Effects on the reproductive parameters of two generations of Rattus norvegicus offspring from dams exposed to heptachlor during gestation and lactation

Heptachlor has been targeted for global elimination because of its toxicity and environmental persistence, in accordance with the Stockholm Convention on Persistent Organic Pollutants (POPs). However, there is no regulation of heptachlor use in Mexico, where relatively high levels have been found in maternal breast milk. The aim of this study was to determine the effects of heptachlor on the reproductive system of offspring of two consecutive generations of rats (F1 and F2) from dams orally administered heptachlor during midgestation and lactation. Female offspring were analyzed for different phenotypic, reproductive, and molecular parameters. In the F1 generation, heptachlor treatment induced decreased body weight at weaning, increased female anogenital distance, and delayed vaginal opening. In both generations, serum progesterone levels decreased and estradiol levels remained unchanged, while overexpression of the progesterone receptor was observed in uterine epithelial cells on estrus day. In the F2 generation, expression of the estrogen receptor α increased in the glandular epithelium. Finally, heptachlor treatment did not affect apoptosis in the uterine epithelial cells. Overall, the results indicate that heptachlor induced female reproductive alterations when administered to dams during the perinatal period. Accordingly, exposure to heptachlor may represent a risk for the reproductive health of humans. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 856–868, 2017.     

Effects of exposure of rat dams to 1-bromopropane during pregnancy and lactation on growth and sexual maturation of their offspring

1-Bromopropane (1-BP) exhibits neuroreproductive toxicities in adult rats and humans. Here, we determined the effects of exposure of rat dams to 1-BP during pregnancy and lactation on the growth and sexual maturation of their offspring. In Experiment 1, 40 rats were exposed to 0, 100, 400 and 800ppm 1-BP during pregnancy and lactation for 8h/day. Ten rats that were not placed in chambers throughout the experiment served to observe the effect of separation of dams from offspring. In Experiment 2, three groups of 10 pregnant rats each were exposed to fresh air in three chambers and 10 other rats were exposed to 800ppm 1-BP during pregnancy and lactation for 8h/day. After delivery, offspring of the exposed and non-exposed dams were swapped so that they were nursed by the opposite dams. In Experiment 1, the survival rate and body weight of offspring were lower than the non-exposed in 1-BP dose-dependent manner. In Experiment 2, the survival rate and body weight of offspring (Group A) nursed by exposed dams and those (Group B) of exposed dams were significantly lower than non-exposed groups. The body weight of Group A was lower than that of Group B, although the two groups showed a significant equal decrease in the survival rate. The number of dead offspring from Group A was significantly higher. Our results indicate that exposure to 1-BP during pregnancy and lactation has comparable effects on survival rate, but exposure during lactation has a more adverse effect on growth of offspring than that during pregnancy. Moreover, exposure during lactation is associated with reduced early survival of third generation (F2) rats.     

Pathological assessment of the nervous and male reproductive systems of rat offspring exposed maternally to acrylamide during the gestation and lactation periods - a preliminary study

To evaluate the developmental effects of exposure to acrylamide (ACR) on the nervous and male reproductive systems, pregnant Sprague-Dawley rats were given ACR at 0, 50, 100 or 200 ppm in the drinking water from gestational day 10 to postnatal day 21 and histopathological assessment of offspring was performed at weaning and postnatal week 11. Neurotoxicity was quantitatively assessed with reference to nerve fiber density, percentages of degenerated and small caliber axons in the sciatic nerves, and numbers of aberrant dot-like structures immunoreactive for synaptophysin in the cerebellar molecular layer. Although maternal neurotoxicity was evident from 100 ppm, no changes suggestive of neurotoxicity or testicular toxicity were observed in offspring. However, lowering of body weights was dose-dependently observed from birth at the dose levels of > or =50 ppm in males and > or =100 ppm in females. Maternal malnutrition was apparent at >/=100 ppm during the lactation period. Therefore, poor lactational ACR-exposure due to maternal toxicity might account for the lack of ACR-induced offspring toxicity other than retarded body growth.     

Exposure to allethrin-based mosquito coil smoke during gestation and postnatal development affects reproductive function in male offspring of rat

The threat of zika virus looms throughout the world and the use of allethrin-based mosquito coils to prevent mosquito bites during and postpregnancy is on the rise. The aim of this study was to analyze the toxic effects of exposure to allethrin-based mosquito coil smoke in rats under conditions that reflect human settings. Pregnant female rats were exposed to mosquito coil smoke and same was continued to the male pups up to 111?days postparturition (21-day weaning plus up to 90?days postweaning). Increased oxidative stress, distorted antioxidant enzyme status, downregulation of genes involved in spermatogenesis, sperm maturation and steroidogenesis was observed. Daily sperm production, total sperm count and acrosome reaction was compromised. Results of our study indicate the toxic effects of exposure to allethrin-based mosquito coil smoke in male offspring and calls for preventing mosquito coil use during pregnancy and postnatal development. Community-based programs that will encourage general population to use classical methods such as use of mosquito nets, keeping the surroundings clean and use of natural mosquito repellents should be conducted.     

Exposure to cadmium during gestation and lactation affects development and function of Leydig cells in male offspring

Toxic effects of maternal exposure to Cadmium (Cd) on Leydig cells of male offspring arises much concern recently, but its toxic effects on the development of Leydig cells and androgen synthesis have not been elucidated. In this study, female rats were exposed to Cd during gestation and lactation, and the development of Leydig cells in the first filial‐generation (F1) male rats was investigated. The steroidogenic signaling pathway and biomarkers related to the development of Leydig cells were detected to disclose how maternal Cd‐exposure caused reproductive damage. F1 male rats with maternal Cd‐exposure gained a low relative weight of testis and declined levels of steroid hormones. Maternal Cd‐exposure interrupted the development of Leydig cells with high expression of SRD5α and cell morphology of immature Leydig cells in adulthood, inhibited the activation of cyclic adenosine monophosphate/ protein kinase A signaling pathway and down‐regulated the steroidogenic enzymes. These results would help to disclose the origin of male sexual dysfunction in the developmental stages of Leydig cells.     

Impairment of adult male reproductive function in rats exposed to ethanol since puberty

The objective of this work was to evaluate reproductive function in adult male rats exposed to ethanol since puberty. Male Wistar rats, 50 days old, received a liquid diet with 36% of the daily calories derived from ethanol or an isocaloric control diet for 55 days. The ethanol treatment impaired sexual behavior and only 22% of these rats reached ejaculation. The fertility of ethanol-treated animals was significantly reduced, mainly after natural mating. Serum testosterone levels, daily sperm production and sperm count in the epididymis were also significantly diminished after ethanol treatment, associated with an acceleration of the sperm transit time in the cauda epididymidis, decrease in sperm motility and increased percentage of abnormal shaped sperm cells. The results showed that chronic consumption of ethanol beginning at puberty impairs the reproductive function of adult male rats.     

Pulmonary Cyp1A1 and CYP1A2 levels and activities in adult male and female offspring of rats exposed during gestation and lactation to 2,3,7, 8-tetrachlorodibenzo-p-dioxin

The levels and activities of pulmonary microsomal CYP1A1 and CYP1A2 in 40-day-old male and female, and 120-day-old male offspring of pregnant rats treated with five weekly 0.1 microg/kg doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during gestation and lactation were compared with those in age-matched offspring of untreated dams. The CYP1A1-preferential activity, ethoxyresorufin O-deethylase (EROD), was comparably induced 5.3- and 6.4-fold in 40-day-old male and female offspring, respectively, but was not induced in 120-day-old male offspring, of TCDD-treated dams. Similarly, CYP1A1 protein was induced in 40-day-old female or male offspring of untreated dams but was undetectable in 120-day-old offspring of untreated or treated dams. CYP1A2 activity, as measured by the bioactivation of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) to mutagens in the Ames assay, was elevated 11.1- and 5.5-fold in 40-day-old female and male offspring, respectively, of TCDD-treated dams, but was unaffected by TCDD exposure in 120-day-old offspring. CYP1A2 protein was undetectable in 40-day-old male or female offspring of untreated dams or in 120-day-old male offspring of treated or untreated dams; it was detected in 40-day-old offspring of treated dams, at a level that was higher in females than in males. The results show that gestational and lactational exposure to TCDD causes long-lasting and gender-preferential induction of CYP1A1 as well as CYPIA2 in the lungs of rat offspring.     

Maternal nicotine exposure during gestation and lactation induces cardiac remodeling in rat offspring

The aim of this study was to evaluate the effects of maternal nicotine exposure on heart morphology and fibrosis in rat offspring. Nicotine was administered to pregnant Sprague-Dawley rats by using a subcutaneous osmotic mini-pump at a dose of 6 mg/kg/day from Gestational Days 7–21 or Gestational Day 7 to Postnatal Day 14. A control group received an equal volume of saline by the same route as nicotine. Rats born to prenatal nicotine-treated dams exhibited significantly greater cell width of cardiomyocytes, fewer cardiomyocyte nuclei number, higher β-myosin heavy chain and transforming growth factor (TGF-β1) expression, and higher collagen deposition in heart compared with rats born to normal saline-treated dams on Postnatal Days 7 and 21. Postnatal nicotine exposure further enhances these effects. We conclude that TGF-β1 may be involved in the pathogenesis of cardiac remodeling induced by maternal nicotine exposure during gestation and lactation in rat offspring.     

Effects of a maternal diet supplemented with chocolate and fructose beverage during gestation and lactation on rat dams and their offspring

1. Consumption of a high-fat and high-energy diet during pregnancy leads to a risk of long-term consequences on fetal development, as well as on the postnatal health of offspring. To investigate the effects of such a diet on fetal programming, we established a high-energy intake pregnant rat model using chocolate and fructose beverage as supplements to a normal chow diet. 2. Pregnant Sprague-Dawley rats were assigned to either chow (control) or a diet supplemented with chocolate and fructose beverage throughout gestation and lactation. The male F(1) pups received normal chow diet after weaning. Physiological or pathological changes in dams and pups (e.g. glucose and lipid metabolism) were evaluated. 3. The results showed that dams offered the high-fat (mainly from chocolate) and high-calorie diet during gestation consumed more energy and gained more weight than chow-fed dams. Over-consumption of chocolate reduced chow intake in dams, leading to low maternal protein supply. As a result, pups from these dams exhibited reduced birth weight that lasted until adulthood. The high-energy diet during lactation led to increased total body fat, as well as impaired liver function, in offspring; thus, the lactational diet is suggested to be a stronger determinant of offspring fat metabolism than gestational diet. 4. The results of the study suggest that over-supply of carbohydrates, such as chocolate and fructose, either during gestation or lactation has a negative impact on the well-being of offspring.     

Carbamazepine-exposure during gestation and lactation affects pubertal onset and spermatic parameters in male pubertal offspring

Carbamazepine (CBZ) is an anti-epileptic drug that acts on Leydig cells, affecting steroidogenesis and causes fetal malformation. The aim of this study was to investigate the effects of CBZ on male sexual maturation and other male parameters. Rat dams were treated with CBZ during pregnancy and breastfeeding. The anogenital distance (AGD) and the anogenital index (AGI) were obtained. Testicular descent and preputial separation were also evaluated. The offspring was euthanized at PND 41 and 63. The accessory glands were weighed and the testes were collected for histopathological, morphometric and sterological analyses. The numerical density of Leydig cells and hormone dosage were obtained. CBZ caused an increase of AGI and a delay of testicular descent and of preputial separation. CBZ also caused a decrease of testosterone level and of sperm count and an increase of abnormal sperm. These results indicate that CBZ delays puberty onset and affects steroidogenesis and sperm quality.     

Chronic oxytocin treatment during late gestation and lactation impairs development of rat offspring

The nonapeptide oxytocin (OT) plays an important role in timing and course of parturition, and in milk ejection during lactation. Exogenously enhanced OT levels were reported to impair body development of rat offspring at birth and during postnatal stages. In the present study, this effect was further investigated by giving pregnant rats of postcoital day 17 a SC implant that delivers small amounts of OT for 2 months (approximately threefold enhancement of OT levels), and by introducing a crossfostering protocol for the offspring. A slightly reduced body weight of 5 to 7% was again observed in pups born to OT-implanted dams. When reared postnatally by OT-treated mothers, pups lost weight gain (− 7 to −10%). During the weaning period, however, body size caught up with that of control animals. When nursed by an untreated mother, this recovery took place before that period. Growth of control offspring was also hampered when placed with OT-treated mothers, but these pups failed to recover from low body weights which lasted up to at least 70 days of age (−7%). Daily urine production of the pups born of and reared by the OT-treated mothers was reduced at 1 month of age, but this effect was only transient and had disappeared at 70 days of age. Notwithstanding, the recovery of body growth, brain sizes, and cerebellar DNA, i.e., cell content was reduced in the pups born and reared by OT-treated mothers, indicative of a lasting effect on brain development. Such late brain changes were not observed in pups of OT-treated mothers nursed by untreated mothers, and in control pups nursed by both types of mothers. In conclusion, one might say that physiologically enhanced levels of maternal OT can induce subtle alterations in brain development that are potentially indicative for lasting physiological and behavioral changes in the offspring.     

Reproductive effects of deltamethrin on male offspring of rats exposed during pregnancy and lactation

The effects of low doses of deltamethrin administered to female rats on the reproductive system of male offspring were examined. The dams (n=10-12/group) were treated daily by oral gavage with 0, 1.0, 2.0, and 4.0 mg deltamethrin/kg from day 1 of pregnancy to day 21 of lactation. Maternal and reproductive outcome data and male sexual development landmarks were assessed. Fertility, sexual behavior, and a large number of reproductive endpoints, such as organ weights, sperm evaluations, testosterone concentration, and testicular histology were examined on adult male offsprings. No signs of maternal toxicity were detected at the dose levels tested. Significantly adverse effects were only seen on testicular and epididymal absolute weights and the diameter of seminiferous tubules in the group treated with the highest dose of deltamethrin (4.0 mg/kg). The results indicate that in utero and lactational exposure to deltamethrin may induce subtle changes in reproductive behavior and physiology of male offspring rats at dose levels that do not cause maternal toxicity.     

Impaired female sexual behavior of rat offspring exposed to Solanum lycocarpum unripe fruits during gestation and lactation: lack of hormonal and fertility alterations

Solanum lycocarpum St. Hil (Solanaceae) is an invasive and native shrub very common in the Brazilian savanna. It is well known that this plant contain steroidal glycoalkaloids that can be transformed into an intermediate for steroidal drugs production, like oral contraceptives. In this way, it is very possible that these glycoalkaloids and its aglycone, once in the body by ingestion of S. lycocarpum fruits, may act disrupting to the endocrine system. Rat offspring were exposed to S. lycocarpum unripe fruits (10% in the diet) from gestational day (GD) 06 to post-natal day (PND) 07. The female exposed offspring showed, at adult age (PND 100), impaired sexual behavior. However, the fecal hormonal metabolite levels, measured at PND 30, PND 60 and PND 90, and the fertility (PND 120) of male and female experimental offspring were normal. We can assume that the steroidal glycoalkaloids, solamargine and solasodine, present in the fruit, are degradated, once inside the organism, to the steroidal alkaloid solasodine, which may penetrate, by simple diffusion, the placental and/or the hematoencephalical barriers and impact the fetuses. Finally, S. lycocarpum fruit may act as phytohormones, promoting perhaps some neural alterations that at adult age may impair the sexual behavior of the experimental female without impairing the fertility and sexual hormone synthesis. These changes observed can be the direct consequence of the toxic actions of the steroidal alkaloid on the female offspring during fetal development.     

Reproductive effects of endosulfan on male offspring of rats exposed during pregnancy and lactation.

1. The reproductive effects of endosulfan on the male offspring of rats were examined. Dams were treated orally with 0, 1.5 or 3.0 mg endosulfan/kg from day 15 of pregnancy to postnatal day (PND) 21 of lactation. The male offspring rats were investigated at PND 65 or 140, corresponding to the pubertal and adulthood stage of development. 2. The dose of 3.0 mg endosulfan/kg induced a decrease in maternal body weight during pregnancy, but litter size and mean birth weight were not affected. Similarly, the age at testis descent and preputial separation was not affected on the male offspring. 3. The daily sperm production (x10(6)) was permanently decreased in the highest dose group when investigated at puberty and at adulthood. At the lowest dose, however, the daily sperm production was significantly reduced only at puberty. 4. Histologically, the percentage of seminiferous tubules showing complete spermatogenesis was significantly decreased at puberty. This finding may explain the decrease in daily sperm production observed in the endosulfan-exposed male rats. 5. The results of this study show that low doses of endosulfan have no apparent effect on developmental landmarks or on the weight of reproductive and accessory sex organ. Daily sperm production was the most susceptible endpoint in the male offspring exposed to endosulfan during pregnancy and lactation. To further understand the reproductive effects of endosulfan on male rat offspring, additional reproductive and toxicokinetic studies should be carried out to determine the extent of endosulfan exposure in male rat offspring in utero and during lactation.     

Behavioural changes in the offspring of rats exposed to diazepam during gestation

Primiparous pregnant Sprague-Dawley dams were administered a single daily s.c. injection of diazepam (0.1 and 1 mg/kg) or vehicle over gestation days 14-20. No differences in neonatal mortality and weight gain were found between the control and diazepam-exposed pups. Conversely, male pups prenatally treated with this benzodiazepine exhibited subtle behavioural alterations either during early postnatal life or during adulthood. In particular, a significant decrease in the locomotor activity of the diazepam-treated groups was found at the end of the second postnatal week (14-16 days). Furthermore, the administration of diazepam during gestation produced marked changes in the length of ultrasonic calls of rat pups removed from their nest. Finally, adult male rats (120 days of age) prenatally exposed to diazepam showed a notable impairment in copulatory activity as well as a significant decrease in the duration of ultrasonic (22 kHz) post-ejaculatory calls emitted during sexual behaviour. These findings suggest that late gestational exposure to diazepam induces both short- and long-term behavioural changes in rat offspring, changes characterized by altered activity patterns and emotional-motivational responsiveness to environmental challenges.     

Limited lactational transfer of acrylamide to rat offspring on maternal oral administration during the gestation and lactation periods

To evaluate the developmental exposure effects of acrylamide (ACR) on the nervous and male reproductive systems, pregnant Sprague-Dawley rats were given ACR at 0, 25, 50 or 100 ppm in the drinking water from gestational day 6 to postnatal day (PND) 21 and histopathological assessment was performed at PND 21. Exposure levels in offspring were examined by measurement of free ACR and hemoglobin (Hb)-ACR adducts on PND 14, and compared with maternal levels on PND 21. Additionally, a group of offspring that received ACR at 50 mg/kg by intraperitoneal injections directly three times a week from PND 2 to 21 was subjected to analysis for comparison with maternal exposure groups. Although maternal neurotoxicity was evident at 100 ppm, no changes suggestive of neurotoxicity or testicular toxicity were observed in their offspring except for growth retardation evident as lowered body weights. In contrast, offspring given ACR intraperitoneally exhibited obvious neurotoxicity, but not testicular damage. Free ACR in serum and milk was detected in neither dams nor their offspring. The level of ACR-Hb adducts in offspring was one tenth or less than that in dams. In summary, although preweaning rats have susceptibility to ACR-induced neurotoxicity, the internal level of ACR in offspring exposed through maternal oral administration is insufficient to induce neurotoxicity and testicular toxicity due to limited lactational transfer.     

Consumption of xenoestrogen-contaminated fish during lactation alters adult male reproductive function.

Little information exists on the transfer of endocrine-disrupting effects through the food chain. The transfer of chemicals, particularly from the aquatic ecosystem, that can cause such effects on fish-eating predators must be established. Fish from the St. Lawrence River are exposed to xenoestrogens causing male reproductive dysfunction. The objective of this study was to determine if lactational exposure to contaminated fish could alter the development of the male reproductive system in rats. Three experimental groups were used: rats (dams) gavaged with (a) distilled water (control), or (b) homogenized fish from a reference site (Iles de la Paix) or (c) homogenized fish from a xenoestrogen-contaminated site (Ilet Vert). Pups were exposed via lactation and sampled on either day 21 (weaning) or day 91 (adults). There was no effect on the body weights or in the male reproductive organ weights between groups except for adult epididymal weight, which was significantly decreased in the xenoestrogen group. Adult sperm concentrations and sperm motility parameters were all significantly decreased in the xeonestrogen group as compared to the reference and control groups. Furthermore, the distribution of stages of spermatogenesis was altered in the xenoestrogen group, indicating an effect on the kinetics of spermatogenesis. Immunoreactivity of connexin43 (Cx43), a gap-junctional protein, was markedly decreased in the seminiferous epithelium of the xenoestrogen group, suggesting that the intercellular coordination of testicular function may be affected. These data indicate that contaminants from xenoestrogen environments may pass through the food chain and exert effects on male reproductive functions.     

Proteomic analysis of hippocampal proteins of F344 rats exposed to 1-bromopropane

1-Bromopropane (1-BP) is a compound used as an alternative to ozone-depleting solvents and is neurotoxic both in experimental animals and human. However, the molecular mechanisms of the neurotoxic effects of 1-BP are not well known. To identify the molecular mechanisms of 1-BP-induced neurotoxicity, we analyzed quantitatively changes in protein expression in the hippocampus of rats exposed to 1-BP. Male F344 rats were exposed to 1-BP at 0, 400, or 1000 ppm for 8 h/day for 1 or 4 weeks by inhalation. Two-dimensional difference in gel electrophoresis (2D-DIGE) combined with matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) were conducted to detect and identify protein modification. Changes in selected proteins were further confirmed by western blot. 2D-DIGE identified 26 proteins with consistently altered model (increase or decrease after both 1- and 4-week 1-BP exposures) and significant changes in their levels (p < 0.05; fold change ≥ ± 1.2) at least at one exposure level or more compared with the corresponding controls. Of these proteins, 19 were identified by MALDI-TOF-TOF/MS. Linear regression analysis of 1-BP exposure level identified 8 differentially expressed proteins altered in a dose-dependent manner both in 1- and 4-week exposure experiments. The identified proteins could be categorized into diverse functional classes such as nucleocytoplasmic transport, immunity and defense, energy metabolism, ubiquitination-proteasome pathway, neurotransmitter and purine metabolism. Overall, the results suggest that 1-BP-induced hippocampal damage involves oxidative stress, loss of ATP production, neurotransmitter dysfunction and inhibition of ubiquitination-proteasome system.     

Absence of structural or functional alterations in male and female reproductive organs of F1 and F2 generations derived from female mice exposed to 3'-azido-3'-deoxythymidine during pregnancy

To investigate the effects of in utero exposure to 3'-azido-3'-deoxythymidine (AZT) on male and female reproductive system development, pregnant CD-1 mice were given daily intragastric doses of 25.0 mg AZT during days 12 through 18 of gestation. The offspring were examined at birth, as well as at pubertal, young adult and adult stages of development, for reproductive organ endpoints including anogenital distance, onset of testicular descent, latency to vaginal opening, and proportion of time for each of the stages of estrous cycle. These reproductive endpoints remained mostly unchanged in AZT-treated offspring as compared to the controls. Males and females exposed in utero to AZT (F1 generation) were fertile when mated to untreated females and males, respectively, and their liveborn F2 offspring showed no adverse effects for any of the reproductive parameters tested. Thus, no evidence of developmental reproductive toxicity was noted either in the F1 mice exposed to AZT during the critical period of male and female reproductive system development, or in the F2 mice born of matings between the AZT-exposed F1 mice and unexposed animals.     

Reproductive and developmental toxicity in F1 Sprague-Dawley male rats exposed to di-n-butyl phthalate in utero and during lactation and determination of its NOAEL

Di-n-butyl phthalate (DBP) is one of the commonly used plasticizers in China. DBP can enter the environment and organisms through various routes and then affect reproductive and developmental processes of the organism and its descendants (mainly affecting male offspring). It is known that animals are sensitive to exposure of DBP in utero and during lactation. In the present study, pregnant rats were treated with different doses of DBP (0, 50, 250, and 500 mg/kg body weight/day) by daily gavage from GD1 to PND21. The developmental condition of F1 rats and the reproductive system of mature F1 male rats were monitored. DBP had no obvious effect on pregnant rats; however, it reduced several parameters including birth weight, number of live pups per litter, body weight gain and male anogenital distance. Severe damage to the reproductive system of mature F1 male rats included testicular atrophy, underdeveloped or absent epididymis, undescended testes, obvious decline of epididymal sperm parameters, total sperm heads per g testis, decrease of organ/body weight ratio of epididymis and prostate, and was observed in the group treated with 250 mg/kg BW/day and higher. These results showed that the male reproductive system was the main target organ of DBP exposure. The NOAEL (no observable adverse effect level) for developmental toxicity of DBP was established based on pup body weight and male reproductive lesions at 50 mg/kg BW/day. Accordingly, the RfD for human exposure to DBP through oral intake was recommended as 500 mg/kg BW/day.