Safety evaluation of certain food additives - ipcs inchem

434 ALKOXY-SUBSTITUTED ALLYLBENZENES
hydroxymethyl eugenol and at 10 4 –10 5 mol/l for 1-hydroxyestragole. The UDS
activity and cytotoxicity of the parent substances occurred at concentrations
approximately an order of magnitude greater than those for their corresponding
metabolites. Additionally, cytotoxicity was observed at slightly higher concentrations
than those needed to induce UDS, although the differences were minimal. A clear
non-linear relationship and threshold were established between dose for both
substances and their metabolites and UDS activity. In an earlier study, similar
results were obtained for methyl eugenol, safrole and estragole (Howes et al.,
1990a). UDS studies with 1.78–178 μg methyl eugenol/ml (10–1000 μmol/l) and
1.62–162 μg safrole/ml (10–1000 μmol/l) in rat, mouse and human hepatocytes
gave uniformly positive results; in each case, however, as the concentration of the
test material increased, the level of LDH leakage also increased, indicating cytotoxic
effects (Burkey et al., 1998, 1999, 2000; Sipes et al., 1999). In another study with
1.25–20 μg methyl eugenol/ml, the incidence of UDS was marginally increased
compared with controls but did not reach statistical significance, so it was
determined to be an equivocal result (San & Reece, 2003).
In several in vitro studies, 0.032–549 μg safrole/ml showed no increase in
UDS in rat hepatocytes, HeLa cells and human fibroblast cells (San & Stich, 1975;
Martin et al., 1978; Agrelo & Amos, 1981; Agrelo & Severn, 1981; Martin &
McDermid, 1981; Robinson & Mitchell, 1981; Klaunig et al., 1984; Probst & Hill,
1985). Safrole was shown to increase UDS in primary rat hepatocytes and HeLa
cells at concentrations of 0.000 162–1620 μg/ml (Michalopoulos et al., 1978;
Althaus et al., 1982; Williams, 1984; Barrett, 1985; Glauert et al., 1985; Martin &
Campbell, 1985; Williams et al., 1985; Howes et al., 1990b). It should be noted that
at concentrations greater than 162 μg/ml, safrole is highly cytotoxic (Burkey et al.,
2000).
2.3.2 In vivo
In an in vivo study, hepatocytes isolated 4 or 12 h after rats received a 500,
1000 or 2000 mg/kg bw dose of estragole were evaluated for UDS. Only at the high
dose were the net nuclear grain counts greater than 5 (Müller et al., 1994).
In vivo UDS studies with safrole yielded equivocal results. No increase in
UDS was observed when rats were administered 200 and 1000 mg safrole/kg bw
by corn oil gavage and the hepatocytes were isolated at 2 and 12 h (Mirsalis et al.,
1982). In a study reported with little further detail, no UDS was observed when mice
were administered up to 50% of the LD50 of safrole (Robertson, 1978). Increases in
UDS levels were observed when 640 mg safrole/kg bw was administered to mice
via intraperitoneal injection of a corn oil suspension and the mice were sacrificed
3 h post-treatment (Friedman & Staub, 1976). Similarly, when 500 and 1000 mg
safrole/kg bw were administered by single gavage dose to rats, increased UDS
levels were observed at 24, 39 and 48 h post-dosing (Uno et al., 1994). UDS levels
were increased 24 and 48 h post-administration when rats were dosed with 250,
500 or 1000 mg safrole/kg bw (Ohtsuka et al., 1998).
Given that estragole, methyl eugenol and safrole have been shown to form
DNA adducts when laboratory rodents were exposed to high dose levels, it is not
surprising that both substances and their 1-hydroxy metabolites induce UDS. In