Safety evaluation of certain food additives - ipcs inchem

MISCELLANEOUS NITROGEN-CONTAINING SUBSTANCES (addendum) 571
(iii) Conclusions from genotoxicity studies
Bacterial mutagenicity testing of isothiocyanates in standardized bacterial
reverse mutation assays using a variety of strains has shown no consistent evidence
of mutagenicity. In the absence of metabolic activation, both positive (Yamaguchi,
1980; Eder et al., 1982; Brooks et al., 1984; Kassie & Knasmüller, 2000; Kassie et
al., 2001a, 2001b) and negative (Neudecker & Henschler, 1985; Mortelmans et al.,
1986) results have been reported, usually at or near cytotoxic concentrations (see
also Annex 1, reference 177). In studies with metabolic activation, positive results
were reported in modified Ames assays using preincubation conditions conducive
to depletion of metabolic detoxication pathways (Neudecker & Henschler, 1985;
Uda et al., 1992). Furthermore, equivocal evidence of isothiocyanate genotoxicity
has been reported in in vitro clastogenic assays (sister chromatid exchange,
chromosomal aberrations, micronuclei) (Kasamaki et al., 1982; Kasamaki &
Urasawa, 1985; Galloway et al., 1987; Musk & Johnson, 1993; Kassie et al., 1999,
2001a, 2001b; Kassie & Knasmüller, 2000).
Conditions in most of these studies provided the opportunity for either direct
interaction with DNA or indirect formation of DNA adducts due to oxidative stress
and subsequent cytotoxicity. Numerous studies have shown that test conditions
(e.g. cytotoxicity, ionic strength, low pH) and culture conditions (e.g. hypo- and
hyperosmolality, high pH) can induce increased DNA damage (Zajac-Kaye & Ts’o,
1984; Brusick, 1986; Bradley et al., 1987; Galloway et al., 1987; Seeberg et al.,
1988; Morita et al., 1989; Scott et al., 1991), leading to DNA fragmentation, which
can cause false-positive responses in genotoxicity assays (Meintières & Marzin,
2004). The metabolism of isothiocyanates in vitro and in vivo may lead to depletion
of GSH levels and a release of nucleocytolytic enzymes that induce DNA damage.
At low dose levels, it is likely that isothiocyanates exhibit limited genotoxic activity.
(d) Reproductive and developmental studies
(i) Benzyl isothiocyanate (No. 1562)
In a two-generation reproductive toxicity study, benzyl isothiocyanate was
administered via gavage to Sprague-Dawley rats at levels of 0, 12.5, 25 or 50 mg/kg
bw on pregnancy days 1–5 (preimplantation) or 7–13 (post-implantation). The body
weights of rats treated with the flavouring agent before fetal implantation were
recorded on days 1, 5, 10 and 16, whereas rats treated post-implantation were
weighed on days 7, 11, 15 and 20 of gestation. Pregnant females were observed
for vaginal bleeding and signs of clinical toxicity. Pre- and post-implantation rats
were euthanized on days 16 and 20, respectively, and the numbers of implantation
sites, viable fetuses and fetal resorptions were analysed following necropsy. The
weights of viable fetuses and their placentas were documented, and the viable
fetuses were observed for external malformations. Maternal liver, kidney and
spleen weights were also recorded. Pregnant rats treated with benzyl
isothiocyanate prior to implantation were observed to display hypoactivity, perinasal
staining, piloerection and hunched posture. There was also a dose-dependent
decrease in the body weights of these rats during the treatment period. Early fetal
resorptions, number of implantation sites and relative weights of liver, kidney and