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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



    SALATRIM
    (Short- and long chain acyltriglyceride molecules)

    First draft prepared by
    Mr J. M. Battershill,
    Dr J. B. Greig,
    Department of Health, Skipton House,
    80 London Road, London, SE1 6LW, U.K.

    Dr J. R. Lupton,
    Department of Animal Nutrition,
    Texas A&M University, 218 Kleberg,
    College Station, TX 77843-2471, USA

    1.   Explanation
    2.   Biological data
         2.1  Biochemical aspects
              2.1.1  Biotransformation 
                     2.1.1.1  Rats
                     2.1.1.2   In vitro studies
                     2.1.1.3  Caloric availability in rats and humans
              2.1.2  Effects on enzymes and other biochemical parameters
         2.2  Toxicological studies
              2.2.1  Acute toxicity studies
              2.2.2  Short-term toxicity studies
                     2.2.2.1  Rats
                     2.2.2.2  Minipigs
              2.2.3  Long-term toxicity/carcinogenicity studies
              2.2.4  Reproductive toxicity studies
              2.2.5  Special studies on gut microflora
              2.2.6  Special studies on genotoxicity
         2.3  Observations in humas
              2.3.1  Clinic-based studies
              2.3.2  Non-clinic-based study (free-living)
    3.   Comments
    4.   Evaluation
    5.   Appendix 1 Caloric availability of salatrim triglycerides
         5.1  Explanation
         5.2  Definitions and chemistry
         5.3  Caloric value determination
              5.3.1  Biochemical aspects
                     5.3.1.1  Caloric value of SCFA
                     5.3.1.2  Caloric value of stearic acid
                     5.3.1.3  Determining the caloric value of salatrim
                              products based on a rat growth assay
                     5.3.1.4  Determining the caloric value of salatrim
                              products based on the stearic acid
                              absorption coefficient 
    6.   References

    1.  EXPLANATION

         Salatrim fats are a family of structured triacylglycerides that
    differ from triglycerides normally encountered in the diet in that
    they contain at least one long-chain fatty acid (LCFA; principally
    stearic acid) and one or two short-chain fatty acids (SCFAs; acetic,
    propionic and/or butyric acid). Salatrim fats are intended for use as
    low-calorie fats in soft sweets, coatings (e.g., wafers and
    confections), dairy products (including spreads) and shortening in
    biscuits. These materials have not been previously evaluated by the
    Committee. Table 1 provides details on the nomenclature of salatrim
    products.

        Table 1. Typical molar ratios of short- and long-chain acid sources used to prepare 
    the salatrim family of edible oils1

                                                                                         

    Salatrim family    Short-chain source     Long-chain source            Mole ratio
                                                                                         

    Salatrim 4CA       tributyrin             hydrogenated canola oil      2.5:1

    Salatrim 4SO       tributyrin             hydrogenated soybean oil     12:1

    Salatrim 23CA      triacetin              hydrogenated canola oil      11:1:1
                       tripropionin

    Salatrim 23SO      triacetin              hydrogenated soybean oil     11:1:1
                       tripropionin

    Salatrim 32CA      tripropionin           hydrogenated canola oil      11:1:1
                       triacetin

    Salatrim 43SO      tributyrin             hydrogenated soybean oil     11:1:1
                       tripropionin

    Salatrim 234CS     triacetin              hydrogenated cottonseed oil  4:4:4:1
                       tripropionin
                       tributyrin

    Salatrim 234CA     triacetin              hydrogenated canola oil      4:4:4:1
                       tripropionin
                       tributyrin

    Salatrim 234SO     triacetin              hydrogenated soybean oil     4:4:4:1
                       tripropionin
                       tributyrin
                                                                                         

    1  The salatrim family name defines the sources of the short-chain and long-chain fatty 
       acids with the numerals representing the carbon chain lengths of the short-chain 
       acids in decreasing proportion in the mix; the letters define the oil that provides 
       the source of the long-chain fatty acids. (e.g., in salatrim 43SO tributyrin and 
       tripropionin are the SCFAs and the LCFA source is hydrogenated soybean oil. The molar 
       ratio of the mix that is used to prepare the salatrim is 11 parts tributyrin : 1 part 
       tripropionin : 1 part hydrogenated soybean oil).
    
        The products that have been used in safety evaluation studies are
    listed in Table 2. There are only very minor differences in
    composition between salatrim products prepared from different
    long-chain fatty acid sources. However, different batches of a product
    may have used different molar ratios of the starting products.

        Table 2.  Materials used in metabolism and toxicity studies

                                                                                         

    Ames tests                                   4CA, 23CA, 23SO, 32CA, 234CA, 234CS

    In vitro mammalian tests                     23CA

    In vivo bone marrow micronucleus assays      234CA, 234SO

    In vitro metabolism                          4CA, 23CA, 32CA, 234CA
    (porcine pancreatic lipase)

    Metabolism in rats                           23CA

    90-day feeding studies (rats)                4CA, 23CA, 32CA, 23SO, 234CA, 234CS1

    28-day-old minipigs                          23SO

    Effects on gut microflora: rats              23CA, 32CA

    Studies I & II in volunteers                 23CA

    Studies III & IV in volunteers               23SO

    Free-living study in volunteers              4SO, 23SO, 43SO
                                                                                         

    1 Plus supplementary 17-day test of effects on transaminases.
    
        The results of these studies were published in the Journal of
    Agricultural and Food Chemistry, volume 42, issue 2, 1994 (Finley 
     et al., 1994a,b,c,d; Hayes & Riccio, 1994; Hayes  et al.,
    1994a,b,c,d,e,f,g; Klemann  et al., 1994; Scheinbach  et al., 1994).
    The summaries given below contain information from the published
    accounts and also from the full, unpublished study reports.

    2.  BIOLOGICAL DATA

    2.1  Biochemical aspects

        In an initial review of the metabolism of fats, LCFAs and SCFAs,
    it was proposed that SCFAs in salatrim would be released following
    hydrolysis of the triglyceride in the stomach. A proportion of SCFAs

    released would be absorbed in the stomach and utilized as an energy
    source (predominantly butyrate), while the remaining SCFAs released in
    the stomach would be taken up by the liver. Hydrolysis of LCFAs from
    salatrim fats would predominantly occur in the small intestine. The
    absorption of stearate would be very limited. A proportion of absorbed
    stearic acid would be converted to oleic acid. Any SCFAs released in
    the small intestine would enter the hepatic portal vein (Hayes 
     et al., 1994a).

        Experiments using salatrim 23CA administration to rats were
    designed to test this hypothesis. Salatrim 23CA was chosen because it
    contains triacylglycerides unique to the salatrim family whereas
    triglycerides containing butyrate are commonly consumed as part of the
    diet.

    2.1.1  Biotransformation

    2.1.1.1  Rats

        Salatrim products are not absorbed intact, as they are hydrolysed
    and metabolized in an identical manner to triacylglycerides present in
    the diet. Monoacylglycerides containing stearate derived from the
    hydrolysis of salatrim products can be absorbed from the small
    intestine. Administration of a single oral dose of
    14C-stearate-salatrim (1.4 g/kg bw) to male Sprague-Dawley rats
    resulted in approximately 0.4% of the radiolabel being present in fat
    72 hours after dosing, of which half was in oleate (Musick & Peterson,
    1993; Hayes  et al., 1994b).

        A single oral dose of 1.4 g/kg bw of salatrim 23CA 14C-labelled
    in either the acetate, propionate, stearate or glycerol moiety or 1.4
    g/kg bw triolein 14C-labelled in either the oleate or glycerol moiety
    was administered to groups of 5 male Sprague-Dawley rats by gavage.
    Radiolabel elimination was followed for 72 hours. Acetate and
    propionate from salatrim were exhaled as CO2 (82.2% and 89.3% of the
    dose, respectively). Stearate was released from salatrim 23CA and
    oleate was released from radiolabelled triolein, with 21.5% of
    the14C-labelled stearate and 44.3% of the labelled oleate being
    exhaled as CO2. Experiments using salatrim or triolein 14C-labelled
    in the glycerol moiety resulted in approximately 75% of the dose being
    exhaled as CO2. Faecal excretion of radiolabel following
    administration of salatrim labelled in the acetate, propionate or
    glycerol moiety and glycerol-labelled triolein approximated to 4-5% of
    the dose. Greater faecal excretion of radiolabel was noted when
    14C-stearate-labelled salatrim (54.8%) and 14C-oleate-labelled
    triolein (38.4%) were administered to rats. Less than 4% was excreted
    in the urine in these studies and this consisted of radiolabel that
    had presumably been incorporated into intermediary metabolism. It was
    noted that urinary excretion of radiolabel was greater in animals
    treated with salatrim radiolabelled at the SCFA moiety compared with
    experiments where salatrim or triolein was radiolabelled in the LCFA
    moiety. Approximately 10% of the radiolabel in experiments using

    stearate- or glycerol-labelled salatrim and oleate- or
    glycerol-labelled triolein was recovered from the carcass. Very small
    amounts of radiolabel were found in the liver, blood and fat in these
    studies (total <1.8%). Pre-feeding a diet containing 10% salatrim
    23CA for 2 weeks prior to dosing with radiolabelled test materials did
    not affect the metabolism of salatrim or triolein. A slightly higher
    faecal excretion of radiolabel was reported in all the investigations
    using animals prefed salatrim. The authors suggested that competition
    for metabolism by dietary salatrim and radiolabelled salatrim
    administered by gavage was responsible for this observation.

        The authors concluded that the results of these experiments showed
    that the absorption, distribution and elimination of salatrim are
    identical to those of other triglycerides found in the diet and that
    the data supported the observation that stearate was less well
    absorbed than oleate. No conclusions can be derived regarding the
    absorption of salatrim 23CA and its component fatty acids since no
    direct measurements of absorption were undertaken. It is also noted
    that the dosing solutions for this study were prepared by mixing
    radiolabelled triolein (control fat) in the test fat (salatrim)
    matrix, further negating the value of this study with respect to the
    assessment of absorption of salatrim. The data does, however, support
    the view that the metabolism of salatrim 23CA is similar to triolein
    (Musick & Peterson, 1993 ;Hayes  et al., 1994b).

    2.1.1.2   In vitro studies

        The hydrolysis of a number of salatrim products by porcine
    pancreatic lipase was studied over a 30-minute time course. Chloroform
    stock solutions (100 mg fat/ml) of salatrim 4CA, 23CA, 32CA and 234CA
    were incubated at 37C for periods of 2, 5, 10 or 30 minutes.
    Predominant triacylglycerides, diacylglycerides, monoacylglycerides,
    LCFAs and SCFAs were measured by gas chromatography/ mass
    spectrometry. There was a consistent pattern of hydrolysis with each
    of the salatrim products tested, which consisted predominantly of a
    peak in diacylglyceride formation after about 2 minutes with a
    concurrent rapid rise in free stearate over 5 minutes. The rate of
    stearic acid formation dropped during the remainder of the 30-minute
    time period. Experiments with salatrim 23CA and 32CA showed that the
    hydrolysis of the triglyceride containing two short chain fatty acids
    (i.e. di-short triglyceride) was more rapid and more complete than
    that of the corresponding di-long triglyceride, which contained two
    stearate esterifications. The hydrolysis of triacylglycerides
    containing butyrate was more rapid than that of those containing
    acetate. The authors concluded that salatrim molecules undergo
    lipolysis in a predictable manner.

        The authors speculated that the higher rate of SCFA release as
    compared to LCFAs was due to the higher hydrophilicity of SCFA-rich
    triacylglycerides within fat droplets and the more rapid diffusion of
    released SCFAs from the active site of the enzyme into the aquatic
    phase surrounding fat droplets. The authors concluded that a rapid

    release of SCFAS would occur in the stomach and upper intestine
    (Phillips, 1992; Sequeria & Gordon, 1993)

    2.1.1.3  Caloric availability in rats and humans

        A description of studies concerned with this topic can be found in
    the Appendix.

    2.1.2  Effects on enzymes and other biochemical parameters

        Twenty-four rats of each sex (Crl:CD BR VAF strain) were fed 10%
    dietary salatrim 23SO for 17 days. An additional 24 rats of each sex
    were fed 10% dietary corn oil, and 12 rats of each sex received only
    the basal diet throughout the study. The rats were observed twice
    daily, and body weights were recorded weekly. Blood was collected and
    serum concentrations of aspartate aminotransferase (AST), alanine
    aminotransferase (ALT) and gamma-glutamyltransferase (GGT) were
    determined at 12 and 4 days prior to initiation of the study and on
    days 3, 6, 9, 13 and 17 after initiation of the study. Neither
    salatrim nor corn oil had any effect at any study interval on serum
    activity of aspartate aminotransferase, alanine aminotransferase, or
    gamma-glutamyl transferase activities (Kiorpes, 1993a; Hayes  et 
     al., 1994c).

    2.2  Toxicological studies

    2.2.1  Acute toxicity studies

        No information was available.

    2.2.2  Short-term toxicity studies

    2.2.2.1  Rats

     a) Salatrim 4CA

        Diets containing 0 (unsupplemented controls), 2, 5 or 10% salatrim
    4CA or 10% corn oil were fed to groups of 20 Crl:CD BR VAF rats of
    each sex for 13 weeks. Salatrim-containing diets were supplemented
    with varying levels of vitamins A, E, D and K according to the level
    of salatrim 4CA incorporation. A vitamin control group was also
    included in the study (supplemented control group), the level of
    supplementation being equivalent to that used in the 10% salatrim
    diet. A further group of 10 rats of each sex were fed
    salatrim-containing diets for 5 weeks and were also used for interim
    histopathology and measurement of minerals in bone tissue (Ca, Cu, Fe,
    Mg, P, Na, Sr and Zn in defatted femur at 10% salatrim or corn oil
    only). Blood and urine samples were collected at 4 and 13 weeks for
    clinical chemistry, urinalysis and haematology. Levels of bone
    minerals were also measured at 13 weeks. Animals were observed for
    signs of toxicity on a daily basis and body weight gain and food
    intake were measured weekly. At autopsy, the adrenals, brain, liver,
    kidneys and testes were weighed. The 10% dose represented the highest

    concentration believed by the authors to avoid excessive dilution of
    micronutrients.

        No treatment-related deaths occurred. No effects on body weight
    were documented in salatrim-fed animals compared to either
    supplemented or unsupplemented controls (but increased weight gain was
    noted for most weeks of the study in animals fed on corn oil).
    Decreased food consumption was noted in males fed 10% salatrim 4CA and
    males and females fed corn oil, compared to either control group.
    There were no effects on haematology, clinical chemistry or urinalysis
    data compared to either control group. Serum levels and urinary
    clearance of minerals were unaffected by treatment with salatrim or
    corn oil. No treatment-related changes in the levels of fat-soluble
    vitamins in serum or liver were documented at 10% salatrim 4CA
    compared to vitamin-supplemented controls that received the same level
    of vitamins in the diet (the occasional differences noted in single
    sexes at week 5 were not confirmed at termination). No definite
    conclusions can be drawn regarding any potential effects on
    fat-soluble vitamin absorption since an appropriate unsupplemented
    salatrim group was not included in the study. Concentrations of
    strontium and zinc in bone (defatted femur) were higher in both sexes
    at 10% salatrim compared to either control group, while sodium level
    was higher only in females compared with the unsupplemented control
    group. In the 10% corn oil group, the level of strontium in bone was
    higher in both sexes compared to either control group, while the level
    of zinc in bone was lower in males compared with unsupplemented
    controls. No treatment-related effects on organ weights or
    histopathological changes were documented at the interim and final
    necropsies. A number of animals fed 10% corn oil exhibited
    hepatocellular vacuolation. The authors concluded that changes in the
    levels of minerals in bone were directly related to the quantity of
    unsaturated fatty acids in the salatrim diet fed to animals. The NOEL
    was 10% salatrim 4CA, equivalent to 6.4 g/kg bw per day in males and
    7.3 g/kg bw per day in females (Williams, 1992a; Hayes  et al., 
    1994d).

     b) Salatrim 23CA and 32CA

        Diets containing 0 (control), 2, 5 or 10% salatrim 23CA or 32CA or
    10% corn oil were fed to groups of 20 Crl:CD BR VAF rats of each sex
    for 13 weeks. After 13 weeks of treatment, blood and urine samples
    were collected from a subgroup of 10 rats of each sex per group for
    haematology, serum, and urine chemistry and urinalysis determinations.
    Blood was obtained from the remaining 10 rats of each sex per group
    for the measurement of fat-soluble vitamins in serum. Animals were
    observed for signs of toxicity on a daily basis and body weight gain
    and food intake were measured weekly. The 10% dose represented the
    highest concentration believed to avoid excessive dilution of
    micronutrients. Adrenals, brain, kidneys, liver and testes were
    weighed at autopsy. The caecum of each rat was exposed and ligated in
    three places: at the distal ileum, proximal colon, and approximately
    the distal one-third of the blind end. The distal portion of the
    caecum was collected for histological examination. The remaining

    ligated portion of the caecum from each of the rats was used for a
    special study of effects on gut microflora (see section 2.2.5 below).
    Levels of minerals (as for salatrim 4CA) were measured at all dose
    levels in samples of bone (defatted femur) from 10 animals per group
    at necropsy.

        No treatment-related deaths occurred. Mean body weight gain and
    feed consumption were similar to untreated control animals. No
    toxicologically significant effects on haematology or clinical
    chemistry were reported. A dose-related trend toward slightly
    increased urinary phosphorus clearance was noted for rats fed salatrim
    fats. A statistically significant increase in urinary phosphorus
    clearance was noted for males treated with 10% salatrim 23CA and for
    males and females treated with 10% salatrim 32CA. No changes in the
    levels of fat-soluble vitamins in serum or liver were documented
    (except for a reduced level of vitamin A in animals fed corn oil). The
    mean strontium concentration in bone was significantly higher in
    females treated with 10% salatrim 23CA compared to untreated controls.
    A slight and statistically non-significant increase in the mean
    concentration of strontium in bone was also documented in males fed
    10% salatrim 23CA. The mean zinc concentration in bone was
    significantly higher in females fed 10% salatrim 23CA or 10% salatrim
    32CA compared with control females. The mean zinc concentration in
    bone was significantly lower in the males given the 10% corn oil diet
    than in the controls. No treatment-related effects on organ weights
    were noted. An increased incidence of renal mineralization was noted
    microscopically in females of the groups receiving corn oil and 5% and
    10% salatrim 23CA and salatrim 32CA when compared with controls. The
    incidence and severity of this lesion was similar in each of these
    groups of triacylglycerol-treated females. No treatment-related renal
    mineralization was noted in any group of corn oil-treated or
    salatrim-treated males. The authors concluded that changes in levels
    of minerals in bone and renal mineralization were directly related to
    the quantity of unsaturated fatty acids in the salatrim diet fed to
    animals. A number of animals fed 10% corn oil exhibited hepatocellular
    vacuolation. The Committee concluded that these two salatrim products
    did not induce any toxicologically significant effects. Dietary
    salatrim 23CA or 32CA or corn oil at 10% equated to 6-7.5 g/kg bw per
    day (Williams, 1992b; Hayes  et al., 1994c).

     c) Salatrim 234CS and Salatrim 234CA

        Diets containing 0 (control), 2, 5 or 10% salatrim 234CS or 234CA
    or 10% corn oil were fed to groups of 20 Crl:CD BR VAF plus rats of
    each sex for 13 weeks. After 13 weeks of treatment, blood and urine
    samples were collected from a subgroup of 10 rats of each sex per
    group for haematology, serum and urine chemistry and urinalysis
    determinations. Blood was obtained from the remaining 10 rats of each
    sex per group for the measurement of fat-soluble vitamins in serum.
    Animals were observed for signs of toxicity on a daily basis and body
    weight gain and food intake were measured weekly. The 10% dose
    represented the highest concentration believed to avoid excessive
    dilution of micronutrients. Adrenals, brain, kidneys, liver and testes

    were weighed at autopsy. Levels of minerals (as for salatrim 4CA) were
    measured at all dose levels in samples of defatted bone (femur) from
    10 animals per group at autopsy.

        No treatment-related deaths occurred. Mean body weight gain and
    feed consumption in salatrim-treated groups were similar to untreated
    control animals, although a slightly higher weight gain was reported
    for corn oil controls and females fed 10% salatrim 234CS. A
    significant decrease in food consumption was noted in both sexes fed
    10% corn oil. No toxicologically significant effects on haematology or
    clinical chemistry parameters were reported. Slight, but not
    statistically significant, increases in urinary phosphorus clearance
    were noted in all 10% salatrim treatment groups. The mean serum
    vitamin A level was significantly higher than for untreated controls
    in male rats fed 2% salatrim 234CA or 10% corn oil. Mean liver vitamin
    A concentrations were significantly lower than controls in males fed
    10% salatrim 234CA and males and females fed 10% salatrim 234CS or 10%
    corn oil. Mean serum 25-hydroxy vitamin D concentrations were
    significantly lower than those of controls in females fed 2 or 10%
    salatrim 234CA, 2 or 10% salatrim 234CS, or 10% corn oil. The authors
    noted that there were inconsistencies in the results between the sexes
    with regard to the effects of these salatrim products on fat-soluble
    vitamin levels. They considered that corn oil had induced a similar
    reduction in serum 25-hydroxy vitamin D levels and a larger increase
    in serum vitamin A levels compared to salatrim-treated animals. The
    authors concluded that the salatrim products tested did not
    substantially alter fat-soluble vitamin absorption. The mean
    concentration of sodium in bone was significantly lower in females fed
    2% salatrim 234CA, and the mean concentration of zinc in bone was
    significantly higher in males fed 2% salatrim 234CA and also in
    females fed 10% of both salatrim fats compared to untreated controls.

        Reduced liver- and brain-to-body weight ratios in females fed 10%
    salatrim 234CS were considered by the authors to be related to
    increased terminal body weight. Macroscopically, no treatment-related
    effects were observed in salatrim-treated rats. Microscopically, an
    increased incidence of renal mineralization was noted in females fed 5
    or 10% salatrim 234CA when compared with the incidence of renal
    mineralization in untreated control females. A slightly higher
    incidence of renal mineralization also was noted for females fed 5 or
    10% salatrim 234CS compared with untreated controls. Except for the
    groups treated with 10% salatrim 234CA, the renal mineralization was
    similar in appearance in all groups. In the females fed 10% salatrim
    234CA, the severity of renal mineralization was reported to be
    slightly greater than in other groups. The authors concluded that
    changes in levels of minerals in bone and renal mineralization were
    directly related to the quantity of unsaturated fatty acids in the
    salatrim diet fed to animals. A number of animals fed 10% corn oil
    exhibited hepatocellular vacuolation. The Committee concluded that
    these two salatrim products did not induce any toxicologically
    significant effects. Dietary salatrim 234CS or 234CA or corn oil at
    10% equated to 7-8 g/kg bw per day (Williams, 1992c; Hayes  et al., 
    1994g).

    2.2.2.2  Minipigs

     a) Salatrim 23SO

        Diets containing 0, 3, 6 or 10% salatrim 23SO or 10% corn oil were
    fed to groups of 4 Hanford minipigs for 28 days. Pigs were 3.5-7
    months old and weighed 17.2-30.4 kg at initiation of treatment. A
    control group was fed the basal diet alone. The group fed 10% corn oil
    served as a reference for the high-fat content of salatrim diets. All
    diets (including the diet used for the untreated control group) were
    supplemented with 2% (w/w) corn oil. This supplementation was
    considered by the authors to be necessary to avoid possible induction
    of essential fatty acid deficiency caused by dietary dilution with the
    test fat.

        Each pig was given 500 g of the appropriate diet twice each day.
    Test diets were prepared biweekly and stored frozen (-20  10C) until
    removed from the freezer and dispensed into food containers. After
    being removed from the freezer, diets were maintained at room
    temperature for 1-6 days (average 3.3 days) before being fed to the
    pigs. Evidence of significant degradation of the test diet was
    reported in one stability trial where samples of the 3% and 10% test
    diets were stored frozen for 63 days. The authors considered that the
    storage conditions used in the study would result in minimal
    degradation (approximately 7%) of the test diets. The additional
    stability trials from the original unpublished account of the minipig
    study support the view that limited degradation occurred during 2
    weeks of frozen storage. However, the available data suggest that
    degradation may have occurred during storage at room temperature and
    so stability may have differed significantly between batches of diet
    used. Thus, it is difficult from the available information to estimate
    the precise dose levels given to the minipigs.

        Blood was collected from the vena cava of each pig at 2 weeks and
    3 days before initiation of salatrim feeding and at days 3, 7, 14, 21
    and 29 after initiation of feeding. The pigs were fasted overnight
    before blood collection. Haematology and clinical chemistry variables
    were determined on these samples. After 28 days of treatment all pigs
    were subjected to gross necropsy. Adrenals, brain, kidneys, liver,
    ovaries, spleen, testes, thymus and thyroid were weighed. The entire
    femur not used for histopathology was removed and stored frozen at -20
     10C. Dry weight and percentage ash of femurs were determined. Each
    femur was assayed for Ca, P, Sr and Zn concentrations by inductively
    coupled plasma spectrometry.

        No treatment-related effects were noted during daily physical
    examinations. All pigs survived to the scheduled terminal sacrifice.
    In both sexes, mean body weights, body weight gains and feed
    consumption for pigs in the groups receiving salatrim 23SO and corn
    oil were comparable to untreated control pigs. Haematological and
    clinical chemistry evaluation revealed no treatment-related effects.
    The variability of AST and ALT levels in individual animals was
    relatively large in this study. At 2 weeks prior to initiation of

    treatment, serum levels of AST were significantly higher for males in
    the group destined to be fed 10% salatrim diet and serum levels of ALT
    were lower for males destined to be fed 6% salatrim diet. Serum ALT
    was also significantly lower for males fed the 6% salatrim diet at the
    day 7 interval. The reason for this variability was unexplained but
    could represent random variation. The mean serum cholesterol level in
    females given 6% salatrim 23SO at day 3 was lower than that of
    untreated controls. Also at day 3, the serum levels of low-density
    lipoprotein in females given 6 or 10% salatrim 23SO or 10% corn oil
    were lower than those of untreated controls. At day 29, mean serum
    cholesterol and high-density lipoprotein cholesterol levels were
    higher in female pigs fed 10% corn oil diets when compared with
    untreated controls. No biologically significant findings were reported
    with respect to serum and liver vitamin A and E data. No differences
    in percentage ash or bone concentrations of calcium, phosphorus or
    strontium were noted between treated and untreated control pigs of
    either sex in this study.

        There were no differences between the organ weights of pigs fed
    salatrim 23SO and those of untreated control pigs. Macroscopically, no
    treatment-related effects were observed in any of the pigs treated
    with either salatrim or corn oil. Microscopically, a slight increase
    in the severity of focal vacuolation in hepatocytes was noted for one
    male fed 10% corn oil and one male fed 10% salatrim. The authors
    considered that since this slight increase only occurred in two male
    pigs and was not observed in any females, it was a spurious finding or
    perhaps a non-specific fat effect as it occurred only in pigs given
    10% fat diets (corn oil or salatrim). The 10% dietary salatrim 23SO or
    corn oil levels equated to 3.3-3.7 g/kg bw per day. The Committee
    concluded that 10% salatrim 23SO produced neither toxicologically nor
    nutritionally significant effects (Kiorpes, 1993b; Hayes  et al., 
    1994e).

    2.2.3  Long term toxicity/carcinogenicity studies

        No information was available.

    2.2.4  Reproductive toxicity studies

        No information was available.

    2.2.5  Special studies on gut microflora

        Diets containing 10% salatrim 23CA or 32CA or 10% corn oil were
    fed to groups of 20 Crl:CD BR VAF rats of each sex for 13 weeks. At
    necropsy, following a 24-h fasting period, the caecum was exposed and
    ligated with cotton thread in three places: at the distal tip,
    proximal to the ileocecal junction, and distal to the exit into the
    colon. The caecal tip was removed for histology, and the remainder of
    the caecum was removed and frozen at -20C until use. Upon thawing of
    each caecum, its contents were thoroughly mixed by kneading within the
    caecum and then removed. A portion of the caecal contents from five
    male rats from each dietary group was examined by scanning electron

    microscopy for changes in the dominant bacterial morphotypes. Caecal
    contents from all animals were analysed for caecal pH, bile acids,
    neutral sterols (cholesterol and its secondary metabolite coprostanol)
    and phytosterols. The following primary bile acids were measured:
    cholic and alpha- and -muricholic acids. The secondary bile acids
    deoxycholic, lithocholic, hyodeoxycholic, omega-muricholic and
    unsaturated omega-muricholic acids were also measured. Primary
    phytosterols measured were 24-ethylcholesterol and
    24-methylcholesterol. Secondary phytosterol metabolites detected were
    24alpha-methylcoprostanol (campestanol), 24-methylcoprostanol,
    24alpha-ethylcoprostanol (stigmastanol) and 24-ethylcoprostanol
    (sitostanol).

        The authors noted that wide inter-animal variation in the levels
    of the bile acids occurred. No significant differences in caecal pH or
    in the level of secondary bile acids as a percentage of total bile
    acids were reported. Increased coprostanol levels were documented in
    male rats fed salatrim 23CA, 32CA and corn oil, but no affect on the
    ratio of coprostanol to cholesterol was documented. Increases in the
    levels of all four secondary phytosterols were documented in rats fed
    corn oil, whereas the level of only one (24alpha-methylcoprostanol)
    was increased in rats fed salatrim 32CA. In general, salatrim-fed rats
    of both sexes produced slightly less of the three remaining secondary
    phytosterols than chow-fed rats, while rats in the corn oil-fed group
    produced more. No evidence of any alteration in the population of
    bacterial morphotypes was reported, although the authors considered
    that inter-animal variation limited the sensitivity of scanning
    electron micrographs to the detection of major changes only. This
    study indicated that salatrim fats have less effect than corn oil on
    the intestinal microflora of rats (Scheinbach  et al., 1994).

    2.2.6  Special studies on genotoxicity

        Results of tests on the genotoxicity of salatrim fats are given in
    Table 3.



        Table 3.  Results of genotoxicity tests on salatrim fats

                                                                                                                                        

    Test system                 Test object                Test material/Dose levels       Result                Reference
                                                                                                                                        

    Ames test                   S.typhimurium              Salatrim 4CA, 23CA, 32CA,       Negative              Hayes & Riccio, 1994
                                TA98, TA100                23SO, 234CA, 234CS
                                TA1535                     0-1000 g/plate                 (Preincubation 
                                TA1537                     +/- S-9 (4 %, 10%)1             assay method)
                                TA1538

    Mammalian cell gene         CHO cells.                 Salatrim 23CA                   Negative              Hayes et al., 1994f
    mutation                    6-thioquanine              31-1000 g/ml
                                resistance                 +/- S-91, 2

    Chromosome                  CHO cells.                 Salatrim 23CA                   Negative              Hayes et al., 1994f
    aberrations (in vitro)      Metaphase analysis         0-1000 g/ml
                                                           +/- S-91

    UDS (in vitro)              Hepatocytes                5-1000 g/ml                     Negative              Hayes et al., 1994f
                                                           +/- S-91

    Bone marrow                 Rats fed 10% salatrim      Salatrim 234CS and 234CA        Negative              Hayes et al., 1994f
    micronucleus test           in diet for 13 weeks       (approximately 7-8 g/kg 
    (in vivo)                                              bw/day)
                                                                                                                                        

    1  Maximum dose level restricted by salatrim precipitation at 1000 g/ml (or 1000 g/plate).
    2  No evidence of cytotoxicity.
    


    2.3  Observations in humans

    2.3.1  Clinic-based studies

        Four clinical safety studies using controlled diets were
    undertaken. These studies involved:

    *   An acute tolerance test using a double-blind cross-over design.
        Dose levels of 45 g and 60 g salatrim 23CA were administered.

    *   A 7-day test using a double-blind protocol. Dose levels of 45 g
        and 60 g salatrim 23CA were administered.

    *   A 4-way triple cross-over test using 30 g or 60 g salatrim 23SO as
        the test materials, hydrogenated soybean oil as the control and
        hydrogenated coconut oil as the wash-out vehicle. This study used
        a latin square treatment sequence to administer the test, control
        and wash-out diets.

    *   Acute bolus dose of salatrim 23SO to measure effects on serum
        ketones.

     a) Study I: Acute tolerance

        This study utilized a randomized, double-blind, cross-over design,
    in which subjects received salatrim 23CA and control (coconut oil)
    materials for 1 day. Ten subjects (six males and four females between
    the ages of 18 and 65, with a mean age of 38.3 years) participated in
    the study. The test and reference materials, either 60 g/day (for
    eight individuals consuming the 2500-kcal diet) or 45 g/day (for two,
    both female, consuming the 1800-kcal diet), were introduced into the
    diet in the form of vanilla sandwich cookies and chocolate bonbons (or
    bars) each containing 5 g of either salatrim or control fat. On day 4,
    five subjects received the test material, while five subjects received
    the control material. The substitution of test material was reversed
    on day 8. Thus there were two treatment groups in this study, one on
    day 4 (group I) and one on day 8 (group II). Changes in clinical
    parameters on day 5 or 9, respectively, might be suggestive of a
    treatment-related effect. A maintenance diet (either 1800 or 2500
    kcal/day) including the control material was administered on all other
    study days. A standardized 4-day meal plan was repeated for three
    cycles during the study.

        A summary of results taken predominantly from the unpublished full
    report of this study is presented in this review. Following
    administration of salatrim 23CA, there was an increase in mean levels
    of serum lactate dehydrogenase (LDH) and GGT activities in both the
    two treatment groups (i.e. on day 5 for group I and day 9 for group
    II) and an increase in mean levels of serum ALT, AST and alkaline
    phosphatase activities in group 2 (i.e. on day 9 of the study).
    Changes in these parameters were modest and group means did not exceed
    the reference ranges. A slight increase in mean serum cholesterol
    levels was reported at the end of the study for both treatment groups.

    The authors of the published report considered that the small size of
    the treatment group did not permit a conclusion regarding the
    palatability of the salatrim foods. However there was a statement in
    the unpublished report that subjects rated salatrim cookies and
    chocolate candies lower than the identical control food carriers. Mild
    adverse gastrointestinal symptoms were reported in a number of
    individuals (e.g., flatulence, nausea, diarrhoea). There was no
    evidence in the published report, when the data were analysed by time
    of onset, that these symptoms were related to consumption of salatrim
    23CA (GHBA, 1993; GHBA/Hazelton, 1993; Finley  et al., 1994b).

     b) Study II: 7-day test

        This study utilized a randomized, double-blind design, in which
    subjects received either the test salatrim 23CA or control (coconut
    oil) materials over a 7-day period. The test and control materials,
    either 60 g/day (for those on a 2500-kcal diet) or 45 g/day (for those
    on a 1800-kcal diet), were introduced into the diet in the form of
    cookies, bonbons (or bars) and chocolate ice cream. Thirty-six
    subjects (19 males and 17 females between the ages of 18 and 65, with
    a mean age of 33.4 years) participated in the study. All subjects
    received a maintenance diet (either 1800 or 2500 kcal/day) containing
    the control material on days 1-7. On days 8-14, 18 subjects (12 male,
    6 female) received the test material (either 60 g/day, males; or 45
    g/day, females); 18 additional subjects (9 male, 9 female) continued
    on the maintenance diet with food carriers containing control fat. One
    female subject on test material withdrew for personal reasons,
    unrelated to the test, on day 10. On days 15-24, all subjects returned
    to the maintenance diet. A standardized 7-day meal plan was followed
    for three cycles throughout the study.

        Except where stated, the results summarized here have been taken
    from the published report.

        An increase in mean serum ALT (19%) and AST (< 41%) levels
    above the value prior to exposure to salatrim 23CA was recorded during
    the treatment period. Three individuals showed ALT values above the
    normal maximum of 35 milliunits/ml and one subject exhibited a raised
    AST value above the normal maximum of 50 milliunits/ml during the
    treatment period. Lactate dehydrogenase activity also increased during
    the exposure period to salatrim 23CA even though all values remained
    within the normal range throughout the test. The authors considered
    that serum AST and LDH levels declined steadily to control levels. The
    unpublished report states that all parameters declined to near
    baseline levels after withdrawal of salatrim 23CA. Mean corpuscular
    volume, monocytes, serum calcium, carbonate and GGT were all
    significantly altered in the salatrim-exposed individuals. All values
    remained within the normal range, and none of the changes were
    considered by the authors to be clinically relevant.

        During the pre-test period there was a significant increase in
    total cholesterol level which was associated with the ingestion of
    hydrogenated coconut oil. During the test period (days 8-14) there was

    a significant drop in the total and low-density lipoprotein
    cholesterol in the group receiving salatrim 23CA, whereas values for
    the control group remained elevated. No significant changes in
    urinalysis parameters were reported. Large increases in the faecal
    excretion of fats and stearic acid were documented when salatrim 23CA
    was added to the diet.

        The group fed salatrim 23CA reported more headaches and
    gastrointestinal symptoms during the test period. Nausea, abdominal
    pain and headaches were the most frequent symptoms reported. The
    authors considered that the effects reported were mild and did not
    cause anyone to drop from the study or require clinical intervention.
    Data from the unpublished report shows that 14/17 individuals (8 male,
    6 female) consuming salatrim 23CA (78%, compared to 56% in controls)
    reported one or more adverse effect regardless of relationship to
    study material. Flatulence and nausea were reported by 61% (compared
    to 22% in controls) and 67% (compared to 17% in controls) of subjects
    consuming salatrim 23CA, respectively. Headache was reported in 56% of
    subjects consuming salatrim 23CA (compared to 11% in controls).
    Adverse gastrointestinal tract symptoms were moderate in 8/17
    individuals consuming salatrim 23CA and mild in the remainder. None of
    the reported symptoms was considered to be severe. Data from the
    unpublished report also suggested that salatrim carrier foods were
    considered by the subjects to be of lower palatability than control
    carrier foods. The taste of salatrim-containing food carriers may have
    resulted in volunteers being able to distinguish between the various
    food products used in the trial and hence the study may have been
    unblinded (GHBA, 1993; GHBA/Hazelton, 1993; Finley  et al., 1994b).

     c) Study III: 4-day triple cross-over test

        The subjects received test vehicles (chocolate raisin/crisp bars
    and hot chocolate drink) prepared with salatrim 23SO at 30 g/day (plus
    30 g of control fat) or  60 g/day or 60 g/day hydrogenated soybean
    material (control) for a 4-day period. Before and after receiving the
    test or control vehicle, the subjects were given 60 g hydrogenated
    coconut oil vehicle for 4 days. This latter vehicle was prepared from
    the same coconut oil used in studies I and II summarized above and
    served as a wash-out medium between the salatrim and control
    treatments. A standardized 8-day meal plan was repeated three times
    throughout the study.

        No clinically significant differences were reported between 30
    g/day salatrim 23SO and control. When subjects ingested 60 g/day
    salatrim, a statistically significant increase was observed in mean
    serum ALT, AST and LDH activities and a decrease in mean serum
    cholesterol levels was noted. The shifts in these clinical parameters
    were well within the normal ranges for these assays and were
    considered, by the authors, to be clinically unimportant. All values
    approached pre-test levels after subjects were transferred to the
    coconut oil wash-out diets. Total stool weight was significantly
    higher in females and all subjects (males and females combined)
    following consumption of 60 g salatrim/day compared to 30 g

    salatrim/day and 60 g soybean oil/day. Stool water, total fat and
    stearic acid were significantly higher in females and males following
    consumption of 60 g salatrim/day compared to 30 g salatrim/day and 60
    g soybean oil/day. Increased stool softening and reports of abnormal
    stools were documented by females given 60 g salatrim/day compared to
    30 g salatrim/day and 60 g soybean oil/day and by all subjects (i.e.
    males and females) given 60 g salatrim/day compared to 30 g
    salatrim/day. A small but statistically significant increase in mean
    serum -hydroxybutyrate level was documented in the 60 g salatrim/day
    group using combined male and female data (0.20.10 mmol/litre,
    compared to 0.10.06 mmol/litre in controls). Adverse gastrointestinal
    effects (abdominal pain, diarrhoea and nausea) were associated with 60
    g salatrim/day and were reported by 10 female and 5 male volunteers.
    The authors considered that given the lower body weights of the female
    subjects, there might have been a relationship between exposure level
    and body weight which could have been responsible for the higher level
    of complaints in female subjects. These data support the conclusion
    that levels of < 30g salatrim/day did not cause any significant
    gastrointestinal symptoms. The Committee noted the limited duration of
    salatrim exposure in this study.

        Volunteers considered that the hot chocolate drink containing
    salatrim was acceptable but disliked the other salatrim food carriers.
    No evidence of carry-over in subjective assessments of food carriers
    was reported. The taste of the salatrim-containing food carriers may
    have unblinded the study (Besselaar Clinical Research Unit, 1993a;
    Finley  et al., 1994b).

     d) Study IV: Acute study of effects on ketones

        A randomized, blind study was conducted with 42 subjects (6 per
    group) to determine the effect of a single dose of salatrim 23SO,
    hydrogenated soybean oil or medium-chain triglyceride Neobee M-5 (MCT)
    on serum levels of acetate, acetoacetate and hydroxybutyrate
    (monitored for up to 4 hours after the exposure). All fat samples were
    delivered in 1 cup chocolate-flavoured beverage in the morning
    following a 10-h fast. The subjects were randomly assigned to one of
    seven treatment groups; salatrim 23SO: 7.5, 10, 12.5 or 15 g; control
    hydrogenated soybean oil: 7.5 or 15 g; MCT: 15 g. Assignments were
    made to allow balance of groups based on age and gender. A slight
    increase in serum acetate was seen in subjects receiving 15 g of
    salatrim. No increases were observed in serum ketones at any level of
    salatrim feeding. As expected, slight increases in acetoacetate and
    -hydroxybutyrate were observed in subjects receiving MCT. Adverse
    gastrointestinal effects were reported in 5 individuals: a single
    individual from each of the 10 g, 12.5 g and 15 g salatrim groups, and
    1 each from the MCT and soybean control groups. The authors considered
    that salatrim was not ketogenic at the dose levels used in this study
    (Besselaar Clinical Research Unit, 1993b; Finley  et al., 1994b).

    2.3.2  Non-clinic-based study (free-living)

        The design was a randomized, double-blind, multiple-dose, parallel
    comparison of the fat replacement by salatrim 23SO, 4SO or 43SO oils
    with a control soy oil. At least 24 subjects per group, comprised of
    at least 12 females and 12 males, were recruited for this study (two
    control groups and one group each exposed to 30, 45 or 60 g of 23SO,
    60 g of 4SO, or 60 g 43SO). Two control groups were included in the
    study to help account for the anticipated diversity in clinical values
    in a typical population. The ages ranged from 19 to 63 with a mean age
    of 35.2 years. Total fat intake from the delivery vehicles for all
    individuals receiving test or control material was 60 g/day. The total
    duration of the study was 6 weeks. In weeks 1 and 6 all subjects
    received control fat (soy oil). In weeks 2-5, the subjects received
    either control or test fats as assigned per group. The food products
    were changed weekly on a 2-weekly cycle to assure variety.

        Each week of the study, subjects were supplied with food products
    for consumption during the coming week. Each day, five products were
    to be integrated into the subjects' daily diet; four of the products
    contained 15 g of control or test oil. One product (either crackers or
    cornflakes) did not deliver test or control oil and was used as a
    "dummy" carrier. In addition to the food provided by the test
    vehicles, subjects were free to consume a normal diet, the only
    restriction being that the amount of alcohol consumption was limited
    to no more than two 6-oz glasses of wine or two 12-oz servings of beer
    per day.

        After screening, subject selection and initial check-in, which
    included drug usage and pregnancy testing (day 0), subjects reported
    to the clinic on the morning of day 1 to receive products and daily
    diaries for reporting food consumption and health over the next 7
    days. Body weight was also recorded, and blood was drawn for analysis.
    Subjects returned in the morning every 7 days thereafter (days 8, 15,
    22, 28 and 36) to receive food products for the next week, to return
    daily diaries, to record body weight, and to have blood drawn. On the
    final day of the study (day 43), subjects returned to the clinic to
    hand in daily diaries, to be weighed, and to have final samples of
    blood drawn. All subjects reported to the clinic following a 10-h
    fast. The subjects' daily health was assessed in terms of the presence
    of a number of general categories. The clinical phase of the study was
    conducted at Harris Laboratories, Inc, Lincoln, Nebraska 65801, USA.

        Daily diaries were used to record all foods consumed, to rate the
    palatability of the provided foods, and to record side effects and the
    quality of daily life. Daily food records included the type and amount
    of all foods and beverages consumed and the time of day at which each
    item was consumed. Food carriers used in this study were ice cream,
    chocolate milk, pudding and yoghurt produced in the Cornell University
    Dairy by conventional processes. Cinnamon raisin muffins, chocolate
    cake, lemon cake, and waffles were prepared at the Swanson Co, Omaha,
    Nebraska, prior to the study and held frozen until they were dispensed
    to the subjects. Chocolate milk was prepared in multiple batches every

    2 weeks as needed. All other products were produced in a single lot
    prior to the initiation of the study. Formulations for the products
    were adjusted so that each individually packaged serving would deliver
    15g of control oil or salatrim in each unit.

        The food carriers were rotated on a 2-week basis:

    *   Weeks 1, 3, 5:  Muffins, chocolate cake, ice-cream, yoghurt

    *   Weeks 2, 4, 6:  Waffles, Lemon cake, chocolate milk/pudding

    Control diets were administered in weeks 1 and 6.

        Of the 183 subjects starting, 149 completed the study. The results
    of clinical assessments were reported for the subjects who completed
    the study. The Committee noted that there were differences in the
    number of individuals dropping out at various salatrim dose levels
    between the published and unpublished reports. The data given in this
    summary pertain to the unpublished clinical report prepared by the
    study authors. A total of 34 individuals dropped out of the study;
    1/26 (control 1), 3/27 (control 2), 4/27 (23SO 30 g), 5/27 (23SO 45
    g), 2/25 (23SO 60 g), 7/26 (4SO 60g), 12/25 (43SO 60g). Excluding
    control subjects who dropped out of the study, 20 out of the 31
    individuals who dropped out of the study and who had received salatrim
    in their diet recorded adverse effects due to the test material as the
    reason for leaving the study. In this study, the salatrim food
    products were reported by the authors to be well tolerated.

        Transient increases in the mean serum AST and ALT levels over time
    were observed in the controls and all salatrim groups. However, in the
    60 g salatrim groups, the magnitudes of the increases in ALT and AST
    levels from the day 8 baseline were greater than that observed in the
    control groups. The authors considered that by the end of the 4-week
    exposure periods, the ALT and AST activities in all groups approached
    values equivalent to those recorded on day 8. There is no clear
    evidence of a reversal in enzyme levels and appropriate statistical
    tests are required to evaluate the data further. None of the group
    means ever exceeded the normal clinical limits for AST or ALT. A small
    transient reduction in mean serum cholesterol was recorded in the
    unpublished report at 60 g salatrim 23SO or 43SO per day.

        The consumption of 60 g salatrim/day was associated with reports
    of stomach cramps and nausea in a substantial number of subjects. The
    authors calculated the percentage of days in the exposure period in
    which adverse effects were reported [number of individuals with
    adverse effect  number of days with effect / 28  total number of
    individuals]. At 60 g salatrim per day, stomach cramps and nausea were
    reported during approximately 17-21% and 19-25%, respectively, of the
    28-day exposure period depending on which salatrim product was under
    evaluation. At 45 g/day and 30 g/day, stomach cramps and nausea were
    reported during approximately 8-9% and 10-11%, respectively, of the
    28-day exposure period. A similar analysis of other symptoms was not
    presented in the published report. The authors considered that in

    subjects consuming 30 g/day salatrim 23SO, there were no reports of
    nausea that impaired daily function. Three individuals consuming 30 g
    salatrim 23SO/day experienced stomach cramps or nausea for at least 10
    days of the trial (i.e. 3/23 who completed the study) compared to one
    individual in the combined control groups (i.e. 1/49 who completed the
    study) (Harris Laboratories, 1993; Sourby, 1993; Finley  et al., 
    1994c).

    3.  COMMENTS

        The Committee evaluated studies on the caloric value of salatrim,
    being aware that short-chain fatty acids supply fewer kilocalories per
    gram than long-chain fatty acids. However, the claim of reduced
    absorption of stearic acid has not been proven for humans. Because
    there is no specific formulation for salatrim, it is not possible to
    assign a single caloric value to this product. The Committee noted
    that the specifications for salatrim that were elaborated at the
    present meeting permit formulations that include a triglyceride
    mixture with up to 0.87 gram of stearate per gram of fat. The
    biological data available do not provide information on materials with
    such compositions. If future studies determine that stearic acid is
    poorly absorbed from the product, the Committee considered that the
    consequences of this will need to be determined.

        In evaluating the safety of salatrim, the Committee considered
    various studies. An  in vitro study with porcine pancreatic lipase
    demonstrated that a wide range of the salatrim triacylglycerides are
    hydrolysed rapidly. In rats, the  in vivo metabolism of a specific
    salatrim indicated that it was metabolized in an analogous manner to
    triolein.

        Salatrim products do not contain any structural alerts for
    potential mutagenicity. There was no evidence of genotoxicity in an
    adequate range of  in vitro and  in vivo studies.

        Five 90-day feeding studies in rats, each using a different
    salatrim formulation administered at concentrations of up to 10% in
    the diet, showed no toxicologically significant effects. A 28-day
    study in minipigs of a specific salatrim formulation was carried out
    at dose levels of 0, 3, 6 or 10% in the diet, and also showed no
    toxicologically significant effects. These studies were not optimized
    to detect potential nutritional effects, nor was the minipig study of
    sufficient duration. The Committee concluded that, with these
    limitations, the studies did not provide an adequate basis for a
    nutritional or toxicological evaluation.

        Because of the high projected intake of salatrim products (90th
    percentile levels for "all ages" and for 3-5 year olds are 37 and 26
    grams per day, respectively) and given that no systemic effects were
    seen in animal studies, the Committee paid particular attention to the
    results of five studies in humans. Of these, one was a free-living
    trial, the other four were clinic-based with varied experimental
    designs.

        In the four clinic-based studies the experimental protocols
    provided intakes of up to 60 g salatrim/person per day for periods of
    1, 4 or 7 days. Although these studies provided some indication that
    the consumption of salatrim diets was associated with an increased
    incidence of mild gastrointestinal symptoms and significantly elevated
    serum enzymes, the treatment periods were short and the numbers of
    study participants were few.

        The design of the free-living study was as a randomized,
    double-blind, multiple-dose, parallel comparison of fat replacement by
    salatrim 23SO, 4SO or 43SO oils with a control soy oil. At least 12
    females and 12 males were recruited for each of two control groups and
    five groups fed 30, 45 or 60 g per day of 23SO, 60 g per day of 4SO,
    or 60 g per day of 43SO. The total duration of the study was 6 weeks.
    In weeks 1 and 6 all subjects received control fat. In weeks 2-5, the
    subjects received either control or test fats as assigned.

        One hundred and eighty-three subjects started the study; 34
    dropped out, of which four were controls.  Twenty of those who dropped
    out had received salatrim and recorded adverse effects as the reason
    for leaving the study.  The Committee noted inconsistencies between
    the published and unpublished reports of the study in that there were
    differences recorded in the numbers of subjects dropping out.

        The consumption of 60 g per day salatrim was associated with more
    reports (compared to controls) of stomach cramps and nausea in a
    substantial number of subjects. Transient elevations of the levels of
    certain liver enzymes (alanine aminotransferase and aspartate
    aminotransferase) were recorded. Owing to the short duration of the
    study, the high drop-out rate, and the modest number of participating
    subjects, the Committee concluded that it was not possible to evaluate
    whether these observations were clinically significant.

    4.  EVALUATION

        The Committee concluded that the available studies did not provide
    an adequate basis for evaluating the safety and nutritional effects of
    salatrim. The Committee recommended that additional, appropriately
    designed studies be performed to assess fully both the toxicological
    and nutritional consequences of salatrim ingestion.


    5.  APPENDIX 1  CALORIC AVAILABILITY OF SALATRIM TRIGLYCERIDES

    5.1  Explanation

        Salatrim is a family of structured triglycerides prepared with
    combinations of short- and long-chain fatty acids and capable of
    serving as a total replacement for conventional fats and oils in many
    foods. The authors claim that it provides about half the energy
    content of the fats and oils it replaces. An evaluation has been
    requested of the caloric availability of salatrim triglycerides

    (Howlett, 1997). In the request it is stated that the reduced caloric
    content of salatrim has been recognized for nutritional labeling
    purposes in Japan and the USA.

    5.2  Definitions and chemistry

        According to the specification there is no specific requirement
    for amounts of short-chain fatty acids (SCFA) and the amount of
    stearic acid in the final product. Unless this is specified it is not
    possible to assign a single caloric value to salatrim.

    5.3  Caloric value determination

    5.3.1  Biochemical aspects

        The reduced energy content of salatrim triglycerides, as opposed
    to conventional fats and oils, is purportedly due to the lower caloric
    value of short-chain fatty acids (SCFA) and the reduced absorption of
    stearic acid. The following discussion evaluates the evidence for
    these two purported effects.

    5.3.1.1  Caloric value of SCFA

        It is claimed that acetic acid, propionic acid and butyric acid
    contribute less kcal/g than longer-chain fatty acids. Based on their
    heats of combustion (CRC Handbook of Chemistry and Physics,
    1992-1993), these fatty acids contribute 3.5 (acetate), 4.9
    (propionate) and 5.8 (butyrate) kcal/g as compared to the standard
    physiological fuel value for lipids of 9 kcal/g. The Committee agrees
    with that statement, but notes that there is no specific fatty acid
    composition of salatrim designated, so no specific caloric value can
    be attributed to salatrim. Also, since SCFAs contribute less energy
    per gram than LCFA, one would predict that as the SCFA/LCFA in a
    salatrim product increases, the energy content of the salatrim product
    would decrease. In fact, in the studies reported by the petitioner
    that relate the ratio of SCFA to LCFA in salatrim to weight gain in
    rats, the opposite is true (Klemann  et al., 1994). Thus, as the
    amount of SCFA in salatrim increases the energy contribution of that
    salatrim product increases. A ratio of 0.51 SCFA to LCFA is considered
    to provide 2.56 kcal/g, whereas a ratio of 1.99 SCFA to LCFA is
    considered to provide 6.39 kcal/g (Klemann  et al., 1994). In
    general, SCFAs provide less kcals/g than do LCFA. However, the ratio
    of SCFA to LCFA in a triglyceride may also influence the caloric
    availability, and, unless a specific salatrim product is provided, it
    is not possible to assign an overall energy content.

    5.3.1.2  Caloric value of stearic acid

        Based on its heat of combustion (Weast, 1992-1993) stearic acid
    should provide 9.5 kCal/g. It is contended that stearic acid in
    salatrim contributes a much lower energy value because stearic acid is
    poorly absorbed (Klemann  et al., 1994). Studies on the absorption of
    stearic acid are described below.

     a) Rats

     i) Stearic acid absorption, balance study

        It has been claimed that reduced stearate absorption has been
    confirmed in rodent and human absorption/excretion balance studies.
    Forty rats (10 per group) were provided with NIH-07 diet supplemented
    with 10% corn oil (controls) or the same diet supplemented with 5, 10
    or 15% salatrim 23SO. Rats were acclimated to the diets for 5 days,
    and this was followed by 5 days of faecal collection. Stearic acid in
    the faeces was determined according to AOCS Method Ce 1-62 (AOCS,
    1990). The method of analysing intake of stearic acid was not
    described. On the 5% salatrim diet, 0.56 g of stearic acid was
    consumed/day and 0.40 g of stearic acid was excreted in the faeces
    (Finley  et al., 1994a). This represents 28.6% absorption. In the
    case of the 10% salatrim diet, 1.03 g of stearic acid was consumed/day
    and 0.67 was excreted in the faeces; this represents 34.9% absorption.
    With the 15% salatrim diet, 1.49 g of stearic acid was consumed, 1.01
    g was excreted for a total absorption of 32.2% (Finley et al., 1994a).

        Stearic acid appears to be poorly absorbed from salatrim 23SO in
    the rat according to this assay. However, actual absorption data are
    not definitive since the method of determining stearate intake is not
    described and no account is taken of the microbial contribution to
    stearate formation from other 18 carbon fatty acids. It should also be
    noted that this stearic acid excretion study was only performed on one
    salatrim formulation. Other data from different salatrim products
    strongly suggest that stearic acid absorption is dependent upon the
    ratio of short-chain to long-chain fatty acids in the product (Klemann
     et al., 1994).

     ii)    Stearic acid absorption, radiolabel study

        Radiolabelled salatrim fats that mimic salatrim 23CA lot A014 were
    synthesized and purified. The resulting radiolabelled fats were
    designated salatrim APS. Radiolabelled 14C-triolein was used as a
    reference fat. Non-radiolabelled salatrim 23CA Lot A014 was used in
    this study to dilute the radiolabelled salatrim and triolein to the
    appropriate specific activity. Rats were dosed with 1.4 g of fat/kg
    body weight of either radiolabelled salatrim APS or radiolabelled
    triolein as a single oral dose by gavage. After dosing they were
    individually housed in glass metabolism cages designed for collection
    of expired CO2, urine and faeces. One group of rats received salatrim
    23CA at 10% by weight of the diet for 2 weeks prior to administration
    of the radiolabelled fats to see if pre-feeding the fat influenced its
    disposition. Each test group contained 5 rats. After a 72-hour sample
    collection period, rats were sacrificed and radiolabel in all tissues,
    urine, faeces, and CO2 was determined in duplicate samples. Faecal
    samples from the rats fed salatrim APS with radiolabelled stearate
    were analysed for recovery of labelled stearate. A comparison of
    radiolabel from triolein (control fat) with radiolabel from the

    stearate carbonyl in salatrim in faecal material was plotted over time
    (Hayes  et al., 1994b).

        Although the amount of label from stearate is numerically higher
    than that from oleate at 12 hours, these numbers are not statistically
    significant. Percentage of radioactive dose recovered in faeces from
    oleate was 38.4% and from stearate was 54.8%. This would suggest that
    oleate in triolein is 62% absorbed whereas stearate in salatrim 23CA
    is 45.2% absorbed. It is of interest to note that in other studies
    where the authors compare the calculated caloric value of various
    salatrim products to other fats, they use an absorption coefficient of
    99% for oleic acid (Klemann  et al., 1994). The advantage of the
    radiolabelled stearate study over the stearate balance study not using
    radiolabel described above (and in Finley  et al., 1994a) is that
    excretion of stearate is not overestimated in the radiolabel study
    because microbially derived stearate is not measured. The disadvantage
    of the radiolabel study is that it only used five rats per group,
    instead of ten. However, an additional 5 rats per group, differing
    only in that they were pre-fed salatrim 23CA for 14 days prior to
    dosing, were also tested for labelled stearate excretion. Values were
    similar for animals fed the basal diet and those fed salatrim 23CA,
    which should allow for pooling of the excretion data. These data
    suggest that in male Sprague-Dawley rats, the stearic acid in salatrim
    23CA is approximately 45% absorbed.

     b) Humans

     ii)    Stearic acid excretion in humans

        This study had a non-cross-over design with 18 subjects per group
    and a total of two groups. There was a 7-day pre-trial period during
    which all subjects consumed products containing hydrogenated coconut
    oil, a 7-day period during which one group was exposed to either 45 or
    60 g of salatrim 23CA (lot 14)/day, depending on their total caloric
    need (1800 kcal/day or 2500 kcal/day), while the other group received
    products made with hydrogenated coconut oil, and a final 10-day period
    during which all subjects received products made with hydrogenated
    coconut oil. Faecal samples for each subject were collected and pooled
    for the last 3 days of each 7-day test period. Subjects consuming the
    1800 kcal/day ingested 27.4 g stearic acid (the method for arriving at
    this value was not provided) and excreted 7.6 g of stearic acid per
    day for a net absorption of 72.4%. Subjects consuming 2500 kcal/day
    consumed 34.2 g stearic acid/day and excreted 12.3 g stearic acid/day
    for a net absorption of 63.5%. From this study, the authors concluded
    that "In the human clinical study, between 27.6 and 36.5% of the
    stearic acid in salatrim was shown to be absorbed (Abstract),
    resulting in an apparent caloric availability of between 4.7 and 5.1
    kcal/g. These results show that salatrim exhibits similar caloric
    reduction in both rats and humans" (Finley  et al., 1994a).

        No analysis was presented of the amount of stearic acid consumed
    in the diets, or how this was sampled. This is important because the
    absorption of stearic acid is calculated as amount consumed minus

    amount excreted, divided by the amount consumed. Without an accurate
    determination of the amount consumed it is difficult to assess stearic
    acid absorption.

        There are points of disagreement with these conclusions. Firstly,
    the absorption data for humans, as stated in the abstract (Finley 
     et al., 1994a), are incorrect by the authors' own data (shown in
    Table 6 of the study). Absorption of stearate in humans is 63.5 to
    72.4%, not the reported values of 27.6 and 36.5%. Additionally, no
    weight gain data are presented for humans and thus no conclusions can
    be made as to the caloric value of salatrim from this study.

     ii) Stearic acid absorption assessed by researchers other than the
    data submitter

        Researchers other than the petitioner have also measured the
    absorption of stearic acid in humans. Olubajo  et al. (1986) report
    the absorption of stearic acid in a study of 30 men aged between 34
    and 61 years to be between 82 and 88%. Jones  et al. (1985) measured
    absorption of 13C-labelled stearic, oleic and linoleic acids in six
    healthy men. They reported absorption coefficients of 78.0% for
    stearate, 97.2% for oleate and 99.9% for linoleic acid (Jones  et 
     al., 1985). In an in-patient, metabolic-ward investigation, Denke &
    Grundy (1991) fed four different fats (butter, beef tallow, cocoa
    butter and olive oil) as part of liquid diets. Each diet was fed for 3
    weeks. Dietary intakes and faecal excretion rates of three major fatty
    acids (palmitic, stearic and oleic) were determined and used to
    estimate the absorption of these fatty acids. The highest absorption
    rates were noted for oleic acid (approximately 99%). Palmitic acid was
    absorbed at a rate between 95 and 97% whereas absorption of stearic
    acid was slightly lower (90 to 94%) (Denke & Grundy, 1991). In the
    study of Denke & Grundy (1991) the authors made an important
    observation on the methodology for measuring stearic acid absorption.
    They stated that initial analyses relied on methodology in which the
    fatty acid content of a sample was based on extracting total lipids
    from a faecal sample and then determining the proportion of the lipid
    sample that was stearic acid based on gas chromatography analysis. The
    authors stated that: "Unpublished observations in our laboratory
    suggest that faecal fatty acids can be overestimated by 100% when
    calculated by percent of extractable lipid weight. Current results
    strongly suggest that stearic acid is relatively well absorbed". The
    study of Denke & Grundy (1991), which reports stearic acid absorption
    of 90 to 94%, measured the actual mass of stearic acid by adding a
    known amount of 17:0 to the sample. Bonanome & Grundy (1989) evaluated
    the absorption of stearic acid, relative to other fatty acids, in a
    group of 10 normal volunteers. Subjects were fed a meal with a high
    amount of stearic acid, or one containing a relatively low stearic
    acid content. Plasma chylomicrons were isolated at 2,4, 6 and 8 h
    after ingestion of the meals. Fatty acid patterns of chylomicron
    lipids were determined and comparisons were made between the fatty
    acid composition of the chylomicrons and the ingested lipids. The
    percentages of palmitic acid (16:0) and stearic acid, relative to

    other fatty acids, were only slightly lower in the lipids from
    chylomicrons than those in the meal. The authors concluded that the
    absorption of stearic acid is similar to that of palmitic acid and
    that both of these fatty acids are absorbed almost as well as oleic
    acid. Stearic acid absorption in humans may be as low as 63.5 to
    72.4%, as reported by the petitioner (Finley  et al., 1994a).
    However, a more realistic value may be as high as 90 to 94% (Bonanome
    & Grundy, 1989; Denke & Grundy, 1991). The rat is apparently not a
    good model for the human with respect to stearic acid absorption,
    since most studies on rats show lower coefficients of absorption of
    stearic acid.

    5.3.1.3  Determining the caloric value of salatrim products based on a
    rat growth assay

        The primary method by which the data submitter estimated the
    caloric value of various salatrim products is by use of a rodent
    growth assay. The basic protocol in which this assay was described and
    validated is reported in Finley  et al. (1994d). A basal diet
    (NIH-07) is provided to all rats. Basal feed consumption is restricted
    daily to 50% of the feed consumption of rats fed  ad libitum. Test
    and control rats all consume the same amount of basal diet but with
    different amounts of corn oil or test fat added to the basal diet. Ten
    rats are used per group. A regression curve is calculated for the
    weight gain of the rats fed different levels of corn oil (considered
    to supply 9 kcal/g) and the caloric value of the salatrim test fat is
    calculated based on the weight gain of the animals fed salatrim
    compared to that of those fed corn oil. The formula used to determine
    the kcal/g for the test fat is:

            kCALX  =  3D (BWGX - INT)
                                            
                           SLP  KX)

    where KCALX is the estimated kilocalories per gram of test fat, BWGx
    is the mean body weight gain for rats on test fat, INT is the
    intercept from standard curve regression, SLP is the slope of standard
    curve, and KX is the test fat added to the diet (grams per 100g of
    diet) (Finley  et al., 1994d). In fact, this equation is not
    accurate; KX is really the kcals/100g of diet, not the amount of
    fat.

        The rationale behind the growth assay employed is that rats are an
    appropriate model for humans and that balance studies are not
    conducted because they are "cumbersome and do not lend themselves to
    evaluating large numbers of materials"; radiolabelled studies are not
    conducted because they are "expensive and time-consuming and are not
    practical as a routine screening tool" (Finley  et al., 1994d). There
    are a number of problems with using this rodent growth assay to
    determine the caloric content of salatrim products for humans.
    Firstly, and most importantly, no human data are provided on decreased
    weight gain with equivalent amounts of salatrim substituted for
    traditional fats. As shown above, stearic acid appears to be less well

    absorbed in rats than in humans and thus rat studies are not
    appropriate to determine the caloric content for humans of
    triglycerides containing stearic acid. In addition, rats and humans
    have a requirement for the essential fatty acids linoleic acid and
    linolenic acid. Salatrim, consisting of short-chain fatty acids and
    stearic acid provides no essential fatty acids. The basal diet is low
    in fat (4.5% by weight) and also low in essential fatty acids. In
    addition, consumption of the basal diet is limited to 50% of 
     ad libitum fed animals. Although it is understood that the
    development of essential fatty acid deficiency occurs over time, and a
    14-day feeding period is unlikely to result in clinical signs of
    essential fatty acid deficiency, the greater the amount of salatrim in
    the diet, the lower the proportion of the essential fatty acids in the
    total diet. The requirement for essential fatty acids for rats or
    humans is considered to be 1-2% of the total energy. According to this
    estimation, all salatrim diets would be deficient in essential fatty
    acids, and the condition would be exacerbated at the higher levels of
    salatrim consumption. Typically, when salatrim products were tested in
    the rat growth assay, the test fats were provided at 21% by weight
    (i.e. the highest level at which the corn oil was fed). There is an
    additional concern with the testing of most salatrim products only at
    the 21% level. As fat is added to the diet, weight gain increases, but
    not at as rapid a rate at the higher levels of supplementation as it
    does at the lower levels. For example, using the data from study T-216
    and the data on the corn oil control animals, although the overall
    regression curve assumes that corn oil provides 9.0 kcals/g, at the 5%
    level of corn oil supplementation the authors' equation would yield a
    caloric value for corn oil of 9.8 kcal/g and at the 21% level (the
    level at which the test fat was administered) the corn oil would yield
    a caloric value of 8.6 kcal/g. Thus, by testing the salatrim products
    at the highest fat level the data are skewed towards a lower caloric
    value for the test fat. Selection of the level of salatrim
    supplementation required a thorough justification which was not
    provided. In summary, it must be questioned whether the growth assay
    in rats provides meaningful data for the caloric contribution of
    salatrim in human diets.

    5.3.1.4  Determining the caloric value of salatrim products based on
    the stearic acid absorption coefficient

        The growth assay in rats was used to determine the absorption
    coefficient for stearic acid. Eleven salatrim compositions were
    generated by the interesterification of different starting molar
    ratios of tributyrin and hydrogenated canola oil. The molar short- to
    long-chain ratios of the compositions varied between 0.51 and 1.99.
    The caloric content of these 11 salatrim 4CA samples was determined
    using the 14-day rodent growth method. Rats were fed 50% of the basal
    diet of the control rats plus 21% salatrim by weight of the diet.
    Weight gain was calculated over the 14-day period and the weight gain
    of rats on each of the 11 salatrim diets were compared to the weight
    gains of rats on 21% corn oil. Corn oil is considered to supply 9
    kcal/g. A previously developed regression equation (Finley  et al., 
    1994b) was used to estimate the number of kcals/g for each salatrim

    product by comparing it to a standard curve generated for different
    amounts of corn oil added to the basal diet.

        Stearic acid absorption was estimated using the energy (in kcal/g)
    for the various salatrim products based on the rodent growth assay,
    and the ratio of short-chain to long-chain fatty acids in the test
    salatrim products. A table of the assayed composition of one salatrim
    product (with the ratio of 0.51/1 for SCFA/LCFA) was constructed. For
    each portion of the triglyceride the component of the triglyceride,
    the mass fraction of that component, the gross energy from the rat
    study and the absorption coefficient of that component were provided.
    All of the absorption coefficients were derived from the literature,
    and the only "unknown" was considered to be the absorption of stearic
    acid. From this matrix and the 11 different ratios of SCFA to LCFA,
    together with the estimated values of kcal/g from these different
    products, an absorption coefficient for stearic acid was derived. This
    absorption coefficient ranged from 0.15 with the lowest ratio of SCFA
    to LCFA to 0.70 with the highest ratio of SCFA to LCFA (Klemann et
    al., 1994).

        These absorption calculations for stearic acid reinforced the
    Committee's view that it is not possible to assign a general caloric
    value for salatrim without knowing the specific fatty acid composition
    of the product.

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    of 30 g, 45 g, and 60 g, Salatrim 4SO at 60 g, and Salatrim 43SO at 60
    g compared to a 60 g exposure level of control soybean oil. understudy
    are 23SO at exposure levels of 30 g, 45 g, and 60 g, Salatrim 4SO at
    60 g, and Salatrim 43SO at 60 g compared to a 60 g exposure level of
    control soybean oil. Unpublished report from Nabisco Foods, East
    Hanover, NJ, USA (Submitted to WHO by Cultor Food Science).

    Williams, K.D. (1992a) Final report: 13 week dietary toxicity study
    with A7200 in rats. Unpublished report No. HW 6270-157 from Hazelton,
    Wisconsin (Submitted to WHO by Cultor Food Science).

    Williams, K.D. (1992b) Final report : 13 week dietary toxicity study
    with A8200 and A8200 in rats. Unpublished report No. HWI 6270-162 from
    Hazelton, Wisconsin (Submitted to WHO by Cultor Food Science).

    Williams, K.D. (1992c) Final report: 13 week dietary toxicity study
    with A9100 and A7800 in rats. Unpublished report No. HW 6270-168 from
    Hazelton, Wisconsin (Submitted to WHO by Cultor Food Science).

    


    See Also:
       Toxicological Abbreviations