Ordinal dose-response modeling approach for the phthalate syndrome.

BACKGROUND: The phthalate syndrome (PS) is a collection of related male reproductive developmental effects, ranging in severity, that have been observed in rats after gestational exposure to developmentally-toxic phthalates. For statistical purposes, the PS is defined as a single endpoint and one dose-response analysis is conducted, rather than conducting multiple analyses on each individual endpoint. OBJECTIVE: To improve dose-response modeling approaches for the PS and other syndromes of effects by accounting for differing severity levels among the endpoints. METHODS: Ordinal dose-response modeling was performed on PS data from a published study of diisobutyl phthalate (DIBP) gestational exposure to male Sprague-Dawley rats. To incorporate PS endpoint severity, the endpoints were categorized into ordinal levels based on the expected impact of male developmental endpoint's on fertility. Then, a benchmark dose was estimated for each ordinal level. A bootstrap procedure was used to account for the nested nature of the data, and a sensitivity analysis was performed to assess the bootstrap results. A comparison of the estimates between the ordinal and the dichotomous model was performed. RESULTS: The ordinal version of the log-logistic model applied to the data categorized by PS endpoint severity level provided benchmark dose estimates that were closer to each other in value and had lower variability than the traditional dichotomous application. The sensitivity analysis confirmed the validity of the bootstrap results. CONCLUSION: The ordinal dose-response modeling method accounts for severity differences among dichotomous PS endpoints, can be expanded in the future to include more severity levels, and can be used in both single and cumulative phthalate risk assessments.

Toxicokinetics of N-ethyl-2-pyrrolidone and its metabolites in blood, urine and amniotic fluid of rats after oral administration.

The toxicokinetics of N-ethyl-2-pyrrolidone (NEP), an embryotoxic organic solvent, has been studied in Sprague-Dawley rats after oral exposure. NEP and its metabolites 5-hydroxy-N-ethyl-2-pyrrolidone (5-HNEP) and 2-hydroxy-N-ethylsuccinimide (2-HESI) were measured in plasma of pregnant and non-pregnant rats, and fetuses after NEP administration by gavage for 14 consecutive days at 50 mg/kg/day, and in plasma of non-pregnant rats after a single NEP administration. Additionally, amniotic fluid and 24-h urine samples of the pregnant rats were analyzed for NEP metabolites. Furthermore, 24-h urine samples from a repeated dose 28-day oral toxicity study in female (non-pregnant) and male rats administered developmentally non-toxic (0, 5, and 50 mg/kg/day) or toxic (250 mg/kg/day) doses of NEP were analyzed. Median peak plasma concentrations in non-pregnant rats after a single dose and repeated doses were 551 and 611 (NEP), 182 and 158 (5-HNEP), and 63.8 and 108 µmol/L (2-HESI), respectively; whereas in pregnant rats and fetuses 653 and 619 (NEP), 80.5 and 91.7 (5-HNEP) and 77.3 and 45.7 µmol/L (2-HESI) were detected. Times to reach maximum plasma concentrations for NEP, 5-HNEP, and 2-HESI were 1, 4, and 8 h, respectively, and were comparable to N-methyl-2-pyrrolidone (NMP) and its corresponding metabolites. In pregnant rats, plasma elimination of NEP and metabolite formation/elimination was much slower compared to non-pregnant rats and efficient placental transfer of NEP was observed. Our data, overall, suggest differences in the toxicokinetics of chemicals between pregnant and non-pregnant rats which need to be addressed in risk assessment, specifically when assessing developmental toxicants such as NEP.

The pyrethroid insecticides permethrin and esfenvalerate do not disrupt testicular steroidogenesis in the rat fetus.

The present study investigated the effects of maternal exposure to the widely used pyrethroid insecticides, permethrin and esfenvalerate, on fetal testicular steroidogenesis. Pregnant Sprague-Dawley rats were administered permethrin at doses of 1, 10, 50, or 100 mg/kg/day, or esfenvalerate at 0.1, 1, 7.5 or 15 mg/kg/day, by gavage, from gestation day (GD) 13 to 19. Testicular testosterone production and the expression of several key genes necessary for cholesterol and androgen synthesis and transport were assessed in GD 19 male fetuses. Dams treated with 100 mg/kg/day of permethrin or 15 mg/kg/day of esfenvalerate showed clinical signs of neurotoxicity. The highest dose of esfenvalerate also resulted in reduced maternal body weight gain throughout the treatment period. In the fetal testes, mRNA expressions of HMG-CoA synthase and reductase, SR-B1, StAR, P450scc, 3ßHSD, P450 17A1, and 17ßHSD were not affected by exposure to either pyrethroid. No significant change was observed in ex vivo testosterone production. In conclusion, in utero exposure to permethrin or esfenvalerate has no effect on the testosterone biosynthesis pathway in the fetal rat testis up to maternal toxic doses.

Recent biomonitoring reports on phosphate ester flame retardants: a short review.

Organophosphate triesters (PEFRs) are used increasingly as flame retardants and plasticizers in a variety of applications, such as building materials, textiles, and electric and electronic equipment. They have been proposed as alternatives to brominated flame retardants. This updated review shows that biomonitoring has gained incrementally greater importance in evaluating human exposure to PEFRs, and it holds the advantage of taking into account the multiple potential sources and various intake pathways of PEFRs. Simultaneous and extensive internal exposure to a broad range of PEFRs have been reported worldwide. Their metabolites, mainly dialkyl or diaryl diesters, have been used as biomarkers of exposure and have been ubiquitously detected in the urine of adults and children in the general population. Concentrations and profiles of PEFR urinary metabolites are seen to be variable and are highly dependent on individual and environmental factors, including age, country regulation of flame retardants, and types and quantities of emissions in microenvironments, as well as analytical procedures. Additional large biomonitoring studies, using a broad range of urinary diesters and hydroxylated metabolites, would be useful to improve the validity of the biomarkers and to refine assessments of human exposure to PEFRs.

Evaluation of the effects of α-cypermethrin on fetal rat testicular steroidogenesis.

Pregnant Sprague-Dawley rats were administered the insecticide α-cypermethrin at doses of 0.1, 1, 5, or 10mg/kg/day, or di-isobutyl phthalate (DIBP) at 250mg/kg/day, by gavage, from gestation day (GD) 13 to 19. Testicular testosterone production and the expression of several key genes related to cholesterol and androgen synthesis and transport were assessed in GD 19 male fetuses. Dams treated with 10mg/kg/day of α-cypermethrin showed clinical signs of neurotoxicity and reduced body weight gain. α-Cypermethrin had no significant effect on post-implantation loss, fetal weight, incidence of male fetuses per litter, or anogenital distance of the male fetuses. In the fetal testes, mRNA expressions of HMG-CoA synthase and reductase, SRB1, StAR, P450scc, 3ßHSD, P450 17A1, and 17ßHSD were not affected by exposure to α-cypermethrin. Testosterone production by the fetal testis was significantly reduced at 5 and 10mg/kg/day of α-cypermethrin, although to a much smaller extent than in DIBP-exposed fetuses.

Evaluation of the effects of deltamethrin on the fetal rat testis.

Pregnant Sprague-Dawley rats were administered deltamethrin, at doses 0.1, 1, 5 or 10 mg kg(-1) day(-1) , or di-n-hexyl phthalate (DnHP) (250 mg kg(-1) day(-1) ), by gavage, from gestational day 13 to 19. Maternal toxicity was observed at 10 mg kg(-1) day(-1) , as evidenced by transient clinical signs of neurotoxicity and reductions in body weight, body weight gain and corrected weight gain. Deltamethrin had no statistically significant effect on the incidence of post-implantation loss, fetal weight or anogenital distance in the male fetuses. Unlike DnHP, deltamethrin induced no changes in the expression of several genes involved in cholesterol transport or in the steroid synthesis pathway in the testes of gestational day 19.5 male fetuses (SRB1, StAR, P450scc, 3ßHSD, P450 17 A1, 17ßHSD). Fetal testicular levels of P450scc and P450 17 A1 protein were also unaffected by deltamethrin. No statistically significant differences were observed in the ex vivo fetal testicular production of testosterone and androstenedione after deltamethrin exposure, whereas DnHP markedly reduced these parameters. The deltamethrin metabolite, 3-phenoxybenzoic acid, was detected in amniotic fluid. In summary, our results demonstrate that in utero exposure to deltamethrin during the period of sexual differentiation had no significant effect on the testosterone synthesis pathway in the male rat fetus up to a maternal toxic dose. Copyright © 2016 John Wiley & Sons, Ltd.

The estrogenic and androgenic potential of pyrethroids in vitro. Review.

Synthetic pyrethroids are used worldwide as insecticides. Their metabolites are regularly detected in the urine of adults and children from the general population. There is increasing concern that they may induce sex-hormone disrupting effects. The present work reviews available published information on the (anti)estrogenic and (anti)androgenic activity of pyrethroids in in vitro screening tests. In recent years, a large number of pyrethroids have been evaluated using various common testing methods. In tests using recombinant yeast or mammalian cells, the pyrethroids were found to be essentially negative or weakly estrogenic. More inconsistent results were found regarding their estrogenic action in proliferation tests. Conflicting findings were also reported across studies and/or assays which evaluated their anti-estrogenic or anti-androgenic potential. Some studies have suggested that certain pyrethroids may have potential antagonist activity. However, no strong interaction with the estrogenic or androgenic pathway was reported. The present review confirms the interest in performing a screening battery and in adopting an integrative approach for identifying the potential of different compounds from a chemical family to interfere with the endocrine system.

Pyrethroids: exposure and health effects--an update.

Synthetic pyrethroids are present in numerous commercial insecticide formulations and have extensive indoor and outdoor applications worldwide, including agricultural, public, residential, and veterinary usages for pest control. Pyrethroid use has increased continuously in recent years. The aim of this review is to provide updated and comprehensive information on human exposure and potential hazards associated with this class of pesticides. An initial keyword search in the PubMed database was conducted to identify relevant articles. Were taken into considerations only the studies published in the last decade that have assessed exposure and health effects of pyrethroids in human populations. Literature review shows that exposure evaluations increasingly focus on biomonitoring and that a large number of recent epidemiological studies pertain to the effects of pyrethroids on male fertility and prenatal development. The main metabolites of pyrethroids have frequently been detected in urine samples from the general population, confirming widespread exposure of children and adults to one or more pyrethroids. Non-occupational exposure to pyrethroids mainly occurs through ingestion of residues in food, or ingestion of or dermal contact with contaminated house dust or surface-adhering particles, following domestic use. Although clinical features resulting from acute accidental exposure to pyrethroids are well described (e.g., paraesthesiae, and respiratory, eye and skin irritation), information regarding their chronic effects at low concentrations is both limited and controversial. Several recent epidemiological studies have raised concerns about potentially adverse effects on sperm quality and sperm DNA, reproductive hormones, and pregnancy outcomes. Early neurobehavioural development after in utero exposure is discussed. Further research is needed to clarify the possible risks associated with long-term environmental exposure to pyrethroids.

Adverse effects of diisooctyl phthalate on the male rat reproductive development following prenatal exposure.

In a first study, rats were given diisooctyl phthalate (DIOP, CAS 27554-26-3) at 0, 0.1, 0.5, and 1g/kg/day, by gavage, on gestation days 6-20 (GD). There was a significant increase in resorptions at 1g/kg/day and a reduction in fetal weights at 0.5 and 1g/kg/day. Malpositioned testes were observed in fetuses at 1g/kg/day, and supernumerary lumbar ribs and ossification delay at 0.5 and 1g/kg/day. In a follow-up study, DIOP administered on GD 12-19 reduced fetal testicular testosterone at 0.1g/kg/day and above. Finally, postnatal reproductive assessment was conducted in adult male offspring prenatally exposed to DIOP on GD 12-21. Abnormalities of reproductive system (e.g. hypospadias, non scrotal testes, and hypospermatogenesis) were observed in a few adult males at 0.5g/kg/day, and with a high incidence at 1g/kg/day. Thus, DIOP displayed an antiandrogenic activity and disrupted the male reproductive development.

Dose-dependent alterations in gene expression and testosterone production in fetal rat testis after exposure to di-n-hexyl phthalate.

In utero exposure to the phthalate ester plasticizer di-n-hexyl phthalate (DnHP) is known to affect the development of the male reproductive system and induce alterations in androgen-dependent tissues of male rat offspring. Male reproductive malformations produced by several phthalates have been causally linked to decreased testosterone production during the gestational period. This study was designed to evaluate the dose-response relationship for the effects of DnHP on the synthesis and production of testosterone in the fetal rat testis. Pregnant Sprague-Dawley rats were administered the vehicle (olive oil) and either DnHP (5 to 625 mg kg(-1) per day) or diethylhexyl phthalate (DEHP) (50 or 625 mg kg(-1) per day), by gavage, from gestation day (GD) 12 to19. Fetal testes were assessed on GD 19. DnHP reduced ex vivo testosterone production and down-regulated the expression of several genes required for cholesterol transport and steroid synthesis (i.e. SR-B1, StAR, P450scc, 3ßHSD and P450c17). These inhibitions were dose dependent. A no-effect level was established at 5 mg kg(-1) per day and a lowest-effect level at 20 mg kg(-1) per day. mRNA levels of SR-B1, StAR, P450scc and 3ßHSD were not similarly decreased in the adrenals. In conclusion, DnHP shares the same mode of action as DEHP in disrupting fetal testicular androgen synthesis. Alterations in testosterone production and in key steroidogenic gene expressions were apparent at lower doses than those causing postnatal reproductive malformations after gestational exposure during the critical period of male sexual differentiation. This suggests that they can be considered early biomarkers of DnHP-induced fetal testicular effects in rats.

Prenatal developmental toxicity studies on diundecyl and ditridecyl phthalates in Sprague-Dawley rats.

This study evaluates the developmental toxicity of two high molecular weight dialkyl phthalate esters, diundecyl phthalate (DUDP) and ditridecyl phthalate (DTDP). Sprague-Dawley rats were administered 0, 0.25, 0.50, or 1g/kg/day of DUDP or DTDP, by gavage, on gestation days 6-20. DUDP and DTDP had no adverse effects on maternal body weight and food consumption. The number of live fetuses, percent of post-implantation loss and of resorptions, fetal sex, and fetal body weights were not affected by either phthalate. There was no evidence of teratogenicity, whatever treatment. Small decreases in the anogenital distance of male fetuses were noted at 0.5 and 1g DUDP/kg/day. The incidence of fetuses with supernumerary lumbar ribs was significantly higher than control at 0.5 and 1g DUDP/kg/day. Thus, DTDP was not developmentally toxic up to 1g/kg/day and there were signs of DUDP-induced fetal effects at 0.5 and 1g/kg/day.

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.

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.

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.

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.

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.

Developmental toxic effects of ethylbenzene or toluene alone and in combination with butyl acetate in rats after inhalation exposure.

First, the developmental toxic potential of n-butyl acetate (BA) was examined in Sprague-Dawley rats following whole body inhalation exposure, 6 h day(-1), from day 6 to 20 of gestation, at concentrations of 0, 500, 1000, 2000 and 3000 ppm. Maternal toxicity was evidenced by significant decreases in body weight gain at 2000 and 3000 ppm, and by reduced food consumption at 1000 ppm and higher concentrations. The effects on prenatal development were limited to a significant decrease in fetal weight at 3000 ppm. Thus, inhaled BA was not a selective developmental toxicant. In the second part of this study, the developmental toxic effects of simultaneous exposures to ethylbenzene (EB) and BA, or to toluene (TOL) and BA were evaluated. Pregnant rats were administered EB (0, 250 or 1000 ppm) and BA (0, 500 or 1500 ppm), or TOL (0, 500 or 1500 ppm) and BA (0, 500, 1500 ppm), separately and in combinations, using a 2 x 2 factorial design. The maternal weight gain was reduced after exposure to 1000 ppm EB, to 1500 ppm BA, or to 1500 ppm TOL, either alone or in binary combinations. A significant reduction of fetal weight was associated with exposure to 1000 ppm EB alone, to either mixtures of EB with BA, or to 1500 ppm TOL alone or combined with BA at either concentration. No embryolethal or teratogenic effects were observed whatever the exposure. There was no evidence of interaction between EB and BA or between TOL and BA in causing maternal or developmental effects.

Concentrations of N-methyl-2-pyrrolidone (NMP) and its metabolites in plasma and urine following oral administration of NMP to rats.

The primary aims were to study the metabolism in rats and to determine the biological levels after one oral developmentally toxic dose of N-methyl-2-pyrrolidone (NMP), a widely used industrial chemical. Non-pregnant female Sprague-Dawley rats were given an oral single dose of either a non-toxic dose of 125 mg NMP/kg (group 1) by gavage or a developmentally toxic dose of 500 mg/kg (group 2). Blood plasma (7 rats per time point) and urine (10 rats per time point) were sampled up to 72 h after administration and analyzed using mass spectrometry. In both plasma and urine NMP, 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP), N-methylsuccinimide and 2-hydroxy-N-methylsuccinimide (2-HMSI) and 2-pyrrolidone (2-P) were identified. In urine 48% of the administered dose was recovered as 5-HNMP and 2-5% as 2-HMSI. The total recovery in urine was 53-59%. The peak concentrations for NMP in plasma were 1.2 and 6.9 mmol/l, 0.42 and 0.76 mmol/l for 5-HNMP, 0.07 and 0.31 mmol/l for MSI and for 2-HMSI the concentrations were 0.02 and 0.05 mmol/l for groups 1 and 2, respectively. In summary, the same metabolites were found in rats as in humans and the biological levels were reported for NMP and its metabolites after oral exposure to a developmentally toxic dose and one non-toxic dose of NMP.

Effects of aliphatic nitriles in micromass cultures of rat embryo limb bud cells.

The relative effects of a series of eight saturated (acetonitrile, propionitrile and n-butyronitrile) and unsaturated (acrylonitrile, allylnitrile, methacrylonitrile, cis-2-pentenenitrile, and 2-chloroacrylonitrile) aliphatic nitriles were evaluated using an in vitro test for embryotoxicity, the rat limb bud micromass assay. The concentrations that produced 50% inhibition (IC50) of viability and differentiation of the cultured embryonic cells were of the same order of magnitude, whatever tested compound. The IC50 values spread over a wide concentration range from 7-11 microM to 150 mM. Acetonitrile and 2-chloroacrylonitrile were the least and most potent compounds, respectively. The tested chemicals were evaluated using different criteria proposed to identify teratogens in the micromass system, based on either active concentrations or specific inhibition of cell differentiation. A few incorrect classifications were obtained with both nonteratogens and teratogens, when comparing the activity in limb bud cell cultures with the data available on their in vivo teratogenic potential in rats. The concordance between the in vitro and in vivo responses of this set of nitriles was judged insufficient to consider the micromass assay valuable for predicting the in vivo teratogenic outcome of this class of compounds.

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.