Physical and physicochemical factors effecting transport of chlorohydrocarbon gases from lung alveolar air to blood as measured by the causation of narcosis.

This systematic investigation examines gas transport in the lung for two sets of chlorohydrocarbons (CHCs): the chloromethanes (C1) and chloroethanes (C2). The C1 series includes chloromethane, methylene chloride, chloroform, and carbon tetrachloride, and the C2 series includes chloroethane, 1,2-dichloroethane, 1, 1, 2-trichloroethane, and 1, 1, 2, 2-tetrachloroethane. Most CHC gases cause narcosis. The comprehensive narcosis work of Lehmann and colleagues on CHCs was used as a basis for the narcosis endpoint in the present examination. The sites for narcosis are located in the brain (midline cortex and posterior parietal area), the spine, and at many peripheral nerve sites. Central nervous system (CNS) exposure executes a multisite, neural transmission set of inhibitions that promotes rapid loss of consciousness, sensory feeling, and current and stored memory while providing temporary amnesia. Absorption into the system requires dissolution into many lipid membranes and binding to lipoproteins. Lipophilicity is a CHC property shared with many anesthetics according to the Meyer-Overton Rule. Many structurally different lipid chemicals produce the narcosis response when the lipid concentration exceeds -67 mM. This suggests narcotic or anesthetic dissolution into CNS membranes until the lipid organization is disrupted or perturbed. This perturbation includes loading of Na(+)- and K(+)-channel transmembrane lipoprotein complexes and disrupting their respective channel functional organizations. The channel functions become attenuated or abrogated until the CHC exposure ceases and CHC loading reverses. This investigation demonstrates how the CHC physical and chemical properties influence the absorption of these CHCs via the lung and the alveolar system on route to the blood. Narcosis in test animals was used here as an objective biological endpoint to study the effects of the physical factors Bp, Vp, Kd (oil: gas) partition, Henry's constant (HK), and water solubility (S%) on gas transport. Narcosis is immediate after gas exposure and requires no chemical activation only absorption into the blood and circulation to CNS narcotic sites. The three physical factors Bp, K(d) (oil: air), and S% vary directly with unitary narcosis (UN) whereas Vp and HK vary inversely with UN in linear log-log relationships for the C2 series but not for the C1 series. Physicochemical properties of C1 series gases indicate why they depart from what is usually assumed to be an Ideal Gas. An essential discriminating process in the distal lung is the limiting alveolar film layer (AFL) and the membrane layer of the alveolar acini. The AFL step influences gas uptake by physically limiting the absorption process. Interaction with and dissolution into aqueous solvent of the AFL is required for transport and narcotic activity. Narcotics or anesthetics must engage the aqueous AFL with sufficient strength to allow transport and absorption for downstream CNS binding. CHCs that do not engage well with the AFL are not narcotic. Lipophilicity and amphipathicity are also essential solvency properties driving narcotics' transport through the alveolar layer, delivery to the blood fats and lipoproteins, and into critical CNS lipids, lipoproteins, and receptor sites that actuate narcosis. AFL disruption is thought to be strongly related to a number of serious pulmonary diseases such acute respiratory distress syndrome, infant respiratory distress syndrome, emphysema, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pneumonia, pulmonary infections, and idiopathic pulmonary fibrosis. The physical factors (Bp, Vp, Kd [oil: gas] partition, Henry's constant, and water solubility [S%]) combine to affect a specific transport through the AFL if lung C > C(0) (threshold concentration for narcosis). The degree of blood CHC absorption depends on dose, lipophilicity, and lung residence time. AFL passage can be manipulated by physical factors of increased pressure (kPa) or increased gas exposure (moles). Molecular lipophilicity facilitates narcosis but lipophilicity alone does not explain narcosis. Vapor pressure is also required for narcosis. Narcotic activity apparently requires stereospecific processing in the AFL and/or down-stream inhibition at stereospecific lipoproteins at CNS inhibitory sites. It is proposed that CHCs likely cannot proceed through the AFL without perturbation or disruption of the integrity of the AFL at the alveoli. CHC physicochemical properties are not expected to allow their transport through the AFL as physiological CO(2) and O(2) naturally do in respiration. This work considers CHC inspiration and systemic absorption into the blood with special emphasis on the CHC potential perturbation effects on the lipid, protein liquid layer supra to the alveolar membrane (AFL). A heuristic gas transport model for the CHCs is presented as guidance for this examination. The gas transport model can be used to study absorption for other gas delivery endpoints of environmental concern such as carcinogens.

An improved system for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay.

A gas exposure system using rotating vessels was improved for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay. This system was composed of 12 square culture vessels, a device for preparation of air containing test gas, and positive and negative control gases at target concentrations and for supplying these gases to the culture vessels, and a roller apparatus in an incubator. Chinese hamster lung cells (CHL/IU) were grown on one side of the inner surface of the square culture vessel in the MEM medium. Immediately prior to exposure, the medium was changed to the modified MEM. Air in the culture vessel was replaced with air containing test gas, positive or negative control gas. Then, the culture vessels were rotated at 1.0 rpm. The monolayered culture cells were exposed to test gas during about 3/4 rotation at upper positions and alternatively immersed into the culture medium during about 1/4 rotation at lower positions. This system allowed the chromosomal aberration assay simultaneously at least at three different concentrations of a test gas together with positive and negative control gases with and without metabolic activations, and duplicate culture at each exposure concentration. Seven gaseous compounds, 1,3-butadiene, chlorodifluoromethane, ethyl chloride, methyl bromide, methyl chloride, propyne, and vinyl chloride, none of which has been tested to date, were tested on CHL/IU for the chromosomal aberration assay using this gas exposure system. All the compounds except chlorodifluoromethane showed positive responses of the structural chromosomal aberrations, whereas polyploidy was not induced by any of these gases. This improved gas exposure system proved to be useful for detecting chromosomal aberrations of gaseous compounds.

Analysis of chloroethane toxicity and carcinogenicity including a comparison with bromoethane.

Chloroethane (CE) gas carcinogenicity is analyzed and determined from a National Toxicology Program (NTP) bioassay where an inhalation concentration of 15,000 ppm CE gas in air produced the highest incidence of an uncommon-to-rare tumor ever observed by the NTP. Persistently inhaled CE produces endometrial cancers in female mice. The first-tumor-corrected uterine endometrial incidence (I) in B6C3F1 mice is 90%, but no significant tumors occurred in F344 rats. The endometrial cancers dispersed by 1) migrating locally to the adjacent myometrium, 2) then migrating to the bloodstream by intravasation, 3) entering 17 distal organs by extravasation and adapting to the new tissue environment. Distal cancers retained sufficient endometrial cell features to be recognized at each metastatic site. CE produced one of the highest metastasis rates ever observed by NTP of 79%. Comparing CE with bromoethane (BE), a structural analogue, it was found that BE too produced rare murine endometrial cancers yielding the second highest NTP incidence rate of I = 58% with a similar high malignancy rate of 56%. Because of the historical rarity of endometrial tumors in the B6C3F1 mouse, both of these SAR haloethanes seem to be evoking a strong, related carcinogenic potential in B6C3F1 mice, but not in F344 rats. The question of whether humans are similar to mice or to rats is addressed here and in Gargas, et al., 2008. The powerful carcinogenesis caused by these halohydrocarbons may have been caused by excessive and metabolically unresolved acetaldehyde (AC) which is directly generated by Cyp2E1 in the oxidative elimination of CE. With >95% AC metabolic production, as predicted from pharmacokinetic (PK) studies depending on CE exposure, AC is the main elimination intermediate. AC is a known animal carcinogen and a strongly suspected human carcinogen. Also, CE causes incipient decreases of tissue essential glutathione pools [GSH] by Phase II conjugation metabolic elimination of CE (and BE), by glutantione transferase (GST), in most organs (except brain) exposed to high circulating CE and it metabolites. In three laboratories, an excessive stress reaction of hyperkinesis was observed only during 15,000 ppm gas exposure but not when the exposure ceased or when exposure was presented at 150 ppm. Test rodents other than the female mice did not exhibit a pattern of visible stress nor did they have a carcinogenic response to CE gas. Unremitting stress has been documented to contribute a feedback to the hypothalamus which stimulates the hypothalamic-pituitary-axis (HPA), which in turn, induces the adrenal glands. Because estrus and estrogen and progesterone levels were unaltered by CE gas, the adrenal over stimulation, causing high steroid output, may be the penultimate step in this extraordinary carcinogenic response. High adrenal production of corticosteroids could adversely promote endometrial cells to cancers in mice - a mechanism that has already been observed in humans.

Cytogenetic effects of 1,1-dichloroethane in mice bone marrow cells.

The major concern for the halogenated compounds is their widespread distribution, in addition to occupational exposures. Several chlorinated alkanes and alkenes were found to induce toxic effects. In this study, we investigated the genotoxic potential of 1,1-dichloroethane in the bone marrow cells obtained from Swiss-Webster mice, using chromosomal aberrations (CA), mitotic index (MI), and micronuclei (MN) formation as toxicological endpoints. Five groups of three male mice each, weighing an average of 24 +/- 2 g, were injected intraperitoneally, once with doses of 100, 200, 300, 400, 500 mg/kg body weight (BW) of 1,1-dichloroethane dissolved in ethanol. A control group was also made of three animals injected with ethanol (1%) without the chemical. All animals were sacrificed 24 hours after the treatment. Chromosome and micronuclei preparations were obtained from bone marrow cells following standard protocols. Chromatid and chromosome aberrations were investigated in 100 metaphase cells per animal and percent micronuclei frequencies were investigated in 1,000 metaphase cells per animal. 1,1-dichloroethane exposures significantly increased the number of chromosomal aberrations and the frequency of micronucleated cells in the bone marrow cells of Swiss-Webster mice. Percent chromosomal aberrations of 2.67 +/- 0.577, 7.66 +/- 2.89, 8.33 +/- 2.08, 14.67 +/- 2.51, 20.3 +/- 3.21, 28 +/- 3.61; mitotic index of 9.4%, 7.9%, 6.2%, 4.3%, 3.0%, 2.6% and micronuclei frequencies of 3.33 +/- 0.7, 7.33 +/- 0.9, 8.00 +/- 1.0, 11.67 +/- 1.2, 15.33 +/- 0.7, 18.00 +/- 1.7 were recorded for the control, 100, 200, 300, 400, and 500 mg/kg BW respectively; indicating a gradual increase in number of chromosomal aberrations and micronuclei formation, with increasing dose of 1,1,-dichloroethane. Our results indicate that 1,1-dichloroethane has a genotoxic potential as measured by the bone marrow CA and MN tests in Swiss-Webster mice.

Bromoethane, chloroethane and ethylene oxide induced uterine neoplasms in B6C3F1 mice from 2-year NTP inhalation bioassays: pathology and incidence data revisited.

Chloroethane, bromoethane and ethylene oxide represent a unique set of chemicals that induce endometrial neoplasms in the uterus of B6C3F1 mice following an inhalation route of exposure. The results of the NTP's chronic bioassays with these three compounds resulted in an unusually high incidence of uterine epithelial neoplasms in B6C3F1 mice (chloroethane 86%, bromoethane 56%) and a lower incidence for ethylene oxide (10%). The uterine neoplasms were classified as adenomas, adenocarcinomas, and squamous cell carcinomas for bromoethane, and as adenocarcinomas for both chloroethane and ethylene oxide. The adenocarcinomas and squamous cell carcinomas were invasive into the myometrium and the serosa, and metastasized to a wide variety of organs. Metastatic sites included most commonly the lung, lymph nodes, and ovary at unusually high rates of metastases (79% for chloroethane and 38% for bromoethane). Because of the dramatically high rates of uterine neoplasms (induced by chemicals given by the inhalation route) and metastases, a re-evaluation of the pathology and incidence data was undertaken. The earlier results were confirmed. The mechanism of uterine carcinogenesis by chloroethane, bromoethane and ethylene oxide is unclear.

Acute, subacute, and subchronic oral toxicity studies of 1,1-dichloroethane in rats: application to risk evaluation.

1,1-Dichloroethane (DCE) is a solvent that is often found as a contaminant of drinking water and a pollutant at hazardous waste sites. Information on its short- and long-term toxicity is so limited that the U.S. EPA and ATSDR have not established oral reference doses or minimal risk levels for the volatile organic chemical (VOC). The acute oral LD(50) in male Sprague-Dawley (S-D) rats was estimated in the present study to be 8.2 g/kg of body weight (bw). Deaths appeared to be due to CNS depression and respiratory failure. In an acute/subacute experiment, male S-D rats were given 0, 1, 2, 4, or 8 g DCE/kg in corn oil by gavage for 1, 5, or 10 consecutive days. The animals were housed in metabolism cages for collection of urine and sacrificed for blood and tissue sampling 24 h after their last dose. There were decreases in body weight gain and relative liver weight at all dosage levels, as well as increased renal nonprotein sulfhydryl levels at 2 and 4 g/kg after 5 and 10 days. Elevated serum enzyme levels, histopathological changes, and abnormal urinalyses were not manifest. For the subchronic study, adult male S-D rats were gavaged with 0.5, 1, 2, or 4 g DCE/kg 5 times weekly for up to 13 weeks. Animals receiving 4 g/kg exhibited pronounced CNS depression, with more than one-half dying by week 11. The 2-g/kg rats exhibited moderate CNS depression. One 2-g/kg rat died during week 6. There were very few manifestations of organ damage in animals that succumbed or in survivors at any dosage level. Decreases in bw gain and transient increases in enzymuria were noted at 2 and 4 g/kg. Serum enzyme levels and blood urea nitrogen were not elevated, nor were glycosuria or proteinuria present. Chemically induced histological changes were not seen in the liver, kidney, lung, brain, adrenal, spleen, stomach, epididymis, or testis. Hepatic microsomal cytochrome P450 experiments revealed that single, high oral doses of DCE did not alter total P450 levels, but did induce CYP2E1 levels and activity and inhibit CYP1A1 activity. These effects were reversible and regressed with repeated DCE exposure. There was no apparent progression of organ damage during the 13-week subchronic study, nor appearance of adverse effects not seen in the short-term exposures. One g/kg orally (po) was found to be the acute, subacute, and subchronic LOAEL for DCE, under the conditions of this investigation. In each instance, 0.5 g/kg was the NOAEL.

[Combined effects caused by chronic exposure to vinyl chloride and dichloroethane]. / Kombinirovannoe deistvie vinilkhlorida i dikhlorétana pri dlinel'nov postuplenii v ornanizm.

The authors studied solitary and combined effects of vinyl chloride and dichloroethane in chronic (4 months) inhalation experiment using concentrations of 50 and 500 mg/m3. The experiment revealed general toxic effects including disorders of central nervous system and liver. Those disorders are caused by reductive-oxidative reactions disturbances and dystrophic processes. The authors determined safe combinations of vinyl chloride and dichloroethane concentrations for associated action.

Examination of low-incidence brain tumor responses in F344 rats following chemical exposures in National Toxicology Program carcinogenicity studies.

Neoplasms in the brain are uncommon in control Fischer 344 (F344) rats; they occur at a rate of less than 1% in 2-yr toxicity/carcinogenicity studies. Furthermore, only 10 of nearly 500 studies conducted by the National Toxicology Program (NTP) showed any evidence of chemically related neoplastic effects in the brain. Generally, the brain tumor responses were considered equivocal, because the characteristics of potential neurocarcinogenic agents (such as statistically significant increased incidences, decreased latency and/or survival, and demonstration of dose-response relationships) were not observed. A thorough examination, including comparisons with a well-established historical database, is often critical in evaluating rare brain tumors. Chemicals that gave equivocal evidence of brain tumor responses were generally associated with carcinogenicity at other sites, and many chemicals were mutagenic when incubated with metabolic activating enzymes. Other factors that were supportive of the theory that marginal increases in brain tumor incidence were related to chemical exposure were that (a) some of the tumors were malignant, (b) no brain neoplasms were observed in concurrent controls from some studies, and/or (c) brain tumors were also seen following exposure to structurally related chemicals. In 2-yr studies in F344 rats (studies conducted by the NTP), equivocal evidence of carcinogenicity was observed for the following 9 chemicals: isoprene, bromoethane, chloroethane, 3,3'-dimethylbenzidine dihydrochloride, 3,3'-dimethoxybenzidine dihydrochloride, furosemide, C.I. direct blue 15, diphenhydramine hydrochloride, and 1-H-benzotriazole. Glycidol was the only chemical evaluated by the NTP with which there was clear evidence of brain tumor induction in F344 rats. Clarification of the potential neurocarcinogenic risks of chemicals that produce equivocal evidence of a brain tumor response in conventional 2-yr rodent studies may be aided by the use of transgenic mouse models that exhibit genetic alterations that reflect those present in human brain tumors as well as by the use of in utero exposures.

Early changes in sex hormones are not evident in mice exposed to the uterine carcinogens chloroethane or bromoethane.

Chloroethane and bromoethane have been shown to cause a marked uterine tumor response in B6C3F1 mice exposed for 2 years. These chemicals are nearly unique in this regard among the nearly 400 chemicals studied by the National Toxicology Program, and the reasons for this carcinogenic activity are unclear. The possible relationship of changes in blood concentrations of sex hormones to this response was evaluated by examining the estrous cycle of mice prior to and during a 21-day exposure to concentrations of the haloethanes which resulted in the tumorigenic response in the 2-year studies. Serum concentrations of estradiol and progesterone were determined at the termination of the exposures and compared to exposure group and stage of the estrous cycle. No consistent patterns of change were found in estrous cyclicity or in blood concentrations of sex hormones. Thus, the findings suggest that early changes in circulating sex hormones are not important contributing factors in the uterine neoplasia caused by these chemicals.

Genotoxicity studies with chloroethane.

In a recent 2-year inhalation study with F344 rats and B6C3F1 mice conducted as part of the U.S. National Toxicology Program (NTP, 1989), chloroethane (ECl) at an exposure concentration of 15,000 ppm induced a high incidence of endometrial uterine carcinomas only in female mice but not in rats, leading to the conclusion of "clear evidence of carcinogenicity" for the mouse. In order to elucidate whether a genotoxic effect may be a critical factor for the carcinogenicity of ECl in the mouse, we have performed three genotoxicity tests: (1) in vitro HPRT test with CHO cells according to a specially developed gas protocol, (2) in vivo/in vitro UDS with female B6C3F1 mice at an exposure concentration of 25,000 ppm (6 h/day, 3 days), (3) in vivo micronucleus assay with male and female B6C3F1 mice exposed to 25,000 ppm ECl according to the same schedule. In the in vitro HPRT test a mutagenic potential of ECl was detected in the presence as well as in the absence of S9 mix. In contrast, both in vivo test systems failed to detect any indications of genotoxicity of chloroethane at an exposure concentration even higher than that of the NTP study. It is suggested that in vivo the genotoxic potential of ECl is so low that an assumed genotoxic damage is below the detection limit of the test systems used. This leads to the conclusion that genotoxicity may not be a key factor in the induction of the uterine carcinomas in the B6C3F1 mouse.

Ethyl chloride: 11-day continuous exposure inhalation toxicity study in B6C3F1 mice.

Groups of seven B6C3F1 mice per sex were exposed for 23 hr/day to 0, 250, 1250, or 5000 ppm ethyl chloride (EtCl) for 11 consecutive days to evaluate the potential toxicity of EtCl under near-continuous exposure conditions. On the day following the last exposure, a neurobehavioral observation battery was performed, samples were obtained for clinical chemistry and hematology, and necropsies were conducted. Histopathologic examination was subsequently performed. The only observed effects were increased relative liver weights and a slight increase in hepatocellular vacuolation (glycogen or fat) in 5000 ppm-exposed mice. Exposures to EtCl were well tolerated despite the unusually long exposure periods.

Ethyl chloride: a two-week inhalation toxicity study and effects on liver non-protein sulfhydryl concentrations.

Male and female Fischer 344 rats (6/sex/exposure concentration) and male beagle dogs (2/exposure concentration) exposed to 0, 1600, 4000 or 10 000 ppm ethyl chloride (EtCl) for 6 hr/da, 5 da/wk for 2 weeks showed no toxicologically significant treatment-related effects on body weights; clinical chemistry, hematology, or urinalysis parameters; neurology (dogs only were examined); gross pathology or histopathology. The only treatment-related differences in organ or relative organ weights (in rats or dogs) were slight, but statistically significant increases in liver to body weight ratios of male rats exposed to 4000 or 10,000 ppm EtCl (4.9 and 7.5% respectively). Liver non-protein sulfhydryl (NPSH) concentration was measured in male Fischer rats and male B6C3F1 mice that were exposed for 6 hours to 0, 1600, 4000 or 10,000 ppm EtCl (mice were exposed to 0 or 4000 ppm EtCl only). Liver NPSH, measured 1/2 hr post exposure, was less than control values in 4000 ppm exposed rats (88% of control value), 4000 ppm exposed mice (64%), and 10,000 ppm exposed rats (89%). The slight decreases in rat liver NPSH seem consistent with the increased liver to body weight ratios. The toxicity data indicate that 2-week repeated exposures to EtCl concentrations that were up to 10 times the current A.C.G.I.H. T.L.V. (1000 ppm) caused minimal treatment-related effects in dogs and rats.