    examined in germ-free rats given a single oral dose of 1000 mg/kg bw
    (25 Ci/kg bw) gamma-cyclodextrin. About 73% of the radiolabel was
    excreted in expired air (68% in males, 65% in females). Peak
    exhalation of 14C-carbon dioxide occurred at about 90 min. Low
    concentrations of radiolabel were found in faeces and urine. The
    gastrointestinal tract contained 9.2% in males and 11% in females,
    with the majority in the caecum. The results indicate that
    gastrointestinal microflora play little part in the metabolism of
    gamma-cyclodextrin. 

         Overall, this study indicates that gamma-cyclodextrin is
    metabolized to glucose, other low-molecular-mass sugar metabolites,
    and carbon dioxide by luminal and/or epithelial enzymes of the
    gastrointestinal tract (de Bie & van Ommen, 1994; de Bie et al.,
    1998).

    2.2  Toxicological studies

    2.2.1  Acute toxicity

         The acute toxicity of gamma-cyclodextrin was examined in mice and
    rats; the results are summarized in Table 1. In each study, groups of

    10 animals of each sex were tested at three doses or served as
    controls. There were no deaths at the highest doses tested (Matsuda et
    al., 1983).

        Table 1. Acute toxicity of gamma-cyclodextrin

                                                                                        

    Species    Sex                 Route            LD50          Reference
                                                    (mg/kg bw)
                                                                                        

    Mouse      Male and female     Oral             >16 000       Matsuda et al. (1983)
    Mouse      Male and female     Subcutaneous     > 4 000       Matsuda et al. (1983)
    Mouse      Male and female     Intravenous      > 4 000       Matsuda et al. (1983)
    Mouse      Male and female     Intravenous       10 000       Riebeek (1990a)
    Rat        Male and female     Oral             > 8 000       Matsuda et al. (1983)
    Rat        Male and female     Subcutaneous     > 2 400       Matsuda et al. (1983)
    Rat        Male and female     Intravenous      > 3 750       Matsuda et al. (1983)
    Rat        Male and female     Intravenous      > 3 750       Riebeek (1990b)
    Rat        Male                Intraperitoneal  > 4 600       Riebeek (1990c) 
                                                                                        
    

         In a test for micronucleus formation (see section 2.2.3), groups
    of 15 Swiss mice of each sex were given a single dose of 15 g/kg bw
    gamma-cyclodextrin by gavage and were killed in groups of five at 24,
    48, and 72 h. All of the animals survived until termination (Immel,
    1991). 

         The toxicity of intravenously administered gamma-cyclodextrin was
    examined in groups of five male and five female Crl:CD mice and five
    male and five female Crl:W1(WU)BR rats. The mice were given
    gamma-cyclodextrin (purity, > 98%) at doses of 5000, 7500, or 10 000
    mg/kg bw, while the rats were given doses of 2500, 3750, or 5000 mg/kg
    bw as a solution in sterile saline. The animals were observed for
    clinical signs over 14 days after treatment. They were examined for
    gross pathological changes on day 14 after treatment. 

         Both mice and rats showed dose-related signs of toxicity, such as
    piloerection and sluggishness, within 1 h to a few days after
    treatment. Deaths occurred within a few days in both species, but the
    surviving animals recovered and appeared to be healthy at the end of
    the observation period. Macroscopic examination revealed no gross
    treatment-related alterations (Riebeek, 1990a,b).

         The toxicity of intraperitoneally administered gamma-cyclodextrin
    was examined in groups of three male rats after single doses of 2000
    or 4600 mg/kg bw as a solution in sterile saline. The animals were
    observed for clinical signs during 14 days after treatment and were
    examined grossly for pathological changes on the last day. There were

    no signs of toxicity and no deaths. Macroscopic examination revealed
    no gross treatment-related alterations (Riebeek, 1990c).

    2.2.2  Short-term studies of toxicity 

     Rats 

         Groups of 15 Wistar rats of each sex were given
    gamma-cyclodextrin intravenously as single daily doses of 0, 200, 630,
    or 2000 mg/kg bw for 30 days. A group of five animals of each sex were
    allowed to recover for 27 days after treatment. The animals were
    examined throughout the study, and the body weights recorded weekly.
    Clinical chemical parameters were examined at the end of treatment and
    after the recovery period. Ten animals from each group were killed at
    the end of treatment and the remainder at the end of the recovery
    period. 

         There were no clinical signs of toxicity throughout the study.
    The body-weight gain of males at 630 and 2000 mg/kg bw per day was
    slightly decreased, but the difference was not significant. Food
    consumption was decreased in a dose-related manner only during the
    first few days of treatment. Significantly decreased mean values for
    haemoglobin concentration and haemacrit were seen in males at doses
    > 630 mg/kg bw per day, and significantly decreased erythrocyte
    count in males at 2000 mg/kg bw per day. In females, the erythrocyte
    count, haemoglobin concentration, and haematocrit were significantly
    decreased only at 2000 mg/kg bw per day. The reticulocyte counts were
    significantly increased in both males and females at 2000 mg/kg bw per
    day. These changes were reversed after the recovery period. The only
    significant clinical chemical change was an increase in creatinine and
    urea in both males and females at 2000 mg/kg bw per day, which was
    reversed at the end of the recovery period. Urinalysis revealed a
    significantly increased incidence of haemoglobin in the urine of rats
    at 2000 mg/kg bw per day, which was reversed at the end of the
    recovery period. 

         Pathological examination at autopsy showed that the majority of
    animals at 2000 mg/kg bw per day had light-coloured and, in some
    cases, irregularly coloured kidneys. A dose-dependent increase in
    spleen weight was found in all groups, which was significant in males
    at doses > 630 mg/kg bw per day and in females at all doses. Liver
    weights were significantly increased in females at > 630 mg/kg bw
    per day, and kidney weights were significantly increased at 2000 mg/kg
    bw per day. Lung and adrenal weights were significantly increased in
    both males and females at 2000 mg/kg bw per day. These changes were
    partially reversed after the recovery period, but a significant
    increase in kidney and spleen weights was still present in females at
    2000 mg/kg bw per day. Histopathological examination revealed marked
    vacuolation of renal epithelial cells in the proximate convoluted
    tubules, which may have been related to administration of a substance
    that causes hyperosmolarity. There was also extensive pulmonary
    histiocytosis (massive accumulation of alveolar macrophages) in all
    animals at 2000 mg/kg bw per day. These changes were seen only in some

    animals at 630 mg/kg bw per day and at lesser severity. Considerable
    reversibility was observed after the recovery period. The NOEL was 200
    mg/kg bw per day (Fuchs et al., 1992).

         Groups of 15 Wistar rats of each sex were given
    gamma-cyclodextrin intravenously as single daily doses of 0, 60, 120,
    or 600 mg/kg bw for 90 days. Groups of five animals of each sex were
    allowed to recover at the end of the treatment period. The animals
    were examined throughout the study, and body weights were recorded
    weekly. Haematological and urinary parameters were examined after one
    month, at the end of the study, and on day 37 (males) or 36 (females)
    of recovery. Clinical chemical parameters were examined at the end of
    the treatment period and after recovery. Animals were killed after
    90 days, their organs were examined, and tissues were taken for
    histopathological examination. 

         There were no clinical signs of toxicity. Body-weight gain was
    slightly decreased only in males at the high dose, and food
    consumption was decreased in a dose-related manner only during the
    first few days of treatment. Haematological examination revealed a
    slight decrease in the mean erythrocyte count, haemaglobin
    concentration, and haemacrit in females at one month, and these values
    were significantly decreased at three months only in females at the
    high dose. The reticulocyte counts were significantly increased in
    females at the high dose at three months. At the end of the recovery
    period, all of these values were normal. The only clinical chemical
    changes were a slight decrease in bilirubin concentration in males and
    females at the high dose and a slight increase in alkaline phosphatase
    activity in males at the high dose. At the end of the recovery period,
    all of the values were normal. The slight changes observed in urinary
    parameters were considered not to be treatment-related. 

         Histological examination at autopsy revealed an increased
    incidence of enlarged iliac lymph nodes in males and females at the
    high dose, which was probably related to inflammation at the injection
    site. Increases in the relative weights of lungs, liver, and spleen
    were seen in males and in the relative weights of heart, lungs,
    kidneys, spleen, and adrenal glands in females at the high dose. All
    of these changes were reversed after the four-week recovery period.
    Histopathological examination revealed hyperplasia of the mucosa of
    the urinary bladder, which was reversed after four weeks, and
    pulmonary histiocytosis (macrophage aggregates), which tended to
    reverse after four weeks, in males and females at the high dose. The
    reversibility of the urinary bladder changes indicates that the
    hyperplasia was not preneoplastic. The NOEL was 120 mg/kg bw per day
    (Ehling et al., 1992).

         Groups of five male Wistar rats were fed diets containing
    gamma-cyclo-dextrin at concentrations of 0, 5, 10, 15, or 20%,
    equivalent to 0, 2500, 5000, 7500, or 10 000 mg/kg bw per day, for 14
    days. A control group was fed 20% lactose. The animals were examined
    for clinical signs of toxicity, and food and water consumption was
    monitored throughout the study. On day 14, the animals were killed,

    and blood samples were taken for examination of clinical chemical
    parameters. The animals were also examined macroscopically for
    pathological changes. 

         There were no deaths. The incidence of soft stools was slightly
    higher in treated than control animals, and the mean body weights of
    treated animals were slightly lower than those of controls, although
    this difference was not statistically significant. Food intake was
    slightly lower in controls than in treated groups. Plasma alkaline
    phosphatase activity was increased in animals at 15 and 20%, while
    gamma-glutamyl transferase activity was decreased in all treated
    groups. The activity of aspartame and alanine aminotransferases were
    unchanged, as were the concentrations of total protein, albumin, urea,
    and bilirubin. Enlargement of the caecum was seen at all doses, the
    most significant effect occurring in the group fed 20% lactose. There
    were no macroscopic changes attributable to treatment (Lina & Jonker,
    1989; Lina & Br, 1998).

         Groups of 20 Wistar rats of each sex were fed diets containing
    gamma-cyclo-dextrin at concentrations of 0, 1.5, 5, or 20% (equivalent
    to 0, 750, 2500, or 10 000 mg/kg bw per day) for 13 weeks. A control
    group was fed 20% lactose. In order to examine the reversibility of
    any effects seen, a group of 10 animals of each sex were fed either
    20% gamma-cyclodextrin or 20% lactose for 13 weeks and then control
    diet for one month. As a result of a feeding error, the groups
    receiving 20% gamma-cyclodextrin were discarded, and two additional
    groups were fed 0 or 20% gamma-cyclodextrin for 13 weeks and 10
    animals receiving the 20% dose were fed control diet for an additional
    month at the end of the 13-week period. Animals were examined for
    clinical signs of toxicity, and food and water consumption was
    monitored throughout the study. Ophthalmoscopic examinations were
    conducted in animals at the high dose and those given lactose before
    treatment and at the end of the study. The animals were killed at the
    end of the study, and blood samples were taken for clinical chemistry.
    The animals were also examined macroscopically for pathological
    changes. 

         There were no treatment-related deaths during the study. Soft
    stools were observed in some rats at 5 or 20% gamma-cyclodextrin and
    in those given lactose early in the study, but this effect disappeared
    subsequently. Ophthalmoscopic examination revealed no
    treatment-related effects. The body weights of males at 20%
    gamma-cyclodextrin were slightly but significantly decreased
    throughout treatment; a more significant body-weight decrease was seen
    in the males given 20% lactose. No difference was noted in these
    groups during the recovery period. Food conversion efficiency was also
    slightly decreased in males at 20% lactose. None of the changes in
    haematological parameters was considered to be of toxicological
    significance. Some changes in clinical chemistry were seen in females
    at the high dose, namely increased activity of aspartate and alanine
    aminotransferases, especially in one female. The sodium concentrations
    were slightly higher but within the historical control range. Urinary
    parameters were similar in control and treated groups, apart from a

    significant increase in the urinary calcium concentration at the end
    of treatment in males given 20% gamma-cyclodextrin and rats of each
    sex given 20% lactose; the effect was more pronounced in the latter
    group. The calcium:creatinine ratio was significantly increased in
    rats receiving 20% lactose. The increased calcium appeared to be
    associated with the increased load of osmotically active substances in
    the large intestine; there was no increase in calcium at the end of
    the recovery period. 

         Analysis of organ weights revealed significant increases in both
    absolute and relative caecal weights in rats at 5 and 20%
    gamma-cyclodextrin and 20% lactose in comparison with controls. At the
    end of the recovery period, no difference from controls was seen for
    rats given 20% gamma-cyclodextrin, but the increase was still evident
    in those given 20% lactose. This effect is probably related to the
    retention of an osmotically active substance in the large intestine.
    Relative adrenal weights were increased in males given 20%
    gamma-cyclodextrin and the lactose control group at the end of the
    study. A very slight increase in relative liver weight was seen in
    males at 20% gamma-cyclodextrin and females given lactose. No changes
    in organ weight were seen at the end of the recovery period. 

         There were no gross pathological changes attributable to
    treatment with gamma-cyclodextrin. Microscopic examination revealed
    increased cortico-medullary mineralization in the kidneys of rats at
    1.5 and 5% gamma-cyclodextrin but not at the high dose. This change is
    considered to be relatively common in rats and not related to
    treatment with gamma-cyclodextrin. There was no evidence of
    hyperplasia of the mucosa of the urinary bladder or of pulmonary
    histiocytosis, which were observed after intravenous administration.

         The effects observed in rats after receiving gamma-cyclodextrin
    in the diet at concentrations up to 20% appeared to be largely related
    to the presence of high levels of an osmotically active substance in
    the large intestine and not of toxicological significance. Similar,
    and generally more pronounced, effects were observed when the diet
    contained 20% lactose. On the basis of this study, dietary levels of
    20% gamma-cyclodextrin (equivalent to10 000 mg/kg bw per day) were
    considered to be tolerated without toxicological effects (Lina, 1992a;
    Lina & Br, 1998).

     Dogs

         Groups of four male and four female beagle dogs were fed diets
    containing gamma-cyclodextrin at doses of 0, 5, 10, or 20% (equivalent
    to 0, 1250, 2500, or 5000 mg/kg bw per day). Body weight and food
    consumption were recorded weekly throughout the study. Ophthalmoscopic
    examinations were made at the beginning and end of the study. Blood
    was collected for clinical chemical and haematological examination
    before the start of treatment and at weeks 6 and 13. Urinalyses were
    performed at week 13. During week 14, all animals were killed, their
    organs were examined, and tissues were prepared for histopathological
    examination. 

         There were no deaths during the study and no clinical signs of
    toxicity. Diarrhoea was noted in all groups, increasing in incidence
    and severity with increasing doses of gamma-cyclodextrin, and was more
    severe in females than in males. This effect was attributed to the
    slow digestion and intestinal absorption of gamma-cyclodextrin.
    Ophthalmoscopic examination revealed no differences between treated
    and control animals. Weight gain was comparable in all groups, except
    for a slight, non-significant decrease in males at 20% during the last
    six weeks of the study. Treated dogs had slightly decreased food
    intake during the first two weeks of the study, particularly at the
    high dose. This effect persisted throughout the study only in dogs at
    the high dose. No differences in haematological parameters were seen,
    and clinical chemical parameters were similar in treated and control
    groups. Urinalysis revealed a slight decrease in urinary pH in the
    group receiving 20% gamma-cyclodextrin, which was considered to be
    associated with the intake of poorly digestible carbohydrates and
    increased fermentation in the large intestine. 

         Both the absolute and relative weights of the ovary were
    increased in animals at 10 and 20% gamma-cyclodextrin, but no
    histopathological changes were seen, and the weight of the ovaries in
    the control group was lower than that of other dogs in the same
    laboratory. The changes observed are considered unlikely to be
    toxicologically significant. There was no change in absolute liver
    weight, although the relative liver weight was increased in males at
    the high dose as a result of the slight decrease in body weight at
    this dose. This change is likely to be adaptive and was considered not
    to be toxicologically significant. Caecal weights were increased in
    the dogs at 10 and 20% gamma-cyclodextrin but statistically
    significantly only in females. This change is considered to be related
    to the presence of high levels of an osmotically active substance in
    the large intestine. Pathological examination at the end of the study
    revealed no treatment-related change. Similarly, the histopathological
    findings were unremarkable. On the basis of this study, dietary levels
    of 20% gamma-cyclodextrin (equivalent to 5000 mg/kg bw per day) were
    considered to be tolerated without toxicological effects (Til & van
    Nesselrooij, 1992; Til & Br, 1998).

    2.2.3  Genotoxicity

         The results of assays for genotoxicity with gamma-cyclodextrin
    are shown in Table 2. 

    2.2.4  Developmental toxicity

     Rats

         In a study of embryotoxicity and teratogenicity, groups of 25
    presumed pregnant Wistar Crl:(WI)WU BR rats were fed diets containing
    gamma-cyclodextrin (purity, >98%) at concentrations of 0. 1.5, 5, 10,
    or 20% (equivalent to 0, 2500, 5000, or 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 animals were

    examined throughout the study, and body weight and food consumption
    were recorded. The rats were killed on day 21 and examined for
    parameters of reproductive performance. 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. Maternal body-weight gain
    was similar in all groups except for a slight reduction at the 20%
    dose on days 0-16 of gestation. Necropsy of the dams showed no adverse
    effects that could be related to treatment. The number of viable
    litters, the number of 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 gross, skeletal, or visceral
    abnormalities. Under the conditions of this assay, gamma-cyclodextrin
    was not teratogenic (Verhagen & Waalkens-Berendsen, 1991).

     Rabbits

         In a study of embryotoxicity and teratogenicity, groups of 16
    presumed pregnant New Zealand white rabbits were fed diets containing
    gamma-cyclodextrin (purity, > 98%) at concentrations of 0, 5, 10, or
    20% (equivalent to 0, 1500, 3000, or 6000 mg/kg bw per day) on days
    0-29 of gestation. The animals were examined throughout the study, and
    body weights and food consumption were recorded regularly. The animals
    were killed on day 29 of pregnancy, and the fetuses were examined for
    signs of toxicity, external malformations, and soft-tissue defects and
    were stained for detection of skeletal anomalies. 


        Table 2. Results of assays for the genotoxicity of gamma-cyclodextrin

                                                                                                

    End-point        Test object                Concentration     Result        Reference
                                                                                                

    Bacterial gene   S. typhimurium TA1535,     2-20000           Negativea     Blijleven (1991)
       mutation      TA1537, TA 1538,           g/plate
                     TA98, TA100

    Chromosomal      Human lymphocytes          10-5000           Negativea     de Vogel & van 
      aberrationb                               g/ml                           Delft (1996)

    Micronucleus     CD-1 mouse bone            15 g/kg bw        Negative      Immel (1991) 
      formationb     marrow                     (single dose)
                                                                                                

    a   With and without exogenous metabolic activation
    b   Two assays conducted: treatment times, 6, 24, and 48 h; harvest times, 24 and 48 h
    
         No deaths occurred during the study. Diarrhoea was seen in
    several treated animals. Maternal body weight was significantly
    decreased at the 20% dose during the first week of treatment but not
    thereafter. Necropsy of the does showed no adverse effects that could
    be related to treatment. The number of viable litters, the number of
    corpora lutea, and the mean number of implantation sites were similar
    in all groups. One abortion occurred in the control group and one in
    rabbits at the 10% dose. Fetal length and body weight were similar in
    all groups. Examination of the fetuses revealed no treatment-related
    increase in gross, skeletal, or visceral abnormalities. An increase in
    the incidence of haemorrhagic fluid in animals at the 5 and 20% doses
    was considered unlikely to be treatment-related. Under the conditions
    of this assay, gamma-cyclodextrin was not teratogenic
    (Waalkens-Berendsen & Smits-van Prooije, 1992).

    2.2.5  Special studies

    2.2.5.1  Ocular irritation and dermal sensitization

         gamma-Cyclodextrin was not irritating or corrosive to the eyes of
    albino rabbits (Prinsen, 1990). In a skin sensitization assay in
    guinea-pigs, a 30% solution of gamma-cyclodextrin induced no signs of
    sensitization (Prinsen, 1992). 

    2.2.5.2  Cell membrane effects

         The interactions between alpha-, beta-, and gamma-cyclodextrin
    and membrane phospholipids, liposomes, and human erythrocytes were
    studied  in vitro. gamma-Cyclodextrin and the 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. -Cyclo-dextrin but
    not gamma-cyclodextrin or alpha-cyclodextrin increased the release of
    42K+, 86Rb+, and 137Cs+ from erythocytes by passive transport at
    a concentration of 1.7  10-2 mol/L (Szejtli et al., 1986).

         gamma-Cyclodextrin induced haemolysis of human erythrocytes
     in vitro in isotonic saline after 30 min, with a threshold
    concentration of  16 mmol/L (approximately 20 mg/L). A similar effect
    was seen with alpha-cyclodextrin at 6 mmol/L and with -cyclodextrin
    at 3.5 mmol/L. At 15 mmol/L, gamma-cylodextrin caused a 2% release of
    cholesterol from erythrocytes, indicating that cyclodextrin-induced
    haemolysis is associated with removal of membrane components from
    erythrocytes (Irie et al., 1982). Other studies have confirmed that
    gamma-cyclodextrin causes erythrocyte haemolysis at concentrations of
    15-30 mmol/L (Okada et al., 1988; Yoshida et al., 1988; Leroy-Lechat
    et al., 1994).

         In a study on the differential effects of cyclodextrins on human
    erythrocytes  in vitro, alpha-, -, and gamma-cyclodextrin were
    incubated at increasing concentrations with erythrocytes for 30 min.
    The cyclodextrins induced a change in shape, from discocyte to
    spherocyte, but with -cyclodextrin haemolysis occurred before the

    change was complete. gamma-Cyclodextrin was less potent than either
    alpha- or -cyclodextrin in releasing potassium and haemoglobin from
    erythrocytes and in solubilizing various components of erythrocytes,
    including phospholipids, cholesterol, and proteins. A concentration of
    4 mmol/L gamma-cyclodextrin was required to induce release of
    phospholipid, and > 20 mmol was required for release of cholesterol
    or protein (Ohtani et al., 1989). 

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

         Potential reductions in the luminescence of a strain of  E. coli
    after incubation with various concentrations of cyclodextrins were
    examined. Luminescence was reduced by 20 and 50% by -cyclodextrin at
    concentrations of 0.03 and 0.5%, respectively. With
    gamma-cyclodextrin, a concentration of 2% was required to reduce the
    luminescence by 20%, and a 50% reduction could not be achieved even at
    the highest concentration of 10% gamma-cyclodextrin (Br & Ulitzur,
    1994).

    2.2.5.3  Impurities

     Cyclodextrin-glycosyl transferase 

         The gene coding for cyclodextrin-glycosyl transferase is derived
    from a strain of the  Bacillus firmus-lintus complex. This is a
    ubiquitous group of aerobic, gram-positive, alkalophilic
    microorganisms which form sub-terminal endospores. Both  B. firmus 
    and  B. lintus are considered to be non-pathogenic. After isolation
    from this strain, the gene was cloned into pUC18, isolated again and
    inserted into an expression and secretion vector derived from pJF118EH
    by introduction of a DNA fragment coding for an appropriate signal
    peptide. The vector, pJF118EH, is derived from pBR322, a widely used
    vector which is considered to be safe. All genetic modifications were
    made by directed mutagenesis by standard techniques. The gene that has
    been introduced for production of the signal peptide is cleaved off
    during exportation of cyclodextrin-glycosyl transferase and degraged
    with the  E. coli cell.

         A strain derived from  E. coli K12 was used as the host for the
    cyclodextrin-glycosyl transferase 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 cyclodextrin-glycosyl transferase, the
    recombinant  E. coli strain was cultured in a standard medium. The pH
    of the fermentation broth was adjusted by adding 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. The bacteria were then removed by fermentation and
    the supernatant filtered and concentrated to the crude
    cyclodextrin-glycosyl transferase preparation. 

         Groups of 20 Wistar rats of each sex were given an aqueous
    solution of a cyclodextrin-glycosyl transferase preparation at doses
    of 0, 5, 10, or 20 ml/kg bw per day, equivalent to 0, 65, 130, or 260
    mg/kg bw per day of total organic solids (TOS), by gavage for 13
    weeks. The animals were examined for clinical signs of toxicity, and
    food and water consumption was monitored throughout the study.
    Ophthalmoscopic examinations were conducted in rats at the high dose
    before treatment and at the end of the study. Haematological
    parameters were examined before treatment, on days 30 and 60, and at
    the end of the study. The animals were killed at the end of the study,
    and blood samples were taken for examination of clinical chemical
    parameters. The animals were also examined macroscopically for
    pathological changes, and samples were taken for histopathological
    examination. 

         Two animals (one control male and one female at the low dose)
    died as a result of dosing accidents. There were no clinical signs of
    toxicity in the treated animals which survived. Ophthalmoscopic
    examination of animals at the high dose revealed no treatment-related
    changes. There was no significant difference in body weight between
    treated and control rats, and food consumption was unchanged in the
    treated group. There were no treatment-related haematological,
    clinical chemical, or urinary changes. The organ weights of control
    and treated groups were similar, and there were no treatment-related
    gross pathological changes. Histopathological examination revealed an
    increased incidence of pulmonary changes in the treated animals which
    were considered to be related to aspiration of test material. The NOEL
    was 20 ml/kg bw per day, equivalent to 260 mg/kg bw per day of TOS,
    the highest dose tested (Jonker, 1994). 

         The cyclodextrin-glycosyl transferase preparation was tested for
    mutagenic activity in  S. typhimurium strains TA1535, TA1537, TA98,
    and TA100 with and without metabolic activation at concentrations of
    19-1500 g/plate or 62-500 g/plate. The assay was slightly
    complicated by the growth-stimulating effects of some of the
    components of the crude preparation, but the overall result confirmed
    that cyclodextrin-glycosyl transferase had no mutagenic activity in
    these strains. 

     8-Cyclohexadecen-1-one 

         8-Cyclohexadecen-1-one is a colourless, waxy, solid product. The
    commercial preparation has a purity of > 98%. The acute toxicity of
    8-cyclo-hexadecen-1-one was examined in five male and five female mice
    at a dose of 5 ml/kg bw (4.6 g/kg bw) and in five male and five female
    rats at doses of 5 and 10 g/kg bw. The animals were observed for 14
    days. Two of the five mice died during the study, but there were no
    deaths among the rats. It was concluded that the LD50 was > 4.6 g/kg

    bw in mice and > 10 g/kg bw in rats (Dickhaus & Heisler, 1985;
    Spanjers, 1986).

         Groups of five CD-1 mice of each sex were fed diets containing
    8-cyclo-hexadecen-1-one at doses of 0, 500, 1500, or 7500 mg/kg diet
    (equivalent to 0, 75, 230, or 1100 mg/kg bw per day) for four weeks.
    The animals were examined for clinical signs of toxicity, and food and
    water consumption were monitored throughout the study. Haematological
    and clinical chemical parameters were examined before treatment and at
    the end of the study. The animals were also examined macroscopically
    for pathological changes, and samples were taken for histopathological
    examination. There were no deaths and no clinical signs of toxicity.
    The body-weight gain of mice at the high dose was decreased, but food
    and water intake were similar in treated and control groups. There
    were no treatment-related haematological changes. The only clinical
    chemical change was a decrease in the albumin:globulin ratio in males
    at the high dose. An increase in relative liver weight was seen in
    males and females at the high dose. No treatment-related gross
    abnormalities were seen, but microscopic examination revealed effects
    in the cytoplasm of four males at the high dose. The NOEL was 1500
    mg/kg diet, equivalent to 230 mg/kg bw per day (Lina, 1992b).

         Groups of five male and five female Wistar rats were fed diets
    containing 8 cyclohexadecen-1-one at doses of 0, 600, 3000, or 15 000
    mg/kg diet (equivalent to 0, 30, 120, or 750 mg/kg bw per day) for
    four weeks. The animals were examined for clinical signs of toxicity,
    and food and water consumption was monitored throughout the study.
    Haematological and clinical chemical parameters were examined before
    treatment and at the end of the study; the animals were also examined
    macroscopically for pathological changes, and samples were taken for
    histopathological examination. There were no deaths and no clinical
    signs of toxicity. 

         A number of effects were seen in animals at the high dose.
    Body-weight gain and food intake were decreased, and the red blood
    cell counts and packed cell volume were increased. Clinical chemistry
    revealed increased plasma urea and creatinine concentrations in males
    and decreased albumin in females, and urinalysis showed increased
    urinary density in animals of each sex. The absolute and relative
    liver weights were increased in animals of each sex at the high dose
    and also in males at the intermediate dose. The absolute and relative
    adrenal weights were decreased in females at the high dose. Gross
    examination revealed no remarkable findings, but microscopic
    examination showed treatment-related hepatocellular cytoplasmic
    alterations in animals of each sex at the high dose. There was no
    evidence of degeneration or necrosis. The NOEL was 600 mg/kg diet,
    equivalent to 30 mg/kg bw per day (Lina et al., 1986). 

         8-Cyclohexadecen-1-one was tested for mutagenic activity in
     S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with
    and without microsomal activation. There was no increase in the number
    of revertants of any of the strains tested (Wilmer, 1986). 

         8-Cyclohexadecen-1-one was also tested for its capacity to induce
    micronuclei in animals treated  in vivo. Groups of 30 CD-1 mice of
    each sex were given the compound by gavage at a dose of 3 or 5 ml/kg
    bw (equivalent to 2.8 and 4.6 g/kg bw). Five animals of each sex were
    killed at 24 and 48 h and two of each sex at 72 h after treatment.
    Examination of the bone marrow showed no increase in the number of
    normochromatic erythrocytes over that in control mice (Willems, 1986).


    3.  ESTIMATE OF DIETARY INTAKE

         gamma-Cyclodextrin can be used as a carrier for flavours,
    sweeteners, and colours, and it has been proposed for use in this
    manner in dry mixes for beverages, soups, dressings, gravies, sauces,
    puddings, gelatines, and fillings and also in instant coffee and
    instant tea, coffee whiteners, compressed sweets, chewing gum,
    breakfast cereals, savoury snacks, crackers, and spices and
    seasonings. It is also proposed for use as a carrier for vitamins and
    polyunsaturated fatty acids in dry food mixes and in dietary
    supplements, as a flavour modifier in soya milk, and as a stabilizer
    in bread spreads, frozen dairy desserts, baked goods, bread,
    fruit-based fillings, fat-based fillings, processed cheese, and dairy
    desserts. 

         The estimated daily intake of gamma-cyclodextrin from its use in
    food has been calculated by the dietary survey approach (Amann et al.,
    1998), which is based on food consumption data from the 1989-91
    Continuing Survey of Food Intakes by Individuals, in which data were
    collected from a representative sample of individuals residing in
    households in the United States. Each individual was surveyed over
    three successive one-day periods, and the foods consumed were coded
    into one of about 6000 different categories. For the purposes of the
    calculation, it was assumed that each food (or food component) that
    contains gamma-cyclodextrin contains this substance at the highest
    feasible concentration. When gamma-cyclodextrin was used as a
    component of the food, the intake of that component was calculated
    from data on food consumption. The estimated daily intake was
    calculated for each food in which gamma-cyclodextrin may be used from
    data on one- and three-day food intake. Intakes were calculated both
     per capita and per user. Users were defined as individuals who
    consumed a food of at least one of the categories concerned on at
    least one occasion. 

         The largest amounts of gamma-cyclodextrin are consumed with soya
    milk and dairy desserts. Relatively high intakes (> 2 g/day) also
    result from its use in bread spreads and fruit-based fillings. The
    mean one-day intake of gamma-cyclodextrin from all its food uses was
    estimated to be 4.1 g. The 90th percentile user was estimated to
    ingest about 8.8 g on any one day. The three-day averages are 4 g for
    the mean intake and 7.5 g for the 90th pecentile consumer. The intake
    of gamma-cyclodextrin from chewing gum was estimated from a separate
    survey on chewing-gum intake in the United States, and the average
    consumer was estimated to ingest about 0.07 g gamma-cyclodextrin per

    day. These data represent a 'worst-case' scenario and are based on the
    assumption that gamma-cyclodextrin is used simultaneously in all
    possible applications and at the highest feasible concentrations. The
    realistic average daily intake of gamma-cyclodextrin would therefore
    be lower than the levels indicated above. 


    4.  COMMENTS

         The Committee noted that the close structural similarity between
    gamma-cyclodextrin and -cyclodextrin allows some comparisons to be
    made of the metabolism and the toxicity of these two compounds. The
    metabolism of gamma-cyclodextrin is different from that of
    -cyclodextrin, as demonstrated both  in vitro and  in vivo. In rats
     in vivo, gamma-cyclodextrin (at single doses up to 1000 mg/kg bw) is
    rapidly metabolized to glucose, presumably by the luminal and/or
    epithelial enzymes of the gastrointestinal tract. In contrast to the
    metabolism of -cyclodextrin, there was little involvement of the
    gastrointestinal microflora. Similarly, in contrast to -cyclodextrin,
    much less gamma-cyclodextrin was absorbed and only very low levels
    could be detected in the urine. After intravenous injection,
    gamma-cyclodextrin (at single doses up to 600 mg/kg bw) was rapidly
    cleared from the blood and was excreted, largely unchanged, in the
    urine. Although no studies of metabolism in humans  in vivo were
    available, gamma-cyclo-dextrin, unlike -cyclodextrin, can be readily
    hydrolysed by human salivary and pancreatic amylases  in vitro.

         Short-term (28- and 90-day) studies of toxicity indicate that
    gamma-cyclodextrin has little toxicity when given intravenously or
    orally to rats or orally to 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. This result suggests that the metabolism of
    gamma-cyclodextrin is less efficient at doses higher than those used
    in the studies of metabolism. The results of the studies in rats
    treated intravenously indicate that gamma-cyclodextrin is well
    tolerated, even when given systemically, and may be less toxic than
    -cyclodextrin.

         Studies conducted in rats and rabbits with gamma-cyclodextrin at
    doses up to 20% of the diet did not indicate any teratogenic effects.
    Similarly, the results of a battery of studies of genotoxicity were
    negative. Long-term studies of toxicity, carcinogenicity, and
    reproductive toxicity have not been conducted, but, given the rapid
    metabolism of this substance to glucose and its lack of genotoxicity,
    the Committee concluded that such studies were not required for an
    evaluation. 

          In vitro, gamma-cyclodextrin, like -cyclodextrin, sequestered
    components of the membranes of erythrocytes, causing haemolysis. The
    threshold concen-tration for this effect was, however, higher than
    that of -cyclodextrin. Furthermore, gamma-cyclodextrin was not

    detected in blood after dietary administration of high doses to
    animals  in vivo. 

         It was considered unlikely that interaction of gamma-cyclodextrin
    with lipophilic vitamins would impair their bioavailability, because
    of the rapid digestion of gamma-cyclodextrin  in vivo. Also, there
    was no evidence that vitamin deficiency was induced in experimental
    animals given high doses of gamma-cyclodextrin.

         The Committee considered studies on the short-term toxicity and
    genotoxicity of the enzyme, cyclodextrin-glycosyl transferase, used in
    the production of cyclodextrins, and of the complexant,
    8-cyclohexadecen-1-one, used to optimize formation of
    gamma-cyclodextrin. The data indicated that these substances are
    unlikely to be of toxicological concern in final preparations of
    gamma-cyclodextrin that comply with the specifications. The Committee
    also considered information on genetic modifications in the organism
    used to produce the enzyme, which raised no concern.

         Although no studies of human tolerance to gamma-cyclodextrin were
    submitted to the meeting, the Committee was aware that such a study
    was available. Although it was unable to review the data, the
    Committee was reassured by the relatively low toxicity of this
    compound in animals and the fact that it is less toxic than
    -cyclodextrin, for which studies of human tolerance were available.
    Also, its rapid metabolism to maltose, maltotriose, and glucose
     in vitro by human salivary and pancreatic amylases supports the
    conclusion that, as in laboratory animals, it would be metabolized to
    innocuous metabolites before absorption by humans.


    5.  EVALUATION

         The Committee concluded that there were sufficient data to
    allocate a temporary ADI 'not specified' but that the study of human
    tolerance known to have been conducted should be reviewed in order to
    confirm the absence of adverse gastrointestinal symptoms at normal
    levels of intake. These data should be submitted for consideration by
    the Committee by 1999.


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    See Also:
       Toxicological Abbreviations
       Cyclodextrin, gamma- (WHO Food Additives Series 44)