Perchlorate is an important oxidizer used in propellants, pyrotechnics, and as a gas generator in commercial airbags, fireworks, and roadside flares. It is highly water soluble interferes with thyroidal iodide uptake and is an environmental contaminant. By changing the reaction chemistry, 5-aminotetrazole (5-AT) and nitrates replace perchlorate in some propellants. The short term toxicity of 5-AT was evaluated. Using a modified Ames assay, 5-AT was not mutagenic with or without S9 metabolic activation. 5-AT was considered &#;slightly toxic&#; with an EC 50 of 28.8 mg 5-AT/L for a 15 minute exposure in Aliivibrio fischeri. In the in vitro sodium iodide symporter test, 5-AT did not inhibit the uptake of iodine. In the acute rat oral test, no adverse effects and no mortalities were observed at the limit dose of mg 5-AT/kg. In the 14-day sub-acute study, there were no clinical signs of toxicity or morbidity up to 623 mg 5-AT/kg-day; the highest dose tested. No differences were observed in hematology, clinical chemistry, organ weight, body weight, food consumption, histopathology, or DNA damage (peripheral blood micronucleus assay) of treatments compared with controls. The No Observed Adverse Effect Level (NOAEL) was 623 mg 5-AT/kg-day, the highest dose in the subacute oral bioassay.
Finding replacements for perchlorate that will reduce or eliminate the health risks from environmental exposures has been identified as a research goal in the U.S. military and is one of the missions of the Army Environmental Quality Technology (EQT) program (AERTA, ; AR 40&#;5; AR 70&#;1; AR 200&#;1; DoDI, ). Perchlorate is an effective oxidizing compound that is currently used by the U.S. Army in weapon systems. Perchlorate is known to interfere with iodide uptake in the thyroid gland and is persistent and mobile in the environment. Regulatory authorities, therefore, are developing contaminant levels protective of the general population. Perchlorate salts (e.g., potassium, sodium, ammonium) fully ionize in aqueous solutions. The perchlorate anion has been found in many water sources associated with military installations. Consequently, critical training activities are threatened due to perchlorate contamination. Thus, there is a need to identify a suitable replacement for perchlorate. Commercially, 5-aminotetrazole (5-AT) is used in gas generator formulations for airbags, fireworks, and roadside flares. 5-AT, a compound with high-nitrogen content, derives its energy from a high heat of formation and releases benign nitrogen gas (N 2 ) upon reaction with an oxidizing agent, such as a metal nitrate ( Sabatini and Moretti, ). A literature review found a single report for 5-AT toxicity, which reported an intraperitoneal (i.p.) LD 50 of mg 5-AT/kg in the mouse, and no established regulatory values for 5-AT have been reported (CIDPL, ). For the data presented here, a tiered toxicity testing strategy was used to characterize aquatic toxicity, bacterial mutagenicity, short term mammalian oral toxicity, and peripheral blood DNA damage of 5-AT (American Society for Testing and Materials (ASTM), ). Data from these tiered experiments were used to calculate the oral LD 50 , estimate aquatic toxicity and predict genotoxicity. These results were used to compare the toxicity of 5-AT to other compounds being considered as replacements for energetics (e.g., perchlorate) currently in use.
5-Aminotetrazole (5-AT) is a white medium grain crystalline powder. Three lots with corresponding certificates of analyses were acquired from Sigma-Aldrich (St. Louis, MO, USA) for the experiments. For the Ames and Microtox &#; assays, 5-AT anhydrous (97 percent pure, CAS &#;61-5, Lot # MKBFV; Sigma Aldrich, St. Louis, MO, USA) was used. Due to product discontinuation, 5-aminotetrazole monohydrate, 97 percent; (MW 103.08 g/mol; CAS &#;54-3; product number A, lot number STBBV;) was used for the animal study. 5-AT Lots STBBV and STBGV were used for the sodium-iodide symporter inhibition assay (NIS-IA). The monohydrate was preferred due to the instability of the anhydrous 5-AT. 5-AT (85.07 g/mol) constitutes 82.5 percent of the 5-AT·H2O formula weight. All experiments accounted for the percent formula weight of 5-AT and data are reported for 5-AT not 5-AT·H2O. 5-AT monohydrate was water soluble (12 g/L).
The vehicle (solvent) for the Ames, Microtox &#; and NIS-IA assays was DMSO (CAS 67&#;68-5; Sigma Aldrich, St. Louis MO, USA). The vehicle for the rat dosing studies was food grade Mazola&#; corn oil purchased locally.
All purity confirmation and concentration verification analysis of the dosing solutions and stability analyses were performed by Army Public Health Center (APHC) Laboratory Sciences Directorate (LAB)-Client Services Division (CSD)-Method Development Section (MDV). Purity and concentration determination of 5-AT in either DMSO or corn oil and stability of 5-AT in corn oil were accomplished by high performance liquid chromatography with ultraviolet detection (LC-UV; Dionex U, Bannockburn, IL). The samples were isocratically analyzed using an Agilent Eclipse XDB C-18 column and quantitated at 232 nm. Results were reported in mg/mL. To confirm concentration of dosing solutions, the lowest concentration used in each assay was verified by LC-UV and the concentrations of the higher treatments were determined by back calculation.
A microplate Ames assay (Xenometrics MPF Ames Assay &#;, Gewerbestrasse 5 Allschwil, CH- Switzerland) was used for bacterial mutagenicity testing. The test uses the standard Salmonella typhimurium and Escherichia coli strains (E. coli tested as a mixture) (Claxton et al., ). The MPF Ames Assay was purchased as a kit that contained all necessary reagents, positive controls for both &#;S and +S, media and bacterial strains; see . The assays were conducted in a manner consistent with the standards found in Title 40 Code of Federal Regulations (CFR), Part 792, Good Laboratory Practices.
The assay method has been previously described (Williams et al., ). Briefly, the day before the assay was performed, the bacterial strains were thawed and expanded overnight in growth medium at 37°C in an orbital shaker at 250 rpm, supplemented with ampicillin per the manufacturer&#;s protocol. Based on the tested maximal water solubility of 5-AT (10 mg/mL), a 23.8 mg/mL stock solution (25X) of 5-AT in DMSO was prepared on the day of the assay. S. typhimurium and E.coli bacterial suspensions were diluted with incubation medium, distributed in triplicate into 24 well plates (Corning Costar, Fisher Scientific, Pittsburgh, PA, USA) and incubated with either a DMSO vehicle control, the appropriate strain specific positive control (see ) or 6 serial &#; log dilutions of the 23.8 mg 5-AT/mL stock solution for 90 minutes at 37°C 250 RPM. Separate preparations containing Aroclor-induced S9 rat liver extracts were prepared and incubated in parallel for either 90 minutes for S. typhimurium or 20 minutes for E. coli. After the timed exposure incubation, the bacterial suspensions were diluted 11-fold with incubation (indicator) medium and 48 &#; 50 μL aliquots for each suspension/treatment were distributed to 384-well plates (Corning Costar, Fisher Scientific, Pittsburgh, PA, USA) to yield 6 384-well plates (3 -S9 / 3 + S9) for each bacterial strain. The plates were then incubated at 37°C for 48 hours. The indicator medium contains a pH sensitive dye that shifts from purple to yellow in the presence of metabolically active bacteria. After the 48-hour incubation, the wells were scored for reversion (i.e., a mutagenic event) by assessing the color of the medium.
Concurrent with the mutagenicity assay, a separate replicate for each of the strains/treatments (without S9 extract) was incubated to determine 5-AT cytotoxicity after 90 minutes of exposure for the S. typhimurium and mixed E. coli strains. Cytotoxicity was measured using ATP concentration as an endpoint (BacTiter-Glo, Promega, Madison WI, USA). Luminescence was measured using a Synergy HT Multi-detection Microplate Reader and Gen5 software (BioTek Instruments Inc. Winooski, VT, USA). Data were expressed as percent control relative to the vehicle control samples.
The Microtox® assay utilizes a manufacturer-prepared diluent (AZF), acute reagent of freeze dried Allivibrio fischeri (NRRL-B-) (AZF) and reconstitution solution (AZF), purchased from Modern Water Inc. (New Castle, DE). As recommended, zinc sulfate (ZnSO4·7H2O) was used as a standard or positive control and acquired from Sigma-Aldrich (St. Louis, MO). The assays were carried out in the Microtox Model 500 Analyzer® with MicrotoxOmni&#; software. (Microtox Model 500 Analyzer® and MicrotoxOmni&#; software are initially registered trademarks of AZUR Environmental and currently owned by Modern Water Inc.). The manufacturer&#;s protocol with modifications was followed for the 5-AT toxicity test. Three independent replicate assays were performed. For each test day, 100 mg/mL (100X) 5-AT test solutions were prepared in DMSO and then diluted in reconstitution solution for the test. Raw luminescence values were recorded at 5, 15, and 30 minutes by the Microtox analyzer. The EC50 values at 5, 15, and 30 minutes were calculated by the MicrotoxOmni software and further fitted to Hill Function using GraphPad PRISM 5®, a registered trademark of GraphPad Software, Inc. (San Diego, CA, USA).
FRTL-5 rat thyroid follicular cells (, Millipore-Sigma, St. Louis, MO) were cultured in Coon&#;s modified F-12 medium supplemented with 5% FBS (fetal bovine serum) (Gibco, Waltham, MA), 2mM L-glutamine, 10mU/mL TSH (thyroid stimulating hormone), 10μg/mL insulin, 10nM hydrocortisone, 5μg/mL transferrin, 10ng/mL Gly-His-Lys, 10ng/mL somatostatin and penicillin-streptomycin at 37°C and 5% CO2. Cells were grown to 80&#;90% confluence in T75 flasks (Corning, Corning, NY) before trypsinization with 0.25% trypsin/EDTA (Gibco, Waltham, MA). For the radioactive iodide uptake (RAIU) assay, FRTL-5 cells were seeded into 96-well plates (Corning Costar, Corning, NY) at a density of 3.0× 104 cells per well in 200μL media and incubated 48 hours before experimentation. RAIU assays were conducted as previously described (Hallinger et al., ) with minor modifications. All control chemicals were initially solubilized in DMSO at 10 mM: sodium perchlorate (NaClO4; RAIU assay positive control), sodium nitrate (NaNO3; RAIU assay EC80 control), sodium thiocyanate (NaSCN; RAIU assay EC20 control), 2,4-dichlorophenoxyacetic acid (2,4-D; RAIU assay negative control), and 2,3-dichloro-1,4-napthoquinone (DCNQ; cell viability assay positive control for cytotoxicity). On the day of the assay, carrier-free 125I (Perkin-Elmer, Waltham, MA; specific activity: 17.4Ci/mg) was diluted with Hanks&#; balanced salt solution (HBSS) supplemented with 10mM HEPES (uptake buffer; pH 7.4) at 10μCi/mL. The assay was initiated by gently washing cells twice with 200μL uptake buffer using a 96-well Tecan HydroSpeed plate washer (Tecan, Männedorf, Switzerland). Uptake buffer (89μL) was then added back to each well. Chemical samples (1uL at 100X) were added to each well to achieve a 1X chemical/1.0% final DMSO concentration with subsequent addition of 10μL of 125I dilution for a final reaction volume of 100μL/well. Cells were incubated at room temperature for 2 hours and then washed twice with 200μL ice-cold uptake buffer. All uptake buffer was aspirated from wells and 100uL 1N NaOH was added to each well for 10 minutes and then transferred to 12×75mm tubes to measure intracellular radioactivity with a Wizard gamma counter (Perkin Elmer, Shelton, CT). RAIU assay raw data, measured in counts per minute (CPM), were normalized per plate with the mean CPM of DMSO control wells and expressed as percent control (% control). Chemicals that elicited an RAIU inhibition greater than 20% of DMSO control were considered active for NIS inhibition {Hallinger, #22}. Normalized active chemical concentration responses (n=3; 3 biological replicates with the mean of 2 technical replicates for each concentration) were fit using a four-parameter logistic curve model with top and bottom asymptotes constrained to 100 and 0, respectively (GraphPad Prism 6.0, La Jolla, CA).
Cell viability assays were conducted in parallel with the RAIU assays. FRTL-5 cells were seeded and grown and washed with uptake buffer in 96-well solid white assay plates (Corning, Corning, NY) as described for RAIU assay. Uptake buffer (99uL) was then added back to each well with the addition of 1μL of 100X chemical samples. Cells were incubated at room temperature for 2 hours and Cell Titer-Glo (Promega, Fitchburg, WI) reagent (100μL) was then added to each well and plates were shaken for 5 minutes at 700rpm. Plates were incubated at room temperature for 10 minutes and then read on a FLUOstar Omega plate reader (BMG Labtech, Cary, NC) using an endpoint luminescence protocol (top read) to measure relative light units (RLU) indicative of ATP concentration. Assays were replicated in three independent runs containing different passages of the cells (i.e., 3 independent biological replicates). Cell viability raw data, measured in RLU, were normalized per plate with the mean RLU of DMSO control wells and expressed as percent % control. Chemicals that produced a 20% reduction in cell viability relative to DMSO control were considered cytotoxic and subjected to the same curve fitting as RAIU data.
Testing was conducted in compliance with DOD and Federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, National Academy Press, Washington D.C. . The studies reported herein were performed in animal facilities fully accredited by AAALAC International.
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All studies were conducted using young adult Sprague-Dawley Crl:CD(SD) rats obtained from Charles River Laboratories (Wilmington, MA, USA). Female rats were used for the acute study and at the time of their arrival the animals were 43&#;50 days old. Male and female rats were used for the 14-day study and at the time of their arrival the animals were 38&#;42 days old. Rats were pair-housed (same sex) in suspended polycarbonate boxes with Diamond Dry Bedding (product number C; Diamond Star Products, WI, USA). The APHC Quality Systems Office audited critical phases of this study. For histopathology, tissues were preserved, packaged and shipped to Battelle Columbus, OH for processing, slide preparation, staining and histopathological evaluation. Tissues and slides were returned to the APHC for archiving.
The sequential stagewise probit (SSWP) method was used to estimate the oral median lethal dose (ASTM, ; Feder et al., ). The SSWP method potentially reduces the number of animals used for the LD50 test by first testing one animal per dose across the dose range and then subsequently testing additional animals at doses that bracket the LD50. If no deaths are observed in the first round, the method defaults to a &#;limit test&#; where the limit dose is tested with 3 additional animals in the second round. The published i.p. LD50 ( mg/kg), the lower bound of GHS category 5 ( mg/kg) and guidance from Schlede et al {Schlede, #27} were considered in setting the initial 4-dose test. The dosing strategy for the test is described in . The use of a single animal per dose group in the first round to gauge mortality is justified considering that mortality is verifiable (>99%) when additional animals are tested in subsequent rounds of the test {Schlede, #27}. The animals were fasted overnight prior to dosing and fasted weights for each animal were obtained. The doses were prepared for each animal based on individual fasted body weight. The vehicle was corn oil for both stages. The dose volumes were 90 percent of the maximum of 10 mL/kg body weight. Following the administration of 5-AT by gavage, the rats were observed daily for 14 days. The surviving animals were euthanized 14 days after dosing and submitted for gross pathological examination.
The dose levels for the subacute study were based on the lack of clinical signs in the acute phase at the highest dose tested &#; mg 5-AT/kg and and the i.p. LD50 was mg 5-AT/kg. The two values were averaged and used to calculate the nominal highest dose for the subacute study: 700 mg/kg-day (= 1/2 log mg/kg). Sprague-Dawley rats (54 male and 42 female; approximately 40 days old upon arrival) were used for this phase of the study. The animals were sorted into 7 treatment groups (six 5-AT treatments and one vehicle control) of six male and six female rats each based on body weight such that the weight range was evenly distributed between the groups. An additional 12 male rats were used as positive and negative controls for the micronucleus assay. These rats were housed concurrently with animals from the subacute study.
Three stock solutions were prepared at the nominal concentrations 80, 20 and 5 mg 5-AT/mL corn oil. The stability of 5-AT in corn oil was confirmed via LC-UV over a 19-day period prior to the 14-day study. 5-AT was sufficiently stable so that the stock solutions were used for the duration of the 14-day study. Samples from these stock solutions (top, middle and bottom of the container) were taken and analyzed to verify homogeneity of the concentrations prior to the first day of dosing. The doses were delivered daily for 14 days to each rat via gavage. The volume of the dosing solution delivered to each rat was calculated from the body weight of the corresponding animal. Stock solutions were sufficiently concentrated so that the dose volume did not exceed 10 mL/kg body weight. The nominal dose groups were 700, 350, 175, 88, 44, 22, and 0 mg 5-AT/kg body weight. Rats were pair housed (same sex) for the duration of the 14-day study. Body weights were recorded on days 0 (first dosing day), 3, 7, 13 (last dosing day), and 14 (terminal body weights-fasted). Dosing volumes for each animal were adjusted based on body weights taken on days 0, 3, and 7 so that the rats continued to receive the intended dose. Food consumption based on change in feed container weights was monitored weekly. Water was provided ad libitum and intake was not monitored. Animals were observed daily for clinical signs of toxicity, morbidity and mortality.
Approximately 24 hours after the 14th dose, each rat was anesthetized via CO2 gas. Blood was collected via cardiac puncture for clinical chemistry and hematology analyses, and rats were immediately euthanized via terminal CO2 asphyxiation. The brain, heart, kidneys, liver, spleen, epididymides and gonads (i.e., testes or ovaries/uterus) were removed and weighed and gross necropsies were completed. The following parameters, by test group, were analyzed and compared to the controls: (a) body weight; (b) weight gain; (c) food consumption; (d) absolute organ weight; (e) organ-to-body weight ratio; and (f) organ-to-brain weight ratio. Collected organs were preserved in 10% buffered formalin.
Blood aliquots were placed in EDTA, serum-gel, and sodium citrate microtubes for analysis of hematology, clinical chemistry, and hemostasis, respectively. Hematology analyses [white blood cell count (WBC K/mL), WBC differential (K/mL and percent for neutrophils (NEU-%N)), lymphocytes (LYM-%L), monocytes (MONO-%M), eosinophils (EOS-%E), basophils (BASO-%B)), red blood cell count (RBC M/mL), hemoglobin (HGB g/dL), hematocrit (HCT %), mean cell volume (MCV fL), mean cell hemoglobin (MCH pg), mean cell hemoglobin concentration (MCHC g/dL), RBC distribution width (RDW %), platelets (PLT K/mL), mean platelet volume (MPV fL)] were performed with a Cell-Dyne Hematology Analyzer (Abbott Laboratories, Abbott Park, Illinois ). Clinical chemistry samples were allowed to clot at room temperature, centrifuged, and the serum analyzed on VetTest and VetLyte Analyzers (IDEXX Laboratories, Inc., Westbrook, ME) for the following parameters: albumin (ALB g/dL), alkaline phosphatase (ALK P U/L), alanine aminotransferase (ALT U/L), aspartate aminotransferase (AST U/L), blood urea nitrogen (BUN mg/dL), calcium (Ca mg/dL), cholesterol (CHOL mg/dL), creatinine (CREA mg/dL), glucose (GLU mg/dL), globulin (GLOB g/dL), phosphorus (PHOS), total bilirubin (TBIL mg/dL), total protein (TP g/dL), sodium (Na mmol/L), potassium (K mmol/L), and chlorine (Cl mmol/L). Samples for prothrombin time were centrifuged for collection of plasma and analyzed on a BFT-II analyzer (Siemens Health Care Diagnostics, Inc., Tarrytown, NY).
The micronucleus (MN) assay (or, MNA)-positive and negative control male rats were housed, maintained and necropsied with the animals from the subacute study. The positive control rats were dosed twice with 200 mg/kg ethylmethyl sulfonate (EMS), a known DNA damaging agent (CAS 62&#;50-0, Sigma M, Lot # BCBKV) 48 and 24 hours prior to euthanasia. The negative control rats were untreated and served as a negative control for both the EMS and the vehicle (corn oil) treated rats. Saphenous blood from the MNA control rats and male rats from the three highest dose groups with no mortalities was collected the day of necropsy. Blood collected in support of the MNA was processed according to the MicroFlow Basic Kit ® (Litron Laboratories, Rochester NY, USA) instructions. The prepared samples were then shipped to Litron Laboratories for analysis. The experimental method used by Litron is summarized as follows. On the day of analysis, the fixed blood samples were thawed and then washed with physiological buffer containing 1% FBS. Cells were isolated by centrifugation, and the cell pellets were stored at 2 °C to 10 °C until staining. An aliquot (20 μL) of each washed blood sample was added to 80 μL of a solution containing 1 mg/mL RNase, 10 μL/mL reticulocyte (RET) specific anti-CD71-FITC, and 5 μL/mL platelet specific anti-CD61-PE. The samples were incubated in the staining solution for 30 ± 10 minutes at 2°C to 10°C and 30 ± 10 minutes at room temperature. After incubation, the cells were kept at 2°C to 10°C until analysis. A propidium iodide (PI) solution (2 mL ± 0.5 mL) was added to each sample immediately before flow cytometric analysis to stain all DNA, including MN in the cells. Each blood sample was analyzed by high-speed flow cytometry (488 nm) using CellQuest software, version 5.2 (Becton Dickinson, San Jose, CA). Platelets were gated out of the analysis and 20,000 RETs (CD71+) were evaluated for the presence of MN. Normochromatic erythrocytes (NCEs), MN-NCEs, RETs and MN-RETs were counted for each sample.
Descriptive statistics and statistical analyses were performed using SigmaPlot for Windows Version 12.3, (© Systat Software, Inc. ). One-factor analysis of variance (ANOVA) was used for data collected at the end of the study. The available parametric statistical tests included Dunnett&#;s, Holm-Sidak and Bonferroni and the most rigorous test was chosen for each analysis. A non-parametric test was conducted for data that were not normally distributed or of equal variance (i.e., Kruskal-Wallis rank sum analyses and subsequent Dunn&#;s test).
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