Developmental toxicity of diglyme by inhalation in the rat.

Diglyme (Diethylene glycol dimethyl ether, CAS No. 111-96-6) is a glycol ether which has been used in solvent formulations. To assess the potential developmental toxicity of this chemical, groups of pregnant Crl:CD BR rats were exposed to either 0 (control, room air only), 25, 100, or 400 ppm diglyme by inhalation for 6 hrs/day for Days 7 through 16 or gestation (day on which the copulation plug was detected was designation Day 1 G). All female rats were euthanized on day 21G and the fetuses were examined. An additional group of rats was treated with 25 ppm 2-methoxethanol (2ME) to serve as a positive control and for comparison of relative potencies. Maternal toxicity evident as depressed feed consumption at 400 ppm and increased liver weights at 100 ppm. There were no dams in the 400 ppm group with live fetuses (all litters consisted on resorbed conceptuses). Embryo viability was unaffected by concentrations of diglyme as high as 100 ppm. 2ME produced increased liver weights and depressed feed consumption at 25 ppm. Embryo-fetal toxicity was evident as a concentration-related decrease in fetal weight at diglyme concentrations as high as 100 ppm (and with 2ME). There were no fetuses derived from the 400 ppm diglyme-treated dams. A low incidence of structural malformations was observed in all diglyme groups (as well as with 2ME). The incidence of variations, (primarily delayed skeletal ossification and rudimentary ribs) was increased in the 25 and 100 ppm diglyme groups. The incidence and severity in the diglyme and 2ME groups exposed to 25 ppm was essentially the same suggesting similar potency for producing structural variations. In this study, diglyme was embryolethal at 400 ppm; a level that otherwise was only marginally toxic to the dam. Maternal and fetal toxicity also were demonstrated at 100 ppm. Although the fetal defects detected following diglyme exposure at 25 ppm were not significantly different from control values (with the exception of the incidence of skeletal developmental variations), the pattern, type, and incidence of variations were similar to those seen at 100 ppm, suggesting that 25 ppm was an effect level that approaches the lower end of the developmental toxicity response curve. Therefore, the no-observable-effect level (NOEL) for diglyme exposure in the dam is 25 ppm and a NOEL was not clearly demonstrated for the conceptus.

Developmental toxicity of Exell, a surfactant mixture, in rats and rabbits.

A surfactant mixture (C.A.S No. 104559-06-2), is used in combination with foliar-acting herbicides to improve herbicidal performance by increasing plant uptake. It was administered by gavage as an aqueous solution to groups of 25 assumed pregnant Crl:CD BR rats from days 7-16 of gestation at daily dosage levels of 0, 3, 8, 20 or 50 mg/kg body weight. The dams were euthanized on Day 22 of gestation and the offspring were weighed and examined for external, visceral, and skeletal alterations. The surfactant mixture was maternally toxic at levels of 20 mg/kg/day and above as evidenced by reduced maternal weight gain and by an increased incidence of lung noise (wheezing and/or rattling) and sneezing. In addition, maternal food consumption was reduced in the high dosage group. There was no evidence of developmental toxicity at any dosage level. In the rabbit study, groups of 20 artificially inseminated adult Hra:(NZW)SPF rabbits were similarly dosed from Days 6-18 of gestation at daily dosage levels of 0, 12, 35, or 100 mg/kg body weight. The dose were euthanized on Day 29 of gestation and the offspring were weighed and examined for external, visceral, and skeletal alterations. Dosages of 35 and 100 mg/kg/day resulted in reduced maternal body weight gain, food consumption and fetal body weight. Thus, in the rat, the maternal and developmental no-observed-adverse-effect levels (NOAELs) were 8 mg/kg/day and greater than 50 mg/kg/day, respectively. In the rabbit, the maternal and developmental NOAELs were 12 mg/kg/day. This surfactant mixture was, therefore, not demonstrated to be uniquely toxic to either the rat or rabbit conceptus.

Developmental toxicity of inhaled N-methylformamide in the rat.

The developmental toxicity of N-methylformamide (MMF), an industrial chemical intermediate used in the production of agrichemicals, was examined in pregnant rats. MMF was administered by nose-only inhalation, 6 hr daily on Days 7-16 of gestation (the day copulation was confirmed was termed Day 1 of gestation, Day 1G) at exposure concentrations of 0, 15, 50, or 150 ppm. Dams were regularly monitored throughout gestation for body weight gain, feed consumption, and clinical signs. Cesarean sections were performed on Day 22G and the offspring were examined. Maternal toxicity was evident in dams exposed to 50 or 150 ppm; one dam exposed to 150 ppm died on Day 14G (considered to be treatment-related) and dams in the 50 and 150 ppm groups exhibited concentration-related clinical findings. Clinical signs of wheezing and rattling were observed both during and after the exposure period. The 150 ppm group also showed significant decreases in weight gain and feed consumption. A significant increase in the mean number of resorptions per litter at the 150 ppm level indicated an embryolethal effect. Developmental toxicity was apparent by a significant decrease in mean fetal body weight and increases in fetal malformations (subcutaneous cysts on the head, microphthalmia, anophthalmia, fused ribs and/or vertebra, and distended brain ventricles) and variations (misaligned and fused sternebrae) due to retarded development at 150 ppm. Significant fetal body weight decreases were also present at 50 ppm. Thus, in this study, the no-observable-adverse-effect level for both dam and fetus was 15 ppm MMF, indicating that for the parameters included in this study, the conceptus is not uniquely sensitive to MMF.

1-Methyl-2-pyrrolidone (NMP): reproductive and developmental toxicity study by inhalation in the rat.

A two-generation reproduction study with a developmental toxicity component was conducted. For the reproduction phase, male and female rats inhaled 0, 10, 51, or 116 ppm NMP daily for 6 hr/day, 7 days/week from 34 days of age to the end of the mating period for the males (100 exposure days) and till weaning for the females (about 143 exposure days, but interrupted from Day 20 of gestation to Day 4 Postpartum). On Day 70 postpartum, one male and one female selected from each litter later mated with newly obtained, nonexposed adults of the opposite sex to produce an F2 generation. For the developmental phase, rats of both sexes inhaled 0 or 116 ppm NMP as outlined above, but euthanization of the females occurred on Day 21 of gestation followed by fetal examination for structural alterations. The indices of reproductive performance for the NMP-exposed rats did not differ significantly from those obtained for the control rats. Rats exposed to 116 ppm had a detectable decrease in response to sound. No other signs of NMP-related toxicity were detected among the parental rats. An exposure-related but slight decrease in fetal weight was detected only among the F1 offspring whose parents both inhaled NMP at 116 ppm. This slight effect also appeared at birth among the pups of the reproductive phase where it persisted till 21 days after birth when NMP inhalation by the mother ceased. Thereafter, the body weight of the offspring was comparable to the control values. No detectable or developmental effects appeared in the 10 or 51 ppm groups.

Developmental toxicity of dibasic esters by inhalation in the rat.

Dibasic esters (DBE) are a mixture of 3 carboxylic acid esters which are used in the paint and coatings industry. In this study, groups of pregnant Crl:CD BR rats were exposed to either 0.16, 0.4, or 1.0 mg DBE/L by inhalation for 6 hr/day from Days 7 through 16 of gestation (day in which copulation plug was detected was designated Day 1G). A control group of chambered pregnant rats was exposed simultaneously to air only. All female rats were euthanized on Day 21G and the fetuses were examined. A suppression of both food consumption and the rate of body weight gain was seen in the 0.4 and 1.0 mg/L groups during the first 6 exposure days. Staining on the fur and perineal area was seen in rats exposed to 1.0 mg/L and liver weight decreases, although not statistically significant, occurred in the 2 high exposure groups. None of the reproductive parameters were altered in any of the groups and no fetal effects were detected. DBE is not a developmental toxin in the rat following inhalation exposures as high as 1.0 mg DBE/L during the period of organogenesis.

Assessment of the effectiveness of animal developmental toxicity testing for human safety.

Evaluations of studies for four well-known human developmental toxicants clearly suggest that a margin of exposure of 1/100th the NOAEL for the most sensitive animal species tested provides adequate safety for the human conceptus. The lowest reported human teratogenic exposures occurred at doses at least one log above the estimated "safe" or acceptable daily exposure based on the most sensitive animal species, that is, 1/100th animal NOAEL. (The MOE ranged from < 1 to 10.). The data and analyses are consistent with the conclusion that, regardless of the type of in utero effect produced in animals, the margin of safety of 100 is likely to protect the human conceptus in utero from developmental perturbation, and it is a scientifically reasonable and conservative default number.

Application of the Hydra attenuata assay for identifying developmental hazards among natural waters and wastewaters.

This study concerns application of the Hydra attenuata assay to detect the developmental toxicity potential of various aqueous samples. First, the assay was modified for testing aqueous samples because water quality has a major impact on aquatic toxicity testing and the results thus obtained. Ranges of sample pH, salinity (conductivity), and hardness were examined for their adverse effects upon the hydra. Adult hydra were unaffected morphologically by pH 5.5-9.5, and the artificial embryo ("embryo") developed normally in a pH range of 6.25 to 8.25. For water hardness, the minimal affective concentration was 1000 mg/liter (as CaCO3) in adults and 625 mg/liter in the embryos; the NOAELs for these were 750 mg/liter in the adult and 250 mg/liter CaCO3 in the embryo. Salinity in excess of 5 ppt was lethal to adults and embryos, indicating the assay may not be applicable to marine or highly saline samples. Finally, grab samples were tested from rivers in Maryland, Pennsylvania, New Jersey, and Delaware, some of which are impacted by industrial and agricultural activities, as well as several samples of industrial wastewaters from one major facility. The assay functioned normally with these diverse samples and yielded results that can be used in assessing the potential developmental hazard of these materials.

Developmental toxicity of dimethylacetamide by inhalation in the rat.

Dimethylacetamide (DMAC) is a widely used industrial solvent. It has been reported to be teratogenic when given to rats by injection or following dermal application. Most of these studies employed large single doses and did not examine both the fetal and the maternal response. In this study, groups of pregnant Crl:CD rats were exposed to 32, 100, or 282 ppm DMAC by inhalation for 6 hr/day from Days 6 through 15 of gestation (day on which copulation plug was detected was termed Day 1G). A control group of chambered pregnant rats was exposed simultaneously to air only. All female rats were euthanized on Day 21G. At 282 ppm, both maternal weight gain during the exposure period and fetal weight were significantly decreased and accompanied by a significant dose-response trend. These effects were not seen in rats inhaling either 32 or 100 ppm. Fetal resorptions were not increased in any of the groups exposed to DMAC. Fetal incidences of external, visceral, or skeletal variations and malformations were similar between the test and control groups. Therefore, both fetal and maternal toxicity were noted at 282 ppm and the no-observed adverse-effect level under these experimental conditions was 100 ppm for both the dam and the conceptus. DMAC was not demonstrated to produce malformations in the rat fetus even at a level that was toxic to the dam.

Influence of symmetrical polychlorinated biphenyl isomers on embryo and fetal development in mice. II. Comparison of 4,4'-dichlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, 3,3',5,5'-tetrachlorobiphenyl, and 3,3',4,4'-tetramethylbiphenyl.

Outbred albino (CD-1) mice were given the following biphenyl isomers by gavage in cottonseed oil on Days 6-15 of gestation: 4,4'-dichlorobiphenyl (DCB) at 16, 32, and 64 mg/kg/day; 3,3',4,4'-tetrachlorobiphenyl (3,4-TCB) at 1,2,4,8,16,32, and 64 mg/kg/day; 3,3',5,5'-tetrachlorobiphenyl (3,5-TCB) at 64 mg/kg/day; and 3,3',4,4'-tetramethylbiphenyl (TMB) at 64 mg/kg/day. The mice were killed on Day 18 of gestation, necropsies were performed on the dams, and the fetuses were examined for external, visceral, and skeletal malformations. Although DCB was toxic to the dams at 64 mg/kg/day, developmental toxicity was not detected. 3,4-TCB administration was followed by a significant (p less than 0.01) increase in the average percentage of malformed fetuses per litter at 4 (7.2%), 8 (9.8%), 16 (25.4%), 32 (50.0%), and 64 (75.0%) mg/kg/day versus the vehicle control group (1.1%). None of the dosages tested was lethal to any of the dams. Significant decreases in maternal weight gain were observed at 16 mg/kg/day and above; however, the differences from the control value most likely were due to significant decreases in the mean number of live fetuses per dam, as the result of reductions in the number of implants per dam, and significant increases in the incidence of resorptions. Vaginal bleeding and other evidence of abortifacient effects also were present in several dams in groups receiving 3,4-TCB at 16 mg/kg/day and above. Cleft palate and hydronephrosis (significantly increased at dosages of 4 mg/kg/day and above) were the predominant malformations detected. Thus, 3,4-TCB was found to be toxic to the conceptus at dosages of 4 mg/kg/day and above. Neither 3,5-TCB nor TMB showed indications of maternal or developmental toxicity at 64 mg/kg/day.

The embryo-fetal toxicity and teratogenic potential of ammonium perfluorooctanoate (APFO) in the rat.

Ammonium perfluorooctanoate (APFO, greater than 95% pure) was administered to Sprague-Dawley rats from Days 6 through 15 of gestation by inhalation as a dust (whole body exposure) for 6 hr/day at 0, 0.1, 1, 10, and 25 mg/m3, or by gavage at 100 mg/kg body wt/day in corn oil. Maternal deaths occurred in the groups given the highest level of APFO by each route and overt toxicity was evident among the surviving dams of these groups and among those of the 10-mg/m3 group. The fetuses were examined for external, visceral, and skeletal alterations and for APFO-related macroscopic and microscopic alterations of the eyes. In the postpartum period, pups from additional control and experimental dams were examined externally and ophthalmoscopically, and the usual fertility and viability indices were calculated. A teratogenic response was not demonstrated. Toxic effects on the conceptus were noted only in the groups given the highest level of APFO by each route. Hence, APFO was not demonstrated to represent a unique hazard to the conceptus of the rat.

Inhalation toxicity of epichlorohydrin: effects on fertility in rats and rabbits.

The effects of inhaled epichlorohydrin (ECH) on the fertility of Sprague-Dawley rats and New Zealand white rabbits were studied. Groups of 10 male rabbits, 30 male rats, and 30 female rats were exposed to 0, 5, 25, or 50 ppm of ECH vapor for 6 hr/day, 5 days/week for 10 weeks, and were held for a 10-week postexposure females. Exposed male rats were mated with unexposed females at several intervals during and after the exposure period. In addition, female rats which had been exposed for the 10-week period were mated with unexposed males and allowed to deliver their young. Exposure to 50 ppm of ECH vapor for 10 weeks resulted in transient infertility in the male Sprague-Dawley rats; recovery of fertility in rats occurred during the second week after termination of exposure. Male rats exposed to 25 ppm of ECH were able to impregnate unexposed females; however, fewer implantations were observed in these females than in the females mated to control males suggesting that fertility was adversely affected in this group as well. This effect also was reversed by the second week following termination of exposure. The incidence of resorptions in the unexposed female rats which were bred to the exposed males was not adversely affected. Among female rats exposed to ECH, no adverse effects were observed on estrus cycle, pregnancy rate, parturition, or the number and viability of the offspring. No discernible effects were noted on the volume of the ejaculate or on the motility, viability, concentration, or fertility of spermatozoa from male rabbits exposed to up to 50 ppm of ECH. Histologic examination of tissues from an interim and final termination of the exposed animals indicated that the most severely affected organ following inhalation exposure to 25 or 50 ppm of epichlorohydrin in both rats and rabbits was the nasal turbinates. These lesions, interpreted to be a result of irritation from the test material, were no longer present in animals which were held for the 10-week postexposure period. No adverse effects were observed among rats or rabbits exposed to 5 ppm of ECH for 10 weeks.

1,1,3,3-tetrachloroacetone: teratogenicity study in mice and rabbits.

The teratogenicity of tetrachloroacetone (TCA) was evaluated in CF-1 mice and New Zealand white rabbits. Mice were given 0, 5, 15 or 50 mg/kg/day of TCA by gavage on days 6 through 15 of gestation. Rabbits were given 0, 1, 5, or 10 mg/kg/day on days 6 through 18 of gestation. The incidence of malformed fetuses was not significantly increased among mice given 5, 15 or 50 mg/kg/day of TCA. Sixteen fetuses in 3 litters in the 50 mg/kg/day group had cleft palate; they were from three dams which showed the most severe signs of toxicity, including depressed weight gain, decreased water consumption, and gastric ulceration. Dosage with 5 mg/kg/day, a level which was not toxic to the pregnant female, was not toxic to developing mouse fetuses. Fetal effects in rabbits were limited to a slight, but not statistically significant or dose related increase in the incidence of malformed fetuses. On the basis of these results, TCA does not appear to represent a unique hazard to the conceptus.

Teratogenic evaluation of epichlorohydrin in the mouse and rat and glycidol in the mouse.

Pregnant outbred albino rats (CD) and mice (CD-1) were given epichlorohydrin by gastric intubation on d 6-15 of gestation. The rats were killed on d 21 (d 18 for mice) and the offspring checked for gross, visceral, and skeletal malformations. Epichlorohydrin caused a significant reduction in the weight gain of pregnant rats at 80 mg/kg.d as compared with the control group treated only with the vehicle. However, there was no evidence of teratogenicity in the rat fetuses even at a dose level (160 mg/kg.d) that caused the death of some of the treated dams. Epichlorohydrin also did not produce a statistically significant increase in the average percent of malformed mouse fetuses, even at 160 mg/kg.d, a dose that killed 3 of 32 treated dams. The 120 and 160 mg/kg.d levels did cause a significant (p less than 0.05) reduction in the average fetal weight as compared with controls. In addition, the 120 mg/kg.d dose produced the statistically significantly increase in the liver weight of the pregnant mouse. These observations indicate that the 120 and 160 mg/kg.d dose levels were toxic toward the dams and their unborn offspring. In a similar mouse study, glycidol showed no evidence of teratogenicity. There was a significant increase in the number of stunted fetuses at 200 mg/kg.d, but all of these were present in a single litter. Further, the same dose killed 5 of 30 dams.

Teratogenic evaluation of 2-nitro-p-phenylenediamine, 4-nitro-o-phenylenediamine, and 2,5-toluenediamine sulfate in the mouse.

Pregnant outbred albino (CD-1) mice were given 2-nitro-p-phenylenediamine (2NPPD; 32-256 mg/kg/day), 4-nitro-o-phenylenediamine (4NOPD; 16-1024 mg/kg/day) or 2,5-toluenediamine sulfate (2,5TDS; 16-64 mg/kg/day) by subcutaneous injection on Days 6-15 of gestation. The mice were killed on Day 18, the general health and reproductive health of the dams evaluated, and the fetuses examined and processed in order to characterize external, visceral, and skeletal malformations. The 32 and 128 through 256 mg/kg/day dose levels of 2NPPD produced a significant (p less than 0.05) reduction in average weight gain by the dam during pregnancy. There was also a significance reduction in average fetal weight at 128 mg/kg/day and above. Although 2NPPD administration led to a significant increase in the average percentage of malformed fetuses at 160 mg/kg/day and above, these effects occurred only at dosages which produced significant maternal toxicity. Teratogenic effects were observed with 4NOPD, as administration of this dye led to a significance increase in the average percent of malformed fetuses at 256 mg/kg/day and above. A significant reduction in average weight gain by the dam during pregnancy also was observed at these dose levels, as well as significant reduction in average fetal weight. Although 2,5TDS killed 4 of 31 dams at 48 mg/kg/day, and 9 of 11 dams at 64 mg/kg/day, this compound did not cause a significant increase in the average percent of malformed fetuses. There were indications of embryotoxicity, however, as dosages of 32 mg/kg/day and above led to significant reductions in average fetal weight.

Effects of 2.45 GHz CW microwave radiation on embryofetal development in mice.

The embryofetal toxicity and teratogenicity of plane-wave 2.45 GHz continuous wave (CW) microwave radiation at different intensities were investigated in the CD-1 mouse. Mice were exposed on days 1-15 of gestation to an incident power density of 5 mW/cm2 (specific absorption rate of 6.7 mW/gm) and either on days 1-6 or 6-15 of gestation to 21 mW/cm2 (specific absorption rate of 28.14 mW/gm) or to 30 mW/cm2 (specific absorption rate of 40.2 mW/gm) for 8 hours daily. Exposure either on days 1-6 or 6-15 of gestation to a power density of 21 or 30 mW/cm2 caused an increase in colonic temperature of exposed dams of 1 degree C and 2.3 degrees C, respectively. To distinguish between "thermal" and "nonthermal" effects of 21 or 30 mW/cm2, groups of mice were also exposed to elevated ambient temperature to raise their body temperature to the level of those animals exposed to microwave. Ambient temperatures of 30 degrees C and 31 degrees C increased the deep colonic temperature to that obtained with the 21 and 30 mW/cm2 microwave exposure, respectively. The temperature-exposed mice were handled in exactly the same manner as the microwave-exposed mice. A significant reduction in maternal weight gain, either during treatment on days 1-6 or 6-15 of gestation was observed in females of all handled groups. Handling plus exposure to elevated ambient temperature (30 degrees C or 31 degrees C) during days 6-15 of gestation increased this reduction in maternal weight gain. A significant decrease in implantation sites per litter and reduction in fetal weight was noted in the group exposed to 30 mW/cm2 during days 1-6 of gestation. Exposure of mice to a power density of 30 mW/cm2 (days 6-15 of gestation) resulted in a slight, but significant increase in the percentage of malformed fetuses, predominantly with cleft palate, when compared to all other groups.

Embryotoxicity of various noise stimuli in the mouse.

Different noise exposure paradigms were studied to determine their teratogenic and embryo-fetotoxic potential in the CF-1 mouse. Female mice were exposed from days 1-6 or from days 6-15 of gestation to one of three noise exposure paradigms which differed widely in level, spectral, and temporal characteristics. Paradigms for noise exposure were chosen to represent semi-continuous exposure to extremely high-intensity noise (jet engine noise at 126 dBA, from noon to midnight); to represent startling type noise composed of alarm bells, jet engine noise, or narrow band warning devices at 110 dBA, with pseudorandom onset and duration of each controlled by a microprocessor (exposure time of 18% over each 24 hour period); and finally to represent very high frequency noise (18-20 kHz tones, derived from a device commercially marketed for repelling rodents, with exposure from noon to midnight). On day 18 of gestation the females were sacrificed, their reproduction status determined, and the concepti were examined for toxicity and for external, visceral, and skeletal alterations. Maternal plasma corticosterone levels were measured at different periods of gestation. Significantly decreased pregnancy rate was noted in all groups exposed to noise except in the group exposed to the very high frequency noise from days 6-15 of gestation. Significant embryolethal effects occurred in the group exposed to the extremely high intensity jet noise paradigm, from days 1-6 of gestation, and significant fetolethal effects occurred in the group exposed to the very high frequency noise paradigm from days 6-15 of gestation. No significant noise-related changes were noted in the incidence of structural alterations or in the concentration of plasma corticosterone.

Influence of formaldehyde and Sonacide (potentiated acid glutaraldehyde) on embryo and fetal development in mice.

Pregnant outbred albino mice were given formaldehyde or Sonacide (potentiated acid glutaraldehyde) by gavage on days 6--15 of gestation. The mice were killed on day 18, the general health and reproductive status of the dam evaluated, and the fetuses examined and processed in order to characterize external, visceral, and skeletal malformations. Although formaldehyde (stock solution containing 12--15% methanol as a preservative) was lethal to 22 of 34 dams treated with 185 mg/kg/day, and one of 35 dams treated with 148 mg/kg/day, these doses did not produce statistically significant (two-sided p < 0.05 versus controls) teratogenic effects in the fetuses of the surviving dams. Sonacide was also judged not to be teratogenic to the mice employed in this study, in spite of the fact that relatively high doses were employed. The highest doses of Sonacide studied (5.0 ml/kg/day, which is equivalent to 100 mg/kg/day of glutaraldehyde) killed 19 of 35 dams and caused a significant reduction in the mean weight gain of the surviving mothers. In addition, this dose produced a significant increase in the number of stunted fetuses.