Search

To illustrate the process of addressing adverse preclinical findings (APFs) as outlined in the first part of this review, a number of cases with unexpected APF in toxicity studies with drug candidates is discussed in this second part. The emphasis is on risk characterization, especially regarding the mode of action (MoA), and risk evaluation regarding relevance for man. While severe APFs such as retinal toxicity may turn out to be of little human relevance, minor findings particularly in early toxicity studies, such as vasculitis, may later pose a real problem. Rodents are imperfect models for endocrine APFs, non-rodents for human cardiac effects. Liver and kidney toxicities are frequent, but they can often be monitored in man and do not necessarily result in early termination of drug candidates. Novel findings such as the unusual lesions in the gastrointestinal tract and the bones presented in this review can be difficult to explain. It will be shown that well known issues such as phospholipidosis and carcinogenicity by agonists of peroxisome proliferator-activated receptors (PPAR) need to be evaluated on a case-by-case basis. The latter is of particular interest because the new PPAR α and dual α/γ agonists resulted in a change of the safety paradigm established with the older PPAR α agonists. General toxicologists and pathologists need some understanding of the principles of genotoxicity and reproductive toxicity testing. Both types of preclinical toxicities are major APF and clinical monitoring is difficult, generally leading to permanent use restrictions.

Unexpected adverse preclinical findings (APFs) are not infrequently encountered during drug development. Such APFs can be functional disturbances such as QT prolongation, morphological toxicity or carcinogenicity. The latter is of particular concern in conjunction with equivocal genotoxicity results. The toxicologic pathologist plays an important role in recognizing these effects, in helping to characterize them, to evaluate their risk for man, and in proposing measures to mitigate the risk particularly in early clinical trials. A careful scientific evaluation is crucial while termination of the development of a potentially useful drug must be avoided. This first part of the review discusses processes to address unexpected APFs and provides an overview over typical APFs in particular classes of drugs. If the mode of action (MoA) by which a drug candidate produces an APF is known, this supports evaluation of its relevance for humans. Tailor-made mechanistic studies, when needed, must be planned carefully to test one or several hypotheses regarding the potential MoA and to provide further data for risk evaluation. Safety considerations are based on exposure at no-observed-adverse-effect levels (NOAEL) of the most sensitive and relevant animal species and guide dose escalation in clinical trials. The availability of early markers of toxicity for monitoring of humans adds further safety to clinical studies. Risk evaluation is concluded by a weight of evidence analysis (WoE) with an array of parameters including drug use, medical need and alternatives on the market. In the second part of this review relevant examples of APFs will be discussed in more detail.

Currently, the majority of substances tested in lifetime bioassays in rodents are not mutagenic and, therefore, at the most weakly carcinogenic, generally by epigenetic mechanisms. It thus appears obvious that only marginal increases of tumour incidences can be expected in lifetime bioassays and that, therefore, every aspect of a potential carcinogenic effect must be thoroughly evaluated. This paper describes a series of key factors, which should be looked at in order to exclude that the lifetime bioassay in question is flawed for design, technical or qualification reasons. It also provides some hints whether there is indeed a real effect and not just a variation of the spontaneous tumour incidences. Tumour findings must be seen in the context of the animal model, the pharmcokinetics and pharmcodynamics of the test substance, as well as any other observation in the present or other studies with the test substance, including non-tumour findings and--in particular--potential precursor lesions and effects on feed intake and survival. The possibility that the observed carcinogenic effects may be species-specific and not relevant for man is discussed. It is also important to check what findings are reported with similar substances or substances with the same pharmacological effect. Data from additional investigations on material of the same study and/or mechanistic studies are often needed to support the final risk assessment.

Formalin-fixed, paraffin-embedded liver specimens from 27 2-year-old Wistar rats, including 10 normal livers, 11 hepatocellular adenomas, 2 hepatocellular carcinomas, and 4 cystic cholangiomas, were immunostained using the streptavidin/peroxidase method and the PC10 monoclonal antibody (Mab), which recognizes an epitope on the proliferating cell nuclear antigen (PCNA). The following PCNA labeling index (LI) mean values were found for the above four groups of liver specimens: normal livers, 0.43 +/- 0.31%; hepatocellular adenomas, 1.51 +/- 0.59%; hepatocellular carcinomas, 24.80% +/- 10.28%; and cystic cholangiomas, 0.61 +/- 0.21%. Our findings indicate that PCNA LI clearly separates liver malignancies from other benign liver tumors, as well as from normal, non-neoplastic, liver tissues. Although the mean PCNA LI values seemed to reflect histological grading (i. e. normal, neoplastic benign, neoplastic malignant), overlapping between normal livers and hepatocellular adenomas was observed in five cases (i. e. in 2 normal livers and 3 hepatocellular adenomas, where the PCNA LI values varied between 0.74% and 0.96%). It thus appears that PCNA immunohistochemistry represents a promising tool for investigating liver cell proliferation in laboratory rats, and permits distinguishing between benign and malignant liver parenchymal tumors.

SDZ MNS 949, 6,7-dimethoxy-3-methyl-1-(3',5'-bis (methoxyethoxy) phenyl)-isoquinoline, a bronchodilating anti-inflammatory drug that inhibits phosphodiesterase, had been proposed for the treatment of bronchial asthma. Groups of 14 male and 14 female Wistar rats were administered doses of 12, 50, and 130 mg/kg/day in feed for 26 weeks. Periodic radiographic examinations were performed in addition to clinical observations, clinical chemistry measurements and urinalysis. At study termination full necropsy and histopathological examinations were performed on all animals. The principal clinical signs observed were unilateral edematous, red and painful swelling of the distal hindlimbs in 8 of 28 high dose animals, and abdominal swelling in 19 of 28 high dose animals. At radiographic examination periosteal new bone formation was predominantly along the tibia. Lesions at necropsy included dilated small and large intestines. Microscopically, enteritis was observed, and the periosteal new bone formation was confirmed. Hematological findings consisted of thrombocytosis and lymphocytosis, especially in high dose animals. The clinical, radiographical and histological findings in treated rats were consistent with the diagnosis of "hypertrophic osteopathy" or "Marie's Disease".

Many compounds giving a positive result in animal carcinogenicity studies through mechanisms involving secondary carcinogenesis pose little or no risk to humans. This article provides an overview of current understanding, with particular reference to renal tumors in male rats with alpha 2mu-globulin nephropathy, urinary bladder neoplasia in rodents, mesovarian leiomyomas induced in rats by beta 2-receptor stimulants, carcinoid tumors in the rodent stomach induced by prolonged suppression of acid secretion, thyroid follicular cell tumors in rodents, canine mammary neoplasia due to administration of progestagens, rodent mammary neoplasia induced by estrogens, uterine endometrial carcinomas of rats induced by dopamine agonists, Leydig cell tumors in the testis of rats, and ovarian tubulostromal adenomas in mice. A positive result on a rodent carcinogenicity study should not automatically preclude further development of a compound; future progress in this field should increase the accuracy of the rodent carcinogenicity study as a tool in human safety assessment.

Peto test procedures for the statistical evaluation of carcinogenicity studies require that each tumor in an animal that died intercurrently (or was sacrificed in extremis) be classified as either fatal, probably fatal, incidental, or probably incidental. There is considerable controversy as to whether or not the cause of death can be established with accuracy in rodent studies. In the present article, the causes of death or ill-being as found in 10 consecutive carcinogenicity studies--5 studies with 2400 OFA (Sprague-Dawley-derived) and Wistar rats and 5 studies with 2400 OF1 and NMRI mice--were re-examined. A cause of death or moribund state had been established in more than 80% of the cases in rats and in more than 70% in mice. These causes were, in rats, mainly pituitary tumors, chronic progressive nephropathy (males), mammary gland tumors (females), and subcutaneous tumors (males); in mice, mainly hemolymphoreticular tumors, lung tumors, liver tumors (males), and glomerulonephropathy. The criteria used for determining the tumorous or non-tumorous lesions as the cause of death were based on in-life and pathological findings. The validity of such procedures, the possibility of improving criteria in the future, and the usefulness of establishing causes of death in safety assessment are discussed.

Calcitonin, a calcium-lowering hormone, has been associated with an increased incidence of nonfunctioning pituitary tumors in rats. In this study, rats were treated with calcitonin (80 IU/kg/d) for 52 weeks. After treatment with calcitonin, immunohistochemistry and in situ hybridization analyses demonstrated that most pituitary tumors expressed the glycoprotein hormone alpha-subunit. Expression of the alpha-subunit was identified rarely in hyperplastic lesions of control animals. Serum levels of GH, PRL, ACTH, LH, and FSH were unchanged in calcitonin-treated rats relative to controls. However, TSH levels were increased 2.1 fold after chronic treatment with calcitonin in both male and female rats (P less than 0.001). The level of glycoprotein hormone alpha-subunit was markedly increased (20-fold) in male rats with smaller elevations in female rats. Time course studies demonstrated that increases in serum alpha-subunit levels could be detected by 24 weeks of treatment and that elevations in alpha-subunit were present in the majority of animals by 40 weeks of treatment with calcitonin. The authors conclude that high doses of calcitonin, administered to rats for 6 months or longer, increases the incidence of alpha-subunit-producing pituitary tumors.

During a routine long-term drug safety study, lasting approximately 2 1/2 yr, male Wistar rats, treated with a prolactin-inhibiting compound, developed an excess of Leydig cell tumors (LCTs). Most tumors were typical for the rat but a small number showed an unusual variation and some appeared malignant. The variation consisted of glandular and/or tubular structures within the tumor mass which occasionally anastomosed and contained an eosinophilic periodic-acid Schiff (PAS) positive material. In a few of these variants, malignant features such as cellular atypia, capsular, and lymphatic invasion and necrosis were seen. No metastases were detected. Detailed morphological and immunohistochemical investigations were conducted in order to establish the cell of origin of these variants. Glandular/tubular structures were found to stain with varying intensity for vimentin and cytokeratin, but were always negative for beta-tubulin. The results indicated that the cell of origin of these LCT variants was indeed the Leydig cell and that glandular and/or tubular structures within LCTs represented a form of Leydig cell metaplasia.

The administration of lipid-lowering drugs to rodents, notably those related to clofibrate, rapidly provokes a hepatic response characterized by hepatomegaly, proliferation of smooth endoplasmic reticulum and proliferation of peroxisomes in hepatocytes. In some studies hepatocellular carcinoma has been found in rats or mice exposed for their entire life-span to high dose levels of various fibrates. In the present study liver biopsy samples were obtained from 38 hyperlipidemic patients, 28 of whom had been receiving fenofibrate for between 2 months and approximately 3 years (mean values: males 1.79, females 1.98 years). The remaining 10 patients had never been treated with a lipid-lowering drug. Examination of the biopsy samples by a variety of optical techniques and by electron microscopy failed to reveal any difference between the groups. Peroxisomes were relatively rare, there being no evidence of the clear proliferation seen in rodent studies. Other microscopic features of interest were some variation of nuclear size, mitochondria containing paracrystalline inclusions, dilated endoplasmic reticulum associated with reduced amounts of rough endoplasmic reticulum, and the presence of lipid droplets in the liver cells. However, these variations from normal were in general not much more apparent in samples from the fenofibrate-treated patients than in the untreated group. Light- and electron-microscopic observations did not suggest liver intoxication or a carcinogenic pattern.

The i.v administration of lentinan to the Beagle dog induced changes in the cytoplasm of macrophagic cells in the liver, spleen, kidney, lungs, lymph nodes, small intestine. Electron-lucent or filamentous inclusions were demonstrated in the liver, kidney and spleen. A dose level of 0.5 mg/kg/day was without adverse effect.

Single doses of antigen suspension (alum-precipitated canine serum proteins) were administered intratracheally to SPF rats. After periods of 1, 2 and 3 weeks rats were killed and their lungs examined histologically. After an initial macrophage and perivascular lymphoid reaction, dose-related increases were found in the amount of bronchus-associated lymphoid tissue (BALT) and in the amount of DNA within BALT cells, indicating increased cell division. Immunoglobulin-containing cells were demonstrated within BALT 3 weeks after the exposure to antigen. A prominent and extensive bronchial lympho-epithelium was seen overlying BALT follicles in antigen-treated rats, while in control animals the respiratory epithelium overlying BALT was predominantly normal ciliated epithelium. The significance of these findings is discussed in the light of the possible defensive role of BALT in cases of respiratory disease of man and animals.

Male and female Beagle dogs and Cynomolgus monkeys were exposed to anaesthetic (1.5 MAC) and subanaesthetic (1/100 MAC) levels of enflurane and halothane for 3 hours on alternate days for 4 weeks. One-half of the animals were killed following the last exposure and the remainder after 4 weeks of recovery. The animals' condition was assessed during anaesthetic periods by measuring respiration, ECG, blood pressure, temperature and EEG. Haematology, urinalysis and clinical chemistry parameters were evaluated. Gross and microscopic pathological examinations were conducted at the end of the exposure and recovery periods. Two female monkeys in the mid- and high-dose halothane groups died during the study. No deaths were observed in the enflurane group. No quantitative differences were observed in respiration rate, heart rate, blood pressure and EEG activity of animals anaesthetized with enflurane or halothane. Muscle twitches were observed in some mid- and high-dose dogs inhaling enflurane, but not in monkeys. A number of liver function tests became abnormal in mid- and high-dose halothane-treated dogs and high-dose halothane-treated monkeys. This was not observed with enflurane. Histopathologic alterations were confined to the liver of animals exposed to halothane. In dogs, the lesions were characterized by centrilobular hepatocyte degeneration and/or necrosis, fibroblastic proliferation, hepatocyte enlargement, fat deposition and glycogen depletion; and in mid- and high-dose monkeys by moderate to marked hepatocyte vacuolation and fat deposition. Except for one high-dose dog, these lesions were not seen in animals killed after 4 weeks of recovery. No histopathologic alterations were observed with enflurane.

The toxicity of 1-amino-3-chloro-2-propanol is associated with acute histopathological change in the medulla oblongata, characterised lesions of focal oedema. Continued administration results in neurological scars. Lesions can be induced at dose levels of 50 mg . kg-1 day-1. Clinical manifestations of neurological involvement are periods of slight incoordination and loss of balance in a few animals only.

The usefulness of measuring serum bile acid concentrations by RIA in a number of acute experimental liver injuries of rats was assessed by comparing the concentrations with the results of some of the routinely employed methods of examining hepatotoxic changes. Centrilobular liver cell injury produced by CCl4 revealed leakage of GPT and GDH and to a lesser extent AP; along with minimal increase in serum bile acid levels. Serum bilirubin concentration remained unchanged. Surgical bile duct ligation resulted in marked rises in AP, GPT and GDH and total bilirubin levels and levels of serum bile acids. Intravenous injection of MnSO4 induced focal necrosis of liver and bile canalivular dilation associated with elevated GDH and GPT concentrations. AP and bilirubin levels were unchanged. Bile acid levels were raised among female rats. 2,4-Xylidine induced hepatotoxicity revealed bile duct hyperplasia, liver cell enlargement, liver cell necrosis, biliary canalicular dilation and proliferation of endoplasmic reticulum. GDH and GPT levels were raised along with bile acid concentrations. This study suggested that assay of bile acid concentration is a sensitive indicator of several acute hepatic injuries.

One hundred and eighty Wistar-strain rats were exposed to differing concentrations of tobacco smoke, for periods of up to 20 months, in order to examine the response of the pulmonary immune system. The amount of bronchus-associated lymphoid tissue (BALT) in the lungs of exposed rats increased initially over the first 5 weeks of exposure, subsequently falling to below control levels by the fourteenth week and eventually increasing again to a level slightly higher than that of the controls by the twelfth month, at which level it was maintained until the twentieth month. Quantitative immunohistochemical assay of bronchial immunoglobulin levels (only assessed over the initial 14 weeks of exposure) revealed a transitory enhancement of levels followed by a depression, the speed of response being apparently dose-related. Alveolar macrophage activity, indicated by lysosomal enzyme activity, increased relative to the control animals over the same 14-week exposure period. The significance of these observations is discussed and a tentative explanatory hypothesis is advanced.

Chronic relapsing experimental allergic encephalomyelitis (CR-EAE) was induced in rats with an emulsion of guinea-pig spinal cord tissue (GPSC) in complete Freund's adjuvant (CFA) enriched with Mycobacterium tuberculosis H37RA (Tbc). 78% of the sensitized rats developed a CR-EAE showing 2 to 3 clinical relapses during the first 40 days. After 60-80 days, approximately half of the rats with CR-EAE had a further relapse which was followed by complete recovery in only 35% of the cases. The remaining 65% of these animals showed a progressive state of the disease, characterized by paralysis or severe motor deficit, eventually leading to death. CR-EAE in rats showed some similarities to multiple sclerosis in man (MS) and it may be a useful model for the study of this disease.

The accepted animal toxicity studies indicate that the ganglioside mixture extracted and purified from the bovine brain cortex (Cronassial) is without detectable toxicity. It did not induce any adverse effects on any of the characteristics of reproduction and it is not antigenic.

In juvenile wild rats, bronchus-associated lymphoid tissue (BALT) development was similar to that seen in adult specified-pathogen-free rats. In adult wild rats the BALT was widespread. In one animal infected with a mycoplasma-like organism, a region of bronchoepithelium overlying a large BALT nodule was seen, through which lymphocytes appeared able to pass to make direct contact with the bronchial lumen: the significance of this observation is discussed. There was no evidence of infection in lungs from any of the specified-pathogen-free animals, where small foci of BALT were seen.

Year-long intermittent exposures of rats, mice, hamsters, and dogs to hydrazine were conducted using concentrations of 0.05, 0.25, 1.0, and 5.0 ppm. Rats were held 18 months postexposure; hamsters, 1 year postexposure; mice, 15 months postexposure; and dogs, 38 months postexposure. Male and female rats exhibited dose-dependent incidences of benign nasal adenomatous polyps and smaller numbers of malignant nasal epithelial tumors after 1 year of exposure to hydrazine and 18 months postexposure holding. Nasal tumors were often associated with chronic irritation and were most frequent in male rats, with an incidence of greater than 50% in the highest exposure group. Hamsters exposed to 0.25-ppm and higher concentrations showed pathologic changes characteristic of degenerative disease, including amyloidosis. After exposure to 0.5 ppm hydrazine, hamsters developed a 10% incidence of benign nasal polyps compared to 0.5% in controls. Small numbers of colon neoplasms and thyroid parafollicular cell adenomas were found in hamsters, but only in the highest concentrations tested. Lung adenomas appeared to be marginally increased in mice exposed to 1.0 ppm hydrazine, the highest concentration tested in this species. No consistent clinical or pathological effects were seen in dogs during or after exposure to hydrazine at any concentration. Using amyloidosis as a criterion, a no-effect level was not achieved in hamsters. In rats, there appeared to be a marginal production of nasal tumors at 0.05 ppm, while mice showed no effects at 0.25 ppm. This study has demonstrated that the nasal respiratory epithelia of rats and hamsters are the most sensitive tissues to the tumorigenic action of hydrazine following inhalation exposures. This is similar to the reaction of rats to formaldehyde, another highly reactive water-soluble compound.

Beagle dogs were given chloroform in a toothpaste base orally in gelatin capsules on 6 d/wk for 7 1/2 yr, followed by a 20-24 wk recovery period. Groups of 16 males and females received 0.5 ml/kg/d of the vehicle (toothpaste without chloroform) and 8 dogs of each sex remained untreated. Treated groups comprised 8 dogs of each sex remained untreated. Treated groups comprised 8 dogs of each sex, receiving doses equivalent to 15 and 30 mg CHCl3/kg/d in the toothpaste vehicle; another group of the same size received an alternative non-chloroform toothpaste (0.5 ml/kg/d). Eleven of the 96 dogs died during the study, only two of these being in the CHCl3-treated groups. The only significant toxic response during treatment was a moderate rise in serum enzyme levels (e.g. SGPT), reaching a peak in the sixth year of the study and probably corresponding to minimal liver damage. Few Palpable growths were noted while the dogs were alive. "Fatty cysts" were seen in the liver of several dogs at post mortem possibly associated with the chloroform treatment but the distribution of a nodular change in the liver was not obviously dose related. A small number of macroscopic and microscopic neoplasms were seen; one dog in each chloroform-treated group had a malignant tumour but there were no tumours in the liver or kidney of any dog. Overall, exposure to chloroform in a toothpaste base was not associated with any effect on the incidence of any kind of neoplasm. From this and related studies in mice and rats, it is concluded that repeated exposure to chloroform (3.5 percent) in toothpaste is unlikely to result in any hazard to human health.

The fine structure of rhesus monkey renomedullary interstitial cells was studied by electron microscopy. These stellate cells contained variable numbers of lipid droplets, moderate numbers of mitochondria, moderate amounts of rough endoplasmic reticulum, and prominent Golgi zones. In rare instances, apparent release of lipid droplets into the interstitium was observed. The most prominent feature of the interstitial cells was larger nuclear pseudoinclusions which were observed in a high proportion of the animals examined.

The pattern of development of bronchus-associated lymphoid tissue (BALT) in specified-pathogen-free and conventional (non-barrier maintained) rats over the initial 4 weeks of life appeared to be similar. BALT first appeared around the 2nd week of life and increased in amount over the following 2 weeks. Overlying large nodules of BALT the bronchial epithelium becomes infiltrated by lymphocytes to form a lymphoepithelium. This transformation occurs earlier in conventional rats, possibly because of the differing antigen levels to which they are exposed.

A number of instances have been reported in the scientific literature in which acute intoxication with halogenated oxyquinolines has led in some species to convlusions, often followed by death. The toxicity of repeated doses of clioquinol has been investigated extensively in the dog. The clinical syndrome induced in this species is characterized by anorexia, weight loss, extremem muscle weakness and emaciation. In some animals surviving this impairment of condition for several weeks, neuropathy of the central nervous system, but not of the peripheral nerves ensued. It is suggested that these toxicological manifestations are less dependent on the dose-level than on the degree of absorption. Some suggestions regarding the aetiology of the lesions are made.

A mononuclear cell leukemia in Sprague-Dawley rats is described in which liver, spleen, and lung involvement was a constant feature. The cell was 16 to 25 microns in diameter with round, oval or indented nucleus, and the cytoplasm contained bright red granules. This is the first report of such a leukemia in Sprague-Dawley rats and a comparison with mononuclear cell leukemia in other strains of laboratory rats is made.

Six young Beagle dogs had non-inflammatory pancreatic atrophy associated with lack of weight gain and low serum protein levels. Histologically there was severe atrophy with loss of acinar architecture and absence of islets of Langerhans (type 1), or partial atrophy with ppreserved islets (type 2). The correlation between histological type and clinical severity was poor.

The administration of a new piperidine antispasmodic agent (HSR-902) to the dog by the oral and intravenous routes, induced clinical signs attributable to parasympathetic blockage. The only significant toxicological finding was rarefied appearance and enlargement of the hepatocytes at the high dose level (50 mg/kg/day); this was shown to be reversible.

The relationship between the acute toxicity of orally-administered chloroform and its long-term tumorigenic potential was studied in male mice of the CFLP outbred Swiss albino mouse strain. A single dose of approximately 18 mg CHCl3/kg had no detectable acute toxic effect on the liver or kidneys and did not stimulate regenerative activity, whereas both toxicity and subsequent tissue regeneration were observed with single doses of about 60 mg/kg or higher. The severity of the toxic effects and regenerative changes was greater when corn oil was used as a vehicle for chloroform than when the vehicle was a toothpaste base. In earlier long-term studies in mice of the same strain, kidney tumours occurred in males given 60 mg/kg/day throughout life but not in mice given 17 mg/kg/day. The tumour response was greater when the 60-mg/kg/day dose was given in an oily vehicle than when it was given in toothpaste. The findings are consistent with the hypothesis that early acute toxic change and subsequent repair are a sine qua non for tumorigenesis in the kidney and liver in response to chloroform.

Segmental aplasia of the vaginal mucosa was discovered in 3 beagle bitches at the end of a routine toxicological experiment. Anomalies of Müllerian duct development in the bitch are discussed.

The histology and ultrastructure of the rat laryngeal epithelia are described. Five epithelial types were identified. Stratified squamous epithelium was found over most of the epiglottis, arytenoid projections and lateral ventricles. The vocal folds were covered by a low squamoid type of epithelium. Respiratory epithelium, similar to that found elsewhere in the respiratory tract, occupied all the mucosa caudal to the vocal folds, small areas at the base of the epiglottis and along the inner aspects of the arytenoid projections. Two forms of relatively unusual pseudostratified cuboidal epithelium were present in the ventrolateral aspect at the level of the arytenoid projections and within the ventral pouch. Non-myelinated, intro-epithelial nerve fibres were found throughout the larynx, and were abundant in areas at the base of the epiblottis covered by respiratory epithelium and to a lesser extent in the cuboidal epithelium of the ventral pouch. Globule leucocytes were frequently found within respiratory epithelium, less frequently in cuboidal epithelium and only rarely in squamous areas.

Using an image analysing computer a variety of ultrastructural features from micrographs of alveolar macrophages have been quantified. Macrophages from rats exposed to smoke from conventional tobacco cigarettes, for 6 months, revealed statistically significant changes when compared to controls. The macrophages were larger, rounded with fewer pseudopodia and contained increased numbers of inclusions. There were no statistically significant changes in macrophages from rats exposed to smoke from a tobacco substitute.