Frédéric Gallissot

Assessment of the Developmental Toxicity, Metabolism, and Placental Transfer of Di-n-butyl Phthalate Administered to Pregnant Rats

The developmental toxicity and placental transfer of di-n-butyl phthalate (DBP) were evaluated in Sprague-Dawley rats given a single oral dose of DBP on Gestational Day 14. In the developmental toxicity study, dams were dosed with 0, 0.5, 1, 1.5, or 2 g DBP/kg and were necropsied on GD21. Increased incidence of resorptions and reduced fetal body weight were observed at 1.5 and 2 g/kg. Higher incidences of skeletal variations were found at doses > or = at 1 g/kg. No embryotoxic or teratogenic effects were observed at a dose of 0.5 g/kg. In the placental transfer study, dams were dosed with 0.5 or 1.5 g [14C]DBP/kg. Maternal and embryonic tissues were collected at intervals from 0.5 to 48 h. Embryonic tissues accounted for less than 0.12-0.15% of the administered dose. Levels of radiocarbon in placenta and embryo were one-third or less of those in maternal plasma. No accumulation of radioactivity was observed in the maternal or embryonic tissues. From HPLC analyses, it was shown that unchanged DBP and its metabolites mono-n-butyl phthalate (MBP) and MBP glucuronide were rapidly transferred to the embryonic tissues, where their levels were constantly lower than those in maternal plasma. MBP accounted for most of the radioactivity recovered in maternal plasma, placenta, and embryo. Unchanged DBP was found only in small amounts. These findings support the hypothesis that MBP, a potent teratogen, largely contributes to the embryotoxic effects of DBP.

Developmental toxicity of N-methyl-2-pyrrolidone administered orally to rats

The developmental toxicity of N-methyl-2-pyrrolidone (NMP) was studied in Sprague-Dawley rats after oral administration. Pregnant rats were given NMP at doses of 0 (distilled water), 125, 250, 500, and 750 mg/kg/day, by gavage, on gestational days (GD) 6 through 20. Significant decreases in maternal body weight gain and food consumption during treatment, and a reduction in absolute weight gain were observed at 500 and 750 mg/kg. The incidence of resorptions per litter was significantly higher than control at 500 mg/kg, and rose to 91% at 750 mg/kg. Examination of the foetuses revealed treatment-related malformations, including imperforate anus and absence of tail, anasarca, and malformations of the great vessels and of the cervical arches. The incidence of malformed foetuses per litter, and of litters with malformed foetuses was significantly increased at 500 and 750 mg/kg. At 250 mg/kg, one foetus showed malformations similar to those recorded at higher dosages. There was a dose-related decrease in foetal body weights (male, female, and total) that reached statistical significance at 250 mg/kg. A significant increase in incomplete ossification of skull bones and of sternebrae was also present at 500 and 750 mg/kg. In summary, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was 250 and 125 mg/kg/day, respectively. Thus, oral administration of NMP produced developmental toxicity below maternally toxic levels.

Diisobutyl phthalate impairs the androgen-dependent reproductive development of the male rat.

Diisobutyl phthalate (DIBP) is the branched isomer of DBP; DBP side chains have a four-carbon backbone (C4), whereas DIBP has its four-carbon alkyl side chains rearranged into a three-carbon backbone (C3) with a methyl branch. Di-n-butyl phthalate (DBP), and several other ortho-phthalate esters with side-chain lengths of C4-C6, are known to disrupt the androgen-dependent sexual differentiation in the male rat. This study was performed to determine whether in utero exposure to DIBP would induce permanent and dose-responsive alterations of male reproductive development. Pregnant Sprague-Dawley rats were administered olive oil (vehicle control), DIBP or DBP, by gavage on gestation Days 12-21, at doses of 125, 250, 500, 625mgDIBP/(kg day) and 500mgDBP/(kg day). DIBP caused no overt maternal toxicity, nor reduced litter size. Male offspring displayed reduced neonatal anogenital distance (Postnatal day 1, PND) at 250mgDIBP/(kg day) and higher doses, and dose-related retention of areolas/nipples (PND 12-14). Preputial separation (onset of puberty) was delayed in male offspring at 500 and 625mgDIBP/(kg day). Hypospadias, cleft prepuce, and undescended testis were observed in males (11-12 or 16-17 weeks old) exposed in utero to 500 and 625mgDIBP/(kg day). Histopathological lesions were also present in adult testes, mainly consisting in seminiferous tubule degeneration. Our results show that DIBP can cause severe and specific adverse effects on the male rat reproductive development, with a pattern similar to that of DBP. However, DIBP appeared slightly less potent than DBP in inducing malformations.

Differential developmental toxicities of di-n-hexyl phthalate and dicyclohexyl phthalate administered orally to rats.

The objective of this study was to evaluate the developmental toxic potential of di‐n‐hexyl phthalate (DnHP) and dicyclohexyl phthalate (DCHP) in rats. Pregnant Sprague–Dawley rats were exposed to DnHP or DCHP at doses of 0 (olive oil), 250, 500 and 750 mg kg–1 per day, by gavage, on gestational days (GD) 6–20. Maternal food consumption and body weight gain were significantly reduced at 750 mg kg–1 per day of DnHP and at the two high doses of DCHP. Slight changes in liver weight associated with peroxisomal enzyme induction were seen in dams treated with DnHP or DCHP. DnHP caused dose‐related developmental toxic effects, including marked embryo mortality at 750 mg kg–1 per day, and presence of malformations (mainly cleft palate, eye defects and axial skeleton abnormalities) and significant decreases in fetal weight at 500 and 750 mg kg–1 per day. Significant delay of ossification and increase in the incidence of skeletal variants (e.g. supernumerary lumbar ribs) also appeared at 250 mg kg–1 per day. DCHP produced fetal growth retardation at 750 mg kg–1 per day, as evidenced by significant reduction of fetal weight. DnHP and DCHP induced a significant and dose‐related decrease in the anogenital distance of male fetuses at all doses, and there was a significant increase in the incidence of male fetuses with undescended testis at 500 and 750 mg kg–1 per day of DnHP. In conclusion, DnHP showed clear embryolethality and teratogenicity, but not DCHP. There was evidence that both phthalates could alter the development of the male reproductive system after in utero exposure, DnHP being much more potent than DCHP. Copyright

Developmental toxicity of N-methyl-2-pyrrolidone in rats following inhalation exposure

The developmental toxicity of inhaled N-methyl-2-pyrrolidone (NMP) was studied in Sprague-Dawley rats. Pregnant rats were exposed whole body to NMP vapours at concentrations of 0, 30, 60 and 120 ppm, 6 h/day, on gestational days (GD) 6 through 20. Maternal body weight gain was significantly decreased at 60 and 120 ppm on GD 6-13 and maternal food consumption was reduced at 120 ppm on GD 13-21. No significant difference in the gestational weight change corrected for the weight of the gravid uterus was observed, whatever NMP concentration. There were no adverse effects on embryo/fetal viability or evidence of teratogenicity at any concentration tested. Fetal toxicity indicated by reduced fetal weight was observed at 120 ppm. Thus, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was 30 and 60 ppm, respectively.

Developmental toxicities of ethylbenzene, ortho-, meta-, para-xylene and technical xylene in rats following inhalation exposure.

The developmental toxicities of ethylbenzene, o-, m-, p-xylene and technical xylene were studied in Sprague-Dawley rats after inhalation exposure. Animals were exposed to either of these agents at 100, 500, 1000 or 2000 ppm, for 6 h/day, during days 6-20 of gestation. All the agents tested caused maternal toxicity expressed as a reduction in maternal body weight gain at 1000 and 2000 ppm. Decreased corrected weight gain and food consumption were observed at 1000 and 2000 ppm ethylbenzene, o-, m- or p-xylene, and at 2000 ppm technical xylene. No evidence of teratogenic effects was found after exposure to any of these agents up to 2000 ppm. Fetal toxicity evidenced by significant decreases in fetal body weights occurred at concentrations of 500 ppm or greater of o-xylene or technical xylene, and 1000 ppm or greater of ethylbenzene, m- or p-xylene. A significant increase in the mean percentage of fetuses per litter with skeletal variations was also noted at 2000 ppm ethylbenzene, o- and p-xylene. In summary, all tested agents produced developmental toxicity at 1000 and 2000 ppm, concentrations that also produced significant maternal toxicity. With o-xylene and technical xylene, developmental toxicity also occurred at 500 ppm, in the absence of maternal toxic effects. However, the only indication of a treatment-related effect was a slight decrease in fetal weight.

Effects of in utero exposure to di-n-hexyl phthalate on the reproductive development of the male rat

Recently, the plasticizer di-n-hexyl phthalate (DnHP) has been demonstrated to be teratogenic and adversely affect the reproductive tract in male rat fetuses. This study was undertaken to determine the long-term effects of an in utero exposure to DnHP on the reproductive development of the male offspring. Di-2-ethylhexyl phthalate (DEHP), another phthalate ester known to disrupt the androgen-dependent sexual differentiation in the male rat, was used as a positive control. Pregnant Sprague-Dawley rats were administered DnHP or DEHP, by gavage on gestation Days 12-21, at doses of 0, 50, 125, 250, or 500 mg DnHP/kg-d and 500 mg DEHP/kg-d. DnHP had no significant effect on maternal body weight gain and pup weights during lactation. The proportion of live pups on postnatal day 1 was slightly, but not significantly, lower than control at 250 and 500 mg DnHP/kg-d. Male offspring displayed reduced anogenital distance on postnatal day 1 (PND) at 125 mg DnHP/kg-d and above, and areola/nipple retention before weaning and at adulthood at 250 and 500 mg DnHP/kg-d. At necropsy on PND 70-78 or PND 111-120, severe malformations of the reproductive tract were observed in young adult males at 125 mg DnHP/kg-d and higher doses. They mainly consisted of hypospadias, underdeveloped testis, and undescended testis. Additionally, histopathological examination revealed seminiferous tubule degeneration at the two high doses. Our results showed that prenatal exposure to DnHP caused permanent and dose-related alterations of the male rat reproductive development, with a similar profile as DEHP.

Prenatal developmental toxicity studies on di-n-heptyl and di-n-octyl phthalates in Sprague-Dawley rats

This study evaluates the developmental toxicity of two dialkyl phthalate esters, di-n-heptyl phthalate (DHPP) and di-n-octyl (DnOP) phthalate, which have straight-alkyl side chains of seven and eight carbons, respectively. Sprague-Dawley rats were administered 0, 0.25, 0.50, or 1g/kg/day of DHPP or DnOP, by gavage, on gestation days 6-20. DHPP and DnOP had no adverse effect on maternal feed consumption and body weight gain, or on the incidence of post-implantation loss and fetal body weight. There was no increase in the incidence of fetal malformations or external and visceral variations, whatever treatment. A significant increase in rudimentary lumbar ribs was observed at all doses of DHPP and DnOP. The anogenital distance of the male fetuses was significantly decreased at the highest dose of DHPP. This parameter was not affected by DnOP. Thus, the lowest-observed-adverse-effect level (LOAEL) for developmental toxicity was 0.25 g/kg/day for DHPP and DnOP.

Comparative embryotoxicities of butyl benzyl phthalate, mono-n-butyl phthalate and mono-benzyl phthalate in mice and rats: in vivo and in vitro observations.

The embryotoxic effects of butyl benzyl phthalate (BBP) and its two main metabolites mono-n-butyl (MBP) and mono-benzyl (MBzP) phthalate were evaluated in OF1 mice and Sprague-Dawley rats, in vivo and in whole embryo culture. In vivo, pregnant mice and rats received a single oral dose (0.9-5.4 mmol/kg) of either of these compounds on GD 8 and 10, respectively, and their fetuses were examined externally on GD 18 and 21, respectively. In mice, BBP, MBP and MBzP caused concentration-related embryolethality and malformations. In rats, MBP and MBzP did not show developmental toxicity. Some teratogenicity and a slight increase in post-implantation loss were observed after BBP administration, but mice were more susceptible to its toxic effects than were rats. In vitro, GD 8 mouse embryos and GD 10 rat embryos were cultured for 46 h in the presence of the test compounds (0.5 to 3-5mM). The cultured mouse embryos did not appear intrinsically more sensitive to MBP and MBzP, than the rat embryos. Altogether, these results suggest that the species sensitivity observed in vivo after an oral administration of BBP, MBP or MBzP during early organogenesis, might be due to maternal factors, i.e. toxicity and/or kinetics.

Developmental toxic potential of di-n-propyl phthalate administered orally to rats.

The objective of this study was to evaluate the developmental toxic potential of di‐n‐propyl phthalate (DnPP) in rats. Pregnant Sprague–Dawley rats were given DnPP at doses of 0 (olive oil), 0.5, 1 and 1.5 g kg−1 per day, by gavage, on gestation days 6–20. Benchmark doses were calculated for the effects of DnPP on fetal weight and anogenital distance of the male fetuses. Maternal body weight gain was significantly reduced at 1.5 g kg−1 per day, over gestation days 6–9. DnPP‐treated dams also showed a statistically significant increase in liver weight and a mild but statistically significant peroxisomal enzyme induction at 1 or 1.5 g kg−1 per day. Male and female fetal body weights were significantly reduced at 1.5 g kg−1 per day. There was a statistically significant decrease in the anogenital distance of the male fetuses at 1 and 1.5 g kg−1 per day, and three males (of 75) showed malpositioned testis at the high dose. The mean percentage of fetuses per litter with cervical and thoracic rudimentary ribs was significantly increased at 1 and 1.5 g kg−1 per day. Delayed ossification was seen at 1 g kg−1 per day (phalanges) and 1.5 g kg−1 per day (hyoid, sternebrae, and phalanges). No treatment‐related effects on prenatal viability or on fetal external or visceral malformations or variations were observed at any dose. Thus, there was no evidence of teratogenicity up to the high dose of 1.5 g kg−1 per day. The no‐observed‐adverse‐effect level (NOAEL) for developmental toxicity was 0.5 g kg−1 per day. Copyright