Hazard characterization of carcinogenicity, mutagenicity, and reproductive toxicity for short chain primary nitroalkanes.

Nitroalkanes are organic aliphatic hydrocarbon compounds with a nitro moiety that are commonly used as solvents or intermediates to synthesize a variety of organic compounds due to their inherent reactivity. In June 2020, a harmonized classification and labeling (CLH) proposal was submitted to the European Chemicals Agency (ECHA) for the following harmonized carcinogenicity, mutagenicity, and reproductive toxicity ("CMR") classifications for nitromethane (NM), nitroethane (NE), and 1-nitropropane (1-NP): NM Carc. 1B and Repr. 1B; NE Repr. 1B; and 1-NP Repr. 2. In this assessment, a weight of evidence (WoE) evaluation of studies on animal carcinogenicity and reproductive and developmental toxicity, genotoxicity, and mode of action for these three nitroalkanes was performed to critically assess the relevance of the proposed CMR classifications. Overall, the WoE indicates that NM, NE, and 1-NP are not carcinogenic, genotoxic, nor selective reproductive or developmental toxicants. Based on our analysis, classifying NM, NE, and 1-NP as Category 2 reproductive toxicants is most appropriate. Furthermore, not classifying NE and 1-NP with respect to their carcinogenicity is appropriate based on the available studies for this endpoint coupled with negative results in genotoxicity studies, metabolism data, and in silico predictions. We determined that the classification for NM of Carc. 1B is not appropriate, based on the fact that rat mammary and harderian tumors are likely not relevant to humans and lung and liver tumors reported in mice were equivocal in their dose-response and statistical significance.

Organochlorine pesticide acetofenate and its hydrolytic metabolite in rabbits: Enantioselective metabolism and cytotoxicity.

Acetofenate (AF) is a chiral organochlorine pesticide used for controlling hygiene pests. In this study, the metabolism of AF in rabbits in vivo and in vitro was investigated and the primary chiral metabolite acetofenate-alcohol (AF-A) was analyzed. The cytotoxicity of AF and AF-A was also determined. AF in rabbits in vivo was eliminated so rapidly that AF could not be detected within 10min after intravenous administration at 20mg/kg (body weight), and AF-A was quickly formed. In vitro metabolism assay, using plasma and liver microsomes, showed that AF was also quickly metabolized to AF-A and the metabolic process was significantly enantioselective with preferential degradation of (-)-AF and formation of (-)-AF-A. The cytotoxicity of AF and AF-A were investigated by assessing cell proliferation, apoptosis and generation of reactive oxygen species. The results showed that AF and AF-A induce enantioselective cytotoxicity. This study will be helpful for improving knowledge about the metabolism and toxicity of AF on an enantiomeric level and providing evidence to understand the potential environmental risk.

Prognostic value of serum zinc levels in patients with acute HC/zinc chloride smoke inhalation.

Hexachloroethane (HC)/zinc chloride (ZnCl, smoke bomb) exposure in the military setting results in lung injury which is uncommon and has been rarely described in previous studies. The aim of this study is to investigate the correlation between the serum zinc in patients with HC/ZnCl smoke inhalation lung injury and disease severity. A total of 15 patients with HC/ZnCl-related conditions were recruited in this study. The serum zinc level and the pulmonary function tests and liver function tests including total lung capacity (TLC), forced vital capacity (FVC), forced expiratory pressure in 1 second (FEV1), alanine aminotransferase (ALT), and aspartate transaminase (AST) were analyzed. Eleven cases had mild clinical manifestations. Four cases rapidly developed features typical of severe adult respiratory distress syndrome. The level of serum zinc was increased, but FVC, FEV1, and TLC was decreased significantly in the moderate and severe cases. In addition, the serum zinc level correlated well with the TLC, FVC, and FEV1 (r = -0.587, -0.626, -0.617, respectively; P = .027, .017, .019, respectively). The 4 cases in moderate and severe group had delayed impairment of liver functions after the accident. This study suggested that the serum zinc level may be associated with the severity of lung and liver injuries after HC/ZnCl smoke inhalation.

In vitro studies on the tumorigenic potential of the halonitromethanes trichloronitromethane and bromonitromethane.

Epidemiological data indicate that chronic exposure to water disinfection by-products (DBPs) may result in increased risk of cancer. However, the real carcinogenic potential of individual DBPs is not well known. In this study, we assessed the in vitro carcinogenic potential of trichloronitromethane (TCNM) and bromonitromethane (BNM), two halonitromethanes (HNMs) commonly found in DBPs' mixtures at comparably high concentrations. Human lung BEAS-2B cells were exposed for 8weeks to TCNM and BNM, and the acquisition of different in vitro cancer-like features was evaluated. The results indicate that long-term exposure to non-cytotoxic doses of TCNM and BNM did not cause carcinogenic transformation as indicated by the absence of morphological changes, no effects on cell growth, no changes in the level of matrix metalloproteinases (MMPs) secretion, and no increased anchorage-independent cell growth capacity. Furthermore, TCNM- and BNM-exposed BEAS-2B cells were unable to enhance tumour growth directly or by indirect influence of the surrounding stroma. Our results indicate that the carcinogenic effects of DBP mixtures cannot be attributed to the evaluated HNMs. This is the first study evaluating the cell transformation effects of TCNM and BNM under a long-term exposure scenario using suitable hallmarks of the cancer process.

Comparative toxicity of chloro- and bromo-nitromethanes in mice based on a metabolomic method.

Halonitromethanes (HNMs) as one typical class of nitrogenous disinfection byproducts have been widely found in drinking water. In vitro test found HNMs could induce higher cytotoxicity and genotoxicity than trihalomethanes and haloacetic acids. However, data on toxic effect from in vivo experiment is limited. In this study, bromonitromethane (BNM), bromochloronitromethane (BCNM) and trichloronitromethane (TCNM) were chosen as target HNMs, and exposed to mice for 30 d. Hepatic toxicity and serum metabolic profiles were determined to reveal toxic effects and mechanisms of the three HNMs. Results showed the three HNMs significantly decreased relative liver weight, indicating liver is one of the target organs. Further, the three HNMs exposure damaged hepatic antioxidant defense system, and increased oxidative DNA damage. Nuclear magnetic resonance based metabolomics analysis found amino acid metabolism and carbohydrate metabolism were disturbed by HNMs exposure. Some metabolites in these metabolisms are related to oxidative stress and damage. Combined with above results, BNM had the highest toxicity, followed by BCNM and TCNM, indicating bromo-HNMs had higher toxicity than chloro-HNMs. Induction of oxidative stress is one of the toxicity mechanisms of HNMs. This study firstly provides the insight into in vivo toxicity of HNMs and their underlying mechanisms based on metabolomics methods, which is very useful for their health risk assessment in drinking water.

A fugacity-based toxicokinetic model for narcotic organic chemicals in fish.

A novel dynamic fugacity-based model is described, developed, and tested that simulates the uptake of narcotic organic chemicals in fish from water as occurs in aquatic bioconcentration and toxicity tests. The physiologically based toxicokinetic model treats the time course of chemical distribution in 4 compartments (tissue groups) in the fish, including the liver, in which biotransformation may occur. In addition to calculating bioconcentration and toxicokinetics, 5 possible toxic endpoints are defined corresponding to chemical concentration, fugacity, or activity reaching a critical value that causes 50% mortality. The mathematical description of multicompartment uptake is simplified by expressing the equations in the fugacity format. The model is parameterized and tested against reported empirical data for the bioconcentration of pentachloroethane in rainbow trout and for uptake and mortality from aquatic exposures to naphthalene and 1,2,4-trichlorobenzene in fathead minnows. Model performance is evaluated, and it is concluded that with suitable parameterization it has potential for application for assessment of both bioconcentration and toxicity expressed as median lethal concentrations, critical body residues, and chemical activity as a function of time to death.

Citizen scientist lepidopterists exposed to potential carcinogens.

Lepidopterists use substantial volumes of solvents, such as chloroform, 1,1,2,2-tetrachloroethane and xylene, in their traps when collecting faunistic and phenological data. A majority of them are citizen scientists and thus in part not identified by occupational healthcare as being at risk due to solvent handling. We surveyed the extent of solvent use, the frequency and extent of potential exposure and the safety precautions taken in trapping and catch handling by Finnish lepidopterists. Chloroform and 1,1,2,2-tetrachloroethane were the most frequently used anaesthetics. Potential for exposure prevailed during trap maintenance and exploration and catch sorting. Adequate protection against vapours or spills was worn by 17% during trap exploration. Subjects completed a median of 100 trap explorations per season. Dermal or mucosal spills were recorded at a median rate of one spill per ten (chloroform) to 20 (1,1,2,2-tetrachloroethane and xylene) trap explorations. Median annual cumulative durations of 8 and 20 h of exposure to chloroform and 1,1,2,2-tetrachloroethane at levels above odour detection threshold were reported. Subjective adverse findings possibly related solvents had been noticed by 24 (9.8%) lepidopterists. All the events had been mild to moderate. No factor predicting unsafe procedures or adverse reactions was recorded despite thorough statistical testing.

Enantioselectivity in estrogenicity of the organochlorine insecticide acetofenate in human trophoblast and MCF-7 cells.

Previous studies have showed that some chiral pesticides with estrogenic activity possess enantioselectivity in endocrine disruption. Despite the assessment of enantioselectivity in the estrogenic potential of chiral pesticides, which deserve particular attention, there has been limited research into their molecular mechanisms of human health risk. In this study, the role of enantioselectivity in the endocrine disruption and potential human maternal-fetal health risk of acetofenate (AF), an organochlorine insecticide, were investigated in both MCF-7 and JEG-3 cells. The two in vitro assays showing a clear enantioselectivity in the estrogenic activity with S-(+)-AF showed stronger effects than R-(-)-AF and rac-AF. Moreover, the racemate's estrogenicity was in between that of enantiomers. Our results also demonstrated that S-(+)-AF possesses the strongest potential effects in disruption of hormone secretion, maternal immune tolerance, and steroidogenesis in the trophoblast. The results suggest that assessment of the health risk of chiral contaminants should consider the role of enantioselectivity.

Isolation and characterization of an Alcaligenes sp. strain DG-5 capable of degrading DDTs under aerobic conditions.

In this study, an Alcaligenes sp. strain DG-5 that can effectively degrade dichlorodiphenyltrichloro-ethanes (DDTs) under aerobic conditions was isolated from DDTs-contaminated sediment. Various factors that affect the biodegradation of DDTs by DG-5 were investigated. About 88 %, 65 % and 45 % of the total DDTs were consumed within 120 h when their initial concentrations were 0.5, 5 and 15 mg L⁻¹, respectively. However, almost no degradation was observed when their concentration was increased to 30 mg L⁻¹, but the addition of nutrients significantly improved the degradation, and 66 % and 90 % of the total DDTs were degraded at 336 h in the presence of 5 g L⁻¹ peptone and yeast extract, respectively. Moreover, the addition of 20 mM formate also enhanced the ability of DG-5 to transform DDTs, and its DDT transformation capacity (T(c)) value was increased by 1.8 - 2.7 fold for the pure (p,p'-DDT or o,p'-DDT only) and mixed systems (p,p'-DDT, o,p'-DDT, p,p'-DDD and p,p'-DDE). Furthermore, it was found that competitive inhibition in the biodegradation by DDT compounds occurred in the mixed system.

Hematological abnormalities of acute exposure to hexachloroethane smoke inhalation.

There has been no human epidemiological data regarding potential hematological effects of hexachloroethane-zinc oxide (HC/ZnO) inhalation. This is the first epidemiological study to investigate whether HC/ZnO inhalation exposure can induce hematological abnormalities in exposed soldiers. Twenty soldiers, who were exposed to a high concentration of HC/ZnO smoke for 3-10 min in a narrow tunnel (0.6 m in width) during military training, were recruited as the exposed group (n = 20). Another 64 soldiers, who were not visiting the explosion areas, were recruited as controls. Venous blood was collected from all participants for analyses of hematological parameters. After adjustment for potential confounders, there were significant differences in weekly mean hemoglobin (HB), red blood cell count (RBC), and hematocrit (HCT) between exposed soldiers and controls (p < .01) at the first follow-up through the first 4 weeks following exposure. In addition, mean white blood cell count (WBC) of exposed soldiers was significantly higher than that of controls at the 4-week follow-up. Further analysis showed HB, RBC, and HCT changes during the 1st week after exposure and reach lowest levels during the 2nd week. HB, RBC, HCT, and WBC returned to normal within 11 weeks after the HC/ZnO smoke exposure. All other hematological parameters remained unchanged at 72 weeks after the event. The authors concluded that inhalation of HC/ZnO smoke can induce acute, temporally related hematological abnormalities.

Enantioselective induction of oxidative stress by acetofenate in rat PC12 cells.

In a non-chiral environment, the enantiomers of a racemate possessed the identical physico-chemical properties, but in the biological systems they possessed different activities. Considering that the involvement of oxidative damage has been implicated in the toxicities of various pesticides, this study investigated the possibility of enantioselective oxidative stress and cytotoxicity induction by acetofenate (AF) which contains an asymmetrical center on PC12 cells. The results of the cytotoxicity assay indicated that S-(+)-AF presented more toxic effects than R-(-)-AF and (+)-AF. It also demonstrated that S-(+)-AF possessed the strongest effects in induction of reactive oxygen species (ROS) production, decrease in superoxide dismutase (SOD) and catalase (CAT) activities, and increase in malondialdehyde (MDA) level. These results suggested that AF and its enantiomers could induce enantioselective cytotoxicity in PC12 cells mediated by oxidative stress. Therefore, the assessment in environmental safety and new chiral pesticide development should consider enantioselectivity.

Genotoxicity analysis of two halonitromethanes, a novel group of disinfection by-products (DBPs), in human cells treated in vitro.

Halonitromethanes (HNMs) constitute an emerging class of disinfection by-products (DBPs) produced when chlorine and/or ozone are used for water treatment. The HNMs are structurally similar to halomethanes, but have a nitro-group in place of hydrogen bonded to the central carbon atom. Since little information exists on the genotoxic potential of HNMs, a study has been carried out with two HNM compounds, namely trichloronitromethane (TCNM) and bromonitromethane (BNM) by using human cells. Primary damage induction has been measured with the Comet assay, which is used to determine both the repair kinetics of the induced damage and the proportion of induced oxidative damage. In addition, the fixed DNA damage has been evaluated by using the micronucleus (MN) assay. The results obtained indicate that both compounds are genotoxic, inducing high levels of DNA breaks in the Comet assay, and that this DNA damage repairs well over time. In addition, oxidized bases constitute a high proportion of DNA-induced damage (50-75%). Contrarily, no positive effects were observed in the frequency of micronucleus, which measures both clastogenic and aneugenic effects, neither using TK6 cells nor peripheral blood lymphocytes. This lack of fixed genetic damage would minimize the potential mutagenic risk associated with HNMs exposure.

Genotoxic evaluation of two halonitromethane disinfection by-products in the Drosophila wing-spot test.

Few studies on the genotoxicity of halonitromethanes (HNMs) have been done. This limited information on their potential genotoxic risk gives special relevance to the collection of new data on their potential genotoxic activity. In the present study we have analyzed the genotoxicity of two HNMs namely bromonitromethane (BNM) and trichloronitromethane (TCNM) in the in vivo wing somatic mutation and recombination test in Drosophila, also known as the wing-spot assay. This test is based on the principle that loss of heterozygosis and the corresponding expression of the suitable recessive markers, multiple wing hairs (mwh) and flare-3 (flr(3)), can lead to the formation of mutant clones in larval cells, which are then expressed as spots on the wings of adult flies. BNM and TCNM were supplied to third instar larvae (72+/-4 h-old) at concentrations ranging from 0.1 to 2 mM. The results showed that none of the three categories of mutant spots recorded (small, large, and twin) increased significantly by the treatments, independently of the dose supplied, indicating that the selected HNMs exhibit a lack of genotoxic activity in the wing-spot assay of Drosophila melanogaster. These results contribute to increase the genotoxicity database on the HNMs.

Modeling the effects of microbial competition and hydrodynamics on the dissolution and detoxification of dense nonaqueous phase liquid contaminants.

The real significance and engineering potential for bioenhanced dissolution of chlorinated ethene dense nonaqueous phase liquid (DNAPL) contaminants are currently not well understood, in part because they can be influenced by a complex set of factors, including microbial competition for growth substrates. Mathematical simulations were performed to evaluate the effects of competition between Dehalococcoides ethenogenes and Desulfuromonas michiganensis for the electron acceptor tetrachloroethene (PCE) on the distribution of dehalorespirers, PCE dissolution, and the extent of PCE detoxification. The modeling results demonstrate that the outcome of competition between populations for growth substrates can have a significant impact on bioenhancement and, thus, on DNAPL source zone longevity and identify the key factors in determining the outcome of competition and its effects on DNAPL dissolution. The potential for bioenhancement is greatest at lower groundwater velocities. At higher velocities, kinetic properties play a key role in determining which population dominates and where, and the amount of bioenhancement that is realized. Engineered bioremediation techniques that maintain multiple dehalorespiring populations may offer the best approach for optimizing the twin cleanup goals of reduced source zone longevity and complete detoxification while maximizing the utilization of added electron donors.

Induction of macrophage apoptosis by an organochlorine insecticide acetofenate.

Acetofenate (AF) is a widely used insecticide in China and other regions of southeastern Asia. A previous study showed that AF caused adverse developmental effects in zebrafish. Macrophages, which play a key role in inflammation, host defense, and reactions against a spectrum of autologous and foreign invaders, are crucial for innate immunity. However, cytotoxicity and apoptosis of macrophages caused by organochlorine pesticides (OCPs) have so far received little attention. In this study, we used AF as a model chemical to investigate the cytotoxic effects of OCPs on mouse macrophage cell line RAW264.7. Results from cell viability and apoptosis assays showed that AF induced apparent apoptosis in RAW 264.7 cells. Furthermore, AF induced intracellular reactive oxygen species (ROS) generation and DNA damage and resulted in the alteration of a series of signaling molecules including up-regulation of p53 and cytochrome c protein levels, decline of the Bcl-2/Bax protein ratio, and activation of the caspases cascade through caspase-9 and caspase-3. These results, for the first time, revealed that the increase of endogenous ROS and DNA damage comediating OCP-induced apoptosis in macrophages may be by the mitochondria and p53 signal pathway. Our results suggested that macrophages are involved in AF-induced adverse immune effects. Considering the ubiquitous environmental presence of OCPs, this study provided new information on the potential long-term physiological and immunological effects due to chronic exposures to OCPs.

Enantioselectivity in the immunotoxicity of the insecticide acetofenate in an in vitro model.

Chiral pesticides with an asymmetrical center in their molecular structures possess enantioselectivity, not only in their pesticidal activities toward targeted organisms but also in toxicities to nontargeted organisms. Despite the fact that chiral pesticides deserve particular attention because of their ubiquitous presence in living and working environments, there has been limited research into their enantioselectivity in chronic toxicity. The immunotoxicity of chiral pesticides with respect to enantioselectivity has not been studied before. In this study, the role of enantioselectivity in the immunotoxicity of acetofenate (AF), an organochlorine insecticide, was investigated in an in vitro macrophage cell line model. Results of the cytotoxicity assay showed a clear dose-dependent growth inhibition effect of AF with enantioselectivity on RAW264.7 cells. S-(+)-AF was clearly more toxic to macrophages than R-(-)-AF and rac-AF. This work also demonstrated that S-(+)-AF possesses the strongest effects in induction of intracellular reactive oxygen species, DNA damage, and upregulation of p53 gene expression. These results, for the first time, show stark selectivity between enantiomers in their ability to induce macrophage-involved immunotoxicity of AF. These results suggest that assessment of the environmental safety and health risk of chiral contaminants should consider the role of enantioselectivity in immunotoxicity. In addition, our study will improve the knowledge of the role of enantioselectivity in immunotoxicity of chiral contaminants.

Hepatic injuries of hexachloroethane smoke inhalation: the first analytical epidemiological study.

UNLABELLED: There has been no human epidemiological data regarding the hepatic injuries of hexachloroethane-zinc oxide (HC/ZnO) inhalation. This is the first epidemiological study to investigate whether HC/ZnO inhalation exposure can induce hepatic dysfunction in exposed soldiers. Twenty soldiers, exposed to high concentration of HC/ZnO smoke for 3-10 min in a narrow tunnel (0.6 m in width) during military training, were recruited as exposed group and they were divided into high-exposed group (n=10) and low-exposed group (n=10) by the distance from the explosion locale as a surrogate of exposure condition. Another 64 soldiers, not visiting the explosion areas, were recruited as referents. Venous blood was collected for liver function analyses. After log transformation of alanine aminotransferase (ALT) and adjustment for potential confounders, serum ALT in high-exposed soldiers was statistically significantly higher than those of referents for the 3 weeks following exposure. The serum ALT in low exposed soldiers was statistically significantly higher than those of referents at the 3rd week following exposure. The mean ALT levels also showed decreasing gradients by the distance from exposure locale. In addition, the proportions of abnormality on ALT (>40U/L) were also significantly different among three exposure conditions. Follow-up study showed that the hepatic dysfunction started from 1 to 2 weeks and peaked from 3rd to 5th week after exposure. ALT level was then returned to normal within 6-8 weeks after removing from HC/ZnO smoke exposure. No sequelas in hepatic dysfunction were found until 72 weeks follow-up. CONCLUSION: We concluded that inhalation of HC/Zn smoke can induce acute, dose-dependent and definite temporal relationship hepatic dysfunction.

Enantioselectivity in zebrafish embryo toxicity of the insecticide acetofenate.

Enantioselectivity in separation and toxicology of chiral xenobiotics have become one of the frontier topics interfacing chemistry and toxicology. In this study, enantiomers of insecticide acetofenate (AF) were separated on selected chiral columns by HPLC, and enantioselectivity in developmental toxicity was evaluated using the zebrafish embryo-larval assays. The AF enantiomers were baseline separated on Chiralcel OD, Chiralpak AD, and Sumichiral OA-2500I columns under optimized conditions. Pure enantiomers were obtained on Chiralcel OD. Optical rotatory dispersion (ORD) and circular dichroism (CD) detectors were used to determine the elution order and CD spectra of the enantiomers. The absolute configuration of enantiomers was identified as S-(+)-AF and R-(-)-AF by the octant rule from force-field calculations and CD spectra. The individual enantiomers were used in 4-day zebrafish embryo-larval bioassays, and a series of developmental end points were measured and compared. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate the expressions of estrogen receptor alpha (ERalpha) in zebrafish embryo exposed to varying enantiomers. While the enantiomers showed no difference in acute toxicity, significant enantioselectivity was observed in developmental toxicities such as yolk sac edema and pericardial edema. The data of qRT-PCR showed that there was about 3.2-fold induction in the mRNA levels of ERalpha between fish exposed to (+)-AF and (-)-AF. The results suggest that enantioselectivity may occur at the developmental level even in the absence of selective acute toxicity and should be considered when evaluating ecotoxicological effects of chiral contaminants.

In vivo and in vitro evaluation of the acute toxicity, the genotoxicity, and the irritation potency of two hexachloroethane-based pyrotechnic smokes.

The two hexachloroethane (HC)-based smoke formulations studied consisted of HC/Zn/2,4,6-trinitrotoluene (TNT) and HC/Zn. In the in vitro tests, human bronchial epithelial cells were exposed to the smokes at various concentrations. The responses studied were acute toxicity (viability of cells, trypan blue exclusion method) and genotoxicity (DNA single-strand breaks, COMET assay). The tests were conducted in a laboratory-scale chamber (V = 150 L) and in a container (V = 55 m3). Both smoke formulations appeared to be acutely toxic and genotoxic. For the 0.5- and 1-g burning experiments the responses were more pronounced with HC/Zn/TNT than with HC/Zn smoke. To study the irritation potency of the smokes, the mouse bioassay according to ASTM E 981-84 was applied. The respiratory parameters measured were tidal volume (VT), airflow during expiration at 0.5 VT (VD), time of pause after expiration (TP), time of breaking after inspiration (TB), and the respiratory frequency (BPM; breaths per minute). In the single-exposure experiments, HC/Zn/TNT smoke induced concentration-dependent sensory irritation in mice and the occupational exposure limit (TLV) was estimated to be 4 mg/m3. In the repeated-exposure experiments, HC/Zn/TNT smoke induced sensory irritation at the beginning of the exposure. Pulmonary irritation tended to dominate when the exposures were repeated. With HC/Zn smoke we were unable to generate sufficient high exposure concentrations. In the repeated-exposure experiments, indications of sensory and pulmonary irritation were seen at concentrations used. No evidence of apoptotic cell death was found in caspase-3-like protease activity assay.