Summary of General Toxicological and Biological Parameters

Methanol is not foreign to the bodies of mammals, including man, as it occurs naturally as a prod-uct of endogenous biochemical processes. As described in Section 1, methanol is a natural constitu-ent in fruits, vegetables, and fermconstitu-ented drinks common in the American diet. Human exposure to methanol also results from consumption of liquids that contain the direct food additives aspartame and DMDC. Thus, methanol is present in human blood; mean background blood levels are some-what variable and may range from 0.6 (31) to 2.6 mg/L (36). Although gender differences have not been routinely evaluated, at least one study has reported higher baseline blood levels of methanol in females than males (35).

The absorption, distribution, metabolism, and excretion of methanol are generally understood in humans, monkeys, rats, and mice (1, 2). There are sufficient data from human studies and other species to demonstrate rapid absorption following exposure by inhalation, dermal, and oral routes.

Following absorption, methanol distributes rapidly and uniformly to all organs and tissues in direct relation to their water content. Methanol elimination in expired air and urine is somewhat propor-tional to methanol concentration in blood, but accounts for a minor portion (3.1%) of the dose at concentrations that do not saturate metabolic pathways. At saturating doses these routes of elimina-tion may become more significant (45). In mammals, methanol is eliminated primarily by metabo-lism through a series of oxidation steps to sequentially form formaldehyde, formate, and carbon dioxide (Figure 2-1, pg 21).

The disposition and metabolism of methanol appear to be similar regardless of the route of admin -istration (oral, dermal, or inhaled). However, due to the fact that respiration rates are the inverse of size, smaller species are predicted to accumulate higher blood methanol concentrations than larger species when exposed to similar methanol concentrations (45). As noted in Table 2-10, this projec -tion is confirmed by data obtained following inhala-tion exposures to high concentra-tions of metha-nol (≥10,000 ppm) where blood methanol concentrations observed in mice were 2−5 times higher than those of rats exposed to the same concentrations. Species differences are less obvious at lower exposure levels as noted in Table 2-10. At 5,000 ppm the differences between blood methanol levels in rats and mice were generally 2-fold or less; at 1,000 ppm rat and mouse blood levels were simi-lar. The limited data indicate that at 200 ppm rat, monkey, and human blood methanol levels were similar.

The fate of methanol in pregnant animals has been subject to limited research. Available data in-dicate little or no difference in methanol toxicokinetics as a function of pregnancy in non-human primates (52). In pregnant mice and rats there was an indication that penetration of methanol to the fetal compartment decreased in inverse proportion to higher dose, possibly as a result of decreased blood flow (45).

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Inhaled Dose (ppm-hours)

191-1.25 3.81.9 b3.6 b 200-2.511 5 a8.7 a 200-419 6.5 d14.3 d 200-611 31 273.1−7.4 c,v3.9 c7.0−8.1 f5.4−13.2 c5.4−13.2 c8.7−9.5 f 400-87413.4 t 600-2.533 11 a8.7 a 800-8133 31 g 1,000-781997 e (NOAEL) 1,000-8428 83 i 1,200-6385 184 27 c38 c5.4–13.2c,f5.4–13.2 c 1,800-2.598 35 a10 a 2,000-6642 308 308 80 c64 c5.4–13.2c,f5.4–13.2 c 2,000-71,638537 e (LOAEL) 2,500-8 2,3401883 i 3,000-211,375 80 l30 l 4,500-61,444555–1,260 n 5,000-6680−873 v 5,000-74,095 1,869 1,650 e1,000 – 2,170 k (NOAEL) 5,000-84,680 2,139 3,580i1,047 i 5,000-212,293 5,250 l1,210 l 7,500-76,143 3,178 e

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Estimated Dosesx,y (mg/kg bw)Blood/Plasma Methanol in mg/L (range as reported in multiple studies) yBlood Formate in mg/L (range as reported in multiple studies) y MouseRatMonkeyHumanMouseRatMonkeyHumanMouseRatMonkeyHuman 10,000-67,020 1,468− 2,080 o,v 10,000-78,190 3,738 4,204 e1,840 – 2,240 k (LOAEL) 10,000-89,360 4,280 6,028 i1,656 i 15,000-610,530 7,140 o 15,000-712,285 5,616 7,330 e3,169 – 3,826 m 15,000-8 14,040 6,420 11,165 i2,667 i 20,000-7 7,476 5,250 – 8,650 k 20,000-88,560 3,916 i Oral Dosing Lethal Dose - Bolus9,500 u3,000 u Human Lethal Dose300 –1,000 6.0 – 9.0 mg/kg Aspz6.0 – 9.0 q2.4–3.6 q 34 mg/kg Asp3.4 p≤ 4 p 100 mg/kg Asp10 p12.7 p 150 mg/kg Asp15 p21.4 p 200 mg/kg Asp20 p25.8 p8–22 p a Burbacher et al. (52)e Rogers et al. (96)i Perkins et al. (62)m Stanton et al. (100)q Davoli et al. (39)u Gilger & Potts (73) b Cook et al. (31)f Lee et al. (33)j Stern et al. (97)n Weiss et al. (95)r Batterman et al. (34)v Cooper et al. (101) c Horton et al. (53)gBatterman et al. (34)kNelson et al. (98)o Dorman et al. (66)s Batterman&Franzblau(35)w Lee et al. (50) d Osterloh et al. (40)h Pollack & Brouwer (45)l NEDO (99)p Stegink et al. (11)t Franzblau et al. (36) x Inhalation doses in mg/kg bw were estimated by the Methanol Institute (102) and verified by CERHR to ensure that calculations were accurate and reasonable assumptions were used. y Blank cells in tables signify no known information for a particular dose and species. z Asp=Aspartame

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There are marked species differences in the rate of methanol metabolism and these differences are important in interpreting methanol toxicity data. Although metabolism of methanol to formalde-hyde utilizes different enzymatic pathways, this step occurs at similar rates in primates and rodents (1). Formaldehyde is rapidly oxidized (half-life of ~1 minute) to formate in all species. It is the rate at which formate is oxidized to CO₂ that accounts for the pronounced species difference in the tox-icity of methanol (primates are more sensitive than rodents to the acute effects of methanol expo-sure). In rodents the catalase-peroxide system and enzymes utilizing folate as a co-enzyme provide considerable capacity to catalyze this reaction whereas primates depend heavily on the pathway involving folate. Because primates naturally have lower folate concentrations than do rodents they have considerably less capacity to metabolize formate. Formate is oxidized to CO₂ in rodents at twice the rate seen in primates. As a result, the rate of formate oxidation in rats exceeds the maxi-mal rate at which methanol is converted to formate: 1.6 versus 0.9 mmol/kg/hour, respectively (2).

In contrast, when primates receive moderately high doses of methanol, the formation of formate can exceed the oxidation of formate: ~1.5 versus 0.75 mmol/kg/hour, respectively. The net result is that primates may accumulate levels of formate that exert toxicological consequences at doses far lower than those needed to produce equivalent effects in rodents.

A calculated estimate of the methanol concentration that saturates the human folate pathway is 11 mM or 210 mg/kg (2). It should be noted, that whereas exposure of healthy humans to up to 200 ppm methanol for varying periods of time demonstrates time and concentration-dependent increases in blood methanol, no increases in blood formate were detected (31, 33, 40). Short-term exposures of non-human primates to concentrations of methanol ranging from 200 to 1,800 ppm resulted in increases in the levels of blood methanol from approximately 2.4 mg/L prior to exposure to 35 mg/L following exposure to 1,800 ppm. There was no increase in blood formate at any dose in these studies.

There is limited information on the effects of chronic methanol exposure on toxicokinetics in hu-mans. Leon et al. (38) reported there were no significant increases of blood methanol levels above 10 mg/L in 53 healthy adults who for 24 weeks consumed daily doses of aspartame that yielded a methanol equivalent dose of ~7.5 mg/kg. Information from non-human primates (52) indicates that long-term exposure (exposure for 2.5 hours each day for ~300 days) resulted in an increase in methanol clearance rates with no increase in blood formate at exposure levels up to 1,800 ppm.

From these data it is reasonable to conclude that inhalation of methanol at doses up to 1,800 ppm is unlikely to result in elevated blood formate levels in healthy humans. However there are no toxi-cokinetic data on chronic methanol exposures in humans with marginal folate tissue concentrations – a condition that is of concern for susceptible populations. There are limited data to indicate that a single 2-hour exposure of folate deficient non-human primates to 900 ppm methanol vapor did not increase blood formate levels (54).

Finally, it is to be noted that several pharmacokinetic models have been developed for the extrapola-tion of methanol data (45, 57, 62). These models are of value in better understanding the dose and metabolite effects of high doses of methanol in rat and mouse studies. The Horton et al. (53) model is a careful attempt to develop PBPK models for methanol in rats, monkeys, and humans. The au-thors included some lower methanol exposure conditions for the rodent studies, which increases confidence in extrapolating results to humans. The importance of having models account for

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tive respiratory uptake so as not to overestimate lung absorption was reported by Fisher et al. (56).

The Panel notes that Environ (67) performed a comparative analysis of the Perkins et al. (57) and Horton et al. (53) models that provides insights as to the model features and differences. The Expert Panel concludes that the existing pharmacokinetic models may be useful for future quantitative or semi-quantitative assessments of the risks posed by methanol exposure. However, such modeling was outside the scope of this Panel and would require further evaluation of the strengths and limita-tions of the models.

The Panel concluded that the toxicokinetic data pertaining to methanol are of sufficient breadth, depth, and quality to contribute in a material way to evaluating the potential for methanol to pose a risk to human reproduction. There is convincing evidence that formate is the metabolite responsible for methanol toxicity associated with systemic clinical signs, metabolic acidosis, and ophthalmic effects. Since humans and other primates oxidize formate less efficiently than rodents and other lab-oratory animal species, they accumulate formate at lower doses of methanol than do other species.

General Toxicity

The primary sources of information used by the Panel on the general toxicity of methanol were the reviews of IPCS (1) and Kavet and Nauss (2).

Human Data

Information about methanol toxicity in humans from high levels of exposure is available from acute intoxications (poisonings) in the general population, occupational exposures, and labora-tory studies. The minimal lethal dose for methanol in untreated humans has been reported as a range of 300 –1,000 mg/kg bw (1). Typical findings in acute methanol toxicity are temporary mild central nervous system depression followed by an asymptomatic period with a duration of 12 –24 hours that is followed by metabolic acidosis. Ocular toxicity also develops in parallel with these effects. In severe poisonings, abdominal pain and difficulty breathing can occur and progress to coma and death due to respiratory failure. Five epidemiological studies reported symptoms such as headaches, dizziness, blurred vision, nausea, and/or eye irritation in workers exposed to methanol at concentrations exceeding the occupational limit of 200 ppm (1, 2). Two well controlled studies exposed healthy adults to 200 ppm methanol for 75 minutes, leading to a blood methanol of 1.9 mg/

L (31), or 4 hours leading to a blood level of 6.5 mg/L (32), and performed a variety of neurophysi-ologic and neurobehavioral tests. Most results were negative. However, small effects were seen with some evoked potentials and cognitive measures in both studies. The Expert Panel was unable to develop a level of confidence that the effects were methanol related due to the low magnitude of the responses and because the single dose designs did not allow an assessment of dose response.

Experimental Animal Studies

Studies in animals have examined methanol toxicity following acute or repeat dosing. The lethal dose in rats and rabbits was reported to be 2 –3 times higher than the lethal dose reported for mon-keys and 6 –10 times higher than the lethal dose reported for humans (See Table 2-7, pg. 40). Al-though primates, including humans, experience acidosis and adverse visual effects following acute exposure to methanol, those effects do not occur in most laboratory animals such as rats, mice, rab-bits, dogs, and minipigs. For this reason, non-human primates are the most relevant animal models for studying the acute effects of methanol exposure, which are generally thought to be due to

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mate-induced toxicity. However, non-primate species may be appropriate animal models for studies that examine the direct alcoholic effects of methanol. A number of studies identified the eye, brain, and liver as target organs in rats, dogs, and rabbits. The Expert Panel reviewed 3 short-term studies in which Sprague-Dawley rats were exposed with methanol vapors at concentrations up to 5,000 ppm for 6 hours/day for 4 weeks (77-79). These authors reported nasal irritation but no consistent signs of systemic toxicity. Histological examination inconsistently revealed thyroid and liver effects in rats exposed to 300 and 2,500 ppm methanol respectively; reproductive organ lesions were not observed. No signs of systemic toxicity or histological abnormalities were observed in Macaca fas-cicularis monkeys exposed with up to 5,000 ppm methanol vapors for 6 hours/day for 4 weeks, but it does not appear that reproductive organs were examined (77). No effects on weight gain or overt toxicity were noted in female M. fascicularis monkeys exposed to up to 1,800 ppm methanol vapors for about 11 months (52).

Sufficiency Statement

The Panel concluded there are sufficient data to characterize the general toxicity of methanol in humans and laboratory animals, including non-human primates. The general toxicity of methanol has been characterized in humans exposed to low doses in the laboratory and through observation of individuals accidentally or deliberately exposed to high doses. These data confirm that humans and other primates, in contrast to other species, are uniquely sensitive to the toxic effects of metha-nol at lower doses as a result of formate toxicity and metabolic acidosis that result from a slow rate of formate metabolism and clearance. In comparison to non-primate species, the accumulation of formate and resulting acidosis effectively limit the methanol dose tolerated by humans.

Genetic Toxicity

Results of in vivo genetic toxicity assays in mice have been mixed, with both negative and positive results in micronuclei formation and chromosomal aberration assays and negative results in SCE and urine mutagenicity assays (1, 80). Negative results were obtained in the majority of in vitro assays that examined mutations in bacteria and yeast, DNA repair in bacteria, and SCE and cell transformation in mammalian cells; positive results were obtained in a chromosomal malsegrega-tion assay in yeast only in the absence of metabolic activamalsegrega-tion and in a mutamalsegrega-tion assay in mamma-lian cells only with metabolic activation (1). IPCS concluded that “The structure of methanol (by analogy with ethanol) does not suggest that it would be genotoxic.”

Carcinogenicity

There are no reliable data for evaluating carcinogenicity.

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