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        INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

        WORLD HEALTH ORGANIZATION



        SAFETY EVALUATION OF CERTAIN
        FOOD ADDITIVES AND CONTAMINANTS



        WHO FOOD ADDITIVES SERIES 40





        Prepared by:
          The forty-ninth meeting of the Joint FAO/WHO Expert
          Committee on Food Additives (JECFA)



        World Health Organization, Geneva 1998



    MICROCRYSTALLINE CELLULOSE

    First draft prepared
    by Dr J.B. Greig
    Department of Health,
    Skipton House, 80 London Road, London, SE1 6LW, UK

         1.   Explanation
         2.   Biological data
              2.1  Biochemical aspects
                   2.1.1   Absorption, distribution and excretion
                           2.1.1.1   Rats
                           2.1.1.2   Humans
                   2.1.2   Presorption in animal species
              2.2  Toxicological studies
                   2.2.1   Acute toxicity studies
                   2.2.2   Short-term toxicity studies
                           2.2.2.1   Rats
                   2.2.3   Long-term toxicity/carcinogenicity studies
                           2.2.3.1   Rats
                   2.2.4   Reproductive toxicity studies
                           2.2.4.1   Rats
                   2.2.5   Special studies on embryotoxicity and
                           teratogenicity
                           2.2.5.1   Rats
                   2.2.6   Special studies on genotoxicity
                   2.2.7   Special studies on sensitization
                   2.2.8   Special studies on skin and eye irritation
                   2.2.9   Special studies on effects of cellulose fibre
                           on tumour growth
              2.3  Observations in humans
                   2.3.1   Toxicity consequent to substance abuse
                   2.3.2   Changes in gastrointestinal function and
                           nutrient balance
         3.   Comments
         4.   Evaluation
         5.   References

    1.  EXPLANATION

         Microcrystalline cellulose was evaluated at the fifteenth,
    seventeenth and nineteenth meetings of the Committee (see Annex 1,
    references 26, 32 and 38). At the nineteenth meeting an ADI "not
    specified" was allocated. In the light of concern about possible
    persorption and consequential adverse effects of fine particles, the
    substance was re-evaluated at the present meeting.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

    2.1.1  Absorption, distribution and excretion

    2.1.1.1  Rats

         Four rats were fed 14C-labelled microcrystalline cellulose at 10
    or 20% of their diet. No evidence of degradation or digestion was
    noted. Faecal recoveries of radioactivity ranged from 96-104% and were
    complete for all labelled material. No radioactivity appeared in the
    urine (Baker, 1966).

         A study was specifically designed to investigate the possibility
    that persorption of microcrystalline cellulose might induce
    toxicological effects. Groups of male and female Sprague-Dawley CD
    rats (20 per group) from Charles River Laboratories were administered,
    by gavage, suspensions of a special fine particle-size
    microcrystalline cellulose (median particle size 6 µm). The rats were
    dosed orally daily for 90 consecutive days at a level of 5000 mg/kg bw
    per day by means of a 25% suspension in tap water. The animals were
    killed on study days 91-94 and necropsies were carried out under
    conditions that reduced the possibility of contamination of tissues
    with fine particulates. The birefringent microcrystalline cellulose
    particles were not detected in any organ or tissue, including
    gut-associated lymphoid tissue, liver, lung, spleen and brain. The
    size limit for detection of the particles was considered to be < 1 µm
    (Kotkoskie  et al., 1996; FMC Corporation N.V., 1996).

    2.1.1.2  Humans

         One human subject received 150 g of microcrystalline cellulose
    daily in two portions for a 15-day adaptation period. He then received
    14C-labelled microcrystalline cellulose (47.6 µCi) in two portions on
    one day. Supplementation of the diet with unlabelled microcrystalline
    cellulose continued for 10 days. Twenty-four-hour faecal and urine
    collections were examined for radioactivity. No radioactivity appeared
    in the urine or in the expired CO2. All administered radioactivity
    (98.9 ± 3.0%) was recovered from the faeces within two days (Baker,
    1968).

         Metabolism of a preparation of 14C-labelled cellulose by four
    volunteers has been shown to be increased by the consumption, for a
    period of 3 months, of an additional 7 g/per day of dietary fibre. In
    six subjects with an ileostomy, the cumulative excretion of 14CO2
    was lower than in controls. In two constipated subjects metabolism
    appeared to be more extensive and occurred over a longer period
    (Walters  et al., 1989).

         Examination of the stools of one male and one female patient
    given 30 g microcrystalline cellulose as dry flour or gel for 5´ weeks

    showed the presence of undegraded material of the same birefringence
    as the original microcrystalline cellulose administered. No
    significant effects on the human gastrointestinal tract were noted
    during the administration (Tusing  et al., 1964).

         Most (87%) of the radiolabel associated with 131I-labelled
    alpha-cellulose fibres (retained by a sieve with pores of 1 mm diam)
    was excreted by 4 male and 4 female volunteers within 5 days of
    ingestion. Less than 2% of the faecal radiolabel was unbound; urinary
    excretion of unbound radio-iodine accounted for another 1.9% of the
    total dose (Carryer  et al., 1982).

         Other studies have been carried out to demonstrate the
    relationship between persorbability and size and consistency of
    granules. Using quartz sand, the upper limit for persorbability was
    shown to be 150 µm. Starch granules must be structurally largely
    intact to possess the property of persorbability. Persorbed starch
    granules may be eliminated in the urine, pulmonary alveoli, peritoneal
    cavity, cerebrospinal fluid, via lactating milk and transplacentally
    (Volkheimer  et al., 1968).

         In another study, dyed plant foods (oatmeal, creamed corn) were
    fed to human subjects, and blood and urine were examined for coloured
    fibres. Dyed fibres were shown to be present (Schreiber, 1974).
    Lycopodium spores and pollen grains have also been shown to be
    persorbed by humans (Linskens & Jorde, 1974).

         Mean intake of dietary microcrystalline cellulose in the USA has
    been estimated to range from 2.7 g/person per day (children 2 years of
    age) to 5.1 g/person per day (young adult males). For heavy consumer
    intake of microcrystalline cellulose (90th percentile) the values are
    5.4 to 10.2 g/person per day for the same age groups (CanTox Inc.,
    1993).

         The mean intake of dietary microcrystalline cellulose in the
    United Kingdom has been estimated as 0.65 g/person per day. The
    highest mean intake, 0.90 g/person per day, was for children aged
    10-11 (the youngest group for which data were available). For heavy
    consumer intake of microcrystalline cellulose (90th percentile) the
    values ranged from 1.13 g/person per day for adults age 16-24 to 1.83
    g/person per day for males age 10-11 (Egan & Heimbach, 1994).

    2.1.2  Persorption in animal species

         Rats, pigs and dogs were used to study the persorption of
    microcrystalline cellulose. The animals were not fed for 12 hours
    prior to oral administration of the test compound. Rats, dogs and pigs
    were given 0.5, 140 and 200 g, respectively, of the test compound.
    Venous blood was taken from the animals 1-2 hours after administration
    of the test compound, and examined for particles. Persorbed particles
    were demonstrated in the blood of all three species. The average
    maximum diameter for persorbed particles was greater in rats than in
    dogs or pigs (Pahlke & Friedrich, 1974).

         Orally administered poly(DL-lactide-co-glycolide) microspheres of
    diameter 1-10 µm were specifically taken up into the Peyer's patch
    tissue of the gut of BALB/c mice. Those larger than or equal to 5 µm
    diameter remained in the Peyer's patches; those < 5 µm were
    translocated to the mesenteric lymph nodes and spleen (Eldridge 
     et al., 1989).

         These microspheres degrade slowly and can be used for the
    delivery of antigens to the gut-associated lymphoid tissue of BALB/c
    mice, males and females, and the initiation of a secretory IgA
    response in other organs and tissues (Eldridge  et al., 1990).

         The use of particles with differing sizes, i.e. styrene
    divinylbenzene microspheres (5.7 ± 1.5 µm, mean ± SE) and carbonized
    styrene divinylbenzene microspheres (15.8 ± 1.4 µm), indicated that
    smaller particles are more readily translocated to the lungs and
    mesenteric lymph nodes than are those of a larger size. Unlike earlier
    workers, the authors considered that the orally administered particles
    do not reach the peripheral blood but are transported to the lungs via
    the lymphatic system (LeFevre  et al., 1980).

         Orally administered titanium dioxide particles of nominal size
    500 nm are taken up by gut-associated lymphoid tissue of female
    Sprague-Dawley rats and are translocated to liver, spleen, lung and
    peritoneal tissues (Jani  et al., 1994).

         The same research group has investigated the persorption of
    polystyrene microspheres with diameters of 50 nm to 3 µm administered
    by gavage for 10 days to female Sprague-Dawley rats at a dose of 1.25
    mg/kg bw per day. The extent of persorption of the 50 and 100 nm
    particles was 34 and 26%, respectively; the uptake of larger particles
    was less than 14%. The 50 nm particles were detected in stomach, small
    intestine, colon, liver, spleen, kidney and blood. The 3.0 µm
    particles were detected only in the stomach, small intestine and colon
    (Jani  et al., 1990).

         Fluorescent polystyrene microparticles of nominal diameters 0.15
    and 1.0 µm were injected intraduodenally in the region of a Peyer's
    patch in male Wistar rats in which a cannula had been implanted in the
    mesenteric lymph duct. Uptake and detection in lymph was rapid for
    both sizes of particle, with the time of maximal uptake being 65 min
    after the start of the infusion (Jenkins  et al., 1994).

         Sprague-Dawley rats, 10 per group, aged 25 days, 5 months or 15
    months, were dosed with a suspension of fluorescent polystyrene
    microparticles of nominal diameter 2.247 µm for 5 days. There was no
    significant difference between age groups in the number of particles
    that were transferred into gut-associated lymphoid tissue (Simon 
     et al., 1994)

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

         Acute toxicity studies of various cellulose preparations are
    summarized in Table 1.

        Table 1. Acute toxicity of microcrystalline cellulose in animals

                                                                                        

    Species        Sex           Route              LD50              Reference
                                                    (mg/kg bw)
                                                                                        

    Rat 1          M             oral               > 3160            Pallotta, 1959
    Rat 2          M & F         oral               > 5000            Freeman, 1991a
    Rat 3          M & F         oral               > 5000            Freeman, 1996d
    Rat 1          M             intraperitoneal    > 3160            Pallotta, 1959
    Rat 4          M & F         dermal             > 2000            Freeman, 1991b
    Rat 5          M & F         dermal             > 2000            Freeman, 1996e
                                                                                        

                                                    LC50
                                                    (mg/litre)
                                                                                        

    Rat 6          M & F         inhalation         > 5.35            Signorin, 1996
                                                                                        

    1 Test material Cellan 300, five male rats per group.
    2 Based on no deaths in 10 rats of each sex administered 5000 mg/kg of Avicel RCN-15.
    3 Based on no deaths in 5 rats of each sex administered 5000 mg/kg of Avicel AC-815.
    4 Based on no deaths in 5 rats of each sex treated with 2000 mg/kg of Avicel RCN-15.
    5 Based on no deaths in 5 rats of each sex treated with 2000 mg/kg of Avicel AC-815.
    6 Based on no deaths in 5 rats of each sex exposed to 5.35 mg/litre of Avicel AC-815.
    

        In the studies summarized in Table 1, there was no evidence of
    toxicity of microcrystalline cellulose preparations administered
    either orally or dermally to rats at doses of 5000 or 2000 mg/kg bw,
    respectively. The observations seen at necropsy in animals treated
    intraperitoneally with Cellan 300 at 3160 mg/kg bw are consistent with
    an irritant reaction caused by the presence of foreign material. An
    inhalation toxicity study showed only transient effects at a
    concentration of 5.35 mg/litre.

        Groups of five male Sprague-Dawley rats received a single oral
    dose, by stomach tube, of 10.0, 31.6, 100, 316, 1000 or 3160 mg/kg bw
    of a suspension of Cellan 300 (refined alpha-cellulose) in either
    distilled water or Mazola corn oil. The animals were observed for 7

    days following administration. No differences were observed among the
    groups as regards the average body weight, appearance and behaviour
    compared to untreated rats. No observable gross pathology was revealed
    at autopsy in animals dosed with either suspension. Therefore, the
    acute oral LD50 was >3160 mg/kg (Pallotta, 1959).

        Similar single doses of refined alpha-cellulose were given i.p. in
    distilled water suspension to five male rats. During 7 days
    observation there were no abnormalities in the rats given 316 mg/kg bw
    or less. At 1000 and 3160 mg/kg bw inactivity, laboured respiration
    and ataxia were observed 10 min after administration and, at 3160
    mg/kg bw, ptosis and sprawling of the limbs were observed. These
    animals appeared normal after 24 hours and for the remainder of the
    observation period. At sacrifice body weights were higher than normal
    and gross autopsy revealed adhesions between the liver, diaphragm and
    peritoneal wall and congestion of the kidneys. Masses resembling
    unabsorbed compound were also observed and these were found to a small
    extent in the mesentery of the animals administered 316 mg/kg bw.
    There were no deaths and therefore the acute i.p. LD50 was >3160
    mg/kg bw (Pallotta, 1959).

        Ten male and ten female Sprague-Dawley rats fasted overnight were
    fed Avicel RCN-15 (a mixture of 85% microcrystalline cellulose with
    15% guar gum) at a dose level of 5000 mg/kg bw mixed with parmesan
    cheese. Six of ten males and five of ten females consumed the mixture
    within 24 hours. After a 14-day period during which all rats gained
    weight normally they were killed. There were no gross lesions at
    necropsy. Under the specified conditions of administration the LD50
    was >5000 mg/kg bw (Freeman, 1991a).

        An acute inhalation toxicity study using a preparation of Avicel
    AC-815 (composed of 85% microcrystalline cellulose and 15% calcium
    alginate) with mass median aerodynamic diameter of 8.48-8.61 µm (range
    of measures) was dispersed and delivered at a mean concentration of
    5.35 mg/litre in a nose-only inhalation exposure chamber to 5 male and
    5 female Crl:CDBR VAF Plus rats for a period of 4 hours. The rats were
    observed over the 14 days after removal from the chamber. The only
    signs of toxicity were on removal from the chamber and consisted of
    chromodacryorrhea, chromorhinorrhea and, in one male rat, decreased
    locomotion; these signs had resolved by the next day. After 14 days no
    gross lesions were observed at necropsy (Signorin, 1996).

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

        Groups of four male rats were kept on diets containing 0.25, 2.5
    or 25% of various edible celluloses for 3 months. No differences were
    observed among the groups with regard to growth and faecal output.
    Histopathology of the gastrointestinal tract revealed no
    treatment-related abnormalities (Frey  et al., 1928).

        Three groups of five male rats received 0.5 or 10%
    microcrystalline cellulose in their diet for 8 weeks. Growth was
    comparable to controls but the 10% group showed slightly lower body
    weights. Haematology, serum chemistry and vitamin B1 levels in blood
    and faeces showed no differences from controls (Asahi Chemical
    Industry Co., 1966).

        Groups of five male weanling Sprague-Dawley rats received 0, 5, 10
    or 20% of acid-washed cellulose in their diet during three consecutive
    nutrient balance trials over a period of 17 days. Absorption of
    magnesium and zinc were significantly lower in the animals that were
    receiving the 10 and 20% cellulose diets. Histopathology of the
    gastrointestinal tract revealed increased mitotic activity and the
    presence of increased numbers of neutrophils in crypt epithelial
    cells, particularly of the duodenum and jejunum (Gordon  et al.,
    1983).

        A mixture of four types of Elceme (in the ratio of 1:1:1:1) was
    fed to groups of Wistar rats for 30 days at a dietary level of 50%,
    and for 90 days at a dietary level of 10% (Elceme is a
    microcrystalline cellulose, and the four types are identified by
    particle size, namely, 1-50 (powder), 1-100 (powder), 1-150
    (fibrillar), 90-250 (granulate)). All test animals were observed for
    food intake and weight gain. For animals in the 10% group, urinalysis,
    haematological tests and serum biochemical tests were carried out at
    weeks 6 and 13 of the test. A complete autopsy including
    histopathology was carried out at the end of the study. Animals in the
    50% group were subjected to a persorption test, on the last day of the
    study, by addition of a cellulose staining dye (Renal, Wine-red) to
    the food of the test animals at a level equivalent to 5% of the
    Elceme. The animals were sacrificed 24 hours after administration of
    the diet, and a careful histological examination was made of the
    gastrointestinal tract, spleen, liver, kidney and heart for stained
    particles.

        Animals in the 10% group gained significantly less weight than
    those in the control group; the marked decrease commenced in the third
    or fourth week of the study. Food intake was similar in test and
    control groups. Urinalysis, haematological values and biochemical
    values were similar for test and control group 1. At autopsy some of
    the rats on the test diet had distended stomachs, which often
    contained considerable amounts of the test diet. The absolute liver
    and kidney weights and the ratio of the weight of these organs to
    brain weight was increased in test animals when compared with control
    animals. No compound-related pathology was reported. Animals in the
    50% group showed considerable less weight gain than control animals in
    spite of a marked increase in food consumption. No persorption of dyed
    fibres was observed (Ferch, 1973a,b).

        Randomly bred rats of both sexes were divided into groups that
    received a control diet or the control diet with 330 mg/kg
    microcrystalline cellulose for a period of 6 months. Six rats in each

    group were then killed, their organs were examined, and tissues were
    taken for histopathology. No effects of the treatment were observed
    (Yartsev  et al., 1989).

        Groups of Crl:CD(R) BR/VAF/Plus rats (20/sex per group) were
    administered 0 (control), 25 000 or 50 000 mg/kg Avicel RCN-15 in the
    diet for 90 days. A few test animals were noted as having
    chromodacryorrhea/ chromorhinorrhea, but this was not considered to be
    biologically significant. In some early weeks the rats increased diet
    consumption, probably to allow for the increased dietary fibre
    content. Body weight gain was unaffected. During the study and at
    necropsy there was no evidence of treatment-related changes. Clinical
    chemistry, haematology and organ weights were unaffected by treatment.
    Histopathology of 34 organs or tissues, including gastrointestinal
    tract and gut-associated lymphoid tissue of the ileum, provided no
    evidence of toxicity of microcrystalline cellulose. The calculated
    daily consumption of microcrystalline cellulose was 3769 mg/kg bw per
    day for males and 4446 mg/kg bw per day for females. The author noted
    that the NOEL exceeded 50 000 mg/kg diet (Freeman, 1992a).

        Groups of Sprague-Dawley CD rats (20 rats/dose per sex) from
    Charles River Laboratories were administered 0 (control), 25 000 or 50
    000 mg/kg Avicel CL-611 in the diet for 90 days. (Avicel CL-611 or
    Avicel(R) Cellulose Gel is composed of 85% microcrystalline
    cellulose and 15% sodium carboxymethyl cellulose). There were no
    differences in weight gain of the males; a body weight gain decrement
    in females was attributed to a decreased caloric intake. No adverse
    effects attributable to the treatment were observed. At necropsy organ
    weights of the test groups were normal other than changes to adrenals
    of males receiving 50 000 mg/kg and to absolute brain and kidney
    weights in females receiving 25 000 mg/kg, but these were not
    attributed to the treatment. Histopathology of 36 organs or tissues
    from the control and high-dose groups, including gastrointestinal
    tract and gut-associated lymphoid tissue of the ileum, provided no
    evidence of toxicity of the microcrystalline cellulose. The mean
    nominal consumptions, averaged over weekly periods, of Avicel CL-611
    by males and females of the top-dose groups ranged from 2768 to 5577
    and 3673 to 6045 mg/kg bw per day, respectively (Freeman, 1994a).

        Microcrystalline cellulose (Avicel) was used as a positive control
    in a short-term toxicity study (approximately 13 weeks) of Cellulon, a
    cellulose fibre. Sprague-Dawley Crl:CB (SD) BR rats, 20 rats/sex per
    group, received a diet containing 0, 5 or 10% of the appropriate fibre
     ad libitum. Animals were checked daily, and body weights and food
    consumption were monitored weekly. Haematology (10 parameters) and
    clinical chemistry (14 parameters) were performed on blood samples
    taken from 10 rats/sex per group. All animals were necropsied, and
    gross observations and the weights of liver, testes with epididymes,
    adrenals and kidneys were recorded. Histological examination was
    carried out on tissue sections from control and high-dose groups.

        Food consumption was increased in the groups fed cellulose fibre,
    although there were no differences in body weight between the

    fibre-fed and control groups. This effect was attributed to the
    altered nutritional value of the diet. From the haematology and
    clinical chemistry there was only one significant difference of a
    microcrystalline cellulose group from the control value; this was in
    the group of female rats fed 5% microcrystalline cellulose in which
    there was an elevation of the haematocrit. There was no evidence of a
    dose response.

        Study of the necropsy results and the histological observations
    indicated that there was no evidence of any treatment-related effects
    of microcrystalline cellulose during the 13-week feeding study in rats
    at either 5 or 10% in the diet (Schmitt  et al., 1991).

        Groups of Sprague-Dawley (CD) rats (20 rats/dose per sex) from
    Charles River Laboratories were administered, by gavage, suspensions
    of a special, fine particle size, microcrystalline cellulose (median
    particle size 6 µm). The dose levels were 0 (control), 500, 2500 or
    5000 mg/kg per day as a 25% suspension in tap water. Dosing was
    performed daily for 90 consecutive days. No treatment-related deaths
    occurred during the study and the only treatment-related clinical sign
    (pale faeces) was not attributed to toxicity. There were no
    toxicologically significant effects in treated animals with respect to
    body weight, absolute and relative organ weights (5 organs weighed),
    food consumption, clinical chemistry measurements, haematology
    measurements or opthalmoscopic examinations. In animals that had
    received 5000 mg/kg per day there were no treatment-related lesions
    detected histopathologically (in 36 tissues including gut-associated
    lymphoid tissue, liver, lung, spleen and brain) nor was there any
    macroscopic or microscopic finding of microemboli or granulomatous
    inflammatory lesions (Kotkoskie  et al., 1996).

    2.2.3  Long-term toxicity/carcinogenicity studies

    2.2.3.1  Rats

        Three groups of 50 male and 50 female rats received in their diet
    for 72 weeks either 30% ordinary cellulose or dry microcrystalline
    cellulose or micro-crystalline cellulose gel. Appearance and behaviour
    was comparable in all groups. No adverse effects were noted. The body
    weights of males given microcrystalline cellulose gel were higher than
    those of the controls. Food efficiency, survival and haematology were
    comparable in all groups. The liver and kidney weights of males
    receiving microcrystalline cellulose gel were higher than the
    controls. Gross and histopathology showed some dystrophic
    calcification of renal tubules in females on microcrystalline
    cellulose but all other organs appeared unremarkable. Tumour incidence
    did not differ between the groups (Hazleton Labs, 1963).

        The Committtee was aware of a study in which a microcrystalline
    cellulose preparation, of which 90% of the particles had a diameter 
    < 20 µm, was fed to male and female rats at 0 (control), 30, 100 or
    200 g/kg diet. The high mortality during the course of the study, the
    evidence of confounding infection, the limited number of animals for

    which there was histopathological examination, and the absence of
    details of the first year of feeding do not provide adequate
    reassurance as to the ability of this study to detect other than gross
    effects (Lewerenz  et al., 1981).

        A review article makes reference to what may be the earlier, first
    year portion of the above study; however, inadequate referencing did
    not permit a firm conclusion (Steege  et al., 1980).

    2.2.4  Reproductive toxicity studies

    2.2.4.1  Rats

        Groups of eight male and 16 female rats were used to produce P,
    F1a, F1b, F2 and F3 generations after having been fed on diets
    containing 30% microcrystalline cellulose flour or gel or ordinary
    cellulose as a control. The presence in the diet of such an amount of
    non-nutritious material, which contributed no calories, had an adverse
    effect on reproduction. Fertility and numbers of live pups were
    relatively depressed, and lactation performances in all three
    generations, as well as survival and the physical condition of the
    pups, were unsatisfactory throughout the study. The new-born pups
    appeared smaller, weak and showed evidence of disturbed motor
    coordination. Liver weights were increased in the group receiving
    microcrystalline cellulose gel in all generations but other organ
    weights showed no consistent patterns. At autopsy female rats of all
    generations showed kidney changes comprising pitting, occasional
    enlargement and zonation of the cortex. Other organs showed no
    consistent changes. No teratological deformities were seen (Hazleton
    Labs, 1964).

    2.2.5  Special studies on embryotoxicity and teratogenicity

    2.2.5.1  Rats

        Seventy-two rats (Sprague-Dawley CD) divided into eight groups
    were fed a mixture of four types of Elceme in the ratio of 1:1:1:1 in
    the diet at a level of 0, 2.5, 5 or 10% for 10 days, between days 6
    and 15 of pregnancy. Rats of four test groups were killed on day 21 of
    pregnancy and the following parameters studied: number of fetuses and
    resorption sites, litter size and average weight of rats, average
    weight of fetuses and average backbone length. Fetuses were also
    examined for soft tissue or skeletal defects. The remaining groups
    were allowed to bear young, which were maintained to weaning (21
    days). The following parameters were studied: litter size, weight of
    pups at days 7 and 21, and there was a histological study of the
    offspring. Although there is some suggestion that administration of
    dietary Elceme resulted in a dose-dependent increase in resorption
    sites, as well as a change in sex ratio, and possible defects such as
    opaque crystalline lenses, the data has not been presented in a manner
    that permits a meaningful interpretation. However, the author
    concluded that Elceme is non-teratogenic (Ferch, 1973a,b).

        Groups of 25 presumed pregnant Crl:CD(R) BR VAF/Plus rats were
    administered 0 (control), 25 000 or 50 000 mg Avicel RCN-15/kg diet
    (equal to 2.1 and 4.5 g/kg bw per day, respectively)  ad libitum on
    days 6 to 15 of gestation. Animals received basal diet at all other
    times. In the group receiving 50 000 mg/kg the food consumption on
    days 6 to 15 was significantly higher than that of controls, probably
    because of the increased fibre content. On day 20 of gestation the
    dams were killed by carbon dioxide inhalation and the following
    parameters studied: number and distribution of implantation sites,
    early and late resorptions, live and dead fetuses and corpora lutea.
    External, visceral and skeletal examinations of the fetuses were also
    performed. There was no evidence of any adverse effects of the test
    material on either the dams or the fetuses. Due to a protocol error
    fetal sex was not recorded (Freeman, 1992b).

        Groups of 25 presumed pregnant Charles River Sprague-Dawley CD
    rats were administered 0 (control), 25 000 or 50 000 mg Avicel
    CL-611/kg (equal to 2.2 and 4.6 g/kg bw per day, respectively) diet
     ad libitum on days 6 to 15 of gestation. Animals received basal diet
    at all other times. In the test groups the food consumption on days 6
    to 15 was significantly higher than for controls, probably because of
    the increased fibre content. The parameters studied and examinations
    performed were the same as in the study of Freeman (1992b). There was
    no evidence of any effects of the Avicel treatment on the fetuses, and
    there was no evidence of a change of sex ratio in the pups or of eye
    defects. Under the conditions of the study, the maternal and fetal
    NOEL was > 50 000 mg/kg diet (equal to 4.6 g/kg bw per day) (Freeman,
    1994b).

    2.2.6  Special studies on genotoxicity

        Various microcrystalline cellulose preparations have been tested
    for genotoxicity in several different assay systems. The results, all
    of which were negative, are summarized in Table 2.



        Table 2. Results of genotoxicity assays of microcrystalline cellulose

                                                                                                                  

    Test system               Test cells                    Concentration          Results         Reference
                                                                                                                  

    Reverse mutation 1,2      Salmonella typhimurium        50-5000 µg/plate       negative        Batt, 1992
                              TA98, TA100, TA1535, 
                              TA1537, TA1538

    Reverse mutation 1,3      Salmonella typhimurium        10-5000 µg/plate       negative        Lawlor, 1996
                              TA98, TA100, TA1535, 
                              TA1537, TA1538

                              Escherichia coli              10-5000 µg/plate       negative
                              WP2uvrA

    Forward mutation 1,2      Mouse lymphoma                100-1000 µg/ml         negative        Cifone, 1992
                              L5178Y cells, TK locus

    Forward mutation 1,4      Mouse lymphoma                125-1000 µg/ml         negative        Cifone, 1994
                              L5178Y cells, TK locus

    UDS with confirmatory     Rat liver primary cell        10-1000 µg/ml          inactive        McKeon, 1992
    assay 2                   cultures

    In vivo mammalian         Bone marrow polychromatic     5000 mg/kg bw,         negative        Murli, 1992
    micronucleus assay 2,5    erythrocytes of ICR mice      oral

    In vivo mammalian         Bone marrow polychromatic     5000 mg/kg bw,         negative        Murli, 1994a
    micronucleus assay 6      erythrocytes of CD-1          oral
                              (ICR) mice

    In vivo mammalian         Bone marrow polychromatic     5000 mg/kg bw,         negative        Murli, 1994b
    micronucleus assay 4      erythrocytes of CD-1          oral
                              (ICR) mice
                                                                                                                  

    Table 2.  Continued...

    1  With and without rat liver S9 metabolic activation
    2  Test material: Avicel RCN-15
    3  Test material: Avicel AC-815
    4  Test material: Avicel CL-611
    5  Test material: Avicel RCN-15
    6  Test material: Avicel PH101 Pharmaceutical
    


        In the reverse mutation assays the microcrystalline cellulose
    formulations produced a heavy precipitate on the plate at the highest
    concentration. Solubility also affected the forward mutation assays
    and it was not possible to include concentrations of the test material
    that were cytotoxic. In the  in vivo mammalian micronucleus assays it
    is improbable that there was appreciable persorption of the test
    materials, and, therefore, there was little exposure of the bone
    marrow cells. In the test in which Avicel RCN-15 was used it was
    administered admixed with the diet of male and female ICR mice. Only
    mice that had consumed all the diet within 10 hours were retained in
    the study and were killed after 24, 48 or 72 hours. Because one group
    of control mice had 0 micronuclei per 1000 polychromatic erythrocytes,
    the comparison with the test group was statistically significant. This
    was not considered to be a valid observation.

        There is no evidence that microcrystalline cellulose is genotoxic.

    2.2.7  Special studies on sensitization

        Avicel RCN-15 was determined to be non-sensitizing when topically
    applied to ten male and ten female Hartley guinea-pigs (Freeman,
    1991e).

        Avicel AC-815 was determined to be non-sensitizing when topically
    applied to ten male Hartley guinea-pigs (Freeman, 1996c).

    2.2.8  Special studies on skin and eye irritation

        Avicel RCN-15 was judged to be minimally irritating after
    instillation into the eyes of four male and two female New Zealand
    White rabbits (Freeman, 1991c).

        Avicel AC-815 was judged to be minimally irritating after
    instillation into the eyes of four male and two female New Zealand
    White rabbits (Freeman, 1996a).

        Avicel RCN-15 was judged to be non-irritating after a 4-hour
    occlusive contact with the skin of three male and three female New
    Zealand White rabbits (Freeman, 1991d).

        Avicel AC-815 was judged to be non-irritating after a 4-hour
    occlusive contact with the skin of three male and three female New
    Zealand White rabbits (Freeman, 1996b).

    2.2.9  Special studies on effects of cellulose fibre on tumour growth

        The effect of artifical diets containing varied concentrations of
    either wheat bran or pure cellulose fibre on the induction of mammary
    tumours by  N-nitrosomethylurea (i.v., 40 mg/kg) was studied in
    female F344 rats. The wheat bran diet appeared to possess
    anti-promotion properties that pure cellulose lacked. The
    concentrations of serum estrogens, urinary estrogens and faecal

    estrogens did not vary in a consistent, statistically significant
    manner (Cohen  et al., 1996).

        The effect of a high-fibre diet containing 45 000 mg/kg Avicel PH-
    105 on the development of colon tumours was investigated in male
    Wistar rats that were injected with 1,2-dimethylhydrazine
    dihydrochloride (25 mg/kg, s.c., once weekly for 16 weeks). The test
    and control diets were administered for 2 weeks prior to the first
    injection of the carcinogen. There was a reduction in the number of
    animals bearing colon tumours and a statistically significant
    reduction in the number of colon tumours/rat in the high-fibre dietary
    group. However, for small bowel tumours and tumours of the ear canal
    there was no significant difference between the dietary groups Freeman
     et al., 1978).

        A later study by the same authors demonstrated that there was no
    significant effect of increasing the level of cellulose in the diet to
    90 000 mg/kg (Freeman  et al., 1980).

    2.3  Observations in humans

    2.3.1  Toxicity consequent to substance abuse

        Intravenous abuse of drugs available in tablet form has led to the
    detection of excipients, e.g., talc, magnesium stearate or
    microcrystalline cellulose, in the tissues of a series of 33 fatality
    cases of intravenous drug addicts. Microcrystalline cellulose (21
    cases) and talc (31 cases) were detected most frequently and, in some
    cases, were associated with granulomatous lesions (Kringsholm &
    Christoffersen, 1987).

        In one case intravenous abuse of the drug pentazocine, possibly
    for longer than six months, led to a fatal pulmonary granulomatosis
    (Zeltner  et al., 1982).

        In other cases of pentazocine abuse, in addition to pulmonary
    granulomas there were granulomas at the injection site and in the
    right ventricular endocardium. The principal vascular lesion was
    thrombosis (Tomashefski  et al., 1981).

    2.3.2  Changes in gastrointestinal function and nutrient balance

        A number of clinical studies using refined cellulose as roughage
    in the human diet for the treatment of constipation showed no
    deleterious effects. Groups of 18 children received regular amounts of
    edible cellulose instead of normal cereal for three months. The only
    effect noted was an increase in bowel movements but no diarrhoea or
    other gastrointestinal disturbances were seen (Frey  et al., 1928).

        Eight male and eight female volunteers supplemented their normal
    diet with 30 g microcrystalline cellulose per day as either dry powder
    or gel (15% aqueous) for 6 weeks followed by 2 weeks without
    supplementation. No adverse findings were reported regarding

    acceptance or body weight but most subjects complained of fullness and
    mild constipation. Haematology was normal in all subjects. Biochemical
    blood values showed no differences between treatment and control
    periods, nor was there evidence of liver or kidney function
    disturbance. Urinalysis produced normal findings. The faecal flora
    remained unchanged. The cellulose content of faeces increase five to
    eight times during the test period. Microscopy revealed the presence
    of microcrystalline cellulose (Hazleton Labs, 1962).

        In another study, eight healthy males received 30 g
    microcrystalline cellulose daily as supplement to their diet for 15
    days. D-xylose absorption varied between pretest, test and post-test
    periods, being lower during microcrystalline cellulose ingestion. The
    absorption of 131I-triolein was unaffected by microcrystalline
    cellulose ingestion. No change was noted in the faecal flora nor was
    there any significant effect on blood chemistry during ingestion of
    microcrystalline cellulose. Examination of urine, blood and faecal
    levels of vitamin B1 during microcrystalline cellulose ingestion
    showed no difference from control periods (Asahi Chemical Industry
    Co., 1966).

        Twelve men consumed diets containing fibres from various sources
    for periods of 4 weeks. There was no significant difference between
    values of serum cholesterol, triglyceride and free fatty acid levels
    measured after consumption of the basal diet, compared with the values
    measured after consumption of a diet containing cellulose fibres (90%
    cellulose, 10% hemicellulose; James River Corp., Berlin, New
    Hampshire, USA). There were no significant differences in plasma VLDL
    and HDL cholesterol or in the ratio of HDL/VLDL+LDL cholesterol.
    However, the increase in plasma LDL cholesterol after the cellulose
    diet was significant (Behall  et al., 1984).

        A similar study in a group of four men and six women could detect
    no effect of a diet containing added alpha-cellulose (15 g daily) on
    serum total cholesterol, triglycerides, HDL cholesterol and the ratio
    of HDL to total cholesterol. The cellulose was well tolerated (Hillman
     et al., 1985).

        A double-blind cross-over trial of the effects of guar gum and
    microcrystalline cellulose on metabolic control and serum lipids in 22
    Type 2 diabetic patients has been carried out. The fibre preparations
    were given at 15 g/day for a 2-week period and then at 5 g/day for the
    remaining 10-week period of each treatment phase. There was no effect
    of the microcrystalline cellulose diet on fasting blood glucose level,
    glycosylated haemoglobin, serum HDL-cholesterol, serum triglycerides,
    serum zinc or ferritin, or urinary magnesium excretion (Niemi 
     et al., 1988).

        The effect of various dietary fibres, including microcrystalline
    cellulose (40 g), on the uptake of vitamin A (approximately sixty
    times the daily requirement) from a test meal was investigated in 11
    female subjects aged 19 to 22. All the dietary fibres significantly

    increased the absorption of the vitamin A over a period of 9 hours
    (Kasper  et al., 1979).

        A study of apparent mineral balance in a group of eleven men
    revealed that there was no significant effect of cellulose, added to
    the diet at 7.5 g per 1000 kcal for 4 weeks, on the mineral balance of
    calcium, magnesium, manganese, iron, copper or zinc. However, in this
    report the source of the cellulose fibre was not specified (Behall 
     et al., 1987).

        The addition of nutritional grade cellulose (21 g) to the daily
    diet of healthy adolescent girls resulted in reduction of the serum
    calcium, phosphorus and iron concentrations. The authors suggested
    that high-fibre diets may not be advisable (Godara  et al., 1981).

        A study of only three men on a low-fibre diet claimed changes in
    mineral balance consequent on the consumption of additional cellulose
    fibre, 10 g of Whatman No. 3 filter paper daily, in the diet
    (Ismail-Beigi  et al., 1977).

        Microcrystalline cellulose (5 g) did not appear to inhibit the
    uptake of iron in women who were neither pregnant nor lactating
    (Gillooly  et al., 1984).

        A group of twenty women, aged 27-48, who were given 20 g packs of
    alpha-cellulose to be consumed daily for three months, were included
    in a study of the effect of indole-3-carbinol on estrogen metabolite
    ratios. Because the control group and the group fed indole-3-carbinol
    received capsules, the cellulose group could not be blinded; in
    addition, an unspecified number of subjects in this group dropped out
    as they found that the cellulose suspension was unpalatable. However,
    the authors suggest that the estrogen metabolite ratio in the high-
    fibre group was not different from that in the control group (Bradlow
     et al., 1994).

    3.  COMMENTS

    Persorption of microcrystalline cellulose was reported in various
    species, which included rats, in early studies. A recent study in
    which a special fine particle size preparation of microcrystalline
    cellulose (median diameter of particles 6 µm) was administered orally
    to rats (5 g/kg bw per day) for 90 days has failed to confirm the
    earlier observations. In this study precautions were taken to ensure
    that, at autopsy, there was no cross-contamination of the tissues with
    fine particulate matter.

        In various parenteral studies of the acute toxicity of
    microcrystalline cellulose in animals there have been signs consistent
    with a tissue response to foreign particles. Similarly,
    microcrystalline cellulose has been associated with the formation of
    granulomas in human lung when it has been injected intravenously
    during drug abuse. No such lesions have been described as a

    consequence of oral ingestion of microcrystalline cellulose by rats or
    humans.

        In 90-day toxicity tests during which microcrystalline cellulose
    was administered to rats in the diet at concentrations of 2.5 to 50%,
    increased consumption of food to compensate for the content of this
    material was observed. Although this may have some effects on mineral
    absorption there was, in general, no compound-related systemic
    toxicity. The NOEL exceeded 50 g/kg diet, at which dose level the mean
    intakes of microcrystalline cellulose by male and female rats were 3.8
    and 4.4 g/kg bw per day, respectively.

        A two-year feeding study of microcrystalline cellulose in rats was
    brought to the attention of the Committee. Despite a lack of evidence
    of toxic effects, the Committee considered that the execution and
    reporting of the study were not adequate to identify a NOEL.

         In vitro and  in vivo genotoxicity studies were negative.

        In a three-generation reproductive toxicity study in rats that had
    been reviewed by an earlier Committee, there were some effects of
    using 30% microcrystalline cellulose in the diet; these had been
    considered to be a consequence of the quantity of material reducing
    the energy density of the diet. However, in recent embryotoxicity and
    teratogenicity studies in rats there was no evidence of
    compound-related effects at dietary levels up to 50 g of
    microcrystalline cellulose per kg diet (equal to 4.6 g/kg bw per day),
    given on days 6 to 15 of pregnancy.

        In some human studies there have been reports of alterations to
    gastrointestinal function following ingestion of microcrystalline
    cellulose. The changes do not appear to be related to systemic
    toxicity.

    4.  EVALUATION

        The Committee concluded that the toxicological data from humans
    and animals provided no evidence that the ingestion of
    microcrystalline cellulose can cause toxic effects in humans when used
    in foods according to good manufacturing practice.

        It is recognized that small particles of other materials may be
    persorbed and that the extent of persorption is greater with
    sub-micrometre particles. Despite the absence of any demonstrated
    persorption of microcrystalline cellulose in the recent study in rats,
    the Committee, as a precautionary measure, revised the specifications
    for microcrystalline cellulose at the present meeting to limit the
    content of particles less than 5 µm in diameter. The Committee
    retained the ADI "not specified" for microcrystalline cellulose
    conforming to these specifications.

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    See Also:
       Toxicological Abbreviations
       Microcrystalline cellulose (WHO Food Additives Series 1)
       Microcrystalline cellulose (WHO Food Additives Series 5)
       MICROCRYSTALLINE CELLULOSE (JECFA Evaluation)