R. W. Tyl

Determination of a No-Observed-Effect Level for Developmental Toxicity of Ethylene Glycol Administered by Gavage to CD Rats and CD-1 Mice

Previous studies have indicated that ethylene glycol (EG) is a developmental toxicant in rats and mice primarily when ingested. This study was designed to establish no-observed-effect levels (NOELs) for developmental toxicity of EG administered by gavage in both rodent species. Dams were administered EG on Gestation Days 6-15; rats were given 0, 150, 500, 1000, or 2500 mg EG/kg/day; mice were dosed with 0, 50, 150, 500, or 1500 mg EG/kg/day. In rat dams given 2500 mg EG/kg/day, water consumption was increased during treatment and body weights were reduced throughout gestation; liver and kidney weights were increased at euthanization (Gestation Day 21). Relative liver weights were also increased at 1000 mg/kg/day. Effects observed in rat fetuses at 2500 mg/kg/day included the following: hydrocephaly; gastroschisis; umbilical hernia; fused, duplicated, or missing arches, centra, and ribs; poor ossification in thoracic and lumbar regions; and reduced body weights. Reduced body weights, duplicated or missing ribs, centra, and arches, and poor ossification were also observed in rat fetuses at 1000 mg/kg/day. In mice, there was no apparent treatment-related maternal toxicity. In mouse fetuses (Gestation Day 18), effects were observed at 1500 mg/kg/day and included reduced body weights, fused ribs and arches, poor ossification in thoracic and lumbar centra, and increased occurrence of an extra 14th rib. At 500 mg/kg/day, slight reductions in fetal body weight and increased incidences of extra ribs were observed. Under conditions of these studies, NOELs for developmental toxicity were 500 mg/kg/day for rats and 150 mg/kg/day for mice, indicating that mice were more susceptible than rats to the teratogenic effects of EG.

Developmental Toxicity Evaluation of Inhaled 2–Ethoxyethanol Acetate in Ficher 344 Rats and New Zealand White Rabbits

Pregnant Fischer 344 rats and New Zealand white rabbits were exposed to 2-ethoxyethanol acetate (EEA; CAS No. 111-15-9) vapor by inhalation on Gestational Days 6 through 15 (rats) or 6 through 18 (rabbits) at concentrations of 0, 50, 100, 200, or 300 ppm, 6 hr/day. The animals were terminated on Gestational Day 21 (rats) or 29 (rabbits) and fetuses were examined for external, visceral, and skeletal malformations and variations. In rabbits, exposure to 100-300 ppm resulted in maternal toxicity: decreased weight gain at 100-300 ppm, clinical signs at 200-300 ppm, alterations in hematology at 100-300 ppm, reduced gravid uterine weight at termination at 200-300 ppm, and elevated absolute liver weight at 300 ppm. Developmental toxicity was observed at 100-300 ppm: an increased incidence of totally resorbed litters at 200-300 ppm, an increase in nonviable fetuses at 300 ppm, and a decrease in viable implants (live fetuses) per litter at 200-300 ppm. The incidence of fetal malformations (external, visceral, and skeletal) was increased at 200-300 ppm. The incidence of total malformations was 100% at 300 ppm and significantly increased at 200 ppm. Reduced fetal ossification was observed at 100-300 ppm. In rats, exposure to 100-300 ppm also resulted in maternal toxicity: reduced weight gain and reduced food consumption at 200-300 ppm and elevated relative liver weight and alterations in hematology at 100-300 ppm. Absolute maternal liver weight was increased at all EEA exposure concentrations; relative liver weight was increased at 100-300 ppm. Developmental toxicity was observed at 100-300 ppm: increased nonviable implantations/litter (300 ppm), reduced fetal body weight/litter (200-300 ppm), and increased incidence of external (300 ppm), visceral, and skeletal (100-300 ppm) variations indicative of toxicity. The incidence of visceral, skeletal, and total malformations was increased at 200-300 ppm. In conclusion, in both species, inhalation exposure to EEA during organogenesis produced maternal toxicity at 100-300 ppm and developmental toxicity at 100-300 ppm, including teratogenicity at 200-300 ppm. At 50 ppm in both species, there was no evidence of maternal or developmental toxicity, including teratogenicity.

Evaluation of the Developmental Toxicity of Ethylene Glycol Aerosol in the CD Rat and CD-1 Mouse by Whole-Body Exposure

Ethylene glycol (EG) is a major industrial chemical, shown to be teratogenic at high doses by gavage in rodents. Since one route of industrial exposure is to the aerosol at high concentrations, timed-pregnant CD rats and CD-1 mice were exposed, whole-body, to a respirable aerosol of EG (mass median aerodynamic diameter, 2.3 microns) on Gestational Days (GD) 6 through 15 for 6 hr per day at target exposure concentrations of 0, 150, 1000, or 2500 mg/m3 (analytical concentrations of 0, 119 +/- 13, 888 +/- 149, and 2090 +/- 244 mg/m3, respectively), with 25 plug-positive animals per species per group. Clinical observations and maternal body weights were documented throughout gestation for both species. Maternal food and water consumption was measured in rats only throughout gestation. At scheduled necropsy (GD 21 for rats, GD 18 for mice), maternal animals were evaluated for body weight, liver weight, kidney weight, gravid uterine weight, number of ovarian corpora lutea, and status of implantation sites, i.e., resorptions, dead fetuses, live fetuses. Fetuses were dissected from the uterus, counted, weighed, sexed, and examined for external, visceral, and skeletal malformations and variations. All rat dams survived to scheduled termination. Minimal maternal toxicity was indicated by a significant increase in absolute and relative liver weight at 2500 mg/m3. Food and water consumption, maternal body weights and weight gain, and maternal organ weights (other than liver) were unaffected by exposure. Gestational parameters were unaffected by exposure, including pre- and post-implantation loss, live fetuses/litter, sex ratio, and fetal body weight/litter. There was no treatment-related increase in the incidence of any individual malformation, in the incidence of pooled external, visceral, or skeletal malformations, or in the incidence of total malformations by fetus or by litter. There were no increases in the incidence of external or visceral variations. Evidence of fetotoxicity, expressed as reduced ossification in the humerus, the zygomatic arch, and the metatarsals and proximal phalanges of the hind-limb, was observed at 1000 and 2500 mg/m3. All mouse dams survived to scheduled termination. One dam at 2500 mg/m3 was carrying a totally resorbed litter at termination. Maternal toxicity was observed at 1000 and 2500 mg/m3, expressed as reduced body weight and weight gain during and after the exposure period, and reduced gravid uterine weight. (Maternal effects may have been due, in part or whole, to effects on the conceptuses; see below.)(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of Exposure to Water Aerosol or Air by Nose-Only or Whole-Body Inhalation Procedures for CD-1 Mice in Developmental Toxicity Studies

This study was performed to evaluate the effects of nose-only restraint versus whole-body exposure procedures in the absence of test chemical, and to determine the appropriate control environment (water aerosol or air) for subsequent developmental toxicity studies of test materials administered as aerosols. Timed-pregnant CD-1 mice, 30/group, were exposed to high concentrations of water aerosol or to air by whole-body or nose-only inhalation procedures on Gestational Days (GD) 6 through 15 for 6 hr per day. The group exposed to air by whole-body procedures was designated as the control group. Clinical observations and maternal body weights were recorded throughout gestation. At scheduled necropsy on GD 18, maternal animals were evaluated for body weight, gravid uterine weight, liver weight, number of ovarian corpora lutea, and status of uterine implantation sites. Fetuses were counted, weighed, and sexed and were examined for external, visceral (including craniofacial), and skeletal alterations. Indices of maternal toxicity were affected in both nose-only groups. Maternal body weights were reduced during and after the exposure period; maternal weight gain was reduced during the exposure period. Clinical signs observed, from animals struggling during restraint, were resolved by GD 18. At sacrifice on GD 18, maternal body weights and maternal gestational weight gains (both corrected for gravid uterine weights) and absolute liver weights were reduced in both nose-only groups. Four females died (13.3%, all pregnant) in the air nose-only group, and maternal liver weight (relative to body weight) was reduced in the aerosol nose-only group. Gestational parameters were unaffected by any of the treatments. There were no statistically significant differences in the incidences of any individual malformations or malformations by category (external, visceral, or skeletal) or of total malformations. However, exencephaly, low set ears, cleft palate and ventricular septal defect were observed only in both aerosol-exposed groups (whole-body and nose-only exposed). The incidences of individual external or visceral variations or of variations by category or of total variations were unaffected. The incidence of one skeletal variation, poorly ossified supraoccipital skull bone, was significantly increased in the aerosol nose-only group relative to the air whole-body controls. There were also increased incidences (not statistically significant) of extra (14th) ribs in both aerosol groups. Therefore, maternal restraint (in both nose-only groups) during organogenesis produced indications of maternal toxicity, but restraint did not appear to affect normal embryo/fetal morphologic development.(ABSTRACT TRUNCATED AT 400 WORDS)

Workshop on the qualitative and quantitative comparability of human and animal developmental neurotoxicity, work group III report: Weight of evidence and quantitative evaluation of developmental neurotoxicity data☆☆☆

Work Group III discussed the qualitative and quantitative evaluation of developmental neurotoxicity data for risk assessment purposes. The Work Group concurred with the assumption that developmental neurotoxicity may result from as little as a single exposure, dependent on dose and gestational time of exposure. Maternal toxicity, during the pregnancy and/or lactational period, may confound interpretation of effects observed in the offspring, but the majority of the group agreed that if significant effects were observed in a developmental neurotoxicity study, these effects should be presumed relevant to potential risk in humans. Others in the group indicated that these effects should be designated developmental toxicity but not necessarily developmental neurotoxicity. Concurrent toxicity (in the same organism, at the same dose, etc.) was also discussed, with the same concern as with maternal toxicity, i.e., are the results observed from developmental neurotoxicity per se or from other toxicity impacting on and confounding the neurotoxicity test results. A majority of the group regarded any effect observed in a neurotoxicity test as consistent with developmental neurotoxicity. The minority reserved this designation only for neurotoxic results observed at the lowest adverse effect level and in the absence of other developmental toxicity. Discussion on weighting tests in the battery focussed on apical tests which require the most integrated functions with the caveat for confounding effects from other toxicities. Developmental neurotoxicity data were viewed as potentially useful in establishing a reference dose. The interpretation of statistically significant findings was discussed with caveats for Type I errors, consistency, biological plausibility of findings, and the concern that current draft guidelines and test rules require adult toxicity with the risk of confounding.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental toxicity evaluation of inhaled nitrobenzene in CD rats

Pregnant CD (Sprague-Dawley) rats were exposed to nitrobenzene vapor (CAS Registry No. 98-95-3) at 0, 1, 10, and 40 ppm (mean analytical values of 0.0, 1.06, 9.8, and 39.4 ppm, respectively) on gestational days (gd) 6 through 15 for 6 hr/day. At sacrifice on gd 21, fetuses were evaluated for external, visceral, and skeletal malformations and variations. Maternal toxicity was observed: weight gain was reduced during exposure (gd 6-9 and 6-15) to 40 ppm, with full recovery by gd 21, and absolute and relative spleen weights were increased at 10 and 40 ppm. There was no effect of treatment on maternal liver, kidney, or gravid uterine weights, on pre- or postimplantation loss including resorptions or dead fetuses, on sex ratio of live fetuses, or on fetal body weights (male, female, or total) per litter. There were also no treatment-related effects on the incidence of fetal malformations or variations. In summary, during organogenesis in CD rats, there was no developmental toxicity (including teratogenicity) associated with exposure to nitrobenzene concentrations that produced some maternal toxicity (10 and 40 ppm) or that produced no observable maternal toxicity (1 ppm).

Dominant lethal assay of 2,4-pentanedione vapor in Fischer 344 rats.

2,4-Pentanedione (2,4-PD: CAS No. 123-54-6) is a volatile industrial chemical of moderate acute toxicity, centrally neurotoxic by repeated exposure to high vapor concentrations, fetotoxic, and clastogenic. Its wide use and known toxicology indicated the conduct of a dominant lethal assay. Male Fischer 344 rats, 20 per group, were exposed to 2,4-PD vapor concentrations (mean ± SD) of 0, 99.1 ± 2.2, 412 ± 12.6 and 694 ± 9.1 ppm, for 6 hr/day for 5 consecutive days. The day following the final exposure they were bred to unexposed female Fischer 344 rats, 2 per week for 8 consecutive weeks. Weight loss occurred with males during 2,4-PD exposure for the 412 and 694 ppm groups, with compensatory increased weight at 694 ppm, for the first two weeks postexposure. No histopathological change was seen in brain, testes or thymus from high concentration males sacrificed after eight weeks of mating. Minor transient reproductive and gestational effects were present at 412 and 694 ppm. At week 2 there was a reduction, not statistically significant, in the number of corpora lutea and total and viable implants per dam at 694 ppm, and a slight increase in preimplantation loss. At week 3 the number of pregnant females was slightly reduced at 412 and 694 ppm, causing a lowered female fertility index. At week 4 there was a slight reduction in the number of total and viable implants per litter and a significant preimplantation loss at 694 ppm. The dominant lethal factor (FL%) was increased slightly at 694 ppm for weeks 2 and 4. Thus, the “no observable effect” level for dominant lethal effects was 99 ppm. The results, although not statistically significant, are dose-related and compatible with a transient slight dominant lethal effect at the spermatid stage of spermatogenesis.

Evaluation of the Developmental Toxicity of Ethylene Glycol Monohexyl Ether Vapor in Fischer 344 Rats and New Zealand White Rabbits

Timed pregnant Fischer 344 rats and New Zealand White rabbits were exposed to vapor from ethylene glycol monohexyl ether (EGHE, CAS No. 112-25-4) for 6 hr/day on gestational days (gd) 6 through gd 15 (rats) or gd 6 through gd 18 (rabbits) at analytically measured concentrations (as means +/- SD) of 20.8 +/- 0.90, 41.1 +/- 1.77, or 79.2 +/- 10.8 ppm; control animals were exposed to air alone. Monitors for maternal toxicity were body weight, food and water consumption, clinical signs, and hematology. At sacrifice (gd 21 rats, gd 29 rabbits) maternal weight, liver weight, and gravid uterine weight were measured. Gestational parameters monitored were numbers of corpora lutea, preimplantation losses, viable implants, early and late resorptions, and dead fetuses. Live fetuses were sexed, weighed, and examined for external, visceral, and skeletal malformations and variations. Rabbit maternal toxicity occurred at 79.2 ppm as transient weight gain reduction during the exposure period. For maternal rats at 79.2 ppm, there were transient decrease in body weight and body weight gain during exposure, reduced food consumption, increased water consumption, and excess lacrimation. At 41.1 ppm, maternal body weight gain was reduced during the exposure period only. There were no treatment-related effects with respect to hematology, necropsy, or gestational parameters and no significant change in the incidence of malformations or variations (expressed as total, individual, external, visceral, or skeletal). Thus, exposure of rats and rabbits to EGHE vapor during the period of organogenesis produced maternal toxicity at near-saturation vapor concentrations (79.2 ppm), but no evidence for developmental toxicity or teratogenicity. The no-effect vapor concentrations for maternal toxicity were 41.1 ppm for rabbits and 20.8 ppm for rats.

Developmental toxicity study in Fischer 344 rats by whole-body exposure to N,N-dimethylethanolamine vapor

Timed‐pregnant Fischer 344 rats were exposed whole body to N, N‐dimethylethanolamine vapor for 6 h per day on gestational days 6–15 at mean (±SD) analytically measured concentrations of 10.4 ± 0.86, 29.8 ± 2.14 and 100 ± 4.9 ppm. Dams were sacrificed on gestational day 21. There was no maternal mortality in any exposed groups. Maternal toxicity observed in the 100 ppm group included reduced body weight during and after exposures, reduced weight gain during exposure and ocular changes (darkened, cloudy and hazy eyes, slight corneal vascularization and fixed, dilated pupils). Ocular effects were also noted in the other two exposure groups; the effects were quite marked at 30 ppm but only minimal and transient at 10 ppm. There were no effects of treatment on any gestational parameters, including pre‐ and postimplantation loss or sex ratio. Fetal body weights per litter were statistically significantly increased at 100 ppm relative to controls. There were no increases in the incidences of total malformations by category (external, visceral or skeletal) or individually. The incidence of six skeletal variations out of 120 noted differed in exposed groups relative to that of control. Four of these variations were decreases in incidence; only one fetal variation, the split (bipartite) cervical centrum, was elevated at 100 ppm relative to controls. In the absence of any other indications of delayed ossification or fetal body weights, the observed fetal variation does not suggest a consistent pattern of fetal toxicity. Hence, the no‐observed‐adverse‐effect level is around 10 ppm for maternal toxicity and at or above 100 ppm for embryofetal toxicity and teratogenicity.