Unexpected brain lesions in lactating Sprague-Dawley rats in a Two-generation Inhalation Reproductive Toxicity Study with pentafluoropropane (HFC-245fa).

The study presented was conducted following the reproductive study guideline OECD Guideline 416 Two-Generation Reproduction Toxicity Study. Sprague-Dawley rats were exposed to 2000, 10,000 and 50,000 ppm of HFC-245fa. There was an unexpected mortality of lactating dams in the medium and high dose group beginning at day 10 of lactation. Statistically significant histopathological alterations were observed in the cerebellum of a total of 9/30 females of the high dose group of the F0-generation and in 10/27 females of the high dose group of the F1-generation. In contrast there were no brain lesions found in males or non-pregnant females of all dose groups. Neuronal necrosis and degeneration in the cerebellar cortex were observed as the most severe finding. Furthermore vacuolation of the neuropil in different degrees was diagnosed in 7/30 females of the F0-generation and in 9/30 females of the F1-generation. Acute hemorrhages - in particular perivascular - occurred in 5/30 females of the F0- and in 5/30 females of the F1-generation indicating a disturbed vascular integrity. The main lesions found in the cerebrum were glial scars in the corpus callosum and restricted to 2/30 females of the F0-generation of the high dose group. The increased incidence of myocardial fibrosis and mononuclear cell infiltration in males - indicating myocarditis - was only seen in the F0-generation of the high dose group. Females of the F1-generation of the high dose group showed an increased incidence of minimal myocardial fibrosis. In summary, histopathology revealed that the brain, particularly the cerebellum, and to a minor degree the heart turned out to be the toxicological target organs of the substance. Presumably substance-related energy deprivation may be responsible for the observed changes. One of the metabolites, 3,3,3-trifluoropropanoic acid has been shown to be capable of causing this effect.

Evaluation of reproductive/developmental and repeated dose (subchronic) toxicity and cytogenetic effects in rats of a roofing asphalt fume condensate by nose-only inhalation.

A Type III Built-up Roofing Asphalt (BURA) fume condensate was evaluated for subchronic systemic toxicity and reproductive/developmental toxicity screening in Wistar rats, by OECD protocol 422 and OECD cytogenetic protocol 474. Animals were exposed by nose-only inhalation to target concentrations of 30, 100 and 300 mg/m³ total hydrocarbons (actual concentrations, 30.0, 100.1 and 297.3 mg/m³). The study was performed to assess potential hazards from asphalt fumes to which humans could be exposed during application. No adverse effects were seen for spermology, reproductive or developmental parameters or early postnatal development of offspring from day 1 to 4 postpartum. BURA fume condensate did not induce any significant increases in micronucleus frequency in polychromatic erythrocytes of rat bone marrow nor was neurobehavioral toxicity observed at any dose. Systemic effects were slight and seen at doses above those measured at work sites. The systemic NOAEC of 100 mg/m³ for males was based on decreased body weight gain, food consumption and increased absolute and relative lung wet weight correlated with slight histological changes in the lung, primarily adaptive in nature at 300 mg/m³. The female NOAEC of 30 mg/m³ was based on a statistically significant increase in relative wet lung weight at higher doses, correlated with slight histopathologic effects in the lungs at the highest dose. However, no increase in relative lung weight was seen in breeding females at 100 mg/m³.

Formation of fatty acid conjugates of ciclesonide active metabolite in the rat lung after 4-week inhalation of ciclesonide.

Ciclesonide, an inhaled corticosteroid (ICS) with prolonged anti-inflammatory activity, is being developed for the treatment of asthma. Fatty acid conjugation of ICS is thought to be related to prolonged ICS activity. In vitro studies demonstrated that ciclesonide is converted to an active metabolite, desisobutyryl-ciclesonide (des-CIC), which undergoes reversible fatty acid conjugation. We tested the in vivo metabolism of ciclesonide in the lung by exposing rats to inhaled ciclesonide (0.16 mg/kg/day) for 1h daily over 4 weeks. Lungs (n=6 per time point) were extracted with ethanol 2, 5, and approximately 27 h after the final treatment. Ciclesonide and des-CIC concentrations were determined using solid-phase extraction and reverse-phase high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS). Concentrations of fatty acid ester conjugates were indirectly assessed using enzymatic de-esterification before LC/MS/MS. At 2 and 5 h, fatty acid conjugates of des-CIC were the major metabolites (61 and 55%, respectively). Ciclesonide, des-CIC, and fatty acid conjugates of des-CIC were clearly present in lung samples the day after the last inhalation. This in vivo study confirmed ciclesonide activation to des-CIC and formation of fatty acid conjugates. The presence of des-CIC fatty acid conjugates at >24 h after dosing suggests that ciclesonide is appropriate for once-daily dosing.

Cross-fostering inhalation toxicity study with HCFC-123 in lactating Sprague-Dawley rats.

A study was performed in Sprague-Dawley rats (Crl:CD BR) to differentiate between effects of hydrofluorocarbon 123 (HCFC-123) on the lactating dam or on the fetus using fostering and cross-fostering of the offspring. Pregnant and/or lactating dams without the pups present were exposed to the test substance (1000 ppm) or clean air by whole-body inhalation for 6 h/day from day 6 to 19 post conceptionem (p.c.) and from day 5 to 21 post partum (p.p.). Pups were cross-fostered to new dams within the first 2 days after birth. Treatment of the mothers with HCFC-123 led to decreases in serum glucose, cholesterol, and triglycerides and increases in absolute and relative maternal liver weights. Decreased litter and individual pup weight and decreased serum triglycerides were observed in the pups of treated foster mothers. Treatment of the mothers with HCFC-123 did not influence milk production based on the body weight difference of the dam before suckling and 60 min after beginning of suckling using 12-pup "standard litters" of untreated dams. Total fat, glucose, and protein contents in the milk were also not influenced by the treatment. Trifluoroacetic acid (TFA), a main metabolite of HCFC-123, was observed in urine samples of standard litters that had been nursed by treated dams. In conclusion, the effects on offspring due to HCFC-123 treatment consisted of decreased pup weight and decreased serum triglycerides at weaning. All effects were due to treatment of the lactating dams, as no prenatally induced effects were found. Since milk production and nutritional constituents of the milk were not influenced, but significant amounts of the main metabolite were found in pup urine, an effect of HCFC-123 or its metabolite on the pups via maternal milk is considered to be a possible cause for their decreased weight gain.

Embryotoxicity study of monomeric 4,4'-methylenediphenyl diisocyanate (MDI) aerosol after inhalation exposure in Wistar rats.

One of the uses of MDI is as an alternative to formaldehyde in the manufacture of furniture, its main route of exposure to humans being by inhalation. There have been no previous studies on the potential prenatal toxic effects of this compound. To close this gap in information, gravid Wistar rats, Crl:(WI)BR, were exposed by whole-body inhalation to clean air (control) and to 1, 3, and 9 mg/m3 MDI, respectively, for 6 hr per day from Days 6 to 15 post conception (p.c.). Rats were killed on Day 20 p.c. and the following results were obtained: Treatment caused a dose-dependent decrease in food consumption in all substance-treated groups during exposure, returning to normal values after cessation of treatment. The lung weights in the high-dose group were significantly increased compared to the sham-treated control animals. Treatment did not influence any other material and/or fetal parameters investigated (maternal weight gain, number of corpora lutea, implantation sites, pre- and postimplantation loss, fetal and placental weights, gross and visceral anomalies, degree of ossification), although a slight but significant increase in litters with fetuses displaying asymmetric sternebra(e) was observed after treatment with the highest dose of 9 mg/m3. Although the relevance of an increase of this minor anomaly in doses which cause toxic effects in dams (reduced food consumption, increased lung weights) is limited and the number observed is within the limits of biological variability, a substance-induced effect in the high-dose group cannot be excluded with certainty. Consequently, a no embryotoxic effect level of 3 mg/m3 was determined.

Investigation of a potential cotumorigenic effect of the dioxides of nitrogen and sulfur, and of diesel-engine exhaust, on the respiratory tract of Syrian golden hamsters.

Syrian golden hamsters (480 males and 480 females) allocated into 24 groups were exposed 19 hours per day and 5 days per week for 6, 10.5, 15, or 18 months to total diesel exhaust, diesel exhaust without particles, a mixture of nitrogen dioxide (5 parts per million [ppm]2) and sulfur dioxide (10 ppm), or clean air. Two exposure groups from each test atmosphere were also treated by a single subcutaneous injection of either 3 mg or 6 mg of diethylnitrosamine/kg of body weight to evaluate an enhancing effect of diethylnitrosamine on exposure-related changes. Morphological evaluation was done by histopathology. Minor changes of the larynx and trachea were investigated by scanning electron microscopy, which showed a loss of ciliated cells in all exhaust-exposed groups. After exposure to diesel exhaust with or without particles, focal metaplasia and dysplasia of the respiratory epithelium were seen in the oldest animals by scanning electron microscopy. In the same specimens, attached mucous droplets indicated changes in mucous cells and mucous viscosity. Only the exposure to total diesel exhaust significantly increased the tumor rate in the upper respiratory tract of male hamsters treated with 6 mg of diethylnitrosamine per kg of body weight. At the lower diethylnitrosamine dose, no exposure-related effects on the tumor rates could be observed. The results from this study and from our other inhalation experiments appear to be insufficiently conclusive to demonstrate that diesel-engine exhaust should be classified as a cocarcinogen or enhancer for the test system used.

The effect automotive exhaust exposure on the carcinogenicity of dipentylnitrosamine (DPN) in the respiratory tract of rats.

In this investigation rats were exposed to gasoline engine and diesel engine exhaust, respectively, and additionally treated with DPN in order to check the syncarcinogenic effects.

Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions.

A long-term exposure study with hamsters, mice and rats inhaling filtered and unfiltered diesel engine exhaust was carried out to investigate effects of chronic toxicity and, predominantly, carcinogenicity in the respiratory tract. The level of diesel exhaust in the exposure chambers corresponded to a concentration close to 4 mg m-3 in the unfiltered diesel exhaust. Satellite groups of animals were additionally treated with BaP, DBahA or nitrosamines in order to check for syncarcinogenic effects. In hamsters and rats, alveolar lung clearance and mechanical lung function tests as well as biochemical and cytological measurements in lung lavage fluids showed significant changes only after exposure to unfiltered diesel exhaust and, predominantly, in rats. No lung tumors were found in hamsters. Spontaneous tumor rates occurred in mice and both types of diesel exhaust increased the incidence of adenocarcinomas in the lungs. In rats, only the unfiltered diesel exhaust caused a lung tumor incidence. It amounted to 16% with no tumors in the controls. The heavy load of particulate matter in the lungs of rats was caused by an exposure-related impairment of the alveolar lung clearance and may have been instrumental in the induction of squamous cell tumors. However, an effect of particle-associated PAH cannot be excluded. Syncarcinogenic effects of diesel exhaust after initial carcinogen treatment were found only in the respiratory tract of rats.

Lung tumours in rats and mice after inhalation of PAH-rich emissions.

Rats and mice were exposed to a PAH-rich emission to test the carcinogenic activity. The BaP content of the exposure atmosphere was only 2-3 times higher than the concentration of about 30 micrograms/m3 measured in older coke plants. Although only half of the rats exposed to the exhaust have been investigated histologically up to now, the lung cancer incidence already amounts to 11%, no lung tumours were found in the control animals. Also the mice exposed to the exhaust with and without additional treatment with BaP, DBahA or urethane to induce a "base tumour rate" in the lung, showed a clear exhaust exposure-related carcinogenic effect. The lung tumour multiplicity as well as the incidence was significantly higher. The epidemiological findings of an increased lung cancer risk in coke oven workers can now be supported by the results of this animal inhalation study.

The effect of cigarette smoke exposure in European hamsters.

The effects of 2 types of research cigarettes, differing in their total smoke delivery and condensate were examined. Two groups of European hamsters were exposed in a smoking chamber to the cigarette smoke of both types of cigarettes for 130 weeks. Exposure related alterations were found in the respiratory tract and forestomach. However, smoke exposure over this period had no effect on mortality and led to no neoplastic response.

The carcinogenic effect of nitrosopiperidine administered in the drinking water of Syrian golden hamsters.

Syrian golden hamsters (Mesocricetus auratus W.) were used to test the long-term carcinogenic effect of nitrosopiperidine (NP). Groups of 30 females and 30 males were given 0.05%, 0.025% and 0.006% NP in their drinking water for life. The animals developed neoplasms in the larynx, pharynx, trachea and forestomach (papillary polyps, papillomas and epidermoid carcinomas) and in the liver (hepatocellular adenomas, carcinomas). In addition, cholangiocellular and endothelial liver tumours and colon adenocarcinomas were observed. The overall tumour frequency was dose-dependent and higher in males than in females.