Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO)

doi:10.1016/j.mrgentox.2010.11.004

Mutation Research/Genetic Toxicology and Environmental Mutagenesis

Volume 719, Issues 1–2, 3 February 2011, Pages 35-40

https://doi.org/10.1016/j.mrgentox.2010.11.004 Get rights and content

Abstract

3-Nitro-1,2,4-triazol-5-one (NTO) is an energetic explosive proposed for use in weapon systems, to reduce the sensitivity of warheads. In order to develop toxicity data for safety assessment, we investigated the genotoxicity of NTO, using a battery of genotoxicity tests, which included the Ames test, Chinese Hamster Ovary (CHO) cell chromosome aberration test, L5178Y TK^+/− mouse lymphoma mutagenesis test and rat micronucleus test. NTO was not mutagenic in the Ames test or in Escherichia coli (WP2uvrA). NTO did not induce chromosomal aberrations in CHO cells, with or without metabolic activation. In the L5178Y TK^+/− mouse lymphoma mutagenesis test, all of the NTO-treated cultures had mutant frequencies that were similar to the average frequencies of solvent control-treated cultures, indicating a negative result. Confirmatory tests for the three in vitro tests also produced negative results. The potential in vivo clastogenicity and aneugenicity of NTO was evaluated using the rat peripheral blood micronucleus test. NTO was administered by oral gavage to male and female Sprague–Dawley rats for 14 days at doses up to 2 g/kg/day. Flow cytometric analysis of peripheral blood demonstrated no significant induction of micronucleated reticulocytes relative to the vehicle control (PEG-200). These studies reveal that NTO was not genotoxic in either in vitro or in vivo tests and suggest a low risk of genetic hazards associated with exposure.

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)

L. LeCampion 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)
D.M. Maron et al.
Revised methods for the Salmonella mutagenicity test
Mutat. Res.
(1983)
G. Reddy et al.
Genotoxicity assessment of two hypergolic energetic propellant compounds
Mutat. Res.
(2010)
D. Clive et al.
Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y^+/− mouse lymphoma cells
Mutat. Res.
(1975)
D. Clive et al.
Validation and characterization of the L5178Y/TK^± mouse lymphoma mutagen assay system
Mutat. Res.
(1979)
D. Clive et al.
Guide for performing the mouse lymphoma assay for mammalian cell mutagenicity
Mutat. Res.
(1987)
K.L. Witt 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)
K.L. Dearfield et al.
Considerations in the US Environmental Protection Agency testing approach for mutagenicity
Mutat. Res.
(1991)
K.L. Dearfield et al.
Genotoxicity risk assessment: a proposed classification strategy
Mutat. Res.
(2002)
R.N. Hill et al.
Thyroid follicular cell carcinogenesis
Fundam. Appl. Toxicol.
(1989)

Z. Cammerer et al.

In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals

Mutat. Res.

(2007)

K.A. Criswell et al.

Validation of a flow cytometric acridine orange micronuclei methodology in rats

Mutat. Res.

(2003)

V.W. Manchot et al.

Justus liebigs Annalen der Chemie

(1905)

K.Y. Lee et al.

3-Nitro-1,2,4-triazol-5-one, a less sensitive explosive (LA-10302-MS)

(1985)

M.J. Stosz et al.

Development of an insensitive explosive containing NTO

R.J. Spear et al.

A Preliminary Assessment of NTO as an Insensitive High Explosive, MRL Technical Report, MRL-TR-89-18

(1989)

M.W. Smith et al.

NTO-based Explosive Formulations: A Technology Review. DSTO-TR-0796. Technical Report

(1999)

M. Beyard

NATO (North Atlantic Treaty Organization)

Energetic Material Compendium, Version 4.0.1

(2010)

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New munition compounds have been developed to replace traditional explosives to prevent unintended detonations. However, insensitive munitions (IM) can leave large proportion of unexploded charge in the field, where it is subjected to photodegradation and dissolution in precipitation. The photolytic reactions occurring on the surfaces of IMX-101 and IMX-104 formulations and the subsequent fate of photolytic products in the environment were thoroughly investigated. The constituents of IMX-101 and IMX-104 formulations dissolve sequentially under rainfall in the order of aqueous solubility: 3-nitro-1,2,4-triazol-5-one (NTO) > nitroguanidine (NQ) > 2,4-dinitroanisole (DNAN) > 1,3,5-hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). A linear relationship between DNAN dissolution and rainwater volume was observed (r²: 0.86–0.99). It was estimated that it would take 16–228 years to completely dissolve these formulation particles under natural environmental conditions in Oracle, AZ. We used LC/MS/MS and GC/MS to examine the dissolution samples from IMX-101 and 104 particles exposed to rainfall and sunlight and found six DNAN photo-transformation products including 2-methoxy-5-nitrophenol, 4-methoxy-3-nitrophenol, 4-methoxy-3-nitroaniline, 2-methoxy-5-nitroaniline, 2,4-dinitrophenol, and methoxy-dinitrophenol, which are in good agreement with computational modeling results of bond strengths. The main DNAN photodegradation pathways are therefore proposed. Predicted eco-toxicity values suggested that the parent compound DNAN, methoxy-nitrophenols, methoxy-nitroanilines and the other two products (2,4-dinitrophenol and methoxy-dinitrophenol) would be harmful to fish and daphnid. Our study provides improved insight about the rain dissolution and photochemical behavior of IM formulations under natural conditions, which helps to form target-oriented strategies to mitigate explosive contamination in military training sites.
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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.
The manufacturing of energetic compounds (explosives), such as 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) and 3-nitro-1,2,4-triasol-5-one (NTO), generates large volumes of wastewater containing organic pollutants, which are also rich in nutrients, mainly nitrogen. These wastewaters are subject to regulatory compliance and require treatment prior to their release into local water bodies. An approach is proposed to recover nutrients and water from wastewaters prior to treatment by growing microalgae with the aim to reduce the energy footprint of the munitions production facility by producing algae-based biofuels. In order to assess the feasibility of implementation of the proposed approach, ten different fully characterized untreated wastewaters with various levels of organic and inorganic carbon and nitrogen content were assessed for microalgae toxicity using Scenedesmus obliquus ATCC^® 11477 as target microorganism. Toxicity experiments were performed in microplates for six days at 25 °C, 120 rpm orbital mixing speed, under a 14:10 h light: dark photoperiod, and 68 μmol photons/m²s of light intensity. The results indicate that the majority of the wastewater streams assessed could be used as nutrient media with no pretreatment while only three of the streams required chemical treatment due to their high growth inhibition characteristics. From the toxicological results obtained, a site specific mixture prepared by blending of the different untreated waste streams was tested for Scenedesmus obliquus growth. It was observed that this microalga can reach 5 × 10⁷ cells/mL over six days of incubation in the wastewater mixture containing up to 24 mg/L RDX and 28 mg/L NTO. This strain exhibited similar growth patterns in the wastewater mixture as compared to the control sample, suggesting the potential feasibility of using untreated, energetic-laden industrial wastewater along with carbon dioxide [CO₂ (atmospheric or flue gases)] for microalgae biomass production.
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 Pollution
Citation 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.
The emerging insensitive munitions compound (IMC) 3-nitro-1,2,4-triazole-5-one (NTO) is currently being used to replace conventional explosives such as 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX), but the environmental fate of this increasingly widespread IMC remains poorly understood. Upon release from unexploded solid phase ordinances, NTO exhibits high aqueous solubility and, hence, potential mobilization to groundwater. Adsorption and abiotic transformation at metal oxide surfaces are possible mechanisms for natural attenuation. Here, the reactions at ferrihydrite and birnessite surfaces of NTO and its biotransformation product, 3-amino-1, 2, 4-triazol-5-one (ATO), were studied in stirred batch reactor systems at controlled pH (7.0). The study was carried out at metal oxide solid to solution ratios (SSR) of 0.15, 1.5 and 15 g kg⁻¹. The samples were collected at various time intervals up to 3 h after reaction initiation, and analyzed using HPLC with photodiode array and mass spectrometric detection. We found no detectable adsorption or transformation of NTO upon reaction with birnessite, whereas ATO was highly susceptible to oxidation by the same mineral, showing nearly complete transformation within 5 min at 15 g kg⁻¹ SSR to urea, CO_2(g) and N_2(g). The mean surface-area-normalized pseudo-first order rate constant (k) for ATO oxidation by birnessite across all SSRs was 0.05 ± 0.022 h⁻¹ m⁻², and oxidation kinetics were independent of dissolved O₂ concentration. Both NTO and ATO were resistant to oxidation by ferrihydrite. However, NTO showed partial removal from solution upon reaction with ferrihydrite at 0.15 and 1.5 g kg⁻¹ SSR and complete loss at 15 g kg⁻¹ SSR due to strong adsorption. Conversely, ATO adsorption to ferrihydrite was much weaker than that measured for NTO.
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)
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Citation 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].
The insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) was recently approved by the U.S. Army to replace cyclotrimethylene trinitramine (RDX) in conventional explosives. As its use becomes widespread, concern about the potential toxicity of NTO increases. NTO can undergo microbial reduction to 3-amino-1,2,4-triazol-5-one (ATO), which is recalcitrant in waterlogged soils. In this study, the acute toxicity of NTO and ATO towards various organisms, including microorganisms (i.e., methanogenic archaea, aerobic heterotrophs, and Aliivibrio fischeri (Microtox assay)), the microcrustacean Daphnia magna (ATO only), and zebrafish embryos (Danio rerio), was assessed. NTO was notably more inhibitory to methanogens than ATO (IC₅₀ = 1.2 mM, > 62.8 mM, respectively). NTO and ATO did not cause noteworthy inhibition on aerobic heterotrophs even at the highest concentrations tested (32.0 mM). High concentrations of both NTO and ATO were required to inhibit A. fischeri (IC₂₀ = 19.2, 22.4 mM, respectively). D. magna was sensitive to ATO (LC₅₀ = 0.27 mM). Exposure of zebrafish embryos to NTO or ATO (750 μM) did not cause lethal or developmental effects (22 endpoints tested). However, both compounds led to swimming behavior abnormalities at low concentrations (7.5 μM). The results indicate that the reductive biotransformation of NTO could enhance or lower its toxicity according to the target organism.
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 Toxicology
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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].
Nitrotriazolone (3-nitro-1,2,4-triazol-5-one; NTO) is an insensitive munition that has demonstrated effects on reproductive organs in adult male rats. NTO was administered to male (0, 250, and 500 milligrams per kilogram per day (mg/kg-day)) and female (0, 500, and 1000 mg/kg-day) Sprague-Dawley rats (15/sex/group) via oral gavage from weaning through post-natal day 53/54 and 42/43, respectively. Age and body mass at vaginal opening (VO) and preputial separation (PPS), as well as all measures of estrous cyclicity were not affected by treatment with NTO. Males treated with NTO exhibited reductions in testis mass associated with tubular degeneration/atrophy. Less pronounced reductions in accessory sex organ masses were also observed in the 500 mg/kg-day group. Treatment with NTO did not affect thyroid hormone or testosterone levels. These findings suggest that NTO is not acting as an estrogen or thyroid active compound, but may indicate effects on steroidogenesis and/or direct testicular toxicity.

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Genotoxicity assessment of an energetic propellant compound, 3-nitro-1,2,4-triazol-5-one (NTO)

Abstract

Graphical abstract

Introduction

Section snippets

Chemical

Bacterial reverse mutation tests

Discussion

Conflict of interest

Acknowledgements

J. Mol. Catal. B: Enzym.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Fundam. Appl. Toxicol.

Mutat. Res.

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