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    SUCROSE ACETATE ISOBUTYRATE (SAIB)

    Explanation

         This food additive has been previously evaluated by the Joint
    FAO/WHO Expert Committee on Food Additives in 1975 and 1977 (Annex I,
    Refs. 37 and 43). However, no recommendation was made for an
    acceptable daily intake by man. No toxicological monograph was
    prepared. The available data are summarized and discussed in the
    following monograph.

    BIOLOGICAL DATA

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

         Male albino rats (Holtzman), about 250 g, were intubated with
    14C SAIB in corn oil at dose levels equivalent to 27 or 100 mg/kg.
    Forty-five to 50% of the dose was absorbed at the highest dose and
    74-82% at the lower dose. Eighty-eight to 90% of the administered dose
    was eliminated in 48 hours and 90-93% in 96 hours. The relative
    proportion of radioactivity eliminated by the various routes varied
    with the dose. At the high-dose level 55-56% of the absorbed activity
    was eliminated as CO2 and 26-28% in the urine. At the lower dose
    63-67% of the observed activity was eliminated in CO2 and 23-25% in
    the urine. Only small amounts of the administered radioactivity were
    retained in the body: 0.1-0.15% (at the high dose) and 0.35-0.38% (at
    the low dose), with the activity being mainly in the urine. Four days
    after administration of the test compounds the level of 14C
    circulating in the whole blood was less than 0.1% of the dose at the
    higher level and 0.3% at the lower level. Chromatography of extracts
    of the 24-hour faeces of rats showed the presence of SAIB and other
    metabolites. Most of the radioactivity in urine was in the form of
    sucrose, although other unidentified substances were also present
    (Fassett & Reynolds, 1962).

         Female rats were administered 14C SAIB by oral intubation at a
    dose level of 50 mg/kg. More than 90% of the radioactivity remained in
    the gastrointestinal tract at 6 hours with approximately 60% in the
    small intestine. Less than 30% of the 14C found in the small
    intestine was present as SAIB, 4.9% of the 14C dose was excreted in
    the urine, and 2.4% in the respired CO2. Twenty-four hours after
    administration of the test compound, 90% of the 14C had been excreted
    and less than 8% remained in the gastrointestinal tract. Fifty per
    cent. of the 14C was excreted as CO2. Faecal material contained 33%
    of 14C, of which 26% was in the form of unchanged SAIB. 14C
    metabolites present in urine were similar to those obtained from urine
    of rats given an equivalent dose of 14C sucrose (Phillips et al.,
    1976).

         Two rats were dosed orally with a single dose of 14C SAIB at
    dose levels equivalent to 5.8 and 11.2 mg/kg; 59% and 52% of the
    14C-labelled SAIB was respired as CO2, 11% and 13% in the urine,
    and 23% and 27% in the faeces within 3 days. The major C compound in
    the faeces was SAIB or highly acetylated sucrose molecules.
    Chromatographic separation of urine showed 1 or 2 major peaks, which
    were not identified. Six and 6.6% of the 14C was retained in the
    carcass. However, no organ incorporated a significantly greater
    amount than the average. Rats dosed orally with a single dose of
    14C-labelled sucrose at a level of 400 mg/kg showed rapid absorption
    and metabolism of the sucrose to 14CO2, the maximum output being
    observed 2 hours post-dosing. Only small amounts of 14C were
    eliminated in the urine and faeces. At sacrifice (3 days post-dosing)
    the carcass retained 9.6-12.9% of the 14C label. The level in fat was
    somewhat higher than in other tissues (Reynolds et al., 1974).

    Metabolism

         Homogenates of the liver and small intestinal mucosa of rats
    prepared in Krebs-Ringer of phosphate buffer (pH 7.4) were incubated
    with 14C SAIB or 14C sucrose. At 0, 1, 2 and 4 hours, portions were
    removed and assayed for metabolites. The rate and extent of hydrolysis
    of SAIB was less than that of the gum mucosal homogenate, and the rate
    and extent of hydrolysis decreased with increasing concentrations of
    SAIB. Sucrose was readily hydrolysed by the mucosal preparation, but
    hardly at all by the liver preparation (Phillips et al., 1976).

         14C-labelled SAIB was incubated under aerobic conditions with
    preparations derived from the contents of 3 regions of the rat gut,
    namely, stomach, small intestine and the caecum. Preparations from the
    proximal region of the small intestine showed the greatest hydrolytic
    activity, the activity of the caecal contents was less than that of
    the small intestine, and the stomach contents showed no hydrolytic
    activity (Phillips et al., 1976).

         A study of disappearance of radioactivity from loops of the small
    intestine of rats after injection of 14C sucrose and 14C SAIB, after
    1-hour absorptive periods, showed that sucrose was rapidly cleared
    from the intestine, whereas the rate of removal of SAIB activity was
    very slow, less than 13% in 1 hour (Phillips et al., 1976).

         Two dogs were fed a single dose of 3 or 4.9 mg/kg of 14C SAIB.
    Twenty-eight and 27% of the 14C-labelled SAIB was eliminated as
    14CO2, 7% and 5% in the urine, and 53% and 46% in the faeces
    within 7 days. The major 14C compound in the tissues was SAIB.
    Chromatography of the urine showed the presence of one major 14C peak
    as well as a large number of other compounds. Some of the 14C
    appeared to be associated with unchanged SAIB (Reynolds et al., 1974).

         Dogs and rats were dosed with a single dose of 14C SAIB. The
    rats eliminated 7-10% of the dose in the urine in 30-46 hours and the
    dogs 2.8-5.2% of the dose in the urine in 29-30 hours. Size exclusion
    chromatography of the dog and rat urine showed that 14C-labelled
    molecules larger than sucrose were not present to any significant
    extent. The nature of the metabolites were not determined, although
    sucrose, glucose and fructose appeared to be absent (Reynolds &
    Zeigler, 1977).

         In another study, the bile ducts of 3 rats and 1 dog were
    cannulated, and the bile was collected following single per os doses
    of 14C SAIB. The rats eliminated 4.5% of the administered dose in the
    bile, within 15 hours post-dosing. Chromatographic separation of the
    metabolites present in the bile showed that they had properties
    similar to sucrose or sucrose with a few acyl groups attached. In the
    case of the dog, which was subjected to 3 separate trials, about 6% of
    the dose was eliminated in the bile, within 15 hours post-dosing.
    Separation of the metabolites showed the presence of SAIB or highly
    acetylated sucrose (Reynolds et al., 1975).

    TOXICOLOGICAL STUDIES

    Special studies on reproduction

         Groups each of 20 Sprague-Dawley strain rats, equally divided by
    sex, were maintained on diets containing 0, 0.38 and 9.38% w/w SAIB.
    During weeks 9-36 of the study the rats were bred 3 times. Pairs of
    rats were selected within each group (10 pairs) and caged together for
    19 days, after which the male was removed. Females were allowed to
    rear young to weaning at 21 days. Litters were weighed at days 1, 11
    and 21 post-partum. All pups were sacrificed at 21 days, sexed and
    examined for gross abnormalities. The parent rats were bred 3 times,
    each female receiving a different male at each mating. Reproductive
    performance was based on number of pups born, conception rate, pups
    per litter, pups weaned, as well as weight of pups on days 1, 11 and
    21. Reproductive performance as judged from the data presented was
    slightly better in the 0.38% group than control. At the 9.38% level,
    less females became pregnant, fewer pups survived to weaning in the
    first breeding, and more survived to weaning in the third breeding
    (Harper et al., 1966).

         Groups each of 15 female and 5 male rats (Holtzman), 60 days of
    age, were maintained on diets containing 0 or 5% SAIB. These diets
    were fed to parents and their offspring throughout the study. After 1
    month on the diet, rats were regrouped, 1 male and 3 females/cage.
    Pregnant females were then housed separately. The length of gestation,
    number of young and live young, number of young weaned and average
    weight at weaning and post-weaning were parameters recorded for
    assessing reproductive performance. Fifty-one days after last
    parturition, another mating was attempted. Progeny of the first

    breeding (F1) were also bred. The parent generation grew well on the
    5% SAIB diet. The first breeding of the test parent generation was
    equal to or superior to controls, in the parameters measured. At the
    second breeding, a somewhat lower percentage of pups from the treated
    groups was reared from birth to weaning compared to controls, and
    those surviving 2 weeks post-weaning weighed less than control. The
    breeding of the F1 generation appeared satisfactory. However, because
    of an outbreak of respiratory disease, many parents and pups died
    during the test. None of the pups from the SAIB group survived post-
    weaning (Fassett et al., 1965).

    Dominant lethal assay in the rat

         Groups each of 20 male rats (Sprague-Dawley) were administered by
    gavage a single dose of either SAIB in corn oil at a dose level
    equivalent to 20, 200 or 2000 mg/kg, Apholate (positive control) at a
    dose level equivalent to 25 mg/kg, or corn oil alone. Two hours after
    treatment, the males were mated with 2 untreated virgin females for
    the following week. Subsequent matings using naive females were
    carried out during the third, fifth and seventh weeks of treatment,
    corresponding to the post-meiotic, meiotic and pre-meiotic stages of
    spermatogenesis. Twelve to 14 days from the mid-week of the
    presumptive mating the females were sacrificed. The uteri were exposed
    and total implantation sites counted, and classified as viable
    foetuses, early deaths or late deaths. The indices evaluated included
    the insemination index (number of males that inseminated a
    female/number of males per group  100), the pregnancy index (number
    of pregnant females/number of females mated  100), the implantation
    index (number of implantation sites/total number of corpora
    lutea  100), the mean total implantation sites and the early deaths
    per pregnancy. Seventy-two hours after treatment, blood was collected
    from 5 males of each group for leucocyte culture for cytogenetic
    studies. Ten metaphases per rat were examined and scored for
    chromosome number, achromatic gaps, chromatid deletion or exchanges,
    chromosome deletions or exchanges and miscellaneous aberrations.

         The mean implants per pregnancy of the high dose of SAIB
    (2000 mg/kg) and the positive control group were statistically lower
    than control groups during the third and seventh weeks of mating. In
    the case of the positive control, this corresponded with a significant
    increase in early foetal deaths during the third mating week, but not
    during the seventh week. None of the SAIB groups had significantly
    increased early deaths during any of the mating periods. Some of the
    groups had statistically lower mean implants than the respective
    controls, but none of these were fewer than the lower limit of 8 per
    pregnancy in mice. The insemination index and pregnancy index of test
    groups were not significantly different from control. The cytogenicity
    study of the cultured leucocytes showed that there was no significant
    increase in chromosome numbers or aberration in any of the groups
    (Krasavage, 1973).

    Special studies on the effects of SAIB on bromosulfophthalein plasma
    clearance

    Rat

         Groups of 5 male Wistar rats were maintained on diets containing
    4% SAIB for 7 days. Bromosulfophthalein clearance was measured at 0
    (pretreatment) and 24 and 48 hours following withdrawal from the
    treated feed. SAIB had no effect on bromosulfophthalein clearance
    (Procter & Chappel, 1971).

    Dog

         Two male and 2 female beagle dogs were fed diets containing 0.1,
    0.3 or 0.5% SAIB. All 4 animals were tested for bromosulfophthalein
    clearance 24 hours after last feeding of SAIB and again after 48
    hours. A rest period of 1 week was allowed between each dietary level.
    No bromosulfophthalein retention occurred at the 0.1% dietary SAIB
    level; 0.3% and 0.5% SAIB resulted in distinct but reversible
    bromosulfophthalein retention (Procter & Chappel, 1971).

    Monkey

         Two groups each of 3 male squirrel monkeys (Saimiri sciureus)
    were fasted overnight and then were treated either with SAIB (1 g in
    2 ml cotton-seed oil) or cotton-seed oil. Twenty-four hours after
    treatment the monkeys were tested for bromosulfophthalein clearance.
    Following a 7-day rest period, the treatment of the groups was
    reversed. Clearance appeared normal in 2/3 of the animals in each
    group (Procter & Chappel, 1970).

    Special studies on the effects of SAIB on indocyanine clearance

         Groups of male rats (Sprague-Dawley) were fed diets containing 4%
    SAIB and 5% corn oil, or 5% corn oil. Indocyanine green (ICG)
    clearance was determined on 2 rats from each group on days 1, 3, 5, 8,
    10, 22, 26 and 36 of the study. The ICG plasma clearance rates in rats
    from the SAIB group was not significantly different from controls
    (Krasavage et al., 1973).

    Acute toxicity
                                                              

                           LD50
    Animal     Route     (g/kg bw)        Reference
                                                              

    Mouse      Oral         2.5     Fassett & Reynolds, 1962
               i.p.         2.5     Fassett & Reynolds, 1962

    Rat        Oral         2.5     Fassett & Reynolds, 1962
               i.p.         2.5     Fassett & Reynolds, 1962
                                                              

    Short-term studies

    Rat

         Groups each of 50 albino rats (Holtzman), equally divided by sex,
    were maintained on diets containing 0, 1.0 or 5.0% SAIB (w/w) for a
    period of 95 days. Haematological studies including haemoglobin, cell
    volume, leucocyte and differential counts were carried out at days 24,
    52 and 87 of the study. Body weight and food intake was determined
    weekly. The animals were necropsied on day 95 and liver and kidney
    weights recorded and a complete histological study made of the tissues
    and organs (liver, adrenal, kidney, urinary bladder, spleen, stomach,
    small intestine, caecum, heart, cerebrum, cerebellum, bone marrow,
    lung, gonad). There was a slight reduction in weight gain in the males
    fed 5% SAIB and a slight liver weight increase in the females fed 5%.
    No compound-related histopathological changes were observed (Fassett
    et al., 1962).

         Four groups each of 20 Sprague-Dawley rats (10 males, 10
    females), body weight 85-100 g, were maintained on diets containing 0,
    0.30, 1.80 or 9.12% SAIB for 13 weeks. The SAIB was dissolved in
    vegetable oil and the final concentration of vegetable oil added to
    all diets was 9.30%. Body weights were determined weekly. Prior to
    sacrifice at 13 weeks, blood samples were taken and Hb content and
    total WBC and differential cell count determined. At autopsy absolute
    and relative organ weights were determined for liver, kidneys, lungs,
    testes, spleen and heart. A microscopic examination was made of liver,
    kidneys, gonads, spleen, adrenals, bone marrow, heart, jejunum, caecum
    and ileum. At the highest test level there was occasional diarrhoea.
    However, there were no significant differences in weight gain between
    control and test animals. Organ weight, blood chemistry and
    histopathology were similar in all groups and showed no compound-
    related effects (Hunt, 1964).

         Groups each of 80 rats (Sprague-Dawley) equally divided by sex
    were maintained on diets containing 0, 2.5, 5.0 and 10% SAIB. Another
    group was administered daily by gavage phenobarbital at a dose
    equivalent to 100 mg/kg bw. This group was positive control for study
    of liver enlargement and microsomal enzyme induction. Each group was
    divided into 2 series (A and B), group A being treated for 6 weeks and
    group B for 12 weeks. Series A and B were further subdivided into
    subgroups of 10 animals of each sex. One subgroup "2" was subjected to
    the Zoxazolamine muscle test following 6 and 12 weeks on the test
    diet, and then 4 weeks after removal from the test diet. The
    other subgroup "E" was subjected to extensive histological and 2
    biochemical studies, which included ornithine carbamoyl transferase,
    carboxylesterase and proximal liver analysis (protein, glycogen, lipid
    and water). Dietary SAIB had no significant effect on the weight gain
    of test animals except at the 2.5% level, where there was slight
    decrease in weight gain of males in the A series and both sexes in the

    B series. There was a marked reduction of weight gain in animals
    treated with phenobarbital. All animals subjected to the Zoxazolamine
    test showed decreased weight gain for 1 week after the test. In
    general, food consumption in the SAIB groups was not affected,
    although phenobarbital-treated groups showed decreased food intake.
    Urinary excretion of ascorbic acid was measured in A and B groups at
    weekly intervals and showed that, whereas phenobarbital administration
    resulted in a marked and prolonged elevation of urinary excretion of
    ascorbic acid, dietary SAIB had no effect on this parameter. The
    Zoxazolamine muscle relaxant test showed that, whereas the
    phenobarbital treatment caused a very marked reduction in the effect
    of the test compound, dietary SAIB had no effect, suggesting that SAIB
    does not induce microsomal enzymes, that would metabolize
    Zoxazolamine. At autopsy, absolute and relative organ/body weight of
    the adrenals, heart, kidney and liver were determined for test and
    control animals. There were no significant compound-related effects in
    the SAIB-treated animals. The animals administered phenobarbital
    showed enlarged livers. However, this effect was reversible in males,
    but not in females, following a 4-week withdrawal period. Gross
    pathological and histopathological examination of SAIB-treated animals
    did not show any compound-related effects. The biochemical studies of
    SAIB-fed animals showed increased glycogen and water content but no
    increase in carboxylesterase levels. In contrast, the livers of the
    phenobarbital-treated rats showed a significant reduction in glycogen
    and water content, an increase in lipid content, and a marked increase
    in carboxylesterase activity (Procter et al., 1971a).

         In another series of studies, rats (Sprague-Dawley) were divided
    into 14 treatment groups with 10 animals of each sex per group and fed
    diets containing SAIB at dietary levels of 1.0, 2.0 and 4.0% (w/w)
    supplemented with 5% corn oil (w/w). The groups were fed the test
    diets for 28 and 56 days continuously or for 28 days followed or
    preceded by 28 days on control diet. Two groups of each sex were fed
    the control diet containing corn oil only for 28 or 56 days
    continuously. At the termination of the study, animals were sacrificed
    and blood collected for determination of serum alkaline phosphatase
    (SAP), ornithine carbamoyl transferase (OCT), blood urea nitrogen
    (BUN), trigylceride, cholesterol and glucose. The animals were
    autopsied, examined for gross pathology and liver weight and relative
    liver/body weight determined. Liver microsomal enzyme activity
    (p-nitroanisole demethylase), glucose-6-phosphatase and
    bilirubin--D-glucuranyl transferase were determined. Histological
    studies were made of the liver tissue. Weight gain and feed
    consumption of test and control groups were similar. There were no
    significant changes in the serum chemistry of the test animals. Gross
    pathology at autopsy was negative and liver weights (absolute and
    relative) were similar for test and control. Microsomal enzyme
    activity was similar for test and control animals, with the exception
    of glucose-6-phosphatase which depressed in male rats on the 4% SAIB
    diet (Krasavage et al., 1973).

    Dog

         Three test groups, each of 8 pure-bred beagles (4 males, 4
    females), were maintained on diets containing 0.2, 0.6 and 2.0% SAIB,
    dissolved in cotton-seed oil. The control group consisted of 12 pure
    bred beagles (6 males, 6 females). The total fat content of the diets
    was adjusted to 12% by addition of cotton-seed oil.

         Physical and clinical examinations were made twice prior to
    commencement of the feeding study and then at week 12. The clinical
    examinations included haemoglobin concentration, haematocrit, WBC and
    differential cell count, blood glucose, BUN, SAP and LDH, and urine
    glucose, albumin, ketone products, pH, specific gravity, and sediment.
    Neurological reflexes were tested and body weight and food intake
    recorded weekly. At the termination of the study (end of twelfth
    week), all dogs were sacrificed and autopsied. Organ and relative
    organ weights were determined for liver, kidneys, spleen, gonads,
    adrenals, pituitary and brain. The following tissues from control and
    2% level group dogs were examined microscopically: liver, spleen,
    stomach, small and large intestines, pancreas, kidney, bladder,
    adrenals, gonads, thyroid, pituitary, thymus, salivary gland, lymph
    nodes, heart, lungs, marrow, aorta, muscle, spinal cord, brain and
    gall bladder. The liver and kidneys of dogs in the 0.2 and 0.6% SAIB
    groups were also examined microscopically.

         There was no significant compound-related effect on food intake
    and weight gain. Haematological and urine parameters of test animals
    and controls were similar and within normal values. Serum chemistry
    indicated a significant increase in SAP of the dogs in the 2% group
    (approximately two times increase over base level). At autopsy there
    was a marked increase in relative and absolute liver weights in the
    0.6% and 2% groups when compared with controls; all other organ
    weights were normal. No compound-related histopathology was observed
    (Anon., 1965).

         In another study, groups each of 12 beagle dogs (equally divided
    by sex) were fed diets containing SAIB at 0, 0.5, 1.0, 2.0 and 4.0%
    for a period of 12 weeks. Test animals on the 4.0% were maintained a
    further 3 weeks on an SAIB-free diet. Body weight and food intake
    were determined during the course of the study. Fasting blood samples
    were obtained at weeks 4, 8 and 12 of the test and blood biochemical
    tests, including blood urea nitrogen, serum glutamic pyruvic
    transaminase, serum glutamic oxalacetic transaminase, alkaline
    phosphatase, lactic dehydrogenase, serum bilirubin, fasting serum
    glucose, bromosulfophthalein and phenosulfophthalein clearance,
    serum electrolytes, total serum protein, albumin and globulin.
    Bromosulfophthalein plasma disappearance curves and curves for the
    plasma clearance of indocyanine green were determined at week 12 for
    male dogs in the 0 and 4.0% groups. Bromosulfophthalein clearance was
    measured in the 4.0% group during the 3-week withdrawal period. In

    addition to these extensive biochemical tests, haematological analysis
    and urinalysis were carried out on weeks 4, 8 and 12 of the study. At
    the end of the test period, the animals were sacrificed and, following
    completion of gross pathological examination, absolute and relative
    organ body weights determined for the brain, heart, liver, lung,
    kidneys, adrenals, gonads, prostate, uterus, pituitary, spleen and
    thyroid. A microscopic examination was made of samples of heart,
    liver, kidneys, adrenals, small and large intestines. Histochemical
    studies were carried out on liver sections of dogs from the 0.5, 1
    and 2% groups, and included succinate dehydrogenase, phosphorylase,
    glucose-6-phosphate dehydrogenase, glucogen, acid phosphatase,
    alkaline phosphatase, adenosine triphosphatase, and Masson's
    trichrome. Additional samples of liver from test animals (not
    including the 5% group) were analysed for protein, glycogen, lipid and
    water, and carboxylesterase activity was also measured. Serum
    ornothine carbomyl transferase was measured in serum samples obtained
    terminally. Electron microscopic studies were carried out on liver
    samples from animals in all groups except the 4.0% group.

         Daily clinical observation revealed no treatment change.
    Growth and food intake appeared normal in all groups. Urine and
    haematological analysis were similar in test and control groups.
    Marked bromosulfophthalein (BSP) retention occurred among all test
    animals during the experimental period, but was not dose related.
    Increased serum alkaline phosphatase values were directly related to
    dose level and period of exposure. Other parameters were not
    significantly different in test and control animals. The marked
    increase in BSP retention was reversed within 3 weeks when the test
    animals in the 4% group were maintained on an SAIB-free diet.
    Indocyanine green clearance rates were reduced in a manner paralleling
    the changes observed in the BSP tests. Male dogs treated with SAIB
    showed a dose-dependent increase in liver weight. No liver enlargement
    was observed in the female dogs. Histochemical studies of liver
    sections did not reveal any changes in the metabolic or substrate
    content of the parenchymal cells of the liver; however, there was a
    marked increase in enzyme activity (alkaline phosphatase, adenosine
    triphosphatase and glucose-6-phosphate dehydrogenase) of the bile
    canaliculi of treated animals when compared with controls. There was a
    slight but statistically significant reduction of protein content of
    liver and slight increase in liver glycogen. Liver lipid was slightly
    increased at the 2% level. An increase in liver carboxylesterase was
    observed in the test groups, particularly in the males, but the effect
    was not dose related. Ornithine carbomyl transferase values were
    similar for test and control animals and within normal range. Light
    microscopic examination of the liver from treated males showed that
    the liver cells were hypertrophied, the bile canaliculi appeared to be
    slightly dilated and the number of bile pigment granules increased.
    Electron microscopic evaluation of the hepatocytes of treated dogs
    showed various changes, the most consistent being an increase in
    smooth endoplasmic reticulum (SER). The effect was observed in both

    treated males and females, but the effect was most pronounced in the
    males. Other changes included pronounced microvillous patterns,
    prominent Golgi bodies and an increased number of microbodies in the
    intracellular pigment granules (Procter et al., 1970).

         In a parallel study, a group of 8 beagle dogs (equally divided by
    sex) were fed SAIB at a dietary level of 2.0% and then maintained for
    6 weeks on an SAIB-free diet prior to sacrifice. In this study the
    dietary SAIB caused a slight weight depression that was reversed on
    removal of SAIB from the diet. The 18-week values for serum alkaline
    phosphatase and for serum bromosulfophthalein clearance showed that
    the effect of SAIB on liver function was fully reversed during the
    6-week withdrawal period. At autopsy, organ weight studies indicated
    that the liver enlargement reported in the previous study was reversed
    following the 6-week withdrawal period. The histochemical studies
    showed that the findings reported for the groups on test compound
    without withdrawal (high activity of bile canaliculi for alkaline
    phosphatase, adenosine triphosphatase and glucose-6-phosphate
    dehydrogenase) were fully reversible. The effect of SAIB
    administration of liver carboxylesterase was also fully reversible
    during this withdrawal period. Electron microscopic examination of the
    liver indicated that the cellular morphology was completely normal,
    following removal of the SAIB from the diet (Procter et al., 1971,
    1973).

         Six male beagle dogs approximately 6 years old were fed a control
    diet containing 5% (w/w) corn oil for 3 weeks and then an experimental
    diet containing 5% SAIB for 28 days. The control diet was fed for the
    next 57 days, 4 of the dogs were returned to the SAIB diet, and
    indocyanine green (ICG) plasma clearance rates and serum alkaline
    phosphatase determined 24 and 48 hours later. The study was terminated
    3 days later. Haematocrits, haemoglobins, white cell and differential
    counts, SGOT, blood glucose, BUN, serum protein, SAP, OCT, and
    triglyceride and cholesterol determinations were made twice prior to
    and at weekly intervals throughout the feeding study. At the
    termination of the study, all dogs were sacrificed, absolute and
    relative liver and kidney weights determined, and tissues from all
    organs examined microscopically. Dogs on diets containing 5% SAIB
    showed a moderate increase in SAP and a prolongation of ICG plasma
    clearance by the liver. Within 5 weeks of withdrawal of SAIB from the
    diet, SAP activity was near normal. The ICG clearance rate appeared
    within normal range 2 weeks after withdrawal of SAIB from the diet.
    After feeding control diet for 8 weeks, the 4 dogs returned to
    SAIB-containing diets for 24 hours showed a significant prolongation
    of ICG clearance rate, but SAP did not appear to be increased. All
    other parameters of test and control animals were similar (Krasavage
    et al., 1973).

         In another study, groups each of 5 male beagle dogs (11-13 months
    of age) were fed diets containing 5.0% SAIB, plus 5% corn oil, or
    corn oil alone for 93 days. Physical appearance, behaviour, food

    consumption and body weight were determined daily throughout the
    study. Indocyanine green plasma clearance rates were determined at
    3-week intervals. Serum bilirubin was measured at week 7 of the study
    and haematological and blood chemistry (haematocrits, haemoglobin,
    BUN, serum protein, SAP andCT, triglycerides and cholesterol) studies
    were carried out at the termination of the study. All dogs were
    autopsied at the termination of the study, liver and kidney weight
    determined, and all tissues examined microscopically. Liver tissues
    was analysed and glycogen, protein and phospholipid content, and
    samples assayed for microsomal enzyme activity (p-nitroanisole
    demethylase, glucose-6-phosphates and bilirubin--D-glycuranyl
    transferase). Liver, kidney, bone, bile and scrapings of the
    intestinal mucosa were analysed for tissue alkaline phosphatase
    activity. Dogs fed SAIB showed a slight increase in SAP, as well as a
    prolonged indocyanine green clearance time, increased relative and
    absolute liver weight. Liver glycogen and phospholipid content were
    increased while liver protein was decreased. Disk electrophoresis and
    isoenzyme inactivation studies of tissue alkaline phosphatase
    indicated that the elevation of SAP was related to the liver
    isoenzyme. The liver content of alkaline phosphatase in SAIB-fed
    animals was twice that of controls. All other parameters studied were
    similar in test and control animals (Krasavage et al., 1973).

    Long-term studies

    Rat

         Groups each of 20 (10 male, 10 female) Sprague-Dawley strain rats
    were maintained on diets containing 0, 0.38 or 9.38% (w/w) SAIB, for
    104 weeks. During this period rats were bred on 3 successive
    occasions. Rats were housed 5 per cage. Body weight and food intake
    were measured weekly. Animals dying during test period were autopsied.
    All animals surviving 104 weeks were sacrificed and autopsied.
    Relative and absolute organ weights were determined for heart,
    kidneys, liver, lungs, ovaries, spleen and testes. Histological
    studies were made of the following tissues from rats of control and
    9.38% SAIB group: adrenals, aorta, colon, heart, illeus, kidneys,
    liver, lungs, lymph node, ovaries, pancreas, skeletal muscle, spleen,
    stomach, testes, thyroid, urinary bladder and uterus.

         SAIB in the diet did not increase the number of mortalities
    (13/20 control, 14/22 9.38% and 15/20 0.38% groups). However, 4 males
    in the 9.38% SAIB group died during the first 10 weeks. Autopsy
    revealed massive multiple haemorrhage in each case. Subsequent
    measurement of systolic pressure in surviving males failed to
    demonstrate any inter-group difference. There were some differences in
    food intake and body weight between the various groups at various
    stages of the study.

         There were no significant differences in body weight of rats of
    the various groups at the end of the first year; however, during the
    second year, male survivors receiving test substance were heavier than
    respective controls. At termination of the study, organ weight and
    relative organ weight of the various groups were comparable with
    respective controls with the exception of the relative organ/body
    weight of the liver and kidney of male rats of the 9.38% group, which
    were lower than controls. Histological studies did not reveal any
    compound-related lesions (Harper et al., 1966).

    OBSERVATIONS IN MAN

         Three groups each of 8 healthy adults (equally divided by sex)
    were administered 0 or 7.0 mg/kg bw or 20 mg/kg bw SAIB as a single
    dose dissolved in a fruit drink, daily for a period of 14 days. Post-
    and pre-treatment measurements of bromosulfophthalein retention as
    well as pre-, mid- and post-treatment clinical chemistry (serum SAP,
    SGOT, SGPT, LDH, total protein, albumin, calcium, cholesterol,
    glucose, BUN, uric acid, total bilirubin, and A/G ratio), haematology
    (haemoglobin, haematocrit, sediment rate, red and white blood cell
    counts, differential and platelets) and urinalysis showed no
    measurable compound-related effects.

         A male adult was given a single dose of 14C SAIB at a dose level
    of 1.18 mg/kg bw. Samples of urine were collected before dosing and at
    0 and 6.2 hours post-dosing, and subjected to various chromatographic
    procedures. Glucose, fructose and the esters of fructose and sucrose
    were not present in the urine. Two unidentified peaks were considered
    to be the principal metabolites of SAIB (Reynolds & Zeigler, 1977).

         When 14C SAIB was incubated with human faecal homogenates less
    than 2% in the SAIB was hydrolysed to sucrose in a 20-hour period.
    Hydrolysis of SAIB by suspensions of human gut bacteria was also very
    low (Phillips et al., 1976).

         Two male subjects were given SAIB (100 mg and 1 g) as a single
    dose. The urinary excretion of sucrose and sucrose esters was less
    than the limit of detection of the assay procedure used (1 ppm
    (0.0001%) sucrose) in any 24-hour period up to 5 days post-dosing. In
    another study, 2 male subjects were given 1 g of SAIB/day for 7 days.
    No urinary excretion of sucrose was detected. No unchanged SAIB or
    metabolites were detected in faecal sample of 1 subject given 100 mg
    of SAIB daily for 7 days. Two subjects were given sucrose
    intravenously (100, 250 and 800 mg in a 10% solution w/v) and the
    urine collected at 3, 12 and 24 hours. Approximately 50% of the
    administered compound was recovered in the urine by 3 hours at all 3
    dose levels, and there was almost quantitative recovery of the lower
    dose by 12 hours (Phillips et al., 1976).

         14C SAIB was incorporated into a simulated non-carbonated soft
    drink and administered to 3 male subjects. The 3 subjects were each
    given 2 or 3 single doses at widely spaced intervals. The first dose
    was administered at a level of ca. 1 mg/kg to each subject, none of
    which had been previously exposed to SAIB. Two of these subjects were
    given a second dose at the same level 7-27 weeks after the first dose
    and following ingestion of unlabelled SAIB at a level of 1 mg/kg for
    7 days. The third subject received a single dose at a level of
    0.18 mg/kg 25 weeks after the first dose. One subject was given a
    third dose at a level of 1 mg/kg 10 weeks after the second dose and
    immediately after ingestion of a high fat meal. All subjects were
    monitored for elimination of radioactivity in the breath and urine for
    at least 30 days post-dosing. A small portion of the radioactivity was
    reportedly eliminated in the urine, the maximum rate of urinary
    excretion occurring within 3 hours and then falling off to 0.5-0.82%
    of the dose/day by 48 hours. Fourteen to 21% of the dose was excreted
    in the urine. Elimination of radioactivity in the breath occurred
    rapidly. Only a small amount of the total dose was eliminated in the
    6-8-hour period post-dosing, but reached maximum levels of elimination
    9-15 hours post-dosing. Forty-four to 66% of the dose was excreted as
    CO2 in 25 days. A small portion of the dose was unabsorbed and
    appeared in the faeces of all subjects. Prior dosing with SAIB or 14C
    SAIB had no effect on the elimination pattern. Chromatographic studies
    of urine showed several radioactive peaks, which have not been clearly
    identified; however, the amount present as free sucrose was estimated
    to be 20% of the radioactivity. Chromatographic studies of extracts of
    faeces showed a presence of a band of radioactive materials that did
    not correspond with those in the urine. In another study, two subjects
    ingested 14C sucrose at a level of 400 mg/kg. Forty-one to 59% of the
    14C sucrose was metabolized to 14CO2 within 48 hours, the maximum
    rate of elimination occurring 3 hours post-dosing. Both subjects
    eliminated small amounts of 14C in the urine (1.9% and 1.7% of the
    dose). Most of the radioactivity appeared to be in urea 14C (Reynolds
    et al., 1972).

         Twenty subjects (10 male and 10 female) between 18 and 22 years
    of age ingested a daily dose of SAIB at a level equivalent to 1 mg/kg
    bw for a period of 14 days. The dose was taken as a bolus each
    morning. Determination of the following blood parameters were made
    prior to treatment and at days 7 and 18 of the test: SGOT, SGPT, serum
    alkaline phosphatase, serum bilirubin, total protein, albumin, uric
    acid, blood urea nitrogen, also erythrocyte sedimentation rate. There
    were no significant differences in any parameters for any 1 individual
    (Hensley, 1973).

         Human subjects were administered a single oral dose of 14C SAIB
    (101.2 mg/kg bw). Forty-one to 66% of the administered 14C dose was
    eliminated as CO2, and 15-21% in the urine in a 25-30-day period and
    10% in the faeces in a 5-day period. The major 14C constituent in the
    faeces was unchanged SAIB. Chromatography of the urine showed two
    major 14C peaks, similar to that observed with the rat (Reynolds et
    al., 1974).

    Comments

         At this time the metabolism of SAIB in experimental animals (dog
    and rat) and man has not been clearly defined. SAIB undergoes partial
    degradation in the gastrointestinal tract and the products of
    hydrolysis following absorption may be eliminated in the bile and
    urine or further metabolized to CO2. Some partially degraded SAIB and
    undegraded SAIB is eliminated in the faeces. Hydrolysis of SAIB occurs
    as a result of the action of enzymes (non-specific esterase) in the
    gastrointestinal tract, rather than the action of gastrointestinal
    flora. Examination of urinary metabolites of SAIB in rat and dog have
    not shown major qualitative differences; however, the chemical nature
    of the metabolites is still not defined. Metabolites in the urine of
    man have not been identified, but are not glucose, sucrose or esters
    of sucrose and fructose. Studies of the metabolites eliminated in the
    bile of rats and dogs following administration of 14C-labelled SAIB
    indicate species differences. In the case of the rat, the metabolites
    have chromatographic properties similar to sucrose or sucrose with a
    few acyl groups attached, whereas, in the case of the dog, metabolites
    were similar to SAIB or highly acylated sucrose.

         Short-term and chronic feeding studies are available for the rat.
    At the highest dose level fed (9.38%), a number of males died during
    the first 10 weeks of the study as a result of massive multiple
    haemorrhage. Subsequent measurement of systolic pressure in surviving
    males failed to demonstrate any inter-group differences. The study
    failed to reveal any compound-related histology. In the case of the
    dog, 12 weeks of exposure to dietary levels of 0.6% or 2% SAIB
    resulted in a significant increase in SAP (serum alkaline
    phosphatase), as well as relative liver weights. The liver changes in
    the dog included increased liver weight and serum alkaline
    phosphatase, as well as changes in liver function as shown by
    bromosulfophthalein (BSP) retention, and indocyanine green clearance
    tests were dose dependent and occurred in the male but not in female
    dogs. The changes were readily reversible when the test animals were
    maintained on an SAIB-free diet. Changes in indocyanine green
    clearance tests were not observed when rats, monkeys or man were dosed
    with SAIB. It is not known if this species difference is related to
    possible differences in metabolism of SAIB, as has been suggested by
    examination of SAIB metabolites in the bile of rats and dogs. Although
    the effect observed in the dog appears to be readily reversible
    following a relatively short period of exposure to SAIB, it is not

    known if continuous exposure to SAIB for longer periods will result in
    development of a pathological lesion. The available data on
    reproduction studies are inadequate because in 1 study there was a
    loss of parents and pups due to respiratory infection and, in another
    study, at the highest dose level fed (9.38%), less females became
    pregnant than controls, fewer pups survived to weaning in the first
    breeding, and none survived to weaning in the third breeding.

         Evaluation of the carcinogenic potential of SAIB is limited
    because only 1 chronic study in rats is available. No other rodent
    species has been tested at this time.

         The additional studies required by the Committee in 1977 are not
    available.

    EVALUATION

         Further work is required before an acceptable daily intake for
    man can be allocated.

    Estimate of acceptable daily intake for man

    Not allocated.

    FURTHER WORK OR INFORMATION

    Required before an ADI can be allocated

    (1) A carcinogenicity/toxicity study in 2 animal species.

    (2) A 2-year dog study with an adequate number of animals and groups
    to demonstrate a no-effect level.

    (3) A multigeneration reproduction/teratology study.

    REFERENCES

    Anon. (1965) Subacute (90-day) feeding studies with SAIB in dogs.
         Unpublished report of Food and Drug Research Laboratories, Inc.
         (Report No. 86501). Submitted to the World Health Organization by
         the Eastman Kodak Co., Kingsport, Tenn., USA

    Fassett, D. W. & Reynolds, R. C. (1962) The fate of sucrose acetate
         isobutyrate in the rat. Unpublished report No. BCH 62-1 of
         Laboratory of Industrial Medicine, Eastman Kodak Co. Submitted to
         the World Health Organization by the Eastman Kodak Co.,
         Kingsport, Tenn., USA

    Fassett, D. W. et al. (1962) Physiologic effects of sucrose acetate
         isobutyrate. Unpublished report of Laboratory of Industrial
         Medicine, Eastman Kodak Co. Submitted to the World Health
         Organization by the Eastman Kodak Co., Kingsport, Tenn., USA

    Fassett, D. W., Roudabush, R. L. & Tarhaar, C. J. (1965) Reproduction
         study in rats fed sucrose acetate isobutyrate (61-115-2).
         Unpublished report of Laboratory of Industrial Medicine, Eastman
         Kodak Co. Submitted to the World Health Organization by the
         Eastman Kodak Co., Kingsport, Tenn., USA

    Harper, K. H. et al. (1966) Chronic toxicity and effect upon
         reproductive function of SAIB in the rat (Final Report).
         Huntingdon Research Lab. (HRC Report No. 1612/66/140). Submitted
         to the World Health Organization by the Eastman Kodak Co.,
         Kingsport, Tenn., USA

    Hensley, W. J. (1973) A brief report on the use of sucrose acetate
         isobutyrate in human volunteers. Unpublished report prepared for
         the Food Additives Sub-Committee of the National Health and
         Medical Research Council, Australia. Submitted to the World
         Health Organization by the Coca-Cola Co., Atlanta, Georgia, USA

    Hunt, H. (1964) Short-term toxicity of SAIB in rats. Report submitted
         to Aktiebolaget Fructus Fabriker, Stockholm, Sweden. Submitted to
         the World Health Organization by the Eastman Kodak Co.,
         Kingsport, Tenn., USA

    Krasavage, W. J. et al. (1973) Biological effects of sucrose acetate
         isobutyrate in rodents and dogs, J. Agr. Food Chem., 21,
         473-478

    Phillips, J. C. et al. (1976) Studies on the metabolism of sucrose
         acetate isobutyrate in the rat and in man, Fd. Cosmet.
         Toxicol., 14, 375-380

    Procter, B. G. & Chappel, G. I. (1970) Studies of the effect on bromo
         sulfophthalein plasma clearance rate in the Squirrel monkey.
         Unpublished report (Project No. 1570), Bio-Research Laboratories,
         Ltd., Quebec, Canada. Submitted to the World Health Organization
         by the Canadian Soft Drink Association, Toronto, Canada.

    Procter, B. G., Dussault, P., Burford, R. G., Rona, G. & Chappel, C.
         I. (1971a) A subacute study of the effect of ingestion of sucrose
         acetate isobutyrate on the liver of the rat. Unpublished report
         (Project No. 966), Bio-Research Laboratories, Ltd., Quebec,
         Canada. Submitted to the World Health Organization by the
         Canadian Soft Drink Association, Toronto, Canada.

    Procter, B. G. Dussault, P. & Chappel, C. I. (1973) Biochemical
         effects of sucrose acetate isobutyrate (SAIB) on the liver,
         Proc. Soc. Expt. Biol. Med., 142, 595-599

    Procter, B. G., Dussault, P., Rona, G. & Chappel, C. I. (1970) A study
         of the subacute oral toxicity of sucrose acetate isobutyrate
         (SAIB) in the Beagle dog. Bio-Research Laboratories, Ltd (Project
         No. 953, Report No. 1), Quebec, Canada. Submitted to the World
         Health Organization by the Canadian Soft Drink Association,
         Toronto, Canada.

    Procter, B. G., Dussault, P., Rona, G. & Chappel, C. I. (1971) A study
         of the subacute oral toxicity of sucrose acetate isobutyrate
         (SAIB) in the Beagle dog. Bio-Research Laboratories, Ltd (Project
         No. 953, Report No. 2), Quebec. Canada. Submitted to the World
         Health Organization by the Canadian Soft Drink Association,
         Toronto, Canada.

    Reynolds, R. C. Astill, B. D., Terhaar, C. J. & Fassett, D. W. (1974)
         Fate and disposition of sucrose-U-14C acetate isobutyrate in
         humans, rats and dogs, J. Agr. Food Chem., 22, 1084-1088

    Reynolds, R. C., Krasavage, W. J., Travis, M. G. & Terhaar, C. J.
         (1975) Elimination of radioactivity in bile of rats and a dog fed
         sucrose-14C (U) acetate isobutyrate. Health and Safety
         Laboratory, report No. BCH-75-6, Eastman Kodak. Submitted to the
         World Health Organization by the Eastman Kodak Co., Kingsport,
         Tenn., USA.

    Reynolds, R. C., Travis, M. G. & Ely, T. S. (1972) Physiological fate
         of sucrose-14C(U) acetate isobutyrate and sucrose-14C(U) in
         humans. Unpublished report (BCH-72-1), Laboratory of Industrial
         Medicine, Eastman Kodak Co. Submitted to the World Health
         Organization by the Eastman Kodak Co., Kingsport, Tenn., USA

    Reynolds, R. C. & Ziegler, D. A. (1977) Metabolites of sucrose acetate
         isobutyrate in the urine of rats, dogs and a man. Unpublished
         report (BC-77-T2), Eastman Kodak Co. Submitted to the World
         Health Organization by the Eastman Kodak Co., Kingsport, Tenn.,
         USA
    


    See Also:
       Toxicological Abbreviations