904. Salatrim (short- and long-chain acyltriglyceride molecules) (WHO Food Additives Series 40)

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 37°C 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 ± 10°C) 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
± 10°C. 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 -20°C 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 ß-muricholic acids. The secondary bile acids
deoxycholic, lithocholic, hyodeoxycholic, omega-muricholic and
unsaturated omega-muricholic acids were also measured. Primary
phytosterols measured were 24ß-ethylcholesterol and
24ß-methylcholesterol. Secondary phytosterol metabolites detected were
24alpha-methylcoprostanol (campestanol), 24ß-methylcoprostanol,
24alpha-ethylcoprostanol (stigmastanol) and 24ß-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 µg/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 µg/ml
resistance +/- S-9^{1, 2}

Chromosome CHO cells. Salatrim 23CA Negative Hayes et al., 1994f
aberrations (in vitro) Metaphase analysis 0-1000 µg/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 µg/ml (or 1000 µg/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 ß-hydroxybutyrate level was documented in the 60 g salatrim/day
group using combined male and female data (0.2±0.10 mmol/litre,
compared to 0.1±0.06 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
ß-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:

kCAL_X = 3D (BWG_X - INT)

SLP × K_X)

where KCAL_X 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 K_X is the test fat added to the diet (grams per 100g of
diet) (Finley et al., 1994d). In fact, this equation is not
accurate; K_X 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.

6. REFERENCES

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pre-exposure to control Soybean oil followed by a 28 day exposure to
salatrim oils or control soybean oil followed by a 7 day exposure to
control soybean oil. Salatrims 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. understudy
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    See Also:
       Toxicological Abbreviations