Mutation Research/Genetic Toxicology and Environmental Mutagenesis
Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO)
Graphical abstract
Introduction
The energetic explosive 3-nitro-1,2,4-triazol-5-one (NTO) was first prepared in 1905 by nitration of 1,2,4-triazole-5-one (TO) [1]. There was renewed interest in learning more about the chemistry of NTO in the late 1960s. The first report on the explosive nature of NTO was published by Lee and Coburn in 1985 [2]. In the past decade, the military services have been evaluating compounds for explosive formulations to replace highly sensitive explosives, such as 1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT). NTO is reported to be less sensitive and more stable than RDX and TNT [3], [4], [5]. By definition, insensitive munitions are those “which reliably fulfill their performance, readiness and operational requirements on demand, and which minimize the probability of inadvertent initiation and severity of subsequent collateral damage to weapon platforms, logistic systems and personnel when subjected to unplanned stimuli” [6]. NTO is presently used in a number of formulations in weapon systems [7]. The US Army Research Development and Engineering Center (USARDEC) is evaluating explosive formulations containing NTO for use in additional weapon systems.
Development of occupational and environmental exposure standards is limited by the lack of toxicity data for NTO. The acute oral LD50 for NTO is reported to be >5 g/kg in rats and mice. In rabbit tests, NTO produced mild skin irritation but was not an eye irritant and did not induce dermal sensitization in guinea pigs [8]. A recently completed oral subchronic toxicity test in rats at doses of 0, 30, 100, 315 and 1000 mg/kg/day showed no compound-related effects on food consumption or body weight. Reduced testicular size was observed in the 315 and 1000 mg/kg/day groups and microscopic changes in testis were observed in all dose groups. The Lowest Observed Adverse Effect Level (LOAEL) was determined to be 30 mg/kg/day in rats, based on microscopic changes in the testis [9]. There are no published studies on the toxicokinetics and metabolism of NTO in animals. However, the fate of NTO was investigated in vitro using rat liver microsomes and bacterial systems. Rat liver microsome catalysis of NTO under nitrogen atmosphere produced primarily amine, 5-amino-1,2,4-triazol-3-one, but, in the presence of oxygen, produced a major product, 5-hydroxy-1,2,4-trizol-3-one urazole, and a minor product, an amine [10], [11]. Addition of NTO to aqueous medium releases protons and lowers pH. The metabolism of NTO by bacteria is pH-dependent in aqueous systems. The maximum microbial reduction occurred at pH 6 in the presence of sucrose, while a ring cleavage occurred at pH 8 [11]. NTO also showed pH-dependent toxicity to Ceriodaphnia dubia. The growth inhibition value (IC 50) was 57 mg/mL after 7 days of exposure when buffered to a neutral pH [12].
There are no published genotoxicity data for NTO. Therefore, we evaluated both in vitro and in vivo genotoxicity of NTO for health and environmental risk assessment, as part of our comprehensive toxicity and safety evaluation program of new energetic compounds.
Section snippets
Chemical
Test article: 3-Nitro-1,2,4-triazol-5-one (NTO, 99.6%) pure; CAS# 932-64-9, lot number BAE 07B 305001, was obtained from Ordnance Systems, Inc., Kingsport, TN (Fig. 1). For the Ames test, Aroclor 1254-induced rat hepatic S9 fraction was obtained from Molecular Toxicology, Inc. (Boone, NC); Oxoid Nutrient Broth No. 2 was obtained from Oxoid LTD, Hampshire, England; positive-control substances: 2-nitrofluorene (2-NF), sodium azide (NaAz), 9-aminoacridine (9-AA), methyl methanesulfonate (MMS) and
Bacterial reverse mutation tests
The results of the range-finding test for strain TA100 indicated that NTO was toxic at 500 μg/plate and above, without activation, and 5 mg/plate, with activation, in regard to relative cloning efficiency. The results of the range finding test for WP2uvrA indicated NTO was toxic at 1 mg/plate and above without activation. With activation, the revertants were significantly decreased only at 5 mg/plate.
Based on the results of the range finding test, NTO was tested at concentrations of 5, 10, 50, 100
Discussion
The U.S. Army is developing insensitive munitions for future weapon systems under the direction of a Department of Defense (DOD) – wide initiative to improve the safety of munitions [27]. NTO is an energetic explosive candidate to replace highly sensitive munitions containing RDX and TNT [4], [5]. Evaluation of chemical and physical properties of NTO showed it is less sensitive when compared to others munitions [28]. It also showed low mammalian toxicity when compared to RDX and TNT [29]. The
Conflict of interest
No conflict of interest.
Acknowledgements
This work was funded by the U.S. Army Environmental Quality Technology (EQT) Ordnance Environmental Program (OEP) of the U.S. Army Research, Development and Engineering Command (RDECOM), Environmental Acquisition and Logistics Sustainment Program. We would like to thank Erick Hangeland and Chandrark Patel for their continued interest and support of this effort.
References (39)
- et al.
Metabolism of 14C-labelled 5-nitro-1,2,4-triazol-3-one (NTO): comparison between rat liver microsomes and bacterial metabolic pathways
J. Mol. Catal. B: Enzym.
(1998) - et al.
Revised methods for the Salmonella mutagenicity test
Mutat. Res.
(1983) - et al.
Genotoxicity assessment of two hypergolic energetic propellant compounds
Mutat. Res.
(2010) - et al.
Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y+/− mouse lymphoma cells
Mutat. Res.
(1975) - et al.
Validation and characterization of the L5178Y/TK± mouse lymphoma mutagen assay system
Mutat. Res.
(1979) - et al.
Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity
Mutat. Res.
(1987) - et al.
Comparison of flow cytometry- and microscopy-based methods for measuring micronucleated reticulocyte frequencies in rodents treated with nongenotoxic and genotoxic chemicals
Mutat. Res.
(2008) - et al.
Considerations in the US Environmental Protection Agency testing approach for mutagenicity
Mutat. Res.
(1991) - et al.
Genotoxicity risk assessment: a proposed classification strategy
Mutat. Res.
(2002) - et al.
Thyroid follicular cell carcinogenesis
Fundam. Appl. Toxicol.
(1989)
In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals
Mutat. Res.
Validation of a flow cytometric acridine orange micronuclei methodology in rats
Mutat. Res.
Justus liebigs Annalen der Chemie
3-Nitro-1,2,4-triazol-5-one, a less sensitive explosive (LA-10302-MS)
Development of an insensitive explosive containing NTO
A Preliminary Assessment of NTO as an Insensitive High Explosive, MRL Technical Report, MRL-TR-89-18
NTO-based Explosive Formulations: A Technology Review. DSTO-TR-0796. Technical Report
Energetic Material Compendium, Version 4.0.1
Cited by (33)
Outdoor dissolution and photodegradation of insensitive munitions formulations IMX-101 and IMX-104: Photolytic transformation pathway and mechanism study
2021, ChemosphereCitation Excerpt :Ecotoxicity studies have shown that DNAN is deleterious to organism health and it is acutely cytotoxic to bacteria and freshwater algae in the mg/L range, and to earthworm and ryegrass plant in the mg/kg range (Dodard et al., 2013; Liang et al., 2013). While NTO was reported to have relatively low toxicological risk to small mammals (Reddy et al., 2011) and aquatic organisms (Stanley et al., 2015; Madeira et al., 2018), its reduced metabolite product, 3-amino-1,2,4-triazol-5-one (ATO), shows both increased and decreased toxicity, depending on the target organism (Madeira et al., 2018). Detonated residues can also undergo attenuation by natural abiotic and biotic processes including adsorption, hydrolysis, photodegradation, and microbial degradation.
Alternative animal toxicity testing of chemical warfare agents
2020, Handbook of Toxicology of Chemical Warfare AgentsAlgae toxicological assessment and valorization of energetic-laden wastewater streams using Scenedesmus obliquus
2018, Journal of Cleaner ProductionCitation Excerpt :Discharge limits are generally facility specific and are regulated by the facility's NPDES (National Pollutant Discharge System) permits. The biochemical, physiological and toxic effects of the ECs on algae growth are not well-understood, and only limited information is available in the current literature (Reddy et al., 2011; Dodard et al., 2013). In addition, it is widely documented that the sensitivity of microalgae to a particular compound depends on the algal species being exposed, and to date no data describing the aquatic toxicity of the EC investigated in this research, on the freshwater microalgae Scenedesmus obliquus (S. obliquus) have been found in the literature.
Adsorption and oxidation of 3-nitro-1,2,4-triazole-5-one (NTO) and its transformation product (3-amino-1,2,4-triazole-5-one, ATO) at ferrihydrite and birnessite surfaces
2018, Environmental PollutionCitation Excerpt :However, in microbially-active soils subjected to periodic or localized anoxia, NTO is biotransformed to 3-amino-1, 2, 4-triazol-5-one (ATO) (Krzmarzick et al., 2015; Le Campion et al., 1998, 1999). ATO likewise exhibits high aqueous solubility (Smith and Cliff, 1999; Spear et al., 1989) and toxicity (Le Campion et al., 1999; Mullins et al., 2016; Reddy et al., 2011). The abiotic transformation of these compounds as mediated by mineral surfaces has not been studied in detail.
Ecotoxicity of the insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) and its reduced metabolite 3-amino-1,2,4-triazol-5-one (ATO)
2018, Journal of Hazardous MaterialsCitation Excerpt :In addition, NTO was not found to bioconcentrate in the test organisms. A battery of genotoxicity assays performed with NTO, including in vivo and in vitro test, suggested that there is a low risk of genetic hazards associated to the exposure to this compound [9]. NTO was ranked as practically nontoxic in aquatic toxicity tests conducted with Ceriodaphnia dubia and the unicellular green algae Selenastrum capricornutum [10].
Peri-pubertal administration of 3-nitro-1,2,4-triazol-5-one (NTO) affects reproductive organ development in male but not female Sprague Dawley rats
2015, Reproductive ToxicologyCitation Excerpt :This theory was supported in a battery of genotoxicity tests in which NTO was not mutagenic in the Ames Salmonella test (TA98, TA100, TA1535, and TA1537), with and without metabolic activation (S9), or in Escherichia coli (WP2uvrA), did not induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells (CHO-W-B1), and was negative in the L5178YTK+/− mouse lymphoma test [12]. NTO was also non-genotoxic in the in vivo rat peripheral blood micronucleus assay at doses up to 2000 mg/kg-day for 14 days [12]. Detection of explosive compounds and their degradation products in soil and groundwater near ammunition plants raises issues of potential effects on surrounding populations [6].