* Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674; Covance Laboratories, Vienna, Virginia 22182
1 To whom correspondence should be addressed at The Dow Chemical Company, 1803 Building, Midland MI. E-mail: gdcharles{at}dow.com
Received August 19, 2004; accepted November 11, 2004
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ABSTRACT |
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Key Words: Genetic toxicity; formaldehyde; mouse lymphoma; oxazolidine.
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INTRODUCTION |
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During the course of conducting regulatory genotoxicity studies in accordance with European Union (EU) Directive 98/8/EC for biocidal product registration, CS-1246 elicited a positive response in the mouse lymphoma assay (MLA). Based on the directive, further triggered genotoxicity evaluation in vivo in the mouse bone marrow micronucleus (MNT) and unscheduled DNA synthesis tests were negative. CS-1246 is known to release FA upon hydrolysis (Fig. 1). Consequently, FA release was hypothesized as the potential mode of action by which positive results were generated in vitro in the MLA, with negative in vivo genotoxicity assessments based on prior demonstrated FA activity (Speit and Merk 2002; Natarajan et al., 1983
; Richardson et al., 1983
; Brusick, 1982
). To evaluate this hypothesis, a methodology was successfully implemented to abrogate the mutagenic response to FA in mouse lymphoma cultures by the inclusion of FA metabolic competency in the form of formaldehyde dehydrogenase (FDH) and its cofactor NAD+.
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MATERIALS AND METHODS |
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Mouse lymphoma (L5178Y/TK+/) gene mutation assay. The ability of CS-1246 to induce mutations at the TK locus of mouse lymphoma cells was evaluated in two independent assays. These studies were conducted in accordance with Organisation for Economic Co-operation and Development (OECD) guidelines (#476, 1997), using the procedures described by Clive et al. (1995). L5178Y TK+/ cells were treated with 8.7104.7 µM of CS-1246 in Fischer's medium (Gibco, Grand Island, NY) with and without a S9 (Moltox, Cary, NC) metabolic activation system. Positive controls, 20-methylcholanthrene (20-MCA) and methyl methanesulfonate (MMS) (Sigma, St. Louis, MO), were used for activation and non-activation assays, respectively. After the addition of the test compounds, test tubes were incubated for approximately 4 h at 37°C in a roller drum. At the end of the incubation period the cells were pelleted, rinsed with Fischer's medium, and resuspended in 20 ml Fischer's-based medium. The tubes were returned to the roller drum and maintained at 37°C during a standard expression period of 2 days. At approximately 24 h after treatment (day 1), the test cultures were counted and diluted to a concentration of approximately 3 x 105 cells/ml with fresh F10P. If the treated cells failed to multiply to a density of 4 x 105 on the first day after treatment, the culture was returned to the incubator without any dilution. On day 2, cultures were again counted for cell density. From these cell counts, the day 2 relative suspension growth (RSG) was calculated:
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Treatment levels with desired degrees of toxicity were selected for cloning. Cultures with <10% RSG on day 2 were not selected for cloning. A total sample size of 3 x 106 cells from each culture was suspended in cloning medium with trifluorothymidine (TFT) and plated into three petri dishes (100 mm), allowed to gel for approximately 15 min at 0°6°C, and returned to the incubator for approximately 12 days to allow for mutant colony formation. The cloning efficiency was determined by serially diluting the sample in cloning medium without TFT and then plating the cells into three petri dishes (100 mm) at a concentration of approximately 200 cells per dish. The dishes were returned to the incubator for approximately 12 days before the number of colonies per dish was counted.
An image analyzer (LAI High-Resolution Colony Counting System, Loats Associates, Inc., Westminster, MD) was used to count and size colonies. The separation of small and large colonies was determined by inspection of colony sizing histograms of each culture.
The parameter relative total growth (RTG) was used to determine the cytotoxicity of various treatments. Calculations for RTG are described below:
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Mutant frequencies were evaluated on the basis of biologically significant criteria (Moore et al., 2003). The test chemical was considered positive when the mutant frequency in at least one dose level of the treated cultures (resulting in
10% relative total growth) was 95 x 106 above concurrent solvent controls (assuming these to be in the range of 35140 x 106). The test material was considered negative in this assay if there was no evidence of increase in mutant frequency at RTG values
10%.
Modified MLA with formaldehyde metabolic competency. An initial mutagenicity assay was conducted for selecting concentrations of the FA to be used in the gene mutation assay. On the basis of previously published work, the cells were treated with various concentrations of FA (16.7333.3 µM) in the absence and presence of S-9 factor (Brusick, 1982). Mutagenic doses of FA selected from the initial mutagenicity assay (100 and 133.3 µM in the absence and presence of S-9 metabolic activation, respectively) were used to assess the ability of formaldehyde dehydrogenase (FDH) (0.1 U) and its cofactor NAD+ (8 mM) to abrogate the increased mutant frequency produced by FA, during the 4-h treatment period. The remaining steps in the assay were performed as described above. With this modification to the standard protocol, an assessment of the ability to abrogate the increased mutant frequency after exposure to CS-1246 was then performed.
In vivo micronucleus. The MNT is a short-term in vivo cytogenetic assay for detecting agents that induce chromosomal breakage and spindle malfunction (Mavournin et al., 1990; OECD guideline 474). CS-1246 was administered to male CD-1 mice (Charles River, Portage, MI) by oral gavage on 2 consecutive days at dose levels of 0 (negative control), 500, 1000, and 2000 mg/kg/day. The dose levels were based on the results of the range-finding test, which showed no substantial difference in toxicity between the sexes. Hence, only males were used for the MNT, and the highest dose of 2000 mg/kg was the limit dose. The concentrations of the test material in the dosing solutions were verified by analytical methods. Groups of mice, 6/dose, were sacrificed 24 h after treatment on the second day of dosing for the collection of femoral bone marrow and evaluation. Mice treated with 120 mg/kg cyclophosphamide monohydrate at one dose were sacrificed 24 h later and served as positive controls. Two thousand polychromatic erythrocytes (PCE) were examined from each animal and the number of micronucleated PCE (MN-PCE) was recorded. As a measure of cell toxicity, the ratio of PCE to normochromatic erythrocytes (NCE) in the bone marrow was determined by examining 200 erythrocytes. A test was considered positive if a statistically significant increase in the MN-PCE frequency was observed at one or more dose levels accompanied by a dose response.
In vivo/in vitro UDS assay. For the in vivo UDS assay, the assay generally followed the procedure as detailed by Kennelly et al. (1993) (OECD Guideline 486) as modified from the original assay developed by Mirsalis, Tyson, and Butterworth (1982)
. F344 rats (5/group), purchased from Harlan (Frederick, MD) were treated once by oral gavage at 0 (negative control) 1000 and 2000 mg/kg (10 ml/kg). N-dimethylnitrosamine (DMN), the positive control was administered ip to 4 rats at 10 mg/kg and 15 mg/kg for the 24 h and 1415 h time-points, respectively. Animals were sacrificed at either 24 h or 1415 h after dosing and their livers were perfused with collagenase to generate primary cultures of hepatocytes. Cultures were made from 3 animals at each sacrifice time from each dose group and a 4 h culture labeling was initiated using 10 µCi/ml 3H-thymidine at 3560 Ci/mmol and slides evaluated after autoradiography. Four animals from the vehicle and test article dose and three from the positive control groups were analyzed. The cells were examined microscopically at approximately 1500x magnification and UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (cytoplasmic count). The net nuclear grain (NNG) count was routinely determined for 50 randomly selected cells on triplicate coverslips (150 total nuclei) for each animal. The average mean net nuclear grain count (± standard deviation) was determined from the triplicate coverslips. A response was considered positive if applied concentrations caused an increase in the group average of the mean NNG count of at least five grains per nucleus above the average control value. As well, an increase in the group average of the percent of nuclei with five or more net grains or at least 20% above the average control value of these nuclei in test culture. The Animal Care and Use Activities required for the conduct of these studies were reviewed and approved by the appropriate Institutional Animal Care and Use Committees.
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RESULTS |
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DISCUSSION |
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To evaluate the hypothesis that the mutagenic activity in the MLA system was the consequence of in situ FA generation from CS-1246, the MLA was subsequently modified to incorporate the rapid removal of FA from the test system by incorporation of an enzymatic FDH metabolic competency (Blackburn et al., 1991). Formaldehyde, as well as CS-1246 in the current study, produced concentration-related mutagenic effects in MLA cultures, mainly as an increase in small colony mutants thought to support a clastogenic mechanism of mutation formation (Speit and Merk, 2002
). Based on the molar yield of FA from the estimated 95% aqueous hydrolysis of mutagenic concentrations (55.969.8 µM) of CS-1246 (Fig. 1), the expected concentrations of FA generated in situ in the MLA (
106133 µM) themselves displayed significant mutagenic activity (Tables 3 and 4). Incorporation of FA metabolic capacity completely abrogated this concentration-related increase in mutants, and it inhibited the cytotoxicity produced by both FA and CS-1246, although it did not significantly affect the mutagenic response by the non-FAgenerating positive control compounds (MMS and 20-MCA).
The positive mutagenic response of CS-1246 in the MLA, and further investigation of its mode of action, suggested that the positive MLA response was attributable to the generation of FA in situ, in light of the potential for FA release by this material (Fig. 1). Based on the mode of action data, these results led us to hypothesize that CS-1246 was not likely to be genotoxic under in vivo conditions. The failure of CS-1246 to induce micronuclei or unscheduled DNA synthesis in the mouse bone marrow or the rat liver, respectively, is consistent with negative results reported for FA in vivo.
As shown for single-cell organisms with enhanced FDH capacity, the inability of FA to induce mutations in whole animals may reflect a functional capacity to oxidize FA to formic acid (Wehner and Brendel, 1993). Even active S9 fractions and/or the inclusion of rat hepatocytes to Chinese hamster V79 cells has been shown to reduce the FA-induced increase in sister chromatid exchange (SCE) to almost background (Basler et al., 1985
). The failure of CS-1246 to induce micronuclei or unscheduled DNA synthesis in the mouse micronucleus and the in vivo/in vitro rat UDS assays, respectively, is consistent with the proposed mode of action based on previous negative in vivo genotoxicity findings for FA and FA-releasing agents (Mackerer et al., 1996
; Natarajan et al., 1983
; Richardson et al., 1983
).
It is important to note that free FA, primarily as a result of serine metabolism, is present in animal and human blood and tissue at a concentration 6080 µM. It is also notable that many foods possess low ppm concentrations of FA (reviewed in Restani and Galli, 1991
). These concentrations are close to the
100 µM concentrations found to further increase mutation frequency in the MLA results reported here (Tables 3 and 4). Background FA concentrations in mammalian tissue and blood are within an order of magnitude of the
50 µM concentrations of Bioban CS-1246 required to statistically increase MLA mutation frequency (Table 1 and 2). Under normal conditions, the level of free FA is very low in animal and human tissues because of its rapid metabolism. This is achieved by several enzyme systems, predominantly via the enzyme formaldehyde dehydrogenase (FDH) (Casanova-Schmitz et al., 1984
; Heck et al., 1985
).
In summary, during the course of evaluating the potential genotoxicity of CS-1246 it was found to be positive in the MLA. Because of the known hydrolytic breakdown products of CS-1246, it was decided to evaluate FA as the basis of its potential mode of action. The incorporation of a FA-metabolizing system into the assay completely inhibited the observed increase in mutation frequency. Recently, the IARC Working Group concluded that there was sufficient evidence in humans and experimental animals for FA carcinogenicity (group 1) (IARC Monograph, 2004). The extensive literature on FA would therefore be of value in assessing the carcinogenic risk to humans and animals from CS-1246 exposure.
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ACKNOWLEDGMENTS |
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