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    WORLD HEALTH ORGANIZATION

    WHO Food Additives Series 1972, No. 1




    TOXICOLOGICAL EVALUATION OF SOME 
    ENZYMES, MODIFIED STARCHES AND 
    CERTAIN OTHER SUBSTANCES




    The evaluations contained in this publication were prepared by the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    16-24 June 19711





    World Health Organization

    Geneva

    1972





                   

    1 Fifteenth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Wld Hlth Org. techn. Rep. Ser., 1972, No. 488; FAO
    Nutrition Meetings Report Series, 1972, No. 50.

    The monographs contained in the present volume are also issued by the
    Food and Agriculture Organization of the United Nations, Rome, as FAO
    Nutrition Meetings Report Series, No. 50A

    (c) FAO and WHO 1972


    STEAROYL LACTYLIC ACID, CALCIUM AND SODIUM SALTS

    Biological data

    Biochemical aspects

    In vitro hydrolysis with lipase proceeded readily to form stearic
    and lactic acid (Hodge, 1961).  Rats fed either salt of this acid
    excreted only traces of lactate in the faecal fat with good
    utilization of stearic acid and calcium (Hodge, 1961).

    Experiments comparing the metabolism of mixed stearic acid and
    14C-lactic acid with calcium 14C-stearoyl lactylate (lactic acid
    labelled) showed 58 per cent. excretion of the 14C of the physical
    mixture and 60 per cent. of the 14C-moiety as 14C02 within 24
    hours. There was no difference in C14-distribution and excretion
    between the two groups.  Thus lactate derived from calcium stearoyl
    lactylate is metabolized normally (Hodge, 1955).

    Acute toxicity

                                                                     

    Animal    Route        LD50             Reference
                      (mg/kg body-weight)
                                                                     

    rat       oral       over 25 000        Schuler & Thornton, 1952
                                                                     

    Short-term studies

    Rat

    Groups of 5 male rats received CaSL at levels of 0.5, 2.0 and 12.5 per
    cent. in their diet for 43 days.  No animal died but the weights of
    liver, heart, brain, stomach and testes were increased at the 12.5 per
    cent. level, relative liver weight was increased at the 2 per cent.
    level and growth was reduced at the 2 and 12.5 per cent. level (Hodge,
    1953).

    In a paired feeding study, groups of 10 rats were given 0 or 5 per
    cent. CaSL for 27 days.  The test group showed slightly lower food
    efficiency.  Liver weight of the test group was increased but the
    histology was normal except for a slight increase in glycogen.  In
    another paired feeding study, groups of 10 male and 10 female rats
    received either 0.5 per cent. CaSL, or 12.5 per cent. of a 41:59
    mixture of calcium lactate and stearic acid.  The test groups grew a
    little better but had raised liver weight.  Histology of livers and
    kidneys was normal in all groups and X-rays of femurs were comparable
    (Hedge, 1953a).

    Groups of 5 male rats received either 41:59 mixtures of calcium
    lactate and stearic acid for 32 days or 41:59 mixtures of sodium
    lactate and stearic acid for 52 days at 0, 0.5, 2 and 10 per cent. of
    the diet.  At the 10 per cent. level the sodium lactate group had a
    slightly reduced growth, but the organ weights of brain, stomach,
    spleen, lung and testes were raised.  Histology was normal.  Some
    organ weights were reduced at the 10 per cent. level in the calcium
    lactate group but histology was normal (Hedge, 1953).

    Groups of 8 male and 8 female rats received either 3.5 per cent.
    cellulose fibre or 3.5 per cent. stearoyl lactylate in their diet for
    90 days.  There was no difference between groups in growth rate, food
    consumption, faecal fat elimination, gross and histopathology (Schuler
    et al., 1952).

    Groups of 10 male and 10 female rats received NaSL at 0, 0.5, 5 and
    12.5 per cent. in their diet for 102 days.  Growth was reduced at the
    highest level.  No abnormalities compared with controls were seen as
    regards urinalysis, haematology, and faecal excretion.  At the highest
    test level the weights of liver, brain, stomach and spleen were
    increased but gross and histopathology were normal (Hodge, 1953).

    In a similar experiment on groups of 10 male and 10 female rats, CaSL
    was fed in the diet at levels of 0.5, 5 and 12.5 per cent. for 98
    days.  Growth was slightly retarded at 5 per cent. and significantly
    reduced at 12.5 per cent. and the relative weights of liver, stomach,
    heart, spleen and brain were increased at 12.5 per cent.  No
    histological abnormalities were seen in kidneys, brain, lung. spleen
    and liver, but at the 12.5 per cent. level lipogranulomata were
    detected in the adipose tissue.  No increase in stainable liver fat
    was seen.  Urinalysis and blood morphology were normal.  Radiological
    studies of femurs were normal and showed that the additional dietary
    calcium had no effect on body load (Hodge, 1953a).

    Groups of 12 rats were fed for 4 weeks diets containing 0 or 5 per
    cent. CaSL or a mixture of calcium lactate, stearic acid and lactic
    acid.  The animals on CaSL grow best with better food efficiency and
    better calcium deposition in the bones than in controls.  The liver
    weights of the CaSL group were greater than those of controls or the
    group on the mixed compounds.  No other pathological changes were seen
    (Wisconsin Alumni Research Foundation, 1955).

    Twenty male rats received 0 or 5 per cent. NaSL for 28 days and 30
    male rats received 0 or 5 per cent. CaSL for 32 days.  Relative liver
    weights were normal compared with controls in the CaSL group but
    raised slightly in the NaSL group.  Groups of 5 rats were sacrificed
    at 32, 60, 90 and 140 days.  Liver weights were normal in the NaSL
    group after 90 days (Hodge, 1954).

    Further experiments were undertaken to elucidate the effect of
    different levels of calcium and sodium on relative liver weights as
    well as the effect of the fat level of the diet on relative liver
    weights.  The relative liver weights became normal when rats returned
    to stock diets.  When diets contained physical mixtures of stearic
    acid, lactic acid and calcium carbonate, they produced comparable
    liver weights (Hodge, 1954).  Similar tests using 5 per cent. CaSL,
    4.3 per cent. stearoyl lactylic acid or 3 per cent. stearic acid in 24
    groups of 5 male rats each at varying levels of dietary fat showed
    slightly reduced body-weight in the groups receiving CaSL or stearoyl
    lactylic acid.  Mortality was not affected by treatment.  The relative
    liver weights were comparable for all groups and liver histology
    revealed no abnormalities (Hedge, 1959).  In a similar experiment 4
    groups of 32 male rats each were fed diets containing 0 or 5 per cent.
    CaSL, 3.11 per cent. calcium stearate or 3.2 per cent. sodium
    stearate.  The group on CaSL grew better than all other groups.  The
    relative liver weights of the controls were higher than all other
    groups (C. J. Patterson Co., 1956).  The chemical composition of the
    liver was determined in groups of 10 male and 10 female rats given 0
    or 5 per cent. CaSL for 1 month.  Only slight changes in glycogen,
    protein and lipid content were noted, lipid and protein being slightly
    increased compared with controls (Hedge, 1955a).

    Groups of 25 rats received diets containing 0, 0.1, 1.0, 2, 3, 4, 5,
    and 7.5 per cent. of calcium stearoyl lactylate for 1 month.  At the
    two highest levels there was growth retardation with relative liver
    weight increase.  Groups of 5 male rats were given diets containing
    either 15 per cent. lard or 10 per cent. lard plus 5 per cent. calcium
    stearoyl lactylate for 30 days.  The test group grew at a lower rate
    but relative liver weights were less than in the controls.  Groups of
    10 rats received diets containing 5 per cent. of calcium palmityl
    lactylate or calcium oleyl lactylate for 30 days.  All test groups
    grew slower and had markedly raised relative liver weights compared
    with 5 per cent. calcium stearoyl lactylate.  Kidney weights were
    normal for all groups and histological examination of liver, kidneys
    and fatty tissues revealed no abnormalities in any of these groups
    (Hedge, 1956).

    The appearance of "lipogranulomata" and the increased relative liver
    weight are related to the excessive intake of abnormal proportions of
    long-chain fatty acids.  The balance between saturated and unsaturated
    fats (S:U ratio) in the human diet is about 0.6 if the diet contains
    30-40 per cent. fat.  Rats fed diets containing 35-50 per cent.
    saturated fatty acid products (palmitic acid, stearic acid
    ethylstearate, monoglycerides and acetylated monoglycerides of
    hydrogenated lard) develop localized fat necrosis with formation of
    "lipogranulomata".  The condition is preventable by simultaneous
    feeding of cornoil and reversible by a return to normal diet (Cox & De
    Eds, 1958; Herting & Crain, 1958; Ambrose et al., 1958).

    Groups of 5 rats were maintained for periods up to 6 months on diets
    containing varying levels of calcium stearyl dilactylate (3 per cent.
    to 25 per cent.) and stearoyl lactylate acid (8 per cent. to 22 per
    cent.).  The total fat content was 20 per cent.  The outcome depended
    on the S:U ratio.  The added fats were chosen to give ratios from
    0.6-4.4.  Growth was depressed with increasing percentage of calcium
    stearoyl lactylate at 16 per cent. and higher levels and with 14 per
    cent. and above for the acid.  Mortality was high at levels of 20 per
    cent. and above.  Relative liver weights were normal at S:U ratios of
    0.6 (17 per cent. fat plus 3 per cent. calcium stearoyl lactylate or
    17 per cent. fat plus 2.6 stearoyl lactylate acid) but rose with
    higher ratios.  Lipogranulomata appeared at ratio values beyond 1.4.
    The iodine number of depot fats reflected the variation in S:U ratio
    of the diet.  Restoration to the basal ration containing 20 per cent.
    fat caused disappearance of lipogranulomata in 4-6 months. No
    histopathological abnormalities were seen (Hodge et al., 1964). In a
    repeat experiment with 40 male and 40 female rats fed 25 per cent.
    alcium stearoyl lactylate or 18 per cent. stearoyl lactylate acid in
    their diet, all animals developed severe lipogranulomata with high
    mortality. Recovery was rapid if animals were placed on basal diet
    containing 20 per cent. fat (half cornoil half lard).  Growth rate
    recovered and any deaths occurring were unrelated to the diet (Hodge,
    1960).

    Dog

    One male and 3 female beagles were fed a diet containing 7.5 per cent.
    calcium stearoyl lactylate; another group of 1 male and 3 females
    served as controls.  After 2 years, no noteworthy differences were
    observed between the 2 groups.  Urinalysis and haematological findings
    remained normal.  No gross or microscopic changes were found
    attributable to administration of calcium stearoyl lactylate.  Liver
    weights were within normal range; nor did the livers differ in
    moisture, protein, lipids, ash or glycogen content.  Other organ
    weights were also normal.  No adverse effects were observed in 1 dog
    receiving sodium stearoyl lactylate in his diet for 1 month at a level
    of 7.5 per cent., then increased to 12.5 per cent. for 2 weeks and to
    15 per cent. for another month.  No changes occurred in the blood;
    organ weights and the microscopic appearance of the tissues were
    normal (Hodge, 1955b).

    Long-term studies

    None available.

    Comments

    Adequate biochemical studies have revealed no differences between the
    metabolism of C14-labelled lactic acid when present as stearoyl ester
    and when mixed with an equivalent amount of stearic acid.  Since all
    the lactic acid derived from stearoyl lactylate enters the metabolic
    pool after complete hydrolysis of the ester, it is justifiable to
    consider conventional long-term studies as unnecessary.  Extensive

    short-term studies in rats have given variable and inconsistent
    results as regards levels producing no-effect on growth or relative
    liver weight.  Taking into account that the dog appears to be a less
    sensitive species, it appears reasonable to accept the 2 per cent.
    level as no-effect level for the rat.  It would be desirable to
    confirm that man metabolizes stearoyl lactylate similarly to other
    species.

    EVALUATION

    Level causing no toxicological effect in the rat

    Two per cent. (20 000 ppm) in the diet equivalent to 1000 mg/kg
    body-weight/day.

    Estimate of acceptable daily intake for man     mg/kg body-weight

         Unconditional acceptance                           0-20

    REFERENCES

    Ambrose, A. M., Robbins, D. J. & Cox, A. J. (1958) Fed. Res., 23, 536

    Cox, A. J. & De Eds, F. (1958) Amer. J. Path., 34, 263

    Herting, D. C. & Crain, R. C. (1958) Proc. Soc. exp. Biol. (N.Y.), 98,
    347

    Hodge, H. C. (1953) Unpublished report dated 2 April 1953, submitted
    by C. J. Patterson Co.

    Hodge, H. C. (1953a) Unpublished report dated 18 July 1953, submitted
    by C. J. Patterson Co.

    Hodge, H. C. (1954) Unpublished report submitted by C. J. Patterson
    Co.

    Hodge, H. C. (1955) Unpublished report dated 30 June 1955 submitted by
    C. J. Patterson Co.

    Hodge H. C. (1955a) Unpublished report dated 28 May 1955 submitted by
    C. J. Patterson Co.

    Hodge, H. C. (1955b) Unpublished report dated 17 June 1955 submitted
    by C. J. Patterson Co.

    Hodge, H. C. (1956) Unpublished report submitted by C. J. Patterson
    Co.

    Hodge, H. C. (1959) Unpublished report submitted by C. J. Patterson
    Co.

    Hodge, H. C. (1960) Unpublished report submitted by C. J. Patterson
    Co.

    Hodge, H. C. (1961) Unpublished report submitted by C. J. Patterson
    Co.

    Hodge, H. C., Maynard, R. A., Downs, W. L. & Panner, B. (1954)
    Toxicol. appl. Pharmacol., 6, 350

    C. J. Patterson Co. (1956) Unpublished report

    Schuler, M. N. & Thornton, M. H. (1952) Unpublished report submitted
    by Midwest Research Institute

    Schuler, M. N., Kodras, R., Allebach, H. K. B. & Gilliam, W. S. (1952)
    Unpublished report submitted by Midwest Research institute

    Wisconsin Alumni Research Foundation (1955) Unpublished report
    submitted by J. C. Patterson Co.
    


    See Also:
       Toxicological Abbreviations
       Stearoyl lactylic acid, calcium and sodium salts (WHO Food Additives Series 5)