Thomas J. Goehl

Establishing aerosol exposure concentrations for inhalation toxicity studies

Criteria for the selection of aerosol concentrations to be used in inhalation studies assessing the toxicity and carcinogenicity of chemical substances were discussed by the authors in a meeting sponsored by the National Toxicology Program. Concepts in the design of aerosol inhalation studies emerged from that meeting and are being communicated through this publication. Inhalation studies assessing the toxicity and carcinogenicity of aerosols have often used maximum exposure levels on the basis of technological feasibility. Evidence has now accumulated that the amount of pulmonary burden of deposited particles impacts on particle clearance above some as yet not well-defined exposure concentration. The sequelae are such that lung clearance decreases with increased particulate burden to the point of approaching complete cessation. This paper focuses on the major determinants in establishing maximal aerosol concentrations for use in inhalation toxicity studies with special emphasis on experimental design features to assess lung retention. The subject matter of this paper is a rapidly developing area in terms of knowledge. Accordingly, the contents of this article are intended as guidelines and not as absolute rules for the conduct and interpretation of inhalation exposure studies.

Effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate in rats and mice

Effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate were studied in male F344 rats and B6C3F1 mice. Benzyl acetate was rapidly hydrolysed to benzyl alcohol and then oxidized to benzoic acid. After gavage administration of benzyl acetate in corn oil at 500 mg/kg (rats) and 1000 mg/kg (mice), high benzoic acid plasma concentrations were observed. In contrast, much lower benzoic acid plasma concentrations were found after dosed feed administration at about 615 mg/kg/day for rats and about 850 mg/kg/day for mice. Results show that although the daily doses of benzyl acetate are comparable, bolus gavage administration effectively saturated the benzoic acid elimination pathway whereas dosed feed administration did not. In contrast, hippuric acid plasma concentrations were similar after both gavage and dosed feed administration due to the depletion of the glycine supply pool. Study results could explain the different toxicity and carcinogenicity responses of benzyl acetate observed in 2-yr chronic gavage and dosed feed studies.

Toxicology Studies of a Chemical Mixture of 25 Groundwater Contaminants I. Chemistry Development

As part of an effort to evaluate the toxicology of a chemically defined mixture of 25 frequently detected groundwater contaminants, we report here the formulation and analytical chemistry of this mixture. Many problems were anticipated, including limitation of solubility, chemical interactions, and extreme volatility in the aqueous solution of 25 chemicals. The final technically achievable stock solution was prepared based on EPA survey concentrations of these chemicals in groundwater around hazardous waste disposal sites, their toxicity information, and solubility of the individual compounds in the matrix of the aqueous solution of these 25 chemicals. Because the anticipated animal studies were to be conducted at various laboratories, for ease of handling and maximum stability, the stock solution was stored or shipped as two substock solutions: an organic substock with 18 neat organic chemicals in a glass vial sealed with minimum headspace and an aqueous substock solution with 6 metals of various salt forms and phenol. The concentrations of the solutions were such that direct mixing of the organic and aqueous substocks produced the desired high dose level for the animal experiments. Analyses of all 25 chemicals in the drinking water mixture required six different chromatographic and spectroscopic methods. Some loss of organic volatiles during mixing of the substocks and during the first 24 hr following preparation did occur. However, the concentrations of acetone, phenol, and all the metals remained constant during preparation. Solutions held under simulated animal cage conditions for 96 hr showed losses of the organic volatiles; the majority of which occurred within the first 24 hr. This study shows that it is possible to conduct animal experiments on an aqueous mixture containing 25 groundwater contaminants. Furthermore, a reasonable estimate of intake of individual chemicals can be achieved provided that dosing solutions are prepared fresh at frequent intervals (e.g., 48 to 72 hr) and that comprehensive analyses are carried out.

Application of Microencapsulation for Toxicology Studies II. Toxicity of Microencapsulated Trichloroethylene in Fischer 344 Rats

Gelatin-sorbitol microcapsules containing 44.1% trichloroethylene (TCE) were prepared and mixed in NIH-07 rodent meal diet and provided at microcapsule concentrations of 0 (untreated control group), 1.25, 2.5, 5.0, or 10% (equivalent to 0, 0.55, 1.10, 2.21, or 4.41% TCE, respectively) to groups of 10 male F344 rats for 14 days. An additional control group received diets containing 5% empty capsules. For comparisons, TCE dissolved in corn oil was administered by gavage to different groups of 10 male rats for 14 consecutive days at dose levels adjusted to correspond to those in the feed study. Treatment-related deaths occurred only in the highest dose group of the gavage study. Body weight gain and feed consumption were reduced in high-dose groups of both the feed and gavage studies. There was no measurable loss of TCE in feed sampled from the cages during the study. Dose-related increases in organ (liver and kidney) weight/body weight ratios, individual cell necrosis in the liver, and hepatic microsomal NADPH cytochrome c reductase and peroxisomal palmitoyl-CoA oxidase and catalase activities were found in both the dosed-fed and gavage groups. Induction of cytochrome P-450 occurred only in the dosed-feed study. There were no significant compound-related pathologic lesions observed in the kidneys, the only other organ examined microscopically. Differences in lethality, cytochrome P-450 levels, and induction of microsomal or peroxisomal enzyme activities were attributed to differences in the method of dosing (gavage versus dosed-feed). The demonstration of no significant loss of TCE from the feed and of similar toxic effects produced by microencapsulated TCE via feed and TCE in corn oil via gavage indicate that microencapsulation can provide an excellent alternative exposure route for studying the oral toxicological properties of volatile chemicals, such as TCE, in rats.

Two-Year and Lifetime Toxicity and Carcinogenicity Studies of Ozone in B6C3F1 Mice

To evaluate the toxicity and carcinogenic potential of long-term exposure to ozone, B6C3F1 mice were exposed by whole-body inhalation to 0, 0.12, 0.5, or 1.0 ppm and 0, 0.5, or 1.0 ppm ozone for 24 or 30 mo (lifetime), respectively. The incidence of alveolar/ bronchiolar adenomas and carcinomas (combined) increased (p < 0.05) in female mice exposed to 1.0 ppm for 24 or 30 mo and marginally increased (p > 0.05) in male mice exposed to concentrations of 0.5 or 1.0 ppm. An increased incidence of nonneoplastic lesions were observed in the nasal cavities and in the centriacinar region of the lung of mice exposed to 0.5 or 1.0 ppm for 24 and 30 mo. Nasal cavity lesions were mild and included hyaline degeneration, hyperplasia, squamous metaplasia, fibrosis and suppurative inflammation of the transitional and respiratory epithelium of the lateral wall, and atrophy of the olfactory epithelium. Lung lesions included replacement of the epithelium of the alveolar ducts and adjacent alveolar septa with epithelium similar to that normally found in terminal bronchioles (metaplasia) and associated alveolar histiocytosis. Based on the results of these studies, we conclude that inhalation exposure of B6C3F1 mice to ozone for 24 or 30 mo (a) is carcinogenic in female B6C3F1 mice exposed to 1.0 ppm of ozone based on an increased incidence of alveolar/bronchiolar adenoma or carcinoma and (b) results in mild, site-specific, nonneoplastic lesions in the nasal cavity and centriacinar lung of male and female mice exposed to 0.5 or 1.0 ppm of ozone for 2 yrs, which persist with continued exposure to 30 mo. It is uncertain whether or not the marginal increase (p > 0.05) of alveolar/bronchiolar neoplasms in male B6C3F1 mice resulted from exposure to ozone.

Toxicology and Carcinogenesis Studies of Ozone and Ozone 4-(N-Nitrosomethylamino)-1-(3-Pyridyl)-1-Butanone in Fischer-344/N Rats

The purpose of this study was to evaluate the toxicity and potential carcinogenicity or cocarcinogenicity of ozone exposure in rats. Fischer-344/N (F-344/N) rats were exposed 6 hr/day, 5 days/wk, to 0, 0.12, 0.5, or 1.0 ppm ozone by inhalation for 2-yr and lifetime exposures. The cocarcinogenicity study included subcutaneous administration of 0, 0.1, or 1.0 mg/kg body weight of 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and inhalation of 0 or 0.5 ppm ozone to male rats. NNK was administered by subcutaneous injections 3 times per week for the first 20 wk with ozone inhalation exposure. The ozone inhalation exposure was for 2 yr (104 wk), including the first 20 wk of NNK treatment and continuing for 84 wk after the last NNK injection. Ozone exposure caused a concentration-related increase in inflammation of the centriacinar region of the lung. There was also increased fibrosis and an extension of the bronchiolar epithelium in these centriacinar regions to involve the proximal alveoli. There was no increased incidence of neoplasms at any site, including the lung, that was associated with ozone exposure. Rats administered 1.0 mg/kg body weight NNK alone had an increased incidence of bronchiolar/alveolar neoplasms, but this effect was not enhanced by ozone exposure. Ozone exposure for 2 yr and lifetime was associated with site-specific toxic alterations in the nasal passage and lung similar to those previously described for short-term exposures. While there was significant attenuation of the pulmonary lesions as compared to short-term exposures, lesions persisted in the lifetime study and there was evidence of a mild progressive fibrosis. We conclude that under the conditions of these studies: (a) ozone exposure is not carcinogenic to either male or female F-344/N rats, (b) ozone does not enhance the incidence of pulmonary neoplasms in F-344/N rats exposed to a known pulmonary carcinogen (NNK), and (c) mild site-specific toxic lesions characteristic of ozone exposure persist in the nasal passage and lung throughout the lifetime of the rat with continued ozone exposure.

Glycine Modulates the Toxicity of Benzyl Acetate in F344 Rats

The influence of supplemental glycine on benzyl acetate (BA; a compound metabolized via the hippurate pathway)-induced toxicity was investigated. Groups of male F344 rats were fed NIH-07 diet containing 0, 20,000, 35,000, or 50,000 ppm BA for up to 28 days. Two additional groups were fed NIH-07 diet with 50,000 ppm BA and 27,000 ppm glycine or 50,000 ppm BA and 32,000 ppm L-alanine; supplemental glycine and L-alanine were equimolar. The L-alanine group served as an amino nitrogen control. A third group was fed NIH-07 diet with 32,000 ppm L-alanine and served as an untreated isonitrogenous control. BA caused increase in mortality, body weight loss, the incidence of abnormal neurobehavioral signs such as ataxia and convulsions, along with astrocyte hypertrophy and neuronal necrosis in the cerebellum, hippocampus, and pyriform cortex of the brain. These effects were reduced significantly by supplementation with glycine but not with L-alanine. These results suggest that the neurodegeneration induced by BA is mediated by a depletion of the glycine pool and the subsequent excitotoxicity.

Comparison of the toxicity of citral in F344 rats and B6C3F1 mice when administered by microencapsulation in feed or by corn-oil gavage

A study of the potential effects of microencapsulation on the toxicity of citral was conducted in 14-day continuous feeding studies with both sexes of F344 rats and B6C3F1 mice. Toxicity by the feeding route was compared with that from bolus doses of the neat chemical in corn oil administrated by gavage. Both sexes of rats and mice were given diet containing 0, 0.63, 1.25, 2.5, 5 and 10% citral microcapsules. These feed formulations were equivalent to daily doses of 0, 142, 285, 570, 1140 and 2280 mg citral/kg body weight for rats and 0, 534, 1068, 2137, 4275 and 8550 mg citral/kg body weight for mice. The daily gavage doses were 0, 570, 1140 and 2280 mg citral/kg body weight for both sexes of rats, and 0, 534, 1068 and 2137 mg citral/kg body weight for both sexes of mice. Citral microcapsules administered in the diet did not cause mortality in mice or rats. Toxicity was confined to decreases in body weight at the 10% concentration in mice, at the 5 and 10% concentrations in rats, and decreases in absolute weights of the liver, kidney and spleen at the 10% concentration in rats. The only histopathological change observed was minimal to mild hyperplasia and/or squamous metaplasia of the respiratory epithelium in the anterior portion of the nasal passages of rats fed 5 or 10% citral microcapsules. By contrast, citral gavage caused mortality in five out of five male and female mice at 2137 mg/kg body weight, and in two out of five male mice at 1068 mg/kg body weight. There were dose-related increases in absolute liver weights of male and female mice. Cytoplasmic vacuolization of hepatocytes occurred in all female mice gavaged with 1068 and 2137 mg citral/kg body weight, and in male mice from the 2137 mg/kg dose group. Necrosis, ulceration and/or acute inflammation of the forestomach occurred in the high-dose mice of both sexes. Inflammation and/or hyperplasia of the forestomach occurred in about half of the male and female mice dosed with 1068 mg citral/kg. Citral gavage at doses that were equivalent to up to 10% in the diet (2280 mg/kg body weight) did not cause toxicity in rats, except for minimal hyperplasia of the squamous epithelium of the forestomach in high-dose males.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of 1,1‐dichloro‐2,2‐BIS[p‐chlorophenyl]ethylene (DDE) on lactation in rats

An inverse correlation between the concentration of DDE in human breast milk samples and the duration of breast feeding prompted the present study of the effects of DDE administration on the lactational performance of primiparous rats. Daily doses of 10 mg p,p-DDE/kg body weight were given to virgin female Sprague-Dawley rats 5 d/wk for 5 wk prior to mating and continued throughout the gestation and lactation periods. Lactation capacity was determined by monitoring neonatal growth and by measuring milk production, milk composition (total protein, total lipid, and lactose), and mammary-gland weight and nucleic acid content on d 9 and 20 postpartum. Gross toxicity was assessed by monitoring clinical signs and body weight of the dams, and by measuring organ weights of the dams on lactation d 9 and 20. Histopathological evaluation of the mammary glands and selected organs in the dams and pups was also performed. The dose level of DDE employed was apparently not toxic to the dams and did not have a pronounced effect on neonatal mortality. No significant differences between DDE-treated and control groups were observed for any of the lactation parameters, even though the concentration of DDE in the milk of treated rats was approximately two orders of magnitude greater than the upper range of the DDE levels measured in human milk samples. These findings indicate that DDE does not adversely affect lactation or neonatal growth in Sprague-Dawley rats at the dose level used in this study.

Toxicokinetics of pentachlorophenol in the F344 rat. Gavage and dosed feed studies

1. The toxicokinetics of pentachlorophenol (PCP) were studied in the Fischer 344 rat using i.v. and oral (gavage, dosed feed) routes of exposure. 2. Only minor sex differences were observed in the elimination kinetics of PCP after i.v. administration at 5 mg/kg. 3. Absorption of PCP from the gastrointestinal tract after gavage doses of 9.5 and 38 mg/kg in aqueous methylcellulose vehicles was first order with an absorption half-life of about 1.3 h. 4. The absorption rate constant of PCP from doses feed was comparable with that obtained from aqueous methylcellulose gavage formulations. 5. Bioavailability of PCP administered in dosed feed was significantly lower than the bioavailability of PCP administered by gavage. 6. Dose proportionality was established to a dosage of at least 38 mg/kg. 7. Daily fluctuation of PCP plasma concentrations was observed during the dosed feed study with peak and trough concentrations occurring in early morning and late afternoon, respectively. 8. The time course of PCP plasma concentrations during the dosed feed study were simulated using a computer model based on linear theory. The simulations were comparable with the experimentally determined concentrations.