SAFROLE
Explanation
Safrole has not previously been evaluated by the Joint FAO/WHO
Expert Committee on Food Additives.
Safrole (1,2-methylenedioxy-4-allylbenzene) is the principal
constituent of oil of sassafras and a minor constituent of many other
essential oils. The related substance, isosafrole (1,2-methylene-
dioxy-4-propenylbenzene), also occurs as a minor constituent of many
essential oils with a distribution similar to that of safrole. Another
related substance, dihydrosafrole (1,2-methylenedioxybenzene-4-
propylbenzene), is not known to occur naturally but is formed in the
production of piperonyl butoxide (International Agency for Research on
Cancer, 1976).
BIOLOGICAL DATA
BIOCHEMICAL ASPECTS
Absorption, distribution and excretion
Safrole was absorbed from the gastrointestinal tract by passive
diffusion, with the absorption kinetics apparently dependent on its
lipid solubility as determined in an in situ perfusion method in the
rat (Fritsch et al., 1975a). In this same procedure, safrole, at a
level of 2 mg/ml of perfusion medium, reduced the absorption of
glucose and methionine but not butyric acid (Fritsch et al., 1975b).
Biotransformation
Basic ninhydrin-positive substances were excreted in the urine of
male rats treated with safrole or isosafrole in doses of 75-300 mg/kg
i.p. These substances were not seen when dihydrosafrole was
administered in similar doses. These substances readily decomposed to
carbonyl-containing compounds. The substances were not identified in
this study (Oswald et al., 1969), but in a later study (Oswald et al.,
1971) the safrole metabolites were identified as tertiary amino
propiophenones, 3-N,N-dimethylamino-1-(3',4'-methylenedioxyphenyl)-1-
propanone (I), 3-piperidyl-1-(3',4'-methylenedioxyphenyl)-1-propanone
(II), 3-pyrrolidinyl-1-(3',4'-methylenedioxyphenyl)-1-propanone (III).
I and III are excreted by the rat and II by both the rat and
guinea-pig. These safrole metabolites are Mannich bases which are
believed to be formed by oxidation of the allyl group to yield a vinyl
ketone which condenses with an available amine (McKinney et al.,
1972). I, II and III were competitive inhibitors of rat liver
mitochondrial monoamine oxidase with benzylamine-HCl as a substrate.
Metabolite III inhibited rat liver, kidney, and brain monoamine
oxidase with tyramine HCl or serotonin as substrates (Bangdiwala &
Oswald, 1976).
Thin-layer chromatography of rat urine and bile following
intravenous injection of safrole, dihydrosafrole and isosafrole showed
the metabolites were largely eliminated in the bile after this route
of administration. Metabolites were not identified (Fishbein et al.,
1967).
C14O2 was excreted by mice and flies treated with safrole or
dihydrosafrole labelled on the methylene C. Microsome preparations
from the liver of treated mice or abdomens of treated flies yielded
formate-C14, indicating a cleavage of the methylenedioxy ring (Casida
at al., 1966).
The major urinary metabolites of safrole administration i.p.
in the rat or guinea-pig were: 1,2-dihydroxyl-4-allylbenzene,
1'-hydroxysafrole (1,2-methylenedioxy-4-(1-hydroxyallyl)
benzene) (HOS), 1,2-methylenedioxy-4-(2,3-dihyroxypropyl)
benzene, 1,2-dihydroxy-4-(2,3-dihydroxypropyl)benzene,
2-hydroxy-3-(3,4-methylenedioxyphenyl) propanoic acid, and
3,4-methylenedioxybenzoylglycine. Safrole oxide, administered i.p.,
produced the same metabolites as safrole, with the exception of the
first two compounds and the benzoylglycine. This indicates that the
metabolites containing a propyl side chain are probably formed by
conversion of the allyl side chain with safrole epoxide as an
intermediate. Some unchanged safrole epoxide was also found after
administration of this compound. A small amount of 1,2-methylenedioxy-
4-(1,2,3-trihydroxypropyl)benzene was found in rats only (Stillwell et
al., 1974).
Small doses of [C14]-safrole were absorbed rapidly and excreted
almost completely via the urine in 24 hours in both man (doses
0.165 mg or 1.655 mg) and the rat (dose 0.63 mg/kg). A large dose
(750 mg/kg) in the rat resulted in a decrease in the rate of
elimination, only 25% being excreted in 24 hours, and plasma and
tissue levels of safrole and its metabolites were elevated for 48
hours. 1,2-dihydroxy-4-allylbenzene was the main urinary metabolite in
both species. The metabolites HOS and 3'-hydroxy-isosafrole were
detected in the urine of the rat but not in man (Benedetti et al.,
1977).
Rats, mice, hamsters and guinea-pigs excreted a conjugated form
of HOS in the urine after treatment with safrole i.p. The HOS
accounted for 30% or more of the safrole dose in male mice and 1-3% of
the safrole dose in the other species. Pretreatment with phenobarbital
or 3-methylcholanthrene (3-MC) increased HOS excretion tenfold in
rats; phenobarbital had no influence on HOS excretion in hamsters or
guinea-pigs. Bile-duct ligation had little effect on the excretion of
HOS. The conjugated HOS was cleaved by commercial beta-glucuronidase
preparations. A small amount of 3'-hydroxy-isosafrole was found in the
urine; it was believed to arise from rearrangement of HOS. When HOS
per se was administered to rats, about 40% was excreted as HOS;
pretreatment with phenobarbital or 3-MC did not markedly alter the
amount excreted. The percentage of safrole or HOS excreted as HOS
after p.o. administration was similar to that observed after i.p.
administration. When safrole was administered in the diet, excretion
of HOS was 5-10% of the daily dose during the first 18 days and 3-4%
thereafter. Concurrent administration of 0.1% sodium phenobarbital
resulted in an average of 7% of the safrole excreted as HOS over an
11-week period. Forty per cent. of an oral HOS dose was excreted as
HOS over a seven-month period (Borchert et al., 1973b).
HOS was not detected in sassafras (Sassafras albidum) oil
(Sethi et al., 1976).
Male mice were treated with 185 µmol safrole/100 g bw. Adult mice
excreted 46% of the dose as HOS, while 21-day-olds excreted only 12%
as HOS (Drinkwater et al., 1976).
Administration of tritiated HOS to rats and mice resulted in
labelled liver macromolecules with specific activity generally in the
order rRNA=protein, DNA; the nucleosides were not the labelled
compounds. Hepatic macromolecules isolated from adult female rats had
55-65% of the specific activities of those obtained from male rats.
The specific activities of liver macromolecules isolated from adult
female mice were 10-20 times greater than those from pre-weanling
(first three weeks of age) male and female mice. HOS was metabolized
by rat and mouse liver cytosols in a 3'-phospho-adenosine
5'-phosphosulfate-dependent reaction to an electrophilic reactant
presumed to be the sulfuric acid ester. HOS was also oxidized by rat
and mouse liver microsomes to 1'-hydroxysafrole-2',3'-oxide in a
reduced nicotinamide adenine dinucleotide phosphate-dependent
reaction. Electrophilic reactivities of various possible safrole
derivatives in vitro with nucleosides was determined to be in the
order of 1'-oxosafrole, 1'-acetoxysafrole (ACOS), 1'-acetoxysafrole-
2',3'-oxide, 1'-hydroxy-2',3'-oxide, safrole-2',3'-oxide,
1'-oxosafrole-2',3'-oxide. Neither ACOS nor 1'-oxosafrole formation
were detected in vitro, but ACOS reacted with guanosine-5'-
monophosphate to yield a reaction product identified as
0,6-(isosafrol,3'-ul) guanylic acid which in turn yields
3'-hydroxyisosafrole. 1'-oxosafrole has not been isolated as a
metabolite, but small amounts of condensation products of the ketone
with amines appear in the urine of animals treated with safrole or HOS
(Wislocki et al., 1976).
HOS and 1'-hydroxyallylbenzene, metabolites of the hepatotoxic
safrole and the non-hepatotoxic allylbenzene respectively, are
metabolized in a similar manner. HOS rapidly rearranged under acid
conditions to 3'-hydroxyisosafrole, whereas the rearrangement of
1'-hydroxyallylbenzene to cinnamyl alcohol was slow. It was postulated
that the methylenedioxy group stabilized a carbonium ion by a
resonance effect and such resonance stability increases the
electrophilicity of the compound and thus its tissue reactivity
(Peele & Oswald, 1978).
By the use of mass spectroscopy, the following metabolites
of safrole were identified in rat liver epithelial cell cultures:
eugenal, 2-allylcatechol, 4-(2',3'-epoxypropyl)catechol, 2',3'-
epoxysafrole, 2',3'-dihydro-2',3'-dihydroxysafrole, HOS, and
3'-hydroxyisosafrole. HOS, 1',2'-epoxyisosafrole, and 1',2'-dihydro-
1',2'-dihydroxysafrole were identified as metabolites of isosafrole in
this system. Cultures of liver cells from female rats produced greater
quantities of the safrole metabolites than those from male rats
(Janiaud et al., 1976; Delaforge et al., 1977). The epoxy-diol pathway
for metabolizing safrole was also found in the adrenal of the rat
(Doumas & Maume, 1977). With 2',3'-epoxysafrole as the parent compound
the cell cultures produced 2',3'-epoxy(4-allyl)catechol (Delaforge et
al., 1977). The following epoxy compounds were identified in the urine
of rats treated with safrole: 1,2'-epoxyisosafrole, 2',3'-
epoxyallylcatechol, 1',2'-epoxyallylphenol, 2',3'-epoxyallylphenol,
and 1'-hydroxy-2',3'-epoxysafrole glucuronide (Delaforge et al., 1977;
Levi et al., 1977).
Effects on enzymes and other biochemical parameters
Safrole, when combined with piperonyl butoxide and other
methylenedioxyphenol derivatives, inhibited liver aminopyrine
demethylase and biphenyl-4-hydroxylase activity in microsome
preparations from mice pretreated with doses of the test compounds
which were not individually inhibitive (Friedman et al., 1971).
Safrole and isosafrole, administered to rats, produced liver
hypertrophy with increases in hepatic biphenyl hydroxylase, nitro
reductase, glucuronyl-transferase, and cytochrome P-450. Some of the
actions of safrole were inhibited by actinomycin D and some were not,
indicating the increased enzyme activity comes both from new enzyme
protein synthesis and changes in existing enzyme. Addition of safrole
in vitro to a rat liver microsomal preparation did not increase
enzyme activities (Parke & Rahman, 1970). Administration of safrole
and isosafrole also gave rise to a new redox difference absorption
spectra in liver preparations which was attributed to induction of a
new hepatic microsomal haemoprotein (Parke & Rahman, 1971). Aryl
hydrocarbon hydroxylase was induced in liver, lung, kidney, and gut in
rats fed a diet containing 0.25% isosafrole. There were also increases
in hepatic biphenyl 2-hydroxylase and 4-hydroxylase activities,
microsomal protein, liver weight, cytochrome P-450 content, and
apparent cytochrome b5 content, and a decrease in hepatic aniline
4'-hydroxylase activity (Lake & Parke, 1972).
Urinary excretion of 3- and 5-hydroxy derivatives of
2-acetoamidofluorene (2-AAF) was increased by pretreatment of young
and adult male rats with three doses of safrole (100 mg/kg per day)
but not by similar treatment with isosafrole. Safrole also caused an
increased excretion of the N-hydroxy derivative in adult rats. A
single dose of 3-MC caused greater increases than safrole. Assay of
hydroxylated derivatives of 2-AAF formed by liver microsomal
preparations from rats pretreated as described showed increases in
amount of ring-hydroxylated derivatives in the order of 3-MC,
isosafrole, safrole and in the amount of N-hydroxylated derivative in
the order 3-MC, safrole, isosafrole. Administration of ethionine
largely inhibited the increased hydroxylation from safrole and
isosafrole and methionine reversed the inhibition, indicating that
safrole or isosafrole pretreatment produces new synthesis of liver
microsomal hydroxylases. Pretreatment of hamsters with safrole had
little effect on hydroxylation by liver preparations of this species,
while pretreatment with isosafrole tended to inhibit both N- and some
ring-hydroxylation (Lotlikar & Wasserman, 1972).
Safrole, administered at a dietary level of 2% for two weeks to
rats, induced a P-450 cytochrome, similar to that seen with 3-MC, and
caused the formation of a stable safrole-metabolite-cytochrome P-450
complex. The same response was obtained when safrole was incubated
with control microsomes, NADPH and oxygen. The safrole metabolite
could be displaced from the complex in vitro by safrole, biphenyl
and ethylbenzene (Elcombe et al., 1975).
Safrole, at a level of 0.67 mM in the medium, inhibited protein
synthesis in isolated adult rat hepatocytes by 75% (Gwynn et al.,
1979). Safrole did not induce unscheduled DNA synthesis in HeLa cells
(Martin et al., 1978). Safrole (640 mg/kg i.p.) inhibited mouse
testicular DNA synthesis, as measured by uptake of tritium-labelled
thymidine, by 60% (Friedman & Staub, 1976). Adult rat liver epithelial
cells, maintained in culture for six months, and dosed with safrole
(0.5 mg in ethanol) four times in eight days, showed high toxicity
followed by many mitoses. This resulted in a mixed type of cell in the
culture. Some of the cells maintained their epithelial structure, but
most appeared transformed (Janiaud & Padieu, 1977).
The induction of resistant hepatocytes in vivo in rats was
investigated as a short-term test for carcinogens. Safrole was
positive in this test but required three doses (150 mg/kg per dose),
while other carcinogens such as 2-AAF and N-butyl-N-nitrosourea were
positive with a single dose (Tsuda et al., 1980).
Sequential biochemical and morphological observations on the
livers of female rats fed 0.25% safrole in the diet for up to 85 weeks
showed an early increase in liver weight and activity of drug-
metabolizing enzymes. With continued administration, the enzyme
inductive effect disappeared; drug-metabolizing activity was inhibited
although liver enlargement continued and concentrations of NADPH-
cytochrome c reductase, cytochrome b5 and microsomal protein were
elevated. Histochemical and morphological alterations became apparent
after the loss of the inductive response (Crampton et al., 1977).
Safrole, in doses of 10 and 295 mg per rat per day,
dihydrosafrole, in doses of 15 and 360 mg per rat per day, isosafrole,
in doses of 15 and 375 mg per rat per day, and sassafras oil, in doses
of 50 and 1130 mg per rat per day, were administered s.c. for the
first seven post-operative days to partially hepatectomized rats. All
four substances significantly increased the amount of liver
regenerated. The same effect was seen when safrole, dihydrosafrole and
isosafrole, at 0.25% of the diet, were fed for 10 days following
partial hepatectomy. Sassafras bark tea, at 1.5 or 7% of the diet,
also increased the amount of liver regenerated (Gershbein, 1977).
TOXICOLOGICAL STUDIES
Special studies on carcinogenicity
Short-term studies
Mouse
Topical application of 1.8 µmol of the following substances in
female mice twice weekly for seven weeks, with subsequent twice weekly
applications of phorbol-12,13-didecanoate for the remainder of the
24-week experiment, did not produce a significant increase in the
incidence of skin papillomas: safrole, HOS, ACOS, 1'-methoxysafrole,
dihydrosafrole, 1'-hydroxydihydrosafrole, 1'-acetoxydihydrosafrole,
1'-methoxydihydrosafrole. Groups of 25-30 were used. A positive
control of 7,12-dimethylbenz(a)anthracene (DMBA), applied once, did
produce a highly significant increase in skin papilloma incidence
(Borchert et al., 1973a).
Groups of 30 female mice were treated topically with the
following substances in one experiment: 1'-hydroxysafrole-2',3'-oxide,
HOS, ACOS (1.5) by moles, 1'-oxosafrole, 1'-oxosafrole (1.5) by moles,
DMBA (0.2) by moles, and in 0.1 ml acetone solvent; and the following
substances in a second experiment: 1'-hydroxysafrole-2',3'-oxide,
safrole-2',3'-oxide, 1'-acetoxysafrole-2',3'-oxide, HOS, ACOS,
1'-oxosafrole, safrole, and solvent. The dose was 30 µmol per
application except where another dose is indicated in parentheses
after the compound name. The dose was applied five times a week for
six weeks and then croton oil (total dose of 0.5 mg in 0.1 ml) acetone
was applied twice weekly until the experiment was terminated at 36
weeks. DMBA was applied only once. DMBA, 1'-hydroxysafrole-2',3'-
oxide, 1'-acetoxysafrole-2',3'-oxide, and safrole-2',3'-oxide were the
only compounds which produced an increased incidence of papillomas.
The incidence was 1/60 in the solvent controls, 5/30 with
1'-acetoxysafrole-2',3'-oxide, 6/30 with safrole-2',3'-oxide, 20/60
with 1'-hydroxysafrole-2',3'-oxide, and 20/30 with DMBA (Wislocki et
al., 1977).
Mice, six to eight weeks of age, were given safrole i.p. three
times a week for four weeks and killed 24 weeks after the first
treatment. The doses of safrole were (total) 0.9 and 4.5 g/kg per
mouse, and groups of 15 mice per sex and dose were used. Safrole did
not induce pulmonary tumours under the conditions of this study,
although a number of chemotherapeutic agents including uracil mustard
were active (Stoner et al., 1973).
Long-term studies
Mouse
Mice were treated with safrole during the lactation period and
examined for the development of tumours through 49-53 weeks. Safrole
was administered s.c. in four doses on the 1st, 7th, 14th and 21st day
of age. Of 12 male mice surviving to weaning, 50% had hepatomas at
49+ weeks with a total dose of 0.66 mg. With a total dose of 6.6 mg
safrole and 31 males surviving at weaning, 58% had hepatomas at
termination. Hepatomas were not observed in safrole-treated females.
The higher safrole dose also resulted in a slight increase in
incidence of multiple pulmonary adenomas over that in the controls,
the occurrence of pulmonary adenocarcinomas, which were not observed
in the controls, and a slight increase in the incidence of lymphocytic
and reticular lymphomas. Doses of safrole of 11 and 110 mg on day 1
were acutely toxic (Epstein et al., 1970).
Perinatal male mice were given s.c. injections of the following
substances on days 1, 8, 15 and 22 of age for a total dose of
3.18 µmol (= 0.52 mg safrole): safrole, HOS, ACOS and 1'-oxosafrole.
The solvent was trioctanoin and a control group receiving trioctanoin
was included in the study. The survivors were necropsied at 16 months.
Group size at necropsy ranged from 31 to 45. Incidence of liver
carcinomas was: safrole - 51% (14% multiple), HOS - 82%
(55% multiple), ACOS - 74% (50% multiple), 1'-oxosafrole - 16%
(0% multiple), control - 13% (5% multiple) (Wislocki et al., 1977).
Preweanling mice were treated with safrole, HOS, ACOS or solvent
(trioctanoin) s.c. in a total dose of 9.45 µmol given in four
injections on days 1, 7, 14 and 21 of age. Groups of 45-50 per sex and
compound were established at weaning. Surviving males were killed at
12-14 months and surviving females at 16 months. The incidence of
liver tumours in the males was 8, 40, 84 and 82% for the controls,
safrole, HOS and ACOS, respectively. These tumours were multiple in
more than half of the HOS- and ACOS-treated mice but in only 17% of
the safrole-treated mice. The incidence of liver tumours in females
was 0, 0, 16 and 9% for the controls, safrole, HOS and ACOS treated
mice, respectively (Borchert et al., 1973a).
In a study similar to the one described above, a total dose of
4.43 µmol of HOS was administered s.c. to preweanling male mice. The
dose was divided and administered on days 1, 8, 15 and 22 of age. The
test groups contained 60 mice and the control group 66. The mice were
sacrificed at 15 months of age. Six control mice had liver carcinomas;
none were multiple. Thirty test mice had liver tumours; these tumours
were multiple in 20 of the mice (Drinkwater et al., 1976).
Safrole, dihydrosafrole and isosafrole were administered to two
hybrid strains of mice orally starting at day 7 of age. The test
substances were administered by stomach tube daily (464, 215 and 464
mg/kg, respectively) until the mice were weaned at four weeks of age
and in the diet thereafter (1112, 517 and 1400 ppm (0.1112, 0.0517 and
0.14%), respectively). Study termination was at approximately 18
months of age. Eighteen mice of each sex and strain were selected at
weaning for retention in the study. Hepatomas were observed in 46
safrole-treated mice, in 19 dihydrosafrole-treated mice and in eight
isosafrole-treated mice. Pulmonary tumours and lymphomas were also
observed in the dihydrosafrole- and isosafrole-treated mice. The
incidences were 18 and three, respectively, with dihydrosafrole and
four and two, respectively, with isosafrole. The overall incidence of
the various tumours in control mice was 14/378 with hepatomas, 30/378
with pulmonary tumours and 18/378 with lymphomas. Hepatoma incidence
tended to be higher in females than males with safrole but higher in
males than females with dihydrosafrole and isosafrole (Innes et al.,
1969). Hyperplasia and carcinomas of the forestomach were increased in
the females of both strains and in the males of one strain, seven with
dihydrosafrole. The incidence was 88 and 78% for the females and 41%
for the males. Maximum incidence in the controls was 28% in the
females of one strain (Reuber, 1979).
Safrole (120 µg/g bw per treatment p.o.) was administered to:
(a) pregnant mice (four times during gestation on days 12, 14, 16 and
18); (b) lactating mothers (12 times every second day following
parturition); (c) four-week-old offspring (180 times twice weekly for
90 weeks); (d) a + b combination treatment; and (e) a + b + c
combination treatment. All survivors were killed at 94 weeks of age.
Exposure to safrole in utero produced renal epithelial tumours in 7%
of the female offspring; none of the other experimental or control
animals had these tumours. Male offspring (34%) nursed by mothers
treated with safrole during lactation had hepatocellular tumours but
not female offspring. However, when safrole was administered post-
weaning, a significant increase in hepatocellular tumours was observed
in females (48%) but not in males (8%). Of the tumours observed in the
females, 86% were hepatocellular carcinomas of which 42% had pulmonary
metastases (Vesselinovitch et al., 1979).
Safrole and HOS were fed in the diet to adult mice at final
levels of 0.50 and 0.55%, respectively, for 12 months. The survivors
were sacrificed at 17 months. A control group was included and the
number of mice per sex per group ranged from 45 to 55. It was
necessary to increase the dietary level of HOS gradually because the
0.55% level was acutely toxic in uninitiated females. The acute
toxicity was associated with marked liver toxicity. Both safrole and
HOS produced depression of weight gain and increased mortality by the
end of 12 months of treatment. No liver tumours were observed in
either the male or female controls. Eleven male and 25 female safrole-
treated mice had liver tumours. No male mice and 30 female HOS-treated
mice had liver tumours. There were also five s.c. angiosarcomas in the
female HOS-treated mice (Wislocki et al., 1977).
The carcinogenicity of dietary safrole was compared to that of
HOS in one experiment and to that of ACOS in a second experiment in
male mice. Groups of 25-40 adult mice were used for the test and
control groups. They were given the test diets for 13 months and then
control diets for an additional three months. In the first experiment,
safrole was fed at 0.4 and 0.5% of the diet and HOS at equimolar
amounts of 0.44 and 0.55%. Mortality between 12 and 16 months was much
higher in the HOS groups than in the safrole groups. Twenty-seven per
cent. of the 12-month survivors of safrole feeding had hepatocellular
carcinomas compared to 14% for HOS and 10% in the controls. In
addition, 20 mice fed HOS (seven on 0.44%, 13 on 0.55%) had
interscapular sarcomas. One control and two safrole-fed mice had
sarcomas in the same area. In the second experiment, safrole was fed
at 0.4% and ACOS at 0.03% and 0.05 or 0.5% (the text and table differ
on this level). Administration of the higher dose was terminated at
seven months because of increased mortality; the 0.03% level also
caused increased mortality. Three hepatocellular carcinomas were
observed in the controls, none in the ACOS-treated groups and four in
the safrole-treated group. No interscapular sarcomas were found in the
second experiment (Borchert et al., 1973a).
Rat
The carcinogenicity of two possible safrole metabolites,
1'-oxosafrole and ACOS, was compared by s.c. injection. 18.6 µmol of
the test compound dissolved in 0.2 ml trioctanoin were injected twice
weekly for 12 weeks. Groups of 18 rats were used and two experiments
performed, with one terminated at 19 months and the other at 21
months. The combined results of the two experiments showed one animal
with injection-site sarcomas with the vehicle, three with
1'-oxosafrole and 11 with ACOS (Wislocki et al., 1977).
The ability of safrole and isosafrole and some derivatives of
these two substances to induce injection-site sarcomas was compared in
two experiments. 18.6 µmol of the test compound in 0.2 ml trioctanoin
was injected s.c. twice weekly for 20 injections and the animals
killed after 17 or 18 months. The test compounds and number of rats
(all males) per compound in the pooled experiments were: controls,
safrole, HOS, ACOS, 3'-hydroxyisosafrole and 3'-acetoxyisosafrole - 36
rats; isosafrole, 3'-methoxyisosafrole, 2'-bromoisosafrole and
1'-methoxysafrole - 18 rats. Eleven injection-site sarcomas were
observed with ACOS, three with HOS and two with 3'-bromoisosafrole.
None were observed in the controls or in the other test groups
(Borchert et al., 1973a).
Groups of 18 male rats were fed, in the diet, 0.22% safrole,
0.25% 1'-oxosafrole or 0.25% HOS for 17 months, 0.55% HOS for 10
months, 0.5% safrole for 22 months, or 0.5% safrole plus 1% sodium
barbital (to stimulate hepatic microsomal enzymes) in the drinking-
water for 22 months. There were separate control groups for the
10-17-month and the 22-month feeding periods plus a control group
which received 1% sodium barbital. All survivors were killed at 22
months. No hepatic carcinomas or forestomach tumours were observed in
the control groups, with 0.22% safrole or 0.25% 1'-oxosafrole. One
hepatic carcinoma was found in the 1% sodium barbital group. In the
0.25% HOS group there were seven rats with hepatic carcinomas and two
with papillomas of the forestomach. In the 0.5% safrole group there
were three hepatic carcinomas; when rats on this safrole level also
received 1% sodium barbital in the drinking-water there were 12
hepatic carcinomas. In the 0.55% HOS group there were 16 with hepatic
carcinomas, two with papillomas and two with carcinomas of the
forestomach (Wislocki et al., 1977).
Three rat feeding studies were performed to compare the
carcinogenicity of safrole, HOS and ACOS administered in the diet for
8.5-11 months. The rats were killed after 12 or 16 months on
experiment. The pooled results are as follows (only male rats were
used): controls, 50 rats, no tumours of liver or forestomach; 0.3%
safrole, 18 rats, no tumours of liver or forestomach; 0.5% safrole, 50
rats, one with hepatic adenoma, one with hepatic carcinoma and no
forestomach tumours; 0.5% HOS, 30 rats, 27 with hepatic carcinomas and
12 with forestomach papillomas; ACOS, 15 rats with doses from 0.41 to
0.68%, none with hepatic carcinomas and 10 with forestomach
papillomas; and 0.41% ACOS, 18 rats, one with hepatic carcinoma, 11
with forestomach papillomas and one with squamous cell carcinoma of
the forestomach. The hepatic tumours in the HOS rats were larger and
more numerous than those in the safrole rats. The livers of the HOS
rats also showed necrosis, fibrosis and deviations from normal
architecture, while the livers of the safrole rats showed little
deviation from normal. The safrole and HOS rats grew more slowly than
the controls but survival was good. ACOS, at equivalent dose levels,
caused greater depression of growth and early deaths (Borchert et al.,
1973a).
Special studies on mutagenicity
Safrole was generally inactive in mutagenicity studies in various
strains of S. typhimurium with or without metabolic activation
(McCann et al., 1975; Poirier & de Serres, 1979; Cheh et al., 1980;
Dorange et al., 1977b; Swanson et al., 1979; Wislocki et al., 1977)
but has occasionally been reported to be weakly positive (Purchase et
al., 1978; Poirier & de Serres, 1979; Green & Savage, 1978). A special
activation system was described which yielded positive results with
safrole in strain TA-1535 (Dorange et al., 1978). Dihydrosafrole and
isosafrole (Wislocki et al., 1977) and HOS (McCann et al., 1975;
Poirier & de Serres, 1979; Dorange et al., 1977a; Drinkwater et al.,
1976; Wislocki et al., 1977) were also negative in this test. ACOS was
generally positive (McCann et al., 1975; Poirier & de Serres, 1979;
Drinkwater et al., 1976; Wislocki et al., 1977). 3'-hydroxyisosafrole,
3'-acetoxyisosafrole and 1'-oxosafrole were not mutagenic for strains
TA-100 and TA-1535 with or without metabolic activation, but the
2',3'-oxides of safrole, HOS, ACOS and 1'-oxosafrole were directly
mutagenic for these strains (Wislocki et al., 1977). The mutagenic
activity of these 2',3'-oxides (Swanson et al., 1979) and of
2',3'-epoxysafrole (Dorange et al., 1977a) for TA-1535 was confirmed.
The safrole ninhydrin-positive metabolite II was positive with
metabolic activation in TA-1530 and TA-1532 (Green & Savage, 1978).
Safrole ninhydrin-positive metabolites I and III were inactive with or
without metabolic activation (Green & Savage, 1978).
Safrole was positive in the following short-term tests for
mutagenesis: mammalian cell transformation in culture, Rabin's test-
degranulation of rough endoplasmic reticulum from rat liver, and
mouse-sebaceous gland suppression test; and negative in the
tetrazolium reduction and tissue reaction to s.c. implants in mice
(Purchase et al., 1978). Safrole was positive in in vitro mutagenic
assays with E. coli and S. cerevisiae and in the in vivo
intraperitoneal host-mediated assay with S. typhimurium strain
TA-1535 or S. cerevisiae (Poirier & de Serres, 1979). Safrole was
positive in the host-mediated assay with strains TA-1950 and TA-1952
(Green & Savage, 1978).
HOS and ACOS were both positive in in vitro tests with
S. cerevisiae; ACOS was positive with E. coli, but HOS was
negative (Poirier & de Serres, 1979). The safrole ninhydrin-positive
metabolite II was positive in the host-mediated assay using
S. typhimurium strains TA-1950 and TA-1952; the ninhydrin-positive
metabolites I and III were negative in the host-mediated assay with
strains TA-1950 and TA-1952 (Green & Savage, 1978).
Special studies on pharmacology
Safrole at a dose of 20 mg/kg bw i.p., approximately doubled the
sleeping time of mice treated with sodium pentobarbital. Isosafrole in
the same dose was slightly less active. Neither substance had a
significant effect on ethanol sleeping time (Seto & Keup, 1969).
Acute toxicity
LD50
Animal Route (mg/kg bw) Reference
Safrole
Mouse Oral 2 350 Jenner et al., 1964
Rat Oral 1 950 Jenner et al., 1964
Dihydrosafrole
Mouse Oral 3 700 Hagan et al., 1965
Oral 4 300 Jenner et al., 1964
Rat Oral 2 260 Jenner et al., 1964
Isosafrole
Mouse Oral 2 470 Jenner et al., 1964
Rat Oral 1 340 Jenner et al., 1964
Short-term studies
Rat
The ability of safrole (650 mg/kg), isosafrole (460 mg/kg) and
dihydrosafrole (770 mg/kg) to induce macroscopic liver damage was
compared in groups of three male and three female rats with doses of
approximately one-third the oral LD50 administered daily by stomach
tube for four days. The changes induced by these three compounds
ranged from slight discoloration with blunting of lobe edges to marked
discoloration, fatty appearance and enlargement. The average rating of
the lesions was the same for the three compounds; there was one death
in the dihydrosafrole group and two in the isosafrole group (Taylor et
al., 1964).
Safrole (250, 500 and 750 mg/kg), dihydrosafrole (250, 500 and
750 mg/kg) and isosafrole (250 and 500 mg/kg) and corn oil were
administered daily by stomach tube to groups of 10 rats. Treatment
time for the test animals ranged from 19 to 46 days, while the
controls were treated for 106 days. There were no deaths with the 250
and 500 mg doses of dihydrosafrole and the 250 mg dose of safrole. A
few deaths, 2/10, 3/10 and 1/10, respectively, resulted from the
administration of 250 mg isosafrole, 750 mg dihydrosafrole and 500 mg
safrole. Many deaths, 8/10 and 9/10, respectively, were seen with the
500 mg dose of isosafrole and the 750 mg dose of safrole.
Macroscopically, all three compounds induced liver enlargement and
adrenal enlargement with yellow discoloration, most marked with
safrole and dihydrosafrole. Microscopically, there was variation in
hepatic cell size, focal fatty metamorphosis, bile-duct proliferation
and architectural irregularity. Adrenals from the rats given safrole
and dihydrosafrole showed an increase in lipid in the cytoplasm of the
cortex. Safrole was administered to mice at doses of 250 and 500 mg/kg
daily by stomach tube for 60 days (number of mice not indicated).
Liver changes similar to those observed in the rat study were observed
(Hagan et al., 1965).
Dog
Groups of two male and two female dogs received a dose of
80 mg/kg per day of safrole orally for 26-39 days or 40 mg/kg per day
for 91-116 days. At 80 mg/kg, one dog died and the others were
sacrificed moribund. These animals exhibited marked weight loss.
Skeletal muscle was atrophied in 2/4 dogs. The liver was slightly
enlarged in 2/4 dogs. Microscopically, the livers showed a heavy
infiltration of fat, occasional bile-duct proliferation and
inflammatory-cell infiltration, slight to moderate streaking with
necrobiosis, and liver cell atrophy. At 40 mg/kg, the dogs exhibited
emaciation and general weakness; the liver was slightly enlarged in
3/4 dogs. Microscopically, there was slight fat infiltration, moderate
cell atrophy and necrobiosis in the liver (Hagan et al., 1967).
Long-term studies
Rat
Rats (number not reported) were fed safrole or natural oil of
sassafras at levels of 390 or 1170 ppm (0.039 or 0.117%) in the diet
for two years. Hepatocarcinomas were found in all the experimental
groups as well as cellular changes in the kidneys, adrenals, thyroid,
pituitary, and testes or ovaries at 24 months. Kidneys and livers from
high level animals sacrificed at 22 months showed evidence of kidney
congestion but no liver tumours. Blood and urine examinations were
within normal limits (Abbott et al., 1961).
Rats were fed riboflavin-deficient diets (four males and 10
females) or riboflavin-deficient diets plus 1% safrole (five males and
14 females) for 3.5-13.5 months. Body weight of both sexes fed safrole
was depressed; liver weight/body weight ratio of these animals was
elevated. The riboflavin-deficient diet produced fatty degeneration,
some increase in connective tissue and extensive ceroid deposits in
the livers of the males. In the females, it produced mild to moderate
fatty infiltration with no increase in fibrous connective tissues
until 13.5 months when fibrous connective tissue obscured the normal
architecture and nodular regeneration appeared. Addition of safrole to
the diet decreased fatty infiltration in both sexes and produced
adenomatous changes much earlier in the females (Homburger et al.,
1962).
Groups of 10 male rats were fed control diets containing 5, 10 or
30% protein and 5% fat or these diets with 0.5% safrole or 0.06%
butter yellow added. Additional groups received a diet containing 30%
protein and 15% fat or a riboflavin-deficient diet with 10% protein
and 5% fat, and these diets with safrole or butter yellow. The rats
were killed when they appeared moribund (55-634 days). Altering the
dietary protein level had little effect on the incidence of hepatoma-
hepatocarcinomas with butter yellow, although the rats on butter
yellow and the riboflavin-deficient diet showed early mortality.
Hepatoma-hepatocarcinoma incidence increased with increasing protein
level in the safrole-treated animals. The safrole rats receiving 5%
protein showed early mortality. The high fat and riboflavin-deficient
diets had little effect on safrole toxicity or tumour incidence
(Homburger et al., 1965).
Groups of 25 male and 25 female rats were fed 0, 100, 500, 1000
and 5000 ppm (0, 0.01, 0.05, 0.1 and 0.5%) safrole in the diet for two
years. Changes in the liver consisting of benign and malignant tumours
(hepatic cell adenoma, hepatocholangioma, hepatic cell carcinoma and
hepatocholangiocarcinoma), enlargement of hepatic cells, variation in
cell size, fatty metamorphosis, architectural irregularity, bile-duct
proliferation, focal cystic necrosis, focal peripheral margination of
cytoplasm and minimal coagulation necrosis were observed. The liver
injury was rated as very slight at 100 ppm (0.01%), slight at 500 ppm
(0.05%), slight to moderate at 1000 ppm (0.1%), and moderate to severe
at 5000 ppm (0.5%). Tumour incidence was significantly increased at
5000 ppm (0.5%), with 14 rats having malignant tumours and five having
benign tumours versus two and one, respectively, in the controls.
Tumour incidence in the other groups was: eight benign on 1000 ppm
(0.1%), two malignant and one benign on 500 ppm (0.05%), and one
benign on 100 ppm (0.01%). Weight gain and survival were significantly
decreased on the 5000 ppm (0.5%) level and these rats also showed a
mild hypochromic anaemia, a slight leucocytosis and a slight
depression of haematopoiesis in the bone marrow (Long et al., 1963).
Safrole, dihydrosafrole and isosafrole were fed in the diet at
levels of 0, 1000, 2500 and 10 000 ppm (0, 0.1, 0.25 and 1%) to groups
of 10 male and 10 female rats and at a level of 5000 ppm (0.5%) to
groups of 25 males and 25 females for two years. Growth was depressed
in both sexes at levels of 2500 ppm (0.25%) and above and in the
females at 1000 ppm (0.1%) with both safrole and dihydrosafrole. With
isosafrole, growth was depressed in both sexes at 5000 and 10 000 ppm
(0.5 and 1%) and in the females at the two lower doses. None of the
10 000 ppm (1%) safrole test rats survived beyond 62 weeks. These
animals showed testicular atrophy, changes in the stomach consisting
of atrophy and atypical regeneration of the mucosa glands with
associated fibrosis and hyalinization of the surrounding stroma, and
changes in the liver including tumour formation. The livers were
enlarged, mottled and irregularly nodular, with single and multiple
tumour masses. Microscopically, there was hepatic cell enlargement
which was usually focal and resulted in nodule formation. The nodules
tended to progress in one of three ways: (1) cystic necrosis, (2)
cirrhosis, and (3) adenomatoid hyperplasia, leading to formation of
benign and malignant tumours of the same types as in the study cited
above. Similar types of liver changes were seen with the lower doses.
The damage was slight at 1000 ppm (0.1%) and lacked tumours and
cirrhosis, moderate at 2500 ppm (0.25%) but lacked cirrhosis, and
severe at 5000 ppm (0.5%) where there were macroscopic cysts and the
incidence of tumours exceeded the incidence at the higher level. There
was a statistically significant increase in malignant hepatic primary
tumours with this level. There was mild hyperplasia of the thyroid at
5000 ppm (0.5%) and an increase in chronic nephritis with this dose
and the lower doses. Dose levels of dihydrosafrole of 2500 ppm (0.25%)
and above induced eosophageal tumours, benign epidermoid papillomas
and malignant papillary epidermoid carcinomas. No tumours of this type
were observed in the controls. At 2500 ppm (0.25%), 20% of the rats
had eosophageal tumours, with 5% of these malignant. At 5000 ppm
(0.5%), 74% of the rats had such tumours with 32% malignant and, at
10 000 ppm (1%), 75% of the rats had tumours with 50% malignant.
Slight liver damage of the same type as seen with safrole was found
with all test levels of dihydrosafrole; a few tumours were seen, but
the increase was not statistically significant. Moderate follicular
atrophy of the spleen was observed at the 5000 and 10 000 ppm (0.5 and
1%) levels and a moderate increase in chronic nephritis at the two
lower dose levels. The 10 000 ppm (1%) level of isosafrole increased
mortality, with none of the rats on this level surviving beyond 11
weeks of treatment. Slight liver damage of the same type seen with
safrole was seen at all test levels of isosafrole. There were five
rats on the 5000 ppm (0.5%) level with primary hepatic tumours, but
this incidence was not significantly greater than in the controls.
There was slight thyroid hyperplasia on the 2500 and 5000 ppm (0.25
and 0.5%) levels and an increased incidence of chronic nephritis on
the 5000 ppm (0.5%) level (Hagan et al., 1965; Hagan et al., 1967).
Dog
Safrole was administered orally to groups of two male and two
female dogs at 5 and 20 mg/kg bw for six years. Liver changes, but no
tumours, were observed with both doses. The changes at the lower dose
were minimal focal necrosis, bile-duct proliferation, fatty
metamorphosis, hepatic cell atrophy and leucocytic infiltration. At
the higher dose, the liver was enlarged with a nodular surface.
Microscopically, it showed mild post-necrotic cirrhosis characterized
by focal or generalized nodules, consisting of enlarged hepatic cells
with slight focal fatty vacuolization, separated by bands of atrophied
hepatic cells and collagen; slight lymphocytic, Kupffer cell and bile-
duct proliferation; and minimal focal necrosis. With this dose the
thyroids were hyperplastic (Hagan et al., 1967).
Safrole was fed to dogs (number and dose not indicated) for seven
years. Liver function tests consisting of bromsulfalein excretion
curve, soluble enzyme content in the central lobe segments, lipid and
glycogen content of the same segments, and nitroreductase activity
were performed during the course of the study and at termination. The
results of these function tests indicated some degree of tissue injury
during the early part of the study, but a functional adaptation to the
test substance by the end of the study, although microscopic
morphological changes were observed at termination (Weinberg &
Sternberg, 1966).
The toxicity of safrole was summarized by Opdyke (1974).
Comments
Small doses of safrole are absorbed rapidly and almost completely
excreted in the urine within 25 hours of exposure in both rat and man.
1,2-dihydroxy-4-allylbenzene is the main metabolite in both species.
Safrole and isosafrole administered to rats produces liver
hypertrophy and induces microsomal enzymes. Safrole is inactive in
mutagenicity studies with various strains of S. typhimurium with and
without activation. Safrole was positive in the in vitro mutagenic
assay with E. coli and S. cerevisiae and in the in vivo
intraperitoneal host-mediated assay.
Administration of safrole to mice, either orally or
subcutaneously, led to a marked increase in the incidence of liver
tumours. Exposure of mice to safrole in utero produced renal
epithelial tumours. In the case of rats, chronic administration of
safrole resulted in progressive dose-dependent liver damage ranging
from hepatic cell enlargement, nodule formation, cirrhosis adenomatoid
hyperplasia leading to benign and malignant tumours. No liver tumours
were reported in dogs fed safrole for six years, but some liver
changes including bile-duct proliferation were observed.
EVALUATION
No ADI allocated.
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See Also:
Toxicological Abbreviations