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
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
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).
Animal Route LD50 Reference
rat oral over 25 000 Schuler & Thornton, 1952
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,
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
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
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,
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).
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
Level causing no toxicological effect in the rat
Two per cent. (20 000 ppm) in the diet equivalent to 1000 mg/kg
Estimate of acceptable daily intake for man mg/kg body-weight
Unconditional acceptance 0-20
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,
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
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
Hodge, H. C. (1959) Unpublished report submitted by C. J. Patterson
Hodge, H. C. (1960) Unpublished report submitted by C. J. Patterson
Hodge, H. C. (1961) Unpublished report submitted by C. J. Patterson
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.