The industry view on long-term toxicology testing in drug development of human pharmaceuticals.

The approach to chronic toxicity testing over the past decade is reviewed and assessed in the light of developing ICH guidelines. The 1990's have seen a general acceptance that studies with a maximal duration of 6 months in rodents are all that is required for adequate safety assessment of developmental pharmaceutical agents. However, controversy has arisen concerning the most appropriate duration for chronic toxicity testing in non-rodents. Initial suggestions that 6 months duration was sufficient have been countered by findings noted in 12-month studies that were not seen in shorter-term studies. Retrospective analysis of available data eventually lead to a subsequent ICH recommendation that studies of 9 months duration would be now acceptable. However, until recently the FDA position on this recommendation was unclear and an analysis of industry practices since the ICH recommendation was made in 1997 has shown that the 9-month guideline is not widely applied. Recent clarification by the FDA will probably result in a continued but limited use of this alternative. An industry view on the future of chronic toxicology testing in rodents and non-rodents is presented.

Strategies for handling unexpected endocrine findings in early safety assessment.

Early assessment of the endocrine toxicity of pharmaceuticals has received increasing scientific attention due to the important issue of endocrine disrupters, and the need to evaluate mechanisms and relevance to humans of early toxicity findings in animals. Pharmaceuticals can directly alter an endocrine organ, or indirectly regulatory systems. The strategy for handling unexpected endocrine findings in early toxicity studies is by definition extremely variable and should be adapted case by case. Using three examples: (i) thyroid hyperplasia; (ii) prolactin-induced mammary gland hyperplasia in rats; and (iii) aromatase inhibition, the usefulness and limitation of serum endocrine analyses in classical toxicological studies, the need for specific mechanistic studies and the major importance of integration between pharmacodynamic, pharmacokinetic and toxicological data will be discussed. Finally, the importance of assessment of relevance and the extrapolation of preclinical findings to humans at an early stage in clinical development is highlighted.

Review of alternative methods of carcinogenicity testing and evaluation of human pharmaceuticals.

Hundreds of pharmaceuticals have been reported to give a positive result in the standard "Chronic Bioassay", which consists of an 18 to 24 month daily administration of the test compound in mice and rats. This is in contrast with 20 pharmaceuticals, which are known to be carcinogenic to humans. The high incidence of apparently false-positive results in the Chronic Bioassay may be related to differences in mechanism of pharmacological action between rodents and humans, but also to the very high dose levels that have to be administered to rodents in accordance to regulatory guidelines. Lack of relevance to man therefore often has to be demonstrated by additional mechanistic studies. Based upon the deficiencies of the Chronic Bioassay and on the increased knowledge on cellular and molecular mechanisms involved in carcinogenicity, extensive discussions have recently taken place between regulatory agencies and industry associations at the occasion of International Conferences on Harmonization (ICH). These discussions have resulted in the possibility to use alternative short-to-medium-term carcinogenicity models in combination with a single two-year carcinogenicity study for evaluation of carcinogenicity. A description of these models is provided in this review as well as possible strategies for carcinogenicity testing and evaluation in the future.

Transverse vaginal septum in the specific-pathogen-free Wistar rat.

A transverse vaginal septum was found in 2% of a substrain of specific-pathogen-free Wistar rats used in reproductive toxicity studies. Macroscopic observation mostly revealed a complete transverse septum. In a few rats an incomplete septum with a small central pupillary gap was found. Microscopically the septum consisted of a central core of connective tissue covered on both sides by epithelium. The rats with vaginal defects were clinically healthy but were unable to breed because the vaginal septum hampered the progress of the spermatozoa. Cranial to the defect, the uterus, cervix, and cranial portion of the vagina were moderately to severely dilated and often filled with mucopurulent fluid and cellular debris. The presence of a transverse vaginal septum is rare in rats and can therefore be easily missed. Congenital, embryologic, and histologic aspects of this anomaly are discussed and compared with existing literature data.

Evaluation of the bovine corneal opacity-permeability assay as an in vitro alternative to the Draize eye irritation test.

The bovine corneal opacity-permeability assay (BCO-P) was evaluated as an in vitro alternative test model for the Draize eye irritancy test. Fifty pharmaceutical and commercially available compounds were tested in the BCO-P assay. The compounds were selected on the basis of their in vivo irritancy potential as determined in previous Draize tests. Liquids as well as solids were tested. Corneal opacity and permeability were measured to determine ocular irritation potential. When two irritancy classifications (non-irritant and irritant) were considered, 96% of the tested chemicals were classified correctly. A 72% concordance was obtained when four irritancy classifications (non-irritant, mild, moderate and severe irritant) were considered. Furthermore, all compounds that were severe eye irritants in vivo were equally scored in vitro. The results of this study show that the BCO-P assay is a competent in vitro test system for the prediction of ocular irritation of chemicals. This test model can be used as a first screen to avoid in vivo testing of severe ocular irritants.

Safety aspects of oral antifungal agents.

Four main targets have to be considered when evaluating the safety of new systemically acting, oral antifungals: the liver, the endocrine system, serum cholesterol and the developing embryo. The major endocrine targets for high levels of the antifungal azoles are the adrenal cortex and the gonads. Endocrine studies demonstrate that itraconazole has little potential for interfering with steroid hormones in man. Available data also indicate that itraconazole has low predictable hepatotoxicity potential in man. In rats, serum cholesterol levels are raised during treatment with itraconazole, especially after chronic exposure. This is, however, a species-specific phenomenon which leads to secondary events at toxic doses, especially in long-term toxicity studies. In man, including patients with existing hypercholesterolaemia, serum cholesterol levels are not raised. Ketoconazole has been shown to be teratogenic at high, toxic doses in pregnant rats. The same observation has been made for itraconazole, and it may also be true for fluconazole. However, all three azoles show no teratogenicity in the rabbit. Studies with itraconazole in adrenalectomised rats and in rats given exogenous arachidonic acid indicate that adrenal effects occurring at toxic dose levels are important mediators of teratogenicity. Since itraconazole does not affect adrenal function at levels used to treat infections in man, the teratogenic risk is estimated to be low. Itraconazole is therefore a promising new drug, especially with regard to the assessment of its safety in the liver and endocrine system. Moreover, it is more potent and has a broader antifungal spectrum than other azole antifungals, and its development is considered to be an important step forward in chemotherapy.

Toxicological profile and safety evaluation of antifungal azole derivatives.

For the development of new systemically acting, oral antifungal azoles, it is of key importance to compare them with ketoconazole, the first available drug in this therapeutic class. Ketoconazole is a major breakthrough although hepatic side-effects as well as interactions with mammalian steroids might rarely occur during prolonged treatment. The prediction of these side-effects is difficult but the potential to interact with mammalian cytochrome P-450 enzymes is considered to be important. Therefore, for the selection of itraconazole a multidisciplinary approach was applied to study this potential. The present paper deals with the toxicological profile of itraconazole and its safety evaluation. In addition, a further comparison with ketoconazole and also with fluconazole is provided, in so far sufficient information is available. For the liver as a potential target organ, the available data indicate that itraconazole is not a predictable hepatotoxic drug in man. The major endocrine targets for overdosing with antifungal azoles are the adrenal cortex and the gonads. Endocrine studies show that itraconazole is not bearing a potential to interfere with steroid hormones in patients, which is a major improvement when compared to ketoconazole. In rats, elevation of serum cholesterol is observed especially after chronic exposure to itraconazole. This species-specific phenomenon leads at toxic dose levels to secondary events, especially in the long-term toxicity studies. In man, including those with existing hypercholesterolemia, serum cholesterol is not adversely affected by itraconazole. In pregnant rats, ketoconazole was shown to be teratogenic at high, toxic doses. The same observation has been made for itraconazole and this also might be true for fluconazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural evaluation of the effects of diclazuril on the endogenous stages of Eimeria maxima and E. brunetti in experimentally inoculated chickens.

The ultrastructural morphology of the different endogenous stages of Eimeria maxima and E. brunetti was evaluated after oral treatment of inoculated chickens with a single dose of 5 mg/kg diclazuril. The drug induced no ultrastructural change in the growth or differentiation of the various schizont stages of both Eimeria spp. In E. maxima, the micromorphological appearance of micro- and macrogamonts developing from the blast from to maturation also remained unaffected by drug treatment. However, in all fertilized macrogamonts the normal pattern of oocyst wall establishment was completely disturbed, resulting in the formation of an abnormally thickened, incomplete oocyst wall and the necrosis of the zygote. In E. brunetti, the growth and nuclear division during microgametogenesis were not affected but differentiation was clearly abnormal. In comparison with the controls, this abnormal differentiation was characterized by a less extensive enlargement of the parasite surface area, aberrant morphological configurations of condensed heterochromatin, intracytoplasmic flagella formation, and glycogen accumulation. Finally, the complete degeneration of all microgamonts ensued. The growth and differentiation leading to mature macrogamonts was not disturbed; however, subsequent oocyst wall formation was largely precluded and the macrogamonts proceeded to degenerate completely. We conclude that diclazuril treatment primarily affected particular stages in the sexual development of both Eimeria spp., resulting in the complete eradication of these coccidian species.

In vivo action of the anticoccidial diclazuril (Clinacox) on the developmental stages of Eimeria tenella: a histological study.

Diclazuril, a new benzeneacetonitrile anticoccidial, has potent activity against various stages of Eimeria tenella. A single treatment of experimentally infected chickens during the prepatent phase (up to day 5) results in a complete interruption of the life cycle and oocyst shedding. The first- and second-generation schizonts show extensive degenerative changes that finally result in a complete loss of the parasitic stage. The degeneration is characterized by loss of internal structure, the appearance of many intracytoplasmic vacuoles, and incomplete merogony. The merozoites themselves show similar degenerative changes, including the presence of numerous small vacuoles in the cytoplasm. Diclazuril is also effective against both the micro- and macrogametocytes that have a ballooned appearance and loose their internal structure completely. In the macrogametocytes, wall-forming bodies either do not develop or disappear rapidly. Development of typical caecal lesions is prevented when treatment with diclazuril is initiated before large numbers of second-generation schizonts appear, i.e., day 3. It is concluded that diclazuril is lethal against both the asexual and the sexual stages of E. tenella. At the proposed use level of 1 ppm in the feed, the life cycle is interrupted at a very early stage and lesion development and oocyst shedding are completely prevented.

In vivo action of the anticoccidial diclazuril (Clinacox) on the developmental stages of Eimeria tenella: an ultrastructural evaluation.

A single 5-mg/kg oral dose of diclazuril affected both the asexual and sexual development of Eimeria tenella in experimentally inoculated chickens. In second-generation schizonts, early growth and nuclear divisions progressed normally, but a marked inhibition of merozoite formation was observed. Exogenesis of merozoites was largely prevented, whereas production of micronemes, amylopectin granules, and dense bodies and the formation of rhoptries, conoid, and pellicle continued. All these subcellular organelles accumulated, together with differentiated nuclei, within the main cytoplasmic mass. In the end, complete necrosis of the schizonts occurred. In macrogamonts, dilation of the rough endoplasmic reticulum around type II wall-forming bodies, fusion of type II wall-forming body contents, disturbance of the normal parallel arrangement of rough endoplasmic reticulum, and disruption of row formation of amylopectin granules became evident. In the microgamonts, normal evagination of microgametes was prevented; the flagellar complex formed within the main cytoplasmic mass and the differentiated nuclei remained present within the parasite body. The macro- and microgamonts also ended up in a stage of complete necrosis. These data indicate that diclazuril treatment primarily affects the normal differentiation of the respective endogenous stages during parasite development. This leads to complete degeneration of schizonts and gamonts indicating the lethal effect of this new anticoccidial compound.

Action of the anticoccidial clazuril on the endogenous stages of Eimeria labbeana and E columbarum in experimentally infected pigeons.

Racing pigeons were artificially infected with a mixed inoculum of Eimeria labbeana (85 per cent) and E columbarum (15 per cent) and treated orally with 2.5 mg clazuril either on day 1, 2, 3, 4, 5, 6 or 7 after infection. The impact of the treatment on the different developmental stages was evaluated by oocyst output and by histological examination of the duodenum and jejunum. The life cycle always became completely interrupted, but maximal effects were noted when treatment was given on day 4, 5 or 6 after infection. Treatment during patency completely interrupted oocyst excretion within three days after dosing. Degenerative changes in schizonts and gametocytes were always observed. The histology revealed a reduced number and abnormal structure of developing merozoites; a ballooned aspect and presence of numerous small vacuoles in the microgametocytes; the absence of typical wall-forming bodies in macrogametocytes and a complete absence of oocysts. It is concluded that clazuril has a coccidiocidal effect on the asexual and sexual developmental stages of both Eimeria species, resulting in a complete interruption of the life cycle.

[Safety of a new anticoccidial agent, clazuril, during reproduction in carrier pigeons]. / Veiligheid van een nieuw anti-coccidiosemiddel clazuril tijdens de kweekperiode bij postduiven.

In a six-month study, the safety of repeated treatments with clazuril, a new anticoccidial agent for the treatment of pigeons, was evaluated during 4 to 5 consecutive reproduction cycles. Clazuril was administered weekly at the recommended therapeutic dose level of 2.5 mg/pigeon and at twice this dose. The results were compared to placebo treatment. Evaluation was based on the following parameters: reproductive behaviour, egg laying, fertilisation, embryonic mortality, hatching, percentage of weaning and time interval between consecutive reproductive cycles. Maternal-paternal toxicity (body weight evolution, feather coat) and tolerance in newborn pigeons (general behaviour, daily gain in body weight, feathering, skeletal anomalies) were also evaluated. In all the treatment groups, reproductive performance was comparable and no drug- or dose-related side-effects were observed. Fertilisation, embryonic mortality and the time-interval between the consecutive reproductive cycles remained within the normal physiological range. Hatched pigeons showed a satisfactory daily weight gain and deaths among these birds were exceptional. Tolerance of clazuril in adult as well as in day-old pigeons was exceptionally good and body weight evolution, feathering and mortality were not adversely affected. No drug-related pathological findings or skeletal anomalies were detected in the animals at autopsy. It can be concluded that repeated treatments with clazuril are entirely harmless for pigeons during reproduction, even at double the therapeutic dose level. Clazuril is well tolerated by newborn and growing pigeons, which means that pigeon house group treatments during reproduction may be performed without any risk.

Spontaneous and experimental myofibrillar hypoplasia and its relation to splayleg in newborn pigs.

The relation of myofibrillar hypoplasia to clinical splayleg was studied. A strain of Belgian Landrace sows was selected for this study because they produced pigs which had no myofibrillar hypoplasia. Myofibrillar hypoplasia could nevertheless by induced experimentally in these animals by dexamethasone treatment of the sows during late pregnancy. The lesion was observed without clinical signs and was compared to the myofibrillar hypoplasia in clinical cases of splayleg. The differences between these 2 groups may account for the appearance of clinical signs. These differences included the maturity of the myofibrils and the degree of autophagolysosomal glycogen breakdown.

Demonstration of pseudorabies viral antigen in thick and ultrathin tissue sections of young pigs using an immunogold method.

Pseudorabies (Aujeszky's) virus antigens were labeled in thick and ultrathin tissue sections of young pig brain and liver tissue, using an indirect immunogold method. Antigens were tagged with 20 nm gold particles. Controls proved the specificity of the reaction in paraffin sections and ultrathin epoxy sections. Immunogold staining was compared with immunoperoxidase staining in paraffin sections. In ultrathin sections stained with the immunogold method, the gold particles were present on viral nucleocapsids and viral envelopes, as well as on a number of other intracellular structures. These included the inner nuclear membrane, the nucleoplasm, intranuclear filaments, the endoplasmic reticulum, and free cytoplasmic polyribosomes. Gold particles were absent on mitochondria and microtubules. In paraffin sections, immunogold labeling for pseudorabies virus antigen was less sensitive than immunoperoxidase staining. Immunogold staining of ultrathin tissue sections can yield additional information on virus-host cell interactions.

Space occupying lesions of cranial nerves in calves with facial paralysis.

Clinical and pathological data of a recently discovered neurological disorder in young calves were studied. The symptoms were those of a paralysis of the nervus facialis and a dysfunction of the nervus vestibulocochlearis. Macroscopically, space occupying lesions were found at the roots of these two cranial nerves and in some cases further on their course into the os petrosum. On histological examination, these legions contained multiple nodules with mainly histiocytic cells, some plasma cells and multinucleated giant cells. Around these nodules, dense bands of connective tissue with fusocellular fibroblasts, some plasma cells and small blood capillaries were present. The nodular lesions were usually in close contact with the nerve. Ultrastructurally, a variety of cells, predominantly histiocytes and some plasma cells, were seen next to Schwann cells which contained degenerating axons.

Branched Mycobacterium-like organisms from swine lymph nodes.

Branched, weakly acid fast organisms were isolated from 5 of 112 caseous lymph nodes derived from slaughter-house pigs. In two cases they were associated with typical Mycobacterium avium strains. The bacteria differed from all other mycobacteria known at present. Limited experiments in pigs with one strain failed to disclose a possible pathogenic role in the host species although this strain was pathogenic for mice. Tests with this and three other strains showed that all four were apathogenic for chickens.