Harmonisation of rat fetal skeletal terminology and classification. Report of the Third Workshop on the Terminology in Developmental Toxicology. Berlin, 14-16 September 2000.

The initial efforts of the Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV) and the Free University of Berlin to standardise terminology in the field of developmental toxicology began in 1995. Procedures were undertaken to harmonise the terminology used by the International Federation of Teratology Societies (IFTS) and the International Programme on Chemical Safety (IPCS). This article reflects these activities and is a report on the Third Workshop on the Terminology in Developmental Toxicology held in September 2000. This Workshop served as a forum to discuss the results of a survey on the classification of skeletal anomalies that had been previously sent to scientists active in the field. Although high agreement was reached among the evaluators for several terms, the use of a number of terms was rather variable. Therefore, the discussions at the workshop among the experts from research institutions, regulatory agencies, and industry were mainly focussed on those terms for which there was disagreement and/or uncertainties and the possible reasons. Pictures provided by the participants for the illustration of "grey zone" anomalies constituted the basis for detailed discussions. In many of the cases with lower agreement, decisions were facilitated by the provision of the corresponding picture. The main reasons for lower agreement were imprecise terms, insufficient knowledge on postnatal consequences, theoretical terms that are unlikely to occur in isolation, and the possibility of observing a range of severity that might be decisive for the classification of either a malformation or variation. The attendees concluded that "grey-zone" anomalies will never disappear completely and that for the assessment, the grade of severity and/or the frequency of the observation can be decisive for the terminology chosen. A Joint IPCS/IFTS Project was proposed to further consensus of terminology and classification and to link these anomalies to pictures at different skeletal sites. In order to support the harmonisation of regulatory decisions, it was proposed to establish a "Clearinghouse" System under the umbrella of the IPCS. The Clearinghouse could be contacted either by the regulatory authorities or by any company to clarify their queries, particularly with regard to registration or authorisation processes. Finally, it was recommended to also carry out a similar survey on "soft tissue anomalies" and "external findings." The results of this survey will be discussed at a Joint IPCS/IFTS Workshop in Berlin in 2002.

Active site residue 297 of Aspergillus niger phytase critically affects the catalytic properties.

The wild-type phytases from the Aspergillus niger strains NRRL 3135 and T213 display a three-fold difference in specific activity (103 versus 32 U/mg protein), despite only 12 amino acid differences that are distributed all over the sequence of the protein. Of the 12 divergent positions, three are located in or close to the substrate binding site. Site-directed mutagenesis of these residues in A. niger T213 phytase showed that the R297Q mutation (R in T213, Q in NRRL 3135) fully accounts for the differences in catalytic properties observed. Molecular modelling revealed that R297 may directly interact with a phosphate group of phytic acid. The fact that this presumed ionic interaction - causing stronger binding of substrates and products - correlates with a lower specific activity indicates that product (myo-inositol pentakisphosphate) release is the rate-limiting step of the reaction.

Absorption of tretinoin in rats and rabbits following oral and dermal application.

To assess the teratogenic potential of Airol cream (0.05% tretinoin, CAS 302-79-4), plasma concentrations were compared after repeated dermal applications of the maximum dose which could be applied reliably and after oral administration of the highest non-teratogenic dose. The test preparation was applied dermally in two equal portions to rats and rabbits at 2 g/animal/d (equivalent to a tretinoin dose of 3.7 mg/kg/d) and at 6 g/kg/d (equivalent to 3 mg tretinoin/kg/d), respectively. After a single oral administration of 2 mg/kg, Cmax and AUC for tretinoin in rat plasma were 285 +/- 14.6 ng/ml and 595 +/- 123 ng h/ml, respectively. Corresponding values for the rabbit were 78.4 +/- 16.9 ng/ml and 126 +/- 25.4 ng h/ml. In both species, plasma concentrations of tretinoin after dermal application were consistently below the assay quantification limit (< 5 ng/ml and < 2 ng/ml for rat and rabbit, respectively), despite marked irritation of the skin. Thus repeated topical application of the test preparation produced plasma concentration of tretinoin which were well below the plasma concentrations produced by a non-teratogenic oral dose of 2 mg/kg in the rat and rabbit.

Cardiovascular alterations in rat fetuses exposed to calcium channel blockers.

Preclinical toxicologic investigation suggested that a new calcium channel blocker, Ro 40-5967, induced cardiovascular alterations in rat fetuses exposed to this agent during organogenesis. The present study was designed to investigate the hypothesis that calcium channel blockers in general induce cardiovascular malformations indicating a pharmacologic class effect. We studied three calcium channel blockers of different structure, nifedipine, diltiazem, and verapamil, along with the new agent. Pregnant rats were administered one of these calcium channel blockers during the period of cardiac morphogenesis and the offspring examined on day 20 of gestation for cardiovascular malformations. A low incidence of cardiovascular malformations was observed after exposure to each of the four calcium channel blockers, but this incidence was statistically significant only for verapamil and nifedipine. All four agents were associated with aortic arch branching variants, although significantly increased only for Ro 40-5967 and verapamil.

Embryotoxic doses of vitamin A to rabbits result in low plasma but high embryonic concentrations of all-trans-retinoic acid: risk of vitamin A exposure in humans.

Retinoid pharmacokinetics were examined in plasma, placenta and embryos of gestational d 12 rabbits following application of an embryotoxic dosing regimen (10 mg retinyl palmitate/kg body wt per day from gestational d 7 to 12). Vehicle-treated or untreated rabbits served as controls. Physiological concentrations of all-trans-retinoic acid (all-trans-RA) and 13-cis-RA in rabbit plasma (5-8.33 nmol/L) were very close to the endogenous levels in human plasma. In addition, we identified endogenous all-trans-RA, 3,4-didehydroretinol and 3,4-didehydroretinoic acid in rabbit embryo. Following the last retinyl palmitate administration, apparent steady-state concentrations of all retinoids were reached in the examined compartments of rabbits. The major polar retinoid in plasma was 9, 13-di-cis-RA, but its embryonic concentrations were only about 6% of those in plasma. In the embryo, retinol and its esters were found at high concentrations; lower amounts of all-trans-4-oxo-RA and the newly identified 14-hydroxy-4, 14-retro-retinol could also be measured. Embryonic concentrations of all-trans-RA were about 100% higher than endogenous levels. The overall exposure of the embryo to this retinoid was, however, substantial. Embryonic area under the concentration time curve values strongly suggest that the embryotoxicity of the applied dosing regimen is mainly due to the action of all-trans-RA. A very remarkable finding of this study is the marginal increase of plasma concentrations of all-trans-RA over their endogenous levels, which is comparable to the human situation after vitamin A intake. This analogy indicates that high vitamin A intake may be associated with a higher risk for teratogenic effects in humans even in the absence of high elevation of plasma all-trans-RA levels.

Single versus multiple dose administration of all-trans-retinoic acid during organogenesis: differential metabolism and transplacental kinetics in rat and rabbit.

Standard teratogenicity testing is usually performed by administration of a test compound daily throughout an extended period of organogenesis (e.g., between Days 6 and 15 in rat and 6 and 18 in rabbit). On the other hand, single dose experiments during a specific period were often demonstrated to be more effective in unveiling a particular teratogenic effect. We have assessed here if toxicokinetics is an important factor for the interpretation of the differences between two administration regimens of all-trans-retinoic acid (all-trans-RA) in two species. The transplacental pharmacokinetics of a low teratogenic dose of all-trans-RA administered orally were compared in a single versus multiple dose regimen in both the Wistar rat and the Swiss hare rabbit. In both species, the single dose animals were treated on Gestational Day 12, while the multiple dose animals received daily doses from Gestational Days 7 through 12. Pharmacokinetic profiles were determined for maternal plasma and embryo after dosing on Gestational Day 12 (for both the single and multiple dose regimens) and analyzed by reverse-phase HPLC. The dose used for both species was 6 mg/kg body wt/day which has recently been reported to be a marginal to low teratogenic dose when administered daily throughout organogenesis. In both rat and rabbit, the AUC of all-trans-retinoic acid in maternal plasma was much reduced (factor of 9 in the rat, factor of 2 in the rabbit) after multiple application as compared to the single administration, presumably due to enzyme induction. A similar, but not as pronounced effect was also observed in the embryo of both species. This diminished effect in the embryo indicates a relative increase of placental transfer at the lower maternal plasma concentration observed after multiple dosing, which may possibly be due to an increased availability of binding sites such as cytosolic retinoic acid binding protein and nuclear receptors in the embryo. In the rat, also the metabolite levels were reduced, while in the rabbit, the metabolites of the 13-cis-configuration were concomitantly increased. Our results suggest that multiple administration of a drug such as retinoic acid, which induces its own elimination pathways, results in substantially lowered drug levels in maternal plasma and embryo.(ABSTRACT TRUNCATED AT 400 WORDS)

Ro 42-1611 (arteflene), a new effective antimalarial: chemical structure and biological activity.

The discovery of the natural peroxides qinghaosu (arteannuin A, artemisinin) (1) and yingzhaosu A (3) from traditional Chinese herbal medicines was a major advance in the search for new antimalarials (Fig. 1). Whereas qinghaosu can be produced from natural sources and has been well studied, yingzhaosu A has never been available for full evaluation as anti-malarial. We have designed a synthesis of the novel ring system present in yingzhaosu A, the 2,3-dioxabicyclo[3.3.1]nonane and prepared a series of yingzhaosu A analogues which were tested against Plasmodium berghei in mice. Structure-activity rules could be established and used for lead optimization. The best anti-malarial activity was observed for analogues having a keto group within the ring system and an aliphatic or aromatic lipophilic tail as ring substituent. The optimized analogues possessed activity comparable to qinghaosu. In spite of the presence of a peroxide ring, the new compounds were chemically stable against common reagents. In contrast to qinghaosu and its derivatives, they were also stable against hydrolytic decomposition and could therefore be expected to show improved pharmacokinetic properties. As one of the best compounds, Ro 42-1611 (arteflene) (26n, Fig. 2) was selected for detailed preclinical evaluation. Ro 42-1611 (arteflene) was found negative in a battery of mutagenicity tests. It had low acute toxicity after oral or subcutaneous administration. In a 4-week oral tolerance study in rats, doses of up to 400 mg/kg/day were well tolerated.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative teratology and transplacental pharmacokinetics of all-trans-retinoic acid, 13-cis-retinoic acid, and retinyl palmitate following daily administrations in rats.

The retinoids are teratogenic in a wide variety of species. In the rat, 13-cis-retinoic acid and retinyl palmitate are significantly less potent teratogens than all-trans-retinoic acid. This investigation questioned whether differing teratogenic potencies of these moieties can be correlated with the concentrations of these drugs and/or metabolites in the embryonic compartment. Approximately equipotent teratogenic doses of these three retinoids were administered and the pharmacokinetics in maternal plasma and embryo of the most prevalent vitamin A metabolites were measured. The glucuronides of the respective retinoids were the predominant metabolites in the maternal plasma, but were not detected in the embryo. Also, the transport of 13-cis-retinoic acid across the placenta occurred to a much lesser extent than the transport of all-trans-retinoic acid. Administration of either all-trans- or 13-cis-retinoic acid causes a depression in the endogenous retinol concentration. This depression is more pronounced in the maternal plasma than in the embryo. The depression of the retinol level in both plasma and embryo after 13-cis-retinoic acid administration (75 mg/kg/day) was greater than the depression after all-trans-retinoic acid (6 mg/kg/day), corroborating the inferential teratological data that the 13-cis-retinoic acid dose was more embryotoxic than the all-trans-retinoic acid dose. Although the dose of all-trans-retinoic acid was less embryotoxic than that of either 13-cis-retinoic acid or retinyl palmitate, the embryonic exposure to all-trans-retinoic acid was considerably larger, as determined by maximum concentration or area under the concentration-versus-time curve, after administration of all-trans-retinoic acid than after either retinyl palmitate or 13-cis-retinoic acid application. These results suggest that embryonic retinoids other than all-trans-retinoic acid--including the administered substances themselves--are important in the teratogenic process induced by 13-cis-retinoic acid and retinyl palmitate.

The high sensitivity of the rabbit to the teratogenic effects of 13-cis-retinoic acid (isotretinoin) is a consequence of prolonged exposure of the embryo to 13-cis-retinoic acid and 13-cis-4-oxo-retinoic acid, and not of isomerization to all-trans-retinoic acid.

Previous studies suggested that the rabbit is much more susceptible to the teratogenic action of 13-cis-retinoic acid (13-cis-RA) than the mouse or the rat, while the teratogenicity of all-trans-RA was comparable in these species. In the present study we investigated if pharmacokinetics can explain these species- and structure-related differences. The embryotoxic and teratogenic potential of all-trans-retinoic acid (all-trans-RA) and 13-cis-RA were evaluated in the Swiss hare rabbit after oral administration of daily doses of the two drugs throughout organogenesis, from gestation day (GD) 6 to 18 (plug day = GD 0). All-trans-RA was given at dose levels of 0.7, 2 or 6 mg/kg body weight per day and 13-cis-RA at 3, 7.5 or 10 mg/kg per day. The doses needed to elicit a minimum teratogenic response were found to be 6 mg/kg per day for all-trans-RA and 10 mg/kg per day for 13-cis-RA. Using these doses, transplacental pharmacokinetics of all-trans- and 13-cis-RA were performed. Pregnant rabbits were treated once daily from GD 7 to 12 and plasma and embryo samples were collected for HPLC analysis at various time intervals after the final dose. The main plasma metabolites of all-trans- and 13-cis-RA were all-trans-beta-glucuronide (all-trans-RAG) and 13-cis-4-oxo-RA, respectively. The elimination of 13-cis-RA and its metabolites from maternal plasma were much slower than of all-trans-RA resulting in accumulation of the 13-cis-isomers in plasma. Marked differences in the placental transfer of the two drugs and their metabolites were observed. All-trans-RA and all-trans-4-oxo-RA were efficiently transferred to the rabbit embryo, reaching concentrations similar to the plasma levels. On the contrary, the 13-cis-isomers reached the embryo to a lesser extent. Despite its limited placental transfer, a considerable embryonic exposure to 13-cis-RA and 13-cis-4-oxo-RA was noticed after treatment with isotretinoin, as indicated by their area-under-the-concentration-time-curve (AUC) values in the embryo, which were in the same range as the corresponding AUC value of all-trans-RA after treatment with the all-trans-isomer.(ABSTRACT TRUNCATED AT 400 WORDS)

Embryolethality of new herbicides is not detected by the micromass teratogen tests.

New herbicidal compounds (11 pyrimidine-diones, 3 benzoates and 1 sulfonamide) were found to be embryolethal but not teratogenic in rats. The range of the embryolethal dose varied from 0.2 to greater than 200 mg/kg. This broad range enabled us to validate whether proposed in vitro teratogen tests can detect the embryolethality of these herbicides. The IC50 values (inhibition concentration 50%) for both differentiation and proliferation of midbrain and limb bud cells of rat embryos were determined and found to be above 50 micrograms/ml in all cases, confirming that the herbicides were not teratogenic. No correlation, however, was observed between the embryolethality in vivo and the activities in these cells. In order to test whether the potential to cause embryolethality could be predicted and detected as a general cytotoxic effect, the inhibition of colony forming ability in V79 cells was determined. The results indicated that cytotoxicity in V79 cells may be useful for preliminary testing of the embryolethal effect of herbicides.

Toxicological investigations with the benzodiazepine antagonist flumazenil.

The benzodiazepine antagonist ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate (flumazenil, Ro 15-1788, Anexate) was investigated in a series of toxicological studies. In a single intravenous injection study in male and female mice and rats, the highest non-lethal (maximum tolerated) doses were found to be between 62.5 and 125 mg/kg (the amounts of flumazenil present in the aqueous ampoules available for intravenous injection in man are 0.5 and 1.0 mg). In intravenous studies of 4 weeks duration, 10 mg/kg/d were systemically well tolerated in dogs and rats. In 13-week oral studies, 80 mg/kg/d were very well tolerated in dogs (capsule administration) and, after 125 mg/kg/d (by feed-admix) in rats, no untoward compound-related findings apart from a 10-15% increase of the liver weights in females were made. In reproductive toxicity studies, flumazenil revealed no drug-related embryotoxic or teratogenic effect and no adverse effects upon fertility of dosed animals themselves or on the peri- and postnatal development of their offspring. There was no indication for mutagenic potential of flumazenil in vitro concerning induction of gene mutations or clastogenic effects. An in vivo DNA repair test using germ cells of male mice did not yield DNA-damaging activities. From all these toxicological investigations it can be concluded that the risk in man given therapeutic doses or even intentional or accidental overdoses of flumazenil is extremely small.

Assessment of DNA damage in germ cells of male rabbits treated with isoniazid and procarbazine.

The 2 hydrazine derivatives isoniazid (INH) and procarbazine hydrochloride (P) were injected intravenously into rabbits. Radioactive thymidine was injected into both testicles. Rabbits were ejaculated repeatedly, sperms were counted and incorporation of [3H] thymidine into sperm head DNA was determined by liquid scintillation counting. In P-treated rabbits (5 and 50 mg/kg) radioactivity was significantly increased in sperms that were in late phases of spermatocyte and early phases of spermatid maturation at the time of treatment. This indicates that DNA repair synthesis, (unscheduled DNA synthesis, UDS) occurred following drug-induced DNA damage in these germ cells. Normal DNA synthesis in spermatogonia was inhibited by the high dose only. INH (50 and 125 mg/kg) did not cause UDS in spermatocytes and spermatids and did not affect normal DNA synthesis in spermatogonia. The results are in agreement with literature data indicating that P is a potent mutagen and carcinogen. INH, on the other hand, has weak mutagenic and carcinogenic activities that are most apparent in mice.

The relation between net calcium, alkali cation and chloride movements in red cells exposed to salicylate.

1. From a 150 mM-NH4 salicylate medium salicylate enters human red cells with a rate constant of 1.9 min-1 at 0 degrees C. 2. Salicylate increases the red cell permeability for Ca2+ (and Mg2+). There is no saturation of the Ca2+ transfer with respect to salicylate up to 150 mM and with respect to external Ca2+ up to 30 mM. 3. Ca2+ entering from salicylate media activates the Ca-sensitive K channel present in human but not in adult ruminant red cells. 4. The increase in K permeability which ensues hyperpolarizes the membrane in Na salicylate media, accelerating further Ca2+ entry and Mg2+ entry and favouring Cl- loss (see Fig. 8). The Ca2+ inward movement is in agreement with the constant field equation if the membrane potential is assumed to equal the K equilibrium potential and if two charges are attributed to the mobile species. 5. The effect of salicylate on Ca2+ permeability and hence its sequelae are reversible upon washing the cells. 6. 3-OH-benzoic acid and 4-OH-benzoic acid do not exert the effect salicylate has on Ca2+ permeability. 7. In 150 mM-Na salicylate media the Cl--salicylate exchange is virtually nil at 0 degrees C. The exchange seen at 19 degrees C is obviously not across the anion exchange mechanism and proceeds at a rate comparable to that for Cl- movement in the nonexchange-restricted mode given by Hunter (1971, 1977) for cells in a normal medium. 8. Ca2+ seems to increase the Cl- permeability seen under these conditions. 9. The possibility that salicylate acts as an ionophore for Ca2+ is discussed.