797. Sucrose acetate isobutyrate (WHO Food Additives Series 32)

SUCROSE ACETATE ISOBUTYRATE

First draft prepared by
Ms E. Vavasour
Toxicological Evaluation Division
Bureau of Chemical Safety, Food Directorate
Health and Welfare Canada
Ottawa, Ontario, Canada

1. EXPLANATION

Sucrose acetate isobutyrate, which is a mixture of esters of
sucrose esterified with acetic and isobutyric acids, was evaluated
at the nineteenth, twenty-first and twenty-sixth meetings of the
Committee (Annex 1, references 38, 44 and 59). At its twenty-first
meeting, the Committee concluded that a complete toxicological
profile was required for the evaluation of this compound, including
carcinogenicity/toxicity studies in two animal species, a 2-year
study in dogs with adequate numbers and dose groups to demonstrate a
no-effect level and to assess the adverse effects of the substance
on liver function, and a multigeneration reproduction/teratogenicity
study. This information was not yet available when the compound was
again reviewed at the twenty-sixth meeting of the Committee, so no
ADI was allocated, although a toxicological monograph summarizing
the available toxicological data was prepared (Annex 1, reference
60). With the exception of the 2-year study in dogs, the requested
data were available for consideration at the present meeting. The
new data that have been made available as well as relevant studies
from the previous monograph are summarized in this monograph.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.1.1 Absorption, distribution, and excretion

2.1.1.1 Rats

Male albino Holtzman rats, about 250 g, were intubated with
¹⁴C-SAIB of specific activity 0.411 µCi/mg (all ¹⁴C-SAIB used this
and the following studies was labelled on the sucrose portion of the
molecule) in corn oil at dose levels equivalent to 27 or 100 mg/kg
bw. The proportion of the administered dose absorbed from the GI
tract was greater at the low dose (74-82%) than at the high dose
(45-50%). Elimination of 88 to 90% of the administered dose
occurred in 48 hours. The relative proportion of radioactivity
eliminated by the various routes varied with the dose. At the high
dose level, 54-56% of the absorbed activity was eliminated as CO₂
and 26-28% in the urine. At the lower dose, 63-67% of the absorbed
activity was eliminated in CO₂ and 23-25% in the urine. Four days
after the administration of the test compound, less than 1% of the
administered radioactivity was retained in the gastrointestinal
tract (from cardiac valve to rectum), blood, liver and kidney.
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 and Reynolds 1962; Reynolds
1972a; Reynolds et al. 1974).

Using the same protocol as in the previous study, male albino
Holtzman rats weighing approximately 250 g received 100 mg/kg bw of
¹⁴C-SAIB (specific activity, 0.38 µCi/mg) in corn oil by gavage.
At termination of the study, 3 or 3.5 hours after dosing, 78-84% of
the radioactivity was recovered from the gastrointestinal contents.
An additional 7-9% of the radioactivity was recovered from the
stomach, intestinal and caecal tissues. Less than 4% of the
radioactivity was excreted in the breath, urine and faeces within
3-3.5 hours after dosing, indicating that little absorption had
taken place. Extracts of the gastrointestinal contents and organs
were found to contain sucrose and partially acylated sucrose esters
in addition to unchanged SAIB (Reynolds 1963).

Two rats were given a single oral dose of ¹⁴C-SAIB (0.36
µCi/mg) in aqueous emulsion at levels equivalent to 5.8 and 11.2
mg/kg bw. Within 3 days, 59 and 52% of the ¹⁴C-labelled SAIB were
recovered in breath as respired CO₂, 11 and 13% recovered in the
urine, and 23 and 27% recovered in the faeces. The rats retained 6
and 6.6% of the ¹⁴C in the carcass; the distribution of
radioactivity among the organs was comparable. Total absorption of
administered SAIB was 71 and 77% of the administered dose when
recovery of label from urine, expired air and whole carcass was

combined. The major ¹⁴C compounds in the faeces were SAIB or
highly acylated sucrose molecules. Chromatographic separation of
urine showed 1 or 2 major peaks, which were not identified. In
comparison, rats given a single oral dose of ¹⁴C-labelled sucrose
in aqueous solution at a level of 400 mg/kg bw showed rapid
absorption and metabolism of the sucrose to ¹⁴CO₂, the maximum
rate of elimination 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. Distribution of radioactivity in the carcass was
similar for rats treated with SAIB or with sucrose. No accumulation
of radioactivity in a particular organ was observed (Reynolds 1972b;
Reynolds et al. 1974).

Female rats received ¹⁴C-SAIB (specific activity 1.0 µCi/mg)
by oral intubation at a dose level of 50 mg/kg bw. More than 90% of
the radioactivity remained in the gastrointestinal tract at 6 hours,
of which approximately 60% was situated in the lumen of the small
intestine. Less than 30% of this amount was present as SAIB. Only
4.9% of the dose was excreted in the urine, and 2.4% was expired as
CO₂ at 6 hours after dosing. 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.
Metabolism to CO₂ accounted for 45% of the excreted radioactivity.
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).

In a series of experiments with sucrose octaisobutyrate (SOIB),
a constituent ester of SAIB, male rats received approx.200 mg/kg bw
¹⁴C-SOIB (specific activity 0.2 µCi/ml) by corn oil gavage.
Radioactivity was not detectable in urine, faeces or breath until 6
hours following dosing, but by 5 days >95% of the dose had been
excreted by these routes. The major route of excretion was the
faeces (78-93% of the dose); consequently, only a small amount of
administered SOIB was absorbed. ¹⁴CO₂ in the breath was the major
route of excretion for absorbed SOIB. Radioactivity in bile samples
collected from an indwelling catheter for 12 hours after dosing was
negligible or only slightly above background and after 48 hours,
only 0.2% of the administered dose had been collected from bile.
Similarly, radioactivity in terminal blood samples collected at 2,
4, 8, 12, and 24 hours was only detectable in 24-hour samples and
included a negligible percentage of the administered radioactivity
(Noker 1982; Noker et al. 1986).

2.1.1.2 Dogs

Two dogs received single doses of 3.0 or 4.8 mg/kg bw ¹⁴C-SAIB
(specific activity 0.36 µCi/mg) in aqueous emulsion by stomach tube.
A large proportion of the administered dose (compared with rats at
the same dose) was eliminated in the faeces (52.5 and 45.5%,
respectively), within 4 days of dosing. Although collection of
¹⁴CO₂ excreted in breath was incomplete, it still represented the
major portion of excreted radioactivity (26-28%). Smaller amounts
were eliminated in the urine (6 and 7% after 7 and 8 days
respectively). Chromatography of faecal extracts indicated the
presence of SAIB and highly acylated sucrose esters. Radioactivity
in urine samples corresponded almost entirely to sucrose esters
while no significant amount of sucrose was detected (Reynolds and
Travis 1972; Reynolds et al. 1974).

¹⁴C-SOIB (specific activity 0.02 µCi/mg) was administered to
male Beagle dogs by corn oil gavage at a dose of approx. 200 mg/kg
bw. Almost no radioactivity was detected as ¹⁴CO₂ during the
first 24 hours after dosing. Throughout the 5-day study, the amount
of radioactivity excreted as ¹⁴CO₂ accounted for 1% or less of the
dose. The major route of elimination was the faeces, accounting for
77 to 94% of the dose. In one of the dogs, essentially the entire
dose was recovered within 5 days, indicating that SOIB was not
incorporated to any great extent in the tissues. In another study
using dogs with indwelling catheters, between 2 and 10% of the dose
was retrieved in bile collections made over 48 hours or more after a
delay of 4-6 hours following dosing. Chromatographic analysis of
the bile samples revealed at least 9 radiolabelled components.
Although the identity of none of them could be established, SOIB was
not present in the samples and all the components were more polar
than the parent compound. The variability in total biliary
excretion between individual animals and in the same animals in
repeat-dose studies was attributed to differences in degree of
absorption of the compound. The results of a separate 12-hour bile
collection study were considered to be possibly inaccurate since the
anaesthesia (halothane-nitrous oxide) used during the collection
period may have resulted in reduced gastric motility (Noker 1984a;
Noker et al. 1986).

2.1.1.3 Monkeys

Male Cynomolgus monkeys weighing approximately 2.5 kg
received a single dose of approx.200 mg/kg bw ¹⁴C-SOIB (specific
activity 0.08 µCi/mg) by oral gavage in corn oil. The major route
of elimination was the faeces, where the amount recovered from
individual monkeys ranged from 61 to 85% of that administered.
Radioactivity was detected in the breath of one monkey at 12 hours
and in the other two monkeys only after 24 hours. During the 5-day
observation period, the total amount of SOIB excreted as ¹⁴CO_{2
accounted for less than 2% of the administered dose. For each

monkey, 1% or less of the dose was found in the urine. Excretion of
radioactivity in the bile was monitored in three monkeys for at
least 52 hours. For all 3 monkeys, very low levels of radioactivity
were excreted into the bile, corresponding to only 0.1-0.2% of the
administered dose. No significant level of radioactivity was
detected in the blood or plasma in the 48 hours following dosing
(Noker 1984b; Noker et al. 1986).

2.1.1.4 Humans

¹⁴C-SAIB (specific activity 0.39 µCi/mg) 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 approx. 1 mg/kg bw to each subject, none of whom 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 bw/dy
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 bw 10 weeks after the second dose
and immediately after ingestion of a high-fat meal. All subjects
were monitored for elimination of radioactivity in expired air and
urine for 30 days or more post-dosing. Elimination of radioactivity
from the lungs occurred rapidly. [Only a small amount of the total
was eliminated in the 6-8-hour period post-dosing, but excreted
radioactivity reached maximum levels 9-15 hours post-dosing.] The
subjects excreted 14 to 21% of the dose in the urine, the maximum
rate of urinary excretion occurring within 3 hours and decreasing by
48 hours. About 10% or less 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 pattern of elimination. 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 and SAIB or
highly acylated esters of sucrose were not detected.
Chromatographic studies of extracts of faeces showed the presence of
radioactive materials which did not correspond with those in the
urine and which were probably highly acylated esters of sucrose and
SAIB. No effect on blood haematology or selected blood chemical
values was detected. In another study, two subjects ingested
¹⁴C-sucrose at a level of 400 mg/kg bw, corresponding to the
concentration of sucrose in the simulated soft drink administered in
the previous study. Forty-two 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 in 48
hours). Most of the radioactivity appeared to have been
incorporated into urea and free sucrose was not detected in urine
samples (Reynolds et al. 1972; Reynolds et al. 1974).

Two male subjects were given SAIB (100 mg or 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
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 samples of one subject given
100 mg of SAIB daily for 7 days. Two subjects were each given
sucrose intravenously (100, 250 and 800 mg in a 10% solution w/v on
different days) and the urine collected 3, 12 and 24 hours.
Approximately 50% of the administered sucrose 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).

2.1.1.5 Combined species

The disposition of SAIB following a single oral dose was
compared in the rat (5.8, 11.2, 27 and 100 mg/kg bw), dog (3.0 and
4.8 mg/kg bw) and human (0.18 and 1.0 mg/kg bw). The authors
concluded that the excretion patterns for humans and rats showed
more similarities than did the excretion patterns for humans and
dogs. Humans and rats (at the three lower doses) absorbed a larger
proportion of the administered SAIB from the intestine and converted
a higher proportion to CO₂. The results of chromatography of
urine extracts indicated that the types of partial sucrose esters
differed in dogs compared with rats and humans. More highly
acylated sucrose molecules were present in the urine of dogs and
more polar esters in the urine of humans and rats (Reynolds et al.
1971; Reynolds et al. 1974).

Comparison of the disposition of SOIB in rats, dogs and monkeys
receiving the same single, oral dose (200 mg/kg bw), showed that the
three species excreted similar amounts of the administered compound
in the faeces and that this was the major route for excretion of
SOIB. However, the three species differed in the disposition of
absorbed SOIB. Measurement of biliary excretion indicated that the
dog excreted substantial amounts of SOIB by this route (3-10% of the
administered dose) compared with rats and monkeys (<0.2% of the
administered dose). In the rat, the preferred route of elimination
of absorbed SOIB was through expired CO₂, representing 3-15% of
the dose; CO₂ represented 0.1-1.7% of the dose in dogs and
monkeys. The authors conclude that the virtual absence of
radioactivity in the blood, urine, expired CO₂ and bile of the
monkey suggests that this species did not readily absorb SOIB.
Chromatographic analysis of the faecal metabolites of SOIB indicated
that this compound was hydrolyzed to a different extent in the gut
of the rat, dog and monkey with the most extensive hydrolysis
occurring in the rat, less extensive in the dog and little
intestinal metabolism in the monkey (Noker et al. 1986).

2.1.2 Biotransformation

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 of dosing. Chromatographic separation of the metabolites
present in the bile showed that they had properties similar to
sucrose or sucrose with 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 of dosing.
Separation of the metabolites showed the presence of SAIB or highly
acylated sucrose (Reynolds et al. 1975).

Homogenates of the liver and small intestinal mucosa of rats
prepared in Krebs-Ringer phosphate buffer (pH 7.4) were incubated
with ¹⁴C-SAIB. At 0, 1, 2, and 4 hours, samples were removed and
assayed for metabolites. The rate and extent of hydrolysis of SAIB
by liver homogenates was less than that of the intestinal mucosa,
and the rate and extent of hydrolysis decreased with increasing
concentrations of SAIB. In another experiment, ¹⁴C-labelled SAIB
was incubated under anaerobic conditions with preparations derived
from the contents of 3 regions of the rat gut, namely, stomach,
small intestine and caecum. Preparations from the proximal region
of the small intestine showed the greatest hydrolytic activity. The
hydrolytic activity of the caecal contents was less than that of the
small intestine, and the stomach contents showed no hydrolytic
activity. An ex vivo study of the disappearance of radioactivity
from loops of the small intestine of rats after introduction of
¹⁴C-sucrose and ¹⁴C-SAIB 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. When ¹⁴C-SAIB was incubated
with human faecal homogenates, 40% was hydrolyzed in a 16-hour
period, with less than 2% completely hydrolyzed to sucrose.
Hydrolysis of SAIB by suspensions of bacteria isolated from human
faeces was even less than that of the faecal suspensions (Phillips
et al. 1976).

Dogs and rats received a single dose of ¹⁴C-SAIB [not
specified in previous monograph]. 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 was not determined, although sucrose, glucose and
fructose appeared to be absent. A male subject 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 after
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 and Zeigler 1977).

2.2 Toxicological studies

2.2.1 Acute toxicity studies

The results of acute toxicity studies with sucrose acetate
isobutyrate are summarized in Table 1.

Table 1. Results of acute toxicity studies with sucrose acetate
isobutyrate.

Animal Route LD50 Reference
(g/kg bw)

Rat Oral >5.0 Fassett and Reynolds 1962;
Reynolds 1972a

2.2.2 Short-term toxicity studies

2.2.2.1 Mice

Five groups of B6C3F₁/CrlBR mice (10/sex/goup) received
approximately 0, 0.625, 1.25, 2.5 or 5.0 g SAIB/kg bw/day in the
diet for 4 weeks. Body weights, food consumption and physical
examinations were recorded at initiation of treatment and weekly
during the study. Feeding of SAIB at these doses for 4 weeks had no
effect on the biological performance of the treated animals. No
treatment-related observations were noted at the terminal necropsy
(MacKenzie 1987).

2.2.2.2 Rats

A three-week feeding study was conducted in which groups of 30
Sprague-Dawley rats (15/sex/group) were fed 0, 5 000 or 50 000 ppm
SAIB in the diet. Five rats/sex/group were sacrificed after 1, 2
and 3 weeks of treatment. Twice weekly determinations of body
weight and food consumption indicated no adverse effect of treatment
on these parameters. Daily examination of the rats during the first
week revealed a high incidence of respiratory disease which was not
related to treatment; no animals died. Gross necropsy at sacrifice
did not reveal any treatment-related changes. Absolute and relative
liver weights were comparable between control and treated groups
(Procter and Chappel 1970a).

In a series of studies, Sprague-Dawley rats were divided into
14 treatment groups of 10 animals/sex/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 or 56
days continuously or for 28 days followed or preceded by 28 days on
control diet. Two groups 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 carbamyl transferase
(OCT), blood urea nitrogen (BUN), triglyceride, cholesterol and
glucose. The animals were autopsied, examined grossly, and absolute
and relative liver weights were determined. Liver microsomal enzyme
activity (p-nitroanisole demethylase), glucose-6-phosphatase and
bilirubin-ß-D-glucuronyl transferase were determined and the liver
was histologically examined. 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 groups. Microsomal enzyme
activity was similar for test and control animals, with the
exception that glucose-6-phosphatase activity was reduced in male
rats on the 4% SAIB diet (Krasavage and Terhaar 1971b; Krasavage
et al., 1973).

Groups of 50 Holtzman albino rats, 25/sex/group, were
maintained on diets containing 0, 1.0 or 5.0% SAIB (w/w) for a
period of 95 days. Haematological studies consisting of Hb, Hct and
total and differential WBC counts were carried out at days 24, 52
and 87 of the study. Body weight and food intake were determined
weekly. The animals were necropsied on day 95 and liver and kidney
weights recorded and a complete histological study made of 15
tissues and organs, including the liver, stomach and small
intestine. There was a slight reduction in weight gain in the males
fed 5% SAIB and a slight increase in the absolute and relative liver
weight of females fed 5% SAIB. No compound-related
histopathological changes were observed (Fassett et al., 1962).

Four groups of 20 Sprague-Dawley rats (10 males, 10 females;
body weight 85-100 g), were maintained on diets for 13 weeks which
contained 0, 0.30, 1.80 or 9.12% SAIB dissolved in vegetable oil, to
a final concentration of 9.3% oil in the diet. Body weights were
determined weekly. Prior to sacrifice at 13 weeks, blood samples
were taken and Hb content and total and differential WBC count were
determined. At autopsy, absolute and relative organ weights were
recorded for liver, kidneys, lungs, testes, spleen and heart and a
microscopic examination was made of 10 tissues and organs including
the liver. At the highest dose 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 (Hint, 1964).

Groups of 80 rats (Sprague-Dawley), 40/sex/group, were
maintained on diets containing 0, 2.5, 5.0 or 10% SAIB. A positive
control group for the study of liver enlargement and microsomal
enzyme induction received phenobarbital daily by gavage at a dose
equivalent to 100 mg/kg bw. Half of each group received treatment
for 6 weeks, and the other half for 12 weeks. Following 6 and 12
weeks on the test diet, one subgroup (10 rats/sex/group) was
subjected to the Zoxazolamine muscle relaxant test and retested 4
weeks after removal from treatment. The other subgroup (10
rats/sex/group) was subjected to extensive histological and
biochemical tests at the end of 6 or 12 weeks, which included serum
OCT and protein, glycogen, carboxylesterase, lipid and water of the
liver. In addition, urinary excretion of ascorbic acid was measured
at weekly intervals during treatment. At autopsy, absolute and
relative organ weights of the adrenals, heart, kidney and liver were
recorded for test and control animals for both subgroups.

Dietary SAIB had no significant effect on the weight gain of
test animals except at the low dose (2.5%) level, where there was
slight decrease in weight gain of males after 6 weeks and in both
sexes after 12 weeks. In general, food consumption in the SAIB
groups was not affected. The SAIB-treated rats did not differ from
negative controls in their response to the Zoxazolamine muscle
relaxant challenge or in urinary excretion of ascorbic acid. In
contrast, in the phenobarbital-treated rats, the response to
Zoxazolamine was markedly reduced and urinary excretion of ascorbic
acid showed a prolonged and marked elevation. On this basis, the
authors concluded that induction of microsomal enzymes did not occur
in SAIB-treated rats. There were no significant compound-related
effects in the organ weights, gross pathology or histopathology in
the SAIB-treated animals. The biochemical studies of SAIB-fed
animals showed significantly increased glycogen and water content in
the livers of the 10% males and females but no increase in hepatic
carboxylesterase levels. In contrast, the animals which had been
administered phenobarbital had enlarged livers and demonstrated a
significant reduction in glycogen and water content, an increase in
lipid content, and a marked increase in carboxylesterase activity of
the liver (Procter et al., 1971a).

Groups of 20 male and 20 female F-344 rats were fed diets which
provided doses of 0, 0.5, 1 or 2 g SAIB/kg bw/day for 52 weeks.
Individual body weights and food consumption were recorded weekly.
Ophthalmoscopic examinations were performed on all animals at
initiation, 6 months and 12 months; a battery of standard
haematology, clinical chemistry and urinalysis parameters were
measured at 6 and 12 months. A bromosulfophthalein (BSP) clearance
test was performed on all animals in the control and high-dose
groups during weeks 23 and 48. No information on the conduct of the
BSP clearance test was supplied ( i.e. dose of BSP, clearance times
used). All surviving animals were sacrificed after 52 weeks of
treatment and the organ weights for heart, kidneys, liver, gonads

and brain recorded. Histopathological examination was conducted on
kidney, liver, lung and gross lesions from all animals in all dose
groups and on 32 additional tissues, including the common bile duct
and organs of the GI tract, from all control and high-dose animals.
Samples of the liver from 3 control and 3 high-dose animals were
also examined by electron microscopy.

Treatment with SAIB did not affect body weight gain, food
consumption or the general health of the animals. In addition,
results from measurement of haematology, clinical chemistry and
urinalysis parameters did not suggest adverse treatment-related
effects. Absolute, relative-to-brain or relative-to-body organ
weights were not affected and no unusual histopathological
observations were noted. Ultrastructural examination of livers
failed to reveal any change in liver cells or in bile canaliculi and
sinusoidal linings as a result of treatment with SAIB. A NOEL of
2 g/kg bw/day was assigned for this study (MacKenzie 1990a).

2.2.2.3 Dogs

Three test groups of 8 pure-bred beagle dogs (4 males, 4
females), were maintained on diets containing 0.2, 0.6 and 2.0% SAIB
dissolved in cottonseed oil for a period of 90 days. 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
cottonseed oil. Physical examinations and clinical studies were
made twice prior to commencement of the study and at week 12. The
clinical studies included measurement of haemoglobin concentration,
haematocrit, total and differential WBC count, blood glucose, BUN,
SAP and LDH, and standard urinary parameters. 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. Absolute and relative organ weights were
determined for liver, kidneys, spleen, gonads, adrenals, pituitary
and brain. Twenty-three tissues, including the liver, gall bladder
and small and large intestines from dogs in the control and 2%
groups were examined microscopically. 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
or weight gain. Haematological and urine parameters of test animals
and controls were comparable and within normal values. Serum
chemistry indicated a significant increase in SAP activity of both
male and female dogs in the 2% group (approximately two times
increase over pretreatment values). At autopsy there was a marked,
dose-related increase in relative liver weights in the 0.6% and 2%
groups of both sexes when compared with controls; all other organ
weights were normal. No compound-related histopathology was
observed (Morgareidge, 1965).

In another study, groups of 12 beagle dogs (6/sex/group) were
fed diets containing SAIB at 0, 0.5, 1.0, 2.0 and 4.0% for a period
of 12 weeks. Test animals in the 4.0% group were maintained for 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 and urine samples were obtained at weeks 4, 8 and 12 and
standard haematological, biochemical and urinalysis tests were
performed. At these intervals, 30-minute bromosulfophthalein (BSP)
and phenosulfophthalein (PSP) retentions were measured. BSP and
indocyanine green (ICG) plasma disappearance curves were determined
at week 12 for male dogs in the 0 and 4.0% groups. BSP clearance
was measured in the 4.0% group during the 3-week withdrawal period.
At the end of the test period, the animals were sacrificed, and
following completion of gross pathological examination, absolute and
relative organ weights were determined for the brain, heart, liver,
lung, kidneys, adrenals, gonads, prostate, uterus, pituitary, spleen
and thyroid. A microscopic examination was made of samples of 6
tissues including the liver and small and large intestines.
Histochemical studies were carried out on liver sections of dogs
from the 0.5, 1 and 2% groups, and included evaluation of succinate
dehydrogenase, phosphorylase, glucose-6-phosphate dehydrogenase,
glycogen, acid phosphatase, alkaline phosphatase, adenosine
triphosphatase, and use of Masson's trichrome stain. Additional
samples of liver from test animals (not including the 4% groups)
were analyzed for protein, glycogen, lipid and water, and
carboxylesterase activity. Serum OCT 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 change in the treated
dogs. Growth and food intake appeared normal in all groups. Urine
and haematological analyses were similar in test and control groups
and of the biochemical parameters, only SAP values demonstrated an
increase in the treated animals which was directly related to dose
level and duration of exposure. Marked bromosulfophthalein (BSP)
retention occurred among all test animals during the experimental
period, but the magnitude of the response was not dose-related. The
marked increase in BSP retention in the 4% group was reversible
following withdrawal of SAIB from the diet for 3 weeks. 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-related increase in absolute and relative liver weight
which was reversible following 3 weeks on a SAIB-free diet. No
liver enlargement was observed in the female dogs. Histochemical
studies of liver sections did not reveal any changes in the
hepatocytes; 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 and a slight
increase in glycogen in the liver. 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. Serum OCT values assayed in terminal
blood samples were similar for test and control animals and within
normal range. Light microscopic examination of the liver from
treated males showed hepatocellular hypertrophy, dilatation of the
bile canaliculi, and an increase in the number of bile pigment
granules. 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. The structural changes found in the bile
canaliculi and the pericanalicular cytoplasmic areas included
moderate dilatation of the canaliculi, pronounced microvillous
pattern, prominent Golgi bodies and an increased number of
microbodies in the intracellular pigment granules. Since effects of
the test material on the liver were observed at all dose levels, a
NOEL was not observed for this study (Procter et al., 1970).

In a parallel study, a group of 8 beagle dogs (4/sex/group) was
fed SAIB at a dietary level of 2.0% for 12 weeks and then maintained
for 6 weeks on an SAIB-free diet prior to sacrifice. SAIB caused a
slight weight depression that was reversible upon removal of SAIB
from the diet. The effect of SAIB on two indicators of liver
function, SAP activity and plasma BSP clearance was completely
reversed following the 6-week withdrawal period. At autopsy, the
liver enlargement reported in organ weight studies, the high
activity of alkaline phosphatase, adenosine triphosphatase and
glucose-6-phosphate dehydrogenase in bile canaliculi reported in the
histochemical studies, and the effect of SAIB administration on
liver carboxylesterase activity were fully reversible following 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.,
1971b; Procter et al., 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 dogs were
returned to control diet for the next 57 days. Haematocrit,
haemoglobin, total and differential WBC counts, ASAT, blood glucose,
BUN, serum protein, SAP, OCT, and triglyceride and cholesterol
determinations were made twice prior to and at weekly intervals
during the feeding study. Four of the dogs then received one day's
allotment of the SAIB diet, and ICG plasma clearance rates and SAP
were determined after 24 and 48 hours. The study was terminated 3
days later. At the termination of the study, all dogs were
sacrificed, absolute and relative liver and kidney weights
determined, and 23 tissues and organs, including liver, gall bladder
and organs of the GI tract, 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 receiving control diet for
8 weeks, the 4 dogs returned to SAIB-containing diets for 24 hours
showed a significant slowing of ICG clearance rate, but SAP did not
appear to be increased. All other parameters measured in test and
control animals were similar (Krasavage and Terhaar, 1971a;
Krasavage et al. 1973).

In another study, groups 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 91 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 (haematocrit,
haemoglobin, BUN, serum protein, SAP and OCT, triglyceride 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 weights recorded, and all tissues (not specified)
examined microscopically. Livers were analyzed for glycogen,
protein and phospholipid content, and samples were assayed for
microsomal enzyme activity (p-nitroanisole demethylase), and for
glucose-6-phosphatase and bilirubin-ß-D-glycuranyl transferase
activities. Liver, kidney, bone, bile and scrapings of the
intestinal mucosa were analyzed for 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 and Terhaar,
1971c; Krasavage et al., 1973).

2.2.2.4 Monkeys

Two male and two female Cynomolgus monkeys received SAIB by
intubation in an orange juice vehicle over a period of 14 days.
Dosing started at 1.25 g/kg bw/dy and increased by a factor of 2
with a 72-h rest period between doses, up to a dose of 20 g/kg bw/d.
The animals were observed daily for signs of adverse effects. Body
weight and food consumption were recorded daily. Seventy-two hours
after the last dose, the animals were sacrificed and complete gross
postmortem examinations were conducted. No deaths occurred during
the study. Moderate amounts of yellow, watery emesis and/or
yellow/tan watery stools were observed in some males and some
females 1 to 5 hours after dosing. Twenty-four hours after dosing,

all the animals passed moderate amounts of loose, tan stools. Gross
postmortem examinations did not reveal any changes which could be
attributed to an effect of treatment (Tierney and Rinehart, 1979).

Twelve Cynomolgus monkeys were assigned to six goups
(1/sex/group) in a range-finding study in which doses of 0, 0.5,
1.0, 2.0, 5.0 and 10.0 g SAIB/kg bw/day were administered by gavage
in orange juice concentrate for 15 consecutive days. Physical
observations, body weight and food consumption were recorded daily.
Thirty-minute BSP retention was determined pretest and prior to
termination. Following gross necropsy, adrenals, heart, kidneys,
liver and all gross lesions were preserved for histopathological
examination. In addition, two sections of liver from control and
high-dose monkeys were examined by electron microscopy. Treatment
with SAIB for 15 days had no effect on body weight or food
consumption. Gross postmortem examinations and light microscopic
evaluations did not reveal evidence of changes attributable to
treatment. Electron microscopy of liver samples from the high-dose
male and female revealed glycogen aggregation surrounded by smooth
endoplasmic reticulum in hepatocytes which did not represent a
significant alteration in ultrastructural organization (Tierney and
Rinehart, 1980a).

In another study, the evaluation of selected clinical chemical
parameters was conducted in eight Cynomolgus monkeys (1/sex/group)
fed doses of 0, 2.0, 5.0 and 10.0 g SAIB/kg bw/day under the same
conditions as in the previous study. Blood samples were collected
pretest and at termination of the study for measurement of ASAT,
ALAT, SAP, BUN, total protein, albumin, globulin, creatinine, total
bilirubin and bromosulfophthalein retention. Although plasma levels
of BSP were presented and the time for clearance indicated, no other
details of the clearance test ( e.g. dose of BSP) were provided.
Gross necropsy was conducted at sacrifice. Treatment with SAIB had
no effect on body weight or food consumption. There were no
differences in the clinical chemistry parameters, including
bromosulfophthalein retention, which could be attributed to
treatment (Tierney and Rinehart, 1980b).

In a 4-week oral toxicity study with Cynomolgus monkeys,
doses of 0, 500, 1 450 and 2 400 mg SAIB/kg bw/day were administered
by corn oil gavage to 4 groups of 1 monkey/sex/group. Clinical
observations were made twice daily and included monitoring of
inappetence. Body weights were determined weekly and a complete
physical examination was conducted prior to treatment and at the end
of 4 weeks. A standard set of haematology and clinical chemistry
parameters (including gamma-glutamyl transpeptidase, SAP, OCT and
30-minute BSP clearance) were measured from blood samples collected
pretest and at four weeks. Gross necropsy was conducted at
sacrifice. Inappetence was noted occasionally throughout the study
for most monkeys, although it was observed on 13 separate days in
the high-dose female. Consequently, this animal experienced a 12%

weight loss over the course of the study. Body weight gains of the
other animals were comparable. No abnormalities related to
treatment were detected from the physical examinations. The results
of haematological and clinical chemistry tests did not indicate an
effect of treatment although lowered serum phosphorous levels were
detected in the high-dose female. For BSP clearance, only semi-
quantitative results were presented, i.e. >95% clearance for all
groups. This could indicate that the dose used was too low to
detect any changes within a standard 30 min. period. The
concentration of BSP in blood was not presented. Gross necropsy did
not reveal any unusual findings (Blair, 1986).

Groups of 4 male and 4 female Cynomolgus monkeys received
doses of 0, 500, 1 450 or 2 400 mg SAIB/kg bw/day by corn oil gavage
for 52 weeks. Clinical observations, including inappetance, were
made twice daily and body weights determined weekly. Complete
physical examinations and separate ophthalmoscopic examinations were
conducted on all monkeys pretest and during months 3, 6, 9, and 12.
Blood samples for determination of a standard set of haematological
and clinical chemistry parameters (including SAP, gamma-glutamyl
transpeptidase, OCT, BSP clearance and bile acid analysis) were also
collected at the same intervals. At termination, gross necropsy was
performed, selected organs were weighed, and 40 tissues and organs
including liver, GI tract and gross lesions were subjected to
histopathological examination.

During the study a few animals had signs of slight anal
staining and slightly soft stool. However, there were no signs of
inappetance or changes relating to administration of the test
material, and none of the animals demonstrated body weight loss over
the course of the study. The physical and ophthalmoscopic
examinations did not reveal any treatment-related abnormalities. In
addition, analysis of the haematological and clinical chemistry
parameters obtained during the study indicated that no effect of
treatment on these parameters was evident. Semi-quantitative
measurements only of BSP clearance were given and blood levels of
BSP were not presented. There were no unusual histopathological
observations indicating an effect of treatment, including the liver.
Since no effects of treatment were observed, the NOEL was the
highest dose tested, 2400 mg/kg bw/day (Blair, 1990).

2.2.3 Long-term toxicity/carcinogenicity studies

2.2.3.1 Mice

A carcinogenicity study was conducted with B6C3F₁ mice in
which 5 groups of 50 mice/sex/group received 0.0, 0.0, 1.25, 2.5 or
5.0 g SAIB/kg bw/day in the diet for 104 weeks. Physical
examination, body weight and food consumption data were collected
weekly. A standard set of haematology parameters was determined for
15 mice/sex from one control group and from the high dose group at

26, 52, 78 and 104 weeks of the study. Necropsy was performed on
all animals dying on test or sacrificed. The weights of liver with
gall bladder, lungs and kidneys were recorded for all animals
sacrificed at 104 weeks. Histopathology was carried out on lungs,
liver, kidneys and gross lesions from all animals and on 44
additional organs and tissues, including the GI tract, from animals
that died on test and the control and high-dose animals from the
terminal sacrifice.

Survival of the mice from all groups in this study ranged from
66 to 80%. No treatment-related effects on body weight gain were
noted. Food consumption was elevated in both male and female mice
receiving SAIB in the diet. The difference was frequently
statistically significant in the high-dose males. There were no
treatment-related differences in the haematology parameters. A
dose-related decrease was apparent in the absolute weights of the
kidneys of male mice which was statistically significant at the mid
and high doses. The relative kidney weights were also lower than
controls but the magnitude of the decrease was not related to dose.
No unusual histopathological results for the kidney were observed.
The authors concluded that this represented a NOEL of 2.5 g SAIB/kg
bw/day (mid dose) for the male mouse. There was an increased
incidence of hyperplasia of the perivascular and peribronchial
lymphoid tissue of the lung in treated female mice which lacked
dose-relationship; the result was not considered by the authors to
be toxicologically significant. There were no treatment-related
increases in the incidence of any tumour type. Based on the reduced
kidney weights in the two top doses, a NOEL of 1.25 g/kg bw/day was
considered appropriate for this study. The NOAEL was the highest
dose tested, 5.0 g/kg bw/day (MacKenzie 1990c).

2.2.3.2 Rats

Groups of 20 (10 male, 10 female) Sprague-Dawley rats were
maintained on diets containing 0, 0.38 or 9.38% (w/w) SAIB for 104
weeks. During this period the rats were bred on 3 successive
occasions. Body weight and food intake were measured weekly. All
animals dying during the test period and those sacrificed at 104
weeks were autopsied. Relative and absolute organ weights were
determined for heart, kidneys, liver, lungs, ovaries, spleen and
testes. Histological examinations were made of 18 tissues,
including the liver, stomach and ileum, from rats of the control and
9.38% SAIB groups.

Feeding SAIB did not increase the overall number of mortalities
(15/20 in control, 13/20 in 0.38% and 14/22 in 9.38% groups) even
though 4 males in the 9.38% SAIB group died during the first 10
weeks. Autopsy revealed massive haemorrhages at multiple sites in
each case. Subsequent measurement of systolic blood 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. At the end of
the first year there were no significant differences in body weights
of rats from the various groups. During the second year, however,
male survivors receiving treatment at either dose weighed less than
the respective controls. There appeared to be a dose-related
increase in the absolute and relative kidneys weights of both the
male and female rats. Due to the disparities in body weight of
treated and control males, and the small number surviving (2 or 3
animals/group), no meaningful conclusions could be drawn from the
organ weight data for male groups at the termination of the study.
Histological studies did not reveal any compound-related lesions
(Harper et al., 1966).

A carcinogenicity study was conducted with F-344 rats in which
5 groups of 50 rats/sex/group received 0.0, 0.0, 0.5, 1.0 or 2.0 g
SAIB/kg bw/day in the diet for 104 weeks. Physical examination,
body weight and food consumption data were recorded weekly. RBC and
total and differential WBC counts were performed on all rats prior
to study initiation and on all surviving rats at termination.
Clinical chemistry parameters were not measured. Necropsy was
performed on all animals dying on test or sacrificed. Organ weights
were determined for brain, heart, liver, kidneys and gonads for all
animals sacrificed at 104 weeks. Histopathology was carried out on
lungs, liver, kidneys and gross lesions from all animals and on 44
additional organs and tissues, including the organs of the GI tract,
from animals that died on test and from the control and high-dose
animals at termination.

The health of the treated and control animals was comparable
throughout the study and survival rates at 104 weeks were similar.
No effect on body weight was seen in the treated male groups; in
females, a small but significant decrement was noted in the body
weights of the high-dose group compared with control groups for the
first 1´ years. Food consumption was not affected by treatment.
Treatment with SAIB had no effect on haematology, organ weights or
macroscopic and microscopic examinations at termination of the
study. There were no treatment-related increases in the incidence
of any tumour type. The NOEL was 2 g/kg bw/day in this study
(MacKenzie 1990b).

2.2.4 Reproduction studies

2.2.4.1 Rats

Groups of 15 female and 5 male Holtzman rats, 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, the rats were regrouped, 1 male and 3
females/cage. Pregnant females were then housed separately. The
parameters recorded for assessing reproductive performance were
length of gestation, number of young and live young, number of young

weaned and average weight at weaning and post-weaning. Fifty-one
days after the last parturition, another mating was attempted.
Progeny of the first breeding (F₁) were also bred. At necropsy,
liver and kidney weights were measured in both F₀ and F₁ parental
animals, and 12 tissues, including liver and stomach, were examined
microscopically. Treatment with 5% SAIB did not affect the
biological performance of the F₀ generation. The reproductive
performance of the test parent generation resulting from the first
breeding was equal to or superior to that of controls, based on 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 the controls. The breeding of the F₁
generation appeared satisfactory. Due to an outbreak of respiratory
disease, many F₁ parents and pups died during the test and none of
the pups from the SAIB group survived post-weaning. No differences
in organ weights or histopathology were detected between treated and
control groups (Fassett et al., 1965).

Groups of 20 Sprague-Dawley rats, 10/sex/group, age not
indicated, were maintained on diets containing 0, 0.38 and 9.38% w/w
SAIB for 5 weeks. Pairs of rats from each dose group (10 pairs)
were selected 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 during weeks 9-36
of the study, 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 the 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
in the control. At the 9.38% level, fewer females became pregnant,
fewer pups were born and fewer pups survived to weaning on the basis
of the three breedings. The observed effects could have been due to
compromised nutritive value of the feed at this high level of
inclusion. Performance of the 0.38% group was comparable to
controls (Harper et al., 1966).

A three-generation reproduction study was conducted with groups
of 30 male and 30 female weanling rats which received 0, 0.5, 1.0 or
2.0 g SAIB/kg bw/day in the diet for 10 weeks (males) or 2 weeks
(females) prior to mating. These F₀ animals were mated for 21 days
on a one-to-one basis to produce the F₁ litters. From these
litters, groups of 30 rats/sex were selected randomly to continue
with the same dosage of SAIB in the diet, and to be mated 10 weeks
after weaning to produce the F_2a and subsequently, the F_2b litters.
The F_2a litters were mated in a similar fashion to produce the F₃
litters and the F_2b litters were used for teratologic evaluation
(see Section 2.2.5.1). The study was completed with the necropsy of
the F_2a females on Day 14 of the F₃ gestation. Rats received the

test diet continuously throughout the premating, mating, gestation,
lactation and weaning stages of the study. Body weight and food
consumption were measured weekly throughout the study with the
exception of the mating periods and the 2-week rest period between
the F_2a and F_2b litters. The following reproductive parameters
were assessed for the F₁ and F_2a litters: mating index, male and
female fertility indices, gestation index, numbers of live- and
stillborn pups, and pup weights, survival and sex ratio on days 0, 4
and 28 of lactation. Male and female fertility indices and numbers
of corpora lutea and implantations were recorded for the F₃
litters. All F₀ and F₁ adult male and female rats were necropsied
with particular attention to reproductive organs and macroscopic
lesions, the males after completion of parturition and the females
after completion of weaning. There were no consistent statistically
significant differences in body weight gains, although body weights
of the mid- and high-dose F₀ females were lower than controls
during the first part of the lactation period, and slightly lower
food consumption was noted for the F_2a males and females during the
premating period. No treatment-related effects were noted on
fertility, gestation or survival indices for any of the generations
(MacKenzie 1990d). The NOEL was the highest dose tested, 2.0 g/kg
bw/day.

2.2.5 Special studies on teratogenicity

2.2.5.1 Rats

The F_2b litters from the reproduction study described above
were subjected to teratologic evaluation. Groups of 30 male and 30
female F-344 rats from the F₁ generation were subjected to
in utero and lifetime exposure to 0, 0.5, 1.0, and 2.0 g SAIB/kg
bw/day in the diet. The animals were bred on a one-to-one basis 2
weeks after completion of weaning of the F_2a litters. Vaginal
smears were taken daily and the presence of a copulatory plug or
sperm in the vaginal smear was taken as positive evidence of mating
and counted as Day 0 of gestation. The F₁ dams were sacrificed on
Day 20 of gestation and examined for the number and distribution of
fetuses, the number of fetuses undergoing resorption and the number
of corpora lutea. Live fetuses were removed from the uterus,
weighed, sexed and examined for gross abnormalities. Approximately
one-half of the pups from each litter were examined for soft tissue
abnormalities and the remainder were examined for skeletal
abnormalities. Weekly measurement of body weight and food
consumption in the maternal animals did not reveal any effect of
treatment on these parameters. Treatment with SAIB also did not
affect mating indices, male fertility indices, female fertility
indices, the number of corpora lutea or implantations, implantation
efficiency, uterine weights, the number of live fetuses, early
resorptions, late resorptions, sex ratios or fetal weights.
External, skeletal and soft-tissue fetal examinations also did not
reveal any treatment-related effects (MacKenzie 1990d).

2.2.5.2 Rabbits

Groups of 16 inseminated New Zealand white rabbits each
received 0, 500, 850 or 1200 mg SAIB/kg bw/day orally by corn oil
gavage in two doses on day 7 to 19 of gestation (the day of
insemination was counted as day 0). On day 29 of gestation, the
dams were sacrificed and the pups delivered by Caesarean section.
The number and location of viable and nonviable fetuses, early and
late resorptions and the number of total implantations and corpora
lutea were recorded. Each fetus was weighed and examined for
external malformations, then dissected and examined for visceral
malformations, and the carcass prepared for subsequent skeletal
examination. Laboured respiration was noted in several of the does
from each of the treatment groups during the last few days of the
treatment period but in none of the control animals. The authors
attributed this to tracheal irritation from the test material.
There were no treatment-related effects on body weight gain or food
consumption measured on gestation days 0, 7, 13, 20 and 29, nor did
macroscopic examination of the does following sacrifice reveal signs
of maternotoxicity resulting from administration of the test
material. The reproductive parameters and the fetal examinations
did not reveal any embryotoxic or teratogenic effects attributable
to treatment (Schardein, 1988).

2.2.6 Special studies on genotoxicity

The results of genotoxicity studies with sucrose acetate
isobutyrate are summarized in Table 2.

2.2.7 Special studies on liver function

2.2.7.1 Rats

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 and Chappel, 1971).

Groups of male rats (Sprague-Dawley) (group size not specified)
were fed diets containing 4% SAIB and corn oil, or 5% corn oil.
Indocyanine green (ICG) clearance was determined on at least 2 rats
randomly selected 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
and Terhaar, 1972; Krasavage et al., 1973).

Table 2. Results of genotoxicity studies with sucrose acetate
isobutyrate.

Test System Test Object Concentration of SAIB Results Reference

Ames test¹ S. typhimurium TA1535, 10-2 000 µg/plate Negative Jagannath & Brusick 1978
TA1537, TA1538, TA98,
TA100, S. cerevisiae D4

Ames test¹ S. typhimurium TA98, TA100 100-10 000 µg/plate Negative Bonin & Baker 1980

Ames test¹ S. typhimurium TA98, TA100, 333-10 000 µg/plate Negative Lawlor & Valentine 1989
TA1535, TA1537, TA1538

CHO/HGPRT forward Chinese hamster ovary cells 10-1 000 µg/ml Negative Young 1985
mutation assay¹

Unscheduled DNA Rat hepatocytes 0.25-1 000 µg/ml Negative Cifone 1985
synthesis assay

In vitro chromosomal Chinese hamster ovary cells 200-2 000 µg/ml Negative Ivett 1985
aberration assay¹

Dominant lethal assay Rats 20, 200, 2 000 mg/kg bw Negative² Krasavage 1973

1 Both with and without metabolic activation.
2 Matings were conducted only every 2 weeks instead of every week.

2.2.7.2 Dogs

Two male and 2 female beagle dogs were fed a one day's dietary
ration containing 0.1, 0.3 or 0.5% SAIB. All 4 animals were tested
for bromosulfophthalein clearance 24 hours after 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 and Chappel,
1971).

Two series of experiments were carried out with 14 young,
adult, male beagle dogs. In the first series, groups of 2 (control)
or 3 (treated) dogs received a single dose of 2 g SAIB/kg bw or
orange juice vehicle by gavage and 30-minute BSP clearance was
measured 2, 4, 6, 10, 12, 18 or 24 hours later. Compared with
pre-treatment measurements, plasma BSP concentrations were increased
at all post-treatment intervals, and were highest between 4 and 6
hours. Approximately 5 hours post-dosing was considered the maximal
BSP retention time in the dog. The second series of experiments was
conducted to establish the range of doses of SAIB which produced
increased BSP retention in the dog five hours after a single
administration. Fifteen-minute BSP retention values were increased
7-10 fold with doses of 25 mg/kg bw to 2 g/kg bw. No dose-response
correlation was apparent. At 5.0 mg/kg bw, only 1 of 3 dogs showed
increased BSP retention and the authors considered this to be close
to a no-effect dose. Throughout the study, none of the dogs showed
a marked change in body weight. There were no significant changes
in SAP values measured in blood samples at the same intervals as
measurement of BSP clearance. Changing the vehicle to corn oil in
the last two studies of the second series of experiments eliminated
the observations of vomiting and orange-coloured, loose stools which
were common in the foregoing experiments (Dickie et al. 1980a).

Young, adult, male beagle dogs were tested for 15-minute BSP
clearance and SAP levels 5 hours after a single oral administration
of either sucrose hexaacetate diisobutyrate (SHADIB) or sucrose
octaisobutyrate (SOIB) (constituent esters of SAIB) in a series of
experiments. The range of doses used was 100-1 000 mg/kg bw of
SHADIB and 5-1 000 mg/kg bw SOIB. In the first 3 experiments, 4
treatment groups of 3 dogs and a vehcile control group of 2 dogs
were used; in the fourth experiment, 2 dose groups of 3 dogs and a
vehicle control group of 1 dog were used. Compared with
pretreatment measurements, a single dose of SHADIB caused a
significant increase (5-7 fold) in BSP retention at all doses
tested. SOIB administration also resulted in an increase of BSP
retention of about 4-5 fold compared with pretreatment values at
doses of 25 mg/kg bw and higher. As in the previous experiment, no
dose-response correlation was observed and mean BSP retention at
5.0 mg/kg bw SOIB showed only a slight increase over control and
pretreatment values. Neither ester appeared to cause any gross

clinical effects and neither affected SAP activity. None of the
dogs showed a marked change in body weight over the course of the
study. The observation of yellow-orange stools and vomiting was
eliminated by changing the vehicle from orange juice to corn oil as
in the previous experiment (Dickie et al. 1980b).

2.2.7.3 Monkeys

Two groups of 3 male squirrel monkeys ( Saimiri sciureus)
weighing approximately 1 kg, were fasted overnight and then received
either SAIB (1 g in 2 ml cottonseed oil) or no treatment.
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 and Chappel 1970b).

The same experiment was repeated using doses of 2 g SAIB in
4 ml cottonseed oil to deliver a dose of approximately 2 g/kg bw.
Unusually high plasma BSP levels were measured in 3 of the control
animals, but these were considered by the authors to be technical
errors since BSP clearance following treatment with SAIB was normal
in all of the animals (Procter and Chappel, 1971).

Thirty-minute BSP retention and SAP were measured in 10 male
Cynomolgus monkeys 5 hours after a single oral dose of 5 g SAIB/kg
bw, 5 g SOIB/kg bw or corn oil and compared with pretreatment values
for these parameters. Treatment had no effect on BSP retention time
or SAP. There were no unusual clinical observations or changes in
body weight during the study. It should be noted that the authors
did not run a time series as in the corresponding dog experiment to
determine the optimum interval for measurement of hepatic function
following adminstration of the test material (Dickie et al.
1980c).

2.3 Observations in humans

Twenty subjects (10 males and 10 females) between 18 and 22
years of age ingested a daily dose of SAIB at a level equivalent to
10 mg/kg bw/dy for a period of 14 days. The dose was taken as a
bolus each morning. The following blood parameters were measured
prior to treatment and at days 7 and 18 of the study: ASAT, ALAT,
SAP, serum bilirubin, total protein, albumin, uric acid, BUN,
erythrocyte sedimentation rate, sodium, potassium, phosphorous,
total CO₂, cholesterol and glucose. There were no significant
differences in any parameters in any individual (Hensley 1975).

In another 14-day study, 12 male and 12 female subjects were
divided evenly by sex into 3 groups, receiving a carbonated drink
only (controls), or a single daily dose of 7.0 or 20.0 mg SAIB/kg
bw/day in a carbonated drink. In addition, four men received 20 mg
SAIB/kg bw/day for 1 or 3 days only in a pilot experiment to provide

early evaluation of possible alterations in normal hepatic function.
The subjects were 21 to 42 years of age. Blood was collected prior
to testing and on days 7 and 14 for haematological (platelets, total
and differential WBC count, ESR, Hct and Hb) and clinical chemistry
(total protein, albumin, A/G ratio, calcium, cholesterol, glucose,
BUN, uric acid total bilirubin, SAP, ASAT, ALAT, and LDH)
parameters. Standard urinalysis parameters were also recorded at
these times. A 45-minute BSP retention test (5 mg/kg bw BSP) was
also conducted on all subjects prior to treatment and after
completion of treatment. Treatment with SAIB did not affect any of
these parameters for any individual (Orr et al. 1976).

Twenty-seven adult subjects, 13 men and 14 women, between the
ages of 18 to 55, received SAIB in an aqueous/orange juice emulsion
daily for 14 days at a dose of 20 mg/kg bw. In the 7 days prior to
treatment, each subject acted as his/her own control by ingesting an
orange juice beverage and placebo emulsion. Blood samples were
collected from each subject on days -6, 0, 7 and 14 of treatment for
measurement of routine haematological and clinical chemistry
parameters, including specific indicators of hepatobiliary function
(SAP, ASAT, ALT, LDH, gamma-glutamyl transferase, total bilirubin,
direct bilirubin, bile acids and serum proteins). No treatment-
related changes were detected in any of these parameters over the
14-day dosing period (Chiang 1988).

3. COMMENTS

Studies on the disposition of sucrose acetate isobutyrate in
rats, dogs, and humans indicated that absorption from the
gastrointestinal tract is delayed for several hours but that
elimination is nearly complete by 4 to 5 days after ingestion.
Extensive metabolism of sucrose acetate isobutyrate occurred in the
gastrointestinal tract, mainly in the small intestine, characterized
by its de-esterification by non-specific esterases to partially
acylated esters and sucrose. Ingested sucrose acetate isobutyrate
was partially absorbed from the gut and partially eliminated in the
faeces. In all three species, the absorbed dose was largely
catabolized to CO₂, and smaller amounts were excreted in the urine
and bile.

The extent of absorption from the gastrointestinal tract was
greater in humans and rats than in dogs in the dosage range of
1-10 mg/kg bw. However, at doses approaching 100 mg/kg bw/day in
the rat, absorption of sucrose acetate isobutyrate was less
extensive, resembling more the situation in the dog.

In studies with sucrose octaisobutyrate, the most lipophilic
component of sucrose acetate isobutyrate, a dose of 200 mg/kg bw
administered to rats, dogs, and monkeys was almost completely
excreted in the faeces, although analysis of faecal metabolites
indicated that the extent of hydrolysis in the gastrointestinal
tract differed in the three species (rat > dog > monkey). In
addition, the small amount of absorbed sucrose octaisobutyrate was
preferentially excreted in the bile of dogs and in the expired air
of rats. Chromatographic analysis of the urinary and biliary
metabolites of sucrose acetate isobutyrate showed that dogs excreted
more highly acylated sucrose molecules, whereas humans and rats
excreted more polar sucrose esters. Consequently, the dog differs
from the rat and human in its disposition of sucrose acetate
isobutyrate in that it absorbs less of the total dose of sucrose
acetate isobutyrate in the 1 - 10 mg/kg bw range, but it is capable
of absorbing more highly acylated sucrose esters and compared with
the rat, excretes a larger proportion of the absorbed dose in the
bile. Data on the excretion of sucrose acetate isobutyrate in the
bile of humans were not available for comparison.

The results of short-term (up to 1 year) toxicity studies in
mice, rats, and monkeys were also available. The conclusions of an
earlier 2-year study in rats were not considered to be reliable
because of the small numbers of survivors at the end of the study.
SAIB administered at dose levels of up to 10% in the diet for 12
weeks or 2 g/kg bw/day for 52 weeks had no toxicologically
significant effect in the rat, nor was any effect evident in the
liver as assessed by liver function tests, liver weights, and
histopathology In the Cynomolgus monkey, oral doses of sucrose
acetate isobutyrate of up to 2.4 g/kg bw/day had no apparent adverse

effect. In humans, up to 20 mg SAIB/kg bw/day for 14 days was also
without effect. In addition, special liver function tests conducted
in rats, monkeys, and humans following oral administration of single
or multiple exposures to sucrose acetate isobutyrate showed no
effect on hepatobiliary excretion.

The available studies clearly showed the liver to be the target
organ in the dog. Serum alkaline phosphatase levels were elevated
and biliary excretory function was impaired. Liver enlargement was
noted in the males and histopathological changes were apparent in
the liver of both sexes. All of these changes were reversible
within 3 weeks of removal of sucrose acetate isobutyrate from the
diet. In addition, histochemical studies revealed increased enzyme
activity in the bile canaliculi, but not in the hepatocytes. In
special studies on liver function biliary excretion was reduced
within 4-6 hours of oral administration of a single dose of sucrose
acetate isobutyrate. The NOEL for this effect was 5 mg/kg bw/day.
The authors of the report concluded that this represented a
functional rather than a toxic effect of sucrose acetate
isobutyrate. Although the effects on the liver of the dog were
reversible, no study of longer than 12-13 weeks duration was
available for evaluation. It was not known whether continuous
exposure to sucrose acetate isobutyrate for a longer period of time
would have resulted in the development of pathological lesions.

The carcinogenic potential of sucrose acetate isobutyrate has
been investigated in mice and rats in long-term toxicity studies at
doses of up to 2 and 5 g/kg bw/day, respectively, with negative
results. Sucrose acetate isobutyrate was not genotoxic in in vitro
point mutation, chromosomal aberration, or unscheduled DNA synthesis
assays. A multigeneration reproduction/teratologenicity study in
rats and a teratology study in rabbits were also negative.

The NOELs from the long-term studies in mice and rats and from
a 1-year study in monkeys were similar (5, 2, and 2.4 g/kg bw/day,
respectively). However, the NOEL for the dog was much lower
(5 mg/kg bw/day based on inhibition of biliary excretory function).

4. EVALUATION

The Committee concluded that a 2-year study in dogs was no
longer necessary since the effects of sucrose acetate isobutyrate
and its constituent esters on the liver of the dog had been well
characterized in liver function tests and 90-day toxicity studies,
and a study of longer duration was unlikely to yield new information
which would assist in setting an ADI.

Three studies in humans, involving a total of 71 volunteers,
were available for consideration by the Committee. The results of
these studies demonstrated that sucrose acetate isobutyrate had no
effect on BSP clearance or indicator enzymes of cholestasis in
humans when administered orally in a single daily dose (20 mg/kg bw)
that dramatically reduced BSP clearance in the dog (25 mg/kg bw as a
single dose). Humans, therefore, did not respond to sucrose acetate
isobutyrate in the same way as dogs. The Committee agreed that the
data suggested that the dog was an inappropriate species on which to
base an ADI, but at the same time noted the absence of data on the
mechanism by which cholestasis is induced in the dog.

Taking this into account, the Committee decided to use the NOEL
of 2 g/kg bw/day for rats, the lowest obtained in a long-term
toxicity study, to allocate a temporary ADI of 0-10 mg/kg bw, using
a safety factor of 200. The submission of information that would
clarify the disparate effects of sucrose acetate isobutyrate on
hepatobiliary function in the dog compared with other species, in
particular humans, is required for review by 1996.

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    See Also:
       Toxicological Abbreviations
       SUCROSE ACETATE ISOBUTYRATE (JECFA Evaluation)