A single study (104) was reviewed in which a variety of occupations and consequent exposure to complex mixtures were determined in women who gave birth to infants with and without cleft lip or palate. The study did not find an association between methanol exposure and oral clefts, but several limitations in the study were noted including: small number of subjects exposed to methanol, lack of individual exposure data, and confounding by other chemical exposures. Because of these limita-tions the Panel judged the study results to be uncertain and concluded there are insufficient human data upon which to evaluate the developmental toxicity of methanol.
Since methanol is metabolized by a folate-dependent pathway, the Expert Panel reviewed a number of epidemiological studies that examined folate supplementation and various birth defects (108-112). In general these studies suggest that periconceptional supplementation with multivitamins containing folic acid decreases the incidences of birth defects including NTDs and orofacial clefts.
These studies suggest that it will be important to consider possible interactions between methanol exposure and folate status in animal studies in view of various interspecies differences such as dif-ferences in folate levels, methanol metabolism, and toxicokinetics.
Experimental animal data
The panel reviewed developmental toxicity studies that were performed in rats, mice, and non-hu-man primates. Results of these reviews are summarized below.
Prenatal Rat Studies. The results of Nelson et al. (98) are sufficient to conclude that inhalation exposure of Crl:Sprague-Dawley rats to 20,000 ppm methanol vapor for 7 hours/day on gd 7–15
Appendix II
causes prenatal developmental toxicity as evidenced by reduced fetal weight, increased litter in-cidence of exencephaly and encephalocele, and skeletal malformations. This dose caused clinical signs of maternal intoxication in early days of exposure but no other maternal effects. Develop-mental toxicity was also observed following exposure to 10,000 ppm for 7 hours/day on gd 1–19 as evidenced by statistically significant reductions in fetal body weight. The Expert Panel des-ignated 10,000 ppm inhaled methanol as a maternal NOAEL and 5,000 ppm as a fetal NOAEL.
Blood methanol levels were determined in non-pregnant rats with exposures similar to the pregnant dams and were reported at 1,840−2,240 mg/L and 5,250−8,650 mg/L in rats exposed to 10,000 and 20,000 ppm methanol respectively. In the study by NEDO (99), maternal toxicity and adverse developmental effects were observed in Sprague-Dawley rats after inhalation of 5,000 ppm metha-nol on gd 7−17 for an average of 22.7 hours/day. Increased numbers of late resorptions, reduced numbers of live fetuses, decreased fetal weight, and increased numbers of malformed fetuses were observed. The observed malformations were similar to those observed by Nelson et al. (98). No adverse effects were observed at 1,000 ppm. Deficiencies in design or completeness of data presen-tation led the Expert Panel to conclude that the studies of Cummings et al. (138) and Youssef et al.
(140) were of limited utility in this evaluation.
Prenatal Mouse Studies. The studies of Rogers et al. (96) are sufficient to conclude that prenatal exposure of Crl:CD-1 mice to methanol vapor at doses of 2,000 ppm or greater for 7 hours/day on gd 6 –15 causes developmental toxicity as evidenced by cleft palate, exencephaly and skeletal mal-formations. The initial appearance of malformations were dose-associated with cervical ribs seen at 2,000 ppm and cleft palate and exencephaly at 5,000 ppm. Effects on the number of live pups per litter and fetal weight were seen at 7,500 and 10,000 ppm, respectively. Methanol blood levels in the 2,000, 5,000, 7,500, 10,000, and 15,000 ppm groups were measured at 537, 1,650, 3,178, 4,204, and 7,330 mg/L, respectively. The developmental toxicity NOAEL was 1,000 ppm. The maternal NOAEL was judged to be 15,000 ppm by the Expert Panel. Rogers et al. (96) also established dose comparability across inhalation and oral gavage exposure by demonstrating that twice daily gavage with 2,000 mg/kg bw/day methanol on gd 6 –15 results in a methanol blood level (3,856 mg/L) and developmental pattern of response similar to that in mice exposed to 10,000 ppm methanol vapor.
No postnatal studies were performed in the mouse.
In Vivo Rodent Mechanisms Studies. A considerable literature (10 reports or publications) was re-viewed relevant to characterizing mode of action of methanol’s effects on developmental toxicity in the rodent. Two laboratories conducted phase-specificity studies in CD-1 mice exposed to tera-togenic concentrations of methanol through inhalation (149, 150). The majority of findings were consistent between laboratories. As expected, methanol exposure during the period of neural tube development and closure (gd 7– 9) resulted in exencephaly. The incidence of cleft palate was also increased following exposure during gd 7– 9, despite the fact that cleft palate closure occurs later in gestation. Gd 7 was found to be the most sensitive day for developmental effects, since treatment on that day resulted in the greatest incidence of resorptions, exencephaly, cleft palates, and vertebral and rib defects. Bolon et al. (94) subsequently identified a putative mode of action (reduced pro-liferation) and targets of toxicity (neuroepithelium, mesoderm, neural crest) for methanol-induced NTDs in embryos of CD-1 mice exposed to 15,000 ppm methanol vapors for 6 hours/day from gd 7– 9. Connelly and Rogers (151) studied whether cervical vertebrae were associated with homeotic shifts and concluded that methanol can alter segment patterning in CD-1 mouse embryos, resulting
Appendix II
Appendix II
Appendix II
in posteriorization of cervical vertebrae.
Dorman et al. (66) reported an important series of experiments designed to investigate the role of methanol and its metabolite, formate, on development using CD-1 mice. Using a dose of sodium formate (750 mg/kg bw) that is equivalent to the formate concentration following inhalation of 15,000 ppm methanol, no exencephaly was observed. However, 15,000 ppm methanol exposure is a dose that produces exencephaly in mouse fetuses, thus suggesting exencephaly in mice requires direct exposure to methanol as opposed to only accumulation of formate. Two studies examined the impact of folate pools on methanol-induced developmental toxicity in CD-1 mice fed diets with adequate or reduced folate levels (80, 105). In dams fed folate-deficient diets, maternal and fetal hepatic folate levels were reduced. Folate deficiency enhanced the toxicity of methanol as noted by increased incidences of cleft palate and exencephaly in mice treated with methanol at 4,000−5,000 mg/kg bw/day. The Expert Panel noted that the folate deficiency studies were limited due to a lack of pair-fed controls. Using in vivo intrauterine microdialysis, Ward (65) collected data in mice and rats to indicate that at doses that are developmentally toxic (100 or 500 mg/kg or 1,000 mg/kg/hour, IV) there is also a reduced uteroplacental blood flow. They postulated that, under these conditions, hypoxia may have a role in the etiology of embryotoxic effects of methanol. Ward and Pollack (61) compared the rate of methanol metabolism in pregnant and non-pregnant mice and rats and fetal mice. Pregnancy appeared to reduce metabolic rate by ~15%; metabolic rate in mouse liver homog-enate was about two-fold greater than rat liver homoghomog-enates. Metabolic rates in fetal homoghomog-enates were only 5% of those seen in adults. These fetal data are consistent with earlier observations on alcohol and aldehyde dehydrogenases in rat fetuses (153).
Postnatal Rat Studies. Stanton et al. (100) exposed Long-Evans rats to 15,000 ppm methanol vapor for 7 hours/day on gd 7–19 and observed a modest but statistically significant reduced bodyweight in pups at birth, weaning, and pnd 35. Effects were not observed postnatally in the pups that were subjected to a range of tests for neurobehavioral function; however, small sample size limits confi-dence in these negative results.
Offspring from Long-Evans dams that drank water containing 2% methanol on either gd 15–17 or 17–19 were observed to have an increased latency to effect nipple attachment or to reach their home nesting site (141). In a later study, Weiss et al. (95) determined neurological function in Long-Evans pups following 6 hour/day exposure to 4,500 ppm methanol vapor to dams from gd 6 through pnd day 21 and to pups from pnd 1–21. No effects were observed on dam weight during gestation, litter size or postnatal pup weight gain to pnd 18. No effects were observed in latency to nipple attach-ment or olfactory sensory capabilities. Changes in motor activity were variable or inconsistent, but performance on an operant tests suggested subtle cognitive effects.
In the aggregate, the data from postnatal assessments of Long-Evans rats give no indication of ma-ternal toxicity or effects on pup viability following prenatal doses of up to 15,000 ppm methanol vapor (100), or pre and postnatal exposure of 4,500 ppm (95). Modest reduction in bodyweight was observed postnatally in pups whose dams had been exposed to 15,000 ppm methanol vapor on gd 7–19 (100). Suckling behavior was affected in a drinking water study (141), but not replicated in an inhalation study (95). While numerous behavioral outcomes were assessed and found to be nega-tive, one significant effect, the failure of methanol-exposed rats to adjust to a change in response
Appendix II
requirements in an operant task, suggested subtle cognitive effects following exposure to 4,500 ppm with peak maternal blood levels reported at 555 mg/L (95).
There is sufficient evidence in Long-Evans rats that extended exposure via methanol inhalation at 4,500 ppm with peak maternal blood levels reported at 555 mg/L, and blood methanol levels in rat offspring at pnd 21 reported at 1,260 mg/L is associated with adverse neurological outcomes.
In Vitro Rodent Studies. To gain a better understanding of mechanisms of toxicity, seven in vitro studies were conducted with methanol or formate. Exposure of rat and mouse embryos to metha-nol demonstrated effects consistent with those observed in vivo with a greater intrinsic sensitivity of mouse versus rat embryos; developmental toxicity in rats and mice was noted with methanol concentrations of ≥8,000 mg/L and 2,000 mg/L, respectively (154). Increased cell death was noted in mouse (dose not clear) and rat (16,000 mg/L) embryo structures associated with malformations following in vivo exposures; however, increased cell death was not noted in neural tube regions, suggesting a mechanism other than cell death for NTDs (155). Treatment of mouse and rat embryos with formate demonstrated effects similar to those of methanol, but the formate concentrations that caused effect (543−1,840 mg/L) were 4–10 fold lower (66, 157, 158). Toxicity appeared to be induced by both the formate ion and resulting acidosis. In a study testing mixtures of methanol and formate in rat embryos, it was found that the effects of the two compounds were less than additive (159). According to Andrews et al. (157) and Brown-Woodman et al. (158), the formate levels that produced toxicity in in vitro studies are not likely to occur in humans following environmental or occupational exposures.
Rat /Mouse Comparison. In comparing similar studies in rodents, the data are sufficient to dem -onstratethatexposuretohighconcentrationsofmethanolvaporcancausesimilarprenataldevel -opmental toxicity and frank malformations. There is good, but limited, data to indicate that the natureandincidenceoffetaleffectscorrelateswithbloodmethanolconcentrationwhenmethanol exposure isbyinhalationorthegavageroute.Micearejudged tobethemoresensitivespecies since effects were noted at lower chamber concentration doses than rats. However, at equivalent chamberconcentrations,micehad highermaternalbloodmethanollevels.Table3-8compares NOAELs from the definitive prenatal developmental toxicity studies in rat and mouse.
Table 3-8. Nominal Exposure Levels to Methanol Vapor and Corresponding Blood Methanol Levels in Rats (98) and Mice (96).
Species Maternal
NOAEL Fetal
NOAEL Maternal
LOAEL Fetal
LOAEL
Sprague-Dawley Rat
10,000 ppm (1,840−2,240mg/L)*
5,000 ppm (1,000−2,170mg/L)*
20,000 ppm (5,250−8,650mg/L)*
10,000 ppm (1,840−2,240mg/L)*
CD-1 Mouse 15,000ppm (7,330 mg/L)*
1,000 ppm
( 97 mg/L)* Unknown 2,000 ppm
(537 mg/L)*
*Maternal blood methanol level
Cross species comparisons as to postnatal effects are not possible as there are only data in rats.
Appendix II
Appendix II
Appendix II
Postnatal Nonhuman Primate Studies. Burbacher et al. (143) studied the effects of methanol on generalandneurobehavioraldevelopmentof M. fascicularis infantswhosemotherswereexposedto methanolvapors(200−1,800ppmfor2.5hours/dayleadingtobloodmethanollevelsof5−35mg/L) throughout gestation. It was reported that duration of pregnancy was reduced in primates exposed tomethanolvapors,andthatC-sectionswereperformedinsometreatedanimalsbutnotincontrols (see Section 4 for discussion). Adult monkeys experienced no effects on weight gain or overt toxic-ityasaresultofmethanolexposure.Normalweightgainandphysicaldevelopmentwasobserved throughthefirstyearofinfantlife.Neurobehavioralperformancewassimilarincontroland metha-nol groups in seven of nine tests. A subtle, statistically significant, dose-related delay in sensorimo-torfunctionwasseeninmalesofthe600and1,800ppmgroupsandinbothsexesat1800ppm when data were combined for both cohorts. Prenatal methanol exposure decreased preference for novelsocialstimuli;however,therewasnoevidenceofadoseresponserelationship.Anadditional studylookingatpostnatalexposuretoaspartamedemonstratednoeffectsongeneralhealth, devel-opment, or learning in M. arctoides monkeys fed with up to 2,700 mg/kg bw/day aspartame (equiv-alentto270mg/kgbw/daymethanol)duringthefirst9monthsoflife(147,148).Thedifferences between effects observed in these nonhuman primate studies may be explained by exposures occur-ringduringdifferentcriticalwindowsofnervoussystemdevelopment(i.e.,prenatalversuspostnatal exposures).Thesenonhumanprimatestudiestakentogethersuggest,thatdespitepresumedhigher levels of blood methanol achieved in the postnatal exposure study, prenatal exposure may be the moresensitiveperiodleadingtoalteredneurologicalfunctioninnonhumanprimates.
The Expert Panel agreed that these neurobehavioral findings in monkeys were not robust and rec-ognized issues regarding the failure to control for multiple comparisons in the statistical analysis.
The findings, however, are important from a qualitative perspective and the biological plausibility for effects on these two early tests of cognitive performance in the Visually Directed Reaching task and novelty preference in the Fagan test warrants further investigation. The Panel recommended that an independent statistical analysis of the Burbacher et al. (143) study might provide additional insights. In addition, the Panel recognizes that monkeys from this methanol study are still being evaluated for latency and persistence in functional deficits.
While the primate data examining the postnatal neurological outcomes raise some concerns it has identified insufficiencies that prevent making a clear determination about human risk.
Both the rodent and primate neurobehavioral outcomes do suggest that alterations in cognitive func-tion are consistent and subtle.
Role of Methanol as the Proximate Teratogen
TheExpertPanelconsideredseveralpossiblemetabolitesasbeingresponsibleformethanol-induced developmental toxicity. The first was that, as with acute methanol toxicity, formate would be the proximateteratogen.Invitroembryoculturestudiessuggestthatformatecaninducestructural abnormalities in rats or mice (157, 158). Data from Dorman et al. (66), however, provide direct evi-dencethatformateisunlikelytoplayasignificantroleinmethanol-inducedteratogenesisinmice in vivo.ThePanelconcludedthatmethanolisthemostlikelyproximateteratogen;however,the biologi-cal basis by which it induces defects remains unknown. Gastrulating and early organogenesis-stage rodent embryos were particularly sensitive to adverse developmental effects of methanol. The Panel
Appendix II
concluded that the available rodent data are relevant for humans despite known differences between specieswithrespecttomethanolmetabolism.TheExpertPanelconcludedthatrodentsareagood modelforhumanexposurestomethanolatlevelswhereformateisnotaccumulated,sincerodents do not accumulate formate even at very high doses of methanol. Therefore, the developmental tox-icityofmethanolalone(withoutformate)canbeanalyzedinrodentsatdosageshighenoughto determine LOAELs and NOAELs. In conclusion, there is sufficient evidence in rodents that inhala-tionofmethanolatdosesof2,000ppmorgreaterinmice(bloodmethanollevelof537mg/L)or 10,000ppmorgreaterinrats(bloodmethanollevelof1,840mg/L)for7hoursperdaythroughout organogenesis does cause developmental toxicity. These data are assumed relevant to consideration ofhumanrisk.
ThePanelconcludedthatthereissufficientevidencetoassumethatmethanolcouldbea develop-mentaltoxicantinhumans.ThePanelalsonotedthatthebloodmethanolconcentrationsthathave been associated with developmental toxicity in rodents are in the range associated with formate ac-cumulation,metabolicacidosis,andothersignsofacutetoxicityinhumans.
Appendix II