SUCROSE ACETATE ISOBUTYRATE (SAIB)
Explanation
This food additive has been previously evaluated by the Joint
FAO/WHO Expert Committee on Food Additives in 1975 and 1977 (Annex I,
Refs. 37 and 43). However, no recommendation was made for an
acceptable daily intake by man. No toxicological monograph was
prepared. The available data are summarized and discussed in the
following monograph.
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Absorption, distribution and excretion
Male albino rats (Holtzman), about 250 g, were intubated with
14C SAIB in corn oil at dose levels equivalent to 27 or 100 mg/kg.
Forty-five to 50% of the dose was absorbed at the highest dose and
74-82% at the lower dose. Eighty-eight to 90% of the administered dose
was eliminated in 48 hours and 90-93% in 96 hours. The relative
proportion of radioactivity eliminated by the various routes varied
with the dose. At the high-dose level 55-56% of the absorbed activity
was eliminated as CO2 and 26-28% in the urine. At the lower dose
63-67% of the observed activity was eliminated in CO2 and 23-25% in
the urine. Only small amounts of the administered radioactivity were
retained in the body: 0.1-0.15% (at the high dose) and 0.35-0.38% (at
the low dose), with the activity being mainly in the urine. Four days
after administration of the test compounds the level of 14C
circulating in the whole blood was less than 0.1% of the dose at the
higher level and 0.3% at the lower level. Chromatography of extracts
of the 24-hour faeces of rats showed the presence of SAIB and other
metabolites. Most of the radioactivity in urine was in the form of
sucrose, although other unidentified substances were also present
(Fassett & Reynolds, 1962).
Female rats were administered 14C SAIB by oral intubation at a
dose level of 50 mg/kg. More than 90% of the radioactivity remained in
the gastrointestinal tract at 6 hours with approximately 60% in the
small intestine. Less than 30% of the 14C found in the small
intestine was present as SAIB, 4.9% of the 14C dose was excreted in
the urine, and 2.4% in the respired CO2. Twenty-four hours after
administration of the test compound, 90% of the 14C had been excreted
and less than 8% remained in the gastrointestinal tract. Fifty per
cent. of the 14C was excreted as CO2. Faecal material contained 33%
of 14C, of which 26% was in the form of unchanged SAIB. 14C
metabolites present in urine were similar to those obtained from urine
of rats given an equivalent dose of 14C sucrose (Phillips et al.,
1976).
Two rats were dosed orally with a single dose of 14C SAIB at
dose levels equivalent to 5.8 and 11.2 mg/kg; 59% and 52% of the
14C-labelled SAIB was respired as CO2, 11% and 13% in the urine,
and 23% and 27% in the faeces within 3 days. The major C compound in
the faeces was SAIB or highly acetylated sucrose molecules.
Chromatographic separation of urine showed 1 or 2 major peaks, which
were not identified. Six and 6.6% of the 14C was retained in the
carcass. However, no organ incorporated a significantly greater
amount than the average. Rats dosed orally with a single dose of
14C-labelled sucrose at a level of 400 mg/kg showed rapid absorption
and metabolism of the sucrose to 14CO2, the maximum output being
observed 2 hours post-dosing. Only small amounts of 14C were
eliminated in the urine and faeces. At sacrifice (3 days post-dosing)
the carcass retained 9.6-12.9% of the 14C label. The level in fat was
somewhat higher than in other tissues (Reynolds et al., 1974).
Metabolism
Homogenates of the liver and small intestinal mucosa of rats
prepared in Krebs-Ringer of phosphate buffer (pH 7.4) were incubated
with 14C SAIB or 14C sucrose. At 0, 1, 2 and 4 hours, portions were
removed and assayed for metabolites. The rate and extent of hydrolysis
of SAIB was less than that of the gum mucosal homogenate, and the rate
and extent of hydrolysis decreased with increasing concentrations of
SAIB. Sucrose was readily hydrolysed by the mucosal preparation, but
hardly at all by the liver preparation (Phillips et al., 1976).
14C-labelled SAIB was incubated under aerobic conditions with
preparations derived from the contents of 3 regions of the rat gut,
namely, stomach, small intestine and the caecum. Preparations from the
proximal region of the small intestine showed the greatest hydrolytic
activity, the activity of the caecal contents was less than that of
the small intestine, and the stomach contents showed no hydrolytic
activity (Phillips et al., 1976).
A study of disappearance of radioactivity from loops of the small
intestine of rats after injection of 14C sucrose and 14C SAIB, after
1-hour absorptive periods, showed that sucrose was rapidly cleared
from the intestine, whereas the rate of removal of SAIB activity was
very slow, less than 13% in 1 hour (Phillips et al., 1976).
Two dogs were fed a single dose of 3 or 4.9 mg/kg of 14C SAIB.
Twenty-eight and 27% of the 14C-labelled SAIB was eliminated as
14CO2, 7% and 5% in the urine, and 53% and 46% in the faeces
within 7 days. The major 14C compound in the tissues was SAIB.
Chromatography of the urine showed the presence of one major 14C peak
as well as a large number of other compounds. Some of the 14C
appeared to be associated with unchanged SAIB (Reynolds et al., 1974).
Dogs and rats were dosed with a single dose of 14C SAIB. The
rats eliminated 7-10% of the dose in the urine in 30-46 hours and the
dogs 2.8-5.2% of the dose in the urine in 29-30 hours. Size exclusion
chromatography of the dog and rat urine showed that 14C-labelled
molecules larger than sucrose were not present to any significant
extent. The nature of the metabolites were not determined, although
sucrose, glucose and fructose appeared to be absent (Reynolds &
Zeigler, 1977).
In another study, the bile ducts of 3 rats and 1 dog were
cannulated, and the bile was collected following single per os doses
of 14C SAIB. The rats eliminated 4.5% of the administered dose in the
bile, within 15 hours post-dosing. Chromatographic separation of the
metabolites present in the bile showed that they had properties
similar to sucrose or sucrose with a few acyl groups attached. In the
case of the dog, which was subjected to 3 separate trials, about 6% of
the dose was eliminated in the bile, within 15 hours post-dosing.
Separation of the metabolites showed the presence of SAIB or highly
acetylated sucrose (Reynolds et al., 1975).
TOXICOLOGICAL STUDIES
Special studies on reproduction
Groups each of 20 Sprague-Dawley strain rats, equally divided by
sex, were maintained on diets containing 0, 0.38 and 9.38% w/w SAIB.
During weeks 9-36 of the study the rats were bred 3 times. Pairs of
rats were selected within each group (10 pairs) and caged together for
19 days, after which the male was removed. Females were allowed to
rear young to weaning at 21 days. Litters were weighed at days 1, 11
and 21 post-partum. All pups were sacrificed at 21 days, sexed and
examined for gross abnormalities. The parent rats were bred 3 times,
each female receiving a different male at each mating. Reproductive
performance was based on number of pups born, conception rate, pups
per litter, pups weaned, as well as weight of pups on days 1, 11 and
21. Reproductive performance as judged from the data presented was
slightly better in the 0.38% group than control. At the 9.38% level,
less females became pregnant, fewer pups survived to weaning in the
first breeding, and more survived to weaning in the third breeding
(Harper et al., 1966).
Groups each of 15 female and 5 male rats (Holtzman), 60 days of
age, were maintained on diets containing 0 or 5% SAIB. These diets
were fed to parents and their offspring throughout the study. After 1
month on the diet, rats were regrouped, 1 male and 3 females/cage.
Pregnant females were then housed separately. The length of gestation,
number of young and live young, number of young weaned and average
weight at weaning and post-weaning were parameters recorded for
assessing reproductive performance. Fifty-one days after last
parturition, another mating was attempted. Progeny of the first
breeding (F1) were also bred. The parent generation grew well on the
5% SAIB diet. The first breeding of the test parent generation was
equal to or superior to controls, in the parameters measured. At the
second breeding, a somewhat lower percentage of pups from the treated
groups was reared from birth to weaning compared to controls, and
those surviving 2 weeks post-weaning weighed less than control. The
breeding of the F1 generation appeared satisfactory. However, because
of an outbreak of respiratory disease, many parents and pups died
during the test. None of the pups from the SAIB group survived post-
weaning (Fassett et al., 1965).
Dominant lethal assay in the rat
Groups each of 20 male rats (Sprague-Dawley) were administered by
gavage a single dose of either SAIB in corn oil at a dose level
equivalent to 20, 200 or 2000 mg/kg, Apholate (positive control) at a
dose level equivalent to 25 mg/kg, or corn oil alone. Two hours after
treatment, the males were mated with 2 untreated virgin females for
the following week. Subsequent matings using naive females were
carried out during the third, fifth and seventh weeks of treatment,
corresponding to the post-meiotic, meiotic and pre-meiotic stages of
spermatogenesis. Twelve to 14 days from the mid-week of the
presumptive mating the females were sacrificed. The uteri were exposed
and total implantation sites counted, and classified as viable
foetuses, early deaths or late deaths. The indices evaluated included
the insemination index (number of males that inseminated a
female/number of males per group × 100), the pregnancy index (number
of pregnant females/number of females mated × 100), the implantation
index (number of implantation sites/total number of corpora
lutea × 100), the mean total implantation sites and the early deaths
per pregnancy. Seventy-two hours after treatment, blood was collected
from 5 males of each group for leucocyte culture for cytogenetic
studies. Ten metaphases per rat were examined and scored for
chromosome number, achromatic gaps, chromatid deletion or exchanges,
chromosome deletions or exchanges and miscellaneous aberrations.
The mean implants per pregnancy of the high dose of SAIB
(2000 mg/kg) and the positive control group were statistically lower
than control groups during the third and seventh weeks of mating. In
the case of the positive control, this corresponded with a significant
increase in early foetal deaths during the third mating week, but not
during the seventh week. None of the SAIB groups had significantly
increased early deaths during any of the mating periods. Some of the
groups had statistically lower mean implants than the respective
controls, but none of these were fewer than the lower limit of 8 per
pregnancy in mice. The insemination index and pregnancy index of test
groups were not significantly different from control. The cytogenicity
study of the cultured leucocytes showed that there was no significant
increase in chromosome numbers or aberration in any of the groups
(Krasavage, 1973).
Special studies on the effects of SAIB on bromosulfophthalein plasma
clearance
Rat
Groups of 5 male Wistar rats were maintained on diets containing
4% SAIB for 7 days. Bromosulfophthalein clearance was measured at 0
(pretreatment) and 24 and 48 hours following withdrawal from the
treated feed. SAIB had no effect on bromosulfophthalein clearance
(Procter & Chappel, 1971).
Dog
Two male and 2 female beagle dogs were fed diets containing 0.1,
0.3 or 0.5% SAIB. All 4 animals were tested for bromosulfophthalein
clearance 24 hours after last feeding of SAIB and again after 48
hours. A rest period of 1 week was allowed between each dietary level.
No bromosulfophthalein retention occurred at the 0.1% dietary SAIB
level; 0.3% and 0.5% SAIB resulted in distinct but reversible
bromosulfophthalein retention (Procter & Chappel, 1971).
Monkey
Two groups each of 3 male squirrel monkeys (Saimiri sciureus)
were fasted overnight and then were treated either with SAIB (1 g in
2 ml cotton-seed oil) or cotton-seed oil. Twenty-four hours after
treatment the monkeys were tested for bromosulfophthalein clearance.
Following a 7-day rest period, the treatment of the groups was
reversed. Clearance appeared normal in 2/3 of the animals in each
group (Procter & Chappel, 1970).
Special studies on the effects of SAIB on indocyanine clearance
Groups of male rats (Sprague-Dawley) were fed diets containing 4%
SAIB and 5% corn oil, or 5% corn oil. Indocyanine green (ICG)
clearance was determined on 2 rats from each group on days 1, 3, 5, 8,
10, 22, 26 and 36 of the study. The ICG plasma clearance rates in rats
from the SAIB group was not significantly different from controls
(Krasavage et al., 1973).
Acute toxicity
LD50
Animal Route (g/kg bw) Reference
Mouse Oral 2.5 Fassett & Reynolds, 1962
i.p. 2.5 Fassett & Reynolds, 1962
Rat Oral 2.5 Fassett & Reynolds, 1962
i.p. 2.5 Fassett & Reynolds, 1962
Short-term studies
Rat
Groups each of 50 albino rats (Holtzman), equally divided by sex,
were maintained on diets containing 0, 1.0 or 5.0% SAIB (w/w) for a
period of 95 days. Haematological studies including haemoglobin, cell
volume, leucocyte and differential counts were carried out at days 24,
52 and 87 of the study. Body weight and food intake was determined
weekly. The animals were necropsied on day 95 and liver and kidney
weights recorded and a complete histological study made of the tissues
and organs (liver, adrenal, kidney, urinary bladder, spleen, stomach,
small intestine, caecum, heart, cerebrum, cerebellum, bone marrow,
lung, gonad). There was a slight reduction in weight gain in the males
fed 5% SAIB and a slight liver weight increase in the females fed 5%.
No compound-related histopathological changes were observed (Fassett
et al., 1962).
Four groups each of 20 Sprague-Dawley rats (10 males, 10
females), body weight 85-100 g, were maintained on diets containing 0,
0.30, 1.80 or 9.12% SAIB for 13 weeks. The SAIB was dissolved in
vegetable oil and the final concentration of vegetable oil added to
all diets was 9.30%. Body weights were determined weekly. Prior to
sacrifice at 13 weeks, blood samples were taken and Hb content and
total WBC and differential cell count determined. At autopsy absolute
and relative organ weights were determined for liver, kidneys, lungs,
testes, spleen and heart. A microscopic examination was made of liver,
kidneys, gonads, spleen, adrenals, bone marrow, heart, jejunum, caecum
and ileum. At the highest test level there was occasional diarrhoea.
However, there were no significant differences in weight gain between
control and test animals. Organ weight, blood chemistry and
histopathology were similar in all groups and showed no compound-
related effects (Hunt, 1964).
Groups each of 80 rats (Sprague-Dawley) equally divided by sex
were maintained on diets containing 0, 2.5, 5.0 and 10% SAIB. Another
group was administered daily by gavage phenobarbital at a dose
equivalent to 100 mg/kg bw. This group was positive control for study
of liver enlargement and microsomal enzyme induction. Each group was
divided into 2 series (A and B), group A being treated for 6 weeks and
group B for 12 weeks. Series A and B were further subdivided into
subgroups of 10 animals of each sex. One subgroup "2" was subjected to
the Zoxazolamine muscle test following 6 and 12 weeks on the test
diet, and then 4 weeks after removal from the test diet. The
other subgroup "E" was subjected to extensive histological and 2
biochemical studies, which included ornithine carbamoyl transferase,
carboxylesterase and proximal liver analysis (protein, glycogen, lipid
and water). Dietary SAIB had no significant effect on the weight gain
of test animals except at the 2.5% level, where there was slight
decrease in weight gain of males in the A series and both sexes in the
B series. There was a marked reduction of weight gain in animals
treated with phenobarbital. All animals subjected to the Zoxazolamine
test showed decreased weight gain for 1 week after the test. In
general, food consumption in the SAIB groups was not affected,
although phenobarbital-treated groups showed decreased food intake.
Urinary excretion of ascorbic acid was measured in A and B groups at
weekly intervals and showed that, whereas phenobarbital administration
resulted in a marked and prolonged elevation of urinary excretion of
ascorbic acid, dietary SAIB had no effect on this parameter. The
Zoxazolamine muscle relaxant test showed that, whereas the
phenobarbital treatment caused a very marked reduction in the effect
of the test compound, dietary SAIB had no effect, suggesting that SAIB
does not induce microsomal enzymes, that would metabolize
Zoxazolamine. At autopsy, absolute and relative organ/body weight of
the adrenals, heart, kidney and liver were determined for test and
control animals. There were no significant compound-related effects in
the SAIB-treated animals. The animals administered phenobarbital
showed enlarged livers. However, this effect was reversible in males,
but not in females, following a 4-week withdrawal period. Gross
pathological and histopathological examination of SAIB-treated animals
did not show any compound-related effects. The biochemical studies of
SAIB-fed animals showed increased glycogen and water content but no
increase in carboxylesterase levels. In contrast, the livers of the
phenobarbital-treated rats showed a significant reduction in glycogen
and water content, an increase in lipid content, and a marked increase
in carboxylesterase activity (Procter et al., 1971a).
In another series of studies, rats (Sprague-Dawley) were divided
into 14 treatment groups with 10 animals of each sex per group and fed
diets containing SAIB at dietary levels of 1.0, 2.0 and 4.0% (w/w)
supplemented with 5% corn oil (w/w). The groups were fed the test
diets for 28 and 56 days continuously or for 28 days followed or
preceded by 28 days on control diet. Two groups of each sex were fed
the control diet containing corn oil only for 28 or 56 days
continuously. At the termination of the study, animals were sacrificed
and blood collected for determination of serum alkaline phosphatase
(SAP), ornithine carbamoyl transferase (OCT), blood urea nitrogen
(BUN), trigylceride, cholesterol and glucose. The animals were
autopsied, examined for gross pathology and liver weight and relative
liver/body weight determined. Liver microsomal enzyme activity
(p-nitroanisole demethylase), glucose-6-phosphatase and
bilirubin-ß-D-glucuranyl transferase were determined. Histological
studies were made of the liver tissue. Weight gain and feed
consumption of test and control groups were similar. There were no
significant changes in the serum chemistry of the test animals. Gross
pathology at autopsy was negative and liver weights (absolute and
relative) were similar for test and control. Microsomal enzyme
activity was similar for test and control animals, with the exception
of glucose-6-phosphatase which depressed in male rats on the 4% SAIB
diet (Krasavage et al., 1973).
Dog
Three test groups, each of 8 pure-bred beagles (4 males, 4
females), were maintained on diets containing 0.2, 0.6 and 2.0% SAIB,
dissolved in cotton-seed oil. The control group consisted of 12 pure
bred beagles (6 males, 6 females). The total fat content of the diets
was adjusted to 12% by addition of cotton-seed oil.
Physical and clinical examinations were made twice prior to
commencement of the feeding study and then at week 12. The clinical
examinations included haemoglobin concentration, haematocrit, WBC and
differential cell count, blood glucose, BUN, SAP and LDH, and urine
glucose, albumin, ketone products, pH, specific gravity, and sediment.
Neurological reflexes were tested and body weight and food intake
recorded weekly. At the termination of the study (end of twelfth
week), all dogs were sacrificed and autopsied. Organ and relative
organ weights were determined for liver, kidneys, spleen, gonads,
adrenals, pituitary and brain. The following tissues from control and
2% level group dogs were examined microscopically: liver, spleen,
stomach, small and large intestines, pancreas, kidney, bladder,
adrenals, gonads, thyroid, pituitary, thymus, salivary gland, lymph
nodes, heart, lungs, marrow, aorta, muscle, spinal cord, brain and
gall bladder. The liver and kidneys of dogs in the 0.2 and 0.6% SAIB
groups were also examined microscopically.
There was no significant compound-related effect on food intake
and weight gain. Haematological and urine parameters of test animals
and controls were similar and within normal values. Serum chemistry
indicated a significant increase in SAP of the dogs in the 2% group
(approximately two times increase over base level). At autopsy there
was a marked increase in relative and absolute liver weights in the
0.6% and 2% groups when compared with controls; all other organ
weights were normal. No compound-related histopathology was observed
(Anon., 1965).
In another study, groups each of 12 beagle dogs (equally divided
by sex) were fed diets containing SAIB at 0, 0.5, 1.0, 2.0 and 4.0%
for a period of 12 weeks. Test animals on the 4.0% were maintained a
further 3 weeks on an SAIB-free diet. Body weight and food intake
were determined during the course of the study. Fasting blood samples
were obtained at weeks 4, 8 and 12 of the test and blood biochemical
tests, including blood urea nitrogen, serum glutamic pyruvic
transaminase, serum glutamic oxalacetic transaminase, alkaline
phosphatase, lactic dehydrogenase, serum bilirubin, fasting serum
glucose, bromosulfophthalein and phenosulfophthalein clearance,
serum electrolytes, total serum protein, albumin and globulin.
Bromosulfophthalein plasma disappearance curves and curves for the
plasma clearance of indocyanine green were determined at week 12 for
male dogs in the 0 and 4.0% groups. Bromosulfophthalein clearance was
measured in the 4.0% group during the 3-week withdrawal period. In
addition to these extensive biochemical tests, haematological analysis
and urinalysis were carried out on weeks 4, 8 and 12 of the study. At
the end of the test period, the animals were sacrificed and, following
completion of gross pathological examination, absolute and relative
organ body weights determined for the brain, heart, liver, lung,
kidneys, adrenals, gonads, prostate, uterus, pituitary, spleen and
thyroid. A microscopic examination was made of samples of heart,
liver, kidneys, adrenals, small and large intestines. Histochemical
studies were carried out on liver sections of dogs from the 0.5, 1
and 2% groups, and included succinate dehydrogenase, phosphorylase,
glucose-6-phosphate dehydrogenase, glucogen, acid phosphatase,
alkaline phosphatase, adenosine triphosphatase, and Masson's
trichrome. Additional samples of liver from test animals (not
including the 5% group) were analysed for protein, glycogen, lipid and
water, and carboxylesterase activity was also measured. Serum
ornothine carbomyl transferase was measured in serum samples obtained
terminally. Electron microscopic studies were carried out on liver
samples from animals in all groups except the 4.0% group.
Daily clinical observation revealed no treatment change.
Growth and food intake appeared normal in all groups. Urine and
haematological analysis were similar in test and control groups.
Marked bromosulfophthalein (BSP) retention occurred among all test
animals during the experimental period, but was not dose related.
Increased serum alkaline phosphatase values were directly related to
dose level and period of exposure. Other parameters were not
significantly different in test and control animals. The marked
increase in BSP retention was reversed within 3 weeks when the test
animals in the 4% group were maintained on an SAIB-free diet.
Indocyanine green clearance rates were reduced in a manner paralleling
the changes observed in the BSP tests. Male dogs treated with SAIB
showed a dose-dependent increase in liver weight. No liver enlargement
was observed in the female dogs. Histochemical studies of liver
sections did not reveal any changes in the metabolic or substrate
content of the parenchymal cells of the liver; however, there was a
marked increase in enzyme activity (alkaline phosphatase, adenosine
triphosphatase and glucose-6-phosphate dehydrogenase) of the bile
canaliculi of treated animals when compared with controls. There was a
slight but statistically significant reduction of protein content of
liver and slight increase in liver glycogen. Liver lipid was slightly
increased at the 2% level. An increase in liver carboxylesterase was
observed in the test groups, particularly in the males, but the effect
was not dose related. Ornithine carbomyl transferase values were
similar for test and control animals and within normal range. Light
microscopic examination of the liver from treated males showed that
the liver cells were hypertrophied, the bile canaliculi appeared to be
slightly dilated and the number of bile pigment granules increased.
Electron microscopic evaluation of the hepatocytes of treated dogs
showed various changes, the most consistent being an increase in
smooth endoplasmic reticulum (SER). The effect was observed in both
treated males and females, but the effect was most pronounced in the
males. Other changes included pronounced microvillous patterns,
prominent Golgi bodies and an increased number of microbodies in the
intracellular pigment granules (Procter et al., 1970).
In a parallel study, a group of 8 beagle dogs (equally divided by
sex) were fed SAIB at a dietary level of 2.0% and then maintained for
6 weeks on an SAIB-free diet prior to sacrifice. In this study the
dietary SAIB caused a slight weight depression that was reversed on
removal of SAIB from the diet. The 18-week values for serum alkaline
phosphatase and for serum bromosulfophthalein clearance showed that
the effect of SAIB on liver function was fully reversed during the
6-week withdrawal period. At autopsy, organ weight studies indicated
that the liver enlargement reported in the previous study was reversed
following the 6-week withdrawal period. The histochemical studies
showed that the findings reported for the groups on test compound
without withdrawal (high activity of bile canaliculi for alkaline
phosphatase, adenosine triphosphatase and glucose-6-phosphate
dehydrogenase) were fully reversible. The effect of SAIB
administration of liver carboxylesterase was also fully reversible
during this withdrawal period. Electron microscopic examination of the
liver indicated that the cellular morphology was completely normal,
following removal of the SAIB from the diet (Procter et al., 1971,
1973).
Six male beagle dogs approximately 6 years old were fed a control
diet containing 5% (w/w) corn oil for 3 weeks and then an experimental
diet containing 5% SAIB for 28 days. The control diet was fed for the
next 57 days, 4 of the dogs were returned to the SAIB diet, and
indocyanine green (ICG) plasma clearance rates and serum alkaline
phosphatase determined 24 and 48 hours later. The study was terminated
3 days later. Haematocrits, haemoglobins, white cell and differential
counts, SGOT, blood glucose, BUN, serum protein, SAP, OCT, and
triglyceride and cholesterol determinations were made twice prior to
and at weekly intervals throughout the feeding study. At the
termination of the study, all dogs were sacrificed, absolute and
relative liver and kidney weights determined, and tissues from all
organs examined microscopically. Dogs on diets containing 5% SAIB
showed a moderate increase in SAP and a prolongation of ICG plasma
clearance by the liver. Within 5 weeks of withdrawal of SAIB from the
diet, SAP activity was near normal. The ICG clearance rate appeared
within normal range 2 weeks after withdrawal of SAIB from the diet.
After feeding control diet for 8 weeks, the 4 dogs returned to
SAIB-containing diets for 24 hours showed a significant prolongation
of ICG clearance rate, but SAP did not appear to be increased. All
other parameters of test and control animals were similar (Krasavage
et al., 1973).
In another study, groups each of 5 male beagle dogs (11-13 months
of age) were fed diets containing 5.0% SAIB, plus 5% corn oil, or
corn oil alone for 93 days. Physical appearance, behaviour, food
consumption and body weight were determined daily throughout the
study. Indocyanine green plasma clearance rates were determined at
3-week intervals. Serum bilirubin was measured at week 7 of the study
and haematological and blood chemistry (haematocrits, haemoglobin,
BUN, serum protein, SAP and°CT, triglycerides and cholesterol) studies
were carried out at the termination of the study. All dogs were
autopsied at the termination of the study, liver and kidney weight
determined, and all tissues examined microscopically. Liver tissues
was analysed and glycogen, protein and phospholipid content, and
samples assayed for microsomal enzyme activity (p-nitroanisole
demethylase, glucose-6-phosphates and bilirubin-ß-D-glycuranyl
transferase). Liver, kidney, bone, bile and scrapings of the
intestinal mucosa were analysed for tissue alkaline phosphatase
activity. Dogs fed SAIB showed a slight increase in SAP, as well as a
prolonged indocyanine green clearance time, increased relative and
absolute liver weight. Liver glycogen and phospholipid content were
increased while liver protein was decreased. Disk electrophoresis and
isoenzyme inactivation studies of tissue alkaline phosphatase
indicated that the elevation of SAP was related to the liver
isoenzyme. The liver content of alkaline phosphatase in SAIB-fed
animals was twice that of controls. All other parameters studied were
similar in test and control animals (Krasavage et al., 1973).
Long-term studies
Rat
Groups each of 20 (10 male, 10 female) Sprague-Dawley strain rats
were maintained on diets containing 0, 0.38 or 9.38% (w/w) SAIB, for
104 weeks. During this period rats were bred on 3 successive
occasions. Rats were housed 5 per cage. Body weight and food intake
were measured weekly. Animals dying during test period were autopsied.
All animals surviving 104 weeks were sacrificed and autopsied.
Relative and absolute organ weights were determined for heart,
kidneys, liver, lungs, ovaries, spleen and testes. Histological
studies were made of the following tissues from rats of control and
9.38% SAIB group: adrenals, aorta, colon, heart, illeus, kidneys,
liver, lungs, lymph node, ovaries, pancreas, skeletal muscle, spleen,
stomach, testes, thyroid, urinary bladder and uterus.
SAIB in the diet did not increase the number of mortalities
(13/20 control, 14/22 9.38% and 15/20 0.38% groups). However, 4 males
in the 9.38% SAIB group died during the first 10 weeks. Autopsy
revealed massive multiple haemorrhage in each case. Subsequent
measurement of systolic pressure in surviving males failed to
demonstrate any inter-group difference. There were some differences in
food intake and body weight between the various groups at various
stages of the study.
There were no significant differences in body weight of rats of
the various groups at the end of the first year; however, during the
second year, male survivors receiving test substance were heavier than
respective controls. At termination of the study, organ weight and
relative organ weight of the various groups were comparable with
respective controls with the exception of the relative organ/body
weight of the liver and kidney of male rats of the 9.38% group, which
were lower than controls. Histological studies did not reveal any
compound-related lesions (Harper et al., 1966).
OBSERVATIONS IN MAN
Three groups each of 8 healthy adults (equally divided by sex)
were administered 0 or 7.0 mg/kg bw or 20 mg/kg bw SAIB as a single
dose dissolved in a fruit drink, daily for a period of 14 days. Post-
and pre-treatment measurements of bromosulfophthalein retention as
well as pre-, mid- and post-treatment clinical chemistry (serum SAP,
SGOT, SGPT, LDH, total protein, albumin, calcium, cholesterol,
glucose, BUN, uric acid, total bilirubin, and A/G ratio), haematology
(haemoglobin, haematocrit, sediment rate, red and white blood cell
counts, differential and platelets) and urinalysis showed no
measurable compound-related effects.
A male adult was given a single dose of 14C SAIB at a dose level
of 1.18 mg/kg bw. Samples of urine were collected before dosing and at
0 and 6.2 hours post-dosing, and subjected to various chromatographic
procedures. Glucose, fructose and the esters of fructose and sucrose
were not present in the urine. Two unidentified peaks were considered
to be the principal metabolites of SAIB (Reynolds & Zeigler, 1977).
When 14C SAIB was incubated with human faecal homogenates less
than 2% in the SAIB was hydrolysed to sucrose in a 20-hour period.
Hydrolysis of SAIB by suspensions of human gut bacteria was also very
low (Phillips et al., 1976).
Two male subjects were given SAIB (100 mg and 1 g) as a single
dose. The urinary excretion of sucrose and sucrose esters was less
than the limit of detection of the assay procedure used (1 ppm
(0.0001%) sucrose) in any 24-hour period up to 5 days post-dosing. In
another study, 2 male subjects were given 1 g of SAIB/day for 7 days.
No urinary excretion of sucrose was detected. No unchanged SAIB or
metabolites were detected in faecal sample of 1 subject given 100 mg
of SAIB daily for 7 days. Two subjects were given sucrose
intravenously (100, 250 and 800 mg in a 10% solution w/v) and the
urine collected at 3, 12 and 24 hours. Approximately 50% of the
administered compound was recovered in the urine by 3 hours at all 3
dose levels, and there was almost quantitative recovery of the lower
dose by 12 hours (Phillips et al., 1976).
14C SAIB was incorporated into a simulated non-carbonated soft
drink and administered to 3 male subjects. The 3 subjects were each
given 2 or 3 single doses at widely spaced intervals. The first dose
was administered at a level of ca. 1 mg/kg to each subject, none of
which had been previously exposed to SAIB. Two of these subjects were
given a second dose at the same level 7-27 weeks after the first dose
and following ingestion of unlabelled SAIB at a level of 1 mg/kg for
7 days. The third subject received a single dose at a level of
0.18 mg/kg 25 weeks after the first dose. One subject was given a
third dose at a level of 1 mg/kg 10 weeks after the second dose and
immediately after ingestion of a high fat meal. All subjects were
monitored for elimination of radioactivity in the breath and urine for
at least 30 days post-dosing. A small portion of the radioactivity was
reportedly eliminated in the urine, the maximum rate of urinary
excretion occurring within 3 hours and then falling off to 0.5-0.82%
of the dose/day by 48 hours. Fourteen to 21% of the dose was excreted
in the urine. Elimination of radioactivity in the breath occurred
rapidly. Only a small amount of the total dose was eliminated in the
6-8-hour period post-dosing, but reached maximum levels of elimination
9-15 hours post-dosing. Forty-four to 66% of the dose was excreted as
CO2 in 25 days. A small portion of the dose was unabsorbed and
appeared in the faeces of all subjects. Prior dosing with SAIB or 14C
SAIB had no effect on the elimination pattern. Chromatographic studies
of urine showed several radioactive peaks, which have not been clearly
identified; however, the amount present as free sucrose was estimated
to be 20% of the radioactivity. Chromatographic studies of extracts of
faeces showed a presence of a band of radioactive materials that did
not correspond with those in the urine. In another study, two subjects
ingested 14C sucrose at a level of 400 mg/kg. Forty-one to 59% of the
14C sucrose was metabolized to 14CO2 within 48 hours, the maximum
rate of elimination occurring 3 hours post-dosing. Both subjects
eliminated small amounts of 14C in the urine (1.9% and 1.7% of the
dose). Most of the radioactivity appeared to be in urea 14C (Reynolds
et al., 1972).
Twenty subjects (10 male and 10 female) between 18 and 22 years
of age ingested a daily dose of SAIB at a level equivalent to 1 mg/kg
bw for a period of 14 days. The dose was taken as a bolus each
morning. Determination of the following blood parameters were made
prior to treatment and at days 7 and 18 of the test: SGOT, SGPT, serum
alkaline phosphatase, serum bilirubin, total protein, albumin, uric
acid, blood urea nitrogen, also erythrocyte sedimentation rate. There
were no significant differences in any parameters for any 1 individual
(Hensley, 1973).
Human subjects were administered a single oral dose of 14C SAIB
(101.2 mg/kg bw). Forty-one to 66% of the administered 14C dose was
eliminated as CO2, and 15-21% in the urine in a 25-30-day period and
10% in the faeces in a 5-day period. The major 14C constituent in the
faeces was unchanged SAIB. Chromatography of the urine showed two
major 14C peaks, similar to that observed with the rat (Reynolds et
al., 1974).
Comments
At this time the metabolism of SAIB in experimental animals (dog
and rat) and man has not been clearly defined. SAIB undergoes partial
degradation in the gastrointestinal tract and the products of
hydrolysis following absorption may be eliminated in the bile and
urine or further metabolized to CO2. Some partially degraded SAIB and
undegraded SAIB is eliminated in the faeces. Hydrolysis of SAIB occurs
as a result of the action of enzymes (non-specific esterase) in the
gastrointestinal tract, rather than the action of gastrointestinal
flora. Examination of urinary metabolites of SAIB in rat and dog have
not shown major qualitative differences; however, the chemical nature
of the metabolites is still not defined. Metabolites in the urine of
man have not been identified, but are not glucose, sucrose or esters
of sucrose and fructose. Studies of the metabolites eliminated in the
bile of rats and dogs following administration of 14C-labelled SAIB
indicate species differences. In the case of the rat, the metabolites
have chromatographic properties similar to sucrose or sucrose with a
few acyl groups attached, whereas, in the case of the dog, metabolites
were similar to SAIB or highly acylated sucrose.
Short-term and chronic feeding studies are available for the rat.
At the highest dose level fed (9.38%), a number of males died during
the first 10 weeks of the study as a result of massive multiple
haemorrhage. Subsequent measurement of systolic pressure in surviving
males failed to demonstrate any inter-group differences. The study
failed to reveal any compound-related histology. In the case of the
dog, 12 weeks of exposure to dietary levels of 0.6% or 2% SAIB
resulted in a significant increase in SAP (serum alkaline
phosphatase), as well as relative liver weights. The liver changes in
the dog included increased liver weight and serum alkaline
phosphatase, as well as changes in liver function as shown by
bromosulfophthalein (BSP) retention, and indocyanine green clearance
tests were dose dependent and occurred in the male but not in female
dogs. The changes were readily reversible when the test animals were
maintained on an SAIB-free diet. Changes in indocyanine green
clearance tests were not observed when rats, monkeys or man were dosed
with SAIB. It is not known if this species difference is related to
possible differences in metabolism of SAIB, as has been suggested by
examination of SAIB metabolites in the bile of rats and dogs. Although
the effect observed in the dog appears to be readily reversible
following a relatively short period of exposure to SAIB, it is not
known if continuous exposure to SAIB for longer periods will result in
development of a pathological lesion. The available data on
reproduction studies are inadequate because in 1 study there was a
loss of parents and pups due to respiratory infection and, in another
study, at the highest dose level fed (9.38%), less females became
pregnant than controls, fewer pups survived to weaning in the first
breeding, and none survived to weaning in the third breeding.
Evaluation of the carcinogenic potential of SAIB is limited
because only 1 chronic study in rats is available. No other rodent
species has been tested at this time.
The additional studies required by the Committee in 1977 are not
available.
EVALUATION
Further work is required before an acceptable daily intake for
man can be allocated.
Estimate of acceptable daily intake for man
Not allocated.
FURTHER WORK OR INFORMATION
Required before an ADI can be allocated
(1) A carcinogenicity/toxicity study in 2 animal species.
(2) A 2-year dog study with an adequate number of animals and groups
to demonstrate a no-effect level.
(3) A multigeneration reproduction/teratology study.
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See Also:
Toxicological Abbreviations