The problem of maternal toxicity in developmental toxicity studies

Guidelines for developmental toxicity studies require that the highest dose(s) should induce some signs of maternal toxicity. However, the interpretation of the results is often difficult when developmentally toxic effects are recorded only at maternotoxic doses, as it is impossible to ascertain whether the developmental effects are maternally mediated or not. In order to avoid this source of misinterpretation we suggest to use in developmental toxicity tests for environmental chemicals the maximum dose unable to produce maternal toxic effects extrapolated by previous short term toxicity studies.     

The Threshold of Toxicological Concern for prenatal developmental toxicity in rats and rabbits

The Threshold Toxicological Concern (TTC) is based on the concept that in absence of experimental data reasonable assurance of safety can be given if exposure is sufficiently low.Using the REACH database the low 5th percentile of the NO(A)EL distribution, for prenatal developmental toxicity (OECD guideline 414) was determined. For rats, (434 NO(A)ELs values) for maternal toxicity, this value was 10 mg/kg-bw/day. For developmental toxicity (469 NO(A)ELs): 13 mg/kg-bw/day. For rabbits, (100 NO(A)ELs), the value for maternal toxicity was 4 mg/kg-bw/day, for developmental toxicity, (112 NO(A)EL values): 10 mg/kg-bw/day. The maternal organism may thus be slightly more sensitive than the fetus. Combining REACH- (industrial chemicals) and published BASF-data (mostly agrochemicals), 537 unique compounds with NO(A)EL values for developmental toxicity in rats and 150 in rabbits were evaluated. The low 5th percentile NO(A)EL for developmental toxicity in rats was 10 mg/kg-bw/day and 9.5 mg/kg-bw/day for rabbits. Using an assessment factor of 100, a TTC value for developmental toxicity of 100 μg/kg-bw/day for rats and 95 μg/kg-bw/day for rabbits is calculated. These values could serve as guidance whether or not to perform an animal experiment, if exposure is sufficiently low. In emergency situations this value may be useful for a first tier risk assessment.     

Acute and subacute toxicological evaluation of scutellarin in rodents

Acute and subacute investigations were carried out to evaluate the safety of scutellarin, an active flavone glycoside that has been used to treating cardiocerebral vascular diseases and cerebral infarction in rodents. For the acute study, scutellarin was administered to mice by gavage at different dose levels. Scutellarin caused dose-dependent general behavior adverse effects, but the LD₅₀ values could not be detected, and the maximum tolerated dose was more than 10 g/kg. In the subacute study, scutellarin was administered orally at doses of 100 and 500 mg/kg daily for 30 days to rats. Body weight, heart rate, blood pressure, biochemical, hematological and urine parameters were determined at the end of the experimental day. Daily oral administration for up to 30 days did not result in death or significant changes in hematology, blood chemistries or urinalysis. However, a 30 day regimen of scutellarin at doses of 100 or 500 mg/kg led to non-dose related decreases in BUN and triglyceride levels. Scutellarin was found to be minimally toxic or non-toxic in rodents. In view of the doses of the components used, the results from acute and subacute toxicity studies suggest that this component has a sufficient margin of safety for therapeutic use.     

Sensitivity of different generations and developmental stages in studies on reproductive toxicity

Numerous studies on reproductive toxicity are expected to be necessary under the EU program on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Therefore, it is important to analyse existing testing strategies including also the recently implemented extended one-generation reproduction toxicity study (EOGRTS, OECD guideline 443). For this purpose the responsiveness of the different generations and developmental stages in studies on reproductive toxicity is analysed and critical targets of reproductive toxicity are identified by using the Fraunhofer FeDTex database.     

Meta-analysis of toxicity and teratogenicity of 133 chemicals from zebrafish developmental toxicity studies

Zebrafish developmental toxicity testing is an emerging field, which faces considerable challenges regarding data meta-analysis and the establishment of standardized test protocols. Here, we present an initial correlation study on toxicity of 133 chemicals based on data in the literature to ascertain predictive developmental toxicity endpoints. We found that the physical properties of chemicals (BCF or log P) did not fully predict lethality or developmental outcomes. Instead, individual outcomes such as pericardial edema and yolk sac edema were more reliable indicators of developmental toxicity. In addition, we ranked the chemicals based on toxicity with the Toxicological Priority Index (ToxPi) program and via a teratogenic ratio, and found that perfluorooctane sulfonate (PFOS) had the highest ToxPi score, triphenyltin acetate had the highest average ToxPi score (corrected for missing data and having more than 4 outcomes), and N-methyl-dithiocarbamate had the highest teratogenic ratio.     

Toxicological evaluation of neem (Azadirachta indica) oil: Acute and subacute toxicity

Neem (Azadirachta indica), popularly known as traditional medicine is a native plant in India. Neem oil is a vegetable oil derived from seeds or fruits of the neem tree through pressing or solvent extraction, and largely used in popular medicine to have antifungal, antibacterial, antimalarial, antiparasitic, anti-inflammatory, as well as immunemodulatory properties in different animal species. In the present study, acute and 28-day subacute toxicity tests were carried out. In the acute toxicity test, the LD₅₀ values of neem oil were found to be 31.95 g/kg. The subacute treatment with neem oil failed to change body weight gain, food and water consumption. Serum biochemistry analysis showed no significant differences in any of the parameters examined under the dose of 1600 mg/kg/day. Histopathological exams showed that the target organs of neem oil were testicle, liver and kidneys up to the dose of 1600 mg/kg/day.     

Reproductive and developmental toxicity of inhaled 2,3-dichloro-1,3-butadiene in rats

Inhalation developmental and reproductive toxicity studies were conducted with 2,3-dichloro-1,3-butadiene (DCBD), a monomer used in the production of synthetic rubber. In the reproductive toxicity study, Crl:CD^®(SD)IGS BR rats (24/sex/group) were exposed whole body by inhalation to 0, 1, 5, or 50 ppm DCBD (6 h/day) for approximately 10–11 weeks total, through premating (8 weeks; 5 days/week), cohabitation of mating pairs (up to 2 weeks, 7 days/week), post-cohabitation for males (7 days) and from conception to implantation (gestation days 0–7 [GD 0–7]), followed by a recovery period (GD 8–21) for presumed pregnant females. Estrous cyclicity was evaluated during premating (last 3 weeks) and cohabitation. Reproductive organs and potential target organs, sperm parameters, and GD 21 fetuses (viability, weight, external alterations) were evaluated. In the developmental study, pregnant Crl:CD^®(SD)IGS BR rats (22/group) were exposed whole body by inhalation to 0, 1, 10, or 50 ppm DCBD (6 h/day) on GD 6–20; dams were necropsied on GD 21 (gross post-mortem only) and fetuses were evaluated (viability, weight, and external, visceral and skeletal exams). During the in-life portion of the studies, body weight, food consumption, and clinical observation data were collected. At 50 ppm, gasping and labored breathing occurred in both studies during the first few exposures; body weight and food consumption parameters were affected in parental animals from both studies, but were more severely affected in the developmental study. Fetal weight was decreased in the developmental study at 50 ppm. Degeneration of the nasal olfactory epithelium was observed in the reproduction study at 50 ppm. There were no effects on reproductive function, embryo–fetal viability, or increases in fetal structural alterations in either study. The no-observed-adverse-effect level (NOAEL) for reproductive toxicity was 50 ppm. The NOAEL for systemic toxicity in the reproduction study was 5 ppm based on adverse effects on body weight and food consumption parameters and nasal olfactory epithelial toxicity at 50 ppm in parental rats. The NOAEL for maternal and developmental toxicity was 10 ppm based on reduced maternal weight gain and food consumption and reduced fetal weight at 50 ppm in the developmental toxicity study.     

Repeated dose and reproductive/developmental toxicity of perfluorododecanoic acid in rats

Perfluoroalkyl carboxylic acids (PFCAs) are a series of environmental contaminants that have received attention because of their possible adverse effects on wildlife and human health. Although many toxicological studies have been performed on perfluorooctanoic acid with carbon chain length C8, available toxicity data on PFCAs with longer chains are still insufficient to evaluate their hazard. A combined repeated dose and reproductive/developmental toxicity screening study for perfluorododecanoic acid (PFDoA; C12) was conducted in accordance with OECD guideline 422 to fill these toxicity data gaps. PFDoA was administered by gavage to male and female rats at 0.1, 0.5, or 2.5 mg/kg/day. The administration of PFDoA at 0.5 and 2.5 mg/kg/day for 42–47 days mainly affected the liver, in which hypertrophy, necrosis, and inflammatory cholestasis were noted. Body weight gain was markedly inhibited in the 2.5 mg/kg/day group, and a decrease in hematopoiesis in the bone marrow and atrophic changes in the spleen, thymus, and adrenal gland were also observed. Regarding reproductive/developmental toxicity, various histopathological changes, including decreased spermatid and spermatozoa counts, were observed in the male reproductive organs, while continuous diestrous was observed in the females of the 2.5 mg/kg/day group. Seven of twelve females receiving 2.5 mg/kg/day died during late pregnancy while four other females in this group did not deliver live pups. No reproductive or developmental parameters changed at 0.1 or 0.5 mg/kg/day. Based on these results, the NOAELs of PFDoA were concluded to be 0.1 mg/kg/day for repeated dose toxicity and 0.5 mg/kg/day for reproductive/developmental toxicity. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1244–1263, 2015.     

Developmental toxicity of perfluorononanoic acid in mice

Perfluorononanoic acid (PFNA) is a ubiquitous and persistent environmental contaminant. Although its levels in the environment and in humans are lower than those of perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA), a steady trend of increases in the general population in recent years has drawn considerable interest and concern. Previous studies with PFOS and PFOA have indicated developmental toxicity in laboratory rodent models. The current study extends the evaluation of these adverse outcomes to PFNA in mice. PFNA was given to timed-pregnant CD-1 mice by oral gavage daily on gestational day 1–17 at 1, 3, 5 or 10 mg/kg; controls received water vehicle. Dams given 10 mg/kg PFNA could not carry their pregnancy successfully and effects of this dose group were not followed. Similar to PFOS and PFOA, PFNA at 5 mg/kg or lower doses produced hepatomegaly in the pregnant dams, but did not affect the number of implantations, fetal viability, or fetal weight. Mouse pups were born alive and postnatal survival in the 1 and 3 mg/kg PFNA groups was not different from that in controls. In contrast, although most of the pups were also born alive in the 5 mg/kg PFNA group, 80% of these neonates died in the first 10 days of life. The pattern of PFNA-induced neonatal death differed somewhat from those elicited by PFOS or PFOA. A majority of the PFNA-exposed pups survived a few days longer after birth than those exposed to PFOS or PFOA, which typically died within the first 2 days of postnatal life. Surviving neonates exposed to PFNA exhibited dose-dependent delays in eye opening and onset of puberty. In addition, increased liver weight seen in PFNA-exposed offspring persisted into adulthood and was likely related to the persistence of the chemical in the tissue. Evaluation of gene expression in fetal and neonatal livers revealed robust activation of peroxisome proliferator-activated receptor-alpha (PPARα) target genes by PFNA that resembled the responses of PFOA. Our results indicate that developmental toxicity of PFNA in mice is comparable to that of PFOS and PFOA, and that these adverse effects are likely common to perfluoroalkyl acids that persist in the body.     

Review of embryo-fetal developmental toxicity studies performed for recent FDA-approved pharmaceuticals

Details of embryo-fetal development (EFD) studies were compiled from published FDA approval documents for 43 small molecule drugs (2014–2015) and 37 monoclonal antibodies (mAbs, 2002–2015). Anti-cancer agents were analyzed separately. Rats and rabbits were the species used for EFD studies on 93% of small molecule drugs. Overall, the rat and rabbit were equally sensitive to maternal and fetal toxicity (including teratogenicity). Dosages equivalent to more than 50-times the human exposure (or 10-times for mAbs) were frequently used, but were unnecessary for 90% of drugs. EFD studies were not required for several recently approved mAbs owing to pre-existing scientific knowledge. The cynomolgus monkey was used for developmental toxicity testing of 75% of mAbs, frequently using an ePPND study design. Studies in pregnant rodents using homologous murine antibodies supplemented or replaced monkey studies under some circumstances. Most anti-cancer small molecules and mAbs were tested for developmental toxicity in at least one species.     

Assessment of the developmental toxicity of deferoxamine in mice

 Deferoxamine (DFO), an efficient chelating agent available for the treatment of iron and aluminium overload, was evaluated for developmental toxicity in Swiss mice. Intraperitoneal injections of DFO were given to pregnant animals at 0, 44, 88, 176, and 352  mg/kg per day on gestational days 6 through 15. Maternal clinical status was monitored daily during and after treatment. Fetal parameters, including external, visceral, and skeletal malformations and variations, were assessed. Mice were killed on day 18. No maternal mortality was observed, but dams exhibited reduced body weight gain during treatment at 88, 176, and 352 mg/kg per day. Body weight at termination, corrected body weight, and food consumption were reduced in all groups. In contrast, the only significant treatment-related embryo/fetal effect was a decrease in the number of live fetuses per litter at 352 mg/kg per day. The no-observable-adverse-effect level (NOAEL) for maternal toxicity of DFO was <44 mg/kg per day, whereas the NOAEL for developmental toxicity was 176 mg/kg per day. In summary, intraperitoneal administration of DFO to mice during organogenesis produced developmental toxicity in the presence of maternal toxicity. Because of the remarkable maternal toxicity of DFO, extreme caution in the use of this drug is recommended during pregnancy. Received: 4 October 1994/Accepted: 6 December 1994     

Acute and subacute (28-Day) toxicity studies of ionic liquid,didecyldimethyl ammonium acesulfamate,in rats

The aim of this study was to investigate acute and subacute oral toxicity of an ionic liquid, didecyldimethylammonium acesulfamate [DDA][Ace], in rats. The compound tested was classified to the fourth toxicity class with a fixed LD₅₀ cut-off value of 500?mg/kg. Organ pathology induced by [DDA][Ace] in acute experiments included exfoliation of the surface layer of the digestive tract and alveolar septa in lung parenchyma. In a subacute experiment, rats were administered 10, 50, and 100?mg/kg/day [DDA][Ace] for 28 days. Reduced body weight gain and reduced food consumption was observed in mid- and high-dose rats. Statistically significant hematology changes were found mostly in high-dose groups of both sexes: increases in hematocrit, mean corpuscular volume, and mean platelet volume. Statistically significant changes in clinical chemistry parameters included increases in the GGT, SDH, and LDH activity and bilirubin concentration, and decreases in triglycerides, glucose, and inorganic phosphorus concentration. No treatment-related microscopic changes were observed. Under the conditions of this study, the lowest-observed-adverse-effect level of [DDA][Ace] was considered to be 10?mg/kg/day.     

Developmental toxicity evaluation of gallium nitrate in mice

Gallium nitrate, a drug with antitumor activity, is presently undergoing clinical trials as a chemotherapeutic agent for the treatment of certain malignancies. Since there are very limited published animal toxicity data available, this study was conducted to investigate the potential adverse developmental effects of this drug. Pregnant Swiss mice were administered intraperitoneally gallium nitrate at 12.5, 25, 50, and 100 mg/kg/day on days 6, 8, 10, 12, and 14 of gestation. Monitors for maternal toxicity were body weight, food consumption and clinical signs. At sacrifice (day 18) maternal weight, liver and kidney weights, and gravid uterine weights were measured. Gestational parameters monitored were numbers of total implants, resorptions, postimplantation losses, and dead fetuses. Live fetuses were sexed, weighed, and examined for external, internal and skeletal malformations and variations. Maternal toxicity was noted in all the gallium nitrate-treated groups. Embryo/fetal toxicity was evidenced by a decrease in the number of viable implants, a reduction in fetal weight, and an increase in the number of skeletal variations (12.5, 25, 50 and 100 mg/kg). No significant increase in the incidence of malformations was observed at 12.5, 25, or 50 mg/kg. The no-observable-adverse-effect level (NOAEL) for both maternal and developmental toxicity of gallium nitrate was <12.5 mg/kg.     

Evaluation of developmental toxicity of microbicide Nisin in rats.

In the present study, we have investigated the developmental toxicity of a naturally occurring peptide, Nisin in rats in order to determine its suitability as a safe vaginal microbicide. Our earlier studies indicated that, Nisin is a dual function microbicide having contraceptive and antibacterial activities. However, as part of the safety evaluation of any vaginal microbicide, it is essential to determine its teratogenic potential in a suitable animal model before it is found suitable to enter clinical trials. Sixty pregnant rats allocated into four groups were orally administered with 10, 25 and 50 mg Nisin/kg/day from day 6 to day 15 of gestation. Individual food/water consumption and body weight changes were measured daily. Nisin did not cause maternal mortality nor did the treated animals show any clinical signs of toxicity when compared to the control animals. There were no biologically significant differences in maternal liver, kidney, thymus, ovary, gravid and empty uterine weights. Mean number of corpora lutea and implantation sites also did not differ in the treated groups when compared to their respective controls. All the fetuses were weighed, sexed and examined carefully for externally visible malformations. No gross external fetal alterations were observed at any dose tested. When stained by the double staining method, no skeletal malformations and visceral defects were observed in the fetuses. The growth and reproductive performance of the F1 progeny was also unaffected. In conclusion, Nisin shows unique clinical potential as a safe prophylactic microbicide to curb the transmission of STIs/HIV and unintended pregnancies.     

GC法测定沙棘果油中棕榈酸、棕榈油酸和油酸的含量

目的建立沙棘果油中棕榈酸、棕榈油酸和油酸含量的测定方法。方法采用毛细管色谱柱DM-WAX(30 mm×0.32mm,0.50μm);载气为N2;流速为1.5mL·min-1;进样量为1μL;分流比为50∶1;FID检测器;进样口温度为210℃;检测器温度为230℃;程序升温:初始温度50℃,维持1min,以25℃·min-1的速率升至180℃,再以1℃·min-1的速率升至200℃,维持10min。结果棕榈酸、棕榈油酸和油酸的线性范围分别为0.101 0~1.010 4(r=0.999 2),0.100 2~1.002 4(r=0.998 4)和0.101 4~1.013 6μg(r=0.999 4),平均回收率分别为97.2%,98.9%和96.8%,RSD值分别为1.87%,1.58%和2.06%。结论建立的方法准确、重复性好,可用于沙棘果油中棕榈酸、棕榈油酸和油酸的含量测定。     

Subacute toxicity assessment of carotenoids extracted from citrus peel (Nanfengmiju, Citrus reticulata Blanco) in rats

The mixture of carotenoids extracted from citrus peel (Nanfengmiju, Citrus reticulata Blanco) was tested for subacute oral toxicity. In this study, dose levels of 0, 200, 500 and 2000 mg/kg body weight/day were administered by gavage to 10 Wistar rats/sex/group for 28 days. No statistically significant, dose-related effect on food consumption, food efficiency, body weight gain, clinical signs or ophthalmoscopic parameters was observed in any treatment group. Urinalysis, hematological, blood coagulation and serum biochemical examination as well as necropsy or histopathology showed that no observed adverse effect was found. These findings suggested that the No-Observed-Adverse-Effect Level for the mixture of carotenoids extracted from citrus peel was at least 2000 mg/kg body weight/day.     

Vulnerable windows for developmental ethanol toxicity in the Japanese medaka fish (Oryzias latipes)

Ethanol (EtOH) is a well-known developmental toxicant that produces a range of abnormal phenotypes in mammalian systems including craniofacial abnormalities, cognitive deficits and growth retardation. While the toxic potential of developmental EtOH exposure is well characterized clinically, the effect of timing on the extent of toxicity remains unknown. Fish models such as the Japanese medaka, Oryzias latipes, provide a convenient system for investigating the effects of developmental EtOH exposure in vivo. In this study, medaka embryo toxicity tests were used to assess temporal variations in developmental EtOH toxicity. Fertilized eggs were collected and incubated during early, middle or late egg development (e.g., 0–3, 3–6 or 6–9 days post-fertilization) with various sub-lethal concentrations of EtOH [0.1% (17.2 mM), 0.5% (86.0 mM) or 1% (172 mM)]. Uptake of EtOH by the embryo was 60–68% of the solution concentration across all windows. Time to hatch, head width, total body length and whole embryo caspase activity were used to assess toxicity. Hatching delays were noted only at the highest concentration of EtOH. Head width was affected at all ethanol levels, regardless of the window of exposure. EtOH-induced decreases in body length, however, appeared to be most pronounced when exposure occurred either during the first or last window. The effect on caspase-3/7 activity also depended on the window of exposure, with increases in caspase noted in embryos treated on days 1 or 2 (first window) and decreases seen in embryos treated on day 6 (second window) or day 8 (third window). In general, these data suggest that critical periods for heightened sensitivity to developmental EtOH exposure may vary according to the specific endpoint used to assess toxicity.     

Acute and 30-day oral toxicity studies of administered carnosic acid

Purpose

Increasing interest in carnosic acid (CA) is due to its pharmacological properties. The aim of this study was to evaluate the acute and 30-day oral toxicity of CA.

Methods

The acute oral toxicity study in Kuming mice design followed the OECD-guidelines 423, and a 30-day chronic oral toxicity study in Wistar rats based on the enhanced OECD test guideline 407 were performed.

Results

The oral lethal dose (LD50) for mice was 7100 mg/kg of body weight in the acute toxicity study. The histopathological changes were observed in the heart, liver and kidney for the survival mice treated with a single dose CA. For the sub chronic toxicity study, CA administered for 30 days produced slightly reductions in the weight gain pattern, which did not reach the significant level when compared with the control values. With respect to serum biochemistry test, decreased total serum protein levels, but conversely increased aspartate aminotransferase (AST) levels were detected in the high-dose and moderate-dose groups. Histopathologically, light pathological changes were observed in the heart, liver, and kidney of rats treated with the high-dose CA.

Conclusion

The present work suggests that a short-term oral administration of CA has a relatively low toxicity profile.     

山莨菪碱对油酸致急性肺损伤家兔脂质过氧化物及肺表面活性物质含量的影响

采用硫代巴比妥酸(TBA)显色法测定油酸致急性肺损伤后血浆及肺组织脂质过氧化物(LPO)的代谢产物丙二醛(MDA)的含量,用薄层层析及无机磷定量法测定肺表面活性物质(PS)的主要功能成分—卵磷脂(PC)和总磷脂(TPL)含量。观察到油酸致肺损伤后血浆和肺组织匀浆MDA含量明显增加;肺组织匀浆PC及TPL含量明显减少。损伤前给予645—2.可减少LPO的生成,且能防止PS的减少。结果提示PS减少与脂质成分自身变化有关,可能是LPO增加致PS合成减少。654—2能抑制这一过程。