Postnatal investigation of prenatally induced effects on the vertebral column of rats reduces the uncertainty of classification of anomalies.

Classification of substances as teratogenic is based on the observation of external, visceral and skeletal anomalies. Characterization of anomalies as variation or malformation is contingent upon their postnatal persistence and adversity to health. Lack of information thereof may result in inconsistent or incorrect classification. The aim of this work is the examination of vertebral skeletal anomalies regarding their postnatal fate on PNDs 7 and 21. The anomalies unossified, asymmetric ossification, bipartite ossification, hemicentric, as well as misshapen, did not persist up to PND21 and should be classified as a variation. The finding, cervical vertebra centrum dumbbell-shaped, should be categorized as a malformation due to its continued presence on PND 21. Lumbar centrum supernumerary sinister/dexter/sinister+dexter should also be classified as a malformation. This study demonstrates that postnatal examination is useful and substantially improves the ability to perform a scientifically sound classification of an anomaly compared to investigations terminated on GD 21.

Assessment strategies and decision criteria for pesticides with endocrine disrupting properties relevant to humans.

There is growing concern that environmental substances with a potential to modulate the hormonal system may have harmful effects on human health. Consequently, a new EU regulation names endocrine disrupting properties as one of the cut-off criteria for the approval of plant protection products, although it currently fails to provide specific science-based measures for the assessment of substances with such properties. Since specific measures are to be presented by the European Commission in 2013 the development of assessment and decision criteria is a key challenge concerning the implementation of this new EU regulation. Proposals of such decision criteria for substances with potential endocrine disrupting properties in human health risk assessment were developed by the German Federal Institute for Risk Assessment (BfR) and discussed at an expert workshop in November 2009. Under consideration of the requirements laid down within the new plant protection product legislation and the scientific discussions during the workshop, a conceptual framework on evaluation of substances for endocrine disrupting properties in a regulatory context is presented in this paper. Central aspects of the framework include assessment of adversity of effects, establishment of a mode/mechanism of action in animals, considerations concerning the relevance of effects to humans and two options for a regulatory decision.

Effects of fentin hydroxide on reproduction of the Japanese quail (Coturnix coturnix japonica).

In a one-generation reproductive study, the fungicidal compound triphenyltin hydroxide (fentin) was administered to adult Japanese quail for 6 weeks at dietary levels of 3 and 30 ppm. Reproduction was significantly impaired in the high-dose group. The principal adverse finding was a marked increase in embryonic mortality, resulting in a lower hatch rate. Furthermore, a reduction in egg production was observed with ongoing duration of treatment. Most of the other reproduction-related parameters were not affected. The in ovo losses are assumed to result from a direct toxic effect of the test substance on chick embryos. At the low dietary level, reproduction was not altered. In contrast to the obvious reproductive toxicity, there was only limited evidence of adverse treatment-related findings in the adult birds. However, because such minor effects as an increase in mean liver weight, which was accompanied by macroscopic liver findings and a decrease in T4 serum concentration, were still seen at 3 ppm, a no-observed-effect level could not be established.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification.

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

Effects of vinclozolin on spermatogenesis and reproductive success in the Japanese quail (Coturnix coturnix japonica).

In a one-generation reproduction study, the major agricultural fungicide vinclozolin was administered to adult Japanese quail for a period of 6 weeks at dietary levels of 125 and 500 ppm. Fertility and reproductive performance were not affected up to the highest concentration, although the examination of additional endpoints in the drakes (spermatid count, histology of the testis) provided some evidence of an inhibition of spermatogenesis at both dietary concentrations. Likewise, there were no indications of systemic toxicity in the adult birds. Plasma hormone concentrations (estradiol, testosterone, progesterone, T3, and T4) showed a large interindividual variance but treatment-related differences between the groups could not be established. There were no clear-cut indications of antiandrogenic effects in quail, although a limited transfer of the test substance into the eggs was proven.

In utero exposure to low doses of bisphenol A lead to long-term deleterious effects in the vagina.

The origins of the "endocrine disrupter hypothesis" may be traced to reports on adolescent daughters born to women who had taken the highly potent synthetic estrogen, diethylstilbestrol, while pregnant, and who developed a rare form of vaginal cancer and adenocarcinoma. Bisphenol A (BPA) is an estrogenic chemical that is highly employed in the manufacture of a wide range of consumer products. Some observational studies have suggested that the amounts of BPA to which we are exposed could alter the reproductive organs of developing rodents. We examined the influence of BPA at low doses to address the questions of (a) whether in utero exposure affects the vagina of the offspring and (b) which mechanisms cause the toxic effects. Gravid Sprague-Dawley dams were administered either 0.1 (low dose) or 50 mg/kg per day BPA, the no observed effect level, or 0.2 mg/kg per day 17 alpha-ethinyl estradiol by gavage. Striking morphological changes were observed in the vagina of postpubertal offspring leading us to examine vaginal estrogen receptor (ER) expression because BPA binds to the ER alpha, which is important for growth of the vaginal epithelium. We show that the full-length ER alpha is not expressed during estrus in the vagina of female offspring exposed to either dose of BPA when compared to the control group, whereas ER alpha expression does not differ from the control group during the diestrus stage. ER alpha downregulation seems to be responsible for the observed altered vaginal morphology.

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.

Embryotoxicity of meglumine antimoniate in the rat.

Meglumine antimoniate (MA) is a pentavalent antimonial (Sb(V)) drug used to treat leishmaniasis. Despite the fact that Sb(V) organic compounds have been used in clinical practice for more than 50 years, information on their safety during pregnancy is still scanty. This study was undertaken to evaluate the embryo/fetotoxicity of MA in the rat. Wistar rats were treated subcutaneously (s.c.) with MA (300 mg Sb(V)/kg body wt/day) on days 6 through 15 of pregnancy or with a higher dose (3 x 300 mg Sb(V)/kg body wt) on day 11 only. A control group treated with saline on days 6 through 15 and an untreated control group were evaluated as well. Cesarean sections were performed on day 21. No maternal toxicity and no reduction of fetal weight were noted in the groups treated with MA. The repeated administration of MA (days 6 through 15), but not the acute treatment (day 11), enhanced embryolethality. Treatment with MA on days 6 through 15 also caused a higher incidence of an atlas bone anomaly that occurs spontaneously at very low frequencies in our rat strain. These findings indicated that repeated administration of MA was embryolethal and teratogenic in rats.

Developmental toxicity of an octyltin stabilizer in NMRI mice.

The octyltin stabilizer ZK 30.434 is a mixture of 80% dioctyltin diisooctylthioglycolate (DOTTG) and 20% of monooctyltin triisooctylthioglycolate (MOTTG) and is used as stabilizer for rigid polyvinylchloride (PVC) materials. One of the applications of such stabilized films is the packaging of foodstuffs. Exposure to humans occurs via migration of DOTTG/MOTTG from PVC materials. In the present study the developmental toxicity of DOTTG/MOTTG in NMRI mice was investigated. Dams were treated orally with doses of 20, 30, 45, 67, or 100 mg/kg/day DOTTG/MOTTG from gestation day 6 through 17 (plug = day 1). Resorption rates were significantly increased and fetal weights significantly reduced in the study group at the 2 highest doses. External anomalies, such as bent forelimbs, cleft palate, and exencephaly were reported in the group treated with 100 mg/kg/day DOTTG/MOTTG, with the 67-mg/kd dose also exhibiting a significant increase in cleft palate. Moreover, an increase in skeletal anomalies was reported in fetuses exposed to 100 mg/kg/day. The doses of 20, 30, and 45 mg/kg/day elicited a significant increase in supernumerary lumbar ribs. It can be concluded that DOTTG/MOTTG is embryo-fetotoxic and induces developmental effects. The study revealed the need for the establishment of different No-Observed Adverse Effect Levels (NOAEL) for the endpoints investigated.

Toxicological evaluation of a tea from leaves of Vernonia condensata.

Teas of Vernonia condensata Baker (Asteraceae) are widely used in Brazil for gastro-intestinal disorders and to treat several other diseases. In this study, we evaluated the acute toxicity, embryotoxicity and mutagenicity of a lyophilized aqueous extract (LAE) from V. condensata leaves. Single doses of LAE, up to 5000 mg/kg body weight, were given orally or intraperitoneally to male and female Swiss albino mice. No toxicity was observed after oral administration. The "Approximate Lethal Dose" after intraperitoneal injections was 3400 mg/kg for males and 5000 mg/kg for females. Embryotoxicity was investigated in Han:NMRI mice. LAE (0, 500 and 2000 mg/kg/day) was given by gavage on days 10, 11 and 12, and dams were submitted to caesarean sections on day 18 of pregnancy. Fetuses were weighed, examined for externally visible malformations, and evaluated for skeletal anomalies. Except for a slight reduction of fetal body weight accompanied by signs of delayed ossification at the highest dose, no other embryotoxic effect was noted in the exposed offspring. LAE-induced mutagenicity was evaluated in the Salmonella/microsome assay without and with S9 mixture. LAE, tested up to 5000 microg/plate, was not mutagenic to tester strains TA97a, TA98 and TA100. Results therefore suggest that V. condensata aqueous extracts present low acute toxicity and pose neither teratogenic nor mutagenic risks.

Developmental toxicity of the HIV-protease inhibitor indinavir in rats.

BACKGROUND: Indinavir is an antiviral agent used for the treatment of HIV infection. We studied its developmental toxicity in rats. METHODS: Pregnant animals were treated orally with 500 mg indinavir/kg body weight (bw) from day 6 to 15 of gestation (once daily) or from day 9 to 11 (twice daily). Fetuses were evaluated for external and skeletal anomalies on day 21 of gestation. In addition, 19 rats were treated from day 9 of gestation to day 24 postnatally with 500 mg indinavir/kg bw once daily; a control group of 17 rats was treated with the vehicle accordingly. Developmental landmarks were recorded. Sixteen offspring each were studied on postnatal days 7, 14, 21, and 35 for hepatic enzyme activity. Liver tissue was examined by electron microscopy. RESULTS: Fetal examination on day 21 of pregnancy showed no treatment-related effects on number, weight, and viability of the fetuses; however, an increased incidence was noted in the supernumerary ribs and variations of the vertebral ossification centers in both indinavir-treated groups. Postnatal evaluation showed delayed fur development, eye opening, and descensus testis. The most striking finding was unilateral anophthalmia, observed in 7 pups (3%) from 2 out of 19 litters exposed to indinavir, but not in controls. Only minor changes in hepatic monooxygenase activities occurred in dams. Electron microscopy of liver samples showed hepatocellular inclusions of lipids and myelin figure-like structures in maternal livers and infiltration with granulocytes in offspring livers. CONCLUSIONS: Further studies on reproductive toxicity, including combinations of three or more antiretroviral agents as used therapeutically, are needed to determine the hazards of such a treatment.

PCDDs, PCDFs, PCBs, and other organochlorine compounds in human milk from Rio de Janeiro, Brazil.

The levels of polychlorinated dibenzo-p-dioxins (PCDDs) and -furans (PCDFs), polychlorinated biphenyls (PCBs), and other organochlorine compounds were determined in a pooled sample of breast milk from 40 mothers (first lactation: 33; second lactation: 7, age: 15-38 years) living in the urban area of Rio de Janeiro County, Brazil, in 1992. Mothers were breastfeeding only one infant and milk was collected between 4 and 6 weeks after delivery. The results showed a dioxin equivalent concentration of 8.1 pg I-TEq/g milk fat. The levels of other chlorinated compounds (micrograms per gram of milk fat) were as follows: PCBs (total), 0.15; alpha-HCH, 0.001; beta-HCH, 0.27;gamma-HCH, 0.005; HCB, 0.012; DDT (total), 1.7; dieldrin, 0.023, and cis-heptachlor epoxide, 0.008. These results suggest that human background contamination by PCDD/Fs, PCBs, and HCB in Rio de Janeiro is lower than that generally found in industrialized countries.

Establishment and characterization of the follicular thyroid carcinoma cell line ML-1.

The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.

Male reproductive toxicity and beta-luteinizing hormone gene expression in sexually mature and immature rats exposed to 2-bromopropane.

1. The reproductive effects of 2-bromopropane (2-BP) in sexually mature and immature male Sprague-Dawley rats were investigated. The animals were randomly divided into three treatment groups and one control group each of which comprised six mature and six immature rats. The treated groups were injected s.c. 200, 600 and 1800 mg/kg of 2-BP on 5 days a week for 5 - 7 weeks and the control group received the vehicle. 2. The absolute and relative testis weights were significantly reduced in 600 and 1800 mg/kg b.w. dose groups in both mature and immature rats. The absolute epididymis, prostate, seminal vesicle, and pituitary weights and the relative epididymis weights, however, were significant only at the highest dose level used in both mature and immature rats. 3. The sperm concentration and sperm viability in epididymal duct decreased and the percentage of abnormal sperm increased in a dose-dependent manner in both mature and immature rats. Additionally, serum testosterone level was significantly decreased in all dose groups in mature rats, and was significantly reduced only in the group treated with the middle and highest dose in immature rats. 4. In both mature and immature rats treated with 200 and 600 mg/kg, the seminiferous tubules were atrophied and all types of germ cells were decreased in number. At the highest dose level, the effect was more marked showing severely atrophied seminiferous tubules and a complete loss of all types of germ cells. 5. The mating, pregnancy and fertility indices were significantly reduced in the 600 and 1800 mg/kg groups. Additionally, at the highest dose group the number of implantations and viable fetuses per litter were reduced and the resorption rate was increased significantly. 6. In the mature rats, the beta-LH gene expression increased significantly in the 1800 mg/kg group when compared to the control group. 7. It can be concluded that 2-BP induces alterations in both neuro-endocrine axis and the reproductive tract under the present experimental conditions. The no observed adverse effect level (NOAEL) in this study could be estimated to be lower than 200 mg/kg/b.w. based on the alteration in testicular morphology as well as on sperm parameters observed at the dose level of 200 mg/kg.

Correlation between maternal toxicity and embryo/fetal effects.

It has been widely debated whether embryo/fetal toxicity is secondary to maternal toxicity. This argument has led to great difficulties for administrative decision makers involved in public health evaluation of drugs or chemicals. The present study sought to characterize whether there is a correlation between maternal toxicity and embryo/fetal toxicity. Developmental data from control and treated animals in our laboratory were collected and evaluated. Maternal toxicity, defined here as maternal body weight change, was statistically correlated with embryo/fetal parameters. The result showed that embryo/fetal parameters did not correlate with the body weight change. It can be concluded that maternal toxicity does not always lead to embryo/fetal toxicity; therefore, findings should be handled on a case by case basis and causal relationships should be established.