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A number of the components of creosote (e.g., PAHs) are known to be mutagenic (IPCS, 1998).
In vitro assays
A summary of in vitro genotoxicity assays carried out for various
creosotes with bacterial (Ames/Salmonella test, assays with
Escherichia coli) and mammalian (mouse lymphoma cell assay, sister chromatid exchange test
with Chinese hamster ovary cells, chromosomal aberrations in human lymphocytes)
test systems (Simmon & Poole, 1978; Bos et al., 1983, 1985; Nylund et al., 1992; IUCLID, 2000). Almost all creosotes tested showed mutagenic activity
after metabolic activation (S9 mix) in the conventional Ames/Salmonella assay with strain TA98.
Further, the nitroreductase overproducing strain YG1021 and the
O-acetyltransferase overproducing strain YG1024 were tested, which have
increased sensitivity to detect mutagenicity of aromatic nitro and amino
compounds. Positive results were also obtained with several other Salmonella
TA or YG strains or with the mouse lymphoma cell assay and the sister
chromatid exchange test with Chinese hamster ovary cells. Negative results have
been observed with the Salmonella tester strain TA1535 (which may be less
sensitive in this case or indicative of other mutation types). Depending on
creosote type tested, the Salmonella assay with tester strain TA100 as
well as the SOS chromotest with Escherichia coli PQ37 gave positive and negative responses, thus indicating differences
in mutagenic activity between different creosotes. There were also differences
in the relative strengths of genotoxic responses. For example, Nylund et al. (1992), testing four creosote samples (about 85 components identified,
96?98% of total composition; see also Table 3: F, G) from different countries,
found the same order of potency when the creosotes were tested both in
the Ames/Salmonella assay using strains
TA98 and YG1024 and in the sister chromatid exchange test with metabolic
activation. The order of potency was: Danish > former Soviet > German >
Polish creosotes.
In a two-stage transformation assay, creosote (no specification
given) enhanced transformation of Syrian hamster embryo cells initiated
with BaP, thus indicating tumour-promoting activities (Sanner & Rivedal,
1988).
Attempts have been made to identify the compounds or groups
of compounds in different creosotes responsible for the mutagenic activity.
As a result, several fractions of different creosotes have also been demonstrated
to be mutagenic to Salmonella typhimurium TA98 in the presence of metabolic activation. The creosotes were fractionated by means of TLC (Bos et al., 1984a) or distillation (Nylund et al., 1992). Three of seven TLC fractions of creosote P1 were highly mutagenic:
one contained unidentified more polar compounds, the second contained BaP,
and the third contained benz[a]anthracene (Bos et al., 1984a). Both fractionation and mutagenicity profiles differed between
the four different creosotes (Danish, German, Polish, former Soviet) tested
by Nylund and co-workers. A common feature in the tests with Salmonella strains TA98
and TA100 (plus S9 mix) was that the mutagenicity appeared in the distillation
fractions having the highest boiling point ranges (>290 °C) and high
concentrations of known mutagenic PAHs (chrysene, benzo[e]pyrene,
benzo[k]fluoranthene, BaP, dibenzo[a,h]anthracene,
benzo[ghi]perylene). Although the intact creosotes contained lower concentrations of the six PAHs than these fractions, their mutagenic response was mostly higher; only a single fraction of one creosote showed slightly higher mutagenicity than the original creosote (Nylund et al., 1992).
Components suggested to be responsible for the mutagenicity
of creosotes include mainly PAHs, but also aromatic amines and certain
azaarenes (e.g., Sundstrom et al., 1986). Comparisons between mutagenic activities and concentrations of
known mutagenic PAHs in several creosotes (see above) and some of their
corresponding fractions suggested synergistic and antagonistic interactions
(Nylund et al., 1992).
The mutagenic action of creosote (Cindu Chemicals, The Netherlands)
vapour (generated at 37 °C) has been attributed to fluoranthene, based
on the so-called taped plate assay with Salmonella strains TA98 and TA100 in the presence of S9 mix (Bos et al., 1987); however, this test was negative with the creosotes examined by
Nylund et al. (1992).
Urine samples of rats injected intraperitoneally with 250 mg creosote/kg body
weight showed elevated mutagenic activity in the Ames test with Salmonella
typhimurium TA98 in the presence of metabolic activation and
beta-glucuronidase (Bos et al., 1984b,c). The same test (metabolic activation not reported) was positive with urine samples from rats treated orally with creosote (lot CX1984; 50 mg/kg body weight per day) over a 5-week period (Chadwick et al., 1995).
Human urine samples collected from wood impregnation plant workers (n
= 6) at the end of shift and tested according to the Ames Salmonella test with strain TA100 did not show any exposure-related increase in mutagenicity
(Nylund et al., 1989). The same was true for urine samples from three workers of another
wood impregnation plant, when tested with Salmonella strain TA98
(plus S9 mix and beta-glucuronidase): spot wipe samples collected from
several contaminated surfaces (n = 5; solvent: acetone or alcohol) in the working environment of this plant gave positive mutagenic results with the tester strain TA98 in the presence of S9 mix; the extraction with acetone revealed higher mutagenic values than extraction with alcohol (Bos et al., 1984b,c).
Several genotoxicity tests performed with creosote-contaminated
soils or sediments gave positive results.
The mutagenic activity (as monitored by the Ames test with
Salmonella
typhimurium TA98, +/- S9 mix) of a creosote/PCP waste sludge (from an active wood treatment facility) applied to soil was found to persist in surface soil at least 350 days after sludge application. During lysimeter experiments, most of the mutagenicity was detected in surface soil extracts, with weaker responses in leachate samples (Barbee et al., 1996). Similarly, the crude fraction of bottom sediment waste collected from a sediment pond of a plant using both PCP and creosote was mutagenic in the Ames assay with Salmonella typhimurium TA98 (plus S9 mix), with a total activity approximately equal to the sum
of the activities of three (i.e., acid, base, neutral) fractions (Donnelly et al., 1987). A weak mutagenicity in the Salmonella Ames assay against strain TA98 (with metabolic activation) was found in the PAH fraction of soil collected from a wood treatment plant (in operation from 1924 to 1987; using 100% creosote, 50% creosote mixed with other oils and oil carrier, PCP, etc.; oil content 3-6 w/w % of soil; PAH content not quantified) and subjected
to Soxhlet extraction with DCM and class component chromatographic separation
(Zemanek et al., 1997).
Soil samples taken in 1996 from a former creosote wood treatment
facility (in operation from 1917 to 1972) with maximum PAH concentrations
of 3000 mg/kg dry soil were tested with the Ames Salmonella assay using the tester strains YG1041 and YG1042. The creosote soil extracts
(extraction agent: DCM) were found to be moderately mutagenic with metabolic
activation (S9 mix) and were non-mutagenic without metabolic activation.
However, some bioremediation techniques resulted in an increased mutagenicity
despite success in reducing the total PAH concentration, probably due to
the presence of nitrogen-containing heterocycles (Brooks et al., 1998; Hughes et al., 1998).
A soil sample from a hazardous waste site contaminated with
creosote (no further details) was assayed by a micronucleus test with Tradescantia.
Cuttings of Tradescantia clone 4430 were exposed for 30 h to different solutions of aqueous soil
extracts (initial total PAH concentration in the soil: 5749 mg/kg, weight
basis not specified). The micronucleus frequencies increased in a concentration-dependent
manner. A further increase in genotoxicity was seen in soil samples (containing
indigenous microflora) incubated for 8 weeks, which was presumed by the
authors to be due to the generation of water-soluble metabolic intermediates
by the microorganisms (Baud-Grasset et al., 1993).
Sediment samples collected in 1994 near a wharf, which was treated before
immersion in the water with creosote (no specification) some months before
(1993), and extracted with DCM, followed with an exchange into DMSO, were tested
in rainbow trout (Oncorhynchus mykiss) hepatocytes using the nick translation assay (NTA) and the alkaline precipitation
assay (APA). Total PAH concentrations in these sediments ranged from 0.14 to
209 mg/kg dry weight, with the number of PAHs varying from 6 to 16. PAH
concentrations and genotoxicity were higher in samples from the intertidal
section than from the subtidal section. Samples closest to the wharf (1
m and 5 m) showed more genotoxicity than those farthest (40 m and 50 m)
from the wharf. Whereas 80% and 60% of the intertidal samples were genotoxic
according to NTA and APA, respectively, only 10% and 30% of the subtidal
samples were positive in the NTA and APA, respectively. There were some
correlations between levels of some PAHs (naphthalene, acenaphthylene,
fluorene, phenanthrene, anthracene, and pyrene) and the NTA results, but
the relevance of this finding remains unclear (Gagne et al., 1995).
In vivo assays
Creosotes
A commercially available coal tar creosote (Lot No. MOP9328,
manufactured by Nakarai, Kyoto, Japan) has been tested in a collaborative
study using the rodent micronucleus assay. CD-1 male mice (n = 5 or more) received two
intraperitoneal injections (with an interval of 24 h) of creosote (in olive oil)
at a concentration of 92.5, 185, or 370 mg/kg body weight. The frequency of
micronucleated polychromatic erythrocytes in bone marrow increased dose
dependently and with statistical significance (24 h after final treatment). A
single intraperitoneal treatment of 370 mg/kg body weight (corresponding to
about 80% of the LD50) also induced micronuclei (Morita et al., 1997).
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