BUTYLATED HYDROXYANISOLE (BHA)
EXPLANATION
BHA was evaluated for acceptable daily intake for man (ADI) by
the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in
1961, 1965, 1973, 1976, 1980, 1982, 1983 and 1986 (Annex 1,
references 6, 11, 32, 41, 53, 59, 62 and 73). Toxicological
monographs were issued in 1961, 1973, 1976, 1980, 1983 and 1986
(Annex 1, references 6, 33, 42, 54, 63 and 74). At the 30th meeting
of the JECFA (Annex 1, reference 73), a temporary ADI of
0-0.3 mg/kg bw was established pending adequate studies in pigs and
monkeys to explore the potential for BHA to cause esophageal
hyperplasia in these species. A multigeneration reproduction study
was also required. Since the previous evaluation, new data have
become available and are summarized and discussed in the following
monograph. In addition, the monograph reviews special studies of
the effect of BHA on the stomach of experimental animals.
BIOLOGICAL DATA
Biochemical
Covalent binding to macromolecules
Metabolites binding irreversibly to proteins were found in an
in vitro study involving liver microsomes + NADPH, liver
microsomes + cumene hydroperoxide, sheep seminal vesicle microsomes
+ arachidonic acid, or horseradish peroxidase + hydrogen peroxide
(Newberne et al., 1986).
In another in vitro study (Cummings et al., 1985),
3- tert-butylhydroxyanisole (3-BHA) metabolism was found to yield
material which covalently bonded to protein, although the amount of
these products was reduced directly in proportion to the amount of
glutathione available.
In a third study (Hirose et al., 1987a), however, no
covalent binding to macromolecules was found in a 9000 g
supernatant fraction derived from forestomach epithelium that had
been incubated with 3-BHA in the absence or presence of NADPH.
Looking at male F344 rats that had been given an intragastric
injection of radioactively labelled 3-BHA, no metabolites were
detected by thin layer chromatography in the forestomach or
glandular stomach epithelium six hours after treatment. Assessing
the macromolecular binding of 2- and 3-BHA, these same authors
reported no binding to forestomach, glandular stomach, liver or
kidney DNA or RNA, while binding to protein was similar at the
four sites. They concluded that the activity of BHA is not related
to any covalent binding property of BHA.
In contrast to this, deStafney et al. (1986) reported that
3-BHA bound in vitro to liver microsomal proteins. In vivo,
3-BHA bound to forestomach microsomal proteins 14-fold more than to
glandular stomach microsomal proteins and 12-fold more than liver
microsomal proteins. With HPLC studies, these workers showed that
there were peaks in the forestomach preparation that were not
present in other preparations. The authors speculated that the
effect of BHA was due to its ability to deplete thiols (accounting
for the noticeable threshold of its effects), and the binding to
tissue proteins or reactive BHA metabolites, possibly oxygen
radicals resulting from the redox cycling of quinones and
hydroquinones.
Excretion
The elimination of BHA was studied in male F344 rats (Hirose
et al., 1987b). Within 48 hours of ingestion of BHA, 87-96% of
the BHA was found to be eliminated via urine, feces or respiration.
The isomers 2-BHA and 3-BHA labelled at either the methoxy or
tert-butyl site were used. For 3-BHA and 2-BHA, respectively,
63.7% and 69.0% of the tert-butyl label was found in the urine
and 28.8% and 18.1% in the feces (expired air was not examined for
these isotopes). For 3-BHA and 2-BHA, respectively, 49.8% and 46.5%
of the methoxy label was found in the urine, 28.3% and 29.6% in the
feces, while 8.3% and 13.7% was found in expired air.
In a second study in which Sprague-Dawley rats were used
(Ansari & Hendrix, 1985), 41% of the methoxy labelled 3-BHA was
found in the urine while 53% was found in feces 48 hours after
administration by gavage.
In beagle dogs, within 48 hours of an J.p. injection of
methoxy labelled 3-BHA, 50-80% of the label was found in the urine
and 15-30% in feces (Takizawa et al., 1985).
Toxicological Studies
Special studies on the effect of BHA on the stomach
Mice
Groups of 10 male NMRI mice received daily doses of 0 or
1000 mg BHA/kg bw for 28 days via intubation of an arachis oil
solution. At the end of this period, macroscopic lesions
were seen in the forestomach of the mice that resembled lesions
in rats (Altmann et al., 1986).
Male B6C3F1 mice were exposed to 0.5 or 1% BHA in their
powdered diet for 104 weeks and groups of 10 animals were examined
at 8 week intervals. Hyperplasia of the forestomach was first seen
in the group exposed to the highest level of BHA after 64 weeks
exposure at which time 30% of the animals showed this effect.
Papillomas were observed in mice at both concentrations at week 80
and were found in 10-40% of the animals thereafter. Carcinomas were
detected beginning with week 88, but not in numbers significantly
different from concurrent controls (a total of one carcinoma among
all the low level groups and two among the high). The carcinomas
seen were all well-differentiated. The authors state that the
number of carcinomas is significant if compared to their historical
control data (0 tumours in 244 mice) (Masui et al., 1986b; Ito
et al., 1986b; Ito & Hirose, 1987).
Rats
Male Fischer 344 (F344) rats were divided into groups of 5 and
were fed diets (corn oil, pellets, or powder) containing 0, 0.1,
0.25, 0.5, 1 or 2% BHA. After 9 or 27 days, they were sacrificed
and their forestomachs examined histologically for proliferative
changes in the squamous epithelium (Clayson et al., 1986). Groups
of 5-15 rats on the same diet regimen were injected with
radioactively labelled thymidine just prior to sacrifice and their
forestomach squamous epithelia examined for incorporation of the label
(the labelling index). Thymidine is taken up by replicating cellular
DNA, the presence of which is indicative of induced cell proliferation.
No effects on the labelling index were seen in rats fed 0.25% BHA or
less for 9 days. Histopathologically, hyperplasia was observed only
at 0.5% BHA and above, with the size of the forestomach area
involved being proportional to the dose. After 9 days of exposure
to 2% BHA in pellets, a localized four-fold thickening of the
mucosa was found along the lesser curvature of the forestomach.
Papillae and irregularly spaced rete ridges, acanthosis and
hyperkeratosis were observed. Many mitotic figures were seen in the
otherwise normal-appearing basal layers. There was also acute
inflammatory cell infiltration in the underlying layers. After
27 days, the now six-fold thickening had spread widely, being
visible most markedly adjacent to the forestomach-fundic stomach
junction. After 9 and 27 days with a pellet diet containing
2% BHA, the labelling index had been increased approximately
eight-fold in the pre-fundic region. With BHA given via corn oil,
the labelling index after 27 days was only increased four-fold;
however, the thickening had increased over twelve-fold in the
prefundic region. In the mid-stomach region, the effect of
corn oil vs. pellet feeding of BHA on the labelling index was
similar (two-fold), while thickening was less pronounced with corn
oil (four-fold vs. nine-fold). There were no significant differences
between the effects of BHA given by pellet and given by powdered
diet.
Male F344 rats in groups of 5 were exposed to 2% BHA in a
powdered diet for 4 weeks in another study (Hirose et al.,
1987c). Severe hyperplasia was observed mainly in the prefundic
region of the stomach near the esophageal orifice. The forestomach
showed whitish-colored thickening of the epithelium around the
orifice of the esophagus and the limiting ridge of the forestomach,
while focal patchiness was observed in the central region.
Significant body weight gain reduction was also observed in these
animals, although the liver weight was significantly increased
compared to controls.
In a third study (Clayson et al., 1986), groups of 5 male
F344 rats were fed 0, 0.1, 0.25, 0.5 or 2% BHA (powder) for 13
weeks. Body weight gain was seen to be significantly reduced in
animals fed 2% BHA. Proliferative lesions also only developed in
the forestomach epithelium of the 2% group. In these animals, there
was thickening in the squamous epithelium and downward proliferation
of the basal cells. Papillae and rete pegs as well as hyperkeratosis
were also observed. The muscular layer of the forestomach, however,
was normal. The labelling index was increased in animals exposed to
no less than 0.5% BHA, being 2.5x normal after 9 days and 5.3x after
91 days in the animals exposed to 2% BHA. One week after animals were
removed to normal for all exposure groups, but the mucosal lesions
reverted more slowly, still being visible even 9 weeks after cessation
of exposure.
As a follow up to this, Clayson et al., (1986) examined the
forestomachs of rats that had been fed 2% BHA for 3 months and then
basal diet for 12 months, and BHA for 6 months followed by basal
diet for 9 months. The forestomachs of both of these groups were
histologically nearly normal, with the 6 month treatment group
showing a few downward projections from the normal-appearing
epithelium. Two rats that had been exposed to 2% BHA for 12 months
and then basal diet for 3 months, however, had squamous cell
carcinoma of the forestomach, while others had papillary growths
showing a high rate of proliferation as shown by thymidine
labelling.
In another study (Masui et al., 1986a; Ito & Hirose, 1987),
10 male F344 rats were given a pellet diet containing 2% BHA for 24
weeks. Another 20 received the same diet for 24 weeks and then were
given food without BHA for 72 weeks to assess the effect of
removing animals from BHA exposure. The forestomach of rats given
BHA for 24 weeks showed epithelial thickening, especially at the
limiting ridge. Only slight thickening, however, was seen near the
limiting ridge in animals exposed to BHA and then removed from
exposure for 72 weeks. Rats exposed to BHA for 24 weeks developed
hyperplasia and papillomas in their forestomachs. These changes
included upward proliferation with thickening of the stratified
squamous epithelium often with interstitial proliferation, and
downward proliferation of basal cells forming elongated ridges.
Acute inflammatory reactions in the lamina propria or submucosa
were also seen. In animals whose exposure was discontinued, upward
proliferation of hyperplasia and papillomas had completely
disappeared. Downward proliferation of basal cells persisted,
however, in all rats, and papillomas were observed in 3 of the rats
examined. In this group, however, no inflammation was observed, no
dysplastic changes were seen in basal cells and no cancer was
observed.
In a similar study (Ito et al., 1986a; Tamano et al.,
1986), other workers reported that after 104 weeks exposure to 0,
0.125, 0.25, 0.5, 1 or 2% BHA given by powdered diet to groups of
50 male F344 rats, body weight gains over the period of observation
were depressed in a dose-related manner, being statistically
significant in rats receiving at least 0.5% BHA, even though there
was no significant difference in food intake among any of the
groups and non outward clinical symptoms. No significant pathology
compared to controls was seen in animals fed BHA at any site other
than the forestomach epithelium and significant forestomach damage
was only seen in animals exposed to more than 0.5% BHA. This damage
took the form of extensive raised lesions and was expressed in a
dose-related manner with the most severely affected region being at
the limiting ridge. At the end of the 104 week exposure period,
100% of the animals exposed to 2% BHA had hyperplasia visible in
the forestomach, 100% had papillomas, and 22% also displayed
squamous cell carcinoma. These carcinomas were well differentiated,
displaying keratinisation. No metastases were discovered, however.
Animals exposed to 1% BHA showed 88% hyperplasia, 20% papilloma,
but no carcinoma was seen. For rats exposed to 0.5%, 0.25% and 0.125%
BHA, 32%, 14% and 2%, respectively, had hyperplasia visible, but no
more severe lesions were reported.
This work was confirmed by a study in which male F344 rats
were exposed to 1 or 2% BHA in a powdered diet for 104 weeks with
animals sacrificed in groups of 10 every 8 weeks during the course
of the study (Masui et al., 1986b; Ito et al., 1986b).
Hyperplasia of the forestomach was observed in a few animals of
both treatment groups at the first observation time, 8 weeks after
the start of the exposure. It was present in almost all animals
examined from the high level exposure group from 16 weeks exposure
onward and from 40 weeks exposure onward for the low level exposure
group. In the high level group, papillomas were first seen at week
8 and were present in virtually all animals examined in this group
week 32. No papillomas were seen in the low level group for the
first 48 weeks, but they were seen in 80-90% of all animals
examined after week 56. The first animal with squamous cell
carcinoma, seen only in the animals exposed to 2% BHA, was observed
at week 48 and a second animal was observed at week 80, with the
incidence slowly increasing thereafter so that by 104 weeks, 14% of
the animals examined displayed this pathology. No changes of any
kind were seen in the glandular stomach of any animal of any group.
A 104 week exposure of male and female F344 rats to 0.5 or 2%
BHA in pellets was also reported (Ito et al., 1982; Ito et al.,
1983; Ito et al., 1985; Ito & Hirose, 1987). There were
approximately 50 rats per exposure group. The mean body weights of
both sexes, were reduced in those animals given 2% BHA with the
difference being significant after 16 weeks on the diet. At the end
of the exposure period, 100% of the males and 98% of the females
exposed to 2% BHA displayed hyperplasia in the forestomach. By
comparison, only 26% of the males and 20% of the females exposed to
0.5% BHA had hyperplasia. For papillomas, 100% of the males and 96%
of the females exposed to 2% BHA displayed this type of lesion,
while 2% of the animals of both sexes exposed to 0.5% BHA. had this
observation. Squamous cell carcinoma was seen only in animals
exposed to 2% BHA and was observed in 35% of the males and 30% at
that dose level. Grossly, most tumours were seen in the area of the
limiting ridge of the forestomach. Tumours were greyish-white with
nodules. Histologically, squamous cell carcinomas were sometimes
well-differentiated and sometimes not. Those that were well-
differentiated were keratinized with nuclear atypia and many
mitotic figures present. Infiltration into the muscular layer and
fatty tissue was seen. Three cases of metastases were observed. No
significant incidence of tumours in any other organ were seen in
any group. These diagnoses were also independently confirmed (Moch,
1986).
Groups of 5-10 male and female Wistar rats were fed 2% BHA in
a powdered diet for 1, 2 or 4 weeks (Altmann et al., 1985;
Altmann et al., 1986). After 1 week, epithelial damage with mild
hyperplasia and hyperkeratosis, and blood-filled cystic swellings
were observed in the forestomach and livers were markedly enlarged.
Progression of the hyperplasia and hyperkeratosis was seen after 2
weeks. Body weight of those fed BHA was also significantly
depressed. At 4 weeks, severe diffuse hyperplasia, acanthosis and
hyperkeratosis in the forestomach mucosa, with the most pronounced
lesions in the area of the limiting ridge, were seen. The effects
seen, including weight differences, almost completely regressed,
however, after an additional 4 weeks without BHA. In a second
experiment, groups of 3 male rats received intubations of 1 g
BHA/kg bw/day in arachis oil for 1, 2, 4, 8, 16 or 32 days. After
only one day, increased mitotic activity, and, after 2 days, mild
hyperplasia were seen in the forestomach. Inflammatory responses
and superficial effects were not prominent, however, and the
authors believe that the hyperplasia of the squamous epithelium did
not result from the initial damage and subsequent regenerative
activity. After the fourth intubation, the entire wall of the
forestomach was thickened, wrinkled and swollen. After 8 days, the
hyperplastic changes regressed. With the arachis oil gavage, the
lesions appeared remote from the limiting ridge. Four weeks
following the cessation of BHA intubation, the forestomach showed
almost complete regression with only mild hyperplasia remaining. At
no time during any of these experiments were changes seen in the
glandular stomach or esophagus and there were no differences
between the sexes.
In a 90 day feeding study (Altmann et al., 1986), groups of
10 male and 10 female Wistar rats received 0, 0.125, 0.5 or 2% BHA
in crystalline form. The highest concentration led to marked
hyperkeratosis and hyperplasia with epithelial dysplasia in some
basal areas of the forestomach. Less pronounced lesions were seen
at 0.5% and only mild lesions were seen in the 0.125% group. In a
second experiment, 0, 0.025, 0.125 or 2% BHA was administered to
groups of 20 male or 20 female rats dissolved in arachis oil.
Again, pronounced hyperplasia was seen in the forestomach in the
high dose group. None of the lower dose levels of BHA had any
effect. In only one of the rats was the esophagus affected. In a
study of reversibility of BHA damage, groups of 5 male and 5 female
Wistar rats fed 2% BHA for 6, 12 or 15 months showed almost
complete reversal of severe lesions of the forestomach after
withdrawal from BHA for 7 months.
In another study (Takahashi et al., 1986), male Wistar rats
(10 per group) were fed 1 or 2% BHA (powder) for 32 weeks. A weight
gain retardation in those animals receiving BHA was reported as
were thickenings in the squamous cell mucosa of the forestomach as
well as the presence of squamous cell papillomas. Neoplasms in
animals receiving 2% BHA occupied most of the forestomach. These
lesions villous nodules that were greyish-white in color. The
surface epithelium showed hyperkeratosis with necrosis at the
superficial layer and long processes of squamous cell epithelium.
The incidence of papillomas was 100% in this group. Four cases
(20% incidence) ofdownward growth into the submucosa were also
seen. Animals on 1% BHA had single or multiple polypoid tumours
in the forestomach with a 40% incidence of papillomas. No lesions
in the glandular stomach or duodenum were seen in any exposure group.
The development of forestomach lesions was found to be
significantly quickened when Wistar rats in groups of 10 were
subjected to a 2/3 partial hepatectomy and then fed 2% BHA in a
powdered diet (Abraham et al., 1986). Under these conditions
carcinomas were seen after only 3 months. No body weight gain
depression was reported in this study, however. While only slight
hyperplasia was seen in the forestomachs of animals after receiving
BHA alone, those that were fed BHA following partial hepatectomy
showed grossly visible tumours of the forestomach. In these latter
animals, the forestomach mucosa was thickened and white in color
with confluent nodular masses. All ten of the animals exposed to 2%
BHA following partial hepatectomy displayed hyperplasia and had
papillomas with marked hyperkeratosis. Half of the animals showed
carcinoma as well. The carcinomas were well differentiated with
dysmorphic changes, nuclear atypia and mitotic activity.
Infiltration of the carcinoma in the muscle layer and fatty tissue
was seen. Granulocyte, lymphocyte and macrophage infiltration of
the submucosa was also reported. All other organs were normal in
appearance.
A group of 30 male Sprague-Dawley rats was given 1% BHA for 3
months (Newberne et al., 1986). At the end of this exposure
period, 66% of the animals had hyperplasia of the forestomach, 26%
also showed papillomas, and 6% also had carcinomas. The labelling
index showing cells of the forestomach that were undergoing active
DNA synthesis in those animals exposed to BHA was over 11 times
higher than in control animals. In a separate experiment, animals
administered BHA by gavage were found to be even more severely
affected than those fed BHA in the diet. In the former group 12/18
animals were found to have carcinomas while only 2/20 in the latter
had similar problems.
Hamsters
A group of Syrian golden hamsters received a diet of 2% BHA
for 28 days (Altmann et al., 1986). At the end of this period,
macroscopic lesions in the forestomach of the hamsters were seen
but were reported to be different from those in rats and mice. No
hyperkeratosis was seen in the hamsters macroscopically, but the
mucosae were less elastic and sometimes thickened and wrinkled.
Macroscopically, mild hyperplasia and hyperkeratosis were observed.
These were more pronounced in the females.
In another study (Hirose et al., 1986d; Ito et al.,
1986b), groups of approximately 30 male Syrian golden hamsters were
fed 1% BHA in a powdered diet for 1 or 3 days or 1, 2, 3, 4 or 16
weeks after which they were injected with radioactive thymidine (to
enable a labelling index to be ascertained), sacrificed and
examined. Animals exposed to BHA showed a body weight decrease
compared to controls, but a liver weight increase. Focal thickening
of the forestomach epithelium with or without ulceration and
occasional covering with a dense keratin-like greyish-white
substance was seen in hamsters exposed for at least 1 week. There
were no abnormalities in any other organs examined. Severity of
hyperplasia gradually increased with time of exposure. Papillomas
were seen beginning with week 4. Neutrophil infiltration was also
observed. An increase in the labelling index paralleled the
severity of the lesions observed.
In a similar study (Hirose et al., 1986b), fifteen hamsters
were given a powdered diet containing 1% BHA for 20 weeks with 3
being injected with radioactive thymidine just prior to sacrifice
for labelling index determination. Again, body weight depression
was seen in animals fed BHA. Thickening of the forestomach
epithelium with white, keratin-like substance was also noted. All
of the animals displayed severe hyperplasia with 60% additionally
showing papillomatous lesions. The labelling index in the
forestomach was almost three times that in control animals. No
changes were seen in any other organ examined.
These observations were extended to 104 weeks exposure to 1 or
2% BHA (powder) in the next study (Masui et al., 1986b; Ito &
Hirose, 1987). Groups of 10 male hamsters were observed at 8 week
intervals. In these animals, hyperplasia of the forestomach was
observed in all animals in both exposure groups starting at 8 weeks
of exposure. Papillomas were likewise seen in almost all animals in
the high level exposure group beginning at week 8, and in the low
level group from week 16. Hyperkeratosis and downward growth of the
tumour into the submucosa was often observed. Squamous cell
carcinomas were observed in both treated groups beginning in week
64. They were well differentiated and an invasion of the liver was
seen in one case. After 104 weeks of exposure, hyperplasia was seen
in 100%, 96% and 17% of the 2%, 1% and control animals, respectively.
Likewise, 95%, 98% and 0% of these groups showed papillomas, and 10%,
7% and 0%, respectively, carcinomas.
Guinea pigs
Guinea pigs fed a diet containing 1% BHA for 20 months did not
exhibit any gross changes in the stomach (Ito & Hirose, 1987).
Dogs
Groups of 29 male and 30 female beagle dogs (also lacking a
forestomach), were fed 0, 1.0 or 1.3% BHA for 180 days (Ikeda
et al., 1986; Moch, 1986). Food consumption and body weight gain
were reduced in the animals receiving the highest level of BHA and
there was a liver weight increase in animals receiving BHA at
either dose. Ultrastructural examination of the livers of animals
on BHA showed proliferation of smooth endoplasmic reticulum and
hepatocytic cytoplasmic myelinoid bodies. Light and electron
examination of the stomach and lower esophagus showed no
proliferative or hyperplastic lesions and no change in the number
of cells.
BHA at 0.25, 0.5 or 1.0% was fed to groups of 3-4 male or
female beagle dogs for six months in a second study (Tobe et al.,
1986). A dose-related retardation of growth was reported. Liver
weights were increased but no histopathological changes were seen
in that organ. There were no alterations in the stomach mucosa and
there was no induced change in the mitotic index of the squamous
epithelium of the distal esophagus.
In a third report, adult beagle dogs fed a diet containing
0-100 mg BHA/kg bw/day for one year exhibited no histopathological
changes in any tissue examined (Ito & Hirose, 1987).
Pigs
Three different experiments were conducted involving pregnant
young adult specific pathogen free (SPF) Danish Landrace gilts
(Olsen, 1983; Wurtzen & Olsen, 1986; Moch, 1987). Combining the
data of the three studies, groups of 9-13 pigs were fed pellets
containing 0, 0.5%, 1.9% or 3.7% BHA (0, 50, 200, or 400 mg BHA/kg
bw/day, respectively) for the first 110 days of pregnancy.
Epithelial changes in the esophageal part of the stomach were
similar for test and control groups. No papillomas and no changes
in the oesophageal part of the stomach were similar for test and
control groups. No papillomas and no changes in the glandular part
of the stomach were reported. Linear yellow-brown, rough epithelium
was seen in the entire length of the esophagus in a few pigs in the
middle and high dose groups. In the first of the three experiments,
one of the three animals in the middle dose group and two of three
in the high dose group had esophageal lesions seen at gross
necropsy. All three of these animals had histopathological
examination of the esophageal lesions seen at gross necropsy and
were diagnosed as having esophageal hyperplasia. In addition, one
other, animal in the middle dose group had esophageal hyperplasia
noted microscopically, although no lesion was seen at time of gross
necropsy. In the second experiment, one of the three animals in the
high dose groups had an esophageal lesion noted at gross necropsy.
In the second experiment, one of the three animals in the high dose
groups had an esophageal lesion noted at gross necropsy. No
histological examination of this animal was performed, however. In
the third experiment, one of the seven animals in the middle dose
group and one of four animals in the high dose group had a lesion
of the esophagus seen at gross necropsy. None of these animals had
histological examination of the reported lesions. The authors
concluded on the basis of the one middle dose level pig and the two
high dose level pigs of the first study that BHA might have an
effect on esophageal epithelium.
Monkeys
Groups of 8 female cynomogus monkeys were given 0, 125 or
500 mg BHA/kg bw by gavage in corn oil 5 times/week for 20 days,
after which the dose was halved and continued for a total of 85 days
(Iverson et al., 1986; Ito & Hirose, 1987). Although there were
transitory dose-related changes in blood clinical values during the
first part of the exposure, no value differed from the normal range
nor were any abnormal fibroscopic observations made during the course
of the exposure. Histopathological examination at the end of the
exposure period showed no treatment related effects, although the
mitotic index in the basal cell layer of the squamous epithelium of
the distal esophagus was elevated by a factor of 1.9. Liver weight was
also increased in animals exposed to BHA in a dose-related manner.
Special studies on the effect of the molecular structure
In Syrian golden hamsters (Hirose et al., 1986d), the
observation was made that, in parallel with its relative power as
an antioxidant, 3- tert-butylhydroxyanisole (3-BHA) is
significantly better able to cause biological damage than
2- tert-butylhydroxyanisole (2-BHA). Using radioactive labelling
in F344 rats, however, 2-BHA has been shown to be incorporated into
cells of the forestomach after 1 week of treatment at approximately
twice the level of 3-BHA (Hirose et al., 1987b). Food grade BHA
is primarily composed of 3-BHA.
Three papers have reported investigations attempting to
discover which part(s) of the chemical structure of BHA is critical
to its actions. By comparing the ability of 13 structurally related
phenolic compounds to cause lesions in the forestomach of Syrian
golden hamsters after 20 weeks exposure, one report (Hirose et
al., 1986b) concluded that the phenolic compounds must have both
at least one hydroxy and exactly one tert-butyl substituent to
have strong activity in inducing forestomach tumours. Other
structural variations produce their own spectra of biological
consequences (Ito & Hirose, 1987). In the other two papers on this
subject (Altmann et al., 1985; Altmann et al., 1986) in which
Wistar rats were fed one of 12 chemicals related to BHA or BHA
itself, the conclusion is made that the critical part of the BHA
structure is the methoxy group.
Special studies on potentiation or inhibition of carcinogenicity
The ability of BHA to interact with known carcinogens has been
studied extensively (Lindenschmidt et al., 1987; Hirose et al.,
1986c; Ito et al., 1986b; Tsuda et al., 1987; Fukushima et
al., 1987; Hirose et al., 1986a; Tsuda et al., 1984; Newberne
et al., 1986; Takahashi et al., 1986; Moore et al., 1986; Ito
et al., 1985; Williams et al., 1986; Masui et al., 1986c; and
Chung et al., 1986). Several strains of rats and mice of both
sexes have been fed BHA at various concentrations for various durations
either before, after, or before, during and after treatment by various
routes with various concentrations of several carcinogens administered
for various lengths of time. The net effect of BHA plus the carcinogen
has been examined in thirteen organs. What has been found is that BHA
potentiates, inhibits, or has no effect on the ability of the
carcinogen to act, depending on the exact chemical being tested and
the circumstances under which the test is being conducted.
COMMENTS
Studies have been conducted that provide additional
information on the proliferative changes observed in the
forestomach of rats fed BHA. No new studies on the effect of BHA on
the stomach and esophagus of species that do not have a
forestomach, e.g., pigs or monkeys, were conducted. The data show
that continuous exposure of the rat forestomach to 2% BHA in the
diet for 6-12 months is necessary to produce squamous cell
carcinoma. The data also show that the induction of mild
hyperplasia can occur at levels of 0.125% of BHA in the diet but
not at a level of 0.1% BHA. After reevaluating the data in pigs, it
was concluded that the evidence that BHA produces hyperplasia in
the esophagus of the pig is questionable. Moreover, these
presumptive effects in pigs were reported to occur at levels of BHA
significantly higher than those that produced the confirmed effects
in the rat forestomach. Considering the absence of any significant
adverse effects in two dog studies, it was concluded that further
investigations in animals without forestomachs are not required.
The human relevance of the rat studies, while inherently
questionable because the target tissue in the rat has no human
counterpart, cannot readily be ignored. Based on the dose
dependence and reversibility of the lesions produced in the rat,
discussed previously in the 1986 monograph (Annex 1, reference 74)
and since confirmed by more recent studies, it was concluded that
an ADI can be established.
EVALUATION
Level causing no toxicological effect
Rat: 0.1% in the diet, equivalent to 50.0 mg/kg bw/day.
Estimate of acceptable daily intake for man
0-0.5 mg/kg bw.
Further work or information
Desired
Submission of the results of an ongoing reproduction study.
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