TOXICOLOGY AND APPLIED PHARMACOLOGY 2, 59-67 (1960)

Oral Toxicities of Laurie Acid and Certain

Laurie Acid Derivatives
O.GARTH FITZHUGH,PAUL J. SCHOUBOE,AEDARTHUR A. NELSON
Division of Pharmacology, Bureau of Biological and Physical Sciences, Food and
Drug Administration, United States Department of Health, Education
and Welfare, Washington, D. C.

Received September 21, 1959

There has been conflicting evidence regarding the hazard of the addi-
tion of synthetic lauric acid-type emulsifiers to food. This has resulted in
questioning the safety of the addition of the mono- and diglycerides of
lauric acid, on the basis of the presenceof the lauric acid moiety (Federal
Register, 1952).
There is relatively little in the literature regarding the effects of feeding
lauric acid and its glycerides. Evans and Lepkovsky (1932) fed glyceryl
laurate (not defined) to rats at a 607%level in a synthetic diet and con-
cluded the material was nontoxic. Mattson et al. (1951) fed mono-, di-,
and triglycerides at levels as high as 250/Oin a synthetic diet for 70 days.
The rats showed on autopsy no histologic evidence of toxicity attributed to
the test compound. Carrol (1958), Deuel et al. (1941), Hoagland and
Snider (1943), and Thomasson (1956) studied the digestibility of either
lauric acid or its glycerides. Their investigations gave no indication that
the ingestion of these compoundswas harmful.
The effect of feeding lauric acid emulsifiers has been investigated more
thoroughly. Harris et al. (1951a, b) studied the effects of feeding sorbitan
monolaurate, polyoxyethylene monolaurate, and polyoxyethylene mono-
stearate to rats at 257, of their diet and to hamsters at 5 and 15% levels.
They reported severe diarrhea, decreased weight gains, and increased
mortality at all feeding levels and noted pathologic changes in rats fed
the emulsifiers. Sorbitan monolaurate was the most deleterious of the
three. Krehl et al. (1955) reported studies on the effects of feeding poly-
oxyethylene mono- and dilaurates and other polyoxyethylene esters. They
concluded that the low palatability of these compounds together with the
fact that the polyoxyethylene moiety has no nutritive value makes it
impractical for the inclusion of polyoxyethylene emulsifiers in foods for
59
60 0. GARTH FITZHUGH ET AL.

humans at other than low concentrations. Eagle and Poling ( 1956) and
Poling et nl. (1956) fed rats, hamsters, and rabbits at dietary levels up
to 2555 of polyoxyethylene-20 sorbitan monolaurate. They reported de-
creased growth rate and increased mortality in all species beginning as
low as the .5? feeding level for hamsters.

METHODS

Compounds tested were lauric acid, Span 201 (sorbitan monolaurate),

G-2129l (polyoxyethylene-20 monolaurate)? and S-1062” (a mixture of
mono-, di-, and triglycerides of lauric acid”). Diets were prepared in
quantities sufficient for 2 weeks by blending the basal diet of a ground
commercial rat biscuit with the test material so that the test material was
the desired percentage by weight of the total diet.
Albino rats of our Osborne-Mendel strain weighing between 40 and
50 g were used in the experiments. All animals were randomized in the
various experimental groups and their controls on a littermate basis except
for the lauric acid study. The rats were housed in individual cages and
were allowed food and water ad libitum. Animal weights were recorded
weekly, and records of mortalities and abnormalities of the rats in regard
to general physical condition, appearance, and behavior were kept. Com-
plete blood counts were taken from 5 or more animals from each group
toward the end of the experimental period. At the end of the test period,
survivors were sacrificed by exsanguination and autopsied. Organ weights
and gross pathology were recorded for the sacrificed animals, and tissues,
except those for the lauric acid study, were fixed in lOc/, formalin solution
for histologic examination. Organs from animals found dead were excluded
from organ weight calculations, but were saved for histopathologic study
if not markedly autolyzed.
RESULTS
Laurie Acid
Five male rats were fed lauric acid at the 10% level of their diet for
18 weeks. A control group of 5 males was fed concurrently. There were
no observable clinical effects, no adverse effects on weight gain, nor was
there any mortality. Gross organ pathology and comparison of individual

1 Trade names of Atlas Powder Co., Wilmington, Delaware.

0 Trade name of Glyco Products Co., Brooklyn, New York.
3 Composition as supplied by the manufacturer: mcnoglyceride 40-45%, diglycer-
ide 4570, triglyceride 870, glycerine 3.5%.
 TOXICITIES OF LAURIC ACID DERIVATIVES 61

organ weights showed no significant differences between the controls and

test animals.
Span 20
Dietary levels of 25, 20, 15, and 0% of Span 20 were fed to rats for
23 weeks. There were 20 rats at each dosagelevel, evenly divided as to
sex. Among observable effects of Span 20 feeding were diarrhea at all
feeding levels and an unkempt appearance. Severity of the diarrhea was
reduced as the experiment progressed.
Table 1 showsweight gains of control animals compared to test animals
over the 23-week study period. It can be seenthat growth of the rats, as

TABLE 1
WEIGHT GAINS OF RATS FED SORBITAN MONOLAURATE (SPAN 20) FOR 23 WEEKS
Dietary h’umher of Average weight gains
lcvcl iYc;) animals SPX anll standard errOI (p)
__-
0 10 M 406.4 2 12.9
10 F 218.0 r+ 10.0
25” 10 M -
10 F -
20 10 M 160.2 k 12.7b
10 F 160.3 k 7.90
15 10 M 192.2 k 17.9h
10 F 164.8 4 13.9”
fl Only one animal of each sex survived.
b p < 0.001.
c p < 0.01.

expressed by their mean weight gains, was severely retarded at the 15

and 20% levels. At the 25% level, mortality was so great it was not
possible to obtain significant mean weight gain values. Only one animal
in each sex at this dosagesurvived the test period. There were no signifi-
cant differences in the mortalities at the other test levels when compared
to number of deaths that occurred among the control group.
The more constant effects attributable to Span 20 feeding, shown on
autopsy, were palenessand enlargement of the liver and great enlarge-
ment of the common bile duct to as much as 2 cm diameter. Figure 1
showsthe progressive enlargement of the common bile duct which occurred
with each increase of dosagelevel. Gangrene of the tail occurred in 5
rats fed at the 25% dosagelevel. Isolated instances of the condition also
occurred at the other levels of Span 20 feeding.
62 0. GARTH FITZHUGH ET AL.

Thirty-one animals were examined for microscopic pathology. Histo-

logically, the liver damage at all feeding levels of Span 20 was pronounced.
It consisted of approximately half fatty change and half inter- and intra-
lobular fibrosis, with the consequent disorganization of the normal lobular
architecture. Sometimes one or the other of the major lesions would
predominate. In addition, there were small amounts of chronic inflamma-
tory cellular exudate (chronic hepatitis), and rarely focal hepatic cell
necrosis. Slight or moderate hepatic cell enlargement, irregularly distrib-
uted, often occurred, especially when the cells were stuffed with fat. There
was little or no bile duct proliferation, and no bile duct dilatation, within
the liver. There was fibrous thickening of the wall of the common bile
duct together with marked enlargement, slight or moderate epithelial
proliferation, and little or no inflammatory changes. The cause of the
common bile duct enlargement was not evident in the microscopic sections.
The incidence of focal nephritis, although not great, was higher than
usual for rats of this age. The histologic type was not definitely different

FIG. 1. Progressive enlargement of the common bile duct with each increase of
dosage level of Span 20.
 TOXICITIES OF LAURIC ACID DERIVATIVES 63

from the “spontaneous,” but there was a tendency for a more internal
cortical tubular involvement, and proteinuria was greater than usual in
relation to tubular atrophy.
The majority of the lungs contained numerous foamy alveolar macro-
phages,in contrast to the small number ordinarily seen. Bone marrow and
spleen were in general somewhat hyperplastic (myeloid and erythroid,
respectively). Heart, pancreas, stomach, small intestine, colon, adrenal,
testis, ovary, uterus, thyroid, leg bones, and leg musclesshowed nothing
attributable to Span 20.
S-1062
Twenty-four rats per group, divided as to sex, were fed for two years
on S-1062 at feeding levels of 25 and 0% of the diet. An additional
group of controls, using hydrogenated cottonseed oil at the same dosage
levels as S-1062, was run concurrently. When the groups of rats fed
S-1062 were compared with the corresponding group fed the hydrogenated
oil, there was no significant difference in weight gains after 26 or 52
weeks. In both instances, however, weight gains were greater than for the
rats fed the basal diet which reflected the higher caloric intake. After
two years there were no significant differences in the total number of
deaths that occurred in the group of rats fed S-1062 than occurred in the
groups fed either hydrogenated vegetable oil or the basal diet. Autopsies
revealed no gross pathology attributable to feeding S-1062. Detailed
histologic examination of all three groups of rats revealed as the only effect
attributable to S-1062 a slight excess of hepatic cell fatty change as
compared to the small amount in the controls fed the basic laboratory
chow, but this excesswas no greater than that observed in the group fed
the hydrogenated vegetable oil at the same dosage level. The same
difference occured to a lesserand questionably significant degree for intra-
hepatic bile duct proliferation.
G-2129
Twenty-four rats per group, evenly divided as to sex, were fed G-2129
at dosage levels of 25, 10, 5, 2, and 0% of their diets for two years.
Table 2 shows weight gains of the rats at the various dosagelevels after
26 and 52 weeks. There was no significant variation in the growth of either
male or female rats at the 2, 5, or 10% dietary levels. At the 25yC8level
there was a significant retardation in the growth of male rats after both
26 and 52 weeks. Mortality data for rats fed G-2129 over the two-year
period showed no significant differences from that of the controls.
64 0. GARTH FITZHUGH ET AL.

TABLE 2
WEIGHT GAIKS OF RATS FED POLYOXYETHYLE~TE-20 MONOIAURATE (G-2 120)
FOR Two YEARS

0 12 M 430.0 & 16.0 506.1 k 20.6

12 F 216.0 _’ 5.4 262.2 _t 9.5

25 12 M 337.1 t 14.Sa 432.5 2 ll.jcL

12 F 230.4 _t 6.4 270.0 t 9.6
10 12 M 410.5 2 S.4 455.3 51 8.3
12 F 232.4 -C 5.3 279.1 21 7.8

5 12 M 409.0 -c 17.9 466.0 +- 22.1

12 F 243.4 i 6.4 285.3 t 4.5
2 12 M 439.0 ? 16.0 475.2 -c- 12.4
12 F 225.8 -t 7.4 263.4 2 6.8

a p < 0.01.

Behavior and appearance of the animals throughout the experiment was

normal.
Gross pathologic examination revealed two or perhaps three abnormal-
ities attributable to G-2129 feeding, namely hepatic cysts and cecal
enlargement, and, as a minor effect at the 2572’ dosage level, three instances
of slight gastric mucosal hyperplasia. The liver cysts were unilocular or
multilocular, often multiple, and varied in size from a few millimeters to
2% X 1% X 19; cm. At the 25’5 level there were 5 animals affected;
at 105% there were 4, but with smaller and fewer cysts than at 259. The
three remaining groups each had 1 animal with a cyst, the largest of 7-mm
diameter. All liver cysts but one were in surviving animals. The cecal
enlargement occurred, in order of descending dosage level, in 17, 4, 3, 1,
and 0 animals, respectively. The most affected ceca had about three times
the normal volume, and the enlargement appeared to be a combination of
distention and some actual increase in mass; weighing of the ceca was not
done. There was no enlargement of the common bile duct.
Of the 120 rats started on this experiment, 61 were sectioned micro-
scopically, 51 of these in a detailed manner. The liver cysts were of bile
duct origin and were lined by columnar epithelium. There was no general-
ized intrahepatic bile duct dilatation, although in a minority of instances
the ducts adjacent to the cysts showed slight proliferation or dilatation.
The hepatic cells were unaffected by G-2129. The ceca of the treated
animals were histologically not different from those of the controls except
 TOXICITIES OF LAURIC ACID DERIVATIVES 65

for evidences of distention (thinner wall, short glands) in portions of the

sections. The only remaining organ showing anything attributable to
treatment was the stomach, which in 4 animals of the 25%) group and 1
of the 10% group showed slight squamous epithelial hyperplasia, with a
low-grade inflammatory process within and under the hyperplastic epi-
thelium, at the junction of the two main portions of the stomach. The
stomach in the remaining three groups showed none of this change. Lung,
heart, spleen, pancreas, small intestine, colon, kidney, adrenal, testis,
ovary, uterus, thyroid, parathyroid, prostate, urinary bladder, leg bones,
leg muscles, and bone marrow showed no effect of G-2129.

DISCUSSIOK

We could find in our experiments no deleterious effects attributable

to the ingestion of lauric acid or its glycerides at reasonable dietary levels.
Although our work with lauric acid was of a preliminary nature there were
no indications that results from an experiment on a larger scale would be
different.
With the lauric acid emulsifiers containing sorbitan or polyoxyethylene
the problem of toxicity and deleterious effects from oral ingestion is more
pronounced. Of the two we have investigated, Span 20 appeared to be
the more deleterious. There were, however, pathologic changes from the
ingestion of G-2129 which are attributable to this material even when fed
at low dietary levels. For example, there was a progressive increase in
incidence of distended ceca as the dosage increased from the lowest to
highest dietary level.
Pathologic changes observed from the feeding of Span 20 and G-2129
were different, indicating that there could be different metabolic pathways
for the physiological disposition of the materials. For example, with
Span 20 there was great common bile duct enlargement, hepatic fatty
change and fibrosis, and a large increase in pulmonary foam cell macro-
phages, whereas with G-2129 these conditions were not present in the
test animals. On the other hand, liver cysts and cecal enlargement devel-
oped in the rats fed G-2129 but were not noted in the animals fed Span 20.
We do not believe that these differences in pathology can be attributed to
the differences in time the animals were fed the test materials. The Span
20 experiment was of sufficient duration that there should have been
some indication of cyst formation if this were to develop. Likewise, since
the observed pathologic changes attributed to the test materials occurred
over ranges of feeding levels which were comparable, the feeding levels
66 0. GARTH FITZHUGH ET AL.

could hardly be a factor. It must be recognized, however, that these

pathologic effects, as well as others noted, may be a result in part of nutri-
tional inadequacies or of dietary imbalance when emulsifiers with non-
utilizable polyol portions are fed at relatively high dietary levels.
The toxicity of the two emulsifiers containing sorbitan or polyoxy-
ethylene can hardly be attributed to the lauric acid moieties of their
compositions. First, there were different pathologic changes resulting
from the ingestion of these two emulsifiers. Further evidence is that the
lauric acid glycerides, even at the 2.5% dietary level, were not toxic al-
though the amount of potentially available lauric acid was greater than
that from either Span 20 or G-2129. Likewise, the 107, level of feeding
of lauric acid was nontoxic although it approximates the theoretically
available lauric acid from Span 20 and was more than that which would
be available from G-2129, at the highest dietary level (25%).
SUMMARY

Subacute toxicity data for lauric acid and sorb&an monclaurate (Span 20) and
chronic toxicity data for lauric acid glycerides (S-1062) and polyoxyethylene-20
monolaurate (G-2129) in rats have been presented.
Sorbitan monolaurate tested at 25, 20, and 15% levels for 23 weeks caused signifi-
cant growth depression beginning at the lowest dietary level and significant increase
in mortality at the 25% level. There was progressive enlargement of the common
bile duct with each increase in dosage, and gangrene of the tail at all feeding levels,
with a greater number of rats affected at the 25% dosage. Histologically, liver
damage was pronounced and lung changes were present.
Polyoxyethylene-20 monolaurate tested at 25, 10, 5, and 270 levels for two years
caused growth retardation in male rats at the 25% level. Mortality data showed no
significant differences from the controls. There was a progressive increase in inci-
dence of liver cysts and distention of the cecum as the dosage increased.
Feeding of lauric acid glycerides at 25% of the diet for two years caused only
minor histological changes in the liver.
In a group of 5 rats, not microscopically examined, feeding of lauric acid at 10%
of the diet for 18 weeks had no deleterious effect.

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