(VCCEP) Tier 1 Pilot Submission for BENZENE - Tera

in dessicator jars, Glatt et al. (1989) were able to induce histidine reversion with metabolic
activation. Jar set-up apparently allowed benzene vapor to be present long enough to be
converted to biologically active metabolites by rat liver metabolism. Of 13 metabolites tested,
only trans-1,2-dihydrodiol and the diol epoxides (with and without activation) were active.
Seixas et al. (1982) and Kaden et al. (1979) induced azaguanine reversion in Salmonella with
metabolic activation. Benzene (0.069M) inhibited cell survival in E. coli WP100 [uvrA-, recA-], a
repair-deficient strain compared to E. coli WP100[uvrA+,rec+], its repair-proficient partner, and
in Bacillus subtilis strain M45 [rec-] compared to H17[rec+] (McCarroll et al., 1981a,b). The
authors suggested a requirement for recombinational mediated repair to benzene-induced DNA
damage.
Mutational events have been reported in mammalian cells cultured from animals exposed in
vivo. Ward et al. (1992) observed dose-related increases in mutations at the hprt locus in
spleen lymphocytes of CD-1 mice exposed to benzene (0.04, 0.1, and 1.0ppm) by inhalation,
7days/week for 6 weeks, at the two lower concentrations but not at 1.0 ppm. However, effects
were not paralleled by clear increases in numbers of labeled cells, and increases in mutant
frequency may have been artificially inflated by variability in the labeling index, and co-exposure
to a high dose of vinblastine. All these factors led the authors of the EU benzene risk
assessment draft (ECB 2003) to consider this study equivocal. Mullin et al. (1995) detected
increased mutant frequencies in the lacI transgene from lung and spleen cells, but not liver, of
C57Bl/6 mice exposed to 300 ppm benzene, 6 hours/day, 5 days/week for 12 weeks.
Mammalian forward mutation assays, as performed, cannot distinguish between true gene
mutations and small deletions. Thus, interpretation of these results is consistent with action of a
cytogenetic agent that causes chromosome breakage, loss, and/or rearrangement.
Benzene metabolites either tested directly or produced by metabolic activation, have induced
various forms of DNA perturbation in mammalian cells. Exogenous metabolic activation was
required to produce sister chromatid exchanges (SCE) in human lymphocyte cell cultures
(Morimoto, 1983), and endogenous activation induced DNA synthesis in rat hepatocytes
(Glauert et al., 1985), DNA adduct formation in rat liver mitochondria (Rushmore et al., 1984),
and RNA synthesis in mitochondria of rat liver, and rabbit and cat bone marrow (Kalf et al.,
1982). Studies in Chinese hamster V79 cells demonstrated that the benzene metabolites 1,2,3-
and 1,2,4-trihydroxybenzene, quinone, hydroquinone, catechol, phenol, 1,2 dihydrodiol, and the
diol epoxides produced genotoxicity ranging from SCE and micronuclei to gene mutation (Glatt
et al., 1989). Trans, trans-muconaldehyde was also strongly mutagenic in V79 cells and weakly
mutagenic in bacteria (Glatt and Witz, 1990); muconaldehyde and its metabolites—6-hydroxy-
2,4-hexadienal and 6-oxo-trans,trans hexadienoic acid—were also active (Chang et al., 1994).
The overall conclusion from a range of in vitro studies is that benzene does not induce toxicity
and leukemogenesis as a gene mutagen. Chromosome alterations and hyperdiploidy were
observed in human lymphocytes after exposure to hydroquinone in vitro (Eastmond et al.,
1994), and Aubrecht and Schiestl (1995) reported that benzene itself induced intrachromasomal
recombination in human lymphoblastoid cultures. Micronuclei were also seen in human cells
exposed in vitro to various benzene metabolites and combinations of metabolites (Zhang et al.,
1993; Eastmond, 1993). Synergistic increases in micronuclei were induced by catechol and
hydroquinone, but not by catechol and phenol, or phenol and hydroquinone (Robertson et al.,
1991). When mice were treated intraperitoneally with mixtures of these benzene metabolites,
synergistic effects resulted only from mixtures of phenol and hydroquinone (Marrazzini et al.,
1994); adding catechol was no more effective than hydroquinone alone in inducing micronuclei.
Similar results were reported by Chen and Eastmond (1995), using an antikinetochore-specific
antibody and fluorescent in situ hybridization (FISH). The relative frequency of chromosome
Benzene VCCEP Submission
March 2006
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