ACUTE TOXICITY OF RDX NITROSO PRODUCTS 427 JOURNAL OF APPLIED TOXICOLOGY J. Appl. Toxicol. 2005; 25: 427–434 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jat.1090 Up-and-down procedure (UDP) determinations of acute oral toxicity of nitroso degradation products of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) Sharon A. Meyer,1,* Adam J. Marchand,1 Jennifer L. Hight,1 George H. Roberts,2 Lynn B. Escalon,3 Laura S. Inouye4 and Denise K. MacMillan5 1 2 3 4 5 Department of Toxicology, University of Louisiana at Monroe, Monroe, LA 71209, USA Department of Clinical Laboratory Science, University of Louisiana at Monroe, Monroe, LA 71209, USA Analytical Services, Inc., Huntsville, AL, 35806, USA U.S. Army Engineer Research and Development Center, Environmental Laboratory, Environmental Risk Assessment Branch, Vicksburg, MS 39180, USA U.S. Army Engineer Research and Development Center, Environmental Laboratory, Environmental Chemistry Branch, Omaha, NE 68102, USA Received 10 January 2005; Revised 11 April 2005; Accepted 12 April 2005 ABSTRACT: Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a widely used military explosive and soil and ground water contaminant of munitions manufacturing and artillery training sites, undergoes microbial nitroreductase metabolism to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). Human occupational and accidental exposures to RDX, as well as acute oral exposures in rats, result in seizures, but little is known about the toxicity of the RDX degradation products. The main objective of the present study was to determine the oral LD50 of the most potent RDX N-nitroso product in female SpragueDawley rats using the recently validated up-and-down procedure (UDP). With only 26 rats, MNX was identified as the most potent metabolite and a maximum likelihood estimate of 187 mg kg−1 (95% confidence interval 118–491 mg kg−1) for its LD50 was established and found equivalent to that of RDX determined with the same protocol. CNS toxicity, manifested as forelimb clonic seizures progressing to generalized clonic-tonic seizures, was the critical adverse effect. Further, confirmation of the UDP LD50 for MNX with a fixed-dose design enabled identification of 94 mg kg−1 as the highest nonlethal dose. An ED50 of 57 mg kg−1 was determined for neurotoxicity, while splenic hemosiderosis and decreased blood hematocrit and hemoglobin concentration occurred with a threshold at 94 mg kg−1 in 14-day survivors. These studies, while providing new toxicity data necessary for the management of RDX-contaminated sites, illustrate the efficiency of the UDP for comparative acute toxicity determinations and its value in guiding further characterization of dose dependency of identified adverse effects. Copyright © 2005 John Wiley & Sons, Ltd. KEY WORDS: N-nitroso toxicity; toxicity testing; neurotoxicity; anemia Introduction Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a highly energetic chemical used in conventional, biological, chemical and nuclear weapons (National Academy of Sciences, 2000). The need for site remediation of munitions manufacturing and artillery training sites with known RDX-contaminated soil and ground water, some of which are entries on the U.S. Environmental Protection Agency (USEPA) National Priorities List (Agency for Toxic Substances and Disease Registry [ATSDR], 1995), has necessitated the evaluation of the health * Correspondence to: Dr. S. A. Meyer, Department of Toxicology, College of Health Sciences, University of Louisiana at Monroe, Monroe, LA 712090470, USA. E-mail: meyer@ulm.edu Contract/grant sponsor: Environmental Quality and Technology Program, Engineer Research and Development Center, U.S. Army Corps of Engineers; contract/grant number: BS04 008D5P; BAA-02-3435. Copyright © 2005 John Wiley & Sons, Ltd. hazards of RDX. RDX is also being evaluated for future regulatory action under provisions of the Safe Drinking Water Act (USEPA, 2005). Knowledge of the toxicity of RDX environmental degradation products also is important for site remediation. Anaerobic microbial metabolism of RDX results successively in nitroreductase-catalysed formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) (Fig. 1). All three nitroso degradation products have been detected in ground water at RDX contaminated sites (Beller and Tiemeier, 2002) and as intermediates of bioremediation processes (Hawari et al., 2000). Acute occupational and accidental exposures to high doses of RDX have identified the central nervous system (CNS) as the critical target tissue as manifested by reversible seizure activity (Woody et al., 1986; Goldberg et al., 1992; Harrel-Bruder and Hutchins, 1995; Testud J. Appl. Toxicol. 2005; 25: 427–434 428 S. A. MEYER ET AL. acceptance of the up-and-down procedure (UDP) for determination of acute toxicity was in response to this goal. Since categorical data generated by the UDP are statistically analysed to yield maximum likelihood estimations of potency, the UDP was employed for these comparative studies. Further, the UDP LD50 for the most potent metabolite MNX was confirmed with a traditional, fixed-dose design, thus enabling dose-response determinations of MNX toxic effects qualitatively identified in the UDP study. Materials and Methods Figure 1. Structures of parent compound hexahydro1,3,5-trinitro-1,3,5-triazine (RDX) and products hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) et al., 1996; Küçükardalı et al., 2003). Seizures are also induced by acute oral exposure of rats to RDX and are causal to lethality, for which an LD50 of ~100 mg kg−1 has been determined (Cholakis et al., 1980; cited in ATSDR, 1995). Transient deposition of radioactivity from [14C]RDX within the brain occurs within hours of acute oral exposure, followed by accumulation in the liver and kidneys. Disposition in liver is due, in part, to retention of an unidentified metabolite(s) (Schneider et al., 1977). Nothing is known of the activities of the nitroso products relative to defined RDX toxic effects, while the recent demonstration of the weak mutagenicity of TNX (George et al., 2001) illustrates the potential for unique toxic effects contributed by the nitroso toxicophore. Effects typical of other N-nitroso compounds include cytotoxicity and genotoxicity from alkylating metabolites and effects resulting from nitroso redox activity, such as methemoglobinemia with consequential splenic hemosiderosis and anemia. Certain N-nitroso compounds are also sources of nitric oxide (Ohwada et al., 2001) and thus associated with the many toxic effects attributed to reactive nitrogen species. Clearly, research into the potential for MNX, DNX and TNX to elicit these toxic effects will provide information essential for remediation of sites contaminated by these military-unique compounds. In addition, comparative assessment of toxic effects of these compounds with parent RDX provides an opportunity to evaluate the relative activity of N-nitro vs N-nitroso functional groups in eliciting these effects. The objectives of the present study were to identify the most potent of the RDX N-nitroso products and compare its LD50 with that of its parent. A current priority of chemical hazard identification has been the implementation of validated procedures that reduce experimental animal usage. Recent Copyright © 2005 John Wiley & Sons, Ltd. Chemicals MNX, DNX and TNX were obtained from Dr Ron Spanggord (SRI Intl., Menlo Park, CA). Purities, determined by HPLC with UV detection, were >99.9% for TNX, 98.4% for MNX with ~1.2% RDX, and 66.4% for DNX with ~25.3% MNX and 7.1% TNX. Compounds were used without additional purification. Because of the highly energetic property of RDX, nitrosamine derivatives were also handled as potential explosives. RDX (>99%) was obtained from Stan Caulder (Naval Surface Warfare Center, Indianhead, MD) and was stored under absolute ethanol. Stored quantities were limited to <1 g and sonication was avoided in the preparation of corn oil-based dosing solutions. Animals and Treatment Female Sprague-Dawley rats were from the in-house breeding colony (School of Pharmacy, University of Louisiana at Monroe [ULM]) and maintained in accordance with the Guide for Use and Care of Animals (National Research Council, 1996). Breeders were from Harlan-Sprague Dawley (Madison, WI). Rats were housed with a 12 h light/dark cycle and free access to tap water and pelleted rodent chow (no.7012, Harlan/Teklad, Madison, WI). One week prior to trials, groups of rats (175–225 g) were housed individually in polycarbonate cages on hardwood bedding (Sani-chips, Harlan/ Teklad, Madison, WI). Study protocols had prior approval by the ULM Animal Care and Use Committee. Comparative Studies For comparison of acute toxicity of MNX, DNX and TNX, a sequential dosing protocol based upon the UDP was used, but modified to incorporate a smaller dose progression factor (1/3 log unit) to better distinguish between potencies of related compounds. Also, because chemicals were available in limited amounts, the progression was J. Appl. Toxicol. 2005; 25: 427–434 ACUTE TOXICITY OF RDX NITROSO PRODUCTS 429 stopped when a dose was identified with >50% lethality with one of the N-nitroso products and <50% lethality with the others and limit dose determinations were not conducted. Instead, the series was initiated with a starting dose of 400 mg kg−1, a dose approximating that yielding 80% mortality with RDX as determined by Schneider et al. (1977). Dosing solutions were made in the minimal amount of dimethylsulfoxide (DMSO, 5% v/v) in corn oil that allowed preparation of a stable emulsion for the highest dose of the least soluble compound (400 mg kg−1 MNX) without sonication. Solutions were prepared by dissolving compounds in DMSO, and then mixing with corn oil (Sigma Chemical Co., St Louis, MO) warmed to 40 °C just before use. Rats were weighed, then rank ordered based upon random numbers assigned by weight. Food was withdrawn overnight before administration of starting doses at 10–11 a.m. by gavage to rat 1 (TNX), rat 2 (DNX) and rat 3 (MNX). Rat 4 received vehicle at 10 ml kg−1, the volume administered to rats receiving test compounds. Qualitative assessments of appearance, righting reflex, forelimb strength and incidence of tremors and/or convulsions were made at 15 min intervals during the first hour after treatment, hourly over the next 8 h and daily thereafter over the 14 day observation period. Since death was observed at 400 mg kg−1 MNX within 48 h, rats 5, 6 and 7 received 187 mg kg−1 TNX, DNX or MNX, respectively. Doses for the third group of rats were increased to 400 mg kg−1 or decreased to 87 mg kg−1 depending upon whether the 187 mg kg−1 rat survived or died, respectively, within 48 h. Doses for subsequent animals were similarly determined by outcome of the previous rat. Moribund rats, as judged with criteria of the Organization for Economic Cooperation and Development (OECD, 2000), and 14-day survivors were euthanized with CO2 and examined for gross pathological effects. Liver, kidney plus adrenal, stomach plus duodenum and spleen were fixed in neutral buffered formalin for histopathology. UDP Studies and Fixed-dose Acute Toxicity Study Having established that the most potent RDX N-nitroso metabolite was MNX, a UDP study was conducted according to the 2002 EPA acute oral toxicity guideline 870.1100 (USEPA, 2002) with a slope factor sigma = 0.33 and a starting dose of 87 mg kg−1. Additional subsequent dose levels were 187 and 400 mg kg−1. Dose progression was stopped when one of the three stopping rules of the AOT425StatPgm program (USEPA, 2003) was satisfied. A UDP trial with RDX was similarly conducted to provide an oral LD50 generated under the same conditions, i.e. preparation of corn oil-based solutions by dilution of compounds dissolved in DMSO. It was anticipated that prior dissolution in DMSO would control for the wide Copyright © 2005 John Wiley & Sons, Ltd. variability in LD50 from other studies that has been attributed to the particle size of RDX solid used to prepare dosing solutions (Yinon, 1990). Dosing solutions of RDX were prepared just before use by drying an aliquot of ethanol slurry and redissolving the weighed, residual solid in DMSO, then warm corn oil as described above. To confirm the MNX LD50 and to provide material for the assessment of dose dependency of endpoints identified in the UDP, a fixed dose protocol was performed as in the 1998 EPA 870.110 guidelines except that the number of dose levels was increased from 3 to 5 to facilitate inclusion of no-adverse-effect- and lowestadverse-effect-levels for outcomes other than lethality. Five groups of six rats (175–225 g) were weighed and housed individually, then randomly assigned to dose. Prior to treatment, food was withdrawn overnight. Treatments were vehicle or MNX at 47, 94, 187, 280 or 420 mg kg−1. Rats were observed for incidence of tremors and/or convulsions frequently over the first 8 h after treatment. Survivors were weighed at 7 and 14 days, then exsanguinated by cardiac puncture while under CO2 anesthesia. Blood was collected in heparinized syringes and transferred to EDTA-containing tubes for hemocytology and determination of creatinine and urea nitrogen. Livers and spleens were weighed and sections taken for histopathology. Blood Analyses and Histopathology Blood urea nitrogen (BUN) and creatinine concentrations were analysed with a Synchron CX® System (Beckman Instruments; Brea, CA). In this system, BUN is measured as the rate of NADH oxidation by a coupled ureaseglutamate dehydrogenase enzymatic reaction. Creatinine was measured as a complex with picrate upon reaction with Jaffe’s reagent. Hemoglobin and red and white blood cell count and size were determined with a CELL-DYN 3500 System (Abbott Laboratories; Abbott Park, IL). Hematocrit is derived from red blood cell size and count. Hemoglobin was measured as absorbance at 540 nm after leukocyte lysis and conversion to hemoglobin-hydroxylamine. Formalin-fixed tissues were embedded in paraffin and 5 µm sections were stained with hematoxylin and eosin. Qualitative examinations for pathology were made on all collected tissues. Sections of spleen were also stained for ferric iron deposits by reacting with potassium ferrocyanide to form Prussian blue and counterstained with Nuclear fast red (Vacca, 1985). Quantitative assessment of Fe3+-laden splenic macrophages was done by counting dark blue staining vesicles of diameter greater than that of yellow-staining erythrocytes in red pulp of spleens stained for Prussian blue (Fig. 2). Slides were coded before observation and at least 20 100× oil immersion fields were counted per animal. J. Appl. Toxicol. 2005; 25: 427–434 430 S. A. MEYER ET AL. 2003). Estimates of the MNX LD50 and ED50 for convulsions were determined from the fixed-dose acute toxicity study by log-probit analysis with SAS PROC PROBIT (SAS, 1989). Effects of MNX on body and organ weights, hematological parameters, and blood urea nitrogen and creatinine of the 14-day survivors were determined by ANOVA with post-hoc comparisons of treatment means against vehicle control done with Dunnett’s test. Count data of large, Prussian blue-stained splenic vesicles/microscopic field were analysed for dose dependence using the Cochran-Armitage trend test (Piegorsch and Bailer, 1997) and comparison of treatment means against vehicle was done by ANOVA plus Dunnett’s test on the square root transformation with 3/8 continuity factor. Results Comparative Lethality of N-nitroso Products of RDX The incidence of lethality was 100%, 67% and 67% at 400 mg kg−1 for MNX, DNX, TNX, respectively (Table 1). No rats died upon treatment with DNX or TNX at 187 mg kg−1, the next dose level in the progression, while two of the three rats treated with MNX died. The most notable clinical symptom observed was the occurrence of convulsions. Forelimb clonic seizures were observed most often and occasionally progressed to severe generalized clonic-tonic seizures requiring euthanasia. Seizures, which occurred as early as 5 min after dosing, were often preceded by vacuous chewing movements and a brown discharge from the mouth. Forelimb strength and righting reflex appeared unaffected by any of the chemicals, although performance of these tests would often precede the onset of convulsions. Seizures occurred at a 100% incidence for MNX and DNX and at 67% for TNX at the highest dose, while at 187 mg kg−1, an incidence of 67% with MNX compared with 33% with DNX or TNX. From these data, it was concluded that MNX Figure 2. Photomicrographs of Prussian blue-stained spleens from rats 14 days after treatment with 187 mg kg−1 MNX (B) or vehicle (A). Large iron-laden lysosomes of macrophages in the red pulp are indicated by arrows Statistical Analysis Maximum likelihood estimates of MNX LD50 and the 95% confidence interval from the UDP study were obtained from the AOT425StatPrg, version 1.0 (USEPA, Table 1. Comparative MNX, DNX and TNX acute toxicitya Chemical Dose (mg kg−1) Group 1 MNX DNX TNX 400 187 87 x|+ 400 187 o|+ 400 187 o|− 2 3 x|+ 4 5 x|+ o|– x|+ x|+ o|− x|+ 6 7 Lethality Convulsions x|+ 2/2 2/3 0/2 2/2 2/3 1/2 o|− 2/3 0/3 3/3 1/3 o|+ 2/3 0/3 2/3 1/3 o|– o|+ o|+ o|− Incidence x|+ o|− a Outcomes are given as lethality|occurrence of convulsions as symbolized by x, death or o, survival and −, absence or +, presence of convulsions. Rats were administered the indicated dose for each chemical in sequence at 48 h intervals. Rats dosed first were those in group 1, those dosed second in group 2, dosed third in group 3, etc. Copyright © 2005 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2005; 25: 427–434 ACUTE TOXICITY OF RDX NITROSO PRODUCTS 431 Table 2. MNX fixed-dose acute oral toxicity study Dose (mg kg−1) 0 47 94 187 280 420 Lethality Convulsions Body weight gain/2 wka (g) Spleen weight at 14 da (% of body wt) PrB+ splenic lysosomesa,b (#/field) 0/5 0/5 0/5 3/5 5/5 4/5 0/5 0/5 4/5 4/5 5/5 5/5 15.8 ± 2.4 17.2 ± 3.7 −8.8 ± 6.3* 17, −18 0.27 ± 0.02 0.26 ± 0.01 0.29 ± 0.02 0.26, 0.23 4.43 ± 0.93 5.20 ± 0.81 7.08 ± 1.21* 13.13, 5.14 0.18 19.11 a −45 Mean ± SEM for n = 5 for doses 0–94 mg kg . Individual values for survivors of doses >94 mg kg are given. * P < 0.05. Prussian blue-stained vesicles of diameter greater than that of erythrocytes in 100× oil immersion fields of splenic red pulp. Significant dose-dependent trend, P < 0.05. −1 −1 b was the most potent of the RDX N-nitroso metabolites with respect to acute oral toxicity. No obvious pathological lesions were noted either grossly or histologically for tissues taken from rats treated with any chemical and euthanized shortly after treatment. Stomachs were distended and filled with fluid and yellow granular material, but no histopathological evidence of tissue damage was observed. For the 14-day survivors, Prussian blue staining of spleens from rats treated with 187 mg kg−1 of the chemicals indicated hemosiderosis for the single MNX survivor and for two of the three TNX-treated rats. Neither spleen from two DNX-treated rats exhibited hemosiderosis. Although the third rat treated with 187 mg kg−1 DNX survived 48 h after dosing and was thus classified as a survivor for the UDP determination of lethality (Table 1), death occurred during night 13 and thus tissue from this animal was not available for histopathology. No other gross or histopathological lesions were noted for the 14-day survivors treated with MNX. UDP Determination of MNX LD50 Relative to RDX Because published values for the LD50 for RDX vary widely due to difficulties with dispersion of this chemical in a suitable gavage vehicle (Yinon, 1990), UDP determinations for RDX and MNX were performed under the same conditions (vehicle, 5% DMSO in corn oil, starting dose, 87 mg kg−1, sigma = 0.33). Maximum likelihood estimates from these determinations of LD50 (with 95% confidence interval), as estimated with the AOT425StatPgm program, were 187 mg kg−1 (118– 491 mg kg−1) for MNX and 187 mg kg−1 (71–295 mg kg−1) for RDX. In both determinations, the 187 mg kg−1 dose was the only dose which gave a partial response, i.e. two deaths and one death of three rats treated with MNX and RDX, respectively. Both rats treated with the high dose of either MNX or RDX (400 mg kg−1) convulsed within several minutes, while convulsions occurred with two of three rats treated with 187 mg kg−1 MNX or RDX. One of two MNX-treated and both RDX-treated rats receiving 87 mg kg−1 convulsed. Copyright © 2005 John Wiley & Sons, Ltd. Table 3. Hematological assessment of MNX acute oral toxicitya Dose (mg kg−1) 0 47 94 187 280 420 Hemoglobin (g dl−1) Hematocrit (%) WBCb (×103 µl−1) 14.8 ± 0.5 14.5 ± 0.3 13.1 ± 0.5** 14.8, 15.6 46.0 ± 1.1 45.4 ± 0.7 42.0 ± 1.7* 46.5, 49.9 5.00 ± 0.65 5.06 ± 0.84 6.15 ± 0.88 3.65, 5.36 17.2 54.2 6.39 a Mean ± SEM for n = 5 for doses 0–94 mg kg−1. Individual values for survivors of doses >94 mg kg−1 are given. * = P < 0.05, ** = P < 0.01. b Leukocyte counts. MNX Fixed-dose Study and Dose-dependence of Toxic Effects The results of acute toxicity of MNX determined with the fixed-dose design are presented in Tables 2 and 3. The highest nonlethal dose observed was 94 mg kg−1. The oral LD50 for MNX determined with a fixed-dose protocol and regression analysis of probit vs log dose yielded a value of 186 mg kg−1 with a 95% confidence interval of 103– 268 mg kg−1. Similar analysis of the incidence of convulsions gave an ED50 of 57 mg kg−1 (13–101 mg kg−1). Further observation of survivors identified nonlethal adverse effects (Table 2). The body weight gain over the subsequent 14 days was significantly decreased for rats treated with 94 mg kg−1 MNX compared with those receiving vehicle. Histopathological examination of hematoxylin/eosin-stained spleens indicated hemosiderosis of red pulp of MNX-treated rats, although the relative spleen weight was not significantly increased. Quantitation of large, iron-laden macrophage lysosomes in splenic red pulp of Prussian blue-stained sections (Fig. 2, Table 2) revealed a statistically significant trend towards greater numbers with increasing dose of MNX. Relative liver weights of rats treated with 47 and 94 mg kg−1 MNX (mean ± SEM = 3.52 ± 0.13 and 3.61 ± 0.15%, respectively) did not differ from that of vehicletreated controls (3.72 ± 0.06%). J. Appl. Toxicol. 2005; 25: 427–434 432 S. A. MEYER ET AL. Hematology of blood of 14-day survivors detected statistically significant decreases in hemoglobin and hematocrit values of rats treated with 94 mg kg−1 MNX from that of vehicle controls (Table 3). Additional hematological effects reported for RDX were not evident in our rodent trials with the nitrosamine product MNX. Transient leukocytosis has been observed with accidental human exposure to RDX (ATSDR, 1995; Küçükardalı et al., 2003), while leukocyte counts were unaffected by MNX in our studies (Table 3). Further, renal toxicity observed in rats chronically treated with RDX (U.S. Army, 1983) was absent in the MNX-treated rats. Blood urea nitrogen and creatinine concentrations were equivalent for control and MNX-treated rats (data not shown). Discussion The primary goal of these studies at the outset was to compare the relative acute oral toxicity of the three Nnitroso degradation products of RDX. Since all three Nnitroso compounds have been detected in ground water of RDX-contaminated sites (Beller and Tiemeier, 2002), knowledge of their relative toxicities is of value for direction of future efforts related to site remediation. Once identified, a second objective was to begin profiling the toxicity of this N-nitroso product by determining its oral LD50 and surveying for target tissue toxicity. To achieve this goal, the recently validated procedure for acute oral toxicity, the up-and-down procedure (UDP) was employed. Acceptance of the UDP by several regulatory agencies has resulted from efforts to minimize animal numbers used for toxicity testing. The greater efficiency of the UDP procedure is enabled by a sequential dosing regime in which each successive dose level is determined by the 48 h outcome of the previous trial. Another advantage of the UDP is that the singular animal treatment offers the opportunity to make detailed clinical observations shortly after dose administration and to collect preliminary data useful for subsequent hazard identification, design of dose-response studies and assessment of mechanisms of toxicity. These features were particularly of value for our comparative studies with RDX parent and N-nitroso metabolites since monitoring the incidence and severity of convulsions, some of which occurred within minutes after dosing, augmented our conclusion based upon lethality that MNX was the most potent of the metabolites. The equivalence of the LD50 for MNX and its parent RDX derived with the UDP, compared with the lesser incidence of lethality of TNX at that dose, suggests that a nitroreductive pathway could function in vivo leading to detoxication for this endpoint. Results with DNX were like those for TNX, even though the DNX preparation contained significant MNX contamination (25%), and thus supportive of nitroreductive detoxication of Copyright © 2005 John Wiley & Sons, Ltd. RDX. An estimated LD50 of 100 mg kg−1 for SpragueDawley rats orally gavaged with a saline slurry of RDX (Schneider et al., 1977) compares favorably with the UDP RDX LD50 obtained with female Sprague-Dawley rats in this study. Both male and female rats were used by Schneider et al. (1977) and no difference in sensitivity to lethality was indicated. Reduction of the N-nitro groups of RDX also appeared to reduce seizure incidence, as evidenced by the lowest value for TNX at 187 mg kg−1 and greater incidence for RDX than MNX at 87 mg kg−1. A greater potency of RDX over MNX is also suggested by the determination of 80% seizure incidence at 25 mg kg−1 RDX by Burdette et al. (1988) compared with our estimate of an ED50 of 57 mg kg−1 for MNX. Archived histological material from the comparative studies allowed an efficient survey for pathological effects in several selected tissues. The well-known toxicity of nitrosamines to the stomach justified examination of this tissue. Exploration for liver and kidney lesions was suggested by previously observed disposition of [14C]RDX residues in these tissues of rats shortly after gavage administration (Schneider et al., 1977) and toxicity to these tissues in chronic dietary trials (U.S. Army, 1983). No overt pathology of these tissues was observed from MNX-treated rats euthanized early after dosing or from those that survived for 14 days. However, preliminary evidence for hematological toxicity was obtained from an obvious hemosiderosis of splenic red pulp of the 14-day surviving rat that had received 187 mg kg−1 MNX. Guided by this observation, we were then able to conclusively demonstrate dose-dependent deposition of ferric iron in spleen of MNX-treated rats using tissue from the fixed-dose design study. Pigmentation of spleens from rats chronically treated with RDX has been previously noted (U.S. Army, 1983), but no evidence exists for this effect with acute rodent exposures (ATSDR, 1995). A preliminary survey for Prussian blue-positive spleens, using material from our UDP determination of RDX LD50, indicated that hemosiderosis was absent. Since hemosiderosis is conclusively present with acute MNX treatment and since two of three spleens from TNX survivors stained heavily for Prussian blue, these results suggest that the N-nitroso may be the toxicophore for this endpoint. Thus, splenic hemosiderosis may be an adverse effect acquired upon reduction of parent RDX. Alternately, a recent observation that phenobarbital-induced rabbit liver microsomes enriched in CYP2B4 catalyses a high rate of nitrite production upon RDX denitration (Bhushan et al., 2003) suggests a mechanism centered on the N-nitro group. Splenic hemosiderosis is often a sequel to erythrocyte damage as a consequence of methemoglobin formation. Many nitrogenous chemicals, such as nitrite (Kohn et al., 2002), can redox couple with heme ferrous iron to yield methemoglobin and, if severe, anemia can result from loss of circulating erythrocytes upon entrapment in the J. Appl. Toxicol. 2005; 25: 427–434 ACUTE TOXICITY OF RDX NITROSO PRODUCTS 433 red pulp of the spleen. Although anemia was absent in rats fed RDX in subchronic studies reported by Levine et al. (1990), human case reports (Stone et al., 1969) have documented transient mild anemia with up to 4% methemoglobin in humans within hours of acute oral exposure to approximately 25 g of the compound C-4 (91% RDX). Transient anemia and methemoglobinemia was also noted in the case reports of Küçükardalı et al. (2003). A greater sensitivity of erythrocytes from humans than rats to products of RDX N-nitro metabolism in vivo may result from their known lower activity of methemoglobin reductase (Smith and Beutler, 1966). A decrease in hematocrit and blood hemoglobin with MNX observed in our fixed-dose studies (Table 3) is supportive of a mechanism involving methemoglobin for this RDX degradation product. A more careful assessment of hemosiderosis in rat spleen with RDX and TNX treatment than possible with the UDP design is needed to better determine whether N-nitro or N-nitroso is the important functional group for this effect. These studies, along with those of George et al. (2001), have identified three toxic effects for the reduced degradation products of the munition RDX; mutagenicity, neurotoxicity and hematological toxicity. Reduction of RDX N-nitro groups confers weak mutagenicity (George et al., 2001), but diminishes lethal neurotoxicity. Hematological toxicity is associated with acute exposure to the mono N-nitroso product MNX and further study is needed to determine whether N-nitro reduction is conducive to this effect. Related toxicities previously reported to the nervous and hematological systems of humans exposed to parent RDX suggest the relevance of hazards of the RDX degradation products, especially MNX, identified here in rodents to risk assessment of RDX contaminated sites. Acknowledgements—This research was supported by BS04 008D5P and BAA-02-3435 under the Environmental Quality and Technology Program, Engineer Research and Development Center, U.S. Army Corps of Engineers. Permission was granted by the Chief of Engineers to publish this information. 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