Makoto Ema

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY STUDIES OF MANUFACTURED NANOMATERIALS

This paper reviews studies in vivo and in vitro on the reproductive and developmental toxicity of manufactured nanomaterials including metallic and metal oxide-based particles, fullerenes (C(60)), carbon black (CB), and luminescent particles. Studies in vivo showed increased allergic susceptibility in offspring of mouse dams intranasally insufflated with respirable-size titanium dioxide (TiO(2)), adverse effects on spermatogenesis and histopathological changes in the testes and changes in gene expression in the brain of mouse offspring after maternal subcutaneous injection of TiO(2) nanoparticles, transfer to rat fetuses of radiolabeled gold nanoparticles and C(60) after maternal intravenous injection, death and morphological abnormalities in mouse embryos after maternal intraperitoneal injection of C(60), and adverse effects on spermatogenesis in mouse offspring after maternal intratracheal instillation of CB nanoparticles. Studies in vitro revealed that TiO(2) and CB nanoparticles affected the viability of mouse Leydig cells, that gold nanoparticles reduced the motility of human sperm, that silver, aluminum, and molybdenum trioxide were toxic to mouse spermatogonia stem cells, that silica nanoparticles and C(60) inhibited the differentiation of mouse embryonic stem cells and midbrain cells, respectively, and that cadmium selenium-core quantum dots inhibited pre- and postimplantation development of mouse embryos. Although this paper provides initial information on the potential reproductive and developmental toxicity of manufactured nanomaterials, further studies, especially in vivo, using characterized nanoparticles, relevant routes of administration, and doses closely reflecting expected levels of exposure are needed.

Rat two-generation reproductive toxicity study of bisphenol A

This study was conducted to determine the low-dose effects of bisphenol A (BPA) in a rat two-generation reproduction study. Groups of 25 male and 25 female Crj: CD (SD) IGS rats were given BPA at 0.2, 2, 20, or 200 microg/kg/day by gastric intubation throughout the study beginning at the onset of a 10- and 2-week premating period, in F0 males and females, respectively, and continuing through the mating, gestation, and lactation periods, for two generations. There were adult (F0, F1, F2) and postnatal day (PND) 22 (F1, F2) necropsies: the oldest F2 males and females being killed at postnatal weeks 7 and 14, respectively. No compound-related clinical signs or effects on body weight or food consumption were observed in any generation. There were no compound-related changes in surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna detachment, incisor eruption, eye opening, testes descent, preputial separation, or vaginal opening in F1 and F2 generations, or behavior in the open field or water filled multiple T-maze in the F1 generation. No test compound-related changes in estrous cyclicity, copulation index, fertility index, number of implantations, gestation length, litter size, pup weight, pup sex ratio, pup viability, or other functional reproductive measures were noted in any generation. A few significant changes in the anogenital distance (AGD) per cube root of body weight ratio were found at 0.2 and 20 microg/kg in F1 males, at 2, 20, and 200 microg/kg in F1 females, and at 20 and 200 microg/kg in F2 females. However, the changes in the AGD were consistently small (within 5% of control values), and no continuous changes in the AGD or AGD/cube root of body weight ratio were detected. There were no compound-related changes in epididymal sperm counts or motility in F0 and F1 males. No compound-related necropsy findings or effects on organ weight including the reproductive organs were found in any generation. Histopathologic examinations revealed no evidence of compound-related changes in any organs including the reproductive organs of both sexes. The data indicate that oral doses of BPA of between 0.2 and 200 microg/kg over 2 generations did not cause significant compound-related changes in reproductive or developmental parameters in rats.

Two-generation reproductive toxicity study of the flame retardant hexabromocyclododecane in rats.

Male and female rats were fed a diet containing flame retardant hexabromocyclododecane (HBCD) at 0, 150, 1500 or 15,000 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through the mating, gestation and lactation periods for two generations. The mean daily intakes of HBCD during the whole period of administration were 10.2, 101 and 1008 mg/kg bw in F0 males, 14.0, 141 and 1363 mg/kg bw in F0 females, 11.4, 115 and 1142 mg/kg bw in F1 males, and 14.3, 138 and 1363 mg/kg bw in F1 females for 150, 1500 and 15,000 ppm, respectively. The incidence of rats with decreased thyroid follicles size was increased in F0 and F1 males and females at 1500 ppm and higher. Serum TSH levels were increased in F0 and F1 females at 1500 ppm and higher, and serum T4 levels were decreased in F0 males and females at 15,000 ppm. The number of the primordial follicles in the ovary of F1 females was reduced at 1500 ppm and higher. There were increases in the absolute and relative weights of the liver in male adults and male and female weanlings at 1500 ppm and higher, and in female adults at 15,000 ppm, and of the thyroid in male and female adults at 15,000 ppm. Decreased body weight and body weight gain associated with reduced food consumption were found in F1 males and females at 15,000 ppm. Decreases were found in the viability index of F2 pups and the body weight of male F1 and F2 pups and female F2 pups at 15,000 ppm. In F2 pups, there were low incidences of the completion of eye opening in males at 15,000 ppm and in females at 1500 ppm and higher, and of completed mid-air righting in females at 15,000 ppm. The data indicate that the NOAEL of HBCD in this study was 150 ppm (10.2mg/kg bw/day). The estimated human intake of HBCD is well below the NOAEL in the present study.

Critical period for adverse effects on development of reproductive system in male offspring of rats given di-n-butyl phthalate during late pregnancy.

The objective of this study was to determine the susceptible days for the adverse effects of di-n-butyl phthalate (DBP) on development of reproductive system in male offspring following maternal administration on successive 3-day period during late pregnancy. Pregnant rats were given DBP by gastric intubation at 1000 or 1500 mg/kg on days 12-14 or 18-20 of pregnancy or at 500, 1000 or 1500 mg/kg on days 15-17 of pregnancy. A significant decrease in the maternal body weight gain and/or food consumption was found in the DBP-treated groups regardless of the days on which DBP at 1000 and 1500 mg/kg was given. A significant increase in the number of resorptions per litter was found in the groups given DBP at 1500 mg/kg on days 12-14 and 15-17 of pregnancy. The weights of male and female fetuses were significantly decreased in the groups given DBP at 1000 and 1500 mg/kg on days 12-14 and 18-20 and at 1500 mg/kg on days 15-17. A significant increase in the incidence of fetuses with undescended testes was found at 1500 mg/kg on days 12-14 and at all doses on days 15-17. A significant decrease in the anogenital distance (AGD) of male fetuses was observed in the groups treated with DBP regardless of the days of treatment. The AGD/body weight ratio in male fetuses was significantly reduced in the groups given DBP on days 15-17, but neither on days 12-14 nor 18-20. The AGD of female fetuses in the DBP-treated groups was comparable to that in the control group. It was concluded that period of days 15-17 of pregnancy was the most susceptible for DBP-induced undescended testes and decreased AGD in male offspring.

Effects of dibutyl phthalate on reproductive function in pregnant and pseudopregnant rats.

The effects of monobutyl phthalate (MBuP) on reproductive function were determined in pregnant and pseudopregnant rats. Rats were given MBuP by gastric intubation at 250, 500, 750, or 1000 mg/kg on days 0 to 8 of pregnancy and pregnancy outcome was determined on day 20 of pregnancy. The effects of MBuP on the uterine function, as a cause of early embryonic loss, were also determined in pseudopregnant rats, with an induced decidual cell response. The same doses of MBuP were given to pseudopregnant rats on days 0 to 8 of pseudopregnancy and the uterine weight on day 9 served as an index of uterine decidualization. MBuP at 1000 mg/kg caused significant increases in the incidences of preimplantation loss in females successfully mated and of postimplantation loss in females having implantations. Uterine decidualization in pseudopregnant rats was significantly decreased at 1000 mg/kg. These findings suggest that early embryonic loss due to MBuP is mediated, at least in part, via suppression of uterine decidualization, an impairment of uterine function.

Biological response and morphological assessment of individually dispersed multi-wall carbon nanotubes in the lung after intratracheal instillation in rats

Biological responses of multi-wall carbon nanotubes (MWCNTs) were assessed after a single intratracheal instillation in rats. The diameter and median length of the MWCNTs used in this study were approximately 60 nm and 1.5 μm, respectively. Groups of male Sprague-Dawley rats were intratracheally instilled with 0.04, 0.2, or 1 mg/kg of the individually dispersed MWCNT suspension. After instillation, the bronchoalveolar lavage fluid was assessed for inflammatory cells and markers, and the lung, liver, kidney, spleen, and cerebrum were histopathologically evaluated at 3-day, 1-week, 1-month, 3-month, and 6-month post-exposure. Transient pulmonary inflammatory responses were observed only in the lungs of the group of rats exposed to 1 mg/kg of MWCNTs. Morphology of the instilled MWCNTs in the lungs of rats was assessed using light microscopy and transmission electron microscopy (TEM). Light microscopy examination revealed that MWCNTs deposited in the lungs of the rats were typically phagocytosed by the alveolar macrophages and these macrophages were consequently accumulated in the alveoli until 6-month post-exposure. The 400 TEM images obtained showed that all MWCNTs were located in the alveolar macrophages or macrophages in the interstitial tissues, and MWCNTs were not located in the cells of the interstitial tissues. There was no evidence of chronic inflammation, such as angiogenesis or fibrosis, induced by MWCNT instillation. These results suggest that MWCNTs were being processed and cleared by alveolar macrophages.

Adverse effects on development of the reproductive system in male offspring of rats given monobutyl phthalate, a metabolite of dibutyl phthalate, during late pregnancy

The objective of this study was to determine the adverse effects of monobutyl phthalate (MBuP), a major metabolite of dibutyl phthalate (DBP), on development of the reproductive system in offspring following maternal administration during late pregnancy, and to assess the role of MBuP in the antiandrogenic effects of DBP. Pregnant rats were given MBuP by gastric intubation at 250, 500, or 750 mg/kg on days 15 through 17 of pregnancy. Maternal body weight gain and food consumption during the administration period were significantly decreased at 500 mg/kg and higher and at 750 mg/kg, respectively. A significant increase in the incidence of postimplantation embryonic loss was found at 500 mg/kg and higher. The body weights of male and female fetuses were significantly lower at 750 mg/kg. A significant increase in the incidence of fetuses with undescended testes was found at 250 mg/kg and higher. A significant decrease in the anogenital distance (AGD) of male fetuses was observed at 250 mg/kg and higher. The AGD/body weight ratio and AGD/cube root of body weight ratio in male fetuses was also significantly reduced at 250 mg/kg and higher. The AGD, AGD/body weight ratio and AGD/cube root of body weight ratio in female fetuses in the MBuP-treated groups were comparable to those in the control group. The present study indicates that MBuP on days 15 to 17 of pregnancy produced adverse effects on the development of reproductive system in male offspring and suggest that MBuP may be responsible for the induction of the antiandrogenic effects of DBP.

Characterization of the developmental toxicity of di-n-butyl phthalate in rats

The objective of this study was to determine the characterization of the developmental toxicity of di-n-butyl phthalate (DBP) in rats. Pregnant rats were given DBP by gastric intubation at a dose of 0.75, 1.0 or 1.5 g/kg on days 7-9, 10-12 or 13-15 of pregnancy. Postimplantation loss was 100% for each period of dosing at 1.5 g/kg. A significant increase in the postimplantation loss was found in dams given DBP at doses of 0.75 and 1.0 g/kg regardless of the days of treatment. No evidence of teratogenicity was detected when DBP was given on days 10-12. Treatment on days 7-9 with DBP at doses of 0.75 and 1.0 g/kg caused a significant increase in the number of skeletal malformations such as deformity of the vertebral column in the cervical and thoracic regions and of the ribs, but neither external nor internal malformations. Treatment with DBP on days 13-15 at doses of 0.75 and 1.0 g/kg resulted in a significant increase in the incidence of fetuses with external and skeletal malformations such as cleft palate and fusion of the sternebrae. The frequency of malformations increased as the dose of DBP was increased. The highest incidence of malformed fetuses occurred after treatment with DBP on days 13-15. It could be concluded that susceptibility to the teratogenicity of DBP varies with the developmental stage at the time of administration.

Further evaluation of developmental toxicity of di-n-butyl phthalate following administration during late pregnancy in rats.

The objective of this study was to further evaluate the developmental toxicity of di-n-butyl phthalate (DBP) administered during the second half of pregnancy. Pregnant rats were fed a diet containing DBP at a dose of 0 (control), 0.5, 1.0 or 2.0% ad libitum on days 11-21 of pregnancy. Average daily intakes of DBP were 331, 555 and 661 mg/kg for the 0.5, 1.0 and 2.0% groups, respectively. No significant changes induced by DBP were detected in the incidence of postimplantation loss and numbers of live fetuses and of resorptions and dead fetuses. The weights of male and female fetuses at 2.0% DBP were significantly decreased. The incidences of fetuses with cleft palate and fetuses with fusion of the sternebrae at 2.0% DBP and fetuses with undescended testes at 1.0 and 2.0% DBP were significantly increased. There were significant decreases in the anogenital distance (AGD) of male fetuses in the 1.0 and 2.0% DBP groups, also. AGD of female fetuses in the DBP-treated groups was comparable to that in the control group. It was concluded that DBP administered during the second half of pregnancy produced adverse effects on the reproductive development in male fetuses.

Effects on development of the reproductive system in male offspring of rats given butyl benzyl phthalate during late pregnancy

The objective of this study was to determine the effects of maternal exposure to butyl benzyl phthalate (BBP) on the development of the reproductive system in male offspring. Pregnant rats were given BBP by gastric intubation at 250, 500, or 1000 mg/kg on days 15 to 17 of pregnancy. A significant decrease in maternal body weight gain and food consumption was found in rats given BBP at 500 and 1000 mg/kg. A significant decrease in the number of live fetuses per litter was found at 1000 mg/kg. The weights of male and female fetuses were significantly decreased in the groups given BBP at 1000 mg/kg. A significant increase in the incidence of fetuses with undescended testes was found at 500 and 1000 mg/kg. A significant decrease in the anogenital distance (AGD) of male fetuses was observed at 500 and 1000 mg/kg. The AGD/cube root of body weight ratio in male fetuses was also significantly reduced at 500 mg/kg and higher. The AGD and AGD/cube root of body weight ratio of female fetuses in the BBP-treated groups were comparable to those in the control group. It was concluded that BBP on days 15 to 17 of pregnancy produced adverse effects on the development of the reproductive system in male offspring.