Teratogenicity Studies Research Articles

Rat two-generation reproductive toxicity and teratogenicity studies of a novel coccidiostat – Ethanamizuril

In the current study, to support the safety assessment of ethanamizuril as a new potent anticoccidial agent of triazine compounds, a reproductive toxicity and teratogenic potential assay of ethanamizuril was investigated. Groups of 30 males and 30 females were administered 0, 0.02, 0.1 or 0.2 mg/ml ethanamizuril by gastric incubation through a 10-week prebreed period as well as during mating, gestation, parturition and lactation in any generation. Compared to the control group, no test compound-related changes in copulation index, fertility index, gestation length, litter size, pup weight, pup sex ratio, pup viability, epididymal sperm counts or motility or other functional reproductive measures were noted in any generation, except few significant changes in high dose group in the number of sperm motility at III level in F0 males and the body weights of GD14 and GD21 in F1 rats. There were no compound-related necropsy findings or effects on organ weight. Histopathologic examinations revealed no evidence of compound-related changes in any organs including the reproductive organs of both sexes. In conclusion, long-term administrated 0.2, 1.0 and 2.0 mg/kg of ethanamizuril by means of oral gavage did not affect the reproduction of Sprague-Dawley rats and the development ability of their offspring under the experimental conditions.

Teratogenicity study in rats of commonly used calcium channel blockers

Background: Calcium channel blockers (CCBs) are popular medicines used to treat hypertension, tachyarrhythmia’s or angina during pregnancy. Lack of adequate safety data has however created an uncertainty in the use of CCBs in pregnancy. Nifedipine has been reportedly associated with a variety of embryotoxic and fetotoxic effects in animals. Therefore, this study was undertaken to establish whether or not the commonly used CCBs (nifedipine and amlodipine) would produce teratogenic effects in rats.Methods: Twenty pregnant rats were randomly assigned to each of the treatment and control groups. Nifedipine and amlodipine were used in three dose levels of 5, 10, 20 mg/kg and 0.5, 1, 2 mg/kg body weight respectively to test its teratogenic effects. The maximum dose of the test drugs used in our study was ten times the maximum recommended human dose. The drugs were administered to the pregnant rats using nasogastric tubes from day 6 through day 15 of pregnancy. The number of live births, stillbirths, litter sizes, crown-rump lengths, birth weights and gross abnormalities of the pups delivered were observed and recorded. Skeletal changes and soft tissue changes were also observed in the pups delivered to treated pregnant rats.Results: It was found that nifedipine and amlodipine did not produce any teratogenic effects in rats at doses 2.5 to 10 times the recommended human dose. None of the pups showed any gross morphological, skeletal or visceral defects.Conclusions: Nifedipine and amlodipine appear to be in therapeutic safe during pregnancy doses.

Toxicity Effect of Bougainvillea glabra (Paper Flower) Water Extracts on Zebrafish Embryo

Background: Bougainvillea glabra, or paper flower, is an ornamental plant known to possess various pharmacological activities. However, there is little information on the potential toxicity of this plant. Therefore, the aim of this study was to assess the acute toxicity and the potential teratogenic activity of water extracts of B. glabra bracts on zebrafish embryos which were selected as the model in this study. Methods: The pink, purple, and dark pink bracts of B. glabra were extracted using the Soxhlet method. Embryos with the same division stage were selected and treated with the extracts (1-300 µg/mL) for 72 h. The mortality of the embryos was recorded and the teratogenicity effect induced by the extracts was identified. The yolk sac area and pigmentation were quantified using Image-J. The data were analyzed by IBM SPSS 22.0. Results: All extracts within the tested concentrations did not induce death in embryo, except the pink bract extract with LC50 of 85.51 µg/mL. In the teratogenicity study, all the extract-treated embryos showed yolk sac edema at different concentrations, and the defect observed was independent of the concentrations. On top of that, the purple bract extract induced hypopigmentation in embryo, significantly at 30 µg/mL compared to control. Conclusion: This study concluded that the water extracts derived from the pink, purple, and dark pink bracts of B. glabra have mild toxicity toward embryo.

Tebukonazol hatóanyag-tartalmú gombaölő szer és ólom-acetát együttes hatásának teratológiai vizsgálata házi-tyúk embriókban

Vizsgálatunk arra irányult, hogy a növényvédelemben széles körben alkalmazott tebukonazol hatóanyagú MYSTIC 250 EC gombaölő szer és egy környezetszennyező nehézfém, az ólom-acetát egyedi és együttes méreghatását tanulmányozzuk házityúk-embriókban. A toxikológiai vizsgálat során kísérleti anyagként 0,01%-os ólom-acetát oldatot, valamint a MYSTIC 250 EC (250 g/l tebukonazol) 0,1%-os, a forgalmazó által ajánlott emulzióját alkalmaztuk. A bemerítéses kezelést a keltetés megkezdése előtt, míg a tojások feldolgozását a várható kelés előtt 2 nappal, a keltetés 19. napján végeztük el. A kórbonctani vizsgálat során lemértük az embriók testtömegét, feljegyeztük az elhalások számát, továbbá rögzítettük a makroszkópos fejlődési rendellenességeket. Statisztikai értékelésnél az élő embriók testtömegeinek összehasonlításához egytényezős varianciaanalízist (One-way ANOVA), az elhalások és a fejlődési rendellenességek számának elemzéséhez Fischer-féle egzakt tesztet alkalmaztunk. A vizsgálati anyagokkal elvégzett egyedi és együttes kezelések eredményeként a kezelt csoportokban az embriók testtömeg értékei szignifikánsan kisebbek voltak a kontroll csoporthoz viszonyítva. Az egyedi és együttes kezelések következtében jelentkező embrióletalitás - a fungiciddel egyedileg kezelt csoport kivételével - szignifikánsan emelkedett a kontroll csoporthoz képest. A fejlődési rendellenességek sporadikusan fordultak elő a kezelt csoportokban, teratogén hatás nem volt igazolható. Kísérletünkben felhasznált 0,01%-os ólom-acetát oldat és MYSTIC 250 EC fungicid 0,1%-os emulziójának egyedi méreghatása toxikus volt a tojásban fejlődő házityúk-embriókra nézve. A kísérleti anyagok együttes alkalmazása során az embriótoxikus dózisú ólom-acetát mellett a növényvédelmi gyakorlatban felhasznált MYSTIC 250 EC gombaölő szeres kezelés fokozta az embriótoxicitást, a toxikus interakció additív jellegű volt.

Importance of the Toxicological Tests in the Application and Safety of Ozone Therapy

Until today, there is not any official registry of the different routes of systemic ozone therapy. With the aim to prove the safety of this therapy, several ways of ozone (O3) applications were evaluated, such as rectal insufflation (RI), major autohemotherapy (M-AHT) and intraperitoneal application (IP). For RI, some toxicological tests were performed such as: acute, sub-chronic, mutagenic (bone marrow chromosomic aberrations and micronucleus), teratogenic and irritation studies (at 40 mg/L and 10 mL during 15 days). For M-AHT, acute toxicological (in rats with 21, 47 and 64 mg/L and a volume of 2 mL with daily evaluation up to 15 days) and mutagenic (chromosomal aberration analysis, micronucleus assay and sister chromatid exchange test) studies were performed. For IP application, subchronic (O3 was administered during 15 days in mice at concentrations of 11, 29 and 35 mg/L and volumes of 80 mL/kg) and genotoxic (cytotoxic and clastogenic activity) studies were evaluated. All the toxicological studies performed for the O3 RI fulfilled the requirements established for an official registry (no deleterious effects were observed). However, more toxicological studies must be done with respect to M-AHT and IP O3 applications in order to prove the complete safety of these ways.

Assaying Reproductive Capacity in Female Rodents.

It is an important part of many rodent studies to assess reproductive capacity in the female. Animal models that involve exposure to organic compounds, teratogenicity studies, or exposure to suboptimal environments during early development often result in deficits in female fertility. In addition to longitudinal physiology assays of fecundity, there are several molecular biology approaches to assessing female reproductive potential that can be performed to provide a "snapshot" of fertility potential at a single time-point. Here we describe some of the most useful ways to assess female reproductive capacity in rodents.

Teratogenicity of valproic acid and its constitutional isomer, amide derivative valnoctamide in mice.

The anticonvulsant valproic acid (VPA) has a known teratogenic effect capable of inducing major congenital malformations and developmental disorders. A comparative teratogenicity study of VPA and its analog valnoctamide (VCD), which is a new generation candidate antiepileptic drug, was carried out using Swiss Vancouver (SWV) mice. Pregnant SWV dams were treated with either a single intraperitoneal injection of VPA (1.8 and 2.7 mmol/kg), VCD (1.8 and 2.7 mmol/kg), or vehicle on E8:12 (gestational day:hour). The numbers of implantation and resorption, viable and dead fetuses, and the presence of gross fetal visceral and skeletal abnormalities were determined (E18). Real-time Polymerase chain reaction (RT-PCR) arrays were used to analyze the expression of 84 genes related to the processes of neurogenesis and neural stem cell differentiation. Significant decreases in pregnancy weight gain and the number of live fetuses were observed when VPA was administered at the high dose, whereas the percentage of exencephalic fetuses was significantly increased in VPA treated compared with an equivalent VCD dosage group. There was a dose-related increase in visceral defects in the VPA-exposed fetuses. Missing skull bones and fused vertebrae in fetuses occurred at the high dose of VPA. Three genes (Mtap2, Bmp8b, and Stat3) were significantly upregulated and one (Heyl) was downregulated in samples from VPA-treated dams. The study demonstrates that the teratogenicity of VPA was significantly greater than that of an equimolar dose of VCD. Four genes (Mtap2, Bmp8b, Stat3, and Heyl) represent candidate target genes for the underlying teratogenic mechanism responsible for VPA-induced malformations.

Genotoxicity and teratogenicity of seabuckthorn (Hippophae rhamnoides L.) berry oil

As botanicals and dietary supplements are used increasingly in many countries, the issue of safety is particularly critical for regulation of food products containing these substances. Seabuckthorn (Hippophae rhamnoides L.) has been used for centuries as a medicine and nutritional supplement in Asia and Europe. However, data regarding to the safety assessment of the plant and its extracts is still rare. This study was to evaluate the potential toxicity of seabuckthorn berry (SB) oil conducted in three genotoxicity studies and a teratogenicity study. Results of the genotoxicity studies indicated that SB oil has no genotoxicity under the experimental conditions of this study. Specifically, SB oil did not display any mutagenic activity on histidine dependent strains of Salmonella typhimurium under exposure concentrations of 8, 40, 200, 1000, and 5000 μg/plate; SB oil did not have significant effect on sperm morphology and have no influence on micronucleus rate of polychromatic erythrocytes in mice at doses of 9.36, 4.68, and 2.34 g/kg body weight. In the teratogenicity study, pregnant rats were treated with 4.68, 2.34, and 1.17 g/kg SB oil from gestation day 7 to 16 and no treatment-related maternal toxicity or embryo toxicity was observed. Taken together, these results support the safe use of seabuckthorn berry oil for potential dietary consumption in food or as a dietary supplement.

Usefulness of zebrafish in evaluating drug-induced teratogenicity in cardiovascular system

To confirm the usefulness of zebrafish for evaluating the teratogenic potential of drug candidates, the effect of O-ethylhydroxylamine hydrochloride (OHY), which induces mutagenesis by methylation, was evaluated in teratogenicity studies in rats and zebrafish. In the rat teratogenicity study, OHY-induced cardiovascular malformations such as increased abnormal vascular structures and ventricular septal defects. In the teratogenicity study using zebrafish-injected microspheres and green fluorescent protein-expressing Tg zebrafish (flk1:EGFP), OHY exposure was associated with the loss or malformation of the mandibular arch, opercular artery, and fourth branchial arch. These results suggested that OHY-induced external malformations in zebrafish eleutheroembryos adequately reflect OHY’s teratogenicity in rat fetuses. Moreover, the zebrafish teratogenicity study incorporating vascular morphological examinations, including those of blood vessels in the heart, head and trunk, is an easy and reliable screening method to detect potential drug-induced teratogenicity and phenotypic characteristics.

STUDI TERATOGENIK PEMBERIAN TRITERPEN TOTAL DARI Centella asiatica (Linn) Urban PADA MENCIT PUTIH

ABSTRACT The main active compound of the Centella asitica (Linn) Urban is total triterpenic. Total triterpenic has been proven to be effective in inflamatorry, infection and diabetic to patient. To examine its safety in pregnancy condition, the influence of total tripenic of Centella asitica had been studied in pregnant Wistar mice. The total tripenic of Centella asitica were administered orally at dose level of 20 mg/kgbw, 40 mg/kg bw , 80 mg/kgbw on day sixth to fifteenth of gestation. The mother was sacrified on day eightteenth of gestation to observe life fetus, resorption, and fetus bodyweight. One third of the fetuses eviscerated for skeletal staining with alizarin red and two third of the fetuses were hardened with Bouin’s fixation solution. No malformation were found in the eviscerated group. The at all doses did not cause any fetus resorption, did not cause malformation in organ and skeleton but cause fetus growth failure. Key words: teratogenicity study, total triterpenic, Centella asiatica . .

OZONE THERAPY: TERATOGENIC STUDY OF OZONE. POSSIBLE INDICATIONS IN OBSTETRICS AND GYNECOLOGY

OZONE THERAPY: TERATOGENIC STUDY OF OZONE. POSSIBLE INDICATIONS IN OBSTETRICS AND GYNECOLOGY

Toxicological assessment of Euglena gracilis strain Eu029 shows no adverse effects in vivo and in vitro

Euglena gracilis is a single-celled organism capable of photosynthesis and heterotrophy. Euglena sp. have long been studied in the laboratory for its metabolic pathways, cell motility, and ease of culture. The safety of E. gracilis strain eu029 (EG029) for use as a food ingredient was assessed in a bacterial reverse mutagenesis assay (Ames), rec assay, in vivo micronucleus assay, acute toxicity study in mice, 13-week toxicology in rats, and a teratology study in mice and rats. EG029 was not genotoxic. The No Observed Adverse Effect Level (NOAEL) in the 13-week study was greater than 1000 mg/kg/day, the highest dose tested. Teratogenicity studies did not find any defects in fetal development or effects to maternal health in rats at 1000 mg/kg/day, the highest dose tested.

Methodology of Genotoxic and Teratogenic Studies in Rats.

The genotoxicity methods applied to rats are tests that can detect any damage, including changes in the number of chromosomes or in the structure of chromosomes, and nucleotide changes with structural abnormality in the DNA of animal cells. However, the method of teratogenicity is used to detect the effects of chemicals which cause congenital defects in living organisms. This study contains information about the effectiveness, reliability, ways of application, and methodology of genotoxic and teratogenic methods applied in vivo in rats.

Mutagenicity and teratogenicity studies of vitacoxib in rats and mice.

Vitacoxib is a new drug candidate for treatment of inflammation, pain and fever as selective cyclooxygenase-2 inhibitors. In the current study, the mice sperm abnormality, mammalian erythrocyte micronucleus and in vivo chromosome aberration, and teratogenicity in SD rats were evaluated. Vitacoxib did not cause an increase in the frequency of structural chromosome aberrations, nor did it produce an increase in the number of micro nucleated polychromatic erythrocytes at dose of 1250–5000 mg/kg body weight (BW). There were no toxicological signs observed in teratogenicity test in female SD rats at dose of 200–5000 mg/kg BW. Based on these results of these studies, vitacoxib does not appear to be observed mutagenicity and teratogenicity.

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae.

Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies.

Pharmacological and Toxicological Study of Maytenus ilicifolia Leaf Extract. Part I - Preclinical Studies.

One of the Brazilians medicinal plants most cited in ethnopharmacological surveys for the treatment of ulcers and gastric diseases was evaluated for its efficacy and toxicity. Maytenus ilicifolia leaf extract (MIE) was acutely and chronically (180days) administered to rats, mice, and dogs. Acute tests were antiulcer effect and toxicological trials (observational pharmacological screening, LD50, motor coordination, sleeping time and motor activity). Chronic tests were the following: weight gain/loss and behavioral parameters in rats and mice; estrus cycle, effects on fertility, and teratogenic studies in rats and mutagenic features in mice, in addition to the Ames and micronucleus test. The following parameters were assessed in dogs: weight gain/loss, general physical conditions, water/food consumption, and anatomopathological examination of the organs subsequent to the 180-day treatment. The results showed a clear antiulcer activity for MIE from 70mg/kg and an absence of toxicological effects in the three animal species, even if given in high doses or over a long period. The present results confirm the antiulcer property and absence of toxicological effects in three animal species of MIE, which is in line with its current popular medicinal use. Copyright © 2017 John Wiley & Sons, Ltd.

The incidence of congenital malformations and variations in Göttingen minipigs.

Knowledge of the incidence of spontaneous congenital abnormalities is critical for the accurate interpretation of findings in teratogenicity studies in any species. In this paper, results of the examination of 1739 neonatal Göttingen Minipigs are presented. Over the 2-year period under consideration, the incidence of external and visceral malformations was less than 0.2 and 0.1%, respectively. The most common external malformations were syndactyly, limb hyperflexion, domed head and scoliosis. The most common internal malformations were undescended testes, ventricular septal defect, diaphragmatic hernia and atrial septal defects. Pentadactyly and variation in the aortic arch's bifurcation (absent truncus bicaroticus) were the most common variations. These data will help support the use of the Göttingen Minipig as a non-rodent species in embryofetal development studies where concerns persist about the availability of background data.

Teratogenic study of phenobarbital and levamisole on mouse fetus liver tissue using biospectroscopy

Biospectroscopic investigations have attracted attention of both the clinicians and basic sciences researchers in recent years. Scientists are discovering new areas for FTIR biospectroscopy applications in medicine. The aim of this study was to measure the possibility of FTIR-MSP application for the recognition and detection of fetus abnormalities after exposure of pregnant mouse to phenobarbital (PB) and levamisole (LEV) alone or in combination. PB is one of the most widely used antiepileptic drugs (AEDs), with sedative and hypnotic effects. When used by pregnant women, it is known to be a teratogenic agent. LEV is an antihelminthic drug with some applications in immune-deficiency as well as colon cancer therapy. Four groups of ten pregnant mice were selected for the experiments as follows: one control group received only standard diet, one group was injected with 120mg/kg of BP, one group was injected with 10mg/kg of LEV, and the last group was treated simultaneously with both BP and LEV at the above mentioned doses. Drugs administration was performed on gestation day 9 and fetuses were dissected on pregnancy day 15. Each dissected fetus was fixed, dehydrated and embedded in paraffin. Sections of liver (10μm) were prepared from control and treated groups by microtome and deparaffinized with xylene. The spectra were taken by FTIR-MSP in the region of 4000–400cm−1. All the spectra were normalized based on amide II band (1545cm−1) after baseline correction of the entire spectrum, followed by classification using PCA, ANN and SVM. Both morphological and spectral changes were shown in the treated fetuses as compared to the fetuses in the control group. While cleft palate and C-R elongation were seen in PB injected fetuses, developmental retardation was mostly seen in the LEV injected group. Biospectroscopy revealed that both drugs mainly affected the cellular lipids and proteins, with LEV causing more changes in amide I and lipid regions than PB. Application of PCA, ANN and SVM methods were able to successfully classify these FTIR spectroscopic data and discriminate between control and treated groups of fetuses, making it a new potential tool for drugs teratogenic investigations.

Evaluation of the embryotoxic and teratogenic effect of the β‐HPC in ovo

Exposure of drugs during the pregnancy may have different effects on the development of the embryo, depending on the period of the exhibition. Injuries during the division of the zygote and implementation that can have effects such as malformations, dysfunctions, alterations in growth, stillbirth and altered postnatal functions are called embryotoxicity. On the other hand, irreversible lesions that are compatible with life, but which may result in structural or functional abnormalities in the born products, it is called teratogenicity. The objective of this study was to evaluate whether the β‐HPC produces embryotoxic and teratogenic effects in ovo. Eggs were incubated for 24 h at 37°C and 60% humidity. We included the following treatment groups (n=5): control, negative control, positive control (caffeine 10 mg/mL, for embryotoxicity test) and 60, 120, 240 and 480 μg/mL of β‐HPC. 1 mL of albumin was removed and was replaced by 1 mL of treatment, and sealed with paraffin. The embryos were obtained after 48 hours of incubation with the treatment. They were fixed, dehydrated and were stained for observation. For teratogenicity study, has followed the same procedure, only the embryos developed fully until hatch. Once outside of the hatched chickens were analyzed to verify the presence of abnormalities. Embryotoxicity results showed that β‐HPC different concentrations did not induce structural malformations in embryos. However, the highest dose induced a higher stage compared to the control. In the results of teratogenicity was observed that β‐HPC, did not induce malformations in the development of the chicken. However, it was observed that the development of the chickens was lower, concentration‐dependent. The highest concentration (480 mg/mL) induced a smaller stadium. In conclusion the β‐HPC did not induce effects embryotoxic and teratogenic effects, however, can detain the development of chicken, concentration‐dependent.

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite.

High nitrate levels in the environment may result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.