Oral Bioavailability of Cyclotrimethylenetrinitramine (RDX) from Contaminated Site Soils in Rats

Abstract

Cyclotrimethylenetrinitramine (RDX), a commonly used military explosive, was detected as a contaminant of soil and water at Army facilities and ranges. This study was conducted to determine the relative oral bioavailability of RDX in contaminated soil and to develop a method to derive bioavailability adjustments for risk assessments using rodents. Adult male Sprague-Dawley rats preimplanted with femoral artery catheters were dosed orally with gelatin capsules containing either pure RDX or an equivalent amount of RDX in contaminated soils from Louisiana Army Ammunition Plant (LAAP) (2300 μg/g of soil) or Fort Meade (FM) (670 μg/g of soil). After dosing rats, blood samples were collected from catheters at 2-h intervals (2, 4, 6, 8, 10, and 12) and at 24 and 48 h. RDX levels in the blood were determined by gas chromatography. The results show that the peak absorption of RDX in blood was 6 h for neat RDX (1.24 mg/kg) and for RDX from contaminated soil (1.24 mg/kg) of LAAP. Rats dosed with RDX-contaminated FM soil (0.2 mg/kg) showed peak levels of RDX in blood at 6 h, whereas their counterparts that received an identical dose (0.2 mg/kg) of neat RDX showed peak absorption at 4 h. The blood levels of absorbed RDX from LAAP soil were about 25% less than for neat RDX, whereas the bioavailability of RDX from FM soils was about 15% less than that seen in rats treated with neat RDX (0.2 mg/kg). The oral bioavailability in rats fed RDX in LAAP soil and the FM soil was reduced with the neat compound but decrease in bioavailability varied with the soil type.

Cyclotrimethylenetrinitramine (RDX) is the most important munitionsderived pollutant encountered at military installations. Contamination to the environment occurs during synthesis, assembly, and destruction and disposal of ordnance, and during troop training. The number of sites contaminated with explosives is not yet known, though the Army alone has over 2000 explosive-contaminated sites that may require assessment. Still more contaminated sites exist at Navy and Air Force facilities (Framework of Action 1995). Cleanup goals for sites contaminated with soil-borne metals and organic compounds often are established on the basis of risk assessments, which typically rely on the estimated oral toxicity of the substances of concern. These toxicity estimates are often based on historical animal data resulting from oral gavage or feeding studies, using the pure compound. However, these studies do not account for natural processes that sequester compounds in the soil, thereby reducing their availability to biological receptors such as animals, plants, or humans. This study was therefore conducted to determine the relative bioavailability of RDX in soil and to develop a reliable in vivo method to derive bioavailability adjustments for risk assessments.

The Army is currently in the process of cleanup of ordnance manufacturing facilities and other sites that have been heavily contaminated with munitions compounds. However, the need for cleanup of low levels of contamination commonly found in training areas remains the focus of debate. Much of the problem arises from lack of information about the behavior of munitions compounds in soil environments.

The toxicity of organic compounds in soil environments is often seen to decrease with time, due to natural processes. Natural attenuation of the toxicity of explosive compounds in soil environments is due to chemical and biochemical degradation of these materials and from sequestration of intact compounds within the soil. Sequestration reduces the toxic effects of soil-borne organic compounds by reducing the bioavailability and hence the uptake of these substances by biological receptors (Alexander 2000). However, the sequestered fraction could be detected by modern analytical methods and is currently considered as a bioavailable toxicant in the risk assessment process. Considering sequestered compounds as fully bioavailable toxicants causes overestimation of exposure and overly conservative cleanup requirements. The U.S. Environmental Protection Agency (EPA) and other members of the regulatory community recognize that reductions in bioavailability should be addressed in the context of the risk assessment process and that consideration of bioavailability will often reduce the extent of cleanup (US EPA 1997). The concept of incorporating bioavailability data in risk assessment has been effectively employed in the case of drinking water contaminants such as trichloroethylene (TCE), cadmium, and mercury (DeSesso, Jacobson, and Scarano 1998). Studies demonstrating reduced bioavailability of soil-borne metals in swine and primates have already been used to raise allowable exposure to soil-borne toxic metals (Casteel et al. 1997; Roberts et al. 2002). Oral bioavailability of soil-borne lead (Pb) in adult volunteers by the stable isotope dilution method was studied by administration in gelatin capsules. The results of these studies showed that about 26% of the capsule-administered Pb was absorbed in the blood in 30 h as compared to 2.5% of Pb in ingested soil matrix (Maddaloni et al. 1998).

Although a considerable body of evidence has shown that explosive compounds become strongly sorbed to soils (Pennington and Patrick 1990; Price, Brannon, and Hayes 1997; Brannon, Price, and Hayes 1998), none of these findings were derived from oral bioavailability studies with mammals and none has been used in the risk assessment process to produce changes in cleanup levels. Recent studies in our laboratory showed that dermal absorption of RDX from contaminated RDX soils was considerably less than absorption of neat compound (Reifenrath et al. 2002; Reddy, Allen, and Major 2008). The reduced dermal absorption was also shown with other chemicals such as phenol (Skowronski et al. 1994) and DDT and benzo(a)pyrene (Wester et al. 1996) when they were administered in a soil matrix. The rodent oral studies reported herein are the first such studies available on the relative oral bioavailability of RDX in contaminated soils.

MATERIALS AND METHODS

Chemical

Neat cyclotrimethylenetrinitramine (RDX) was procured from Dr. William M. Koppes, Naval Surface Warfare Center, Indian Head, MD. The purity of the RDX was further confirmed to be 99.9% by Mr. Michael Hable of our Directorate of Laboratory Sciences (DSL).

Testing Soils

The RDX-contaminated soil sample collected form Louisiana Army Ammunition Plant (LAAP) was provided by Dr. T. F. Jenkins, U.S. Army Cold Regions Research and Engineering Laboratory, Hanover, NH. The second sample of RDX-contaminated soil was provided by the University of Nebraska and identified as field-collected soil from Fort Meade in Maryland, USA. The primary mission of LAAP was to load, assemble, and package explosives into shell castings, to manufacture ammunition. This was placed on the National Priorities List (NPL). The soils were silt loamy and clay soils (Pennington et al. 1999). The Fort Meade facilities were formerly used for motor pool and tank maintenance facilities. The soils of FM were well drained loamy, clay, and sandy (USAEC 2007). The soils were collected about 12 inches deep from the soil surface. The soil moisture was not measured but dried soils were sieved. They were stored in amber bottles and refrigerated. A sample of LAAP soil was analyzed by our Explosives Analysis Team (EAT) and was found to contain 2300 μg RDX/g soil. The soil of Fort Meade was analyzed by the EAT and found to contain 670 μg RDX/g soil. In both cases the soil was sieved to a maximum size of 0.0234 inches (0.5944 mm) using a no. 30 soil sieve. These soil samples were not characterized to determine clay/sand/loam partition.

Animals

Adult male Sprague-Dawley rats (approximately 400 g in weight, 11 weeks old) were obtained from Charles River Laboratories, Raleigh, NC. All rats were implanted with femoral artery catheters at Charles River Labs prior to shipment. The attending veterinarian examined the animals and found them to be in acceptable health. Due to the relatively short patency period of the catheters, the rats were quarantined for only 12 h prior to study initiation. All rats were maintained in a temperature of 64°F to 79°F, at a relative humidity of 30% to 70%, and with a 12-h light/dark cycle. With the exception of an 8-h fasting cycle (4 h prior to dosing and 4 h post dosing), a certified pesticide-free rodent chow (8728C Certified Rodent Diet; Harlan Teklad, Madison, WI) and drinking quality water were available ad libitum. Rats were housed individually in suspended polycarbonate boxes with Harlan Sani-Chip (P. J. Murphy Forest Products, Montville, NJ) bedding. Each rat was uniquely identified by number using cage cards. The animal husbandry was performed in animal facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Research was conducted under an IACUC-approved animal use protocol, and was compliant with Animal Welfare Regulations and requirements set forth in the Guide for the Care and Use of Research Animals (NRC, 1996).

Capsule Filling Procedures

Neat and soil-borne RDX were administered in gelatin capsules, which were carefully filled with neat chemical or with sieved RDX-contaminated soil. Due to the small size of the empty gelatin capsules (size 9, 2.65-mm diameter/8.6-mm length), an exact target weight for the filled capsules could not be obtained consistently. Therefore, the total amount of contaminated soil given to each rat was calculated and used, along with the verified RDX concentration of the soil, to yield the dose of RDX given to each rat. The capsules containing neat RDX were filled by the addition of a known amount of neat compound by adding an appropriate volume of RDX stock solution in acetone to the capsule. An exact amount of the stock solution was then placed in each capsule using a calibrated pipette. The open capsules were then placed in a fume hood overnight to allow the acetone to evaporate before sealing the capsules. The amount of RDX in one capsule from each batch was verified by the EAT of DLS.

Dosing Materials

Rats were dosed orally with gelatin capsules filled with either pure RDX or RDX-contaminated soils. Gelatin capsules (size 9) were administered with the help of a size 9 capsule balling gun and a stainless steel capsule funnel, which were procured from Torpac, Fairfield, NJ. The details of the procedure were described by Lax, Militzer, and Trauschel (1983).

Blood Sampling and Analysis

Blood sampling was done from the preimplanted catheter of each rat at designated time intervals. The catheter tubing used by Charles River Laboratories was designed for sampling using a 21-gauge × 1-inch blunted needle attached to a 1-ml gas-tight syringe. The sampling procedure remained the same for each rat. The stainless steel catheter plug was removed using hemostats and the lock solution was removed from the catheter using a syringe. A 100-μl blood sample was taken from each rat. The catheter was flushed with 0.1 ml of sterile saline. Finally, 0.07 ml of heparinized saline (500 IU heparine/1 ml final solution in saline) was injected into the catheter so that it would remain patent for the next sampling time. The catheter plug was placed back into the tubing and the area was cleaned with an alcohol swab before insertion back into the wound clip. Blood samples collected at different time were placed (100 μl) in a volumetric flask containing 100 ml of water and placed in the refrigerator prior to analysis. Blood sampling from each rat for the pilot study was started at 2, 4, 6, 8, 10, 12, 24, and 48 h post dosing. The blood samples were analyzed by EAT according to methods described by Bishop et al. (2003). Blood samples (100 μl) were added to water (100 ml) as soon as collected and placed in the refrigerator and extracted with 1 ml of isoamyl acetate (anhydrous, 99+ %). Aliquots of isoamyl acetate extractions were analyzed by gas chromatography equipped with an electron capture detector (Bishop et al 2003). The relative bioavailability was determined by comparison of area under the curve (AUC) of blood concentration of RDX over time, for animals receiving equivalent dosage of RDX in neat form and from contaminated soil. Dosage values were obtained by dividing dose values by body weight (BW). Blood concentrations could be determined only on blood samples collected between 2 and 12 h. RDX levels were low to quantify.

Pilot Study

In order to standardize the oral dosing and blood sampling procedures as well as the analytical methods, a pilot study utilizing six male catheterized Sprague-Dawley rats was performed. The three rats receiving neat RDX were dosed with one capsule containing the RDX (1.24 mg RDX/kg BW) and four placebo capsules. The remaining three rats received a total of five capsules containing the RDX contaminated soil for a total dose of approximately 1.24 mg RDX/kg BW. All doses were based on an anticipated rat weight of 400 g. Exact dosages were calculated for each rat based on their weight the day of dosing and the capsule weights once they were filled. Dosing occurred at 10-min increments to allow for time to take blood samples from each rat at the specified times. The exact doses of neat RDX and soil are given in Table 1.

Bioavailability of Louisiana Army Ammunition Plant (LAAP) Soil

In order to have enough rats for statistical significance, a total of 24 catheterized Sprague-Dawley rats were purchased for this study. However, three animals were eliminated prior to the study due to clogged catheters. Dosing procedures remained the same for the final bioavailability study. Eleven rats each received a capsule containing 1.24 mg RDX/kg BW neat RDX and four placebo capsules and the remaining 12 rats received five consecutive capsules containing LAAP soil (grams) for a total dose of 1.24 mg RDX/kg BW. Blood sampling for the final study occurred at 2, 4, 6, 8, 10, and 12 h post dosing. Based on the preliminary results of the pilot study, the RDX concentration in the rat blood was too low to accurately quantify at the 24- and 48-h sampling times.

Bioavailability of Fort Meade Soil

A second bioavailability study with Fort Meade soil sample with a much lower concentration of RDX than the LAAP soil sample was conducted. A total of 22 catheterized Sprague-Dawley rats were used. All dosing procedures remained the same as the previous study using LAAP soil. Eleven rats received a dose of approximately 0.2 mg RDX/kg BW of neat RDX in one capsule and four placebo capsules. The remaining 11 rats received five consecutive capsules containing Fort Meade soil for a total dose of 0.2 mg/kg RDX in grams of soil. Exact dosages were calculated on the day of dosing of neat RDX and RDX soil weights based on the rat weights and reported in Table 1. Blood-sampling procedures remained the same for this phase of the study, occurring at 2, 4, 6, 8, 10, and 12 h post dosing. All analytical methods also remained the same.

RESULTS

Pilot Study

The results of the pilot bioavailability study indicated that oral dosing of rats at a dosage of approximately 1.24 mg RDX yielded quantifiable levels of RDX blood time/concentration curve for a period of 12 h. Blood concentrations at 24 and 48 h post dosing had dropped to levels that were too low to be detectable or accurately quantifiable. No clinical signs of RDX toxicity, such as seizures, and no unscheduled mortality occurred during dosing or blood sampling.

RDX blood concentrations (3.5 μg/ml) for those rats dosed with contaminated soil and neat RDX peaked at approximately 8 h. The small number of rats used in each dose group notwithstanding, for statistical significance, the results of this pilot study indicated that the RDX contamination in the LAAP soil was approximately 25% to 35% less bioavailable for uptake from 2 to 12 h post dosing when compared to the results for neat RDX. The results are presented in Figure 1.

Bioavailability of Louisiana Army Ammunition Plant (LAAP) Soil

The dosing procedure for the final bioavailability study using LAAP soil caused one unscheduled death, which was not treatment related. In addition, two of the catheters implanted by Charles River Labs were no longer patent when they were received. Repeated attempts to flush the catheters were unsuccessful.

Blood concentrations over the 12-h period were monitored from a total of 11 rats dosed with neat RDX and 10 rats dosed with RDX-contaminated soil. RDX blood concentrations for those rats dosed with contaminated soil and neat RDX peaked at approximately 6 h. Similar to the pilot study, RDX blood concentration AUC for those rats dosed with contaminated soil were approximately 25% lower than that for rats receiving neat RDX throughout the 12-h sampling period. The calculated AUC for rats dosed with neat RDX was 3.6, compared to 2.7 for rats dosed with soils containing similar amount of RDX; the difference was significant (p ≤ .0003). Mean blood concentrations are reported in Figure 2.

The area under each animal’s individual time-concentration curve was plotted and calculated using the rectangle method. The AUC values for the rats dosed with neat RDX were then compared to those dosed with RDX-contaminated soil using a t test. These results indicated that the time-concentration curves of the rats dosed with LAAP soil were significantly lower than the curves of those rats dosed with equivalent amounts of neat RDX (p ≤ .003).

Bioavailability of Fort Meade Soil

The dosing procedure for the final bioavailability study using Fort Meade soil did not result in any treatment-related deaths. However, two of the catheters implanted by Charles River Labs were no longer patent when they were received. Repeated attempts to flush the catheters were unsuccessful. Blood concentrations over the 12-h sampling period were gathered from a total of 11 rats receiving neat RDX and 9 rats receiving RDX-contaminated soil. RDX blood concentrations for those rats dosed with contaminated soil peaked at approximately 6 h, whereas blood concentrations for those rats dosed with neat RDX peaked at approximately 4 h. In this phase of the study, the results indicated that the blood concentrations of the rats dosed with contaminated soil was approximately 15% lower at the 4-h sampling period compared to the rats dosed with neat RDX. The calculated AUC for rats receiving neat RDX was 4.63 and 4.41 for dosed with soils containing a similar amount of RDX; the difference was not significant. Average blood concentrations are reported in Figure 3.

DISCUSSION

The bioavailability of a toxicant can be defined as the fraction of administered dose reaching the systemic circulation of the animal (Gibaldi 1991). There are several papers and reports available that describe bioavailability of chemicals in the environment (NRC 2003; Ramesh et al. 2004). The oral bioavailability is calculated from the ratio of area under the blood concentration-time curve (AUC) to the administered dose of a toxicant.

The results of these studies indicated that the oral dosing of rodents followed by repeated blood sampling is a feasible method for determining the relative bioavailability of soil-borne RDX. Initial results utilizing soil with the relatively high RDX concentration of LAAP soils (2300 ppm in soil and approximately 1.24 mg/kg in the rat) exhibited a clear, statistically significant trend, indicating that the adsorption of RDX to the soil particles rendered it less bioavailable for uptake. The RDX blood concentrations of the rats dosed with contaminated soil remained lower than those dosed with neat RDX throughout the 12-h sampling period. This procedure for measuring bioavailability is site specific and will likely yield different results for specific contaminants at each site. The environmental conditions possibly affecting the bioavailability of RDX include, but are not limited to, soil type, soil moisture, soil aeration, organic matter content, soil temperature, pH, climate, and the aging time in the soil.

Development of an environmental risk assessment for human health ideally is supported by data on both oral and dermal exposure to the contaminant. However, the most significant environmental exposure normally stem from ingestion of contaminated soils and drinking water. The absorption of compounds dissolved in drinking water is normally high (in comparison to other means of delivery) and bioavailability adjustments in exposure assessments are not normally used. Conversely, it is known that soils can bind pollutants and reduce absorption. Compounds that are extensively sorbed to soil are less able to elicit toxic effects. Therefore, only that portion of a toxicant equivalent to the relative bioavailability should be assessed in an exposure assessment. If accurate relative bioavailability assessments are required in a risk assessment of pollutants in soil, it is recommended that studies similar to those reported herein be conducted in an appropriate animal model.