Rudolf Bechter

Human health risk assessment of carbamazepine in surface waters of North America and Europe

A human health risk assessment was carried out for environmental exposures to carbamazepine (CBZ) and its major human metabolites, carbamazepine diol (CBZ-DiOH) and carbamazepine N-glucuronide (CBZ-N-Glu). Carbamazepine is an active pharmaceutical ingredient (API) used worldwide as a medicine for treating epileptic seizures and trigeminal neuralgia. Carbamazepine tends to be detected in surface water more frequently, and at relatively higher concentrations, than most other APIs. Predicted no effect levels (PNECs) for CBZ and its major human metabolites were developed for surface waters to be protective of human health from environmental exposures from drinking water and fish consumption. These PNECs were compared to both measured (MEC) and predicted (PEC) environmental concentrations for North America and Europe. PECs were calculated using the geo-referenced models PhATE for North America and GREAT-ER for Europe. The combined PNEC for drinking water and fish consumption for CBZ is 226,000ng/L. Ninetieth percentile MECs ranged from 150 to 220ng/L, while 90th percentile PECs ranged from 333 to 658ng/L. Calculated margins of safety (MOS) therefore range from 340 to 1500. MOS for the major metabolites are significantly higher. This assessment indicates that CBZ and its major metabolites have high MOS (>>1) and thus should have no appreciable risk to human health through environmental exposures based on available human data.

Interlaboratory evaluation of embryotoxicity in the postimplantation rat embryo culture

The embryotoxicity of eight xenobiotic compounds in rat postimplantation whole embryo culture was blindly tested in four laboratories according to a standard protocol. The results show that the four nonteratogens amaranth, penicillin, isoniazid, and saccharin did not affect embryogenesis apart from general toxicity at very high concentrations in culture for amaranth and isoniazid. There was good concordance of results across the laboratories. The four teratogens (retinoic acid, 6-aminonicotinamide, acetylsalicylic acid, and vincristine) induced a variety of specific embryotoxic effects, which were in most cases similar in all laboratories. These results indicate that the definition for specific embryotoxicity used, as well as the culture duration and embryonic age are crucial for concordant scoring. Other methodologic differences did not significantly influence scoring of embryotoxicity. Therefore, within the limits of the end points and embryonic stage represented in the method, embryo culture appears as a useful method for embryotoxicity screening, which can be reproducibly applied in different laboratories.

Flow cytometric evaluation of the effects of doxorubicin on rat spermatogenesis

Histopathologic examination of testicular tissue allows testicular impairment to be investigated. As an alternative to histopathology, flow cytometry (FCM) using a triple staining technique that combines DNA-ploidy with mitochondria stainability and vimentin immunostaining has also been utilized to evaluate testicular damage. In this article we evaluate the effects on spermatogenesis after acute exposure of rats to doxorubicin. Testicular cell suspensions of treated and control animals were analyzed by FCM. This allows several cell types to be identified and quantified, giving a control pattern. Deviations from this control pattern are considered as an indication of testicular damage. Doxorubicin produced a depletion of spermatogonia as early as 3 d after treatment. This effect could be followed through the temporal evolution of spermatogenesis. Comparable results were obtained by histopathology. The presented results show that FCM is a suitable and sensitive method for the detection of testicular damage. The advantages of FCM over other techniques include its rapidity and objectivity.

New and traditional approaches for the assessment of testicular toxicity

In this study, the suitability of several methods for the assessment of testicular damage, including histopathology, flow cytometry (FCM), testicular sperm head counts, and secretion of androgen binding protein (ABP), has been evaluated. Testicular toxicity after acute exposure of adult rats to different doses of the known toxicant 1,3-dinitrobenzene (DNB) was analyzed. The effects showed dose dependence, in spite of the large variability within each dose group. Histopathology and FCM showed germ cell depletion, particularly of round spermatids; testicular sperm head counts were reduced and ABP production was increased. All evaluated methods showed similar sensitivities. The increased testicular ABP levels support the theory that the Sertoli cell is the likely target of DNB induced testicular toxicity, producing subsequent germ cell depletion. The presented results show the suitability of FCM for the analysis of testicular damage and also support the usefulness of including a metabolic marker for Sertoli cell function.

Teratogenicity of arotinoids (retinoids) in the rat whole embryo culture.

Structural modifications of the arotinoid molecule RO 13-7410 led to a difference in the teratogenic potencies of more than five orders of magnitude in mice in vivo and in micromass cultures of rat embryonic limb bud cells (Kistler et al. 1990). Five of these retinoids were selected and tested in rat whole embryo culture to determine the suitability of this in vitro test system for the identification of potentially non-teratogenic derivatives among this class of chemicals. The highest concentrations of the compounds with no effects (NOAEL) on general conceptus growth, on differentiation and on the frequency of dysmorphogenic embryos in vitro were compared with the lowest effective teratogenic doses in vivo (LOAEL) or with the concentrations leading to 50% inhibition of limb bud cell differentiation (IC50) in vitro. NOAELs for the parameters of conceptus development ranged from 10−5 μg/ml (0.03 nM) to 10 μg/ml (28.7 μM) for the compounds tested. These correlated very well with LOAEL and IC50 (R >0.95). The types of dysmorphogenesis in vitro were those typical for retinoids, and for the most part resembled the malformations found in vivo. We conclude that the whole embryo culture system is a useful tool for the preliminary testing of retinoids.

Flow cytometry as a sensitive tool to assess testicular damage in rat

Abstract The aim of this study was to compare results obtained by flow cytometry (FCM) with those obtained from testicular histopathology with regard to testicular damage following acute exposure of adult rats to the known testicular toxicant, methoxyacetic acid (MAA). Special emphasis was given to defining the sensitivity of three-parameter FCM compared with testicular histopathology. Furthermore, the effect on the male reproductive system of a single oral dose of MAA was evaluated with traditional methods, e.g. testicular sperm head counts, and organ weights. Adult, male Han/Wistar rats were randomly assigned to four groups of ten animals to be treated with a single dose of 0, 65, 325 and 650 mg MAA/kg body wt. (p.o., gavage). The animals were killed 2 days after treatment, and testicular and epididymal weights were recorded. One testis and the corresponding epididymis were used for histopathology. The other testis was used partly to determine sonication- resistant, testicular sperm-head counts (SHC), and partly for enzymatic digestion followed by FCM. The results obtained in this study are in agreement with the literature, and show that, in the adult male rat, 2 days after administering a single oral dose of MAA, specific depletion of spermatocytes is evident. Detectable testicular effects were produced by the high (650 mg/kg body wt.) and mid (325 mg/kg body wt.) doses, whilst the low dose (65 mg/kg body wt.) did not produce any noticeable effect. There was a strong correlation between results obtained by FCM and those obtained by testicular histopathology, and no difference in sensitivity between the two methods was observed. In summary, three-parameter FCM represents a sensitive and reliable method for the detection of testicular injury in the rat. It requires only small amounts of tissue, and the sensitivity was shown to be similar to that of histopathology. Moreover, FCM has the advantages of being quick and objective, which permits large numbers of cells to be analysed. The potential use of this method as a fast screening tool for testicular toxicity in routine toxicology studies should be considered.

Bioavailability of therapeutic proteins by inhalation--worker safety aspects.

A literature review and analysis of inhalation bioavailability data for large therapeutic proteins was conducted in order to develop a practical estimate of the inhalation bioavailability of these drugs. This value is incorporated into equations used to derive occupational exposure limits(OELs) to protect biopharmaceutical manufacturing workers from systemic effects. Descriptive statistics implies that a value of 0.05, or 5% is an accurate estimate for large therapeutic proteins (molecular weight ≥ 40kDa). This estimate is confirmed by pharmacokinetic modeling of data from a human daily repeat-dose inhalation study of immunoglobulin G. In conclusion, we recommend using 5% bioavailability by inhalation when developing OELs for large therapeutic proteins.

The value of acute toxicity testing of pharmaceuticals for estimation of human response

The determination of single high doses of active pharmaceutical ingredients (API) is used mostly to fulfill regulatory demands. Oral LD(50) values in animals for over 300 API were compared to the minimal effective therapeutic doses (METD) in humans in order to find a correlation between animal and human data. The highest correlation between human METD and animal LD(50) was found for the dog (R=0.323), the lowest for the rat (0.287). It was determined that acute oral LD(50) of rats have poor correlation with the METD, and cannot be used as a classification criteria into official acute toxic categories. Only 13% of API has been classified as fatal if swallowed according to the EU CLP regulation, none of the substances with very low therapeutic dose have been identified as EU CLP acute toxicity category 1. Substances with very low therapeutic doses, which could potentially have toxic effects in humans, are not identified with the use of oral LD(50) and current classification system. We propose that the acute toxicity based on rat LD(50) dose is not used as a basis for classification of pharmaceuticals, and that the METD is applied as basis for classification.

Comparison of the metabolic activity of yolk sac tissue in the whole embryo and isolated yolk sac culture.

In rat visceral yolk sac tissue cultured from 10.5 to 12.5 days of prenatal age, metabolism of the lipoxygenase inhibitor N-hydroxy-N-methyl-7-propoxy-2-naphtalinethanamine (QA 208-199, QAB) and the accumulation of its metabolites have been shown previously. In this study, the metabolic activities of visceral yolk sac tissues cultured either alone or together with the embryo were compared. The metabolite patterns in medium and visceral yolk sac tissue generated by intact conceptuses or by isolated visceral yolk sac tissues were similar. After 24 as well as 48 h of culture, the major in vivo metabolite, 7-propoxy-naphthalene-2-ylacetic acid (QAA) and other, as yet unidentified metabolites, accumulated in embryo proper and visceral yolk sac tissues. QAB was not found in the embryo proper, and was only found in yolk sac tissues using the higher concentration. In particular the metabolites M5 and M6 exhibited a massive accumulation in the visceral yolk sac, whereas QAA, M3, and M4 accumulated to a much lesser degree. In isolated yolk sacs cultured for 48 h, tissue levels of QAA and M4 were similar to those in yolk sacs of cultured whole conceptuses, whereas M5 and M6 exhibited twofold higher levels in isolated yolk sacs. These findings were in agreement with the distinct increase of myeloid figures containing partly fragmented inclusion bodies in yolk sac tissues. These results suggest that the visceral yolk sac may be the major site of QAB metabolism in cultured rat conceptuses in vitro.

Cancer risk of immunosuppressants in manufacturing

During the chemical and pharmaceutical production of active pharmaceutical substances which are intended for immunosuppressive therapy, the employees may be exposed to these substances via inhalation. Immunosuppressants are linked to development of certain types of cancers e.g., lymphoma or skin cancer in transplant patients. The development of these cancers in patients is linked to the level of immunosuppression needed for transplantation in order to avoid organ rejection. Below these levels, with the immune system functioning uninhibited, cancer is unlikely to develop. An internal workshop was conducted to compare several pharmaceutical substances with the intrinsic property to cause immunosuppression, with the attempt to define the risk of healthy employees to develop cancer due to exposure to immunosuppressive substance at work and to determine the appropriate hazard classification for regulatory purposes. Data are discussed with emphasis on cyclosporine to reason the dose-response relationship and the safe level for occupational exposure. Our review indicates that if the exposure to cyclosporine at the workplace is below the threshold necessary to induce immunosuppression, the risk to develop cancer is negligible. Non-mutagenic immunosuppressants do not contribute to malignancies in occupational setting if their air concentrations do not exceed the immunosuppressive threshold limited with occupational exposure limits (OELs), which is for cyclosporine 17.5μg/m(3).