Safety evaluation of certain food additives - ipcs inchem

376 ALKOXY-SUBSTITUTED ALLYLBENZENES
formation of protein adducts has been directly related to the formation of the
1-hydroxy metabolite in a dose-related manner (Borchert et al., 1973; Gardner
et al., 1995). The 44 kDa adduct was reported in vitro when the 1-hydroxy
metabolite (50 μmol/l) was incubated with rat hepatocytes; at a higher concentration
(500 μmol/l), additional protein adducts were observed (Gardner et al., 1995, 1996).
A marked increase in methyl eugenol–protein adducts occurred when CYP
was induced by an assortment of inducers, including dexamethasone. Autoinduction
of the 1-hydroxylation pathway was reported in hepatic microsomes of
rats given 30–300 mg methyl eugenol/kg bw per day orally for 5 days, but was not
observed in rats given 10 mg/kg bw per day for 5 days (Gardner et al., 1997a).
Dose levels of 100, 300 or 500 mg estragole/kg bw were administered orally
as single doses in 0.5% weight by volume (w/v) methyl cellulose or by repeated
daily doses to male Fischer rats for 5 days. Control animals received an equivalent
volume of 0.5% methyl cellulose solution. Livers were removed from rats
immediately after sacrifice, and hepatic protein adducts were detected by sodium
dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using
antisera raised by immunizing rabbits with 4-methoxycinnamic acid–modified rabbit
serum albumin. A major 155 kDa adduct was expressed in the livers of animals that
received all doses of estragole. Other protein adducts (170, 100, 44 and 35 kDa)
were also detected in the high-dose group. Rats administered estragole for 5 days
at 300 mg/kg bw per day expressed predominately 155 and 44 kDa adducts, with
lower levels of the 100 and 35 kDa adducts detected. Adduct levels also increased
disproportionately with respect to dose and were roughly 250-fold higher in livers of
rats administered a single dose of estragole at 500 mg/kg bw than in animals dosed
with the compound at 100 mg/kg bw. The 155, 100, 44 and 35 kDa adducts were
detected in greatest abundance in liver microsomal fractions, whereas the 170 kDa
adduct was most abundant in the nuclear fraction. Some adducts (i.e. 170, 155, 100
and 35 kDa) were detected in the cytosolic fractions, whereas relatively low levels
of the 44 kDa adduct were detected in the nuclear fractions and not in the cytosolic
fractions (Wakazono et al., 1998).
2.1.7 DNA adducts
(a) Rodent DNA adducts
In studies beginning in the early 1980s (Miller et al., 1983), safrole, myristicin,
methyl eugenol and estragole, their 1-hydroxy metabolites and the corresponding
sulfate esters of the 1-hydroxy metabolites were shown to form DNA adducts in
vivo and in vitro. In early studies, high doses were administered to achieve a
carcinogenic effect, whereas in more recent studies, multiple dose levels have been
administered to evaluate the dose–response relationship.
Dose-dependent formation of safrole–DNA adducts has been studied in rats
(Daimon et al., 1998). Male Fischer rats were given either single oral doses of 0, 1,
10, 100, 250 or 500 mg safrole/kg bw or five successive daily doses of 62.5, 125 or
250 mg safrole/kg bw. Two major DNA adducts (N 2 -(trans-isosafrole-3-yl)-2deoxyguanosine
and N 2 -(safrole-1-yl)-2-deoxyguanosine) and two unidentified