Toxicity Testing of Fat Emulsions for

Intravenous Administration

L. D. JONES, D.V.M.,* M. W. CA5TLEBERRY, D.V.M.,t J. E. CANHAM, M.D4 AND

N. W. KING, D.V.M.*

T HE potential value of nontoxic fat emul- program to determine the tolerance and toxicity
sions suitable for intravenous adminis- of various experimental or commercial emul-
tration in any disease or surgical condition 3 The purpose of this report is to describe
characterized by serious weight loss or emacia- the results of further testing of the toxicity of

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tion was well summarized by Stare et al. various fat emulsions in rats and rabbits.
fifteen years ago.’ Clinicians, physiologists and
MATERIALS AND METHODS
pharmacologists have been keenly aware of the
advantages and the need for providing adequate The outline of the current studies is quite similar
parenteral nourishment to the patient in- to previous studies performed at this Laboratory
which are reported elsewhere .
capable of taking sufficient nutrients orally.
A well tolerated intravenous fat emulsion would Test Emulsions
help to supply the calories and the protein
Two emulsions which are commercially available
sparing effect essential for adequate parenteral
in Europe, Lipofundin#{174} and Intralipid,#{174} and twelve
nutrition. Numerous fat emulsions have been experimentally prepared fat emulsions were evalu-
prepared but few have attained even limited ated in this series of tests. Composition of the four-
acceptability in the treatment of ill or injured teen emulsions utilized is given in Table I. Descrip-
human beings because of various toxic manifes- tion of the preparation and the physical and chemical
tations. These toxic manifestations have been characteristics of experimental emulsions produced
described elsewhere.2 Thus the search for the by Singleton et al. are contained in other reports.4”

component or components in the emulsions Each emulsion was administered to six rabbits
and/or six rats for evaluation. Twelve preparations
responsible for the toxic manifestations and for
were administered at a daily dosage level of 3 gm.
the development of a nontoxic fat emulsion
per kg. of body weight five days a week for three
suitable for intravenous use has continued.
weeks. The 10 and 13 per cent Lipofundin were
Although the ultimate measure of the utility
administered at a daily rate of 2.0 and 2.25 gm. per
of a fat emulsion for intravenous nutrition is kg. of body weight, respectively, over the same time
its acceptance by man, screening of experi- period.
mental and commercial emulsions must, through
necessity, be performed on laboratory animals.
Test Animals

Our Laboratory, as a unit of the U. S. Army The animals utilized were male New Zealand

Surgeon General’s Task Force on Intravenous white rabbits and male rats of the Spragime-Dawley

Fat Emulsion, has maintained a continuous strain. The animals were housed in individual cages
in a temperature regulated room. Commercial
From the U. S. Army Medical Research and Nutri- laboratory chow and water was provided ad libitum
tion Laboratory, Fitzsimons General Hospital, Denver, throughout the experiment. Following purchase,
Colorado. the rabbits were observed for a minimum of two
* Staff Pathologist, Pathology Division; t Chief, weeks prior to the administration of the emulsions.
Pathology Division; Chief, Metabolic Division.
The principles of laboratory animal care as promul- § 15 per cent Lipofundin is not a commercial product
gated by the National Society for Medical Research but was prepared by specific request of U. S. Army
were observed. Medical Research and Development Command.

American Journal of Clinical Nutrition 62 Vol. 16, January 1965

 Toxicity Testing of Intravenous Fat Emulsions 63

TABLE I

Composition of Test Emulsions

Administered
Component and Concentration
to
Emulsion Code
No. *
Aqueous
% Oil Phase % Emulsifying System Rabbits Rats

ii
Phase
%
l.ipofundin K399. la 15 Cottonseed 5.0 Sorbitol Soybean phosphatide, hydrogenated + +
Lipofundin 577K.... lb 10 Cottonseed .5.0 Sorbitol Soybean phosphatide, hydrogenated + +
Intralipid 293009. 2 20 Soybean 2.5 Glycerol Egg yolk phosphatide + +
Intralipid 293011.... 2 20 Soybean 2.5 Glycerol Egg yolk phosphatide +
Intralipid 293012. 2 20 Soybean 2.5 Glycerol Egg yolk phosphatide +
Riker experimental
emulsions
X63225 3 20 Soybean 2. 5 Glycerol 1.2 Egg yolk phosphatide +
X64010 3 20 Soybean 2. 5 Glycerol 12 Egg yolk phosphatide +
SR.4734.107A 4 20 Soybean 2. 5 Glycerol 1.2 Egg yolk phosphatide, highly puri +
fled
SR.4734. 107B 4 20 Soybean 5 . 0 Sorbitol 1.2 Egg yolk phosphatide, highly puri +
fled
SR-141 4 20 Soybean 2. 5 Glycerol 1.2 Egg yolk phosphatide, partially +
purified

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SR.143K 4 20 I Soybean 2. 5 Glycerol 1.2 Egg yolk phosphatide, “postleci. +
thin,” cephalin fraction
SR.143L 4 20 Soybean 2.i Glycerol 1.2 Egg yolk phosphatide, ‘preleci +
thin,” lyso fraction
SR.151 4 20 Soybean 2. 5 Glycerol 1.0 Chromatographically homogeneous + +
lecithin + +
SR.152. . . . 4 20 Cottonseed 2.5 Glycerol 1.0 Chromatographically homogeneous
lecithin

* la = Product of B. Braun, Melsungen, Germany; and produced per special request of Medical Research Branch, Medical Research

lb
2
:i
4
=

=
I)evelopment
Product
Product
Product
Experimental
search
of AB.
of Riker
Command,
of B. Braun,

& I)evelopment
Vitrum,
I.abs.
emulsions
, SGO,
Melsungen,
Stockholm,
Inc.
prepared
l)iv. , New
USA, Washington,
Germany;
Sweden;

by Mr.
Orleans,
W.
l.a.
D. C.
distributed
distributed

S. Singleton,
by Pharmachem
by Riker

Chemist,
Labs.,

Oilseed
Corp.,
Inc.,

Crop
Bethlehem,
Northridge,

Lab., USI)A
Pennsylvania.
California.

Southern Utilization Re-

The test emulsions were administered intravenously All rabbits and rats were held for a minimum of
via the ear veins at the average rate of 1 ml. each ten days for observation and examination following
2.4 minutes. Rectal temperatures were taken completion of the infusion periods. During the
before the infusion, 30 minutes after the infusion second week after infusion, all animals were sacri-
and then at hourly intervals up to live hours after ficed ; at necropsy representative samples of each
the infusion. organ were taken for microscopic studies.
Body weight, rectal body temperature, food in-
take, water intake and urine output were recorded Tissue Technics
throughout the week before the infusion, during the All tissue specimens collected at necropsy were
three week infusion period, and for one week after the
fixed in neutral buffered 10 per cent formalin. Ad-
infusion. During these weeks, the following pro-
ditional specimens of liver and spleen were placed in
cedures were performed on all rabbits at weekly in-
20 volumes of absolute ethyl alcohol for 4 hours, then
tervals : clotting time, hematocrit (packed cell
removed and placed in 70 per cent ethyl alcohol for
volume) , red blood cell and nucleated red blood
an additional 72 hours before further processing. All
cell counts, hemoglobin, reticulocyte counts, white
tissue specimens were processed, embedded in
blood cell counts and differentials. Weekly urinaly-
paraffin and sectioned by the usual method. The
ses included determinations for specific gravity,
alcohol-fixed tissues were stained for iron by the
albumin, pH, acetone, sugar, bile, occult blood and
Gomori method, employing Prussian blue, and
hemoglobin as well as microscopic examination of
counterstaining with Nuclear Fast Red. In addi-
the sediment,
tion, all tissue sections were stained with hemo-
After a preliminary observation period, the rats
toxylin and eosin.6
were weighed and examined daily throughout the
infusion test period and for one week after the in- RESULTS
fusion. The test emulsions were administered to the
rats via the tail vein. The injection time was less A summary of the weight performance and
than 2 minutes. pertinent hematologic findings during the
64 J ones et al.

TABLE II
Summary Performance-Rabbits

Weights (gm.) Blood

Hemoglobin (gm. %) Packed Cell Volume (%)

Emulsion
Initial Final After
Change Infusion
After
Initial Final After Initial Final
Infusion Infusion

Lipofundin K399* 3,538 3,555 +0.4 3,554 19 14.8 13.4 46 39 38

Lipofundin577K 2,979 3,058 +2.7 3,128 15.2 16 16.5 45 42 42
Intralipid 293009 3,054 3,053 0 3,185 16.1 15 15.6 38 40 40
Intralipid2930ll 3,022 3,176 +5.1 3,296 16.2 16.3 17.6 42 42 42
Intralipid 293012 3,598 3,748 +4.2 3,800 16.2 17.2 16.7 45 43 43
SR-151 2,911 3,044 +4.6 3,081 15.8 13.6 15 43 39 43
SR-152 3,062 3,093 +1.0 3,218 14.1 13.6 13.6 44 38 40

NOTE: All figures represent an average for six animals. Initial = prior to first infusion. Final = prior to last infusion. After Infu.
sion = seven days after last infusion.
Values for four rabbits.

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*

TABLE III following the first infusion while a second died

Summary Performance-Rats shortly after the second infusion. These
deaths closely duplicated those observed in a
Weights (gm.)
_______ previous study utilizing this 3 In the
Emulsion remaining four rabbits, prolonged , significant
C’
/0
Initial Final Change
Infusion hyperpyrexia occurred after the infusion. The
average daily food consumption decreased 55

Lipofundin K399. 289 323 +11.8 320 per cent while the water intake decreased 50
Lipofundin 577K. 256 298 +16.4 301 per cent during the first week of infusion.
Intralipid 293009. 366 361 -1.4 . ‘ ‘ Both food and water intake improved during
Riker experilnen-
tal emulsions the last two weeks of infusion. The rabbits
X63225* 297 264 -11.1 273 receiving the 10 per cent Lipofundin (577K)
X64010* 307 291 -5.2 302 also demonstrated significant hyperpyrexia
SR-4734-107A 195 273 +40
SR-4734-107B
..

. 200 260 +30 ::: after the infusion. Occasional significant hy-
SR-141 287 270 -5.9 277 perpyrexia occurred following infusion with
SR-l43K
SR-143L
SR-151
342
347
359
350
350
362
+2.3
+0.9
+0.9
:
352
SR-151.
and
Intralipid
SR-152
(293011,
produced focal
293012) and
inflammatory
SR-

SR-l52 339 347 +2.4 365 reactions at the site of the injections. This
action was most prominent with Intralipid
NOTE : All figures average values for six
animals. 293012. Low grade thrombophlebitis de-
Initial = weight prior to first infusion. Final weight
=
veloped in the ear veins of some rabbits re-
prior to last infusion. After Infusion = weight seven
days after last infusion. ceiving SR-151. All emulsions administered
* Infusions terminated following the tenth infusion. to rabbits except Intralipid 29301 1 produced a
decrease in hemoglobin and/or packed cell
studies are presented in Tables ii and Iii. volume. During infusion with Intralipid
Other pertinent clinical observations will be 293009, the white blood cell count doubled.
reported. Both nucleated red blood cell counts and
reticulocyte counts increased in those rabbits
Rabbits receiving Intralipid 29301 1 . Clotting time
Lipofundin, both 10 and 15 per cent, was not was not significantly altered by any of the
well tolerated by the rabbits. Of the six emulsions administered. During the infusion
rabbits receiving 15 per cent Lipofundin period intermittent traces of albumin and bile
(K399), one rabbit died less than 24 hours appeared in the urine of some of the animals
 Toxicity Testing of Intravenous Fat Emulsions 65

receiving Lipofundin K399 and 577K, Intralipid Histopathological Examination

293012, SR-151 and SR-152. Occult blood Microscopic examination of tissue sections
was occasionally found in the urine from rabbits disclosed deposition of the intravenous fat pig-
receiving SR-151 and 152. Rabbits receiving ment previously described8 in the livers and
Intralipid 293009 demonstrated a progressive spleens of all the rabbits and rats given infu-
decrease in food intake throughout the period sions, with all the emulsions tested. The
of the infusions. There was a variation in the greatest amount of intravenous fat pigment
degree of thermogenicity and weight gain pro- deposition was noted in the animals given cot-
duced by the various production batches of the tonseed oil emulsions whereas the least
Intralipid tested. This variation in the ther-
amount was found in those given infusions of
mogenic response observed with different pro-
SR-151. Lipoid microgranulomas were present
duction batches of Intralipid is similar to that
in the livers and/or spleens of all the test ani-
observed in man.7 The most favorable re-
mals except those given emulsion SR-141.
sponses were observed in the rabbits receiving
Minimal portal fibrosis was found in the livers
Intralipid 29301 1 and 293012.
of animals given Lipofundin. Detailed de-

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scriptions of the histopathologic lesions found
Rats
following prolonged intravenous fat emulsion
Difficulty was encountered in comparing therapy have been reported 10
weight gains between groups of rats because
COMMENTS
of the variation in the initial age and weight of
the rat groups. For example, the greatest The primary purpose of testing intravenous
weight gain observed was in the rats receiving fat emulsions for toxicity is to determine those
SR-4734-107A and SR-4734-107B (Table III). emulsions or emulsion components which may
The average initial age and weight of these rats be least toxic for use in man. The results of the
were considerably lower than that of the other present study demonstrate the difficulty in
rats. Rats receiving the Riker experimental translating findings from one species of animals
emulsions (X63225 and X64010) did quite to another. Lipofundin, which was admin-
poorly with inappetence, marked weight loss, istered at a reduced dosage rate as compared to
development of inflamed necrotizing lesions of all the other emulsions (2.0 or 2.25 gm. per kg.
the tails, diarrhea and a general appearance of as compared to 3 gm. per kg.) , had an adverse
loss of condition. Because of these marked effect upon rabbits but was well tolerated in
adverse effects infusions of both of these rats. Intralipid 29301 1 was well tolerated in
emulsions were terminated after the tenth rabbits, but Intralipid 293009, the only Intra-
infusion. A significant weight loss was also lipid preparation tested in rats, was disappoint-
observed in the group of rats receiving Intra- ing in both rats and rabbits. One of the most
lipid 293009. The hemoglobin and hematocrit poorly understood reactions of prolonged ad-
were determined before and after infusion in ministration of intravenous fat emulsions to
rats receiving SR-151 and 152. Hemoglobin man is the overloading syndrome.’1 Unfortu-
and heinatocrit values decreased following the nately it has not been possible to reproduce all
administration of both emulsions. Rats re- the manifestations of this syndrome in experi-
ceiving SR-152 had intermittent diarrhea. mental animals. Therefore, although toxicity
Over-all physiological observations indicated in animals may indicate that an emulsion is
that the emulsion best tolerated was SR-107A, relatively nontoxic, evaluation of the material
with 10 per cent Lipofundin a close second. in man is necessary before it can be assumed to
be a good therapeutic agent for parenteral nu-
Necropsy Findings trition.
No significant gross lesions attributable to The development of the histopathologic le-
the intravenous administration of fat emulsions sions noted in the spleens and livers of test ani-
were found at necropsy in any of the experi- mals receiving repeated infusions as related to
mental animals. the various components of intravenous fat
 66 Jones et al.

emulsions administered has been discussed else- the period of J)rolonged infusions with intra-
3 Intravenous fat pigment has been ob- venous fat emulsions, there is an increase in the
served in the livers of animals receiving emul- total blood volume and in the plasma volume.
sions containing various oils. Cottonseed oil In preliminary experiments, Flick et al. 13 ob-
emulsions appear to stimulate the greatest served that the use of a complete fat emulsion
deposition of intravenous fat pigment, while the has a potentiating effect upon the recovery of
soybean oil, glycerol and egg yolk phosphatide animals after experimental hypovolemia. Un-
emulsions appeared to produce the least. fortunately, the data presented are too incoin-
Although the marked decrease in the amount plete to determine if this effect was due to an
of intravenous fat pigment deposition seen in effect upon the circulating blood volume or was
the animals given SR-151 may be related to the the result of the caloric value of the emulsion
purification of egg phosphatide to chromato- infused. It is possible that the dichotomy be-
graphically pure lecithin, it should be noted tween increased body weight despite decreased
that the total emulsifier content was decreased food intake in animals receiving repeated in-
in this emulsion. However, based on the fusions of intravenous fat emulsions is partially

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series of animals given SR-151, it would appear due to a retention of body fluids. Studies are
that the emulsifier system of an emulsion is im- necessary to clarify this point.
portant in the deposition of intravenous fat pig-
SUMMARY
ment. It is impossible to dismiss the oil phase
of the emulsion as participating in the pigment Clinical and histopathologic toxicity studies
deposition. This was illustrated in the animals were conducted on fourteen fat emulsions in-
given SR-152 in which moderate to marked in- eluding Lipomul, Intralipid, Lipofundin and
travenous fat pigment deposition was found in cottonseed or soybean oil emulsions prepared
the spleens and livers. SR-152 contained the commercially or experimentally for intravenous
same emulsifier system as SR-151 but the oil use. These emulsions were administered to
phase consisted of cottonseed oil instead of soy- rabbits and/or rats in a daily dose of 2.0 to 3.0
bean oil. Hence, it seems obvious that the gal. of fat per kg. of body weight. Clinical
complete emulsifier-oil chylomicron complex of studies included observation of the animals’
an emulsion is important in stimulating intra- general reaction to the infusion, local reaction
venous fat pigment deposition. Lipoid micro- at the site of injection, variation in body weight,
granulomas were noted in spleens and/or livers and weekly hemograms and urinalyses. The
of all animals tested in this series of experiments animals were sacrificed at least ten days after
except those given SR-141. It is extremely infusion and a detailed necropsy was performed.
difficult to draw any conclusions from this ob- No gross lesions could be attributed to the fat
servation based upon the composition of SR- emulsion . However, histopathologic examina-
141. Possibly, if the number of animals given tion revealed the deposition of intravenous fat
this material had been increased, lipoid micro- pigment in the liver and spleen of every animal.
granulomas would have been observed. The most pigment was found in animals given
Frequently, during these studies, a gain in cottonseed oil emulsions and the least in ani-
weight was observed during the infusion period mals given emulsion SR-131. Lipoid granu-
that was not commensurate with the marked lomas were observed in all test animals except
decrease in food and/or fluid intake. It would those given emulsion SR-141. Mild hepatic
be pleasant to ascribe this weight gain to the fibrosis was observed in animals receiving Lipo-
increased calories derived from the intrave- fundin. Adverse clinical reactions in rabbits
nously administered fats. However, as can be included death in two animals, produced by
determined from the data in Tables im and III, Lipofundin K399, and hyperpyrexia, anorexia,
the test animals demonstrated a decrease in decreased water intake, weight loss, and de-
weight during the first week after infusion when crease in hemoglobin and hematocrit following
the fluid and food intake was increasing. infusion. The best performance in rabbits was
Iacono1’ has demonstrated in rats that, during obtained with Intralipid 293011 and in rats
 Toxicity Testing of Intravenous Fat Emulsions 67

with SR-4734-107A. Although Lipofundin lecithin from egg phosphatides. J. Oil

C7zemists Soc., in press, 1964.
was well tolerated by rats, in general, soybean
6. THOMPSON, S. W. Selected Histochemical aIld
oil-egg yolk phosphatide-glycerol type emul-
Histopathological Methods. Springfield, Ill.,
sions produced the least adverse physiologic and 1964. Charles C Thomas.
histopathologic changes in the test animals. 7. MUELLER, J. F. Personal comnmunication.
8. THOMPSON, S. W. , JOHNSON, F. B. and FORBI S.

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