Abstract
Hexahydro-1,3,5-trinitro-1,3,5-triazine, a polynitramine compound, commonly known as RDX, has been used as an explosive in military munitions formulations since World War II. There is considerable data available regarding the toxicity and carcinogenicity of RDX. It has been classified as a possible carcinogen (U.S. Environmental Protection Agency, Integrated Risk Information System, 2005, www.epa.gov/IRIS/subst/0313.htm). In order to better understand its gentoxic potential, the authors conducted the in vitro mouse lymphoma forward mutation and the in vivo mouse bone marrow micronucleus assays. Pure RDX (99.99%) at concentrations ranging from 3.93 to 500 μg/ml showed no cytotoxicity and no mutagenicity in forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, with and without metabolic activation. This finding was also confirmed by repeat assays under identical conditions. In addition, RDX did not induce micronuclei in mouse bone marrow cells when tested to the maximum tolerated dose of 250 mg/kg in male mice. These results show that RDX was not mutagenic in these in vitro and in vivo mammalian systems.
Keywords
Hexahydro-1,3,5-trinitro-1,3,5-triazine is a cyclic polynitramine, commonly known as RDX. It has been used as an explosive in military munitions formulations since World War II. It has been detected in soils and water at production and disposal sites or at load assembly and pack facilities (Walsh and Jenkins 1992; Major 1999) There are considerable data on toxicity and carcinogenicity of RDX (McLellan, Hartley, and Brower 1992; ATSDR 1995). It has been classified as a possible carcinogen (USEPA 2005b). Review of literature on genotoxicity revealed that there were some data gaps in the evaluation of the genotoxicity of RDX. The U.S. Environmental Protection Agency (EPA) currently follows a three-tiered system in their mutagenicity testing battery of chemicals in the assessment of human health risk. (Dearfield et al. 1991, 2002). In order to complete data gaps of three-tiered test battery approach, we conducted the in vitro mouse lymphoma forward mutation and the in vivo mouse bone marrow micronucleus assays to examine the genotoxic potential of RDX in mammalian in vitro and in vivo mutagenicity test systems.
Numerous studies have been conducted with the Ames Salmonella typhimirium assay with RDX. These studies have consistently yielded negative results for all strains of bacteria tested. Simmon et al. (1977) tested the potential mutagenicity of RDX in S. typhimirium strains TA100, TA1535, TA1537, and TA1538 at concentrations ranging from 0.24 to 14 μg/plate. Similarly, RDX was evaluated in strains TA98, TA100, TA1535, TA1537, and TA1538 at concentrations 0, 1, 10, 100, 300, or 1000 μg/plate with and without metabolic activation by Cholakis et al. (1980). Whong, Speciner, and Edwards 1980 conducted similar studies with RDX in all five Salmonella strains with and without metabolic activation system in concentrations up to 2.5 mg/plate. Tan et al. (1992) tested the possible mutagenicity of RDX and the related compound octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) up to concentrations of 1 mg/plate and showed that these compounds were not mutagenic in two strains of bacteria, S. typhimirium TA98 and TA100.
The mutagenic potential, of RDX has also been evaluated with the Mutatox assay using the bacteria Vibro fischeri, yielding mixed results. In these studies, two of the three replicates showed positive response with and without metabolic activation (no dose-dependent response was observed) (Arfsten, Davenport, and Schaeffer 1994). RDX was also evaluated in Saccharomyces cerevisiae strain D3 with and without metabolic activation and was found to be nonmutagenic in this test system (Simmon et al. 1977). More recently, George, Huggins-Clark, and Brooks (2001) evaluated the mutagenic potential of RDX and its mono-, di-, and trinitroso metabolites using the more modern S. typhimirium microsuspension assay with strains TA98 and TA100 (with and without metabolic activation). RDX was tested in this system at concentrations up to 250 μg/plate and was found to be negative in both strains. Therefore, these results were consistent with the previously reported studies regarding the mutagenic potential of RDX.
The 1-nitroso- and the 1,3-dinitroso metabolites of RDX were also found to be negative for mutagenicity in strains TA98 and TA100 (George, Huggins-Clark, and Brooks 2001). However, the 1,3,5-trinitroso congener was weakly mutagenic in strain TA100 with and without metabolic activation but negative in strain TA98. There were no confirmatory assays conducted to investigate whether or not the weak mutagenicity signal in one tester strain was potentially a false positive response.
Genotoxicity studies conducted in several in vitro test systems using mammalian cell culture also showed negative results. Dilley, Tyson, and Newell (1978) tested unscheduled DNA synthesis (UDS) with RDX at concentrations ranging from 250 to 4000 μg/ml with human fibroblasts (WI-38 cells), with and without metabolic activation. These studies produced negative results, showing that RDX did not cause DNA damage in human fibroblasts. Studies of the cytotoxic and gentoxic effects of energetic compounds on mammalian cells in vitro showed that RDX was not cytotoxic in V79 Chinese hamster lung fibroblast cells or in TK6 human lymphoblastic cells, even at its maximum aqueous solubility limits (50 to 70 μg/ml) (Lachance et al. 1999). Moreover, it was not mutagenic in V79 cells with or without metabolic activation (Lachance et al. 1999). The aqueous extracts of soils contaminated with 2,4,6-trinitrotoluene (TNT), TNT metabolites, and RDX did not produce cytotoxicity in human fibroblasts nor mutagenicity in an hypoxanthine guanine phosphoribosyltransferase (HPRT) test in V79 cells, in any of the tests performed (Berthe-Corti et al. 1998)
An in vivo mammalian mutagenicity study was conducted with RDX in F-344 rats using the dominant lethal assay (Cholakis et al. 1980). Rats were fed a diet containing RDX at doses of 0, 5, 16, or 50 mg/kg/day for 13 weeks and allowed to mate with unexposed virgin females for a 2-week period. The RDX-treated males were subsequently evaluated for potential mutagenic effects in the spermatogenic process (lethal to the embryos or fetus). No significant effects on the number of corpora lutea, implants, or the number live or dead embryos were observed. RDX did not induce a dominant lethal effect at doses up to 50 mg/kg/day in the diet. We did not find other published reports of in vivo tests of RDX genotoxicity. Therefore, we conducted an in vitro mouse lymphoma forward mutation and an in vivo mouse bone marrow micronucleus assay to better address the EPA’s tiered evaluation criterion.
MATERIALS AND METHODS
These genotoxicity studies reported herein were performed in compliance with Good Laboratory Practice (US EPA 2005a), Title 40 of the U.S. Code of Federal Regulations Part 792 and any applicable amendments.
Chemical
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX; CAS no. 121-82-4) was purified to 99.99 % by Dr. W. Koppes, Naval Warfare Center, Indian Head, MD. The predominant HMX contaminant and minor contaminants were removed by recrystalizatioin with acetone and the purity was confirmed by high-performance liquid chromatograph (HPLC).
Cell culture chemicals such as RPMI 1640 medium, Fischer’s medium, antibiotics, and
Mouse Lymphoma Forward Mutation
Mouse lymphoma tk+/tk−-3.7.2C L5178Y cells were obtained from Dr. Donald Clive, Burroughs Wellcome Company, Research Triangle Park, NC, and were maintained in RPMI 1640 medium containing
RDX was dissolved in dimethylsulfoxide (DMSO) at 100 times the highest desired treatment concentration. Dosing was initiated by performing 1:100 dilution of the stock into the medium containing cells. A preliminary dose range-finding assay was conducted with a treatment period of about 4 h, with and without metabolic activation, as described below. One day after treatment, cytotoxicity was measured by determining cell density. Because of the presence of precipitate at concentrations at and above 250 μg/ml, the treatments for the definitive assay were limited to 500 μg/ml. The mouse lymphoma forward mutation assay was performed as described previously (Clive et al. 1987). Briefly, the mutation assays were initiated with 6 × 106 cells and treated for 4 h in a rotary shaker. After the 4-h treatment phase of the assay, the cells were incubated for a 2-day expression period. On day 2, 3 × 106 cells were seeded for identification of mutants with cloning medium containing 5-trifluorothymidine (TFT). About 600 cells were also seeded into cloning medium without TFT for determination of viable colonies. After 10 to 14 days, colonies were counted automatically using the High Resolution Colony Counter (HRCC) System (Loats Associates, Inc). The mouse lymphoma assay produces a bimodal distribution of large and small colonies. The origin of the bimodal distribution of mutant colony sizes is considered to reflect the types of genetic damage, with the large colonies derived from cells with intragenic mutations that affect only the thymidine kinase gene and the small colonies the result of larger mutations that affect cell growth as well as the thymidine kinase gene. Both the small and large colonies were quantified for all cultures. (Hozier et al. 1985)
The evaluation criteria used for the mouse lymphoma assay is as follows. The results are considered positive if dose-dependent increases of twofold or greater in mutant frequency are obtained over the concurrent background mutant frequency (average mutant frequency of the vehicle control cultures). The twofold or greater increase is based on extensive experience that indicates such responses are repeatable in additional trials. The test article is evaluated as negative if a twofold increase in mutant frequency is not observed for (1) a range of doses that extends to toxicities causing 10% to 20% relative total growth (RTG); (2) for relatively nontoxic test articles, a range of doses extending to the maximum concentration of 5 mg/ml or 10 mM (whichever is lower); (3) a range of doses that extends to a level approximately twice the solubility limit in culture medium; (4) the increase(s) are not repeatable in a confirmatory trial.
Mouse Micronucleus Assay
Young adult male and female CD-1 (ICR) strain mice were purchased from Charles River Laboratories, Raleigh NC. They were fed a commercial diet (PMI Feed Inc, Certified Rodent Diet 5002) and, tap water was available ad libitum. They were maintained at room temperature 64°F to 79°F, humidity, and 30% to 70%; light cycle 12-h light/dark. The animals were acclimated for at least 5 days prior to testing.
Test article was dissolved in corn oil (Welch, Holme, & Clark lot no. 12–394). Based upon the result of the dose range-finding study, the estimated maximum tolerated doses was 250 mg/kg after one oral gavage administration using a dose volume of 10 ml/kg. Because no relevant gender difference was observed in the dose range-finding study, only males were used in the definitive micronucleus assay. Therefore, as per Organization for Economic Cooperation and Development (OECD) guideline 474 requirements (OECD 1997), RDX doses of 62.5, 125, and 250 mg/kg were examined in definitive micronucleus assay using the appropriate vehicle and positive-control groups. The positive-control group was dosed with 80 mg/kg cyclophosphamide in sterile deionized water. Bone marrow smears were prepared and allowed to air dry. The slides were then fixed in methanol, stained in May-Grunwald–Giemsa stain and protected by permanently mounted cover slips. The slides were blind scored for micronuclei in polychromatic erythrocytes (PCEs) and norchromatic erythrocytes (NCEs) and determination of PCE/NCE ratios to access possible bone marrow cytotoxicity. The micronucleus frequency (expressed as percent micronucleated cell) was determined by analyzing number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least the first 500 erythrocytes per animal. The criteria for the identification of micronuclei were those of Schmid (1976).
RESULTS
Mouse Lymphoma Assay
The genotoxic potential of RDX was evaluated in the mouse lymphoma assay. RDX formed a transparent colorless solution in the vehicle DMSO. Two independent trials were performed with concentrations ranging from 3.93 to 500 μg/ml in the absence and presence of S9 activation. No cytotoxicity to weak cytotoxicity was induced. Although it is desirable to include highly cytotoxic treatments, higher concentrations were not included in this study because a precipitate was observed in treatment medium at concentrations as low as 250 μg/ml. Higher concentrations would have interfered with the performance of the assay.
In the mutagenicity assays performed with RDX, shown in Tables 1 through 4, no dose-related increases in the mutant frequency were induced that exceeded the minimum criterion for a positive response. Without activation, the background (vehicle control) mutant frequency was 94.1 × 10−6 and the RDX-treated cultures induced mutant frequencies ranging from 79.2 × 10−6 to 105.2 × 10−6. A mutant frequency of 188.2 × 10−6 was required for a positive response. With S9 activation the background mutant frequency was 72.5 × 10−6 and the RDX-treated cultures induced mutant frequencies ranging from 82.5 × 10−6 to 133.5 × 10−6. A mutant frequency of 145.0 × 10−6 was required for a positive response. In the confirmatory assays without activation, the background mutant frequency was 62.8× 10−6 and the RDX-treated cultures induced mutant frequencies ranging from 55.1 × 10−6 to 77.1 × 10−6. A mutant frequency of 125.5 × 10−6 was required for a positive response. With S9 activation the background mutant frequency was 117.5 × 10−6 and the RDX-treated cultures induced mutant frequencies ranging from 104.9 × 10−6 to 172.2 × 10−6. A mutant frequency of 235.0 × 10−6 was required for a positive response.
The positive-control chemicals, MMS and MCA, both induced large increases above the background mutant frequency and the negative-control mutant frequencies were within historical range for all trials in the absence and presence of S9 activation. In addition, a distribution of the colony sizes was determined. The positive controls induced large increases in the mutant frequency and the size distributions of the control and treated cultures were in the expected range. Therefore, under the experimental conditions used, RDX was considered to be negative in this assay.
Micronucleus Assay
The dose range finding study revealed that RDX induced signs of clinical toxicology and mortality at high-dose group. Mice dosed at all dose levels showed neurotoxic signs (hyperactive) and deaths at high dose of 500 mg/kg. Therefore 250 mg/kg dose was selected as maximum tolerated dose in this micronuceuos assay. RDX at doses 62.5, 125, and 250 mg/kg were not cytotoxic to bone marrow (i.e., no statistically significant decrease in PCE:NCE ratios) (Tables 5 to 7). In addition, there were no significant decreases in the PCE:NCE ratios observed at any RDX dose or bone marrow sampling time point. The positive-control article, cyclophosphamide, induced statistically significant increase in micronucleated PCE as compared to that of vehicle control as expected (Table 3). RDX was found to be negative in the in vivo mouse bone marrow micronucleus assay.
DISCUSSION
RDX is extensively used in military applications as a high explosive. The US Army developed extensive data on toxicity and carcinogenicity to evaluate the health and environmental effects of RDX (McLellan, Hartley, and Browen 1992; ATSDR 1995). Three chronic-duration feeding studies have been done with RDX in rodents. The studies done in two strains of rats (Hart 1977; Levine et al. 1983) were negative for cancer. However, equivocal evidence of carcinogenicity was seen in female (but not male) B6C3F1 mice (Lish et al. 1984). The carcinogenicity in female mice was only statistically significant when the incidence of liver carcinomas and adenomas are combined and when the unusually low incidence of cancer in the concurrent control animals was used. On the strength of this evidence, RDX was classified as possible human carcinogen (USEPA IRIS 2005b). The EPA’s new cancer guidelines require brief description of the nature of the carcinogenicity, mode of action, and dose/response assessment method consisting of a narrative statement (USEPA 2005a). Recently certain chemicals (taxophene, pthalates, chloroform) were reclassified on the basis of mode of action and relevance to human carcinogenicity in accordance with the new cancer guidelines. (Goodman et al. 2000; Roberts et al. 2003). In the sprit of this new guidance, we conducted additional studies to better assess the genotoxicity of RDX.
Previous all genotoxicity studies conducted with Ames Salmonella sp. showed negative responses in all tests with and without the S9 metabolic activation system (Simmon et al. 1977; Cholakis et al. 1980; Whong et al. 1980; Tan et al. 1992; George, Huggins-Clark, and Brooks 2001). A single study conducted with another bacterium Vibro sp. showed inconclusive results in mutatox assay. Two of the three replicates showed positive response. In this study details of compound purity was not reported. The degradation products of RDX resulting from bioreductive processes were also tested for genotoxicity. The mononitroso and the dinitrso metabolites were shown to be negative in the Ames Salmonella assay (George, Huggins-Clark, and Brooks 2001; Snodgross 1984). However, the trinotroso metabolite showed weak mutagenicity in only one strain (TA100) with and with out metabolic activation (George, Huggins-Clark, and Brooks 2001). No confirmatory tests were performed to see whether this weak response was potentially a false positive.
The in vitro mammalian cell cytotoxicty and mutagencity tests revealed that RDX is not mutagenic at concentrations tested. Similarly, aqueous soil extracts did not reveal any toxicity or mutagenicity in any of the tests with the HPRT in V79 cells (hamster lung fibroblast) performed by (Berthe-Corti et al. 1998). In another in vitro mammalian assay RDX (250 to 4000 μg/ml) did not induce UDS in human fibroblast with or without metabolic activation (Dilley, Tyson, and Newell 1978). Data presented in this report with RDX in the mammalian mouse lymphoma forward mutation test also showed a negative response with and without metabolic activation. This was also true in the confirmatory test conducted under similar conditions. It has been reported by Snodgrass (1984) that hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) showed positive response in chromosomal aberrations tests conducted with Chinese hamster ovarian (CHO) cells and it increased significantly DNA repair in rat hepatocytes, but showed negative response in dominant lethal test in mice. The details, the experimental data, and the purity of the test compound from this early work (Snodgrass 1984) is no longer available. Thus, the significance of this work cannot be evaluated. The in vivo dominant lethal tests showed RDX was negative in rats (Cholakis et al. 1980).
The in vivo mouse micronucleus assay is considered a good indicator of carcinogenicity. The US EPA Gene-Tox analysis revealed that about 95% of the chemicals that showed carcinogenicity also demonstrated a positive response with this assay. (Kier 1988). Present studies with RDX showed negative response in this in vivo mouse micronucleus assay. The carcinogenicity data on RDX in mice reported by Lish et al. (1984) was reevaluated (Parker et al. in preparation). The reevaluation showed that only a single dose group of RDX in a single gender was associated with a combined incidence of cancer and adenoma that was statistically different from the incidence in the control population (PWG 2001). The validity of data of Lish et al. (1984) is called in question by the fact that the RDX used in the study was of military grade and contained considerable amounts up to 11% of HMX and other impurities.
Footnotes
Tables
Acknowledgements
The authors thank Dr. Howard Bausum for a critical review of the manuscript.
Disclaimer: The views, opinions, and/or findings should not be construed as official Department of Army position, policy, or decision unless designated by other official documentation.