Evaluation of neutralized chemical agent identification sets (CAIS) for skin injury with an overview of the vesicant potential of agent degradation products.

Vesication and skin irritation studies were conducted in hairless guinea-pigs to determine the vesicant and skin irritation potential of chemically-neutralized Chemical Agent Identification Sets (CAIS). The CAIS are training items that contain chemical warfare-related material--sulfur mustard (HD), nitrogen mustard (HN) or lewisite (L)--and were declared obsolete in 1971. Animals were dosed topically with 'test article'--neat HD, 10% agent/chloroform solutions or product solutions (waste-streams) from neutralized CAIS--and evaluated for skin-damaging effects (gross and microscopic). Product solutions from the chemical neutralization of neat sulfur mustard resulted in microvesicle formation. All agent-dosed (HD or agent/chloroform solutions) sites manifested microblisters as well as other histopathological lesions of the skin. Waste-streams from the neutralization of agent (agent/chloroform or agent/charcoal) were devoid of vesicant activity. Cutaneous effects (erythema and edema) were consistent with the skin-injurious activity associated with the neutralizing reagent 1,3-dichloro-5,5-dimethylhydantoin (DCDMH). Chemical neutralization of CAIS was effective in eliminating/reducing the vesicant property of CAIS containing agent in chloroform or agent on charcoal but was inefficient in reducing the vesicant potential of CAIS containing neat sulfur mustard.

Neuromuscular effects of low level exposures to Sarin, pyridostigmine, DEET, and chlorpyrifos.

A study is being initiated to investigate subtle neurobehavioral effects and neuropathology in rats due to exposure to combinations of low levels of Sarin (GB), N,N-diethyl-m-toluamide (DEET), chlorpyrifos (CPF), and pyridostigmine bromide (PB). A similar study is being initiated in rhesus monkeys to investigate neurophysiologic effects and neuromuscular pathology due to exposure to a combination of GB, DEET, CPF, and PB, along with vaccination with botulinum toxoid. A description of these studies is presented.

Efficacy of tacrine as a nerve agent pretreatment.

Tacrine (THA) was evaluated in vitro and in vivo as a pretreatment for nerve agent intoxication. In vitro experiments showed that the primary effect of THA was direct inhibition of purified fetal bovine serum acetylcholinesterase (AChE) with a slight effect on slowing the aging rate of nerve agent-inhibited AChE. THA produced significant behavioral effects at doses above 1.7 mg/kg, i.m., in the mouse and 3.4 mg/kg, i.m., in the guinea pig. At the no observable effect level (NOEL) for mice (1.7 mg/kg), THA was effective (P < or = 0.05) in reducing tabun- and soman-, but not sarin-induced lethality in mice. Experiments in the guinea pig showed that at the NOEL (3.4 mg/kg, i.m.) THA was not effective in decreasing lethality due to soman exposure. Since there was significant overlap between pharmacologically effective doses of THA and those which produce behavioral toxicity, THA was not considered a suitable pretreatment for nerve agent intoxication.

Evaluation of compounds as barriers to dermal penetration of organophosphates using acetylcholinesterase inhibition.

An efficient, objective method for evaluating the efficacy of barrier compounds in preventing dermal penetration of organophosphates (OP) in rabbits was developed using time-dependent reduction in erythrocyte (RBC) acetylcholinesterase (AChE) activity as an endpoint. Anesthetized rabbits, with or without a dermal application of a mixture of high- and low-molecular-weight polyethylene glycols (mean molecular weight of 540 daltons; PEG 540), were exposed to different percutaneous doses of 3 highly toxic OP compounds. Dose-response curves were generated for RBC AChE inhibition as a function of percutaneous dose for each OP test material over time. From data generated, a single dose of each OP was selected to challenge PEG-540-protected and unprotected animals to validate the method as a means of differentiating effective from ineffective barriers to skin penetration. Data for a complete evaluation of a PEG 540 test barrier application were obtained within 4 h and anesthesia was maintained for the entire period.

Stagewise, adaptive dose allocation for quantal response dose-response studies.

A principal design objective of many dose-response studies is to estimate extreme percentiles of a dose-response distribution, e.g., the ED95 dose for a particular drug therapy, as precisely as feasible using the smallest number of experimental subjects possible. Such a design requirement necessitates that allocation of subjects to drug doses be carried out in a stagewise fashion to maximize the information obtained from each subsequent experimental observation in light of what has previously been determined. This paper describes and illustrates specialized methods and associated computer programs to evaluate, on a stagewise basis, the anticipated relative sensitivities of alternative experimental plans in the case of dichotomous responses. Following each stage of experimentation, the current estimates of the dose-response distribution parameters, as well as the uncertainties in these estimates, are updated and are used to assign subjects to experimental dose levels for the next stage of testing. Competing dose allocations are compared with respect to anticipated improvement in estimation precision. The adoption of such a stagewise dose allocation strategy is illustrated by example.

Stagewise, group sequential experimental designs for quantal responses. one-sample and two-sample comparisons.

The use of stagewise, group sequential experimental designs with dichotomous responses in toxicity or drug screening programs is discussed. Such designs represent a compromise between the standard, fixed sample size designs and fully sequential designs. Stagewise group sequential designs place specified numbers of animals on test at each stage, up to a maximum number of stages. The greatest increases in sample size efficiency occur with small numbers of stages, particularly when going from one stage to two. Two-stage designs can result in a 15 to 20 percent reduction in average sample size. Five-stage designs can result in a 30 to 40 percent reduction in average sample size, with no appreciable decrease in Type 1 error or power. Examples of the efficiencies that arose in actual screening programs are given. This paper demonstrates that the routine use of stagewise, group sequential designs in standardized screening protocols can result in substantial savings in animal use with virtually no sacrifice of statistical sensitivity.

Metabolism and nephrotoxicity of indan in male Fischer 344 rats.

Indan, a component of fuels, solvents, and varnishes, is metabolized in male Fischer 344 rats to 1-indanol, 2-indanol, 5-indanol, 1-indanone, 2-indanone, 2-hydroxy-1-indanone, cis-1,2-indandiol, and trans-1,2-indandiol. The metabolites were identified using the techniques of gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The rats treated with indan demonstrated the classic lesions of hydrocarbon-induced nephropathy. The kidney damage produced was less than that found for tetralin and other branched-chain acyclic hydrocarbons.

Metabolism and nephrotoxicity of tetralin in male Fischer 344 rats.

Tetralin, a component of fuels, solvents, and varnishes, is metabolized in male Fischer 344 rats to 1-tetralol, 2-tetralol, 2-hydroxyl-1-tetralone, 4-hydroxyl-1-tetralone, 1,2-tetralindiol, and 1,4-tetralindiol. Rats treated with tetralin demonstrated the classic lesions of hydrocarbon-induced nephropathy.

Identification of urinary metabolites in rats exposed to the nephrotoxic agent 2,3,4-trimethylpentane.

Branched-chain hydrocarbon-induced nephropathy in male rats was examined using 2,3,4-trimethylpentane. Lesions are elected only in male rats, not in females or in controls. Mechanisms of nephropathy may be the interaction of metabolites with the male rat-specific protein alpha 2u globulin. The identified urinary metabolites of 2,3,4-trimethylpentane in male rats given the hydrocarbon by gavage are 2,3,4-trimethyl-1-pentanol, 2,3,4-trimethyl-2-pentanol and 2,3,4-trimethyl-1-pentanoic acid. Of the C8-isomers, 2,3,4-trimethylpentane dosing leads to the highest incidence of kidney damage in male rats.

Gas chromatographic/mass spectrometric studies of the urinary metabolites of male rats given indan.

The identified urinary metabolites of male rats exposed to indan are: 1- and 2-indanone; 1-, 2- and 5-indanol; 2- and 3-hydroxyl-1-indanone; and cis- and trans-indan-1,2-diol. Indan causes kidney damage in male rats in a manner similar to the cyclic hydrocarbons cis- and trans-decalin and JP-10. Lesions produced by indan occur only in male rats and not in female or control rats.

The metabolism of 2,3,4-trimethylpentane in male Fischer-344 rats.

The urinary metabolites of the potent nephrotoxic hydrocarbon 2,3,4-trimethylpentane (2,3,4-TMP) given Fischer-344 male rats by gavage included 1-hydroxy-2,3,4-trimethylpentane, 2,3,4-trimethyl-1-pentanoic acid and 2,3,4-trimethyl-5-hydroxy-1-pentanoic acid. Analyses were performed by gas-liquid chromatography (GC) and gas-liquid chromatography/mass spectrometry (GC/MS). A comparison of the urinary metabolites of 2,3,4-TMP with those of 2,2,4-trimethylpentane (2,2,4-TMP) showed that more monocarboxylic acid was produced with 2,3,4-TMP.

Femoral venipuncture for collection of multiple blood samples in the nonanesthetized rat.

A technique for blood collection via the femoral vein was developed for use in nonanesthetized rats. The technique was useful for single or serial blood collection. Volumes of 0.1 to 1 ml were collected for serum biochemical or venous blood gas determinations. The technique was effective, reproducible, did not require anesthesia, and was less stressful than other methods of blood collection in the rat.

The metabolism of n-octane in Fischer 344 rats.

The urinary metabolites of n-octane in Fischer 344 rats given the hydrocarbon by gavage included 2-octanol, 3-octanol, 5-oxohexanoic acid, and 6-oxoheptanoic acid. The sex of the animals influenced the relative amounts of metabolites formed. Analyses were performed by gas-liquid chromatography (GC) and gas-liquid chromatography/mass spectrometry (GC/MS). This is the first reported finding of keto acids in hydrocarbon oxidative metabolism. No kidney damage was found as a result of n-octane dosing although the 2,2,4-trimethylpentane (iso-octane) isomer does cause kidney lesions in male rats.

Metabolism of nephrotoxic cis- and trans-decalin in Fischer-344 rats.

The cis- and trans-decalin stereoisomers have found many uses as solvents and fuel components. The metabolism of the decalin isomers in male and female Fischer-344 rats and the effects of the decalins on renal damage were evaluated. Only male rats had kidney damage. Metabolism of cis-decalin yielded cis,trans-1-decalol and cis,cis-2-decalol in the urine of both sexes, with the male also producing cis,cis-1-decalol. The urinary metabolites of trans-decalin included trans,cis-2-decalol in both male and female rats and trans,trans-1-decalol in males. Extracts of kidney homogenates from male rats, but not from females, dosed with cis- and trans-decalin yielded cis-2-decalone and trans-2-decalone, respectively. A single male rat treated with trans-decalin produced no 2-decalone in the kidney extract and also showed no renal damage.

Identification of urinary metabolites of the nephrotoxic hydrocarbon 2,2,4-trimethylpentane in male rats.

The compound 2,2,4-trimethylpentane (2,2,4 TMP) is reported to be especially potent in inducing kidney lesions in male rats (1,2). Although the pathology produced by 2,2,4 TMP has been examined (1), there are no reports concerning the metabolism of 2,2,4 TMP by the male rat. The eight principal urinary metabolites of 2,2,4 TMP found in the urine of Fischer 344 male rats are: 2,2,4-trimethyl-1-pentanol, 2,4,4-trimethyl-1-pentanol, 2,4,4-trimethyl-2-pentanol, 2,2,4-trimethyl-1-pentanoic acid, 2,4,4-trimethyl-1-pentanoic acid, 2,4,4-trimethyl-2-hydroxy-1-pentanoic acid, 2,2,4-trimethyl-5-hydroxy-1-pentanoic acid and 2,4,4-trimethyl-5-hydroxy-1-pentanoic acid.

Metabolite identification of missile fuel JP-10 by gas chromatography mass spectrometry.

The identification of the metabolites of the Air Force missile fuel JP-10 was accomplished. 5-Hydroxy-JP-10 was identified as the urinary metabolite of rats, mice and hamsters exposed to JP-10. 5-Keto-JP-10 was a major metabolite found in kidney extracts of male rats exposed to JP-10 but not found in kidney extracts from female rats or from other species. Sex-related differences in the formation of 5-keto-JP-10 in rats were studied.

Suspected tremetol poisoning in horses.

Of 10 horses in a heavily overgrazed pasture, 4 died within 1 week. Clinical signs included muscle tremors, ataxia, reluctance to walk, heavy sweating, and myoglobinuria. Serum creatine kinase, aspartate transaminase, and alkaline phosphatase activities were high. Histopathologic findings were nonspecific. On the basis of clinical signs, clinicopathologic findings, nonspecific histopathologic findings, the condition of the pasture, the identification of numerous white snakeroot plants from which trematone was extracted, and evidence that these plants had been heavily browsed, it was believed that the horses died from ingestion of Eupatorium rugosum.

Teratogenic assessment of three methylated hydrazine derivatives in the rat.

The embryotoxicity and teratogenicity of methylhydrazine, 1,1-dimethylhydrazine, and 1,2-dimethylhydrazine were investigated with pregnant Fischer-344 rats. The compounds were administered ip on d 6-15 of pregnancy. A dose-dependent reduction in maternal weight gains occurred for all three compounds. A dose-related teratogenic effect did not occur for any of the three compounds. Embryotoxicity, manifested as reduced 20-d fetal weights, occurred only in the 1,1-dimethylhydrazine and 1,2-dimethylhydrazine high-dose treatment groups. The results indicate that none of the three methylated hydrazine derivatives are selectively embryotoxic or teratogenic in the rat.

The acute toxicity of perfluorooctanoic and perfluorodecanoic acids in male rats and effects on tissue fatty acids.

The acute toxicities of single ip injections of perfluorooctanoic (PFOA) and perfluorodecanoic (NDFDA) acids were evaluated in male Fischer rats. The LD50/30 day for PFOA was 189 (208-175) mg/kg and for NDFDA was 41 (47-34) mg/kg. All rats treated with lethal doses of PFOA died within the first 5 days; with NDFDA there was delayed lethality, with deaths in the second and third weeks after dosing. Four groups of rats were used for a more detailed study of toxicity and for analysis of fatty acids from liver, testes, and whole blood. One group received a single dose of 100 mg PFOA/kg; a second, a single dose of 2 ml of propylene glycol-water (1:1)/kg (vehicle control); a third, a single dose of 50 mg NDFDA/kg; the fourth was given 2 ml vehicle/kg and pair-fed with the NDFDA group. The first three groups were fed ad libitum. Rats from each group were killed at 2, 4, 8, and 16 days after dosing for fatty acid analysis. Rats dosed with NDFDA lost half their body weight in 16 days and ate virtually no food from Day 7 to Day 14 after dosing. Weight loss was less rapid in pair-fed controls. With PFOA there were transient decreases in food intake and body weight which were reversed by Day 7. Liver weights of PFOA rats were slightly greater than those from vehicle controls. With NDFDA, liver weights were much greater than those from pair-fed controls. In the livers of PFOA rats there were transient increases in oleic and palmitic acids and a decrease in stearic and docosahexaenoic acids. These changes were maximum by Day 2 and nearly resolved by Day 8. With NDFDA, similar changes were observed and arachidonic acid was also greatly decreased. These changes were quantitatively much larger and more persistent. NDFDA has unusually high toxic potency for a perfluorinated hydrocarbon, and some of the toxic effects caused by this acid are remarkably similar to those seen with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The acute toxicity of NDFDA may be due to an ability to interfere with fatty acid metabolism, and studies of its toxicity may be valuable in helping to understand mechanisms of action of TCDD.