A chronic inhalation toxicity/oncogenicity study of methylethylketoxime in rats and mice.

To evaluate the oncogenic potential of methylethylketoxime (MEKO), CD-1 mice (50/sex/group) and F-344 rats (50/sex/group) were coexposed 6 h/day, 5 days/wk for 18 mo (mice) or 26 mo (rats) via whole-body inhalation exposures to target vapor concentrations of 0, 15, 75, and 375 ppm (actual concentrations of 0, 15 +/- 1, 75 +/- 2, or 374 +/- 10 ppm). Satellite groups of rats and mice (10/sex/group/interval) were exposed for 12 mo (mice) and 3, 12, or 18 mo (rats) to evaluate chronic toxicity. Methyl ethyl ketone (MEK), a possible hydrolysis product of MEKO, was present at less than 1%. Treatment-related effects included increased body weight (male rats only), methemoglobin formation, hematology and clinical chemistry changes, increased liver weight, and increased spleen and testes weights (rats only). A high incidence of cataracts and corneal dystrophy occurred in both control and MEKO-exposed rats, with an earlier appearance and slightly higher incidence for these ocular lesions in MEKO-exposed animals compared to controls. Degenerative and reparative changes of the olfactory epithelium in the nasal turbinates, primarily limited to the dorsal meatus, occurred in both rats (75 and 374 ppm) and mice (15, 75, and 374 ppm). In addition, in the mice, liver changes included increased incidences of pigment in reticuloendothelial cells, centilobular hypertrophy, granulomatous inflammation, and a slightly increased incidence of necrosis (75 and 374 ppm). An increase in hepatocellular carcinomas occurred in male mice at 374 ppm. Additional MEKO-related findings in the rat included congestion of the spleen with pigment in reticuloendothelial cells and extramedullary hematopoiesis and a decreased incidence of lymphoreticular mononuclear cell leukemia. Effects observed in the liver of the rats included decreases in the incidence of both peribiliary fibrosis and hyperplasia/proliferation of the biliary duct, an increase of spongiosis hepatis in males, and an increase in the incidence of intracytoplasmic vacuoles and hepatocellular basophilic foci. The effects on the liver were generally most profound in the high-exposure groups and, with the exception of the spongiosis hepatis, occurred in both sexes. An increase in hepatocellular adenomas occurred in the male rats at 75 and 374 ppm, and hepatocellular carcinomas in the male rats at 374 ppm. In both species, the liver tumors appeared relatively late in the life of the animals, with no significant increase in tumors at 12 mo of exposure in mice and at 18 mo of exposure in rats. Lifespan shortening was not observed, as MEKO-exposed animals survived generally as well as, or slightly better than, the controls.

Thirteen-week subchronic rat inhalation toxicity study with a recovery phase of trivalent chromium compounds, chromic oxide, and basic chromium sulfate.

The toxicity of trivalent chromium compounds; chromic oxide and basic chromium sulfate, was investigated in rats in a 13-week nose-only inhalation study that included a 13-week recovery period. Nose-only exposures to insoluble chromic oxide dust at 4.4, 15, or 44 mg/m3 or soluble basic chromium sulfate dust at 17, 54, or 168 mg/m3 (trivalent chromium equivalent concentrations of 3, 10, and 30 mg/m3) were carried out for 6 h/day, 5 days/week. No compound-related mortality occurred. General toxic effects, only observed with high-exposure levels of basic chromium sulfate, included sporadic signs of labored breathing and depressed body weights. No apparent compound-related effects were noted for sperm motility or morphology, for any concentration of either test material. Bronchoalveolar lavage fluid evaluations showed test material in mononuclear cells with chromic oxide and increased neutrophils, protein, lactic dehydrogenase and cellular debris with basic chromium sulfate. The principle effects for both materials were primarily to the respiratory tract. Chromic oxide caused pathological changes in the bronchial and mediastinal lymphatic tissue and lungs, consisting of the presence of pigment-laden macrophages, lymphoid and septal hyperplasia, and interstitial inflammation similar to that observed with other inert dusts. Basic chromium sulfate produced more severe and widespread effects in the nasal cavity, larynx, lungs, and mediastinal lymph node. Effects were characterized by accumulation of foreign material, infiltration of alveolar macrophages, septal cell hyperplasia, and granulomatous and chronic inflammation. Pigment was still present in chromic oxide and, to a lesser extent, in basic chromium sulfate-treated animals after the 13-week recovery period, with partial recovery of the pathological lesions. A NOAEL was not established for either test material, but 4.4 mg/m3 was thought to be near the NOAEL level for subchronic exposure to chromic oxide. The results of this study indicate significant differences in toxicity to the respiratory tract between trivalent chromium compounds chromic oxide and basic chromium sulfate. These are likely related to differences in acidity and water solubility, rather than chromium concentration per se. This conclusion is substantiated by the lack of effect on other internal organs.

Reproductive toxicity evaluation of methylethyl ketoxime by gavage in CD rats.

Methylethyl ketoxime (CAS No. 96-29-7; MEKO; 2-butanone oxime), an antioxidant agent used in paints, resins, and adhesives, was tested for reproductive toxicity in a two-generation study with CD (Sprague-Dawley) rats. Thirty-eight-week-old rats/sex/group (F0) were administered MEKO in water, by gavage, at 0, 10, 100, or 200 mg/kg/day (at a dosing volume of 2 ml/kg), 5 days/week for 10 weeks with vaginal cytology evaluation (VCE) of F0 females during the last 3 weeks of the prebreed period. Animals were mated within groups for 3 weeks with dosing during mating, gestation, and lactation for 7 days/week. F0 parents and F1 weanlings, 10/sex/dose, were necropsied (after a 2-week postwean VCE in F0 females) with hematologic evaluation (including methemoglobin) and histology of adult livers, spleens, and reproductive organs. F1 weanlings, 30/sex/dose, were dosed for 11 weeks and mated as described above. Because of poor reproductive performance, not treatment related, F1 animals with no F2a litters were rebred to produce F2b litters. F1 parents and F2a weanlings, 10/sex/dose, were necropsied and evaluated as described above. Inguinal mammary glands were examined histologically from all nonselected F1 and F2 (a and b) female weanlings. Adult toxicity was observed in both generations and both sexes at all doses. Treatment-related parental deaths occurred at 200 mg/kg/day. At 100 and 200 mg/kg/day, parents exhibited dose-related reduced body weights and weight gains, reduced feed consumption, clinical signs of toxicity, and anemia with concomitant extramedullary hematopoiesis and hemosiderosis in livers and spleens (and increased spleen weights). At 10 mg/kg/day, only adult liver and spleen histologic effects were present. There was no evidence of reproductive organ or mammary glad pathology or of reproductive or postnatal toxicity at any dose tested. There was no adult "no observable adverse effect level" (NOAEL) established; the NOAEL for reproductive and postnatal toxicity was at least 200 mg/kg/day for rats in this study.

Topical treatments for hydrofluoric acid dermal burns. Further assessment of efficacy using an experimental piq model.

Several topical treatments for hydrofluoric acid dermal burns (Zephiran, calcium acetate and magnesium hydroxide antacid soaks, and calcium gluconate gel) were assessed for efficacy in a pig model. Gross appearance and histopathology of treated and untreated burn sites were evaluated. For superficial burns, Zephiran was most effective; calcium acetate, magnesium hydroxide antacid, and calcium gluconate gel were less effective. For deep burns, gross observations showed that calcium acetate and Zephiran were most efficacious, whereas histopathology indicated comparable efficacy of Zephiran, calcium acetate, and calcium gluconate gel for all skin layers. Magnesium hydroxide antacid demonstrated efficacy only for the subdermis. The clinically beneficial effects of both Zephiran and calcium gluconate gel were affirmed. Although results suggest that calcium acetate and magnesium-containing antacids may be beneficial for human hydrofluoric acid dermal burns, these are not established clinical treatments.

Assessing the neurotoxic potential of methyl ethyl ketoxime in rats.

The potential of methyl ethyl ketoxime (MEKO) to produce neurotoxicity following acute and subchronic exposure was studied in rats. A Functional Observational Battery, assessment of motor activity, and neuropathology evaluations were conducted in the context of acute and subchronic toxicity studies. Three independent studies are reported: a pilot time-effect study designed to determine the time course and time to peak effect following a single high dose of MEKO, a single-dose neurotoxicity study, and a subchronic (13-week) repeated-dose neurotoxicity study in rats. An acrylamide-positive control group was included in the acute and subchronic studies for comparison with MEKO. Following an acute oral exposure of MEKO at a dose level of 900 mg/kg, locomotor activity was decreased compared to control with maximum decreases occurring between 30 and 60 min following oral administration. In the acute study, transient treatment-related changes in ease of cage removal, ease of handling, and in posture and gait were observed 1 hr after dosing with 900 mg/kg MEKO, as were significant depressions in motor activity. Following a single 300 mg/kg dose, transient MEKO-related changes in gait and aerial righting reflex were noted 1 hr after dosing. All effects were reversible within 24 hr of dosing. The single 100 mg/kg dose of MEKO was without observable effects. No acrylamide-related behavioral effects were noted following a single 50 mg/kg dose. In the subchronic study, transient treatment-related changes in ease of cage removal, ease of handling, and in posture, gait, and aerial righting were observed at the 400 mg/kg/day dose level when assessments were conducted immediately after dose administration. No consistent behavioral effects were observed prior to daily dose administration even after 13 weeks of exposure, indicating a lack of cumulative behavioral effect. No consistent behavioral changes were noted at doses of 125 mg/kg/day and below. Significant dose-related decreases in red cell mass, and increases in methemoglobin levels, reticulocyte, leukocyte, Heinz body counts, and spleen weights occurred at subchronic MEKO doses of 40 mg/kg/day and higher. No MEKO-related neuropathological changes occurred. Animals receiving acrylamide at 20 mg/kg/day showed expected behavioral and neuropathological changes consistent with peripheral neuropathy. In conclusion, high doses of MEKO can produce transient and reversible changes in neurobehavioral function consistent with central nervous system (CNS) depression. No evidence of cumulative neurotoxicity was detected. The hematopoietic system was effected at doses which did not produce detectable changes in CNS function.

Reliability of using fewer rabbits to evaluate dermal irritation potential of industrial chemicals.

To investigate the feasibility of using fewer than six animals for dermal irritation testing, a retrospective evaluation was conducted on data from 224 studies performed within the last 10 years using standard methodology. For each study, an irritation classification was made from primary dermal irritation indices calculated using irritancy data available for five, four, or three rabbits randomly selected from the original six-rabbit pool. The original six-rabbit analyses resulted in classification of 49 non-irritants, 75 negligible irritants, 66 mild irritants, 24 moderate irritants, and 10 severe irritants. Agreement with the six-rabbit classification was obtained for five-, four-, and three-rabbit groups, respectively, as follows: for negligible irritants, 95, 85, and 69%; mild irritants, 85, 80, and 76%; moderate irritants, 88, 88, and 71%; severe irritants, 100, 90, and 90%. Differences in the dermal irritation indices between groups of six and five, four, and three rabbits were small, with an average difference no larger than 0.3. When disagreements in classification occurred, the test material was most often classified less severe. Using 90% or greater agreement as acceptable criterion for reducing the number of test animals, our results suggest that the use of less than six rabbits would not be suitable for evaluating dermal irritation based solely on a qualitative classification. However, the use of as few as three rabbits would be adequate to evaluate dermal irritancy based on quantitative primary dermal irritancy values.

Hydrofluoric acid dermal burns. An assessment of treatment efficacy using an experimental pig model.

There currently exist various opinions concerning the best therapy for managing hydrogen fluoride (HF) dermal burns. Previously reported animal studies designed to evaluate the efficacy of certain therapies are not completely convincing. Studies initially were conducted to develop a reliable animal model for assessing efficacy of treatment. Evaluation of several animal species, dosing regimens (HF concentrations, exposure periods), and application techniques showed that the most consistent and reproducible dermal lesions were produced with 38% HF applied to the skin of anesthetized pigs for exposures of 9, 12, or 15 minutes using Hill Top Chamber patches. Using this model, the efficacy of six clinically applicable treatments was assessed by subjectively scoring and statistically analyzing photographic and histopathological data obtained from treated and untreated control lesions. Photographic data analysis ranked treatments with respect to effectiveness as follows: iced Zephiran and 10% calcium acetate soaks--highly effective; 2.5% calcium gluconate gel, 5.0% calcium gluconate injection and iced Hyamine soaks--effective; 10% calcium gluconate injection--ineffective. Histopathological data analysis showed the topical treatments (2.5% calcium gluconate gel, iced Hyamine, or iced Zephiran soaks) to be most effective in reducing superficial epidermal damage, and the 5% calcium gluconate injection or 10% calcium acetate soaks to be beneficial to deeper tissues of the dermis and subdermis. Injection of 10% calcium gluconate was ineffective. This study suggests that the anesthetized pig model has good applicability for assessing efficacy of HF dermal burn therapies. In addition, it indicates that further experimentation with 10% calcium acetate soaks is warranted.

Gastric mucosal protection with beta-phenylethylamine (PEA) and other sympathomimetic amines against absolute ethanol in the rat.

The purpose of this study was to investigate the potential of beta-phenylethylamine (PEA), an amphetamine-like compound present in the blood during high stress situations, to protect rat gastric mucosa against absolute ethanol. F-344 rats were pretreated with PEA in saline at several dose levels and at various times prior to oral administration of 1 ml absolute ethanol. PEA at dose levels of 50 and 100 mg/kg significantly reduced the severity of alcohol-induced lesions following oral, but not parenteral, treatment. The duration of protection with PEA was approximately 90 min, with maximum protection observed when PEA was administered 15-30 min before alcohol. Pretreatment with indomethacin did not prevent or reduce the protection induced by PEA. Other sympathomimetic amines such as isoproterenol and ephedrin were similarly cytoprotective against absolute ethanol while amphetamine, phenylephrine, and epinephrine proved ineffective. These results add further support to the role of the sympathetic nervous system in regulating gastric mucosal protection in the rat.

Determination of erythrocyte life span in F-344, Wistar, and Sprague-Dawley rats using a modification of the [3H]diisopropylfluorophosphate ([3H]DFP) method.

The use of [3H]diisopropylfluorophosphate [( 3H]-DFP) to measure erythrocyte survival is well documented; however, the procedure has not been widely used in animal research primarily because it is more labor intensive than other methods. The [3H]DFP method was modified to simplify the preparation of samples for scintillation counting by using whole blood and to allow for small-volume serial blood sampling of individual animals. Eight-week-old F-344, Wistar, and Sprague-Dawley rats received 20 microCi [3H]DFP via the jugular vein. The latter two strains served to validate the procedure as significant data exist on the erythrocyte life spans of these rats. The red cell life span of the F-344 rat has not been previously investigated. Blood samples were collected from a tail vein at 8 days post-labeling and approximately three times per week until Day 33. Whole blood samples were individually prepared for scintillation counting. Blood radioactivity, corrected for sampling loss, was expressed as a percentage of the total activity at the time of initial sampling. Data were analyzed using the least-squares method of linear regression. Results, expressed as a mean erythrocyte life span in days (+/- 1 SE), for each strain were 66.3 +/- 1.6 for F-344, 59.8 +/- 2.2 for Wistar, and 61.0 +/- 1.3 for Sprague-Dawley rats. The results for the Wistar and Sprague-Dawley rats were in good agreement with published data for these strains, thus validating the modified [3H]DFP method for erythrocyte life span determination. In addition, the red cell life span of the F-344 rat was shown to be comparable with other rat strains.

Toxicity of hydroxylamine sulfate following dermal exposure: variability with exposure method and species.

The acute toxicity of hydroxylamine sulfate (HS) and phenylhydrazine hydrochloride (PHZ) were compared in the rabbit and rat following a single 24-hr dermal exposure. The test materials were applied topically and occluded under a plastic or gauze cover or were injected sc. Distilled water served as a control. HS and PHZ produced similar hematotoxic effects consisting of methemoglobin formation, anemia, and reticulocytosis. HS and PHZ proved to be more toxic to the rabbit than to the rat although both chemicals produced similar hematological effects at equivalent dose levels within the same species. HS proved strikingly more toxic when administered under plastic than under gauze despite the fact that both methods included occlusion. PHZ toxicity was less variable with exposure method. HS and PHZ were lethal to the rabbit but no deaths occurred in the rat. The results of this study indicate that HS and PHZ show similar hematotoxicity and, therefore, the clinical data available on PHZ may be useful in predicting the hematological effects of HS on humans.

Toxicity of cyclohexanone oxime. II. Acute dermal and subchronic oral studies.

Dermal exposure of rabbits to cyclohexanone oxime (CHO) for 24 hr at 0, 0.8, 2, and 5 g/kg caused dose-related reticulocytosis on the day after dosing as well as a decrease in hemoglobin in the 5-g/kg females 7 days postdosing. Gavage of rats 5 days a week for 13 weeks at levels of 0, 0.25, 2.5, and 25 mg/kg resulted in a dose-related decrease in erythrocyte number, hemoglobin, and hematocrit, with an accompanying increase in circulating reticulocytes and nucleated erythrocytes, in both sexes. Also seen were corneal opacities and an increased incidence of Howell-Jolly bodies. Results suggested an increased erythropoiesis in the spleen and bone marrow. The data from satellite groups terminated at 30 and 60 days revealed no effect at the lower test level, but results from the end of the study showed a clear cumulative dose response down to the 0.25-mg/kg level. Males were affected earlier and at lower doses than females. These results, along with those of a subacute study with a recovery period, suggest that CHO induces an oxidative attack on erythrocytes which appears reversible upon cessation of exposure.

Toxicity of cyclohexanone oxime. I. Hematotoxicity following subacute exposure in rats.

Cyclohexanone oxime (CHO) was given po to male and female Fischer 344 rats at dose levels of 10, 25, 75, 150, and 300 mg/kg, five times a week for a period of 2 weeks. Control animals received distilled water. All animals given intermediate dose levels (10, 25, 75, and 150 mg/kg) and one half of the animals which were dosed at the high dose (300 mg/kg) as well as one half of the controls were terminated 14 days after administration of the first dose. The remaining rats received no treatment for an additional 14 days and were sacrificed on Day 28 of the study (recovery phase). Dose-related decreases in erythrocyte number, hemoglobin, and hematocrit, with an accompanying increase in reticulocytes and circulating nucleated erythrocytes, were observed in both sexes at Day 14. Methemoglobin levels, determined only at the high dose, were elevated in both sexes at this time. Splenomegaly and hepatomegaly were observed in both sexes at 14 and 28 days. Histopathological examination of the spleen and bone marrow revealed dose-related erythroid hyperplasia at 14 days which subsided by Day 28. The above effects were more pronounced in males. Erythrocyte numbers were only slightly depressed and reticulocytes mildly elevated in males at Day 28. Hematological values were not statistically different from controls in females at this time. These results suggest that CHO induces oxidative damage to the erythrocyte, resulting in a hemolytic anemia accompanied by increased erythropoiesis. The toxic effects appear reversible upon cessation of exposure.

Innovative designs and practices for acute systemic toxicity studies.

Over a four year period our laboratory has conducted 124 acute systemic toxicity studies (64 oral, 39 dermal, and 21 inhalation), altering study and program designs with the objectives of maximizing information while minimizing animal usage. By employing dose selection strategies, probes, lethality limits instead of LD50's, staggered sequential dosing, and by conducting studies in batteries, animal usage was reduced by 48% below the average number currently quoted as necessary for an LD50 study. Simultaneously, use of a neurobehavioral screen, adjunct studies and a flexible study design have led to a significant upgrading in the information generated by these studies. Additionally, the use of a decision tree approach for selecting tissues for histopathology was developed. The use of specific indicators (such as organ weights) for selecting organs for microscopic examination was also evaluated. Our efforts demonstrate that significantly more information can be generated by studies utilizing fewer animals than is now common practice.

Gastric antisecretory and cytoprotective activities of SCH 28080.

SCH 28080 (2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile) is a novel antiulcer agent which has both antisecretory and cytoprotective activities. The antisecretory ED50 values in the pylorus-ligated rat were 3.7 mg/kg p.o. and 2.8 mg/kg i.p., being 7 and 10 times more potent than cimetidine, respectively. In dogs, SCH 28080 was effective in inhibiting acid secretion stimulated by histamine (ED50 of 0.09 mg/kg i.v. and 4.4 mg/kg p.o.), dimaprit, pentagastrin, insulin and feeding. The cytoprotective activity of SCH 28080 was demonstrated by inhibition of ethanol-induced gastric lesions in a dose-dependent manner in rats (ED50:3.0 mg/kg p.o.). SCH 28080 was active in similar dose ranges (1-10 mg/kg) by both p.o. and i.v. routes of administration. This gastric cytoprotective activity was not affected by indomethacin pretreatment. Furthermore, the gastric potential difference was effectively sustained by SCH 28080 (3, 10 and 30 mg/kg p.o.) after intragastric ethanol. SCH 28080 (1-30 mg/kg p.o.) also inhibited gastric ulcers provoked by aspirin, aspirin + acid, indomethacin and stress (cold-restraint) in rats. The data support the concept that it is possible to have combined antisecretory and cytoprotectant actions in a single molecule which is not a prostaglandin.

Erythrocyte survival studies in a rat myelogenous leukemia.

To determine the extent intrinsic erythrocyte defects and/or extrinsic factors were involved in anemia of rats bearing Shay chloroleukemia (SCL), survival of 3H-DFP labeled erythrocytes was studied in leukemic and nonleukemic hosts. Red blood cells labeled before induction of leukemia, were rapidly lost from the peripheral circulation of SCL rats in terminal stages of disease. However, labeled erythrocytes from terminal SCL animals displayed normal lifespans when transfused into nonleukemic controls. Thus the anemia of this leukemia probably resulted from extrinsic factors associated with the leukemic process. Hemorrhage appeared to be primarily responsible for the anemia of this disease.

Influence of prednisolone on ethanol-induced gastric injury in the rat.

The purpose of the present study was to investigate the effect of prednisolone on gastric injury induced by ethanol in the rat. Gastric damage was produced by oral administration of 1 ml of absolute ethanol to rats previously fasted for 24 hr and deprived of water for 19 hr. The severity of the ethanol-induced gastric damage varied considerably within the vehicle-treated group of rats which served as controls. Prednisolone, administered orally as a single dose 15 min before alcohol challenge, significantly decreased the number of rats which developed severe lesions. Prednisolone was effective in increasing the resistance of the gastric mucosa to ethanol when given from 1 to 60 min before alcohol. The steroid proved ineffective when 90 min elapsed between prednisolone and ethanol administration, or when the steroid was given at the same time (0 min) as alcohol. The dose-response curve for prednisolone plateaued at high doses. Our results suggest that a prostaglandin-mediated endogenous cytoprotective potential exists in the rat gastric mucosa. Prednisolone may enhance the degree of mucosal protection afforded by this mechanism.

Effect of corticosteroids on indomethacin-induced intestinal ulceration in the rat.

A single dose of indomethacin induces a severe gastrointestinal syndrome in the rat, characterized by intestinal ulceration, perforation, and death. The mechanism by which indomethacin induces this syndrome is unclear, although it has been suggested that a loss of mucosal integrity leads to inflammation and necrosis of the intestinal wall. The purpose of the present investigation was to study the effect of corticosteroids on indomethacin-induced ulceration. Prednisolone administered orally, even as a single dose (10 mg/rat), significantly reduced the severity of ulceration following indomethacin. This protective effect was most pronounced when prednisolone was administered 1 hr postindomethacin and decreased as the period between indomethacin and prednisolone administration increased. Of the steroids studied, the rank order of efficacy in reducing the severity of indomethacin-induced ulceration was paramethasone acetate > betamethasone > prednisolone. Hydrocrotisone was not significantly effective at the doses ultilized. Our results suggest that corticosteroids exert a cytoprotective effect on intestinal mucosa similar to that produced by prostaglandins.