Genotoxicity assessment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

Hexahydro-1,3,5-trinitro-1,3,5-triazine, a polynitramine compound, commonly known as RDX, has been used as an explosive in military munitions formulations since World War II. There is considerable data available regarding the toxicity and carcinogenicity of RDX. It has been classified as a possible carcinogen (U.S. Environmental Protection Agency, Integrated Risk Information System, 2005, www.epa.gov/IRIS/subst/0313.htm). In order to better understand its gentoxic potential, the authors conducted the in vitro mouse lymphoma forward mutation and the in vivo mouse bone marrow micronucleus assays. Pure RDX (99.99%) at concentrations ranging from 3.93 to 500 microg/ml showed no cytotoxicity and no mutagenicity in forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, with and without metabolic activation. This finding was also confirmed by repeat assays under identical conditions. In addition, RDX did not induce micronuclei in mouse bone marrow cells when tested to the maximum tolerated dose of 250 mg/kg in male mice. These results show that RDX was not mutagenic in these in vitro and in vivo mammalian systems.     

Water quality criteria for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

The occurrence of the munitions compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater surrounding Army ammunition plants may result in contamination of local drinking water supplies. RDX exerts its primary toxic effect in humans on the central nervous system, but also involves gastrointestinal and renal effects. Symptomatic effects following acute exposure include hyperirritability, nausea, vomiting, generalized epileptiform seizures, and prolonged postictal confusion and amnesia. Health effects data were analyzed for RDX, and although no controlled human studies exist concerning the acute or chronic toxic effects of exposure to RDX, sufficient animal toxicity data are available to derive an ambient water quality criterion for the protection of human health. This paper summarizes the available literature on metabolism of RDX and human and animal toxicity. Based on noncarcinogenic mammalian toxicity data, and following the methodologies of the U.S. Environmental Protection Agency, an ambient water quality criterion for the protection of human health of 103 micrograms/liter is proposed for ingestion of drinking water and aquatic foodstuffs. A criterion of 105 micrograms/liter is proposed for ingestion of drinking water alone.     

Delayed myelosuppression with acute exposure to hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and environmental degradation product hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) in rats

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a widely used munitions compound, and hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), its N-nitroso product of anaerobic microbial nitroreduction, are contaminants of military sites. Previous studies have shown MNX to be the most acutely toxic among the nitroreduced degradation products of RDX and to cause mild anemia at high dose. The present study compares hematotoxicity with acute oral exposure to MNX with parent RDX. Both RDX and MNX caused a modest decrease in blood hemoglobin and ~ 50% loss of granulocytes (NOAELs = 47 mg/kg) in female Sprague–Dawley rats observed 14 days post-exposure. We explored the possibility that blood cell loss observed after 14 days was delayed in onset because of toxicity to bone marrow (BM) progenitors. RDX and MNX decreased granulocyte/macrophage-colony forming cells (GM-CFCs) at 14, but not 7, days (NOAELs = 24 mg/kg). The earliest observed time at which MNX decreased GM-CFCs was 10 days post-exposure. RDX and MNX likewise decreased BM burst-forming units-erythroid (BFU-Es) at 14, but not 7, days. Granulocyte–erythrocyte–monocyte–megakaryocyte (GEMM)-CFCs were unaffected by RDX and MNX at 7 days suggesting precursor depletion did not account for GM-CFC and BFU-E loss. MNX added to the culture media was without effect on GM-CFC formation indicating no direct inhibition. Flow cytometry showed no differential loss of BM multilineage progenitors (Thy1.1⁺) or erythroid (CD71⁺) precursors with MNX suggesting myeloid and erythroid lineages were comparably affected. Collectively, these data indicate that acute exposure to both RDX and MNX caused delayed suppression of myelo- and erythropoiesis with subsequent decrease of peripheral granulocytes and erythrocytes.     

Comparison of water quality criterion and lifetime health advisory for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

The U.S. Environmental Protection Agency (USEPA) has recently recommended a lifetime health advisory (HA) for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The purpose of this brief article is to present the basis for the calculation of the HA so that the reader can compare it with a water quality criterion (WQC) that has been proposed (E. L. Etnier, 1989, Regul. Toxicol. Pharmacol. 9, 147-157). In the earlier article, a water quality criterion of 105 micrograms/liter for RDX in drinking water was proposed, and in the present article the methodology by which USEPA calculated a lifetime HA of 2 micrograms/liter is presented. The differences in the derivation of the WQC and the HA are discussed.     

Metabolite profiling of [14C]hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in Yucatan miniature pigs

The study reported herein examined the metabolism of 14C-labeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) resulting from a single oral gavage of 5 ml/kg to male and female Yucatan miniature pigs (43 mg/kg, 56 microCi/kg in 0.5% carboxymethylcellulose in water). Blood, urine, and feces were collected at selected times of 1, 6, 12, and 24 h postdose. At 24 h postdose, liver samples were collected. Blood, plasma, liver, and excreta were analyzed for total RDX-derived radioactivity and metabolites were identified. Urine was the major route of elimination of 14C-RDX-derived radioactivity in both males and females. Relatively low levels of radioactivity were found in gastrointestinal contents and in feces, suggesting nearly complete absorption of 14C-RDX following an oral dose. Analysis of urine by liquid chromatography-mass spectrometry (LC/MS) identified quantifiable levels of two ring-cleavage metabolites, 4-nitro-2,4-diazabutanal and 4-nitro-2,4-diaza-butanamide, as well as parent RDX. The 4-nitro-2,4-diazabutanal, was seen in earlier studies of aerobic metabolism of RDX. The 4-nitro-2,4-diaza-butanamide, an amide, was not previously reported but was tentatively identified in this study. Analysis by a more sensitive method (LC/MS/MS) also showed trace amounts of the RDX metabolites 1-nitroso-3,5-dinitro-1,3,5-triazacyclohexane (MNX) (in both male and female urine) and 1-nitro-3,5-dinitroso-1,3,5-triazacyclohexane (DNX) (in male urine). Analysis of plasma by LC/MS/MS also revealed quantifiable levels of RDX and trace levels of MNX, DNX, and 1,3,5-trinitroso-1,3,5-triazacyclohexane (TNX). None of the liver extracts showed quantifiable levels of RDX or any identifiable metabolites. Most of the radioactivity was in the form of water-soluble high-molecular-weight compounds. RDX when given orally to pigs was rapidly metabolized by loss of two nitro groups followed by ring cleavage.     

Assessing the non-cancer risk for RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) using physiologically based pharmacokinetic (PBPK) modeling

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is an explosive used in military applications. It has been detected in ground water surrounding US military installations and at manufacturing facilities. RDX has been shown to produce hepatotoxicity, testicular, and neurological effects in animals, the latter also in humans. The current chronic oral reference dose (RfD) of 0.003 mg/kg/day was derived based on prostate effects in rats. Here, we provide a reevaluation of the risk associated with RDX exposure by examining old and new data and using physiologically based pharmacokinetic (PBPK) modeling approaches. Candidate non-cancer endpoints in rodents were evaluated and the most plausible mode(s) of action were determined. A PBPK model was used to derive appropriate internal doses based on the mode of action, and then a benchmark dose (BMD) and the lower confidence limit on the BMD (BMDL) were determined using these internal doses in animals. Uncertainty factors (UF) were applied to the animal BMDL or no-observed effect level and a human PBPK model was used to determine a human equivalent dose resulting in the candidate RfDs (cRfDs). A proposed chronic RfD of 0.07 mg/kg/day, based on multiple effects observed in rats, was selected from among the cRfDs.     

Cancer mode of action,weight of evidence,and proposed cancer reference value for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, CAS No. 121-82-4) is a component of munitions formulations, and has been detected in groundwater samples collected at various US military sites. Clean up target levels for RDX may be derived based on consideration of acceptable cumulative human exposure as expressed in toxicity reference values. Evaluations of the cancer weight of evidence and possible modes of action (MOA) for RDX-induced cancer were conducted. It was concluded that the available data provide suggestive evidence of human carcinogenic potential for RDX. While a mutagenic/genotoxic MOA for RDX is unlikely, no alterative MOA is strongly supported by the available data. A nonlinear (threshold) approach to the assessment of human cancer risk was recommended, and a recommended chronic cancer reference dose of 0.08 mg/kg/day was derived. For comparison only, computations using a linear approach were also conducted, yielding a cancer risk specific dose of 0.000235 mg/kg/day for 1 in 10⁵ risk; this value is 2.6-fold higher the current US EPA risk specific dose for 1 in 10⁵ risk. Thus, cleanup standards based on human health risk from RDX exposure could potentially depend on the willingness of risk managers to accept a nonlinear MOA and nonlinear toxicity risk value derivation.     

Reevaluation of a twenty-four-month chronic toxicity/carcinogenicity study of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in the B6C3F1 hybrid mouse

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been widely used as an explosive in U.S. army munitions formulations since World War II. Two-year carcinogenicity studies revealed RDX to be noncarcinogenic in two strains of rats, but a 2-year carcinogenicity study in B6C3F1 mice revealed an increased incidence of hepatocellular neoplasms in females. Based on results of the study in B6C3F1 mice, RDX has been classified as a possible carcinogen. The authors reevaluated the archived histological sections from the B6C3F1 mouse study, using current histopathologic diagnostic criteria and interpretations. The earlier evaluation showed a statistically significant increase in the incidence of hepatocellular adenoma/carcinoma in female mice from the three highest dose groups (7, 35, and 175/100 mg/kg/day). The revaluation yielded a slightly lower incidence at each of the dose levels in female mice. The reduced number of hepatocellular neoplasms was largely due to reclassification of hepatocellular adenomas as foci of cytoplasmic alteration, in compliance with current diagnostic criteria. The reevaluation was reviewed by a pathology working group (PWG), which arrived at a consensus classification of each lesion. Based on the consensus diagnoses of the PWG, only one female group (35 mg/kg/day) showed a significant increase when compared to controls. The incidence of hepatocellular neoplasms for all groups, including the 35 mg/kg/day group, was within the reported incidence range for spontaneous hepatocellular neoplasms in female B6C3F1 mice. The increased incidence of hepatocellular neoplasms in female mice given RDX at 35 mg/kg/day was interpreted as equivocal evidence of a carcinogenic effect.     

Interspecific effects of 4A-DNT (4-amino-2,6-dinitrotoluene) and RDX (1,3,5-trinitro-1,3,5-triazine) in Japanese quail, Northern bobwhite, and Zebra finch

The purpose of this study was to assess the toxicological effects of two munition compounds, 4-amino-2,6-dinitrotoluene (4A-DNT) and 1,3,5-trinitro-1,3,5-triazine (RDX), on three different bird species: two common toxicological model species—the Northern Bobwhite (Colinus virginianus) and the Japanese Quail (Coturnix japonica), and a representative passerine—the Zebra Finch (Taeniopygia guttata). Bobwhite were exposed to 4A-DNT at 0, 8, 15, 30, 60, or 150 mg/kg body weight (bw) d by oral gavage for seven days; because the high dose of 4A-DNT was lethal to bobwhite, the maximum dose was changed to 100 mg/kg bw d for Japanese quail and finches to ensure tissue could be used for future toxicogenomic work. RDX was similarly administered at 0, 0.5, 1.5, 3, 6, or 12 mg/kg bw d. Blood was drawn prior to euthanasia for blood cellularity and chemistry analyses. Finches were clearly least affected by 4A-DNT as evidenced by a lack of observable effects. Bobwhite appeared to be the most sensitive species to 4A-DNT as observed through changes in blood cellularity and plasma chemistry effects. Bobwhite appeared to be more sensitive to RDX than Japanese Quail due to increased effects on measures of plasma chemistries. Finches exhibited the greatest sensitivity to RDX through increased mortality and seizure activity. This study suggests that sensitivity among species is chemical-specific and provides data that could be used to refine current avian sensitivity models used in ecological risk assessments.     

A 90-day repeated dose oral (gavage) toxicity study of perfluorohexanoic acid (PFHxA) in rats (with functional observational battery and motor activity determinations)

Possible toxic effects of perfluorohexanoic acid (PFHxA) were evaluated when administered orally by gavage to rats at levels up to 200 mg/kg/day for 90 days. Lower body weight gains were noted in the 10, 50 and 200 mg/kg/day group males (not dose-responsive) throughout dosing. Other changes included lower red blood cell parameters, higher reticulocyte counts and lower globulin in the 200 mg/kg/day group males and females, higher liver enzymes in males at 50 and 200 mg/kg/day, lower total protein and higher albumin/globulin ratio, and lower cholesterol, calcium in males at 200 mg/kg/day. Minimal centrilobular hepatocellular hypertrophy was present in 200 mg/kg/day group males and correlated with higher liver weights and slightly higher peroxisome beta oxidation activity at the end of the dosing period. Based on liver histopathology and liver weight changes, the no-observed-adverse-effect level (NOAEL) for oral administration was 50 mg/kg/day for males and 200 mg/kg/day for females.     

白首乌C_（21）甾总苷及其醋酸降解产物的急性毒性试验研究

目的比较白首乌C21甾总苷及其醋酸降解产物对小鼠急性毒性的影响。方法分别以1∶0.8等比稀释法配制等比浓度白首乌C21甾总苷组及白首乌总苷醋酸降解产物组的给药剂量,一次性腹腔给药,观察小鼠的死亡率,采用改良寇氏法计算半数致死量（LD50）。结果白首乌C21甾总苷的LD50值为721.2675mg.kg-1,95%的可信限为595.464~847.071mg.kg-1;白首乌总苷醋酸降解产物的LD50值为984.0087mg.kg-1,95%的可信限为833.8096~1134.2078mg.kg-1。结论白首乌总苷醋酸降解产物的LD50值升高,说明醋酸水解降低了白首乌C21甾总苷的毒性,为进一步研究白首乌C21甾总苷的增效减毒作用有显著意义。     

Evaluation of the acute oral toxicity class of tricentric chromium(III) propionate complex in rat

Chromium(III) is an essential element for carbohydrate and lipid metabolism, and various chemical forms of this element are widely used in dietary supplements. Of particular interest is [Cr₃O(O₂CCH₂CH₃)₆(H₂O)₃]⁺ cation (CrProp), that has been proposed as an alternative source of Cr. However, its safety has not been studied completely. In this study, we evaluated the acute toxicity class of CrProp in Wistar rats applying the OECD 423 procedure. Male and female Wistar rats (n = 12, 6 ♀ and 6 ♂) were given by gavage either a single dose of CrProp 2000 mg kg⁻¹ body mass or equivalent volumes of distilled water, and fed Labofeed B diet, and observed carefully for 14 days, than sacrificed to collect samples for biochemical and histologic examination. No death cases were detected, no major abnormalities in animal behaviour, body mass gains, gross organ histology, and blood morphology, and biochemistry were observed, except some changes of liver mass and the activity of ALT in female rats. The results demonstrate that LD₅₀ of CrProp is greater than 2000 mg kg⁻¹ when administrated orally to rat, thus this compound appears to be belong the fifth category in the GHS system or the fourth class (“unclassified”) in the EU classification system.     

The acute oral and intravenous toxicity of p-aminopropiophenone (PAPP) to laboratory rodents

The acute toxicity of p-aminopropiophenone (PAPP) to three species of laboratory animals has been investigated using solutions in dimethylsulphoxide (DMSO) for intravenous and oral (intragastric) administration. In addition, methaemoglobinaemia and Heinz-body haemolytic anaemia are described.     

Repeated sub-chronic oral toxicity study of xylooligosaccharides (XOS) in dogs

In this study, Beagle dogs were administered xylooligosaccharide (XOS, CAS # 87099-0) at doses of 0, 1250, 2500, and 5000 mg/kg/day by oral gavage for 26 weeks. A 4-week recovery period was added to observe delayed or reversible toxicity. Measurements included body weight, food consumption, clinical observations, temperature, electrocardiogram (ECG), urinalysis, blood chemistry, hematology, organ weight, gross necropsy, and histopathological examination. Except for transient diarrhea or vomiting, no treatment-related adverse effects were noted. In the mid-dose groups, transitional diarrhea was observed in the initial 1–2 weeks. In the high-dose groups, diarrhea and/or vomiting were observed episodically over the duration of treatment. However, they disappeared after XOS was withdrawn in the recovery period. Although there was a tendency toward less weight gain in the high-dose group animal group, this is typical in animals and humans fed non-digestible carbohydrates. This chronic toxicity study demonstrated that the no observed adverse effect level (NOAEL) of XOS is 2500 mg/kg body weight (BW)/day. Based on body surface area (conversion factor of 0.54 for dogs to human), this corresponds to daily doses of 1350 mg/kg BW or 81–108 g XOS in human adults weighing 60–80 kg.     

Acute toxicity studies of a novel excipient arabinoxylan isolated from Ispaghula (Plantago ovata) husk

Purpose: The arabinoxylan from Ispaghula (Plantago ovata) husk has been proven scientifically as potential excipient. However, toxicity study of the arabinoxylan is still lacking. The present study was done to investigate the acute toxicity of arabinoxylan in two animal species. Methods: The mice were exposed to (1?g/kg, 5?g/kg, 10?g/kg) and rabbits (2.5?g/kg, 5?g/kg) of arabinoxylan orally and observed for a period of 14 days. On day 15 hematology, serum biochemistry and necropsy was performed in mice relative organ weight calculated and histological examination was carried out. Primary dermal and eye irritation tests were carried out. Cardiac effects of isolated arabinoxylan were studied on frog heart. Results: The acute administration of the arabinoxylan did not produce mortality or significant changes in, water and food consumption however body weights of mice and rabbits decreased initially with a gradual increase till day 14. Internal organs relative weights were found to be normal. Hematological biochemical and histopathological examination did not show any significant (p?Conclusion: This study has shown that acute administration of arabinoxylan may be safe.     

Estimation of acute oral toxicity using the No Observed Adverse Effect Level (NOAEL) from the 28 day repeated dose toxicity studies in rats

Acute systemic toxicity is one of the areas of particular concern due to the 2009 deadline set by the 7th Amendment of the Cosmetics Directive (76/768/EEC), which introduces a testing and marketing ban of cosmetic products with ingredients tested on animals. The scientific community is putting considerable effort into developing and validating non-animal alternatives in this area. However, it is unlikely that validated and regulatory accepted alternative methods and/or strategies will be available in March 2009. Following the initiatives undertaken in the pharmaceutical industry to waive the acute oral toxicity testing before going to clinical studies by using information from other in vivo studies, we proposed an approach to identify non-toxic compounds (LD50>2000mg/kg) using information from 28 days repeated dose toxicity studies. Taking into account the high prevalence of non-toxic substances (87%) in the New Chemicals Database, it was possible to set a NOAEL threshold of 200mg/kg that allowed the correct identification of 63% of non-toxic compounds, while <1% of harmful compounds were misclassified as non-toxic. Since repeated dose toxicity studies can be performed in vivo until 2013, the proposed approach could have an immediate impact for the testing of cosmetic ingredients.     

Evaluation of neurobehavioral abnormalities and immunotoxicity in response to oral imidacloprid exposure in domestic chickens (Gallus gallus domesticus)

ABSTRACT

Domestic chickens (Gallus gallus domesticus) were exposed to imidacloprid by gavage once daily for 7 consecutive days at 0, 0.03, 0.34, 3.42, 10.25, and 15.5 mg/kg/day (n = 20 per group; 5 6-week-old males, 5 6-week-old females, 5 9-week-old males, and 5 9-week-old females). The severity and duration of neurobehavioral abnormalities were recorded. Components of the innate and adaptive immune system were assessed with 7 standard functional assays. Temporary neurobehavioral abnormalities were observed in a dose-dependent manner, including muscle tremors, ataxia, and depressed mentation. Based upon mean clinical severity scores, the no observed adverse effect level (NOAEL) was 3.42 mg/kg/day, and the lowest observed adverse effect level (LOAEL) was 10.25 mg/kg/day. The effective dose value for the presence of any neurobehavioral abnormalities in 50% of the test group (ED₅₀) was 4.62 ± 0.98 mg/kg/day. The ED₅₀ for an adjusted score that included both severity and duration of neurobehavioral abnormalities was 11.24 ± 9.33 mg/kg/day. These ED₅₀ values are equivalent to a 1 kg bird ingesting 29 or 70 imidacloprid treated soybean seeds respectively. Immunotoxicity was not documented, possible causes include the assays were insensitive, relevant immune functions were not examined, or imidacloprid is not immunotoxic at this dosing schedule in this species. Neurobehavioral abnormalities were a more sensitive indicator of the sublethal effects of imidacloprid than immunotoxicity.     

Stage‐dependent toxicity of bisphenol a on Rhinella arenarum (anura,bufonidae) embryos and larvae

The acute and chronic toxicity of bisphenol A (BPA) was evaluated on the common South American toad Rhinella arenarum embryos and larvae by means of continuous and pulse exposure treatments. Embryos were treated continuously from early blastula (S.4) up to complete operculum (S.25), during early larval stages and by means of 24 h pulse exposures of BPA in concentrations ranging between 1.25 and 40 mg L^?1, in order to evaluate the susceptibility to this compound in different developmental stages. For lethal effects, S.25 was the most sensitive and gastrula was the most resistant to BPA. The Teratogenic Index for neurula, the most sensitive embryonic stage for sublethal effects was 4.7. The main morphological alterations during early stages were: delayed or arrested development, reduced body size, persistent yolk plug, microcephaly, axial/tail flexures, edemas, blisters, waving fin, underdeveloped gills, mouth malformations, and cellular dissociation. BPA caused a remarkable narcotic effect from gill circulation stage (S.20) onwards in all the organisms exposed after 3 h of treatment with 10 mg L^?1 BPA. After recovering, the embryos exhibited scarce response to stimuli, erratic or circular swimming, and spasmodic contractions from 5 mg L^?1 onwards. Our results highlight the lethal and sublethal effectsof BPA on R. arenarum embryos and larvae, in the last case both at structural and functional levels. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 146–154, 2014.     

连钱草提取物急性毒性实验研究

目的 通过对连钱草的小鼠灌胃急性毒性实验研究,指导其临床用药的安全性.方法 通过预实验,判断测定LD50的可能性,并据此测定LD50或MTD.结果 MTD结果表明小鼠口服连钱草的最大耐受量相当于50kg人临来日用量的266倍.结论 连钱草的急性毒性很小.     

Megakaryocyte expansion and macrophage infiltration in bone marrow of rats subchronically treated with MNX,N‐nitroso environmental degradation product of munitions compound RDX (hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine)

Hexahydro‐1‐nitroso‐3,5‐dinitro‐1,3,5‐triazine (MNX), environmental degradation product of munitions hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX), causes seizures in rats with acute oral exposure like parent RDX. Our previous studies have additionally reported hematotoxicity with acute MNX exposure manifested as myelosuppression, anemia and splenic hemosiderosis. This study explored whether MNX administered subchronically continued to target bone marrow to elicit peripheral blood cytopenia. Female Sprague–Dawley rats were gavaged daily for 4 or 6 weeks with 47 mg kg⁻¹ day⁻¹ MNX (¼ LD₅₀) or vehicle (5% dimethyl sulfoxide in corn oil) and hematological and clinical chemistry parameters, spleen weights, spleen and bone marrow histopathology and immunohistochemistry with ED1 anti‐CD68 macrophage marker were evaluated 24 h after the last dose. Unexpectedly, no decrease in blood erythroid parameters was seen with subchronic MNX and convulsions and tremors ceased after 2 weeks of treatment. Toxicological effects observed were MNX‐induced increases in blood granulocyte and platelet counts and in bone marrow megakaryocyte and ED1⁺‐macrophage density. MNX was without effect on bone marrow cellularity and picrosirius red stained/collagen fiber deposition. Spleen weight increased modestly with extramedullary hematopoiesis evident, but hemosiderin and relative red and white pulp areas were unaffected. Collectively, this study demonstrated that erythroid effects characteristic of acute MNX exposure were not evident with subchronic exposure. However, megakaryocyte proliferation in bone marrow coincident with thrombocytosis after subchronic MNX exposure suggested continued hematotoxicity, but with a qualitatively different outcome. Granulocytosis and increased bone marrow macrophages implicated an inflammatory component in MNX hematotoxicity. Copyright © 2017 John Wiley & Sons, Ltd.