* Toxicology & Environmental Research and Consulting, 1803 Building, The Dow Chemical Company, Midland, Michigan 48674;
Dow Europe, GmbH, Horgen, Switzerland; and
Eli Lilly & Company, Greenfield, Indiana
Received July 23, 2002; accepted September 30, 2002
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ABSTRACT |
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Key Words: glycol ethers; whole embryo culture; maternal pharmacokinetics.
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INTRODUCTION |
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While it is tempting to view the glycol ethers as a single class of chemicals with common properties, the toxicity profiles across different glycol ether subclasses vary significantly. In the case of developmental toxicity, some of the ethylene glycol ethers have received considerable attention due to their ability to induce fetal malformations (reviewed by Boatman and Knaak, 2001; Hardin, 1983
; Hevelin, 1989
). The prototype ethylene glycol ether is ethylene glycol monomethyl ether (EGME; also known as 2-methoxyethanol), which causes digit, skeletal, and cardiovascular defects, as well as fetal resorptions in mice, rats, and rabbits (Hanley et al., 1984b
; Horton et al., 1985
; Nelson et al., 1984
; Toraason et al., 1985
). EGME is converted by alcohol/aldehyde dehydrogenase (ADH/ALDH) to an alkoxy acid metabolite (Fig. 1
), methoxyacetic acid (MAA), which is considered the proximate developmental toxicant (Brown et al., 1984
; Sleet et al., 1988
; Yonemoto et al., 1984
). Rabbits appear to be the most sensitive laboratory animal species to EGME, with teratogenic effects being induced in 63% of fetuses at 50 ppm (Hanley et al., 1984b
).
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Merkle et al.(1987) were the first to investigate the developmental toxicity of ß-PGME. In an initial study, they tested the acetate of ß-PGME and observed teratogenic effects in Himalayan rabbits and Wistar rats, with rabbits being considerably more sensitive than rats (Merkle et al., 1987
). In a follow-up dose-response study of purified ß-PGME (Hellwig et al., 1994
), rabbits were exposed via inhalation (6 h/day) to 0, 145, 225, 350, or 545 ppm on GD 618, followed by maternal necropsy and fetal examination on GD 29. At 350 and 545 ppm, the percentage of resorbed implantations was increased, as was the incidence of fetal malformations and variations. The predominant malformations observed were enlarged sternum and enlarged rib cartilage (described as a bony plate), missing gall bladder, fused ribs, and digit defects. Maternal toxicity was quite apparent at these dose levels, as evidenced by a 72 g weight loss during the dosing period at 545 ppm. At 350 ppm, rabbits gained only 15% of the control body weight gain during the dosing period. Marginal effects occurred at 225 ppm in the form of a slight, nonstatistically significant increase in bony plate/enlarged cartilage in the presence of slight (12%), nonstatistically significant decreases in maternal body weight gain during the dosing period. The lowest exposure concentration of 145 ppm was devoid of treatment-related maternal or developmental effects.
The striking differences in developmental toxicity profile between commercial ()-PGME and ß-PGME parallel the differences in metabolism of these two isomers, and suggest that MPA is a potential developmentally toxic metabolite of ß-PGME. Therefore, in vitro and in vivo developmental toxicity studies were conducted to thoroughly characterize the developmental toxicity potential of MPA in a highly sensitive test species, the rabbit. Parallel studies with MAA were run as a benchmark compound and positive control. In conjunction with this work, blood time-course studies to describe the formation of MPA from ß-PGME, and the pharmacokinetics of directly administered MPA in rabbits were also conducted. By establishing a critical internal dose of the alleged proximate toxicant, MPA, the risk of human exposure to ß-PGME present as a minor impurity of commercial PGME can then be assessed with greater accuracy and confidence.
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MATERIALS AND METHODS |
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Test animals.
Adult, virgin female New Zealand White (NZW) rabbits ranging in age from 5.06.5 months were obtained from Covance (Denver, PA) and acclimated to the laboratory (AAALAC International accredited) for at least two weeks. The rabbits were housed individually, maintained on 4 (nonpregnant rabbits) or 8 oz/day (bred rabbits) of Purina Certified Laboratory Rabbit Chow No. 5322 and water ad libitum. The animal rooms of the facility were designed to maintain humidity at approximately 4070%, temperature at approximately 22°C and photoperiod at 12 h light:12 h dark. All treated rabbits were identified using uniquely coded alphanumeric metal ear tags.
In Vivo Developmental Toxicity Studies
Study design.
Groups of 20 artificially inseminated NZW rabbits (Gibson et al., 1966) were randomly assigned to treatment groups in either the MAA or MPA developmental toxicity studies (OECD guideline 414). Dose levels for the MAA study were 0, 2.5, 7.5, or 15.0 mg/kg/day, while 0, 10, 26, or 78 mg/kg/day were used for the MPA study. Dose levels were chosen on the basis of previously conducted probe studies. Dose solutions were administered once daily on GD 719 via gavage. Body weights were recorded on GD 0, 720, and 28 (day of insemination was considered GD 0). The dose volume for all test materials was 2 ml/kg body weight and was adjusted daily based on body weight. Feed consumption was determined daily throughout the study.
Maternal and fetal examinations.
On GD 28, all surviving animals were euthanized by iv injection of Beuthanasia-D (Schering, Kenilworth, NJ) via the marginal ear vein and necropsied. Any gross pathologic changes in the maternal rabbits were recorded, as were maternal liver, kidney, and gravid uterus weights. The uterine horns were exteriorized and the following data were collected: (1) the number and position of fetuses in utero, (2) the number of live and dead fetuses, (3) the number and position of resorptions, (4) the number of corpora lutea, (5) the body weight of each fetus, and (6) any gross external alterations. The uteri of animals that appeared nonpregnant were stained with a 10% aqueous solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions. All fetuses were immediately examined by dissection under a low power stereomicroscope for evidence of visceral alterations, determination of sex, and changes in brain structure as assessed by serial sectioning (Staples, 1974
). All fetuses were then preserved in alcohol, eviscerated, stained with alizarin red-S (Dawson, 1926
), and examined for skeletal alterations. In judging the toxicological significance of fetal alterations, the following criteria were considered: (1) statistical significance, and/or (2) historical control values, (3) dose-dependence, (4) occurrence in more than one fetus, and (5) consistency with previously reported developmental effects of EGME (Hanley et al., 1984b
).
Rabbit whole embryo culture studies.
In order to more fully characterize the intrinsic teratogenicity potential of MPA and its parent compound, ß-PGME, rabbit whole embryo culture studies were performed using early somite stage embryos cultured for 48 h according to methods described by Ninomiya et al.(1993) and Pitt and Carney (1999a)
. In brief, untreated, pregnant rabbits were euthanized on the morning of GD 9 via carbon dioxide inhalation, exsanguinated, and the uterine wall was cut open to expose the conceptuses. Healthy, normal appearing conceptuses (approximately 1214 somites) with a visible peripheral visceral yolk sac (VYS) blood vessel, and fused cardiac tubes were considered acceptable for culture. A cut was made around the entire conceptus, immediately parallel and distal to the peripheral VYS vessel in order to isolate the embryo, placenta, and VYS apart from the maternal implantation site. Underlying placental tissue was then trimmed so that only a thin layer remained attached to the VYS and embryo. Each conceptus was then transferred to a culture bottle containing 5 ml of culture medium, and embryos were balanced by litter across treatment groups. The culture medium was composed of 75% (v/v) heat-inactivated rabbit serum, and 25% (v/v) phosphate-buffered saline (PBS), supplemented with penicillin-streptomycin solution (Life Technologies, Gaithersburg, MD). Test materials were dissolved in the PBS portion of the media prior to mixing with serum. Test material concentrations were 0, 1.0, or 5.0 mM of MPA or 5.0 mM MAA in the first embryo culture experiment, and 0, 0.5, or 2.0 mM of ß-PGME in the second experiment. The highest concentration of each compound was based on results of the pharmacokinetic study (see below), and slightly exceeded the peak concentration of each compound following dosing with a high dose (270 mg/kg) of ß-PGME. All media were equilibrated to culture conditions for at least 30 min prior to the start of culture. The culture bottles were placed in a rotating culture bottle unit (BTC Engineering, Cambridge, UK) under continuous gassing with 5% CO2 in air for the first 24 h, and 5% CO2/95% O2 for the remaining 24 h. The gas mixture flow rate (approx. 20 cc/min) and temperature (37°C) remained constant throughout the culture.
After approximately 48 h in culture, embryos were evaluated for viability (based on heart beat), presence of blood flowing through the yolk sac, head length, somite number, morphology score, and incidence of malformations. The morphology score (Pitt and Carney, 1999a,b
) is an index of development in which individual embryonic structures are assigned numerical scores based on their appearance in relation to defined expectations for specific developmental ages. For example, an embryo with two branchial bars would be given a score of 10 for that structure, based on the fact that normal rabbit embryos are expected to have two branchial bars on GD 10. Individual scores for the various embryonic structures were averaged to derive an overall morphological score for each embryo. This approach allowed one to quantify generalized delays in embryo development, as well as helping to identify specific delays in certain organs or tissues. Embryo malformations were defined as specific deviations from the normal pattern of development, clearly distinguishable from generalized delays in development.
Pharmacokinetics study design.
Four groups of three virgin, nonpregnant female rabbits were fitted with auricular artery catheters (Heim, 1989) to allow for serial blood sampling. Feed was removed from the rabbits in the afternoon on the day before dosing, and was returned 4 h postdosing. Target dose levels were 26 mg (0.25 mmol)/kg and 78 mg (0.75 mmol)/kg of MPA, and 67.5 mg (0.75 mmol)/kg and 270 mg (3.0 mmol)/kg of ß-PGME. All doses were administered as aqueous solutions via gavage. Blood samples were collected at 0 (immediately prior to dosing), 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h postdosing. Blood samples (
0.5 ml) were drawn at each time point, and approximately 0.5 ml of heparinized saline was flushed through the catheter to clear the blood from the catheter and provide a "heparin lock." In cases of catheter failure, samples were collected from the marginal ear vein. Following collection of the final blood sample, the animals were euthanized by iv injection of Beuthanasia-D (Schering, Kenilworth, NJ).
Sample analysis.
All blood samples were analyzed for both MPA and ß-PGME using a double derivatization followed by GC/negative chemical ionization/mass spectroscopy (NCI/MS). Weighed aliquots of rabbit blood were either immediately derivatized or frozen until derivatized. For same-day derivatizations, samples of blood were transferred to an HPLC vial containing a weighed amount of ion-pairing agent (IPA) and mixed well. The IPA was a 0.3 N NaOH solution containing 0.1 M tetrabutylammonium hydrogen sulfate and D3-labeled internal standards of MPA and ß-PGME (0.30.7 µg/ml). The samples were derivatized to the pentafluorobenzoyl and pentafluorobenzyl esters by the addition of 4 µl of pentafluorobenzoyl chloride and 60 µl of pentafluorobenzyl bromide, respectively, with heated (46°C) vortexing for 20 min. The esters were then extracted with 1 ml of toluene during another 20-min heated vortex cycle. After centrifuging the samples at 3000 rpm for 10 min, the toluene extracts were transferred to GC vials and analyzed by GC/NCI/MS. Some samples of blood were transferred to HPLC vials containing 10 µl of heparin, mixed well and frozen at 80°C. Prior to derivatization, these samples were removed from the freezer, thawed, and mixed well with the IPA solution. The samples were derivatized using the same procedure described above. Blood samples were shown to be stable after 7 days storage at 80°C (data not shown). Detection limits were 50 ng/g blood for both MPA and ß-PGME.
Pharmacokinetic parameters were calculated using a noncompartmental analysis conducted with the aid of an Excel®-based computer software package (PK Solutions®, 1997, V2.0.2; Summit Research Services, Ashland, OH). Area under the curve (AUC) for MPA and ß-PGME blood concentration-time curves was estimated by PK Solutions® based on the trapezoidal rule as described in Gibaldi and Perrier (1982):
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where t2 and t1 are consecutive time points and Cp1 and Cp2 are blood concentrations of analyte at the respective time points. AUCs over all the time points were summed to give a total AUC for a particular blood time course.
Statistics.
Maternal body weights, body weight gains, organ weights, maternal reproductive parameters, mean fetal body weight/litter, and continuous measures of in vitro embryo development were first evaluated by Bartletts test (p = 0.01) for equality of variances (Winer, 1971). Based upon the outcome of Bartletts test, either a parametric (Steel and Torrie, 1960
) or nonparametric (Hollander and Wolfe, 1973
) ANOVA was performed (p = 0.10). If the ANOVA was significant, a two-sided Dunnetts test or the Wilcoxon Rank-Sum test with Bonferronis correction (Miller, 1966
) was performed, respectively (p = 0.05). The number of corpora lutea, implantations, litter size, and embryo morphology score were evaluated using the nonparametric ANOVA followed by the Wilcoxon Rank-Sum test with Bonferronis correction. Pregnancy rates and percentage data for whole embryo culture were analyzed by the Fisher exact probability test (p = 0.05) and Bonferronis correction was used for multiple testing of groups in comparison to a single control (Siegel, 1956
). Evaluation of the fetal sex ratio was performed by the binomial distribution test (Steel and Torrie, 1960
). The frequency of preimplantation loss, resorptions, and the incidence of fetal alterations were analyzed by the Wilcoxon test (p = 0.05) as modified by Haseman and Hoel (1974)
. Nonpregnant females, females pregnant following staining, or females having totally resorbed litters were excluded from the appropriate analyses. Statistical outliers were identified using a sequential method (Grubbs, 1969
), but only values for feed consumption were routinely excluded unless justified by sound scientific reasons unrelated to treatment.
Good laboratory practices.
All studies and resulting data were audited and found to be compliant with appropriate Good Laboratory Practice (GLP) standards.
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RESULTS |
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Table 2 lists all of the fetal malformations observed in the MPA study, as well as those variations that exhibited a treatment-related increase. Overall, there were 0, 3, 1, and 6 malformed fetuses in the 0, 10, 26, and 78 mg/kg/day MPA groups, respectively, with the incidence of total malformed fetuses in the high-dose group being statistically increased relative to controls. The only specific malformation observed in more than one high-dose fetus was fused ribs, which was seen in three fetuses from three different litters. While not statistically significant, the incidence of fused ribs (4.8% of fetuses, 27.3% of litters) was outside the range of historical control data (maximum of 2.4% of fetuses, 15.4% of litters). Other malformations among the high-dose group were limited to single occurrences and included short tail, spina bifida, ectopic gall bladder, hemivertebrae, scoliosis, and oligodactyly (missing digit I). There were also increased incidences of several fetal variations noted in the high-dose MPA group, including retrocaval ureter, irregular pattern of sternebrae ossification, and delayed ossification of the hyoid, talus, and forelimb phalanges. The incidence of paraovarian cysts (6.3% of fetuses, 27.3% of litters) also was higher than that of contemporary controls, and while not statistically identified, was outside the range of historical controls (maximum of 1.2% of fetuses, 10% of litters) and thus was considered an effect of treatment. There were no treatment-related changes in the incidence of fetal malformations or variations in the middle- and low-dose groups. The few malformations observed showed no dose-response and all incidences were within historical control ranges.
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In contrast to the MPA study, there was a greater incidence and broader spectrum of fetal malformations and variations with MAA, despite the lower dose levels (Table 4). There were 1, 2, 9, and 32 malformed pups in the 0, 2.5, 7.5, and 15.0 mg/kg/day MAA groups, respectively, with the overall incidence of malformed fetuses statistically increased at 7.5 and 15 mg/kg/day. In particular, statistically significant increases in the incidence of three external malformations affecting the distal limb, namely forelimb flexure, oligodactyly, and anonychia (missing toenails), occurred in the high-dose MAA fetuses. In addition, a number of other external malformations (brachydactyly, limb rotation, hindlimb flexure, digit flexure) and variations (kinky tail) appeared to occur at increased incidence. Although these incidences were not statistically different from controls, they were interpreted to be treatment-related based on consideration of historical control ranges, dose-dependency, and/or consistency with the effects of EGME (Hanley et al., 1984b
). In the middle-dose group, slightly increased incidences of forelimb flexure and kinky tail were also observed. There were no treatment-related external alterations among low-dose fetuses, with the incidence of externally malformed fetuses in this group (2/106) similar to that of control fetuses (1/119).
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Increased incidences of several skeletal alterations were evident in the 7.5 and 15 mg/kg/day groups. In the 15 mg/kg/day dose group, an increased incidence of extra ribs (not statistically identified) was the only skeletal malformation considered to be treatment-related. Other skeletal malformations in this group were observed only in single fetuses, thus their relationship to treatment was difficult to ascertain. Treatment-related skeletal variations were numerous in this group and included delayed ossifications of the pubis, forelimb phalanges, hindlimb phalanges, talus, metacarpals, sternebrae, hyoid and centra, irregular pattern of sternebrae ossification, and fused sternebrae. In the middle-dose group, there were no treatment-related skeletal malformations, but several treatment-related variations, such as delayed ossification of the forelimb phalanges, talus, sternebrae, hyoid and pubis, along with fused sternebrae and irregular pattern of sternebrae ossification. No skeletal alterations attributable to treatment were found among the low-dose MAA fetuses.
Rabbit Whole Embryo Culture
MPA.
All measures of embryo development in both MPA dose groups were comparable to control values. This included the percentage of viable embryos, percentage of embryos with an active yolk sac circulation, head length, somite number, morphological score, and percentage of malformed embryos (Table 5). One embryo in the control group had an abnormally small head. In the 1 mM MPA group, one embryo had a misshapen head and also an enlarged pericardium, while another embryo from the same group had a blister over the hindbrain. Low incidences of such malformations in rabbit whole embryo culture are not unusual findings. In particular, small to moderately sized blisters over the head and neural tube have been seen in up to 25% of control rabbit embryos in culture (Pitt and Carney, 1999b
), and thus, are unlikely to be due to treatment. There were no malformed embryos in the 5 mM MPA group.
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ß-PGME.
There were no differences between the ß-PGME groups and controls for any parameter of embryo development (Table 6). One embryo in the control group had an open neural tube in the region of somites 1518, and also had a slightly irregular neural tube in the pontine flexure region of the hindbrain. Another control embryo exhibited a blister over the pontine flexure. In the 0.5 mM ß-PGME group, one embryo had an abnormally small head. In the 2.0 mM ß-PGME group, one embryo exhibited an irregular, zig-zag neural tube, while another had a blister over the telecephalon. A third embryo in this group was grossly malformed, with an abnormally shaped, small head, open neural tube, and missing pericardium.
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DISCUSSION |
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The present in vivo developmental toxicity assessments conducted in NZW rabbits, the species most sensitive to developmental effects of ethylene glycol ethers, showed that MPA caused developmental effects only at maternally toxic dose levels. In fact, the developmental toxicity NOEL of 26 mg/kg/day was higher than that for maternal toxicity (NOEL of 10 mg/kg/day). In contrast, MAA was selectively toxic to the fetus, with a developmental NOEL of 2.5 mg/kg/day, but a maternal toxicity NOEL 7.5 mg/kg/day. These data suggest that, even in the most sensitive species, MPA is about 10-fold less potent than MAA for causing developmental toxicity.
The spectrum of fetal anomalies induced by the high-dose of MPA was more restricted than that of MAA. While both compounds induced fetal resorptions and caused increases in the incidence of retrocaval ureter, paraovarian cysts, irregular pattern of sternebrae ossification, and delayed ossification of the distal limb bones and hyoid (considered fetal variations), MAA induced a host of additional fetal effects that were not increased by MPA (Table 4). These included limb flexures and rotations, digit malformations, diaphragmatic hernia, pale/small spleen, extra ribs, paratesticular cyst, delayed ossification of the skull, sternebrae, pubis and centra, and fused sternebrae. Thus, MPA is not only a weaker developmental toxicant with respect to dose, but is also less potent with respect to the severity and range of effects it induces.
The in vivo studies mentioned above were complemented by rabbit whole embryo culture experiments with MPA, ß-PGME, and MAA in order to compare the intrinsic potency of these materials. Rabbit whole embryo cultures are conducted at a stage of very active development (GD 911), and one that is thought to be highly sensitive to teratogenic insult. Also, the system allows one to precisely control the level of test chemical to which embryos are exposed without the confounding factor of maternal metabolism, elimination, etc. For these reasons, rabbit embryo culture was considered an ideal approach for comparing the intrinsic teratogenic potential of these compounds.
The effects seen in rabbit whole embryo culture were largely consistent with the in vivo studies, in the sense that MAA was strongly teratogenic in vitro, whereas equivalent concentrations of MPA were without effect. This clearly indicated that the intrinsic developmental toxicity potential of MPA is much lower than that of MAA. Rabbit whole embryo culture evaluation of ß-PGME also indicated a lack of treatment-related effects. This finding is consistent with other studies suggesting that developmental toxicity of ethylene glycol ethers is due to their alkoxy acid metabolites, rather than a direct action of the parent compound (Rawlings et al., 1985; Yonemoto et al., 1984
).
In conjunction with in vivo developmental toxicity and rabbit whole embryo culture studies, we also investigated the kinetics of MPA formed from ß-PGME, as well as the kinetics of MPA administered directly, in female (nonpregnant) rabbits. Both compounds were readily absorbed by the oral route (Figs. 2A and 2B
), with peak blood levels reached within 14 h. Levels of MPA formed from ß-PGME peaked at 2.5 h, suggesting rapid conversion of ß-PGME to MPA. Increases in blood ß-PGME and MPA were proportional to dose administered, suggesting linear kinetics, at least in the dose-range examined. Comparison of MPA blood levels following the 67.5 mg/kg ß-PGME dose and 78 mg/kg MPA dose (equimolar doses) demonstrated their pharmacokinetic equivalence, as evidenced by the fact that the semilogarithmic plots of the blood concentration-time courses were virtually superimposable (Fig. 2B
). The latter observation suggests that essentially 100% of the ß-PGME found in blood is converted to MPA.
Whereas parent ß-PGME was cleared rapidly from blood, MPA blood levels declined very slowly, and were still readily detectable 144 h postdosing. This long apparent t1/2 of elimination may indicate plasma binding of MPA and/or resorption of MPA by the kidneys resulting in recycling of MPA, both of which would result in slow elimination from blood. Although a long plasma t1/2 could also be caused by saturation of renal elimination of MPA, one would expect that to occur only at the early part of the time-course, and to change the slope of the high-dose elimination curve compared with the low-dose curve. The overall slow elimination from plasma could also be due to a combination of these different mechanisms. The long elimination half-life for MPA in rabbits is in direct contrast to the imputed value for rats, based on rapid elimination of radioactivity following administration of 14C-ß-PGME, where 90% of the administered dose was eliminated within 48 h (Miller et al., 1986). These pharmacokinetic data suggest that overall systemic exposure to MPA is much greater in rabbits than in rats exposed to equivalent doses of ß-PGME. Thus, pharmacokinetic differences might explain the greater sensitivity of rabbits relative to rats following inhalation exposure to ß-PGME (Hellwig et al., 1994
). Interestingly, MAA appears to be eliminated slowly in humans (t1/2 = 77 h; Groeseneken et al., 1989
), supporting the use of the rabbit as a preferred animal model for this compound.
The combined weight of evidence from the developmental toxicity and pharmacokinetic studies reported herein offer valuable insight regarding the potential risks of developmental effects posed by the ß-isomer contained in commercial PGME. Whether one makes estimates based on delivered doses or internal dose metrics, it is clear that extremely high exposures to commercial PGME would be required to adversely affect development. For example, knowing that the LOEL for MPA of 78 mg/kg/day is pharmacokinetically equivalent to 67.5 mg/kg/day ß-PGME, and assuming that commercial PGME contains 0.5% ß-isomer, then developmental toxicity would require a delivered dose of 13,500 mg/kg/day (67.5/0.005). Making a similar analysis based on the pharmacokinetic values obtained in this study, it appears that developmental toxicity would require MPA blood levels of at least 0.51.3 mM (Cmax) or 19.952.9 mM-h/l (AUC), levels which generally are considered quite high. Based on human exposure data (Anundi et al., 2000; Dentan et al., 2000
; Devanthery et al., 2000
; Hubner et al., 1992
; Jones et al., 1997
; Laitinen, 1997
) and occupational limits set for PGME (ACGIH threshold limit value =100 ppm, STEL = 150 ppm), it is highly unlikely that human exposure to ß-PGME (or MPA) would ever approach such developmentally toxic levels.
In summary, our data on the developmental toxicity and pharmacokinetics of MPA provide a mechanistic foundation to address concerns about the presence of ß-isomer in commercial PGME. The combined weight of evidence consists of negative developmental toxicity studies with the commercial product in several species and routes of exposure, in vivo and in vitro developmental studies showing that MPAs intrinsic toxicity is much lower than that of MAA, and pharmacokinetic data indicating a high threshold based on internal MPA dose. These data collectively increase confidence in the conclusion that MPA formed from small amounts of ß-isomer in commercial PGME represents a negligible risk to the developing fetus.
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ACKNOWLEDGMENTS |
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NOTES |
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