Mode of Mutagenic Action for the Biocide Bioban CS-1246 in Mouse Lymphoma Cells and Implications for Its In Vivo Mutagenic Potential
http://www.100md.com
《毒物学科学杂志》
Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674
Covance Laboratories, Vienna, Virginia 22182
ABSTRACT
The biocidal agent, BIOBAN CS-1246 (7-ethyl bicyclooxazolidine, CAS# 7747–35–5, CS-1246) induced a concentration-dependent mutagenic response in mouse lymphoma (L5178Y TK+/–) cells both with and without the addition of S9 metabolic activation. Previous data indicating the ability of CS-1246 to hydrolyze in aqueous media to generate formaldehyde (FA), led us to investigate the potential role of FA in the CS-1246–induced mutagenic response in the mouse lymphoma assay (MLA). To accomplish this, the MLA on CS-1246 was repeated in the presence of a metabolizing system (formaldehyde dehydrogenase/NAD+), which was shown to successfully inhibit the mutagenic response of formaldehyde in this assay system. Significantly, the observed mutagenicity of CS-1246 was completely abrogated when the cultures were supplemented with formaldehyde dehydrogenase/NAD+, suggesting that the positive MLA response was attributable to the generation of FA in situ. These results demonstrate that in vitro mutagenicity of CS-1246 in the MLA is most likely associated with FA. Negative results from two in vivo assays for genotoxicity were consistent with the known activity of FA in these assays. In the mouse bone marrow micronucleus (MNT), there were no significant increases in micronucleated polychromatic erythrocytes (with evaluation of 2000/animal), after treatment with 0.5, 1, and 2 g/kg/day CS-1246 (6/dose group) for 2 consecutive days and sacrifice 24 h later. Furthermore, in the unscheduled DNA synthesis (UDS) study, male F344 rats (5 /dose group), given a single oral gavage (0, 1, and 2 g/kg) and evaluated at two time points (2–4 and 14–15 h post dosing), did not elicit an UDS response, indicating a lack of DNA reactivity in vivo. Based on the negative in vivo findings, it can be inferred that the FA detoxification mechanisms that exist in intact organisms prevent the likelihood of generating FA at levels capable of causing genotoxicity following exposure to CS-1246 at low, environmentally relevant concentrations. The extensive literature on FA would therefore be of value in assessing the carcinogenic risk to humans and animals from CS-1246 exposure.
Key Words: Genetic toxicity; formaldehyde; mouse lymphoma; oxazolidine.
INTRODUCTION
BIOBAN CS-1246 (7-ethyl bicyclooxazolidine; CAS# 7747–35–5, CS-1246) is a broad-spectrum bactericide used in paints; inks; emulsions; slurries; non-food contact adhesives; consumer, household, and institutional products; and metal-working fluids. Typical use concentrations in the various applications can range from 400 to 2000 ppm. It is believed that biocidal activity of CS-1246 is related to generation of formaldehyde (FA) secondary to hydrolytic degradation (Fig. 1). Based on use patterns, humans are most likely to be exposed to CS-1246 via dermal contact with the biocide itself or through products treated with the biocide. Because of its low volatility, little hazard potential is expected via vapor inhalation. Likewise, secondary exposure via ingestion is also unlikely, because humans are not exposed to residues in food or drinking water.
In animal studies, CS-1246 is slightly toxic by the oral route, is severely irritating to the eyes and skin, and is a weak skin sensitizer. A low incidence of allergic contact dermatitis has been reported in workers under long-term usage conditions as a preservative in metal-working fluids (Dahlquist, 1984; Camarasa et al., 1993). Sub-acute rat studies carried out by the oral route (28 consecutive days of dosing) and dermal route (5 days/week over 21 days) at concentrations up to 1000 mg/kg/day yielded signs of local irritation at the sight of contact but failed to produce specific target organ toxicity. CS-1246 is not teratogenic in rodents (Dow Chemical Company Toxicity Data. http://www.dow.com/webapps/lit/litorder.aspfilepath=biocides/pdfs/noreg/253-01210.pdf&pdf=true).
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+.
MATERIALS AND METHODS
Test material. CS-1246 samples used in these studies were supplied by ANGUS Chemical Company (Buffalo Grove, IL). The purity was determined to be between 96.8% and 98.8% for all lots of test material. The concentrations of the test material in the dosing solutions were analytically verified by gas chromatography with flame ionization detection (GC/FID) and external standard quantitation.
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 8.7–104.7 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:
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:
The mutant frequency per 106 clonable cells was calculated as below:
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 10–6 above concurrent solvent controls (assuming these to be in the range of 35–140 x 10–6). 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 (16.7–333.3 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 2–4 h and 14–15 h time-points, respectively. Animals were sacrificed at either 2–4 h or 14–15 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 35–60 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.
RESULTS
Mouse Lymphoma Assay Results for CS-1246
The genotoxic potential of CS-1246 was assessed with concentrations ranging from 8.7 to 69.8 μM and 8.7 to 104.7 μM in the absence and presence of S9 activation, respectively. Distilled water was used as the negative control. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, MMS and 20-MCA for assays in the absence and presence of S9, respectively. In the mutagenicity assay, CS-1246 induced dose-related and significant increases in mutant frequencies up to 8.7-fold in the absence of S9 and 7.3-fold in the presence of S9 (Tables 1 and 2), as compared to the negative control cultures at the highest evaluated concentration (i.e., 104.7 μM, due to unacceptable toxicity at higher concentration). Hence, under the experimental conditions used, CS-1246 was considered to be positive in this in vitro MLA study.
Mouse Lymphoma Assay Results for Formaldehyde and CS-1246 in the Presence of Formaldehyde Dehydrogenase
The hydrolysis of CS-1246 in aqueous buffered solution and its ability to generate FA has previously been determined (unpublished data). Based on these data, its half-life under the pH and temperature conditions of the MLA is estimated at 1 h. This would allow for 95% hydrolysis of the CS-1246 test material in solution during the course of the 4-h MLA incubation. To test the hypothesis that the positive MLA activity of CS-1246 was due to in situ FA generation, the MLA assay was fortified with a formaldehyde dehydrogenase (Blackburn et al., 1991). In a preliminary mutagenicity assay FA was evaluated at concentrations from 16.6 to 333.3 μM (the acceptable cytotoxicity limit) in the absence and presence of an externally supplied metabolic activation system (S9). Increased mutant frequencies were observed from concentrations of 100 μM and above in both the presence and the absence of S9 activation (data not shown). Based on these results, concentrations of 100 and 133.3 μM in the absence of S9 and 133.3 μM in the presence of S9 were selected for use in a gene mutation assay. In the presence of NAD+ (8 mM) and FDH (0.1 U) together, the increases in mutant frequency were completely abrogated (Tables 3 and 4), returning the cultures so treated to background mutant frequency levels with greatly enhanced relative growth compared to cultures treated with FA alone. Cultures treated with the positive control chemicals (MMS and 20-MCA) had significantly higher mutant frequencies compared to the solvent control, even in the presence of NAD+ and FDH. Consequently, a methodology was successfully implemented to abrogate the mutagenic response of FA in mouse lymphoma cultures by the inclusion of FA metabolic competency in the form of FDH and its cofactor NAD+. With this modified assay methodology, the presence of a FA metabolic system (FDH/NAD+; Table 5) completely inhibited the mutagenic response of CS-1246 (55.9, 62.9, and 69.8 μM), in the MLA cultures.
Mouse Micronucleus Test
Clinical signs were noted only in the 2000 mg/kg/day dose group where 3 mice (out of 8 dosed) died prior to the scheduled sacrifice. CS-1246 did not induce a significant increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes when they were given as a single oral dose of 2000 mg/kg/day (the limit dose) on two consecutive days to male CD-1 mice as compared to the negative controls (Table 6). The positive control animals showed a significant increase in the frequency of MN-PCE as compared to the negative control animals. There were no statistically significant differences in the percent PCE for groups treated with the test material, whereas the mean percent PCE value of the positive control was significantly lower than the negative controls. Hence, CS-1246 was considered negative in this test system under the experimental conditions used.
In Vivo UDS
In this assay for CS-1246, doses were selected based on a range-finding study with dose levels of 400, 800, 1200, 1600, and 2000 mg/kg. The results of the assay are presented in Table 7. Treatment with CS-1246 at 1000 and the limit dose of 2000 mg/kg yielded NNG values less than zero, producing group mean NNG values over the two experimental time periods of 2–4 and 14–15 h, in the range –0.89 to –0.47. None of the treated hepatocyte cultures had an increase in the group average of the percent of nuclei with five or more net grains or a 20% increase above the average values of the nuclei in control cultures. The system was shown to be sensitive to the known DNA damaging agent, DMN and the assay was therefore considered valid, indicating that in vivo dosing of CS-1246 did not result in increased UDS in hepatocytes isolated immediately 2–4 or 14–15 h after dosing.
DISCUSSION
CS-1246 was previously found to be non-genotoxic in a bacterial reverse mutation assay (Ames test) and an in vitro chromosomal aberration assay using Chinese hamster ovary cells (unpublished data). The positive mutagenic response by CS-1246 in the MLA led us to further investigate its mode of action, in light of the potential for in situ FA release by this material (Fig. 1). Formaldehyde has been shown to be mutagenic in mouse lymphoma cultures (Speit and Merk, 2002; Blackburn et al., 1991; Wangenheim and Bolcsfoldi, 1988; Brusick, 1982) both with and without metabolic activation. Craft and Skopek (1986) reported FA to induce mutations at the thymidine kinase locus in TK6 human lymphoblasts. These studies, however, did not attempt to evaluate the ability of FDH activity on ameliorating the effects of FA.
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 (55.9–69.8 μM) of CS-1246 (Fig. 1), the expected concentrations of FA generated in situ in the MLA (106–133 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-FA–generating 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 60–80 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.
ACKNOWLEDGMENTS
Support for this work was provided by The Dow Chemical Company.
REFERENCES
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Covance Laboratories, Vienna, Virginia 22182
ABSTRACT
The biocidal agent, BIOBAN CS-1246 (7-ethyl bicyclooxazolidine, CAS# 7747–35–5, CS-1246) induced a concentration-dependent mutagenic response in mouse lymphoma (L5178Y TK+/–) cells both with and without the addition of S9 metabolic activation. Previous data indicating the ability of CS-1246 to hydrolyze in aqueous media to generate formaldehyde (FA), led us to investigate the potential role of FA in the CS-1246–induced mutagenic response in the mouse lymphoma assay (MLA). To accomplish this, the MLA on CS-1246 was repeated in the presence of a metabolizing system (formaldehyde dehydrogenase/NAD+), which was shown to successfully inhibit the mutagenic response of formaldehyde in this assay system. Significantly, the observed mutagenicity of CS-1246 was completely abrogated when the cultures were supplemented with formaldehyde dehydrogenase/NAD+, suggesting that the positive MLA response was attributable to the generation of FA in situ. These results demonstrate that in vitro mutagenicity of CS-1246 in the MLA is most likely associated with FA. Negative results from two in vivo assays for genotoxicity were consistent with the known activity of FA in these assays. In the mouse bone marrow micronucleus (MNT), there were no significant increases in micronucleated polychromatic erythrocytes (with evaluation of 2000/animal), after treatment with 0.5, 1, and 2 g/kg/day CS-1246 (6/dose group) for 2 consecutive days and sacrifice 24 h later. Furthermore, in the unscheduled DNA synthesis (UDS) study, male F344 rats (5 /dose group), given a single oral gavage (0, 1, and 2 g/kg) and evaluated at two time points (2–4 and 14–15 h post dosing), did not elicit an UDS response, indicating a lack of DNA reactivity in vivo. Based on the negative in vivo findings, it can be inferred that the FA detoxification mechanisms that exist in intact organisms prevent the likelihood of generating FA at levels capable of causing genotoxicity following exposure to CS-1246 at low, environmentally relevant concentrations. The extensive literature on FA would therefore be of value in assessing the carcinogenic risk to humans and animals from CS-1246 exposure.
Key Words: Genetic toxicity; formaldehyde; mouse lymphoma; oxazolidine.
INTRODUCTION
BIOBAN CS-1246 (7-ethyl bicyclooxazolidine; CAS# 7747–35–5, CS-1246) is a broad-spectrum bactericide used in paints; inks; emulsions; slurries; non-food contact adhesives; consumer, household, and institutional products; and metal-working fluids. Typical use concentrations in the various applications can range from 400 to 2000 ppm. It is believed that biocidal activity of CS-1246 is related to generation of formaldehyde (FA) secondary to hydrolytic degradation (Fig. 1). Based on use patterns, humans are most likely to be exposed to CS-1246 via dermal contact with the biocide itself or through products treated with the biocide. Because of its low volatility, little hazard potential is expected via vapor inhalation. Likewise, secondary exposure via ingestion is also unlikely, because humans are not exposed to residues in food or drinking water.
In animal studies, CS-1246 is slightly toxic by the oral route, is severely irritating to the eyes and skin, and is a weak skin sensitizer. A low incidence of allergic contact dermatitis has been reported in workers under long-term usage conditions as a preservative in metal-working fluids (Dahlquist, 1984; Camarasa et al., 1993). Sub-acute rat studies carried out by the oral route (28 consecutive days of dosing) and dermal route (5 days/week over 21 days) at concentrations up to 1000 mg/kg/day yielded signs of local irritation at the sight of contact but failed to produce specific target organ toxicity. CS-1246 is not teratogenic in rodents (Dow Chemical Company Toxicity Data. http://www.dow.com/webapps/lit/litorder.aspfilepath=biocides/pdfs/noreg/253-01210.pdf&pdf=true).
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+.
MATERIALS AND METHODS
Test material. CS-1246 samples used in these studies were supplied by ANGUS Chemical Company (Buffalo Grove, IL). The purity was determined to be between 96.8% and 98.8% for all lots of test material. The concentrations of the test material in the dosing solutions were analytically verified by gas chromatography with flame ionization detection (GC/FID) and external standard quantitation.
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 8.7–104.7 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:
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:
The mutant frequency per 106 clonable cells was calculated as below:
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 10–6 above concurrent solvent controls (assuming these to be in the range of 35–140 x 10–6). 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 (16.7–333.3 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 2–4 h and 14–15 h time-points, respectively. Animals were sacrificed at either 2–4 h or 14–15 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 35–60 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.
RESULTS
Mouse Lymphoma Assay Results for CS-1246
The genotoxic potential of CS-1246 was assessed with concentrations ranging from 8.7 to 69.8 μM and 8.7 to 104.7 μM in the absence and presence of S9 activation, respectively. Distilled water was used as the negative control. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, MMS and 20-MCA for assays in the absence and presence of S9, respectively. In the mutagenicity assay, CS-1246 induced dose-related and significant increases in mutant frequencies up to 8.7-fold in the absence of S9 and 7.3-fold in the presence of S9 (Tables 1 and 2), as compared to the negative control cultures at the highest evaluated concentration (i.e., 104.7 μM, due to unacceptable toxicity at higher concentration). Hence, under the experimental conditions used, CS-1246 was considered to be positive in this in vitro MLA study.
Mouse Lymphoma Assay Results for Formaldehyde and CS-1246 in the Presence of Formaldehyde Dehydrogenase
The hydrolysis of CS-1246 in aqueous buffered solution and its ability to generate FA has previously been determined (unpublished data). Based on these data, its half-life under the pH and temperature conditions of the MLA is estimated at 1 h. This would allow for 95% hydrolysis of the CS-1246 test material in solution during the course of the 4-h MLA incubation. To test the hypothesis that the positive MLA activity of CS-1246 was due to in situ FA generation, the MLA assay was fortified with a formaldehyde dehydrogenase (Blackburn et al., 1991). In a preliminary mutagenicity assay FA was evaluated at concentrations from 16.6 to 333.3 μM (the acceptable cytotoxicity limit) in the absence and presence of an externally supplied metabolic activation system (S9). Increased mutant frequencies were observed from concentrations of 100 μM and above in both the presence and the absence of S9 activation (data not shown). Based on these results, concentrations of 100 and 133.3 μM in the absence of S9 and 133.3 μM in the presence of S9 were selected for use in a gene mutation assay. In the presence of NAD+ (8 mM) and FDH (0.1 U) together, the increases in mutant frequency were completely abrogated (Tables 3 and 4), returning the cultures so treated to background mutant frequency levels with greatly enhanced relative growth compared to cultures treated with FA alone. Cultures treated with the positive control chemicals (MMS and 20-MCA) had significantly higher mutant frequencies compared to the solvent control, even in the presence of NAD+ and FDH. Consequently, a methodology was successfully implemented to abrogate the mutagenic response of FA in mouse lymphoma cultures by the inclusion of FA metabolic competency in the form of FDH and its cofactor NAD+. With this modified assay methodology, the presence of a FA metabolic system (FDH/NAD+; Table 5) completely inhibited the mutagenic response of CS-1246 (55.9, 62.9, and 69.8 μM), in the MLA cultures.
Mouse Micronucleus Test
Clinical signs were noted only in the 2000 mg/kg/day dose group where 3 mice (out of 8 dosed) died prior to the scheduled sacrifice. CS-1246 did not induce a significant increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes when they were given as a single oral dose of 2000 mg/kg/day (the limit dose) on two consecutive days to male CD-1 mice as compared to the negative controls (Table 6). The positive control animals showed a significant increase in the frequency of MN-PCE as compared to the negative control animals. There were no statistically significant differences in the percent PCE for groups treated with the test material, whereas the mean percent PCE value of the positive control was significantly lower than the negative controls. Hence, CS-1246 was considered negative in this test system under the experimental conditions used.
In Vivo UDS
In this assay for CS-1246, doses were selected based on a range-finding study with dose levels of 400, 800, 1200, 1600, and 2000 mg/kg. The results of the assay are presented in Table 7. Treatment with CS-1246 at 1000 and the limit dose of 2000 mg/kg yielded NNG values less than zero, producing group mean NNG values over the two experimental time periods of 2–4 and 14–15 h, in the range –0.89 to –0.47. None of the treated hepatocyte cultures had an increase in the group average of the percent of nuclei with five or more net grains or a 20% increase above the average values of the nuclei in control cultures. The system was shown to be sensitive to the known DNA damaging agent, DMN and the assay was therefore considered valid, indicating that in vivo dosing of CS-1246 did not result in increased UDS in hepatocytes isolated immediately 2–4 or 14–15 h after dosing.
DISCUSSION
CS-1246 was previously found to be non-genotoxic in a bacterial reverse mutation assay (Ames test) and an in vitro chromosomal aberration assay using Chinese hamster ovary cells (unpublished data). The positive mutagenic response by CS-1246 in the MLA led us to further investigate its mode of action, in light of the potential for in situ FA release by this material (Fig. 1). Formaldehyde has been shown to be mutagenic in mouse lymphoma cultures (Speit and Merk, 2002; Blackburn et al., 1991; Wangenheim and Bolcsfoldi, 1988; Brusick, 1982) both with and without metabolic activation. Craft and Skopek (1986) reported FA to induce mutations at the thymidine kinase locus in TK6 human lymphoblasts. These studies, however, did not attempt to evaluate the ability of FDH activity on ameliorating the effects of FA.
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 (55.9–69.8 μM) of CS-1246 (Fig. 1), the expected concentrations of FA generated in situ in the MLA (106–133 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-FA–generating 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 60–80 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.
ACKNOWLEDGMENTS
Support for this work was provided by The Dow Chemical Company.
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