### OECD SIDS METHANOL

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# SIDS Initial Assessment Report

For

SIAM 19

Berlin, Germany, 18-20 October, 2004

1. Chemical Name: Methanol

2. CAS Number: 67-56-1

3. Sponsor Country: United States

4. Shared Partnership with: BASF AG, Germany, Methanol Institute U. S. A., Meth anor v. o. f., Netherlands, Shell Chemicals Ltd., U. K., Stato il ASA, Norway, TOTAL Raffinerie Mitteldeutschland GmbH, Germany, Veba Oil Refining & Petrochemicals GmbH, G ermany 5. Roles/Responsibilities of the Partners: BASF – Initial preparer of documents. U.S. EPA – reviewer and editor of documents, presen ter at SIAM

• 

### Name of industry sponsor /consortium BASF AG, Germany Contact person: Dr. Hubert Lendle GUP/CL – Z 570 D-67056 Ludwigshafen

• 

### Process used The documents were written by BASF AG, Germany and edited by U.S. EPA 6. Sponsorship History

• 

### How was the chemical or category brought into the OECD HPV Chemicals Programme ? ICCA Initiative

7. Review Process Prior to the SIAM: An iterative process was used by which BASF first prepared a draft based on the U.S. EPA HPV Program submission from the Methanol Institute Testing Group. U.S. EPA reviewed the draft and provided comments to BASF. U.S. EPA also edite d later drafts of the documents and inserted relevant infor mation into the documents. Meetings between U.S. EPA and BASF were also used to resolve issues. 8. Quality check process: Within the review process, data in the SIAR were co mpared with data in the SIDS Dossier and for selected endpoints a comparison with original studies was done. U.S. EPA checked the consistency among the document s. 9. Date of Submission: July 26, 2004

10. Date of last Update: July 26, 2004

11. Comments: In a separate evaluation, the U.S. EPA is also asse ssing the hazards of methanol to obtain an Agency-wide consen sus evaluation for the Integrated Risk Information Syst em (IRIS). As

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### part of the IRIS assessment, EPA will review pharma cokinetic models to determine the best method of extrapolatin g animal data to humans; this work will assist in determinin g the relevance of the effects observed in animals to hum ans. As part of the IRIS assessment, an independent Science Advi sory Board, consisting of scientific experts from several exter nal organizations, will review the evaluation.

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# SIDS INITIAL ASSESSMENT PROFILE

CAS No. 67-56-1

Chemical Name Methanol

Structural Formula CH 3 

### OH

SUMMARY CONCLUSIONS OF THE SIAR

Human Health

Toxicokinetics, Metabolism, and Distribution: Methanol is readily absorbed by inhalation, ingesti on and dermal contact and distributes rapidly throughout t he body. Metabolism in humans, rodents, and monkey s contributes up to 98 percent of the clearance, with more than 90 percent of the administered dose exha led as carbon dioxide. Renal and pulmonary excretion cont ributes only about 2 – 3 percent. The metabolism a nd toxicokinetics of methanol varies by species and do se. In humans, the half-life is approximately 2.5 – 3 hours at doses lower than 100 mg/kg bw. At higher doses, th e half life can be 24 hours or more.

The mammalian metabolism of methanol occurs mainly in the liver, where methanol is initially converted to formaldehyde, which is in turn converted to formate . Formate is converted to carbon dioxide and water . In humans and monkeys, the conversion to formaldehyde is mediated by alcohol dehydrogenase. In rodents, the first reaction occurs mainly via a catalase-peroxid ase pathway which is rate-limiting. Methanol in tu rn accumulates in the blood at high doses. In primate s, the last reaction step, conversion of formate to carbon dioxide by the formyl-tetrahydrofolate synthetase, is rate-limiting, leading to a disproportionate inc rease of formate in the blood and sensitive target tissues ( such as CNS and the retina).

In humans, when exposed via inhalation up to an air concentration of 0.065 mg/L, no increase of blood methanol is expected. Up to 0.26 mg/L (single or repeated exposure), the methanol blood l evel is likely to increase 2 to 4- fold above the endogenous methanol concentration in humans, but still remains significantly below 10 m g/L. Air concentrations up to 1.6 mg/L resulted in similar b lood methanol among rats, monkeys, and humans. How ever, above 1.6 mg/L, a steep exponential increase occurs in rats, a smaller exponential increase occurs in monkeys, and humans exhibit a linear relationship between ai r concentrations and blood methanol levels.

Baseline levels of formate in blood are about 3 to 19 mg/L (0.07 – 0.4 mM) in humans. Toxic blood for mate concentrations are reported to be 220 mg/L and high er (> 5 mM formate). Inhalation of about 1.20 mg methanol/L for 2.5 hours contributed only insignificantly to t he internal formate pool in monkeys (in the μMrange); this also held true for folate-deficient co nditions. Also after repeated inhalation of 2.6 mg /L (2000 ml/m 3 ) for 6 hrs/day, 5 days/week, for 1 or 2 weeks, monke ys showed no discernible increase in formate concen tration in blood [estimated body burden 200 to 300 mg/(kg bw * d)], whereas formate accumulation has been observed in primates at methanol doses greater than 500 mg/kg b w given as a bolus. The critical methanol dose tha t saturates the folate pathway in humans is estimated to be ≥ 200 mg/kg bw. Based on data produced in monkeys, m etabolic saturation in humans is also less likely to happen during inhalation if the amount is divided over a p rolonged time and not incorporated as a bolus.

Acute Toxicity: Formate is considered to be the ultimate toxicant in acute methanol intoxication in humans. Acidosis and ophthalmologic changes are typical pri mary effects. However, these effects do not occur in rodents or rabbits., which are able to remove formate more efficiently. In these animals, CNS depression is us ually the cause of defects and finally death.

A blood level of 500 mg methanol/L in acutely poiso ned patients generally is regarded as requiring hem odialysis. This blood concentration can transiently be achieve d in an adult person (70 kg) by ingestion of 0.4 ml methanol/kg (approximately 0.32 mg/kg). Generally, in humans, transient central nervous system (CNS) e ffects appear above blood methanol levels of 200 mg/L and serious ocular symptoms appear above 500 mg/L.

The minimal acute methanol dose to humans that can result in death is considered to be 300 to 1000 mg/ kg by

### OECD SIDS METHANOL

4 ingestion, and fatalities have occurred in untreate d patients with initial methanol blood levels in th e range of 1500-2000 mg/L. However, such high blood methanol levels able to cause death are not .likely to be ac hieved through inhalation exposure. For example, 2.6 or 6 .5 mg/L resulted in methanol blood levels that bare ly exceed 100 and 200 mg/L, respectively, after an 8-hour wor king shift. Exposure to 0.26 mg methanol/L for 4 h ours was without significant physiologic effects in human vo lunteers.

In rats, LC 50

values have been calculated to be 83.2 and 128.8 m g/L after 4 hours. In cats, the LC 50 was 85.4 mg/L after four hours. In monkeys, air concentrati ons of 52 mg/L after 1-4 hours and 13 mg/L after 18 hours led to an unspecified level of mortality. Dermal LD 50 s in rabbits range from 15800 to 20000 mg/kg bw. I n rats, the dermal LD 50 is greater than 45,000 mg/kg bw. In monkeys, four daily dermal doses of 400 mg/kg bw eventually resulted in death. Oral LD 50 s in rats range from < 790 to 13,000 mg/kg bw; in m ice, the values range from 7,300 to 10,000 mg/kg bw; in rabbits, the LD 50 was approximately 14,200 to 14,400 mg/kg bw; and i n monkeys, the values range from 7000 to 9000 mg/kg. Although most of the references for these values provided only l imited details, the values are consistent within species a nd route of exposure.

Irritation/Sensitisation: Methanol exhibited no skin irritation in one stud y in which the chemical was administered under occluded conditions as a 100 per cent concentration. Limited information from anoth er study showed moderate irritation with 500 mg. Available studies show that methanol is a slight eye irritant with doses of 0.05 to 0.1 mL. Some reversibility of effects w as observed. High concentration of methanol vapours may be irritating to mucous membranes. A guinea pig maxim ization assay gave no evidence of contact sensitisa tion after induction and challenge doses of 50 percent.

Repeated-Dose Toxicity: In a whole body inhalation study in monkeys expos ed to 0.013, 0.13, and 1.3 mg/L 21 hours per day 7 days per week for 7, 19, and 29 mon ths, several general clinical signs as well as dege nerative effects in the brain (at 0.13 and 1.3 mg/L), slight peripheral nerve damage (at 0.13 and 1.3 mg/L), ve ry slight degeneration of the optic nerve (concentrations not noted), increased fat granules and slight fibrosis in the liver (all concentrations), and Sudan positive granules i n the kidney were observed (at 0.13 and 1.3 mg/L). Also, a slight myocardial disorder (at 0.13 and 1.3 mg/L) a nd localized effects in the trachea and possible sl ight fibrosis in the lungs (concentrations not noted) were observ ed. Although the statistical significance of the e ffects cannot be verified from the study report, the number of ef fects and systems affected indicate a relationship with methanol.

In another whole body inhalation study in monkeys e xposed up to 20 days for 21 hours per day, coma and lethality were observed at concentrations > 9.1 mg/(L * d). In the brain, necrosis of the basal ganglia an d cerebral edema were observed at 6.5 mg/(L * d) and at 3.9 mg/(L * d), hyperplasia and fibrosis around myelin sheaths of the basal ganglia as well as a slight to moderate incre ase in astroglia cells were observed. The optic ne rve showed atrophy at >3.9 mg/(L * d), along with reduction in myelin fibers. In the liver, fibrosis was observed at 6.5 mg/(L * d) and mild fatty degeneration was observed at 3.9 mg/(L * d). In the kidney, partly vacuolated hyaline degeneration was observed at 6.5 mg/(L * d).

In rats exposed to methanol up to 6.5 mg/L for 6 ho urs per day, five days per week for 28 days, no adv erse effects were observed except local nasal irritation and inc reased relative spleen weights, which were observed only at the middle dose. The estimated blood level of methanol was about 250 mg/L under this condition.

In a whole body inhalation study in mice exposed fo r 12 months to concentrations of 0.013, 0.13, and 1 .3 mg/L 20 hours per day, slight changes in clinical signs, body and organ weights, and some changes in histop athology were observed. In rats exposed in the same manner, slight changes in body weight and organ weights we re observed at the highest dose. The NOEL is 0.13 mg/ L. In rats, gavage doses of 100, 500, and 2,500 mg /(kg bw * d) for 90 days resulted in increased liver enzymes and reduced brain weights at the highest dose resul ting in a NOAEL of 500 mg/(kg bw * d).

Mutagenicity: Numerous in vitro assays (including seven Ames assays, four micronucl eus/cytogenetic assays, a mammalian gene mutation assay, a yeast gene mutat ion assay, a mouse lymphoma test, three cell transformation assays, and a DNA damage and repair assay) were conducted on methanol. The majority of these assays are negative, with the exception of positive results in the mouse lymphoma test and a mitotic recombination assay in Aspergillus, and ambiguous r esults in an Ames assay for strain TA102 and in the DNA damage and repair assay. Only limited details were available for the mouse lymphoma test. Of the ele ven in vivo assays (all micronucleus and cytogenicity assays pl us a Drosophila SLRL assay), all are negative except one cytogenetic assay, which was positive for aneup loidy, sister chromatid exchange, and micronuclei. Limited information was available regarding this positive r esult. Thus, although most studies indicate that m ethanol does not have the potential to be mutagenic, not all stu dies lead to that conclusion.

### OECD SIDS METHANOL

5 Carcinogenicity: Methanol was tested in two long-term whole body in halation studies (24 months in rats and 18 months in mice for 20 and 19 hours per day, respect ively). There was no evidence of a carcinogenic po tential in rats and mice exposed to air concentrations of up t o 1.3 mg/L.

Reproductive/Developmental Toxicity: Monkeys. In monkeys, parents were exposed via inhalation pri or to and during breeding as well as during pregnancy to doses of 0.26, 0.78, and 2.34 mg/L. A late wasting syndrome was observed at the highest dose in 2 out of 7 fema le descendants, associated with signs of severe mal nutrition and gastroenteritis noted at autopsy. Mild neurobe havioral effects in offspring and some vaginal blee ding in mothers were observed at all concentrations. Howev er, due to the normal variance in and the low numbe r of animals, along with pre-term delivery of several mo thers, the observed findings are difficult to inter pret.

Rats. Several inhalation studies in rats resulted in a va riety of effects in offspring due to prenatal and/o r postnatal dosing. In a 2-generation whole body inhalation re productive study in which rats were exposed for 19- 20 hours/day, decreased brain weights in the first and second generation offspring (F1, F2) resulted in a NOAEL of 0.13 mg/L. In a developmental study in which rats were exposed by whole body inhalation on gestation days 1 to 19 at the two lowest doses and days 7 to 15 at the highest dose for 7 hours/day, malformations and fet al weight changes resulted in a NOAEL of 6.5 mg/(L * d) (the lowest dose tested). A second whole-body i nhalation developmental study in which rats were exposed on g estation days 7 to 17 for 23 hours/day, malformatio ns, increased fetal resorptions, and decreased numbers of live fetuses were observed, resulting in a NOAEL of 1.3 mg/(L * d).

Mice. In a study of reproductive effects, there was an in significant increase in morphological anomalies in spermatozoa in male mice at 1000 mg/(kg bw * d) after oral dosing for five weeks. A developmental whole body inhalation study in mice exposed on gestation days 6 to 15 for 7 hrs/day resulted in developmental eff ects including increased exencephaly and cleft palate, f ully resorbed fetuses, decreased numbers of live pu ps, and decreased body weights; this study resulted in a NO AEL of 1.3 mg/(L * d). Oral studies in mice resulted in various malformations at 4000 mg/(kg bw * d) (the LOAEL) and higher; no NOAELs could be estab lished from these studies.

Humans. No epidemiological studies in humans have been loca ted to demonstrate that there is a link between methanol exposure and an increased incidence of fet al malformations or developmental impairment.

Rodent data on reproductive and developmental toxic ity are relevant for humans despite the known diffe rences in methanol metabolism between rodents and humans. Ro dents are adequate models for human exposure to methanol at levels where formate does not accumulat e. However, blood methanol concentrations associat ed with serious teratogenic effects and reproductive toxici ty observed in the rodent studies are in the range of 1000 – 2000 mg methanol/L blood, associated with formate a ccumulation in humans, which is likely to result in metabolic acidosis and visual and clinical effects in humans. In humans, generally, transient central nervous system (CNS) effects appear above blood methanol le vels of 200 mg/L, ocular symptoms appear above 500 mg/L, and fatalities have often occurred in untreat ed patients with initial methanol levels in the ran ge of 1500- 2000 mg/L. Other effects (e.g., subtle, not yet def initive neurological effects observed in primates) may be exhibited at lower inhalation concentrations and lo wer methanol blood levels.

Environment

Methanol is a colorless, clear, highly flammable li quid with a mild alcoholic odor. Physicochemical p roperty values for methanol are: melting point –97.8 0 C, boiling point 65 0 C, density 0.79 g/m 3 , vapor pressure 128 hPa, and log Kow –0.74. Methanol is miscible with water at 20 ° C. It does not undergo hydrolysis. A value of 0.4 61 Pa m3/mol for the Henry’s Law constant indicates th at volatilization is not a significant removal proc ess from the aquatic compartment.

Air is the main target compartment, based on a fuga city model calculation (Mackay Level III) with abou t 73 % of environmental methanol distributing to air and 1 6 % to water. Methanol is degraded in the atmosphe re by photochemical, hydroxyl-radical dependent reactions . The estimated elimination half-life is calculate d to be about 17-18 days with a rate constant of 0.93 x 10 -2 cm 3 /molecule-sec. Methanol is readily biodegradable. Bioaccumulation in fish is expected to be low.

In several 96-hour studies in fish in which methano l concentrations were measured during the tests, LC 50s ranged from 15,400 to 29,400 mg/L. In Daphnia magna , 24-hour and 48-hour EC50s were > 10,000 mg/L

### OECD SIDS METHANOL

6 (highest dose tested). In one 24-hour study, the a ctual EC50 value reported in Daphnia magna was 21,4 00 mg/L. In aquatic plants, an EC50 for growth inhibi tion of Chlorella pyrenoidosa was calculated to be 28,440 mg/L over 10-14 days. No information on analytical monitoring was available for the invertebrate and plant studies. The low toxicity of methanol for aquatic organisms is confirmed by QSAR calculations, resulting in acu te toxicity values for fish, daphnids and algae well a bove 1000 mg/L.

Exposure

The world-production volume of methanol is approxim ately 30 million tonnes annually in 2000, about 11 million tonnes used in countries participating in N AFTA (North American Free Trade Agreement) and abou t 3.3 million tonnes in Western Europe, of which 70% is b eing used for the manufacture of formaldehyde, MTBE and acetic acid. Several additional compounds are synt hesized from methanol.

Methanol occurs naturally in humans, animals and pl ants. The general population is exposed to methanol mainly through consumption of food and beverages and throu gh use of consumer products such as paints, sealers and adhesives that contain methanol as a solvent. Worl dwide, air concentrations of methanol have been mea sured at ranges from 1.05 to 13.1 x 10 -5 mg/L.

Releases into the environment may occur from both n atural and man-made sources. It is however released predominantly from production and use in industrial processes. According to the U.S. Toxics Release I nventory, about 89,225 tonnes of methanol were released on- a nd offsite for facilities in all industries in the U.S. in 2000. The largest emitter was the pulp and paper industry . About 90 percent of the emissions were to the ai r and about 1.7 percent was released to surface water.

The most important routes of potential exposure are inhalation and dermal exposure at the workplace. A mean background body burden of 0.5 mg methanol/kg bw has been estimated based on baseline blood leve ls and elimination kinetics of methanol.



### RECOMMENDATION AND RATIONALE FOR THE RECOMMENDATION AND

NATURE OF FURTHER WORK RECOMMENDED

Human Health: This chemical is a candidate for further work. Me thanol exhibits potential hazardous properties for human health (neurological effects, CNS depress ion, ocular effects, reproductive and developmental effects, and other organ toxicity). Rapid metabolism and ex cretion is noted depending on the dose. In the US (the Integrated Risk Information System), further work i s being performed regarding the use and refinement of pharmacokinetic models for extrapolating animal dat a to humans.

Environment: The chemical is currently of low priority for furt her work, due to its low hazard profile.

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# SIDS Initial Assessment Report

1 IDENTITY

1.1 Identification of the Substance

CAS Number: 67-56-1 IUPAC Name: Methanol Molecular Formula: CH 4 

### O Structural Formula: H 3 

### C-OH Molecular Weight: 32.04 Synonyms: Methyl alcohol, Methyl hydroxide, Methylo l, Monohydroxymethane

1.2 Purity/Impurities/Additives

Purity: ≥ 99.85% (w/w)

Impurities: water < 0.1% (w/w)

chloride ≤ 0.00005% (w/w) sulphur ≤ 0.00005% (w/w)

iron ≤ 0.00001% (w/w)
ethanol ≤ 0.005% (w/w)

acetone ≤ 0.003% (w/w)

1.3 Physicochemical Properties

Table 1: Summary of Physicochemical Properties

Property Value Reference
Physical state liquid Merck Index, 1989; BASF, 1999 a Melting point approx.

− 98 °C Merck Index, 1989; BASF, 1999a
Boiling point 65 °C Merck Index, 1989; BASF, 1999a
Relative density 0.79 g/cm 3 (20 °C) Merck Index, 1989; BASF, 1999a Vapour pressure 127.9 hPa (20 °C) Daubert and Danne r, 1989
Water solubility Miscible (at 20 °C) BASF, 1999a

Partition coefficient noctanol/water (Log Kow) − 0.82 to

− 0.64 Hansch and Leo, 1979;

Rekker, 1977; Sangster, 1989
Henry’s law constant (25 °C) 0.461 Pa • m 3 /mol Gaffney, 1987

Flash point 10 °C (closed cup) BASF, 1972 Auto-ignition point 455 °C BASF, 1979



### Methanol is a colourless, clear, volatile, and high ly flammable liquid. When pure, it has a mild alcoholic odour. Methanol is miscible with water a t all ratios at ambient temperature, but also soluble in many organic solvents. Methanol is expe cted to have no explosive and oxidising

### OECD SIDS METHANOL

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### properties, as indicated by its chemical structure. Vapours may form explosive mixture with air (BASF, 1999b).



## 2 GENERAL INFORMATION ON EXPOSURE

The methanol production process involves the cataly tic steam reforming of a hydrocarbon feedstock, typically natural gas, to give a gaseous mixture of carbon oxides and hydrogen. This mixture is then compressed and reacted over another catalyst to give methanol and by-products as follows:

CO + 2H 2 

### <-> CH 3 

### OHCO 2 

### + 3 H 2 

### <-> CH 3 

### OH + H 2 

### O

The pure product is obtained by fractional distilla tion. All necessary steps are performed in closed systems. The details of this process are outlined in the following paragraphs.

The gas is reformed over a nickel catalyst. The syn thesis gas, containing a mixture of hydrogen, carbon oxides, steam and residual methane leaves th e reformer at about 880°C and 20 bar, and is cooled before being compressed to synthesis pressur e.
The synthesis loop comprises a circulator, converte r, feed/effluent exchanger, separator, and in some cases a heat recovery exchanger. The optimum o perating pressure is typically about 80 bar for large plants, but for smaller plants (less than 600 t/day) a pressure of 50 bar may be economical due to the reduced material requirements. Methanol con version is at a rate of 5% per pass hence there is a continual recycling of the unreacted gases in the synthesis loop.

Synthesis gas is mixed with the unreacted gas and p reheated in a feed/effluent interchanger. The converter effluent is cooled by passing through the feed/effluent interchanger and dependent on the reactor type, a heat recovery exchanger. Prior to the catchpot, the gas is cooled to about 40°C to condense the product methanol. A purge is taken fr om the unreacted gas to remove inerts (N2, CH4, argon, and surplus hydrogen or carbon oxides) from the loop. This purge is used as fuel in the reformer. A purge gas expander can be used to reco ver power from the purge gas as it is lowered to the fuel system pressure.

The crude methanol from the separator contains both water and low levels of by-products, which must be removed to achieve required product purity.

2.1 Production Volumes and Use Pattern

In the year 2000, the world-wide production volume was about 30 million tonnes annually (estimated capacity approximately 35 million tonnes ), which can be split as follows:

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### Table 2: Production Volumes of Methanol (2000)

Region Production Volume (in million tonnes)

North America (based on NAFTA 1 )Production approx. 7
South America (includes Trinidad) approx. 7
Western Europe approx. 3.3
Eastern Europe approx. 0.5
Former Soviet Union approx. 2
Africa and Middle East approx. 5
Asia/Pacific approx. 5

Total approx. 30

1 NAFTA = North American Free Trade Agreement



### Production of methanol is reported to occur at 98 p lants world-wide (Methanol Institute, 2003). Approximately 70 percent of the methanol produced w orldwide serves as feedstock for chemical syntheses, in particular formaldehyde, methyl tert- butyl ether (MTBE), acetic acid, methyl methacrylate, and dimethyl terephthalate. Only a s mall proportion is utilized for energy production, although this use has great potential. The use of p ure methanol as solvent is limited, although it is often included in solvent mixtures. (Ullmann, 2000) . The profile of main uses within NAFTA is summarized below:

Table 3: Main Uses of Methanol in the NAFTA Region

Use Percentage
MTBE/TAME * approx. 36%
Formaldehyde approx. 23%
Acetic Acid approx. 9%
Exports approx. 7%
Solvents approx. 6%
Methyl Methacrylate approx. 3%
DMT * approx. 2%
Gasoline/Fuel approx. 0.2%
Others approx. 14%

DMT = dimethyl terephthalate TAME = tert. Amylmethylether = 2-methoxy-2-methylbu tane MTBE = tert-butylmethyl ether



### MTBE demand is expected to decline in 2004 in the U SA because several states will discontinue its use as a gasoline additive (CMAI, 2001). Methanol is used to synthesize a large number of other organic compounds (see Table 3 “Others”). These co mpounds and their uses are identified in Table 4.

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### Table 4: Additional Compounds Synthesized from Met hanol and Accompanying Uses

Compound Uses

Formic acid Preservatives, pickling agents
Methyl esters of organic acids Solvents, monomers
Methyl esters of inorganic acids Methylation reagen ts, explosives, inorganic acids, insecticides
Methylamines Pharmaceutical precursors, auxiliaries, absorption liquids for gas washing and scrubbing
Trimethylphosphine Pharmaceuticals, vitamins, fragr ances, fine chemicals

Sodium methoxide Organic intermediate, catalyst
Methyl halides Organic intermediates, solvents, propellants
Ethylene Organic intermediates, polymers, auxiliaries



### Likely future uses of methanol include the synthesi s of light olefins [methanol-to-olefin (MTO) process], fuel for Otto engines and gas turbines of power stations, and as a fuel additive and hydrogen carrier for fuel cell applications (CMAI, 2001; Ullmann, 2000; Kavet and Nauss, 1990).

2.2 Environmental Exposure and Fate

2.2.1 Sources of Environmental Exposure

Methanol occurs naturally in humans, animals and pl ants. In addition, methanol is released into the environment from both natural and man-made sources, the latter being the most significant. Natural sources of methanol emissions include volcanic gass es, vegetation, microbes and insects (IPCS/WHO, 1997). Of the man-made sources, methano l is released predominantly from its production and use in industrial processes. Accord ing to the US Toxics Release Inventory, 89,225 tonnes methanol were released on- and offsite for f acilities in all industries in the U.S. in 2000. The largest emitter in 2000 was the pulp and paper industry. About 90% of emissions (79,658 tonnes) were released into the air, and 1.7 % (1531 tonnes) into surface water (U.S. EPA, 2001).

A minor source of environmental exposure may result from use of consumer products such as paints, sealers and adhesives that contain methanol as a solvent.

Urban air levels of methanol world wide in the range of 1.05 – 13.1 x 10 -5 

### mg/L have been reported. In Sweden, methanol was detected at concentrations of 59 – 940 10 -5 

### mg/L at dense traffic sites in Stockholm and 0.5 – 3.0 x 10 -5 

### mg/L at five other sites in and around Stockholm. In three US locations (Boston, MA; Houston, TX; and Lima, OH), median methanol levels of 0.6 – 6.0 x 10 -5 

### mg/L were found in 52 samples (US EPA, 1993). Meth anol was also detected at air concentrations of 0.06 – 0.18 x 10 -5 

### mg/L in Point Barrow, Alaska (IPCS/WHO, 1997).

2.2.2 Photodegradation

Methanol is degraded in the atmosphere by photochem ical, hydroxyl-radical dependent reactions, with a rate constant of 0.932 x 10 -12 

### cm3/molecule

. 

### sec (Atkinson, 1989). A half-life in the troposphere of about 17 – 18 days can be estimated for methanol

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### 2.2.3 Stability in Water

As an alcohol, methanol lacks hydrolysable groups a nd is chemically stable in water.

2.2.4 Transport between Environmental Compartments

With a Henry’s law constant of 0.461 Pa m3/mol, met hanol is not expected to significantly volatilize from the aquatic compartment (Gaffney et al., 1987)

. Methanol is the simplest of the alcohols, having on ly one carbon atom, and is completely miscible in water. The dominant mechanisms of methanol loss from subsurface soil and groundwater are expected to be biodegradation and advection (disper sion and diffusion), with little loss from adsorption on soils due to its high solubility as w ell its low octanol-water partition coefficient (Malcolm Pirnie, 1999).

A distribution calculation performed with the Macka y Level III fugacity model (BASF, 2003) and using emission rates derived from total releases in to water, air and soil (79658, 1531 and 658 tonnes, respectively) reported in the U.S. Toxics R elease Inventory for 2001 predicts that the air is the target environmental compartment for methanol ( see Table 5).

Table 5: Environmental Distribution of Methanol (F ugacity Model)

Environmental Compartment Emissions Calculated from TRI Releases (kg/hr) Resulting Mass

Amount (%)

Air 9093 73.3

Water 175 15.6

Soil 75 11.1

Sediment 0 0.0233



### 2.2.5 Biodegradation

Methanol is readily biodegradable based on the resu lts of standard tests that show 76 – 82 percent and 95 percent removal in standard ready tests afte r 5 and 20 days, respectively (Price et al., 1974; Wagner, 1976). Methanol occurs naturally in the en vironment due to various biological processes in vegetation, microorganisms and other living spec ies. It is well established in the scientific literature that methanol will biodegrade readily in the surface water, subsurface aquatic or soil systems under both aerobic and anaerobic conditions (Malcolm Pirnie, 1999).

2.2.6 Bioaccumulation

Experimental BCFs of < 10 in fish species, includin g Cyprinus carpio and Leuciscus idus

, have been measured (Hansch and Leo, 1985, Gluth et al., 1985, Freitag et al., 1985). . These results are expected because methanol has a high water solubili ty and a low octanol-water partition coefficient (log K ow 

### = - 0.82 to – 0.64) (Hansch and Leo, 1979; Rekker, 1977).

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### 2.3 Human Exposure

2.3.1 Occupational Exposure

The major anthropogenic sources of methanol include 1) its production, storage and use as a solvent, 2) its use as a chemical intermediate, and 3) as trace constituent of exhaust from vehicle engines (IPCS/WHO, 1997). See Section 2.1 for a mo re complete list of uses. During the period January 7, 1998 to November 12, 2003, a total of 95 0 measurements of methanol concentrations were taken at several workplaces at BASF AG in Ludw igshafen, Germany. All concentrations are time-weighted-average values over a reference perio d of 8 hours. The measured exposure is taken during different activities such as production, pro cessing, quality control, pilot plant, research laboratories, filling stations, and others. The 95 th 

### percentile value of the measured concentrations was 0.032 mg/L (32 mg/m3).

In the European Union, the occupational exposure li mit for methanol is 260 mg/m3 (200 ppm) as an 8 hour TWA. In the US, the limit is 200 ppm as an 8 hour TWA and 250 ppm as a STEL.

The National Occupation Exposure Survey (NOES) of 1 981-1983 estimated that about 1,700,000 employees were potentially exposed to methanol in t he US (IPCS/WHO, 1997).

2.3.2 Consumer Exposure

In humans, the average background level of methanol in blood was found to be 0.73 mg/L, and ranged from 0.32 – 2.61 mg/L (Sedivec et al., 1981) . Formate, the major metabolite, ranged from 3 to 19 mg/L (0.07 – 0.4 mM). A mean background body burden of 0.5 mg methanol/kg bw has been estimated from the data of Sedovic et al. (1981) (K avet and Nauss, 1990). Additional body burdens may arise from various everyday exposures.

Methanol occurs naturally in humans, animals and pl ants. The general population may be exposed to methanol via intake of fresh fruits, vegetables and commercial beverages like fruit juices, the artificial sweetener aspartame, beers, wines and di stilled spirits (IPCS/WHO, 1997).

Methanol is also contained as a solvent in products such as paints, sealers, and adhesives, which may be available to consumers and used in car care, hobbies, crafts and home maintenance (National Library of Medicine, 2003).

Additional sources of background body burdens may a rise from metabolic processes such as hydrolysis of various methyl esters, which may be c ontained in food and other consumer products. For instance, L-aspartyl-L-phenylalanine methyl est er (the artificial sweetener aspartame), hydrolyzes to form methanol.

In 2002, the American Association of Poison Control Centers found 1,049 incidences of exposure to methanol reported to poison control centers. This represents a significant reduction from the 2,517 methanol exposure cases reported in 1997. Approxim ately 85 percent of methanol exposures (882 in 2002) resulted from “unintentional misuse” (larg ely ingestion) that was unplanned or not foreseen by the patient. In 2002, over 120 of the methanol exposures were due to “intentional abuse” relating to suspected suicide attempts or ab use to achieve a euphoric or psychotropic effect. The reports for methanol represent only a very smal l fraction of the total “alcohol” exposures (69,215) reported in 2002, and are much fewer than the total ethanol exposures (66,898). In many cases, exposure to alcohols results from the ingest ion of illegal or “bootleg” alcoholic beverages where the patient has unintentionally consumed a be verage that has been “spiked” with elevated methanol or another alcohol (Watson et al., 2003).

### OECD SIDS METHANOL

13 

### As a hydrogen carrier fuel, methanol is likely to b e contained in cartridges that provide fuel to handheld fuel cell devices that power consumer elec tronic devices such as laptop computers and cellular phones. Stringent design and performance standards are now being developed for these methanol fuel cartridges (by the Underwriters Labor atory Standard Technical Panel 2265 and International Electrotechnical Commissions TC 105 W orking Group #8) to preclude direct consumer contact with the methanol fuel. Fuel cell -powered vehicles may also use methanol as a hydrogen carrier fuel. International standards for fueling systems for fuel cell vehicles (Society of Automotive Engineers Standard J2579) are being deve loped to minimize the risk of consumer exposure from methanol and other hydrogen fuels whe n refueling or operating fuel cell vehicles. A dry-connect, “spill-free” methanol fueling nozzle s ystem has been developed to prevent the consumer from coming into direct contact with metha nol during vehicle refueling.



## 3 HUMAN HEALTH HAZARDS

3.1 Effects on Human Health

3.1.1 Toxicokinetics, Metabolism and Distribution

Overview

Methanol is readily absorbed by inhalation, ingesti on and dermal contact, and partitions rapidly and equally throughout the organism in relation to the water content of organs and tissues, regardless of its exposure route. A small amount is excreted unc hanged by the lungs and kidneys. Half-lives of methanol in the body are roughly 2.5 to 3 hours at doses less than 100 mg/kg bw. At high doses (greater than about 1000 mg/kg bw), half-lives can be 24 hours or more (IPCS/WHO, 1997). As much as 90 percent of ingested low doses of methano l (e.g., 2 mg/kg bw) is exhaled as CO 2 

### in rodents and monkeys. Even after very high doses (1 000 mg/kg bw), up to almost 80 percent was recovered as CO 2 

### (Kavet and Nauss, 1990). However, there are prono unced differences in metabolism and biotransformation between rodents an d primates, especially at high (saturating) dose levels.

The mammalian metabolism of methanol proceeds as fo llows:

Step 1 Step 2 Step 3 Methanol  Formaldehyde  Formate  CO 2 

### + H 2 

### O

Step 1. In humans and monkeys, the initial reaction (from m ethanol to formaldehyde) is mediated by alcohol dehydrogenase (ADH). In rodents, the re action occurs mainly through a catalaseperoxidase system. The metabolic conversion to for maldehyde proceeds in both primates and rodents at a similar rate under non-saturated condi tions (Kavet and Nauss, 1990; Medinsky et al., 1997). However, in rodents, the catalase reaction is the limiting step, which means that at a critica l dose, methanol will accumulate and cause effects th at may not occur in humans. In humans, ADH levels are high enough so that accumulation of meth anol does not occur except at dose levels that are lethal or near-lethal.

Step 2. This step occurs as a two-reaction process. Formal dehyde is catalysed by the NADdependent formaldehyde dehydrogenase in the presenc e of glutathione to form the intermediate, Sformylglutathione. The intermediate is then hydrol ysed to formic acid by an associated thiolase.

Step 3. In this step, formate combines with tetrahydrofolic acid (THF) to form 10-formyl-THF through the action of formyl-THF synthetase. Next, 10-formyl-THF is converted to CO 2 

### by formylTHF dehydrogenase. In humans and monkeys, THF leve ls are only 60 percent of that in rat liver

### OECD SIDS METHANOL

14 

### and 16 – 26 percent of that in mouse liver. Also, the amount of formyl-THF dehydrogenase in human and monkey liver preparations is only about 2 5 and 37 percent, respectively, of that in rat liver (Johlin et al., 1987). Therefore, in humans and monkeys, the conversion of formate to CO 2 

### constitutes the limiting step, leading to a disprop ortionate increase of formate in the blood and in sensitive target tissues such as CNS and the retina

. Formate accumulation in primates has been observed at methanol doses greater than 500 mg/kg bw (Tephly and McMartin, 1984, as cited in NTP, 2003). Another study showed that formic acid in the blood of rats and monkeys was similar at doses up t o 600 mg/kg bw, but became substantially higher in monkeys at 3000 mg/kg bw (Katoh, 1989 as cited in NTP, 2003). However, after repeated inhalation of 2.6 mg/L (2000 ml/m 3 

### ) for 6 hrs/day, 5 days/week, for 1 or 2 weeks, mon keys (Rhesus monkey, Macaca mulatta) showed no methanol accumula tion or discernible increase in formate concentration in blood (Horton et al., 1992). The b ody burden can be estimated to be of the order of 200 to 300 mg/(kg bw * 

### d), i.e. below or in the range of the critical thre shold of saturation of the folate-dependent formate metabolism (see also below ). While this amount of methanol could overwhelm the formate metabolism pathway if given a s a bolus, the probability of saturating metabolism with this amount divided over a prolonge d time under inhalation exposure is unlikely in monkeys (Horton et al. 1992).

After a single exposure of 0.26 mg/L (200 ml/m 3 

### ), formate levels remained unchanged in humans, irrespective of physical activity (Lee et al., 1992 ); these findings were confirmed by D ́Alessandro et al. (1994) and Franzblau et al. (1993) in simila r experiments on human volunteers.

The conversion of formate to CO 2 

### is also affected by the endogenous folate pool whi ch, in monkeys is only about 25 – 50 percent of the amount in rats (McMartin et al., 1977). Also, studies show that formate oxidation in retinal tissue is 50 percent l ower in humans than in rats, and is again thought t o be related to lower folate levels in human retinas (about 14 percent of levels in rats) (Martinasevic et al., 1996). This difference in folate may also help explain the specific ocular and optic nerve toxicity of methanol in humans. Furthermore, this implies that humans with a poor folate status are supposed to be more susceptible to methanol action. On the other hand, in monkeys exposed repeatedly to 900 ml/m 3 

### (approx. 1.2 mg/L) methanol for 2.5 h, the increme ntal increase in blood formate was very low and remained significantly bel ow the endogenous formate levels under folatedeficient conditions (see Appendix: Dorman et al. ( 1994) and Medinsky et al. (1997)). The methanol dose that saturates the folate pathway in humans is estimated to be 210 mg/kg bw (Kavet and Nauss, 1990).

Table 6 compares the maximal in vivo conversion rates of the above key steps of methanol metabolism in rats and monkeys (Kavet and Nauss, 19 90; McMartin et al., 1977; Clay et al., 1975). In monkeys, the maximum elimination rate of formate was about 34 mg/(kg * 

### h), whereas in rats it was about 73 mg/(kg * 

### h) (McMartin et al., 1977). Elimination half-lives from blood were 30 – 50 minutes in the monkey and 12 – 23 minutes in rats, depending on the doses (Clay et al., 1975).

Table 6: Comparison of Reaction Rates in Rats and Monkeys

Methanol  Formate (Step 1, 2) Formate  CO 2 (Step 3)

Rat

~ 30 mg/(kg * h) * ~73 mg/(kg * h)

Monkey ~48 mg/(kg * h)

~ 34 mg/(kg * h) *

* Rate limitation of this reaction is evident from th e lower maximal conversion rate.

### OECD SIDS METHANOL

15 

### Species Comparisons among Humans, Other Primates, a nd Rodents
The species-specific kinetics and differences in th e metabolic transformation of methanol and formate in rodents and primates allow a better unde rstanding of the different effects encountered in those species and their relevance for extrapolation across species. They can be illustrated with data from inhalation exposures, which is a relevant expo sure route for humans (see Table 7).

For doses up to 1.5 mg/L (1200 ml/m 3 

### ) for 6 h/d, a similarity in blood methanol levels (including a linear relationship between air and blood concentra tions as well as formate levels) are predicted for rat, monkey and man. Furthermore, no increases in methanol blood levels above background occur in rats and monkeys at 0.065 mg/L (50 ml/m 3 

### ) (Horton, 1992) or even after continuous exposure of rats to 0.13 mg/L (100 ml/m 3 

### ) (NEDO 1987). However, above 1.5 mg/L, the increa se in blood methanol levels is non-linear for rats (resulting i n a steep exponential increase) and for monkeys (with a flat exponential increase), whereas it rema ins linear for humans (Horton et al., 1992).

Table 7: Kinetic and Blood Values after Inhalation in Rodents versus Primates

Rat 1) Mouse Monkey 1 Human 1

Exposure

conc. [mg/L] Half-life

[hrs] Methanol

(blood) [mg/L] (1x/d, 6 h) Methanol

(blood)
[mg/L] (1x/d, 6 h) Half-life

[hrs] Methanol (blood)

[mg/L] (1x/d, 6 h) Methanol (blood)

[mg/L] (1x/d, 6 h)

0.26 0.8 ± 0.2 3.1 ± 0.4 1.1 ± 0.2 3.9 ± 1.0 3-8 2

0.65-0.780 approx. 10 approx. 10 approx. 10

1.6 1.0 ± 0.1 26.6 ± 2.0 approx. 150 3 3.2 ± 1.2 37.6 ± 8.5 approx. 25

2.6 2.1 ± 0.1 79.7 ± 6.1 approx. 500 4 2.9 ± 0.6 64.4 ± 10.7 approx. 50

6.5 no data approx. 400 approx. 2000 4 no data approx. 240 approx. 140

1 Data taken from Horton et al. 1992, Fig. 8 and Tab. 2, unless otherwise indicated

2 From Horton et al. 1992 and other sources (see also above: 3.1.1 Inhalation)

3 Estimated from Perkins et al. 1995, Tab. 3

4 Mouse data are taken from Pollack and Brouwer 1996, Fig 36 

### The rate of methanol accumulation in mice is two- t o three-fold higher than in rats due to their more rapid breathing and higher absorption, even though mice have a two-fold higher elimination rate. These factors possibly contribute to the greater se nsitivity of mice to the teratogenic effects of methanol, which are mediated by the parent compound (Pollack and Brouwer, 1996; Perkins et al., 1995; 1996).

Additional metabolism data in animals and humans ar e discussed in Appendix A.

Conclusion

Methanol is readily absorbed by inhalation, ingesti on and dermal contact, and partitions rapidly and equally throughout the organism in relation to the water content of organs and tissues. A small amount is excreted unchanged by the lungs and kidne ys. Half-lives of methanol in the body are roughly 2.5 to 3 hours at doses less than 100 mg/kg bw.

At high doses disproportionate increases of the par ent compound in the blood are obtained in rodents, but not in humans. On the other hand, in h umans the metabolite formate accumulates at high doses. This important difference mirrors the d ifferent enzymes and enzyme capacities involved

### OECD SIDS METHANOL

16 

### in the oxidative pathway from methanol to carbon di oxide. Specifically, two different rate limiting processes have been identified: in rodents, high do ses (after inhalation of 2.5 – 3.3 mg/L) lead to th e saturation of catalase, resulting in the accumulati on of methanol whereas formate levels remain low, whereas in primates (especially humans), the parent compound is well oxidized and does not accumulate, but formate increases disproportionatel y.
From studies in humans and monkeys exposed to conce ntrations of 0.26 – 2.6 mg/L (administered for 6 to 8 hours), it can be concluded that methano l remains close to 50 mg/L in blood. At inhalation exposures of 2.6 mg/L, rats also exhibit methanol blood levels that are not much higher (at about 80 mg/L), whereas the level in mice was 4 00 mg/L. At a higher inhalation exposure (6.5 mg/L), humans show the lowest blood methanol level (at 140 mg/L), followed by monkeys, rats, and mice, with the level in mice being more than 10 times higher than humans. Formate accumulation in primates has been observed at metha nol doses greater than 500 mg/kg bw.

3.1.2 Acute Toxicity

Studies in Animals

Inhalation. LC 50 

### -values in SD rats of 67,000 ml/m 3 

### (6 hours) and 98,000 ml/m 3 

### (4 hours) were obtained corresponding to 87.5 and 128.2 mg/L, resp ectively (BASF, 1980a,b). Clinical signs of toxicity were aqueous secretion of eyes and nose, l abored breathing, staggering, apathy, and narcosis.

A similar range of toxicity values is reported for the mouse: LC 50 

### (2.25 h) = approx. 79 mg/L (Von Burg, 1994), narcotic doses = 40 and 55 mg/L after 2.2 and 7 h, respectively (Mashbitz et al., 1936; Lehmann and Flury, 1943).

In cats, LC 50 

### values after 6-hour exposures ranged from 26 to 48 mg/L (Von Burg, 1994; Witte, 1931). A shorter duration (4.5 hours) led to an LC 50 

### of 85.4 mg/L (Von Burg, 1994). Studies in Rhesus monkeys indicate lethal concentra tions (percent mortality not reported) at 13 mg/L (after18 hours) and 52 mg/L (after1–4 hours). Blindness associated with optic atrophy was reported. Eventual recovery from this lesion was o bserved (McCord, 1931).

Dermal According to an early report, all rats survived 35, 000 mg/kg bw after occlusive skin application of methanol, while deaths were reported at 45,000 mg/k g bw (Eulner and Gedicke, 1955). In rabbits, a dermal LD 50 

### of about 17,000 mg/kg bw was found (Carnegie-Mello n, 1981). In Rhesus monkeys, four daily doses of 400 mg/kg bw (total 1600 mg/kg bw) caused sickness within 24 hours, and eventually death. After four daily doses of 1000 mg/kg bw (total 4000 mg/kg bw), severe illness took place followed by death on the second day (McCord, 1931).

Oral In rats, mice, rabbits, and dogs, the LD 50 

### values after single oral administration range from about 5,600 to 14,400 mg/kg bw (NIOSH 1985; BASF, 1975; K imura et al., 1971; Smyth et al., 1941; IPCS/WHO, 1997; Munch et al. 1972; Gilger and Potts , 1955).

Rodents, rabbits and dogs suffer from ataxia, narco sis and coma after high methanol doses. However, these animals do not exhibit the acidosis and ophthalmologic changes typically seen in humans at high lethal and sub-lethal doses (Gilger and Potts, 1955).

### OECD SIDS METHANOL

17 

### In Rhesus monkeys, 1000 to 2000 mg/kg bw did not le ad to mortality, while animals receiving 3000 to 8000 mg/kg bw died within two days. Treated ani mals showed acidosis, and some of them exhibited semi-coma and ophthalmologic changes (Gil ger and Potts, 1955).

At higher doses, of those monkeys with severe intox ication, the retina and the optic papilla showed extended oedema, and the pupils were wide and non-r esponsive. Six of eight animals dosed with 6000 mg/kg bw exhibited cystic degeneration of the outer retinal granular layer, and in one animal there was evidence of significant demyelinisation o f the optic nerve. Histological lesions were seen in the putamen and nucleus caudatus in 3 of 8 anima ls. All of these effects were most pronounced at the high doses only after early compensation of acidosis using bicarbonate application, because these monkeys generally did not survive those high doses of methanol but after early treatment with bicarbonate (Potts, 1955; Potts et al., 1955).

Contrary to observations by Gilger and Potts (1955) , there was no evidence of marked acidosis in 12 Rhesus monkeys (28 applications) after sublethal doses up to 6000 mg/kg bw. Specifically, there was no hyperventilation, no increase in urina ry excretion of organic acids, or shift in serum bicarbonate. Blindness was seen in only one surviv ing monkey dosed with 9000 mg/kg bw; the effect was transient four days after exposure. The LD 50 

### was between 7000 and 9000 mg/kg bw (Cooper and Felig, 1961).

In a study of neurobehavioral effects, Male Long-Ev ans rats were gavaged with single methanol doses of 1000, 2000, and 3000 mg/kg bw representing about 10, 20, and 30 % of the LD 50. 

### Ten minutes after dosing the animals were subjected to a fixed wheel running test to assess operant running (FR20). The rats displayed no signs of ove rt intoxication such as gait disturbance, but did exhibit a significant, dose-related reduction in FR 20 response at all dose levels (Youssef et al., 1993). A NOAEL for behavioural changes could not b e derived.

Studies in Humans

Toxicity Syndrome

Oral ingestion dominates as the most frequent route of poisoning, but percutaneous absorption or inhalation of vapors are as effective as the oral r oute in producing methanol acute toxic syndrome.

After such acute poisoning, a blood level of ≥ 500 mg/L indicates treatment by hemodialysis is needed. This blood concentration can be achieved i n an adult person by ingestion of 316 mg (or 0.4 ml) methanol/kg bw (Kavet and Nauss, 1990; p. 26). Generally, transient central nervous system (CNS) effects appear above blood methanol levels of 200 mg/L, ocular symptoms appear above 500 mg/L, and fatalities have often occurred in untreat ed patients with initial methanol levels in the range of 1500-2000 mg/L. The minimal acute lethal dose to humans is in the range of 300 to 1,000 mg/kg bw (Roe, 1982; Kavet and Nauss, 1990; IPCS/WH O, 1997). In general, coma, seizures and prolonged acidosis were poor prognostic signs (Nara qi et al., 1979).

Acute methanol intoxication (including acidosis and visual effects) evolves in a well-defined pattern. First, a mild depression of the CNS occur s and is followed by an asymptomatic latent period commonly lasting 12 to 14 hours. Clinical s ymptoms include headache, dizziness, nausea, and vomiting, abdominal pain, and labored, periodic breathing (Kussmaul breathing) and may progress to coma and death from respiratory failure . Methanol exposure result in ocular effects ranging from mild photophobia, misty or blurred vis ion to markedly reduced visual acuity and total blindness. Severe visual disturbances have been re ported in workers who experienced methanol air levels of about 1.5 mg/L (1200 ml/m 3 

### ) or more.

### OECD SIDS METHANOL

18 

### Ophthalmologic examination reveals hyperaemia of th e optic disc, followed by the appearance of oedema projecting into the surrounding of the retin a from the optic disc, while the papilla itself is not oedematous. As post-mortem signs, focal necrose s have been observed in the basal ganglia in the brain and specifically the putamen, which parti cipates in the control of motor activities.

Survivors of severe methanol intoxication may conti nue suffering from residual lesions of the putamen and from visual and lingual impairment (Kav et and Nauss, 1990; Erlanson et al., 1965; Naraqi et al., 1979). The incidence of permanent o cular abnormalities was found to correlate with the incidence of metabolic acidosis and with the am ount of methanol consumed (Dethlefs and Naraqi, 1978).

Human susceptibility to methanol toxicity may be de creased by concurrent ingestion of ethanol, which slows the entrance of methanol into the metab olic pathway by competitive inhibition of the ADH enzyme. Another important determinant of suscep tibility is the hepatic folate status, which governs the rate of formate detoxification. In gene ral, there is considerable wide interindividual variability of the toxic dose (Kavet and Nauss, 199 0; IPCS/WHO, 1997). Also blood measurements in lethal cases revealed a high variability in form ate and methanol levels (Roe, 1955; Litovitz et al.

, 1988).

Human Exposure Studies

Neurophysiological effects were tested in 12 health y subjects when exposed for 4 hours to 0.026 mg/L (control) and 0.260 mg/L in an exposure chamber in a cross-over design. The EEG was recorded before (as a baseline reference) and at th e end of each exposure with the subject’s eyes closed and opened and during a choice reaction test (color word stress test) (Muttray et al., 2001). During subjects’ exposure to 0.26 mg/L, their score s for pre-narcotic and irritating symptoms were not different from controls. Under the open-eye co ndition and during the choice reaction test, no significant changes were found. Under the closed-e ye condition, theta-band and some delta-band brain waves were significantly less active at the e nd of exposure to 0.26 mg/L than in controls. These changes suggest a slight excitatory effect of 0.26 mg/L methanol. However, the effect was weak, as indicated by the scores of acute symptoms, which did not change.

Osterloh et al. (1996) and Chuwers et al. (1995) st udied neurobehavioral effects of methanol on 26 volunteers exposed to 0.26 mg/L methanol for 4 hour s. The variables assessed were color discrimination and contrast sensitivity, auditory e voked potentials, and neurobehavioral qualities. Overall visual, neurophysiological, and neurobehavi oral test outcomes were not significantly affected, with the exception of some between-subjec t variables. Specifically, slight effects on P300 amplitude (brain waves in response to sensory s timuli) and information processing and psychomotor skills (symbol digit testing) were appa rently noted. However, these effects cannot be adequately evaluated due to the low number of subje cts and high standard deviations for those parameters.

Conclusion
For most experimental animals, rats, mouse, rabbits , dogs, and monkeys, the lethal dose of methanol is high (greater than 2000 mg/kg bw after single oral administration). This also applies for inhalation. Lethal concentrations were found t o be much higher than 10 mg/L for rats, cats and monkeys (exposure range 4 – 7 h). This can be esti mated to also correspond to body doses of >>2000 mg/kg bw. After single dermal exposure, hig h lethal doses are reported in rats and rabbits, but somewhat lower for Rhesus monkeys. Doses betwe en 1000 and 2000 mg/kg bw proved to be lethal. CNS suppression progressing to a narcotic state and possible fatality is the dominant effect in non-primate animals. The acidosis and ophthalmo logic changes typical in humans do not occur in rodents or rabbits. Also, reports of acidosis a nd ophthalmologic changes in Rhesus monkeys are conflicting.

### OECD SIDS METHANOL

19 

### Generally, in humans, transient CNS effects appear above blood methanol levels of 200 mg/L. Ocular symptoms appear above 500 mg/L. The minimum lethal dose in humans has been shown to be 300 to 1000 mg/kg bw, lower than generally obser ved in experimental animals. Fatalities have occurred more commonly in untreated patients with initial methanol blood l evels in the range of 1500-2000 mg/L. In general, coma, seizures and pro longed acidosis were poor prognostic signs. However, exposures up to 0.26 mg methanol/L for 4 h ours have been shown to be without significant physiologic effects in human volunteers

.

3.1.3 Irritation

Skin Irritation

The irritation potential of an unspecified dose of undiluted methanol in rabbits was examined under occlusive conditions after exposure intervals of 1, 5, and 15 minutes and 20 hours. According to Draize scoring, no signs of skin irritation were ob served 24 hours and 8 days after treatment for any of the exposure time periods (BASF, 1975). Another source, however, found that rabbits exposed to 500 mg showed moderate skin irritation (NIOSH, 1 982, as cited in DGMK, 1982). However, because these data were from a secondary source, th ey could not be verified.

Eye Irritation

One hour after instillation of 0.05 ml undiluted me thanol into the eyes of two rabbits, slight erythemas and cornea opacity as well as moderate oe demas associated with secretion were observed. After 24 hours, the effects were assesse d as mild, and after 8 days the animals had no symptoms (BASF, 1975).

In six rabbits, mild to moderate conjunctivitis and edemas as well as mild iritis were produced after instillation of 0.1 ml undiluted methanol into the eyes. Average scores after 24, 48, and 72 hours were approximately 2 for conjunctivae and <1 for ot her effects. Primary irritation subsided after 72 hours, although redness of the conjunctivae persist ed at that time (Jacobs, 1990) 1



### . Exposure to an atmosphere saturated by methanol vap ours at 20 °C produced severe irritation of mucous membranes and milky corneal opacity in rats (time not specified) and eventually led to mortality with all animals dead after 8 h (BASF, 19 75).

Conclusion
Methanol exhibited no skin irritation in one study but moderate irritation in a study that could not be verified. Available studies show that it is a s light to moderate eye irritant (with some reversibility of effects). High concentration of m ethanol vapours may be irritating to mucous membranes. Based on the vapour pressure of about 1 30 hPa at 20 °C, the molecular weight of 32 g/mol and the molar volume of about 24 L/mol, it ca n be estimated that the saturation concentration

was ≥ 150 mg/L and, thus, clearly lethal.

3.1.4 Sensitisation

Studies in Animals

In one study conducted by BASF (1979a), a guinea pi g maximization bioassay gave no evidence of contact sensitisation in 10 female animals after in duction and challenge with a 50 % aqueous methanol solution (0.1 ml). In another BASF study using 24 female animals (two tests with 12

1 Information on effects 8 and 14 days later was not available.

### OECD SIDS METHANOL

20 

### animals each), 1 of 12 (test 1) and 2 of 12 (test 2 ) animals exhibited a slight skin response (score 1 ) after 24 and 48 hours (BASF, 1979a).

Conclusion

In summary, the low number (3 of 34) animals with a score of 1 show no notable sensitisation potential of methanol.

3.1.5 Repeated-Dose Toxicity

Studies in Animals
Several key studies are summarised in Table 8.

Table 8: Animal Studies on Prolonged Exposure to M ethanol (Inhalation, Oral)

Study Design Result Source (Reliability)

Chronic inhalation [IUCLID 5.4, No. 1] Macaca fascicularis monkey 0.013, 0.13, and 1.3 mg/L (10, 100, 1000 ml/m 3 ) for ~21 h/d, 7 d/wk, for 7, 19 and 29 months

8 animals per group, 2, 3 and 3 for indicated intervals, respectively

Recovery group at 1.3 mg/L (1000 ml/m 3 ): 1 + 6 months General: Runny nose at 1.3 mg/L. Crouched stance at 1.3 mg/L.

Brain: Increased hyperplastic responsiveness of the astroglia in some parts of the white substance in timerelated manner (all dose levels), not considered degenerative, absent in recovery groups. Slight degeneration of the inside nucleus of the thalamus was observed at 0.13 and 1.3 mg/L after 7 months or mor e. Slight increases of responsive stellate cells were observed at 0.13 and 1.3 mg/L. The authors conclude that th e tissues around the ventricules at the hypothalamus are very sensitive to methanol. No clear correlation o f the effects was observed with exposure level and durati on. No fibrosis or necrosis noted. Peripheral Nervous System: One monkey at 0.13 mg/L and 2 at 1.3 mg/L showed slight but clear changes i n peroneal nerves; the authors concluded that these e ffects show that methanol causes damage to peripheral nerv es. Visual system: Some degenerative effects observed on the optic nerve and corpus geniculatum laterale at 7 and 19 months (doses not noted). Slight degeneration of the optic nerve suspected at 29 months (1or 2 cases at each dose level). These effects were not interpreted as significant. Liver and kidney: Increase in fat granules with doseresponse from 0.013 to 0.13 mg/L. Some signs of fibrosis at 1.3 mg/L, which were considered borderl ine. In the kidney, Sudan positive granules appeared on the renal tubule epithelium at 0.13 and 1.3 mg/L. Some additional effects (hyalinisation of glomeruli) wer e observed at 1.3 mg/L. The authors concluded that t hese effects were possibly related to methanol exposure.

Cardiac: At 0.13 and 1.3 mg/L, some effects observed on the ECG, which were described by the authors as a slight myocardial disorder. Histologically, a sign ificant increase of Sudan positive granules was noted in th e 1.3- mg group without pathological manifestations (e.g. fibrosis) Lungs/trachea: In the lungs, fibrosis was seen in the interalveolar space. However, no dose respo nse NEDO, 1987 (4)

### OECD SIDS METHANOL

21 was observed, and the effect was also seen in the c ontrols. In the trachea, atrophy of the epithelium of the mu cous membrane and a decrease in goblet cells was seen in four cases (doses were not shown, although effects were not observed in controls). The effect was localized.

It is not clear whether the effects observed were r elated to methanol exposure.
LOAEL = 0.013 mg/L (The LOAEL was determined based on the qualitative discussion of effects.)

Chronic inhalation [IUCLID 5.4, No. 2] Fischer-344 rat (m; f) 0.013, 0.13, and 1.3 mg/L (10, 100, 1000 ml/m 3 ) for ~20 h/d, 7 d/wk, for 12 mo Examinations were comprehensive and included optic nerve, eye, reproductive
organs No significant clinical and histopathological effec ts. However, slight changes of body and organ weights w ere observed within a 5 percent limit in the high-dose groups only.

NOEL = 0.130 mg/L NEDO, 1987 (4)

Chronic inhalation [IUCLID 5.4, No. 3]

B6C3F1 mice (m; f) 0.013, 0.13, and 1.3 mg/L (10, 100, 1000 ml/m 3 ) for ~20 h/d, 7 d/wk, for 12 mo

Examinations were comprehensive and included optic nerve, eye, reproductive
organs Significance of clinical and histopathological effe cts unclear at highest dose, slight changes of body and organ weights within 5 % limit, slight increase in more severe (“moderate”) fatty degeneration

in livers of male mice, but considered incidental because of a high incidence of this lesi on in the control, too. No cases that would have to be grade d as “severe” were found.

NOEL = 0.13 mg/L NEDO, 1987 (4)

Subchronic oral [IUCLID 5.4, No. 4]

SD rat (m; f)
(30 animals/sex/dose) 100, 500, 2500 mg/(kg * d), gavage, 90 d, interim sacrifice: wk 6 (10 animals per sex and dose) Increased liver enzyme levels; reduced brain weight s

NOAEL = 500 mg/(kg * d) U.S. EPA, 1986 [Cited in U.S. EPA, 1995] (4)

Subacute inhalation

[ IUCLID 5.4, No. 5 ] SD rat (m; f) 0.66, 2.7, and 6.6 mg/L (500, 2000, 5000 ml/m 3 ) for –6 h/d, 5 d/wk over 4 weeks

35 tissues were examined No significant histopathological effects, but nasal irritation and ocular discharge were obse rved. Relative spleen weights were increased significantl y only at 2.7 mg/L in females.

NOAEL = 6.6 mg/L (for systemic toxicity) Andrews et al., 1987 (2)

Subacute inhalation [ IUCLID 5.4, No. 6 and No. 7]

Macaca fascicularis monkey 3.9, 6.5, 9.1, and 13 mg/L (3000, 5000, 7000, and 10000 ml/m 3 ) for up to 20 d, 21 h/d and including recovery animals 4 animals per control and exposure group were used. Blood levels at 3.9 and 6.5 mg/L:

- methanol: 80 and 5250 mg/L, respectively

- formate: 30 and 1210 mg/L, respectively Coma and lethality: At ≥ 9.1 mg/L ( ≤ 6 d);

Brain: At 6.5 mg/L (5 and 14 d) , necrosis in basal ganglions of cerebrum (2/3 animals) and cerebral oedema, recovery noted (2/2 animals). At 3.9 mg/L (at 20 d), hyperplasia and fibrosis around myelin sheets of basal ganglia and white substance with slight to moderate increase in the astroglia. NEDO, 1987 (4)

### OECD SIDS METHANOL

22 Visual system: At > 3.9 mg/L: incipient atrophy of optic nerve, reduction in myelinated fibers Liver and kidney: At 3.9 mg/L: mild fatty degeneration At 6.5 mg/L: fibrosis in liver, partly vacuolated, hyaline degeneration in kidneys
LOAEL = 3.9 mg/L (This study could not be critically reviewed because the report did not include a suffi cient amount of quantitative data, statistical results, o r histopathological information.)



### Studies in Humans

In male and female workers exposed to methanol from 0.3 to 7.8 years, highly exposed workers more often complained of blurred vision, headache a nd nasal irritation during or after work. None of the workers reported suffering from photophobia. No retinal changes were observed. Among three workers exposed to about 1.0 – 1.6 mg/L plus one worker exposed to 0.12 to 3.6 mg/L, two showed retarded pupil reflex and one exhibited mild mydriosis, but no permanent eye damage. Forgetfulness and skin sensitivity were other commo n complaints (Kawai et al., 1991; IPCS/WHO, 1997).

In a study published by NIOSH (Frederick et al., 19 84), teacher aides who worked at or near spirit duplicator machines (using a 99 percent methanol du plicator fluid) and duplicating machine operators experienced exposures between 0.48 and 4. 0 mg/L (mean 1.38 ±0.75 mg/L). A health survey based upon a self-administered symptom quest ionnaire suggested that symptoms relevant to methanol toxicity (headache, dizziness, and eye irr itation) were significantly increased among exposed staff as compared with a non-exposed teache r control group at the same institute (Frederick et al., 1984: see also IUCLID, 5.10, entry 21).

Additional studies also showed that headaches were associated with occupations that involve the operation of duplicating machines (NTP, 2003).

Conclusion

In a chronic exposure study in monkeys, some histol ogical findings were observed in the cerebral area after prolonged and almost continuous exposure to doses of up to 1.3 mg/L, although the effects were generally of a mild nature. Several a dditional effects, including dose-related liver effects, were observed in this study. Effects in other organs and systems were also observed. The neurological effects were assumed to be due to meth anol because several such effects were observed, neurological effects were also seen in th e subchronic monkey study, and not yet verifiable neurobehavioral effects were observed in a reproduc tive/developmental study in monkeys (see Section 3.1.7 – Burbacher et al., 1999). A LOAEL of 0.013 mg/L (continuous exposure) was chosen for the chronic study in monkeys. Although the statistical significance of the effects cannot be verified from the study report, the number of ef fects and systems affected indicate a relationship with methanol.

In a subacute 20-d study in monkeys

, 3.9 mg/L (3000 ml/m 3 

### ) was chosen as the LOAEL (subacute, continuous exposure) where neurotoxic lesions appea red to progress in monkeys (according to NEDO, 1987). This exposure concentration correlate d with methanol blood levels 80 mg/L and formate levels 30 mg/L.

In the NEDO (1987) studies of chronic exposure in r ats and mice, the effects are difficult to interpret from the available study and NOELs for co ntinuous exposure could be established at the mid-dose of 0.13 mg/L because adverse effects were explicitly not reported.

### OECD SIDS METHANOL

23 

### In a 28-day study, there was no evidence of adverse effects in rats exposed to methanol up to 6.5 mg/L (6 hours per day for 28 days), except local na sal irritation and increased relative spleen weights, which were observed only at the middle dos e (Andrews et al., 1987). The estimated blood level of methanol is expected to be about 250 mg/L under this condition, as described in Section 3.1.1, Tab. 6.

3.1.6 Mutagenicity

Studies in Animals

In vitro Studies
Methanol has been examined in numerous test systems including bacterial, mammalian and fungal test systems. Most studies failed to demonstrate m utagenic activity, with four exceptions: 1) in Salmonella tester strain TA 102, a questionably pos itive response was observed (DeFlora et al., 1984); 2) in a DNA damage and repair assay with E. coli WP strains (repair proficient and deficient types), the result was considered ambiguous (deFlor a et al. 1984b); 3) in a mouse-lymphoma assay, a significant increase in the mutation rate was rep orted at 7.9 mg/ml methanol (McGregor et al., 1985); and 4) in a test of mitotic chromosomal segr egation in Aspergillus nidulans , a positive result was observed (Crebelli et al., 1989). All other studies produce d consistently negative results (see Table 9).

Table 9: In Vitro 

### Genotoxicity Testing on Methanol

Bioassay Test system

(+/- S9) Test Concentrations Result Source (Reliability)

Bacterial Assays

Salmonella typhimurium TA 1535, 1537, 1538, 98, 100 (+/- S9) ≤ 2.5 mg/plate negative DeFlora, 1981 (2)

Salmonella typhimurium TA97, 102 (+/- S9) ≤ 7.5 mg/plate negative, in TA 102: ambiguous DeFlora et al., 1984a

(2)

Salmonella typhimurium TA 1535, 1537, 1538, 98 (+S9) ≤ 3.6 mg/plate negative Gocke et al., 1981 (2)

Salmonella typhimurium TA 1535, 1537, 1538, 98, 100 (+/- S9) 0.005 – 5 mg/plate negative Shimizu et al., 1985 (2)

Escherichia coli WP2uvrA (reverse mutation) (+/- S9) 0.005 – 5 mg/plate negative Shimizu et al., 1985 (2)

Escherichia coli WP67, WP2, CM871 (increased cytotoxicity in DNA-repair deficient strains as indicator of genotoxicity) 20 mg/microwell (~ 60 mg/mL) ambiguous DeFlora et al., 1984b (2)

Mammalian Cell Culture Systems

Micronucleus Chin. Hamster V79 (-S9) approx. 40 mg /ml negative Lasne et al., 1984 (2)

Micronucleus Syrian hamster embryo cells No data (b ased on prior range-finding) negative Fritzenschaf et al., 1993 (4)

Sister Chromatid Exchange (SCE) Chin. Hamster Ovary cell (CHO)

(-S9) ≤ 1 mg/ml, 1x/d for 8 days negative Obe and Ristow, 1977 (2)

Cell Transformation Mouse embryo fibroblasts

C3H/10T1/2Cl8 (+/-S9), +/-TPA 5 – 100 mg/ml negative Ragan and Boreiko, 1981 (2)

Cell Transformation Baby Syrian Hamster kidney cell s

BHK21 C13 (+/-S9) approx. 8 – 80 mg/ml negative Strobel and Greb, 1981 (2)

### OECD SIDS METHANOL

24 Gene Mutation Chin. Hamster V79, reversion to 8-

Aza, 6TG or Quabain resistance (+/-S9) approx. 16 – 63 mg/ml negative NEDO, 1987 (2)

Chromosomal Aberration Chin. Hamster lung cells (+/-S9) approx. 7 – 28 mg/ml negative NEDO, 1987 (2)

Mouse Lymphoma L5178Y (+S9) 7.9 mg/ml positive McGregor et al., 1985 (4)

Other

Chromosomal malsegregation Aspergillus nidulans diploid strain P1

(fungus) (-S9) 6 % (v/v) positive Crebelli et al., 1989 (2)

Gene mutation Yeast Schizosaccharomyces pombe

(ade 6 locus)(+/-S9) 5 % (v/v) negative Abbondandolo et al. (1980) (4)



### In vivo Studies
The majority of in vivo assays are negative for mutagenicity and clastogen icity. Some of these assays were conducted under specific stress conditi ons using folate-deficient mice (see: Am. Petrol. Inst., 1991). The positive findings resulted from t he same working group and represent two of ten cases (Chang et al., 1983; Pereira et al., 1982) (s ee Table 10).

Table 10: In Vivo 

### Genotoxicity Testing on Methanol

Bioassay Test system Test Doses Result Source

Chrom. Aberration, Micronucleus SCE Mouse, oral 1000 mg/kg bw positive Pereira et al., 1982 (4);

NTP, 2003

SCE C57Bl mouse,

inhal. 6 h/d, 5 d, in primary lung-cell culture 1.04, 5.20 mg/L (800, 4000 ml/m

3 ) negative Campbell et al., 1991

(2)

Micronucleus ICR mouse, single dose, gavage 1050, 2 110, 4210, 8410 mg/kg bw negative NEDO, 1987 (2)

Micronucleus NMRI mouse, single dose, ip. 1920, 320 0, 4480 mg/kg bw negative Gocke et al., 1981 (2)

Micronucleus CD-1 mouse, gavage during pregnancy

(6-10 gd): adult + fetus peripheral blood, folate-sufficient and folate-deficient conditions 2500 mg/(kg bw * d) negative Fu et al., 1996 (2)

Micronucleus Swiss Webster mouse, ip., 4 d, folatesufficient and folate-deficient conditions 300, 600, 1200, 2500 mg/(kg bw * d) negative Am. Petrol. Inst., 1991 (2)

Micronucleus C57Bl mouse,

inhal. 6 h/d, 5 d, in primary lung-cell culture and peripheral blood 1.04, 5.20 mg/L (800, 4000 ml/m

3 ) negative Campbell et al., 1991

(2)

Chromosomal Aberration C57Bl mouse,

inhal. 6 h/d, 5 d, in primary lung-cell culture 1.04, 5.20 mg/L (800, 4000 ml/m

3 ) negative Campbell et al., 1991

(2)

Chromosomal Aberration C57Bl mouse,

inhal. 6 h/d, 5 d, in spermatocytes of pachytene 1.04, 5.20 mg/L (800, 4000 ml/m

3 ) negative Campbell et al. ,1991

(2)

Chromosomal Aberration Mouse, intraperitoneal, total dose split in

3 daily fractions, bone marrow 75 - 300 mg/kg bw positive Chang et al., 1983 (4)

NTP, 2003

### OECD SIDS METHANOL

25 Point or structural mutation Male Drosophila melanogaster SLRL

assay (Sex-linked recessive lethal mutation) 1000 mM in oral feed negative Gocke et al., 1981 (2)



### Conclusion
The majority of in vitro and in vivo assays on methanol are negative for mutagenicity an d clastogenicity although a few of the in vitro and in vivo assays were positive. The positive or ambiguous in-vitro findings are all associated with high methanol concentrations from 0.8 to 6 % (v/v), but are not higher than those that proved to be negative in other tests. Thus, although most studies indicate that methanol does not have the po tential to be mutagenic, given positive results in some tests, the possibility of mutagenicity cannot be ruled out.. Carcinogenicity

Inhalation
Groups of male and female Fisher rats as well as B6 C3F1 mice (52 animals per sex and group) were exposed to 0.013, 0.13, and 1.3 mg/L (10, 100 and 1 000 ml/m 3 

### ) for 24 and 18 months, 20 and 19 hours per day, respectively. Clinical, ophthalmolo gical, clinical-chemical, hematological and pathological/histopathological examination were don e (NEDO, 1987).

No clinical signs of intoxication could be observed in treated rats and mice. No changes were evident in hematological and biochemical parameters in either species. Body weight was slightly, but not significantly, decreased between weeks 51 t hrough 72 in the highest female dose-group of rats (1.3 mg/L). No differences in survival were n oted in the treatment groups compared with the control group.

The following specific tumours appeared at a somewh at higher incidence in high-dose groups of both sexes in rats after week 79 and 104 without cl ear dose-response relationship (see IUCLID, 5.7, entry 1):

- papillary lung adenomas in males (6/52 vs. 1/52 in the control), but not in females;

- adrenal phaeochromocytomas in females (7/52 vs. 2/5 2), but not in males;

- 3/52 and 5/52 metastatic (transition) tumors in mal es and females, respectively, vs. 0/52 in the controls. The statistical analyses did not reveal significanc e. However, the Fisher ́s Exact Test was close to the limit of applicability for testing the data of the phaeochromocytomas. No increase of this tumour type was seen in male rats which showed a high spon taneous tumour rate (7/52 in the control vs. 4/51 in the high –dose group).

In mice, there were no appreciable differences from the control in either numbers of animals with tumors or in degree of malignancy observed.

There was no evidence of an increase in liver tumou rs in rats or in spontaneous liver tumour rate in mice.

After almost continuous inhalation exposure of monk eys, an increased diffuse hyperplastic responsiveness of the astroglia in some parts of th e cerebral white substance was noted at all dose levels of 0.013 mg/L (after 29 months), 0.13, and 1 .3 mg/L (after ≥ 7 months) without histological signs of degeneration. This effect was absent in r ecovery groups after 1 and 6 months (NEDO, 1987).

### OECD SIDS METHANOL

26 

### Conclusion
Overall, tumour frequencies showed no statistically significant differences between treatment groups and controls in either rats or mice. The bio logical significance of the non-statistically significant increase in the incidence of the phaeoc hromocytomas in the aged female high-dose rats is considered to be low. The methanol-related pro liferative effects on the astroglia cells observed in monkeys were of transient nature and not associa ted with cellular neoplastic transformation. The weight of evidence suggests that methanol is not ca rcinogenic.

3.1.8 Toxicity for Reproduction

Studies in Animals
Studies of Reproduction

Key studies in rats and monkeys that address reprod uctive performance (and any resulting developmental toxicity) are summarised in Table 11. The study by Burbacher et al. (1999) of reproductive and developmental toxicity in monkeys is also included here because the dosing schedule is similar to a one-generation reproductiv e toxicity study in rodents.

Table 11: Animal Studies on Reproduction and Devel opment

Study Design Results Source (Reliability)

Two-generation study

Inhalation SD rats (m, f) 0.013, 0.13, and 1.3 mg/L (10, 100, 1000 ml/m 3 ) at 19-20 h/d, 7 d/wk F0: 103-108 d (pre-mating, mating, gestation, lactation); F1: 61-62 d + 145-153 d; F2: 54-56 d Blood methanol levels: F1(9wks of age): 3–3.5 mg/L, 1–4.2 mg/L, and 53 (m ) – 100 (fem.) mg/L, respectively, vs. controls (baseline) 2–3 mg/L. Reproductive performance: No treatment-related alterations in F0 and F1 for all reproductive parameters. Development: . No pathological effects/no histological manifesta tion/no post-natal functional deficiencies (learning, behav iour). However, the following effects were observed: At 1.3 mg/L: Post-natal differentiation apparently influenced: “ descensus testis” 0.5 to 1 d earlier than in male control gro ups of F1- and F2-pups (statistically significant: level not defined). (Note: There is considerable variation also between the control groups of both generations). Absolute and relative brain weights of F1 and F2 we re significantly lower (both sexes) (affected cerebrum, cerebellum); sligh t but not significant shifts were observed in organ weights: thymus and p ituitary were lower, heart, lung, and liver were higher (no quantitative data on organ weights and statistical results included) . At 0.13 mg/L : “Descensus testis” in male F2-progeny about 0.5 d earlier than in male control F2 pups. Detailed data not spe cified and not addressed under “Discussion”.
NOAEL(devel) = 0.13 mg/L (NOAEL based on the absolute and rel ative decreased brain weights in F1 and F2 animals. This study, however, could not be critically reviewed because the report did n ot include a sufficient amount of quantitative data, statistical results, i n particular on organ weights, or histopathological information. NEDO, 1987;

Takeda/ Katoh, 1988 (4)

### OECD SIDS METHANOL

27 Reproductive and developmental toxicity

Inhalation Macaca fascicularis monkeys (adult f)
0.26, 0.78, and 2.34 mg/L (200, 600, 1800 ml/m 3
for 2.5 h/d before breeding (120 d), during breeding (70 d), and during pregnancy (about 165 d). 9-12 monkeys per group Mean blood methanol levels: ~5mg/L (1- to 2-fold), ~10 mg/L (3- to 4-fold), and 40 – 50 mg/L (13- to 16-fold), respectively

Blood formate levels: at baseline level at all exposures Maternal toxicity: none Reproductive performance (menstrual cycles, conception rate, and livebirth delivery rate): not affected, only observati on: dose-unrelated reduction of gestation length in all methanol-expos ed animals of about 6 to 8 d, within normal range of other monkey colonies. C-sections were performed in 2 monkeys at 0.26 mg/L, 2 at 0.78 mg/L , and 1 at 2.34 mg/L who experienced vaginal bleeding, probably due to d etachment of the placenta. An additional C-section was done in one monkey at 2.34 mg/L following 3 nights of unproductive labor. Developmental toxicity: Apparent behavioural effects: Delay in early sensorimotor development was observed in the Visual ly Directed Reaching Test: Ages at achievement of the test (fr om control to highest dose group) were: 24, 32, 43, and 41 days (males) and 34, 33, 28, and 40 days (females). Delay in visual recognition memor y (but no clear dose response was observed). Wasting syndrome: 2/7 female offspring showing growth retardation at 12 and 17 months in the 2.34 mg/L– exposure group. Signs of severe malnutrition and gastroenteritis noted at autopsy.

LOAEL (devel) = 0.26 mg/L Burbacher et al., 1999

Special study on spermatogenesis B6C3F1 mice (male): 10 animals, 4 mo. of age; 1000 mg/kg bw (1x/d, 5 d), oral. Preparation after 5 wk (after testicular spermatid maturation) Sperm morphology: No significant morphological abnormalities: In spermatozoa isolated from dissected cauda epidid ymis for light microscopy, slight, statistically insignificant inc rease in sperm abnormalities (mean factor about 1.7 above neg. con trol, with very high variance), In positive control group (cyclophosphamide, 100/kg ): some 6-fold increase in abnormalities.

NOAEL = 1000 mg/(kg bw * d) Ward et al., 1984 (2)



### Developmental Toxicity Studies
Methanol has been tested in numerous developmental toxicity studies using rat, mouse, rabbit and monkey. However, the number of classic development al investigations following oral administration is limited or poorly documented. Fr equently, single studies are designed to elucidate specific questions based upon previous experimental experience. The key studies are summarized in Table 12.

### OECD SIDS METHANOL

28 

### Table 12: Animal Studies on Pre-Natal Development Following Exposure to Methanol

Study Design Results Source (Reliability)

Developmental Toxicity/Teratogenicity
Developmental Toxicity

Inhalation SD rat, 6.5, 13, and 26 mg/L (5000, 10000, 20000 ml/m 3 )

at 7 h/d, gd 1-19 for the two lowest doses or gd 7- 15 for the highest dose Methanol blood levels: 1000 – 2170 mg/L, 1840 – 2240 mg/L, and 5250 – 8650 mg/L, resp. Maternal toxicity: slight, unsteady gait at the highest concentration Reproductive performance (corpera lutea, implantations, fetal lethality, resorptions): not significantly affected

. Fetal body weight: slight decrease at ≥ 13 mg/L (p<0.05)

Malformations : At 26 mg/L: V isceral defects : 7/15 litters with 15/96 malformed fetuses vs. 0/1 5 litters and 0/107 fetuses in the untreated control Skeletal defects : 14/15 litters with 72/92 malformed fetuses vs. 0/ 15 litters and 0/98 fetuses in the untreated control At 13 mg/L (not statistically significant):

Visceral malformations were 2/15 litters with 2/107 fetuses , and skeletal malformations were 2/15 with 2/115 fetuses

. At 6.5 mg/L (not statistically significant):

Visceral defects were 1/13 litters with 2/90 fetuse s, and skeletal defects were 1/13 with 1/90 fetuses.

NOAEL (maternal) = 13.0 mg/L

NOAEL (devel) = 6.5 mg/L Nelson et al., 1985 (2)

Developmental Toxicity

Inhalation

Crl:CD SD Rats 0.26, 1.3, and 6.5 mg/L (200, 1000, or 5000 ml/m 3 ) for 22.7 hr/d (avg), gd 7-17 36 animals/group Dams: 6.5 mg/L: reduced bodyweight gain; reduced food an d water intake; one died; one sacrificed in extremis. Prenatal Development: 6.5 mg/L: increased late resorptions, reduced no. live fetuses, decreased fetal weight, increased numbers of litters with fetuses that have malformations (ventricular s eptal defect), variations (thymus, vertebrae, ribs), delayed ossif ication. Postnatal Development: 6.5 mg/L: prolonged gestation, reduced post-implantation embryo survival, and number of li ve pups/litter, decreased survival rate on postnatal day 4. Brain, thyroid, thymus, and testes weights were decreased at 8 weeks of age, bu t not overall body weight. Developmental milestones not affected when parturition was taken into account. NOAEL (devel) = 1.3 mg/L NEDO, 1987 (4)

Developmental Toxicity Inhalation CD-1 mice
1.3, 2.6, 6.5, 9.75, 13, and 19.5 mg/L (1000, 2000, 5000, 7500, 10000 or 15000 ml/m 3 ) for 7 h/d, gd 6-15 Mean blood methanol levels: 100 mg/l, 540 mg/L, 1600 mg/L, 3200 mg/L, 4200 mg/L , and 7300 mg/L, with the background level at about 1.6 mg/L Maternal toxicity: none Fetal body weight: Statistically-significant decrease at ≥ 13 mg/L

Malformations: at 6.5 mg/L, increase in exencephaly and cleft pal ate (approx. 10 % fetuses vs. 0.3 % in the control)

Dose-related increase in extracervical ribs or ossi fication sites lateral to the 7 th cervical vertebra at ≥ 2.6 mg/L (p<0.01) (approx. 50 %/litter vs. 26 to 34 % in the controls and approx. 34 %/litter at 1.3 mg/L) Other Developmental Effects: Dose-related statistically significant increase in females with fully resorbed fetuses at 13 mg/L. Doserelated statistically significant decrease in numbe r of live pups/litter at Rogers et al., 1993 (2)

### OECD SIDS METHANOL

29 Study Design Results Source (Reliability)

> 9 .75 mg/L. NOAEL (devel) = 1.3 mg/L (based on cervical-rib defect)

Studies to Address Specific Questions
Developmental Toxicity
Oral CD-1 mice,

4000 (= 2000 x 2) mg/kg bw or 5000 (= 2500 x 2) mg/kg bw gd 7 only (Special test design) Stage-dependent methanol toxicity in organogenesis: gd 7 considered as most sensitive
Malformations in both dose-groups: dose-related, mainly skeletal defects of the skull and neural tube: - increase in cleft palate (at 19 and 14 % vs. 0 % in the control);



### - increase in

extracervical ribs to C7 vertebra: at either dose approx. 30 % of fetuses vs. 0 % in control;
- split, fused or incomplete vertebra C2 about 41% in the high dose fetuses vs. 7.7 in the control;
- 25 instead of 26 presacral vertebrae in 10 % vs. 2.4 % in control.
LOAEL = 4000 mg/kg bw Connelly and Rogers, 1997 (2)

Developmental Toxicity

Inhalation CD-1 mice, 6.5, 13, 19.5 mg/L (5000, 10000, 15000 ml/m

3 );

6 h/d on gd 6 – 15, 7 - 9, or

9 – 11 (Special test design) Stage-dependent methanol toxicity in organogenesis: - neural tube and ocular defects (significant duri ng gd 7-9); - hind-limb anomalies (significant during gd 9-11) ; -cleft palate, renal pelvic dilatation/hydronephro sis and tail anomalies (significant during both gd period 7-9 and 9-11), e vident at ≥ 13 mg/L -Increased resorptions at 19.5 mg/L for exposures that included gd 7, significant only at the top exposure level - Decreased body weight and decreased live fetuses /litter at 19.5 mg/L - At 6.5 mg/L, no teratogenic effects, but incipie nt increase in postimplantation losses (p<0.05) LOAEL (develop) = 6.5 mg/L Bolon et al., 1993 (2)

Developmental Toxicity Oral CD-1 mice, 5000 mg/(kg bw * d) [= 2500 mg/(kg bw*d), 2x/d] gd 6 – 10 (Study in combination with folate-deficiency and folate supplementation) Modulation of methanol toxicity by folate status: Methanol and folate-supplemented mice: 5000mg/(kg * d) methanol produced marginal teratogenic effect (exencephaly) under folate (5/222 vs. 1/282 cases in non-treated, folate-sufficient c ontrol). Folate deficiency only: no significant increase in malformations (cleft palate and exencephaly)

Methanol and folate deficient mice: significant increase in cleft palate (36/235 vs. 5/215 of folate-poor control), no signi ficant increase in exencephaly (8/235 vs. 2/215 of folate-poor contro l).
LOAEL = 5000 mg/(kg * d) Fu et al., 1996; Heid et al. 1992 (2)

Developmental Toxicity

Inhalation Long-Evans rat, 5.85 mg/L (4500 ml/m

3 ) for 6 h/d, gd 6 to post-natal day 21 (Pre- and postnatal study focused on neurotoxic effects) Mean blood methanol levels: in dams: 550 ± 70 and 560 ±90 mg/L (gestation and l actation); in neonates: 1260 ±230 mg/mL [i.e. neonates showed about twice the blood levels of dams persisting up to 4 weeks post- weaning (48 d of age)] Neurotoxicity: No detectable evidence of neuropathology (based on brain histomorphology, expression of neural cell-adhesion mole cules (NCAM) and behavioural testing). There was a weak transie nt effect on the molecular level: reduced NCAM formation, possibly a ssociated with treatment. Performance on operant tests suggested subtle cognitive effects.

NOAEL = 5.85 mg/L Weiss et al., 1996; Stern et al., 1996, 1997 (2)

### OECD SIDS METHANOL

30 

### Studies in Humans The limited data available on methanol exposure dur ing pregnancy in humans and reproductive and developmental effects do not show an association wi th methanol (NTP, 2003). In an epidemiological study, the reproductive effects of various occupations and associated exposures to complex mixtures were examined in women who gave bi rth to infants with and without cleft lip or palate (Lorente et al., 2000). No association was found between methanol exposure and oral clefts. The small number of subjects exposed to methanol, l ack of individual exposure data, and confounding by other chemical exposures did not all ow the authors to draw firm conclusions as to the role of methanol on these outcomes.

NTP (2003) did review several studies that investig ated the association between the periconceptional use of multivitamins containing fo lic acid and birth defects (e.g., neural tube defects and orafacial clefts). These studies sugge st that folate deficiency in humans may lead to greater susceptibility to such defects. Because th e association between methanol and these effects was not directly investigated, no conclusions can b e made regarding causality between methanol. And birth defects based on the results of human stu dies. However, these results should be considered along with the results of studies of inc reased developmental effects in folate-deficient rodents exposed to methanol. As noted by NTP (2003 ), the current evidence suggests that women with low folate status may be more susceptible to t he adverse developmental effects of methanol, but clearly more information is needed to clarify t his issue.

Conclusion
NTP (2003) concluded that rodent data on reproducti ve and developmental toxicity are relevant for humans despite the known differences in methanol me tabolism between rodents and humans. Rodents are adequate models for human exposure to m ethanol at levels where formate does not accumulate. However, blood methanol concentrations associated with serious teratogenic effects and reproductive toxicity are in the range associat ed with formate accumulation, which is likely to result in metabolic acidosis, and visual and clinic al effects in humans (NTP, 2003). Other effects (e.g., subtle, not yet definitive, neurological eff ects observed in primates) may be exhibited at lowe r inhalation doses and lower methanol blood levels.

Reproduction Studies in Animals

No impairment of fertility and reproductive perform ance was found in male and female rats (parent and daughter generation) exposed to continuously hi gh doses of methanol (NEDO, 1987). In male mice after repeated oral dosing at 1000 mg/ (kg bw * 

### d), increases in morphological anomalies in spermatozoa occurred but the increase was insignificant (Ward et al., 1984). The concomitant blood level can be estimated to be in t he range of >500 – 1000 mg/L in mice (see Appendix).

There was no clear evidence of malformations or var iations in the pre- and post-natal development of the progeny of two rat generations (NEDO, 1987). However, slight but significant retardation in brain growth (F1 and F2) led to the conclusion of t he NOAEL (20 h/d) being 0.13 mg/L. This corresponds to a blood level of 1–4 mg/L, which is not statistically different from the 0.013 mg/L group and the untreated control. There were also s ome statistically significant differences in time to decent of the testes, although the biological si gnificance of this difference is not clear because the differences were so slight. Due to experimenta l dose selection, the NOAEL happened to be very low compared with the LOAEL. Studies in monkeys (Burbacher et al., 1999) provide d some evidence of effects by prenatal methanol exposure on the neurobehavioral developmen t of nonhuman primate infants during the first 9 months of life. The interpretation was som ewhat limited by the normal variance and the low number of animals However, based on these neurobeh avioral results and the observance of

### OECD SIDS METHANOL

31 

### neurological effects in other studies, the LOAEL fo r developmental effects was determined to be 0.26 mg/L.

Developmental Toxicity Studies in Animals

High concentrations of methanol may cause specific malformations in organogenesis in pre-natal development of rodents. In the rat, the NOAEL (inh alation for 7 hours per day) was determined to be 6.5 mg/L, based on observance of malformations a s well as slight decreased body weights. This corresponds to a maternal methanol blood level of 1 000 to 2170 mg/L (Nelson et al., 1985).

In the mouse, there is unequivocal evidence of stru ctural malformations in the progeny at about 6.5 mg/L (inh. 7 h/d) and some evidence at 2.6 mg/L (inh. 7 h/d) (Rogers et al., 1993). The corresponding average blood levels of methanol were about 1600 and 550 mg/L. The NOAEL (inh.7 h/d) is suggested to be 1.3 mg/L (the corresponding blood methanol is about 100 mg/L ).

The marginally teratogenic oral dose is reported to be 5000 mg/(kg * 

### d) in mice (Fu et al., 1996). The estimate of the respective methanol blood conce ntration is of the order of >2000 mg/L (see Appendix).

Folate deficiency enhanced the teratogenic effects of methanol in mice, but this was not due to an increase in formate levels (Fu et al. 1996, see Tab le 12).

3.1.9 Specific Toxicity (Ocular Toxicity)

In monkeys ( Macaca mulata ), acute methanol toxicity (after a single dose) do es not yield signs of ocular toxicity (McMartin et al., 1975). On the ot her hand, repeated dosing succeeded in producing ocular lesions after a high initial oral dose of 20 00 mg/kg bw followed by lower doses for up to 6 days, depending on the animal’s acidotic response i n blood. The ranges of blood levels were 1000 – >2000 mg methanol/L and 350 – 550 mg formate/L (8 – 12 mEq/L) (Martin-Amat et al., 1977).

In a subsequent study in monkeys, sodium formate in fusion (without acidosis) produced blindness and marked optic disc oedema between 24 and 48 hour s, while the retina itself including the ganglion-cell layer was histologically completely n ormal. After 10 hours, all animals had accumulated formate in blood between about 10 to 30 mEq/L, while blood pHs had been maintained between 7.4 and 7.6 (Martin-Amat et al., 1978).

In a folate-deficient state, high oral doses of met hanol (1500 – 2500 mg/kg bw) induced changes in the electroretinogram (ERG) and the potassium-induc ed Müller cell depolarisation (KIMD) in rats, whereby formate in blood increased, with concentrat ions ranging from approx. 6 to 15 mM at 48 hours. No adverse ocular effects were noted at 100 0 mg methanol/kg per os despite folate deprivation (Lee et al., 1994; Garner et al., 1995a ). No adverse effects were produced by oral doses of up to 3500 mg/kg bw in rats maintained on normal diet (Garner et al., 1995b). The authors assume that intraretinal metabolism of methanol to formate is necessary for the initiation of methanol-induced retinal toxicity, based on negativ e results following formate infusion (Garner et al., 1995a).

Conclusion

Rats are relatively resistant to methanol-related e ffects on the visual organ in contrast to humans. However, monkeys may exhibit clear signs of ocular damage under selective test conditions in which formate (without associated acidosis) is the causative agent, and where effective formate blood levels were about 450 mg/L and higher ( ≥ 10 mM). Repeated dosing in monkeys may also lead to ocular effects.

### OECD SIDS METHANOL

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### 3.2 Initial Assessment for Human Health

The most prominent route of occupational exposure t o methanol is inhalation. In case of an expanded use of methanol in automotive fuels, inhal ation as well as dermal exposure would become increasingly important to the general population. Methanol is rapidly absorbed by all potential routes of exposure and readily distributed in the b ody.

3.2.1 Acute Toxicity, Skin and Eye Irritation, and Sensit isation

In humans methanol intoxication may lead to ocular lesions and potential lethality. The minimal lethal dose is considered to be 300 – 1,000 mg/kg b w. These effects are not mediated by the parent compound but by a rise in the formate blood levels up to about 5 - >10 mM, which is associated with acidosis. Acidosis may become fatal to the or ganism unless medical treatment is administered.

There is some evidence from studies in monkeys that the acidotic state does not substantially contribute to visual impairment (Martin-Amat et al. , 1978: see above 3.1.5: Oral Studies and Ocular Toxicity). Due to species differences in metabolis m, there is at present no animal model (including monkeys) to completely and satisfactorily reflect t he toxicity syndrome in humans. In fatal human cases after acute methanol intoxication, post-morte m signs of damage were observed in the basal ganglia in the brain, specifically the putamen (whi ch participates in the control of motor activities)

. Survivors of severe methanol intoxication may conti nue suffering from residual lesions of the putamen and visual and lingual impairment (see 3.1. 2: Toxicity Syndrome).

In rats, LC 50 

### values of 128.8 mg/L (after 4 hours) and 87.5 mg/L (after 6 hours) were calculated. In monkeys, lethal concentrations of 52 mg/L after 1-4 hours and 13 mg/L after 18 hours have been observed. A dermal LD 50 

### of 17000 mg/kg bw was calculated for rabbits; four daily dermal doses of 400 mg/kg bw eventually resulted in death. Oral LD 50 

### s range from 5600 to 14400 mg/kg bw in rats, mice, rabbits, and dogs. Monkeys that received ora l doses of 3000 to 8000 mg/kg bw died within two days.

Methanol exhibited no skin irritation in one study but moderate irritation in a study that could not be verified. Available studies show that it is a m ild to moderate eye irritant (with some reversibili ty of effects). High concentration of methanol vapour s may be irritating to mucous membranes. Results of a guinea pig maximization test show no n otable sensitisation potential of methanol.

3.2.2 Repeated-Dose Toxicity

In a chronic exposure study in monkeys, some histol ogical findings were observed in the cerebral area after prolonged exposure to doses of up to 1.3 mg/L, although the effects were generally of a mild nature. Several additional effects, including dose-related liver effects, were observed in this study. Effects in other organs and systems were a lso observed. The neurological effects were assumed to be due to methanol because several such effects were observed, neurological effects were also seen in the subacute monkey study, and no t yet verifiable neurobehavioral effects were observed in a reproductive/developmental study in m onkeys (see Section 3.1.7 – Burbacher et al., 1999). A LOAEL of 0.013 mg/L was chosen for the chronic co ntinuous-exposure study in monkeys. Although the statistical significance of the effects cannot be verified from the study report, the number of effects and systems affected indicate a relationship with methanol.

In a subacute 20-d study in monkeys

, 3.9 mg/L (3000 ml/m 3 

### ) was chosen as the LOAEL (subacute, continuous exposure) where neurotoxic lesions appea red to progress in monkeys (according to NEDO, 1987). This exposure concentration correlate d with methanol blood levels 80 mg/L and formate levels 30 mg/L.

### OECD SIDS METHANOL

33 

### In the NEDO (1987) studies of chronic exposure in r ats and mice, the effects are difficult to interpret from the available study, and NOELs (cont inuous exposure) could be established at the mid-dose of 0.13 mg/L, because adverse effects were explicitly not reported.

In a 28-day study, there was no evidence of adverse effects in rats exposed to methanol up to 6.5 mg/L (6 hours per day for 28 days), except local na sal irritation and increased relative spleen weights, which were observed only at the middle dos e (Andrews et al., 1987). The estimated blood level of methanol is expected to be about 250 mg/L under this condition, as described in Section 3.1.1, Tab. 6.

3.2.3 Mutagenicity and Carcinogenicity

The majority of in vitro and in vivo assays on methanol are negative for mutagenicity an d clastogenicity although a few of the in vitro and in vivo assays were positive or ambiguous. The positive in-vivo findings cannot be evaluated on th e limited data base available. Although most studies are negative for mutagenicity, the possibil ity that methanol can result in mutagenicity cannot be completely ruled out.

Overall, tumour frequencies showed no statistically significant differences between treatment groups and controls in either rats or mice. The bio logical significance of the non-statistically significant increase in the incidence of the phaeoc hromocytomas in the aged female high-dose rats is considered to be low. The methanol-related proli ferative effects on the astroglia cells observed in monkeys were of transient nature and not associated with cellular neoplastic transformation. The weight of evidence suggests that methanol is not ca rcinogenic.

3.2.4 Reproductive and Developmental Effects

No epidemiological studies in humans have been loca ted to demonstrate that there is a link between methanol exposure and an increased incidence of fet al malformations or developmental impairment.

In monkeys, inhalation exposure of parents prior to and during breeding as well as during pregnancy was associated with a late wasting syndro me and not verifiable, mild neurobehavioral effects in offspring as well as some vaginal bleedi ng and unproductive labor in mothers. These recordings resulted in the identification of 0.26 m g/L (the lowest dose tested), as a LOAEL. However, due to the normal variance in and the low number of animals, the observed findings are somewh at difficult to interpret.



### Several inhalation studies in rats resulted in a va riety of effects in offspring due to prenatal and/o r postnatal dosing. In a 2-generation reproductive s tudy, decreased brain weights resulted in a NOAEL(>19 h/d) of 0.13 mg/L. In another study on r ats, malformations and fetal weight changes resulted in a NOAEL(7 h/d) of 6.5 mg/L (the lowest dose tested). A third study resulted in malformations, increased fetal resorptions, and dec reased numbers of live fetuses, resulting in a NOAEL of 1.3 mg/L.

An inhalation study in mice resulted in development al effects including increased exencephaly and cleft palate, fully resorbed fetuses, and decreased numbers of live pups; this study resulted in a NOAEL(7 h/d) of 1.3 mg/L. Oral studies in mice res ulted in various malformations at 4000 mg/kg bw and higher; no NOAELs could be established from these studies.

NTP (2003) concluded that rodent data on reproducti ve and developmental toxicity are relevant for humans despite the known differences in methanol me tabolism between rodents and humans. Rodents are adequate models for human exposure to m ethanol at levels where formate does not accumulate. However, blood methanol concentrations associated with serious teratogenic effects

### OECD SIDS METHANOL

34 

### and reproductive toxicity are in the range associat ed with formate accumulation, which is likely to result in metabolic acidosis, and visual and clinic al effects in humans (NTP, 2003). Other effects (e.g., subtle, not yet definitive neurological effe cts observed in primates) may be exhibited at lower inhalation doses and lower methanol blood levels.

3.2.5 Ocular Toxicity

Rats are more or less resistant to methanol-related effects on the visual organ in contrast to humans. However, monkeys may exhibit clear signs of ocular damage under selective test conditions in which formate (without associated acidosis) is the causative agent, and where effective formate blood levels were about 450 mg/L and higher ( ≥ 10 mM). Repeated dosing in monkeys may also lead to ocular effects.



## 4 HAZARDS TO THE ENVIRONMENT

4.1 Aquatic Effects

Acute Toxicity Test Results

Several data on acute toxicity of methanol to fish, invertebrates, and algae are available. The result s of the most relevant studies are shown in Table 13 and indicate a low toxicity for aquatic organisms Although not all the studies have been performed in sealed vessels and with analytical control of the exposure concentrations the low value for the Henry ’s Law constant indicates that volatilisation is not a significant removal process from the aquatic phase. The low acute toxicity of methanol for aquatic organisms is confirmed by QSAR calculations , resulting in acute toxicity values for fish, daphnids and algae well above 1000 mg/l.

### OECD SIDS METHANOL

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### Table 13: Short-term Toxicity of Methanol in Aquat ic Organisms (Key Studies )

Organism Test type Species Value Rel. code Reference

Fish flow through

flow through

flow through Lepomis

macrochirus

Salmo gairdneri

Pimephales promelas LC 50 (96 h) = 15,400 mg/L

(measur.)

LC 50 (96 h) = 20,100 mg/L (measur.)

LC 50 (96 h) = 28,100 mg/L (measur.) 1

1

1 Poirer et al., 1986

Call et al., 1983

Call et al., 1983

Invertebrates static Daphnia

magna EC 50

(48 h) > 10,000 mg/L (nom.) 2 Kuehn et al., 1989

Algae static

static Scenedesmus

quadricauda

Chlorella pyrenoidosa Toxic Threshold (192 h) =

8,000 mg/L (nom.)

EC 50 (10-14 d) = 28,400 mg/L (nom.) 2

2 Bringmann and Kuehn, 1978

Stratton and Smith, 1988

Bacteria static Pseudomonas putida Toxic Threshold (16 h) =

6,600 mg/L (nom.) 2 Bringmann and Kuehn, 1977



### Acute Toxicity to Fish

In continuous flow-through systems mean LC50(96 h) values for freshwater fish were 15,400 mg/L ( Lepomis macrochirus ) (Poirer, 1986), 20,100 mg/L ( Salmo gairdneri ) and 28,100 mg/L ( Pimephales promelas ) (Call, 1983). These measured effect concentration s are in very good agreement with a LC50(96 h) of 10,630 mg/L estimated with the QSAR Program ECOSAR (v0.99).

Acute Toxicity to Invertebrates

In a reliable standard static test with Daphnia magna no adverse effects were reported at 10,000 mg/L after 48-h exposure (Kuehn, 1989). A QSAR calc ulation performed with ECOSAR (v0.99) indicates a LC50 (48 h) for daphnids of 9375 mg/L.

Acute Toxicity to Algae

The toxicity threshold of methanol for the alga Scenedesmus quadricauda after an exposure of 8 days was reported to be at 8,000 mg/L (Bringmann an d Kuehn, 1978). The average concentration causing 50% inhibition in growth rate was found to be at 28,400 mg/L during an exposure period of 10 – 14 days (Stratton, 1988). A QSAR calculation c onducted with ECOSAR (v0.99) estimates a EC50 (96 h) of 4,982 mg/L for green algae.

Chronic Toxicity Test Results

No experimental data are available.

### OECD SIDS METHANOL

36 

### Toxicity to Microorganisms

The lowest toxic limit concentrations have been rep orted to be at 6600 mg/L in a cell multiplication test for 16 hours using two Pseudomonas species (according to German Standard Procedures DE V L8) (Bringmann, 1973; Bringmann and Kuehn, 1977).

4.2 Terrestrial Effects

No relevant data are available.

4.3 Other Environmental Effects

No data are available.

4.4 Initial Assessment for the Environment
Air is the main target compartment for methanol whe n the distribution is estimated using the releases to different environmental compartments as reported to the Toxics Release Inventory. However, a certain portion of methanol will distrib ute to the water compartment. Methanol is readily degradable by photo-oxidation and biologica lly under aerobic and anaerobic conditions. It is of low toxicity to aquatic organisms and does no t bioaccumulate in appreciable amounts in fish. Based on the weight of evidence of the available da ta, methanol is not considered harmful to the environment under normal circumstances.



## 5 RECOMMENDATIONS

For human health, the chemical is currently a a can didate for further work. Methanol exhibits potential hazardous properties for human health (ne urological effects, CNS depression, ocular effects, reproductive and developmental effects, an d other organ toxicity). Rapid metabolism and excretion is noted. In the US (the Integrated Risk Information System), further work is being performed regarding the use and refinement of pharm acokinetic models for extrapolating animal data to humans..

For the environment, the chemical is currently of l ow priority for further work due to its low hazard profile.

### OECD SIDS METHANOL

37 

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Lee, E.W., Garner, C.D. and Terzo, T.S. (1994). A r at model manifesting methanol-induced visual dysfunction suitable for both acute and long-term e xposure studies. Toxicol. Appl. Pharmacol., 128, 199-206

Lehmann K.B. and Flury F. (1943). Toxicology and hy giene of industrial solvents., Williams und Wilkins, Baltimore (cited in: DGMK 1982)

Leon, A.S., Hunninghake, D.B., Bell, C., Rassin, D. K., Tephly, T.R. (1989). Safety of long-term large doses of aspartame. Arch Intern Med. 149: 2318-24 [cited in NTP, 2003]

Litovitz T. L., et al. (1988). Annual Report of the American Association of Poison Control Centers National Data Collection System. Am. J. Emerg. Med. , 6, 479-515

Lorente C. et al. (2000). Maternal occupational ris k factors for oral clefts. Scan. J. Work Environ. Health, 26, 137-145.

Lyman,W.J. et al. (1982). Handbook of chemical prop erty estimation methods, McGraw-Hill, New York, 5-4

Makar, A.B. et al. (1968). Methanol poisoning in mo nkey. Mol. Pharmacol., 4, 471 [cited in Roe, 1982]

Malcolm Pirnie, Inc. (1999). Evaluation of the fat e and transport of methanol in the environment, prepared for the American Methanol Institute, Janua ry.
Martin-Amat, G., Tephly, T.R., McMartin, K.E. et al . (1977). Methyl alcohol poisoning II. Development of a model for ocular toxicity in methy l alcohol poisoning using the Rhesus monkey. Arch. Ophthalmol., 95, 1847-1850

### OECD SIDS METHANOL

42 

### Martin-Amat, G., McMartin, K.E., Hayreh, S.S., Hayr eh, M.S. and Tephly, T.R. (1978). Methanol poisoning: Ocular toxicity produced by formate. Tox icol. Appl. Pharmacol., 45, 201-208.

Martinasevic, M.K., Green, M.D., Baron, J. and Teph ly, T.R. (1996). Folate and 10- formyltetrahydrofolate dehydrogenase in humans and rat retina: Relation to methanol toxicity. Toxicol. Appl. Pharmacol., 141, 373-381.

Mashbitz N.L. et al. (1936). J. Industr. Hyg. Toxi col., 18, 117 (cited in: DGMK 1982)

McCord, C.P. (1931). Toxicity of methyl alcohol (m ethanol) following skin absorption and inhalation. Industrial and Engineering Chemistry, 2 3, 931-936.

McGregor, D.B., Martin, R., Riach, C.G. and Caspar y, W.J. (1985). Optimisation of a metabolic activation system for use in the mouse lymphoma L51 78Y tk+tk- mutation system. Environ. Mutagen., 7(Suppl. 3), 10 (abstr.).

McMartin, K.E., Makar, A.B., Martin G., Palese, A. and Tephly, T.R. (1975). Methanol poisoning I. The role of formic acid in the development of me tabolic acidosis in the monkey and the reversal by 4-methylpyrazol. Biochem. Med., 13, 319-333.

McMartin, K.E., Martin-Amat, G., Makar, A.B., and T ephly, T.R. (1977). Methanol poisoning V. Role of formate metabolism in the monkey. J. Pharma col. Exp. Ther., 201, 564-572.

Medinsky, M., Dorman, D.C., Bond, J.A., Moss, O.R., Janszen, D.B. and Everitt, J.I. (1997). Pharmacokinetics of methanol and formate in female Cynomolgus monkeys exposed to methanol vapors. Health Effects Institute, Res. Report No. 7 7

Medinsky, M.A. and Dorman, D.C. (1995). Recent deve lopment in methanol toxicity. Toxicology Lett., 82/83, 707-711

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Muttray A. (2001). Int. Arch. Occup. Environ. Heal th, 74, 43-48

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NEDO (1987). Toxicological Research of Methanol as a Fuel for Power Station, Summary Report on Tests with Monkeys, Rats and Mice. New Energy De velopment Organization, TokyoNelson, B.K. and Brightwell, W.S. (1985). Teratological ass essment of methanol and ethanol at high inhalation levels in rats. Fund. Appl. Toxicol., 5, 727-736NIOSH (1985). Dangerous Properties of Industrial Materials Report

NIOSH (1982). Registry of Toxic Effects of Chemical Substances, Edition July 1982 [cited in: DGMK 1982]

NTP (2003). NTP-CERHR Monograph on the Potential Hu man Reproductive and Developmental Effects of Methanol. NIH Publication No. 03-4478. September, 2003

### OECD SIDS METHANOL

43 

### Obe, G. and Ristow, H. (1977). Chromosomal damage b y alcohol in vitro and in vivo. Mutat. Res., 56, 211-213Osterloh, J.D. et al. (1996). Serum conc entrations of methanol after inhalation at 200 ppm. J. Occup. Environ. Med., 38, 571-576 [cited in Bouchard et al., 1998]

Pereira, M.A., Chang, L.W., McMillan, L., Ward, J.B ., Legator, M.S. (1982). Battery of short-term tests in laboratory animals to corroborate the dete ction of human population exposures to genotoxic chemicals. Environ. Mutagen., 4, 317 (abstr.)

Perkins, A.P., Ward, K.W. and Pollack, G.M. (1996). Ventilation of ambulatory rats exposed to methanol vapor in flow-through exposure system: mea surement and input to toxicokinetic models. Inhal. Toxicol., 8, 143-162

Perkins, R.A., Ward, K.W. and Pollack, G.M. (1995). Comparative toxicokinetics of inhaled methanol in the female CD-1 mouse and Sprague-Dawle y rat. Fund. Appl. Toxicol. 28, 245-254

Poirer S.H., Knuth, L.M., Anderson-Buchou, C.D. et al. (1986). Comparative toxicity of methanol and N,N-dimethylformamide to freshwater fish. Bull. Environ. Contam. Toxicol., 37, 615-621

Pollack, G.M. and Brouwer, K.L.R (1996). Maternal-f etal pharmacokinetics of methanol. Health Effects Institute (HEI), Res. Report No. 74

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Ragan, D.L. and Boreiko, C.J. (1981). Initiation of C3H/10T1/2 cell transformation by formaldhyde. Cancer Letters, 13, 325-331.Rekker, R .F. (1977). The Hydrophobic Fragmental Constant, Elsevier Scientific Publishing Company,Am sterdam

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Sedivec, V. et al. (1981). Bioogical monitoring of persons exposed to methanol vapours. Int. Arch. Occup. Environ. Health, 48, 257-271

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### OECD SIDS METHANOL

44 

### Stegink, L.D., Brummel, M.C., McMartin, K.E. et al. (1981). Blood methanol concentrations in normal adult subjects administered abuse doses of a spartame. J. Toxicol. Environ. Health., 7, 281- 290 [cited in NTP, 2003]

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Stern, S., Reuhl, K., Soderblom, S. et al. (1996). Perinatal methanol exposure in the rat I. Blood methanol concentration and neural cell adhesion mol ecules. Fund. Appl. Toxicol., 34, 36-46

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Stratton, G.W., Smith, T.M. (1988). Interaction of organic solvents with the green alga Chlorella pyrenoidosa, Bull. Environ. Contam. Toxicol., 40(5) , 736-742.

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Weiss, B. , Stern, S., Soderblom, S.C. et al. (1996 ). Developmental neurotoxicity of methanol exposure by inhalation in rats. Health Effects Inst itute (HEI), Res. Report No. 73.

Witte R. (1931). Dissertation Wuerzburg [cited in: DGMK, 1982]

Yasugi, T. et al. (1992). Formic acid excretion in comparison with methanol excretion in urine of workers occupationally exposed to methanol. Int. Ar ch. Occup. Environ. Health, 64, 329-337 [cited in DFG, 1999].

### OECD SIDS METHANOL

45 

### Youssef, A.F., Weiss, B., Cox, C. (1993). Neurobeha vioral toxicity of methanol reflected by operant running. Neurotoxicol Teratol.,15, 223-227 (cited in NTP (2003)

### OECD SIDS METHANOL

46 

### APPENDIX A: TOXICOKINETIC STUDIES IN ANIMALS AND H UMANS

Studies in Animals

In Vivo Studies

Oral. After a single oral exposure to rats and mice, abso rption was rapid and nearly complete. In rats, single doses of 100 and 2500 mg methanol/kg l ed to maximum blood concentrations of 100 and 2000 mg methanol/L, respectively. In mice, 250 0 mg/kg bw caused a maximum blood concentration of 2500 mg/L (Pollack and Brouwer, 19 96).

Inhalation

. In monkeys, m 

### ethanol air concentrations of 0.26, 0.78, and 2.24 mg/L (200, 600, 1800 ml/m 3 

### ) resulted in blood concentrations of 5 mg/L (1- to 2-fold), 10 mg/L (3- to 4-fold), and 40 – 50 mg/L (13- to 16-fold above baseline), respectively, after single or repeated exposure for 2.5 hours/day (Burbacher et al., 1999). After repeated inhalation of 2.6 mg/L (2000 ml/m 3 

### 6 hrs/day; 5 days/week; 1 or 2 weeks) monkeys showed no methanol or formate accumulation (Horton et al., 1992).

In rats, three consecutive 6-hour exposures of abou t 5.85 mg/L (4500 ml/m 3 

### ) resulted in higher initial levels of methanol up to 950 mg/L, which th en levelled off to 600 mg/L in blood (Stern et al., 1996).

The mean fractional respiratory absorption of metha nol in rats and mice was 85 percent at air concentrations from 1.3 to 6.5 mg/L (1000 to 5000 m l/m 3 

### ). At higher concentrations (13– 26 mg/L), absorption tended to be lower in rats (60 – 70 percent), but not in mice (Pollack and Brouwer, 1996; Perkins et al., 1995; 1996). The lo wer absorption efficiency from the upper respiratory tract in rats was mainly attributed to a lower breathing rate. The rate of methanol accumulation in mice is two- to three-fold higher t han in rats due to their more rapid breathing and higher absorption, even though mice have a two-fold higher elimination rate. These factors possibly contribute to the greater sensitivity of m ice to the teratogenic effects of methanol, which are mediated by the parent compound (Pollack and Br ouwer, 1996).

Dorman et al. (1994) and Medinsky et al. (1997) inv estigated the influence of methanol on the endogenous formate pool in monkeys under folate-suf ficient (FS) and folate-deficient (FD) dietary conditions. Radiolabelled methanol was measured du ring exposure of female Cynomolgus monkeys ( Macaca fascicularis ) to 0.013, 0.059, 0.260, and 1.2 mg/L (10, 45, 200 , and 900 ml/m 3 

### ) (FS) and 1.2 mg/L (900 ml/m 3 

### ) (FD) for two hours per session. All doses except the highest might be encountered during use of methanol as an automot ive fuel. Reported blood concentrations of methanol 2 hours after the beginning of exposure we re 0.65, 96, 672, and 3,392 mg/L for the FS exposures, and 211 ±71 μM (6.752 μg/L) for the FD e xposure. The corresponding formate levels were 0.07 ±0.02, 0.25 ±0.09, 2.3 ±2.9, and 2.8 ± 1. 7 μM (FS exposures) and 9.5 ± 4.7 μM (FD exposure). Although there was an indication of hig her formate levels under folate deficiency, these increments were far below the endogenous blood form ate level of 100–500 μM.

Other Routes. Intraperitoneal injection of doses of methanol did not lead to formate accumulation in Cynomolgus monkeys ( Macaca fascicularis ) except at a high dose of 3000 mg/kg bw, which is associated with acidosis and lethality. No abnorma lity was seen in the 25- to 600-mg/kg dose groups (NEDO, 1987).

In rats after intravenous administration 97 percent of the clearance of methanol occurred by oxidative metabolism, whereas renal and pulmonary e xcretion contributed only to 0.6 and 2.5 percent, respectively (Horton et al., 1992).

### OECD SIDS METHANOL

47 

### Studies in Humans

In Vivo Studies

Oral - Methanol. Volunteers receiving methanol in amounts of 0.2 ml/ h for 6 hours or 0.5 ml/h for 8 hours, which correspond to total doses per person of 950 mg (14 mg/kg bw) and 2600 mg (40 mg/kg bw), exhibited urinary methanol concentration s of below 8 and 18 mg/L, respectively (Ferry et al., 1980).

In another study, blood levels of 47 to 76 mg/L wer e measured 2 to 3 hours after oral uptake of 71 – 84 mg methanol/kg bw (6.6 – 7.4 ml per person). Me thanol disappearance obeyed first-order kinetics with a half-life of about 3 hours. Only a very small fraction of an oral dose of 50 mg methanol/kg (about 2 percent) was eliminated unchan ged via the respiratory and urinary routes, and about 98 percent was metabolised (Leaf and Zatman, 1952; DFG, 1999; IPCS/WHO, 1997).

Oral - Aspartame. In another study, fasted adults were administered 3 4, 100, 150, or 200 mg aspartame/kg in 300 ml orange juice as a single dos e, which corresponded to methanol portions of about 3.5, 10, 15 and 20 mg/kg bw. Blood methanol levels were below detection (4.0 mg/L) in the lowest dose group. At 100 mg/kg bw, blood methanol and urinary formate were increased in a doserelated manner. However, blood formate levels were not increased at any dose. Peak mean blood methanol levels were 12.7, 21.4, 25.8 mg/L, at 100, 150, and 200 mg aspartame/kg. All levels returned to normal within 24 hours after exposures ceased (Stegink et al., 1981). Studies in which one-year old infants were administered aspartame sh owed similar results (Stegink et al., 1983).

When aspartame was administered in a single dose (5 00 mg/person = approx. 50 mg methanol equivalent) methanol blood levels were not much dif ferent from the endogenous levels. Mean increases close to 1 mg/L were recorded compared wi th pre-exposure levels of 1.4 – 2.6 mg/L (Davoli et al., 1986).

A longer-term study demonstrated that ingesting asp artame in a methanol equivalent of 7.5 mg/(kg bw * 

### d) resulted in blood methanol levels around 10 mg/L in adults (Leon et al., 1989), which is still far below metabolic saturation.

Inhalation. Inhalation studies have shown a net absorption of m ethanol of 60 to 85 percent (IPCS/WHO, 1997). In one study, the absorption hal f-life in a group of 22 volunteers after inhalation was estimated to 0.80 hours (Osterloh et al., 1996). Using data from inhalation exposure towards 1.05 mg/L (800 ml/m 3 

### ) methanol for 30 and 120 minutes, the half-life of methanol in blood was 1.44 hours (Batterman et al., 1998).

After a single exposure of 0.26 mg/L (200 ml/m 3 

### ) methanol, blood levels in exposed humans increased 3.5 to 4-fold above the endogenous blood methanol concentration from 1.8 to 7.0 mg/L at rest and from 1.9 to 8.1 mg/L under light exercise (Lee et al., 1992). Formate levels remained unchanged, irrespective of physical activity. Thes e findings were confirmed by D ́Alessandro et al. (1994) and Franzblau et al. (1993) in similar exper iments on human volunteers. Medinsky and Dorman (1995) also reported similar results. Highe r urinary methanol concentrations of approximately 40 mg/L have been predicted for worke rs exposed to 200 ml/m 3 

### (8-hour shift) (Heinrich and Angerer, 1982; Kawai et al., 1991).

Twenty-six volunteers exposed to 0.26 mg/L (200 ml/ m 3 

### ) methanol for 4 hours showed peak
methanol concentrations in blood of 6.5 ± 2.7 mg/L at the end of the exposure which declined during a 4-hour post-exposure period without reaching pre- exposure levels (1.0 ± 0.6 mg/L for a watervapor control phase and 1.8 mg/L ± 2.6 for a methanol phase); these were based on a re stricted diet without coffee, vegetables, fruit, alcohol, or aspa rtame (NTP, 2003). Formate levels in serum and urine remained unchanged (Osterloh et al., 1996 and Chuwers et al., 1995).

### OECD SIDS METHANOL

48 

### In a pilot study designed to measure neurobehaviora l effects, a single exposure of 0.25 mg/L for 75 minutes resulted in increased plasma and urine meth anol concentrations (3.3 and 2.5 fold, respectively). No change in plasma formate concent rations was observed (average 0.08 mmol/L = 3.6 mg/L) (Cook et al., 1991).

Dermal. Following dermal exposure in human volunteers, aver age skin absorption rates of methanol have been reported to be at 0.192 mg/(cm 2

* 

### min) (Dutkiewicz et al., 1980) and 8.1 ± 3.7 mg/(cm 2

* 

### h) = 0.135 ± 0.062 mg/(cm 2

* 

### min) (Batterman and Franzblau, 1997). According to Dutkiewicz et al. (1980), exposure of one hand to l iquid methanol for 2 minutes would result in a body burden of 170 mg, similar to that resulting fr om exposure to an approximate air concentration of 0.050 mg/L (38 ml/m 3 

### ) for 8 hours (Dutkiewicz et al., 1980). Full expo sure of one hand for 16 minutes is equivalent to blood levels after inhalat ion of 400 ml/m 3 

### for one 8-hour working shift (11 mg/L; corrected for background value) (Franzblau an d Batterman, 1995; Batterman and Franzblau, 1997).

PBPK Modeling

PBPK model simulations show that after a five day c ontinuous inhalation of 0.26 mg/L (200 ml/m 3 

### ), predicted blood and urine levels of methanol (5. 5 mg/L and 8.1 mg/L, respectively) were not higher than after one exposure session, and blo od and urinary formate levels (1.16 mg/L and 1.5 mg/L, respectively) also remained far below reporte d baseline levels (4.9-10.3 and 6.3-13 mg/L, respectively) (Bouchard et al., 2001). These simul ations were consistent with data produced in humans (Sedivec et al., 1981; Osterloh et al., 1996 ; Batterman et al., 1998) and show that such exposure levels do not achieve metabolic saturation

.

### OECD SIDS METHANOL

49 

# I U C L I D

D a t a S e t

Existing Chemical ID: 67-56-1
CAS No. 67-56-1
EINECS Name methanol
EC No. 200-659-6 Molecular Weight 32.04 g/mol
Molecular Formula C H4 O

Producer Related Part Company: BASF AG Creation date: 12-NOV-1992

Substance Related Part Company: BASF AG Creation date: 12-NOV-1992

Memo: master

Printing date: 26-OCT-2006 Revision date: 15-DEC-2004 Date of last Update: 26-OCT-2006

Number of Pages: 327

Chapter (profile): Chapter: 1, 2, 3, 4, 5, 6, 7, 8, 10 Reliability (profile): Reliability: without reliability, 1, 2, 3, 4 Flags (profile): Flags: without flag, confidential, non confi dential, WGK (DE), TA-Luft (DE), Material Safety Dataset, Risk Assessment, Directive 67/548 /EEC, SIDS, BPD Notification, key study, registration BPD B-docs, registration BPD
A-docs

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

50 1.0.1 Applicant and Company Information

Type: lead organisation
Name: BASF AG
Contact Person: Dr. Rolf Sarafin Date: GUP/CR - Z570
Street: Carl-Bosch-Str
Town: 67056 Ludwigshafen Country: Germany Phone: +49 621 60 44712
Telefax: +49 621 60 58043
Email: rolf.sarafin@basf.com Homepage: www.basf.com

18-APR-2006

Type: other: cooperating organisation
Name: Methanol Institute Country: United States

Type: cooperating company
Name: Methanor v.o.f. Country: Netherlands

Type: cooperating company
Name: Shell Chemicals Ltd. Country: United Kingdom

Type: cooperating company
Name: Statoil ASA Country: Norway

Type: cooperating company
Name: TOTAL Raffinerie Mitteldeutschland Gmb H Country: Germany

Type: cooperating company
Name: Veba Oil Refining & Petrochemicals Gmb H Country: Germany

1.0.2 Location of Production Site, Importer or For mulator

-

1.0.3 Identity of Recipients

-

1.0.4 Details on Category/Template

-

1.1.0 Substance Identification

Mol. Formula: C H4 O Mol. Weight: 32.04 g/mol

Remark: INDEX-Number: 603-001-00-X
12-SEP-2005

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

51 1.1.1 General Substance Information

Substance type: organic Physical status: liquid Purity: >= 99.85 - % w/w
Colour: colourless Odour: alcoholic

Method: GC Remark: The Data of the Substance Related Part a re also submitted on behalf of the following compan ies:

Akzo Chemicals International B.V. (NL) Arco Chimie France, SNC Aragonesas, Industrias y Energia, S.A. (E) Bayer AG (D) BP Chemicals LTD (GB) Brenntag AG (D) Cepsa (E) Cheminova Agro A/S (DK) DEA Mineraloel AG (D) DOW Corning Coordination Center ( B) Du Pont de Nemours GmbH (D) Ecofuel S.P.A (I) Elf Atochem (F) Ertisa, S.A. (E)
Hoechst AG
Huels AG ICI Chemicals & Polymers (GB) Krems Chemie Aktiengesellschaft ( A) Leuna Raffineriegesellschaft mbH (D) Methanor v.o.f (NL) (Montefibre) (I) = Enichem Fibre (I) Neste Resins B.V. (NL) Neste OY (FIN) Norsk Hydro A/S (N) Perstorp AB (S) Perstorp SpA, Div. Polyols (I) Praoil (Enichem Group) (I) Rhone-Poulenc Chimie (F) Rhone-Poulenc Rhodia AG (D) Ruetgerswerke AG (D) Ruhr Oel GmbH (D) Shell Nederland Chemie B.V. Union Carbide Benelux NV (B) VEBA Oel AG (D) Wilhelm Grean GmbH & Co.

12-SEP-2005 (1)

Substance type: organic Physical status: liquid
Colour: colourless Odour: alcohol-like

12-SEP-2005 (2)

Substance type: other: Substance classification:
Alcohols Physical status: liquid

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

52 Colour: colourless Odour: ageeable to pungent

12-SEP-2005 (3)

Purity: >= 99 - % w/w

Method: GC.

12-SEP-2005 (4)

1.1.2 Spectra

1.2 Synonyms and Tradenames

Methanol

Methanol (8CI, 9CI)

Methyl alcohol

Methyl hydroxide

Methylol

Monohydroxymethane

1.3 Impurities

EINECS-Name: Chloride as ClMol. Formula: ClMethod: IMPCA 002-92
Remark: <= 0.00005 % w/w (<= 0.5 mg/kg).

12-SEP-2005 (5)

CAS-No: 7704-34-9
EC-No: 231-722-6
EINECS-Name: sulphur Mol. Formula: S

Method: ASTM D 3961-89
Remark: <= 0.00005 % w/w (0.5 mg/kg).

12-SEP-2005 (5)

CAS-No: 7439-89-6
EC-No: 231-096-4
EINECS-Name: iron
Mol. Formula: Fe

Method: ASTM E 394-94
Remark: Total iron. <= 0.00001 % w/w (0.1 mg/kg).

12-SEP-2005 (5)

CAS-No: 7732-18-5

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

53 EC-No: 231-791-2
EINECS-Name: water Mol. Formula: H2 O
Contents: <= .1 - % w/w

Method: ASTM E 1064-92

(5)

CAS-No: 64-17-5
EC-No: 200-578-6
EINECS-Name: ethanol
Mol. Formula: C2 H6 O
Contents: <= .005 - % w/w

Method: ASTM E 346-94 Remark: <= 50 mg/kg.

12-SEP-2005 (5)

EINECS-Name: Acidity (free acid as acetic acid)
Contents: <= .003 - % w/w

Method: ASTM D 1613-91
Remark: <= 30 mg/kg.

12-SEP-2005 (5)

CAS-No: 67-64-1
EC-No: 200-662-2
EINECS-Name: acetone Mol. Formula: C3 H6 O
Contents: <= .003 - % w/w

Method: ASTM E 346-94
Remark: <= 30 mg/kg.

12-SEP-2005 (5)

1.4 Additives

-

1.5 Total Quantity

Quantity: 100000 - 500000 tonnes produced in 1991 Quantity: 100000 - 500000 tonnes imported in 1995 Quantity: 100000 - 500000 tonnes produced in 1995 Quantity: 100000 - 500000 tonnes produced in 1999 Quantity: 10000 - 50000 tonnes imported in 1999 Quantity: 10000 - 50000 tonnes imported in 2000 Quantity: > 1000000 tonnes produced in 2000

Remark: Production volumes in the year 2000:

North America (NAFTA): Production : approx. 7 Mtonnes Import : approx. 4 Mtonnes -> Total supp ly: approx. 11 Mtonnes

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

54 use: Formaldehyde : approx. 23%
DMT * : approx. 2% Acetic Acid : approx. 9% MTBE/TAME * : approx. 36% Methyl Methacryla te: approx. 3% Gasoline/Fuel : approx. 0.2%
Solvents : approx. 6%
Others : approx. 14% Exports : approx. 7%

South America approx. 7 Mtonnes incl. Trinidad

Western Europe approx. 3. 3 Mtonnes Eastern Europe approx. 0. 5 Mtonnes

Former Soviet Union approx. 2 Mtonnes Africa and approx. 5 Mtonnes Middle East

Asia/Pacific approx. 5 Mtonnes

World approx. 35 Mtonnes/a capacity

* DMT = dimethyl terephthalate TAME = tert. amylmethylether = 2-methoxy-2-methylbutane MTBE = tert-butyl methyl ether

Quantity: 50000 - 100000 tonnes imported in 2002

Quantity: > 1000000 tonnes produced in 2002

Flag: confidential

Quantity: 0 tonnes imported in 2005

18-APR-2006

Quantity: 100000 - 500000 tonnes produced in 2005

18-APR-2006

1.6.1 Labelling

Labelling: as in Directive 67/548/EEC Symbols: (F) highly flammable (T) toxic Specific limits: yes R-Phrases: (11) Highly flammable (23/24/25) Toxic by inhalation, in contact with skin and if
swallowed (39/23/24/25) Toxic: danger of v ery serious irreversible effects through inhalation, in co ntact with skin and if
swallowed S-Phrases: (7) Keep container tightly closed (16) Keep away from sources of i gnition - No smoking

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

55 (36/37) Wear suitable protective clothing and gloves (45) In case of accident or if y ou feel unwell, seek medical advice immediately (show the labe l where possible)

12-SEP-2005 (2) (6)

1.6.2 Classification

Classified: as in Directive 67/548/EEC Class of danger: highly flammable
R-Phrases: (11) Highly flammable Specific limits: no

12-SEP-2005 (2) (6)

Classified: as in Directive 67/548/EEC Class of danger: toxic
R-Phrases: (23/24/25) Toxic by inhalation, in contact with skin and if
swallowed Specific limits: yes Conc./Class. 1: C >= T; R23/24/25-39/23/24/25
20%

12-SEP-2005 (2) (6)

Classified: as in Directive 67/548/EEC Class of danger: toxic
R-Phrases: (39/23/24/25) Toxic: danger of very seriou s irreversible effects through inhalation, in co ntact with skin and if
swallowed Specific limits: yes Conc./Class. 1: C >= T; R23/24/25-39/23/24/25
20% Conc./Class. 2: 10% <= T; R20/21/22-39/23/24/25

C < 20%

12-SEP-2005 (2) (6)

Classified: as in Directive 67/548/EEC Class of danger: harmful R-Phrases: (20/21/22) Harmful by inhalation, in conta ct with skin and if
swallowed Specific limits: yes Conc./Class. 1: C >= T; R23/24/25-39/23/24/25
20% Conc./Class. 2: 10% <= T; R20/21/22-39/23/24/25

C < 20% Conc./Class. 3: 3% <= Xn; R20/21/22-68/20/21/22

C < 10%

12-SEP-2005 (6)

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

56 Classified: as in Directive 67/548/EEC Class of danger: harmful R-Phrases: (68/20/21/22) Harmful: possible risk of ir reversible effects through inhalation, in contact wi th skin and if swallowed Specific limits: yes Conc./Class. 1: C >= T; R23/24/25-39/23/24/25
20% Conc./Class. 2: 10% <= T; R20/21/22-39/23/24/25

C < 20% Conc./Class. 3: 3% <= Xn; R20/21/22-68/20/21/22

C <
10%

12-SEP-2005 (6)

1.6.3 Packaging

-

1.7 Use Pattern

Type: type Category: Wide dispersive use

Type: industrial Category: Basic industry: basic chemicals

Remark: Use as Feedstock for Chemical Syntheses

Approximately 70 % of the methano l produced worldwide is used in chemical syntheses: in or der of importance formaldehyde, methyl tert-butyl e ther (MTBE), acetic acid, methyl methacrylate, and dimethyl terephthalate. Only a small proportion is utilized for energy production, although this use has great potential.

Formaldehyde Formaldehyde is the most importan t product synthesized from methanol; in 1988, 40 % of the me thanol produced worldwide was used to synthesize this produ ct. The processes employed are all ba sed on the oxidation of methanol with atmospheric oxygen.

Methyl tert-butyl ether is produc ed by reacting methanol
with isobutene on acid ion exchan gers. Increasing amounts of
methanol are used in this form in the fuel sector. The ether is an ideal octane booster and ha s become extremely important due to the introduction of unleaded grades of gasoline and awareness of the pos sible harmfulness of aromatic high-octane components. In 1988, 20 % of worldwide methanol production was used for MTBE synthesis; annual increase rates of up to 12 % are expected.

Acetic Acid

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

57 Another 9 % of the methanol produ ced is used to synthesize acetic acid, and annual growth ra tes of 6 % are estimated. Acetic acid is produced by carbon ylation of methanol with carbon monoxide in the liquid pha se with cobalt - iodine, rhodium - iodine, or nickel - iod ine homogeneous catalysts .

Other Synthesis Products
In the intensive search after the oil crisis for routes to alternative fuels, processes were developed that allowed fuels to be produced from synthes is gas with methanol as an
intermediate. Mobil in the United States has contributed decisively to the development of such processes, which involve mainly the reaction of me thanol on zeolite catalysts. The most important and , up to now, the only industrially implemented process is methanol to gasoline (MTG) synthesis.

Methanol is used to synthesize a large number of other organic compounds:

Formic acid preservati ves, pickling agents

Methyl esters of solvents, monomers organic acids

Methyl esters of methylatio n reagents, explosives, inorganic acids insecticid es

Methylamines pharmaceut ical precursors,
auxiliarie s, absorption liquids for gas wa shing and scrubbing

Trimethylphosphine pharmaceut icals, vitamins, fragrances , fine chemicals

Sodium methoxide organic in termediates, catalyst

Methyl halides organic in termediates, solvents, propellant s

Ethylene organic in termediates, polymers,
auxiliarie s

(7)

Type: industrial Category: Basic industry: basic chemicals

Remark: Methanol to Olefins (MTO)

One of the larger potential appli cations for methanol is the methanol-to-olefins (MTO) process

. In this process,methanol is conve rted to light olefins, primarily ethylene and propylene. The quantity of methanol that in theory would be required for a 500,000 ton per year ethyle ne plant is 7,000 tons per day, or 2,450,000 tons per year. Obviously,this is larger than any methanol unit currently being built today. Given the generally lower costs o f the traditional cracking

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

58 technology however, MTO will like ly only capture niche markets or serve as "swing" capac ity for existing ethylene producers. Nonetheless, the volum e potential for methanol is huge.

One of the potential benefits of the MTO process is the ability to separate the olefin pr oduction from the natural gas location by building the meth anol and the MTO units in the most financially attractive l ocation.

(8)

Type: use Category: other: use as Feedstock for Chemical Synth eses

Remark: World demand for MTBE from Methanol is e xpected to fall below 8 Mio. t/a equivalent to ap prox. < 16% of the world demand of methanol in 2004.

(8)

Type: industrial Category: Fuel industry

Remark: Use as Energy Source

Methanol is a promising substitut e for petroleum products if they become too expensive for use as fuels.

Methanol as a Fuel for Otto Engin es The use of methanol as a motor fu el has been discussed repeatedly since the 1920s. Use h as so far been restricted to high-performance engines for r acing cars and aeroplanes.

(7)

Type: use Category: Fuel additives

Remark: Biodiesel.

12-SEP-2005 (4)

Type: use Category: other: Other Energy Uses of Methanol

Remark: A use that has been discussed particular ly in the United States and implemented in pilot p rojects is the firing of peak-load gas turbines in power s tations ( peak shaving). Benefits include simple storage a nd environmentally friendly combustion in the gas turbine.
The use of methanol as a fuel in conventionally fired boilers obviates the need for costly flue gas treatment plants but is not yet economically viable.

12-SEP-2005 (7)

Type: use Category: other: Other Energy Uses of Methanol

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

59 Remark: Methanol as a Hydrogen Carrier for Fuel Cell Applications

Methanol is a leading candidate t o provide the hydrogen necessary to power a range of fue l cell technology applications. Taking an estimate from DaimlerChrysler, by 2020, 4.2 million to 12 million n ew fuel cell cars will be sold each year. DaimlerChrysler a lso believes that methanol is the best hydrogen carrier fuel for personal transport. Long before the automotive fuel c ell market reaches maturity, consumers will be using cartridges of methanol to fuel direct methanol fuel cell po wer packs for a range of electronic devices such as cellul ar phones and laptop computers. Methanol also will be used to power portable fuel cell generators, and even re sidential fuel cell systems.

(9)

Type: use Category: Intermediates

Type: industrial Category: Paints, lacquers and varnishes industry

Type: industrial Category: Chemical industry: used in synthesis

12-SEP-2005 (2)

Type: use Category: Laboratory chemicals

Type: use Category: Solvents

Type: use Category: other: RM

Remark: Methanol's low freezing point and its mi scibility with water allow it to be used in refrigerat ion systems, either in pure form (e.g., in ethylene plants) o r mixed with water and glycols. It is also used as an antifreeze in heating and cooling circuits; compared to oth er commonly used antifreezes (ethylene glycol, pro pylene glycol, and glycerol), it has the advantage o f lower viscosity at low temperature. It is, however, no l onger used as an engine antifreeze; glycol-based products are employed instead. Large amounts of methanol are use d to protect natural gas pipelines against the formation o f gas hydrates at low temperature. Methanol is added to natural gas at the pumping station, conveyed in liquid form in the pipeline, and recovered at the end of the pipel ine. Methanol can be recycled after removal of water t aken up from natural gas by distillation.
Methanol is also used as an absor ption agent in gas
scrubbers. The removal of carbon dioxide and hydrogen sulfide with methanol at low temp erature (Rectisol process, Linde and Lurgi) has the advantag e that traces of methanol in the purified gas do not genera lly interfere with further

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

60 processing. The use of pure methanol as a sol vent is limited, although
it is often included in solvent m ixtures.

12-SEP-2005 (7)

1.7.1 Detailed Use Pattern

-

1.7.2 Methods of Manufacture

Orig. of Subst.: Synthesis Type: Production

Remark: The methanol production process involves the catalytic steam reforming of a hydrocarbon feedst ock, typically natural gas, to give a gaseous mixture of carb on oxides and hydrogen. This mixture is then compressed a nd reacted over another catalyst to give methanol and by- products, according to the following reactions.
CO + 2 H2 <-> CH3OH
CO2 + 3 H2 <-> CH3OH + H2O. The pure product is obtained by f ractional distillation. All necessary steps are performed in closed systems.

The gas is reformed over a nickel catalyst. The synthesis gas, containing a mixture of hydr ogen, carbon oxides, steam
and residual methane leaves the r eformer at about 880oC and 20 bar, and is cooled before bein g compressed to synthesis

pressure.

The synthesis loop comprises a ci rculator, converter, feed/effluent exchanger, separato r, and in some cases a heat recovery exchanger. The optimum operating pressure is typically about 80 bar for large plants, but for smaller pla nts (less than 600 t/day) a pressure of 50 bar may be economi c due to the reduced material requirements.

Synthesis gas is mixed with the r ecycle gas and preheated in a feed/effluent interchanger.
The converter effluent is cooled by passing through the feed/effluent interchanger and de pendent on the reactor type, a heat recovery exchanger. Prior to the catchpot, the gas is cooled to about 40oC to condense the product methanol. A purge is taken from the recycle gas to remove inerts (N2, CH4, Argon, and surplus hydrogen or carbon oxides) from t he loop. This is used as fuel in the reformer. A purge gas expander may be used to recover power from the purge gas as it is let down to the fuel system pressure.

The crude methanol from the separ ator contains both water and low levels of by-products, wh ich must be removed to achieve required product purity.

12-SEP-2005 (10)

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

61 1.8 Regulatory Measures

-

1.8.1 Occupational Exposure Limit Values

Type of limit: MAK (DE) Limit value: 270 mg/m3

Country: Germany Remark: Exceeding factor: 4.
If the MAK and BAT values are com plied with, there should be
no risk for the fetus. Cutaneous absorption.
Source: TRGS 900 (Germany), 08/2004

12-SEP-2005 (2) (11)

Type of limit: MAK (DE) Limit value: 200 other: ppm

Country: Germany Remark: Exceeding factor: 4.
If the MAK and BAT values are com plied with, there should be
no risk for the fetus. Cutaneous absorption.
Source: TRGS 900 (Germany), 08/2004

12-SEP-2005 (2) (11)

Type of limit: other: EU-TWA Limit value: 260 mg/m3

Country: Europe

12-SEP-2005 (6)

Type of limit: other: EU-TWA Limit value: 200 other: ppm

Country: Europe

12-SEP-2005 (6)

Type of limit: BAT (DE) Limit value: 30 other: mg per liter urine

Country: Germany Remark: Time of sampling (long term exposure): After several preceding shifts en d of exposure/end of shift.
Examination material: Urine.

12-SEP-2005 (3)

1.8.2 Acceptable Residues Levels

-

1.8.3 Water Pollution

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

62 Classified by: other: VwVwS (Germany), Annex 2 Labelled by: other: VwVwS (Germany), Annex 2 Class of danger: 1 (weakly water polluting)

Country: Europe
Source: VwVwS (Germany), 17.05.1999

12-SEP-2005 (2) (12)

1.8.4 Major Accident Hazards

Legislation: Stoerfallverordnung (DE) Substance listed: yes

Country: Germany Remark: Annex I - No.: 26

Threshold for operating range to § 1 sec. 1:

-Record 1: 500,000 kg

-Record 2: 5,000,000 kg

12-SEP-2005 (3)

1.8.5 Air Pollution

Classified by: TA-Luft (DE) Labelled by: TA-Luft (DE) Number: other: 5.2.5 (organic substances) Class of danger: I

Country: Germany Remark: Following total amounts must not be exce eded in exhaust gas:

mass flow: 0.10 kg/h or

mass concentration: 20 mg/m3

12-SEP-2005 (3)

1.8.6 Listings e.g. Chemical Inventories

Type: EINECS Additional Info: EINECS-No. 200-659-6

12-SEP-2005 (13)

Type: ENCS Additional Info: ENCS No. 2-201

Remark: ENCS CLASSIFICATION: Low Molecular Chain-like Organic Compounds.

12-SEP-2005 (13)

Type: ECL

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

63 Additional Info: ECL Serial No. KE-23193
ECL Toxic Chemical No. 97-1-80

Remark: KOREAN TCCL DESIGNATION: ECL Toxic Chemical No. 97-1-80

12-SEP-2005 (13)

Type: other: SWISS Additional Info: SWISS No. G-2063

Remark: SWISS CLASSIFICATION: Giftliste 1 (List of Toxcic Subst ances 1), 31 May 1999. Toxic Category 3.

12-SEP-2005 (13)

Type: TSCA

12-SEP-2005 (13)

Type: DSL

12-SEP-2005 (13)

Type: AICS

12-SEP-2005 (13)

Type: PICCS

12-SEP-2005 (13)

Type: other: ASIA-PAC

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Type: CHINA Additional Info: Inventory of Existing Chemical Substances (IECSC) , 2004

12-SEP-2005 (14)

Type: other: New Zealand Additional Info: Composite List of Single Component Substances to be considered for Transfer (April 2003)

Flag: non confidential
12-SEP-2005 (14)

1.9.1 Degradation/Transformation Products

EINECS-Name: Thermal decomposition:

### OECD SIDS METHANOL

1. GENERAL INFORMATION ID: 67-56-1 DATE: 26-OCT-2006

64 Vapours may form explosive mixtur e with air.

12-SEP-2005 (2)

1.9.2 Components

-

1.10 Source of Exposure

-

1.11 Additional Remarks

Memo: Characterisation: Highly inflammable liquid. Vapours form explosive mixtures w ith air.
Mixable with water. High volatile.

12-SEP-2005 (3)

Memo: Hazardous reactions: Explosion hazard when product rea cts with metals and forms hydrogen. Reacts with strong oxidizing agen ts. Aluminium, magnesium, toxic vapou rs/gases.

12-SEP-2005 (2)

1.12 Last Literature Search

Type of Search: Internal and External Chapters covered: 1
Date of Search: 12-SEP-2005

Remark: Update 2006 Flag: non confidential
12-SEP-2005

Type of Search: Internal and External Chapters covered: 5.10
Date of Search: 06-NOV-2002

1.13 Reviews

### OECD SIDS METHANOL

2. PHYSICAL CHEMICAL DATA ID: 67-56-1 DATE: 26-OCT-2006

65 2.1 Melting Point

Value: = -97.8 degree C

Reliability: (4) not assignable
Handbook

(15)

Value: = -98 degree C

Reliability: (4) not assignable Manufacturer / producer data with out proof

(16)

2.2 Boiling Point

Value: = 64.7 degree C

Reliability: (4) not assignable
Handbook

(15)

Value: = 64.7 degree C

Reliability: (4) not assignable Manufacturer / producer data with out proof

(16) Value: = 64.6 degree C

Reliability: (4) not assignable Secondary quotation (17)

2.3 Density

Type: density
Value: = .79 g/cm3 at 20 degree C

Reliability: (4) not assignable
Handbook

(15)

Type: density
Value: = .791 - .792 g/cm3 at 20 degree C

Reliability: (4) not assignable Manufacturer / producer data with out proof

(16)

### OECD SIDS METHANOL

2. PHYSICAL CHEMICAL DATA ID: 67-56-1 DATE: 26-OCT-2006

66 2.3.1 Granulometry

2.4 Vapour Pressure

Value: = 129 hPa at 20 degree C

Reliability: (4) not assignable Manufacturer / producer data with out proof

(16)

Value: = 319 hPa at 38 degree C

Reliability: (4) not assignable Secondary quotation (18)

Value: = 552 hPa at 50 degree C

Reliability: (4) not assignable Manufacturer/producer data (16)

Value: = 128 hPa at 20 degree C

Reliability: (4) not assignable Secondary quotation (19)

Value: = 127.9 hPa at 20 degree C

Method: other (calculated)

Method: Value calculated with the following equa tion: Y= exp[A + B/T + ClnT + DT2] and using the following equation coefficients:
A= 8.1768E+01
B= -6.8760E+03 C= -8.7078E+00
D= 7.1926E-06 Remark: Source for accurate physical property da ta. Data are collected from a wide ra nge of sources and critically evaluated. Reliability: (2) valid with restrictions Scientifically verified data, sta ndard reference source for

vapour pressure

19-OCT-2006 (20)

2.5 Partition Coefficient

Partition Coeff.: octanol-water log Pow: = -.82

Method: other (measured)

Reliability: (2) valid with restrictions scientifically verfied data

### OECD SIDS METHANOL

2. PHYSICAL CHEMICAL DATA ID: 67-56-1 DATE: 26-OCT-2006

67 (21)

Partition Coeff.: octanol-water log Pow: = -.77

Reliability: (4) not assignable Manufacturer / producer data with out proof (16)

Partition Coeff.: octanol-water log Pow: = -.74

Remark: Recommended value Reliability: (2) valid with restrictions Scientifically verified data

(22)

Partition Coeff.: octanol-water log Pow: = -.64

Method: other (calculated)

Remark: Value calculated applying the hydrophobi c fragmental constant (f) for small molecules. Reliability: (2) valid with restrictions scientifically verfied data

(23)

2.6.1 Solubility in different media

Solubility in: Water Value: at 20 degree C
Descr.: miscible

Remark: pH value: neutral Reliability: (4) not assignable Manufacturer / producer data with out proof

(16)

Solubility in: Water
Descr.: miscible

Reliability: (4) not assignable Secondary quotation (17)

2.6.2 Surface Tension

Value: = 22.55 mN/m at 20 degree C

Reliability: (4) not assignable Manufacturer / producer data with out proof (24)

### OECD SIDS METHANOL

2. PHYSICAL CHEMICAL DATA ID: 67-56-1 DATE: 26-OCT-2006

68 2.7 Flash Point

Value: = 9.7 degree C Type: closed cup

Method: other: Abel-Pensky

Reliability: (2) valid with restrictions National standard specification w ithout detailed
documentation

(25)

Value: = 11 degree C Type: closed cup

Method: other: DIN 51755

Reliability: (4) not assignable Manufacturer / producer data with out proof (16)

2.8 Auto Flammability

Value: = 455 degree C

Method: other: DIN 51 794

Remark: Autoignition temperature Reliability: (2) valid with restrictions National standard specification w ithout detailed
documentation

(26)

Value: = 385 degree C at 1013 hPa

Remark: Autoignition temperature Reliability: (4) not assignable Manufacturer / producer data with out proof (27)

2.9 Flammability

Result: highly flammable

Reliability: (4) not assignable Manufacturer / producer data with out proof

(16)

2.10 Explosive Properties

Result: not explosive

Remark: because of chemical structure Reliability: (2) valid with restrictions expert judgement

### OECD SIDS METHANOL

2. PHYSICAL CHEMICAL DATA ID: 67-56-1 DATE: 26-OCT-2006

69 (28)

2.11 Oxidizing Properties

Result: no oxidizing properties

Remark: because of chemical structure Reliability: (2) valid with restrictions Expert judgement

(28)

2.12 Dissociation Constant

-

2.13 Viscosity

Value: = .59 mPa s (dynamic) at 20 degree C

Reliability: (4) not assignable Manufacturer / producer data with out proof (16)

2.14 Additional Remarks

Remark: Explosion limits: 5.5 - 36.5 vol.%

Conditions to avoid, substances t o avoid: Hazardous reaction influenced by strong oxidizing agents, (e.g. nitric acid, chromium triox ide, perchloric acid and salts). Vapours may form explosive mixtur e with air. Explosion hazard when product rea cts with metals and forms hydrogen: aluminium, magnesium at elevated temperature.

Hazardous reactions: vapours form explosive mixtures with
air

Hazardous decomposition products: ignitable gases / vapours Reliability: (4) not assignable Manufacturer / producer data with out proof (16)

Remark: Explosion limits: 5.5 - 36.5 vol.% Reliability: (4) not assignable
Handbook (29)

Remark: Explosive limits: 5.5 - 31.0 vol.% Reliability: (4) not assignable Secondary quotation (30)

Remark: Explosive Limits in air: 6.0 - 36.5 vol. % Reliability: (4) not assignable Manufacturer / producer data with out proof (27)

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

70 3.1.1 Photodegradation

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH
Rate constant: = .000000000000932 cm3/(molecule * sec) Degradation: = 50 % after 17.2 day(s)

Method: other (measured) Test substance: as prescribed by 1.1 - 1.4

Remark: Rate Constant: 0.932 (+/- 0.233)*10^-12 cm^3/molecule*sec at
298 K.
Recommended value based on a unit -weighted least squares analysis of several published and scientifically evaluated experimental rate constants. Reliability: (1) valid without restriction scientifically verfied data

(31)

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH

Remark: k(298K)= 7.8*10e-13 or 1.4*10e-13 cm3/mo lecule*sec. Temp. dependence of k/cm3/molecul e*sec: 3.3*10e-12 exp (-380/T). Temp. range/K: 240-300 Reliability: (2) valid with restrictions (32)

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH Conc. of sens.: 1000000 molecule/cm3
Rate constant: = .000000000001 cm3/(molecule * sec) Degradation: = 50 % after 8 day(s)

Remark: Bimolecular reaction constant Reliability: (2) valid with restrictions (33)

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH Conc. of sens.: 500000 molecule/cm3
Rate constant: = .0000000000012 cm3/(molecule * sec) Degradation: = 50 % after 14.6 day(s)

Remark: Reaction at ca. 300 K, smog chamber test Reliability: (2) valid with restrictions (34)

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH Conc. of sens.: 3000000 molecule/cm3

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

71 Remark: Atmospheric transformation: 1 to 5 days Reliability: (4) not assignable Secondary literature (35)

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH
Rate constant: = .000000000000944 cm3/(molecule * sec)

Remark: Recommended value (at 298 K)based on a u nit-weighted least-squares analysis of several experimental absolute rate constants scientifically evaluate d by the author. Reliability: (1) valid without restriction scientifically verfied data

(36)

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH

Remark: The half-life of methanol in the troposp here for this reaction will obviously depend up on the concentration of hydroxyl radicals. Using an avera ge hydroxyl radical
concentration of 1*10^6 molecules /cm^3, half-lifes for the reaction ranged from 6.7-13.4 days. However, using the global diurnal mean troposphe ric concentration of 5*10^5 molecules/cm^3 quoted in s ome references, the half-lifes ranged from 13.4-17.6 days. Reliability: (4) not assignable secondary qoutation (37)

Type: air
INDIRECT PHOTOLYSIS Sensitizer: OH Conc. of sens.: 1000000 molecule/cm3 Degradation: = 50 % after 7.3 day(s)

Remark: Reaction at 300 K and atmospheric pressu re; k=1.1*10^-12 cm^3/molecule*sec Reliability: (4) not assignable secondary quotation (38)

Type: air

Remark: Methanol is listed as hazardous air poll utant under Title III of CAAA (Clean Air Act Amendm ents) with an atmospheric lifetime of 16-18 days. Reliability: (4) not assignable secondary literature (39)

Type: water
INDIRECT PHOTOLYSIS Sensitizer: OH

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

72 Result: Rate constant = 1*E9 l/mol*sec (40)

Type: water
INDIRECT PHOTOLYSIS Sensitizer: OH

Method: other (measured)

Remark: k(OH) = 8.46 * 10e9 liter/mol*sec OH concentration: 5 * 10e-14 mol/ liter temperature: 298 K (41)

3.1.2 Stability in Water

Method: other

Remark: Alcohols are generally resistant to hydr olysis. Reliability: (1) valid without restriction
Handbook

(42)

3.1.3 Stability in Soil

- 3.2.1 Monitoring Data (Environment)

Medium: air

Remark: concentration: 6-60 ug/m3 (3 locations, 52 samples) (35)

Type of measurement: other Medium: other

Remark: Methanol is a naturally occurring compou nd, being a meta- bolite in man, animals and plants

. (43)

Type of measurement: other

Remark: Holzer et al. (1977) identified methanol in forest air in Alabama, 20-35 miles away from th e closest urban activities/ habitation, and ascribed its pres ence to natural sources. (44)

Type of measurement: other

Remark: Owens et al. (1969) identified methanol as one of the volatile compounds emitted by alf alfa, and Isidorov et al. (1985) reported that methanol was given off by evergreen cyprus in a survey of the volatil e emissions from the foliage of 17 trees characteristi c of the forests of Northern Europe and Asia. (45) (46)

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

73 Type of measurement: other Medium: air

Remark: Jonsson et al. (1985) reported the resul ts of a 1 year study (1982 to 1983) of air quality in Stockholm. Samples were collected at four urban sites in central Stockholm and a more rural site 12 km from the ce ntre of the city. The minimum detectable quantity in th e 2-3 litre samples collec- ted was about 0.008 nl/l for a ty pical oxygenated compound, i.e. about 0.01 ug/m^3 methanol. The following methanol levels were reported: 1. Busy street, 8 samples/day col lected over 7 days, 56 samples in total, March-May 1983; average: 34.98 ug/m^3; range: 5.31-94.84 ug/m^3. 2. Quiet street, 8 samples/day co llected over 3 days, plus samples taken in early morning an d at night, 40 samples in total, August-September 1982; ave rage: 10.96 ug/m^3; range: 2.0-28.17 ug/m^3. 3. Busy street, 8 samples/day col lected over 12 days, 96 samples in total, September-Decem ber 1982; average: 10.22 ug/m^3; range: 0.76-34.6 ug/m^3. 4. Quiet street, 8 samples/day co llected over 7 days, plus samples taken in early morning an d at night, 68 samples in total, January-March 1983; averag e: 5.02 ug/m^3; range: 0.59-15.07 ug/m^3. 5. Recreation area, 12 km from ce ntral Stockholm, 8 samples /day collected over 7 days, 56 sa mples in total, May-June 1983; average: 11.21 ug/m^3; rang e: 3.21-22.27 ug/m^3. (47)

Type of measurement: other Medium: other: water and sediment

Remark: Jungclaus et al. (1978) analysed the was tewater and effluent-receiving waters and sed iments for a US chemical plant manufacturing a broad range of chemicals, including pharmaceuticals, herbicides and s urfactants. The plant was
located on a small freshwater riv er draining into a small brackish cove, which eventually f ed into an estuary.
Methanol was found at levels of 1 7-80 mg/l in the
waste-water but not in the efflue nt-receiving waters or associated sediments (no detectio n limits were reported). (48)

Type of measurement: other Medium: other: condensate waters

Remark: Mohr and King (1985) identified methanol in one of four samples of condensate waters take n from a fixed-bed coal gasification process plant. No de tection limit was reported
but methanol was found in the sam ple at a level of 1050 mg/l. (49)

Type of measurement: other Medium: biota

Remark: Pellizzari et al. (1982) collected 42 sa mples of mothers'
milk from women in four urban are as of the USA. They ana- lysed all the samples by GC/MS an d the mass spectra from

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

74 eight samples were later interpre ted manually. Methanol was found in one of these samples , however, no actual level of occurrence was reported. (50)

Type of measurement: other Medium: food

Remark: Methanol has been isolated and identifie d in the volatile components of the following foods tuffs: baked potatoes (Coleman et al., 1981), roasted f ilbert nuts (Kinlin et al., 1972), dried legumes (Lovegren et al., 1979). Of these occurrences, only Lovegren et al. quantified the levels of methanol present, as follows: Lim a beans (seven samples), common beans (five samples), mung beans (one sample) and soya beans (one sample): mean lev el =4.2 mg/kg; range: 1.5-7.9 mg/kg. Split peas (one sample): level =3 .6 mg/kg. Lentils (two samples): mean level =4.4 mg/kg. (51) (52) (53)

3.2.2 Field Studies

-

3.3.1 Transport between Environmental Compartments

Type: adsorption
Media: water - air

Remark: The Henry's Law constant of 1.35*10^-4 a tm*m^3/mole
indicates that volatilization fro m environmental waters may be significant. Reliability: (2) valid with restrictions (54)

Type: volatility
Media: water - air
Method: other: calculated according to HENRYWIN, V 3.10

Result: Henry's Law Constant at 25 °C = 4.27E-6 atm * m3/mol. Reliability: (2) valid with restrictions

(55)

Type: adsorption
Media: water - soil

Remark: Lokke (1984) studied the adsorption of m ethanol onto three different soil types at 6 deg C. Only slight methanol ad- sorption occurred with the two sa ndy soils tested (percen- tage organic matter of 0.09% and 0.1% in the samples) and with the clay soil (percentage or ganic matter was 0.22%).
Methanol solutions of concentrati on 0.1, 1.0, 9 and 90 mg/l were used in 1 hour exposure adso rption studies and adsorp-
tion coefficients of between 0.13 and 0.61 were measured for all soil types and at all concent rations. These coefficients
indicate that methanol has a low adsorptive capacity on
soils. Reliability: (4) not assignable

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

75 Secondary quotation (56)

Type: adsorption
Media: water - soil

Method: QSAR calculation performed with PCKOCWI N v1.66 (2000)
Remark: The very low value of the estimated Koc indicates no significant adsorption of methano l on soils. Result: Estimated Koc = 1 Reliability: (2) valid with restrictions

(57)

Type: volatility
Media: water - air

Remark: Methanol has a estimated Henry's Law Con stant of 4.4*10^-6 atm*m^3/mol at 25 deg C

. Reliability: (4) not assignable (58)

Type: volatility
Media: water - air

Result: H = 0,461 Pa*m3/mol Reliability: (2) valid with restrictions

(59)

3.3.2 Distribution

Media: air - biota - sediment(s) - soil - wate r Method: Calculation according Mackay, Level I

Result: water: 84.59 %
air: 15.38 %
sediment: 0.0068 % soil: 0,0067 % Reliability: (2) valid with restrictions (60)

Media: air - biota - sediment(s) - soil - wate r Method: Calculation according Mackay, Level III

Remark: Input parameters for fugacity model:

Molecular weight: 32.04 Vapor pressure: 129 hPa Henry' Law constant: 0.461 Pa*m3/ mol Log Kow: -0.82 Water solubility: 1E+006 mg/l
Result: compartment mass amount (%) emission (kg/h) air 73,3 9 093 water 15,6 175 soil 11,1 75 sediment 0,0233 0 Reliability: (2) valid with restrictions

(61)

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

76 3.4 Mode of Degradation in Actual Use

3.5 Biodegradation

Type: aerobic Degradation: = 95.6 % after 5 day(s)

Method: other: (BOD of THOD)
GLP: no Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable Test methods and performance unce rtain, limited
documentation available
19-OCT-2006 (62)

Type: aerobic
Inoculum: domestic sewage Degradation: = 53.4 % after 5 day(s)

Method: other: BOB-Test (BOD of THOD)
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Concentration of test substance:2.5 ppm. Mineralized dilution water, settl ed sanitary sewage as seed and incubation at 20 °C. Incubation time: 50 days. Result: Percentage of the Theoretical Oxygen Dem and (BOD of ThOD) at day 5, 10, 15, 20, 30, 40, 50: 53 .4, 62.7, 69.4, 93.4, 97.7 Reliability: (2) valid with restrictions acceptable study meets basic scie ntific principles
19-OCT-2006 (63)

Type: aerobic
Inoculum: domestic sewage, non-adapted Degradation: = 76 % after 5 day(s)
Result: readily biodegradable
Kinetic: 5 day(s) = 76 % 10 day(s) = 88 % 15 day(s) = 91 % 20 day(s) = 95 %

Method: other: closed bottle test (BOD of THOD)
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: A settled domestic wastewater was added (3 ml/bottle) as seed
material to clean BOD bottles. Th e bottles were half filled
with aerated dilution water conta ining the specified minerals
and buffer. Small aliquots of the test chemic al were added to these bottles yielding concentrations o f 3, 7 and 10 mg/l. At least
two of these concentrations were tested in duplicate. Dissolved oxygen (DO) was monitor ed periodically in the individual bottles through the us e of a commercial DO meter fitted with an agitated probe. The bottles were opened for sampl ing and DO measurements about five times during the course of t he 20-day test. Reliability: (2) valid with restrictions

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

77 Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment

19-OCT-2006 (64)

Type: aerobic
Inoculum: domestic sewage, non-adapted Degradation: = 82.5 % after 5 day(s)

Method: other: Respirometric test (BOD of THOD)
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Determination of the reaction kinetic by means of the respirometric dilution method.
This method involves the use of a nutritious matrix, usually but not necessarily domestic sewa ge, which contains a lot of organic substances competing as n utrient with the substrate
under test. Result: ThOD: 1498, 0 mg/g rate constant K: 1.882 1/d BOD at day 5: 1236 mg/g BOD/ThOD at day 5: 82.7% Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment

19-OCT-2006 (65)

Type: aerobic
Inoculum: other bacteria: synthetic sewage Degradation: = 71.2 % after 5 day(s)

Method: other: Respirometric test (BOD of THOD)
GLP: no Test substance: as prescribed by 1.1 - 1.4

Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment (65)

Type: aerobic
Inoculum: activated sludge, industrial
Concentration: 100 mg/l related to Test substance Degradation: > 90 % after 1 day(s)

Test substance: as prescribed by 1.1 - 1.4

Remark: Matsui et al. (1988) examined the biodeg radability of methanol in a batch reactor using activated sludge from
the Fukashiba industrial wastewat er treatment plant, which was acclimated to the waste water (containing various organic compounds) originating fr om the Kashima petro- chemical complex in Japan. Methan ol, at an initial concen- tration of 100 mg/l, was found to be highly biodegradable,
and 91% COD removal and 92% TOC r emoval was achieved after a 1 day acclimation period and af ter 1 day's operation of
the batch reactor. Reliability: (2) valid with restrictions

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

78 acceptable study meets basic scie ntific principles (66)

Type: aerobic Degradation: = 99 %

Method: OECD Guide-line 301 D "Ready Biodegrada bility: Closed Bottle
Test" Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable secondary quotation (67)

Type: aerobic Degradation: = 70.7 - 88.7 % after 5 day(s)

Method: other Test substance: as prescribed by 1.1 - 1.4

Method: standard dilution method (BOD of THOD) a nd sea water
dilution method Remark: BOD5 value for methanol was measured by the sea water dilution method (S.W.D.M) and the standard dilution method (S.D.M).
Concentration of test substance: 2.56 ppm. Result:

S.D.M. S.W.D .M.

BOD5 (g/g) 1.33 1.06 ThOD (g/g) 1.50 1.50 BOD/ThOD (%) 88.7 70.7 Reliability: (2) valid with restrictions
19-OCT-2006 (68)

Type: aerobic
Inoculum: activated sludge, domestic, non-adapted Degradation: = 80 %

Method: other: Respirometric test (Sapromat)
Year: 1973
GLP: no Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable Secondary quotation (IUCLID Data Sheet), Data to EU (69)

Type: aerobic
Inoculum: other bacteria: marine inoculum Degradation: = 69 % after 5 day(s)

Method: other: closed bottle test (BOD of THOD)
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: BOD 5/10/15/20 of THOD = 69/84/85/97 %. concentration: 3-10 mg/l of test substance Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

79 (64)

Type: aerobic
Inoculum: other: contaminated soil
Contact time: 30 day(s) Degradation: ca. 19 % after 30 day(s)

Method: other: see freetext
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: Microcosm studies were done in 0.5l-glas s bottles with caps containing 150 g soil impacted wi th relatively high
concentrations of solvents inclus ive Methanol (MeOH) and approximately 0.4 l groundwater. H2O2 wasadded as an oxygen source to maintain 1 to 2 mg/l ox ygen in solution. In the Microcosms complemented with NH4C l and Na2HPO4 in ratio
COD:N:P= 100:10:2 the result was 19% degradation of MeOH in 30 days. Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, acceptable for

assessment (70)

Type: aerobic
Inoculum: other: contaminated soil Degradation: ca. 100 % after 32 day(s)

Method: other: see freetext
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: The continuous-flow reactor consisted of a 30-gal glass reactor and the center portion wa s filled with site soil and groundwater. Suction and disc harge tubing was enveloped in pea stone, perforated baffles were used to separate the
zones. Aerobiosis were achieved b y addition of H2O2. Result: The half live time for MeOH was : t1/2 = 16 days Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, acceptable for

assessment (70)

Type: aerobic
Inoculum: activated sludge, adapted Degradation: = 50 - 80 %

Remark: Swain and Somerville (1978) monitored th e biodegradation
of methanol in a model activated sludge system. Methanol
was not broken down when added tr ansiently (at levels of 0.23% by volume) to the system op erating with a retention time of 11 hours. However, adapti on of the sludge in the system to 0.1% by volume methanol occurred over a period of several days, and after 2 days acclimation, about 50%
of the methanol was utilised. Aft er 6 days acclimation,
at least 80% of the methanol had been degraded. Reliability: (4) not assignable

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

80 secondary quotation (71)

Type: aerobic

Remark: Goldsmith assessed the potential for met hanol degradation in subsurface soil and groundwate r from sites in Penn- sylvania, New York and Virginia u nder aerobic conditions. Substantial bacterial populations were present in all samples and methanol was readily degraded. A range of methanol concentrations from 1 to 1000 mg/l were used and rates of degradation from 0.8 to 20.4 mg/l/day were recorded in these aerobic studies and in p arallel anaerobic studies using material from the same site s. The rates of degradation were generally greater in the ana erobic samples than in the aerobic. Reliability: (4) not assignable secondary quotation (72)

Type: aerobic

Remark: Further details of the study of Goldsmit h were reported by Novak et al. They stated that methanol at concentrations up to 1000 mg/l was degraded at a ll three sites to such an
extent that contamination would b e reduced to non-measurable amounts within a year. The soils and overlying water used in these tests were not thought t o have been previously contaminated. In one aerobic samp le of soil from a Penn- sylvania site, complete degradati on of 100 mg/l methanol occurred in less than 30 days. Reliability: (4) not assignable secondary quotation (73)

Type: aerobic

Remark: Biodegradation of methanol in not heavil y polluted aerobic aquifer material: half-live 58-26 0 days (location: Clay loam Virginia) Reliability: (4) not assignable Secondary literature (74)

Type: anaerobic
Inoculum: other: bacterial population from a sewage sludge Degradation: = 75 - 80 %

Remark: Bekes et al. (1975) studied the degradat ion of methanol by the bacterial population from a s ewage sludge. Under an- aerobic conditions rapid degradat ion occurred, leading to 75-80% disappearance of the metha nol. Rapid liberation of
methane and carbon dioxide was ob served. Reliability: (4) not assignable secondary quotation (75)

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

81 Type: anaerobic
Concentration: 500 mg/l related to Test substance Degradation: = 66 %

Remark: Chou et al. (1978) studied the acclimati on and degradation of several petrochemical wastewat er components (including methanol) in an anaerobic ferment ation system. Methanol, at a concentration of 500 mg/l, was found to be metabolised by enriched bacterial cultures (deve loped on acetate substrate) after a period of acclimation. Af ter a 6 day lag period, 66% of the methanol was removed rapid ly at a rate of 182 mg/l/ day from serum bottle inocula. Reliability: (2) valid with restrictions 2.1; accpetable study, meets basi c scientific principles (76)

Type: anaerobic Degradation: = 83 - 91 % after 3 day(s)

GLP: no

Remark: Oremland et al. (1982) studied the degra dation of methanol
in anaerobic sediments collected from the upper 15 cm of a salt marsh in San Francisco Bay. The sediments were highly
reduced and contained methane and hydrogen sulphide. The sediments were homogenised anaero bically with San Francisco Bay water and 310-340 umol methan ol/flask was added to make up the inocula. After 3 days incu bation, 83-91% conversion
of the methanol had occurred. The products of methanol degradation were methane, CO2 and water. Reliability: (2) valid with restrictions 2.1; accpetable study, meets basi c scientific principles (77)

Inoculum: other: methylotrophic organisms

Remark: In a survey of methylotrophic organisms, i.e. those micro- organisms recognised by their abi lity to grow on compounds that contain no carbon-carbon bon ds and to assimilate car- bon as formaldehyde or as a mixtu re of formaldehyde and carbon dioxide, Hanson (1980) ide ntified the following strains/genera of microorganism c apable of using methanol as a growth substrate: Pseudomona s sp. (including P. AM1, P. C, P. 8 and P. oleovorans), Me thylobacterium organophi- lum XX, Hyphomicrobium sp., Strep tomyces sp., Rhodopseudo- monas acidophila, Paracoccus deni trificans, Microcyclus aquaticus, Thiobacillus novellus, Micrococcus denitrificans, Achromobacter 1L and Mycobacteriu m (isolated from activated sewage sludge). Hansen also ident ified one strain of methanol-oxidising fungus, Tricho derma lignorium, and the following genera of methanol-oxid ising yeasts: Pichia, Saccharomyces, Hansenula, Rhodoto rula, Kloeckera, Candida, Torulopsis. Reliability: (4) not assignable secondary quotation (78)

Inoculum: other

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

82 Remark: Other organisms identified as being capa ble of utilising methanol as a growth substrate in other reports included: Methanosarcina sp. (anaerobe, tra nsformed methanol to methane), Candida sp. (a yeast is olated from Niger Delta sediment), Mycobacterium sp. (iso lated from various samples of Japanese soil) and a yeast iso lated from palm wine. Reliability: (4) not assignable secondary quotation (79) (80) (81) (82)

3.6 BOD5, COD or BOD5/COD Ratio

3.7 Bioaccumulation

Species: Cyprinus carpio (Fish, fresh water)

Remark: Gluth et al. studied the accumulation of methanol by carp, Cyprinus carpio, exposed to the c hemical for up to 72 hours. The amount of radioactivity deriv ed from the 14-C-labelled chemical was determined in the li ver, kidneys, intestine, muscle, blood and gills of carp e xposed to methanol at 5 mg/l. The initial uptake of metha nol into the different tissue types was the same after 2 4 hours, and levels re- mained constant over 72 hours in the liver, kidneys, gills and intestine but dropped slightl y in the blood and muscle.
Bioaccumulation factors of about 1 were proposed for the fish tissues and organs.

Reliability: (2) valid with restrictions (83)

Species: Leuciscus idus (Fish, fresh water)
BCF: < 10

Remark: The bioconcentration factor of methanol experimentally measured in fish (golden ide) was less than 10. Based on a log Kow of -0.77 the BCF value for methanol can be estimated to be 0.2 from a recom mended regression-derived equation. Reliability: (2) valid with restrictions

(84) (85) (86)

Species: other: Chlorella fusca (green alga) Exposure period: 24 hour(s) at 25 degree C
Concentration: 50 μg/l
BCF: = 28400

Method: other
GLP: no data

Method: Algae with a dry weight of 20 mg/200 ml were exposed in a 500 ml Erlenmeyer flask with glas s stoppers to 14C-labelled methanol in nutrient solution for at least 24 hours at room temperature (19 - 25°C) under ill umination (16 hr/d) and agitation. After 24 hours the algae were rem oved by centrifugation at at 4000 rpm. The 14C activity of the algae was determined by combustion

### OECD SIDS METHANOL

3. ENVIRONMENTAL FATE AND PATHWAYS ID: 67-56-1 DATE: 26-OCT-2006

83 in a Packard Tri Carb Sample Oxid izer and counting in a Packard Tri Carb liquid scintilla tion counter Model 3385.
The bioaccumulation factor on the basis of dry weight is calculated by dividing the chemic al concentration of the algae by the final concentration in the water after 24
hours.

Reliability: (3) invalid The derived high bioconcentration factor is anomalous compared to those for other aquatic organi sms. It is likely due to the fact that methanol is taken up ve ry rapidly and metabolized by the algae. The 14C-label, which i s measured to calculate the BCF value, is therefore incorpora ted into the algae in
metabolic forms other than methan ol. In addition the test was conducte d in a wide temperature range and with an unappropriate exposur e time. (87)

3.8 Additional Remarks

Memo: more information see IPCS report no. 1 96

(88)

Result: Methanol biodegradation has been shown t o take place also under anaerobic conditions (all e lectron acceptors besides oxygen). Microbial growth on methanol with nitrate as the electron acceptor is the second most energ etically favored mode of
methanol metabolism. There are ov er 100 wastewater treatment plants in the United States that currently use methanol as a carbon source to remove nitrate (NO3-) from water by anaerobic denitrification. Reliability: (1) valid without restriction

(89)

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

84 AQUATIC ORGANISMS

4.1 Acute/Prolonged Toxicity to Fish

Type: flow through Species: Lepomis macrochirus (Fish, fresh water) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: yes LC50: = 15400

Method: other
Year: 1986
GLP: no data

Remark: Method: Test procedures followed the bas ic flow-through test method described by the U. S. Env ironmental Protection Agency (EPA-660/3-75-009, 1975). The exposure system for fish cons isted of several modified proportional diluters with diluti on factors ranging from 0.6 to 0.8. Each diluter cycle delive red 1.0 l of water and toxicant, where appropriate, to e ach of five test
concentrations and a control. Eac h test concentration and control was duplicated by dividin g the original volume equally using flow-splitting tank s. The glass exposure tanks containe d 6.3 l water. Diluters were cycled at a rate to yield 5 to 8 tank volumes/day. Exposure samples containing metha nol were analyzed by direct aqueous injection gas-liquid crom atography. The percent recovery of methanol-spiked expos ure water for bluegill test was 101.5 +/- 2.7% (n = 5). Bluegills were held in 530-l fibe rglass holding tanks at their respective test temperature s and fed a commercial formulation of dry food. Groups of 10 bluegills were rando mly assigned to the exposure tanks at the start of ea ch test. Fish were not fed 24 h before or during a test. Average weights of exposed fish w ere obtained by weighing
control fish from each test. Aver age weight of blugills was 3.07 g +/- 1.38 Death was the primary endpoint, a lthough effects on equilibrium, behavior and colorat ion were noted Median effect concentration, EC50 was 12700 mg/l (11800 - 13700). Dead fish were counted and remove d at 1, 3, 6, 12 and 24 h after initial exposure and at 24 -h periods thereafter.
Determination of LC50 and EC50 an d their 95% confidence intervals were made using the Tri mmed Spearman Karber method (Hamilton et al., 1977). Mortalit y data from duplicate exposure tanks were combined befo re LC50 and EC50 determinations were made. Test condition: Water for all tests was untreated and obtained directly from Lake Superior and heated to mean test temperature. Water temperature of the test: 19 .8 +/- 2.3 °C Mean hardness: 46.6 mg/l CaCO3 (4 0.4 - 56.3) Mean alkalinity: 41.7 mg/l CaCO3 (30.0 - 46.0) pH range: 7.04 - 7.97 Mean dissolved oxygen: 78.8 % (54 .3 - 88.9) Test substance: Methanol, no further data. Reliability: (1) valid without restriction 1c: Test procedures in accordance with accepted standard

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

85 methods, sufficiently documented, acceptable for assessment

(90)

Type: flow through Species: Pimephales promelas (Fish, fresh water) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: yes LC50: 28100

Method: other
Year: 1983
GLP: no data

Remark: The test was conducted with Lake Superio r water. The flow -through test used a proportional diluter system, in which there were five toxicant concentr ations plus a control all in duplicate. Individual exposure chambers meas ured either 26x17x15 cm, or
20x35x15 cm and contained 3.1 l a nd 6.3 l of water. The diluter system delivered 0.5 l of fresh water alone to the
controls or 0.5 l of fresh water plus toxicant in the case of the exposure groups every 15 - 19 min. Water temperature was mantained a t 25 °C and a 16-h light photoperiod was used. Ten to 20 f ish of age 30-32 days were placed into each chamber. fish we re not fed during the acute

test. Observations for mortalities and other gross behavioral effects were made at regular inte rvals through 96 hrs. Several water quality parameters (temperature, dissolved oxygen, hardness, alkalinity, aci dity and pH) were monitored throughout the tests.
Toxicant concentrations were meas ured daily in acute tests. All twelve chambers were analyzed at 0 and 96 h, with alternating replicates analyzed a t 24, 48 and 72 h. Methanol concentrations were dete rmined by using Rhodamine B dye as a tracer. The stock soluti on contained 25 mg/l which was delivered to the exposure tan ks. The concentration of
alcohol was calculated from the a nalysis of Rhodamine B, which was measured a spectrofluor ometer. The excitation and emission wavel engths were 554 nm and 578 nm respectively. Standard were pr epared in Lake Superior
water and lake water was used as the reference blank. The exposure tanks contained from 0.1 to 1.3 μg/l Rhodamine

B.

Result: LC50 (24h) = 28400 mg/l LC50 (48h) = 28400 mg/l LC50 (72h) = 28400 mg/l
Test substance: methanol, purity > 99 % Reliability: (1) valid without restriction 1c: Test procedures in accordance with accepted standard methods, sufficiently documented, acceptable for assessment

(91)

Type: flow through Species: Pimephales promelas (Fish, fresh water) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: yes

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

86 LC50: = 28200

Method: other: 5 concentrations and 4 fish/conce ntration were used
Year: 1932
GLP: no data Test substance: other TS

Remark: 11 alcohols with increasing chain length were tested to create a structure activity relat ionship with LC50 and octanol-water partition coefficie nt. Result: A bilinear relationship was found betwee n log LC50 and log P with increasing toxicity with inc reasing log P.
Test substance: Methanol, no further data. Reliability: (2) valid with restrictions

(92)

Type: flow through Species: Salmo gairdneri (Fish, estuary, fresh wa ter) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: yes LC50: = 20100
Method: other
Year: 1983
GLP: no data

Remark: The test was conducted with Lake Superio r water. The flow -through test used a proportional diluter system, in which
there were five toxicant concentr ations plus a control all in duplicate. Individual exposure chambers meas ured either 26x17x15 cm, or
20x35x15 cm and contained 3.1 l a nd 6.3 l of water. The diluter cycled every 1o-16 min, p roviding from 6.8 to 10.6 volume additions per day. Water temperature was mantained a t approximately 12 °C and a 16-h light photoperiod was used. Ten fish were tested per chamber. Fish were not fed during the acute test.
Observations for mortalities and other gross behavioral effects were made at regular inte rvals through 96 hrs. Several water quality parameters (temperature, dissolved oxygen, hardness, alkalinity, aci dity and pH) were monitored throughout the tests.
Toxicant concentrations were meas ured daily in acute tests. All twelve chambers were analyzed at 0 and 96 h, with alternating replicates analyzed a t 24, 48 and 72 h.
Methanol concentrations in the ex posure water were determined by GLC using direct aq ueous injection on a Tenax GC packed column and flame-ioniz ation detection Result: LC50 (24h) = 20300 mg/l LC50 (48h) = 20100 mg/l LC50 (72h) = 20100 mg/l
Test substance: methanol, purity > 99 % Reliability: (1) valid without restriction 1c: Test procedures in accordance with accepted standard methods, sufficiently documented, acceptable for assessment

(91)

Type: flow through Species: Salmo gairdneri (Fish, estuary, fresh wa ter)

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

87 Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: yes LC50: = 20100

Method: other: not indicated
GLP: no data Test substance: other TS

Remark: Method: Flow-through acute toxicity test
EC50: median effect concentration , EC50=13000 mg/kg
Test substance: Methanol, no further data. Reliability: (4) not assignable

(90) Type: other: flow through or static Species: Pimephales promelas (Fish, fresh water) Exposure period: 96 hour(s)
Unit: g/l Analytical monitoring: yes LC50: 29.4 EC50 : 28.9

Method: other
GLP: no data Test substance: other TS

Remark: Test organism: fathead minnow, age 28-29 days, overall mean length 21 +/- 2 mm

Test conditions: Test temperature: 25 degrees cent igrade Dissolved oxygen: 7.3 mg/l Hardness: 43.5 mg/l CaC03 Alkalinity: 46.6 mg/l CaCO3
Tank volume: 6.3 l pH: 7.66

Test concentrations nominal: 4000; 6700; 11200; 18600; 31000 m g/l

EC50: 28900 mg/l/96 h
Test substance: methanol, purity > 99 % Reliability: (2) valid with restrictions 2.1; accpetable study, meets basi c scientific principles (93)

Type: semistatic Species: Poecilia reticulata (Fish, fresh water) Exposure period: 7 day(s) Unit: mg/l Analytical monitoring: LC50: = 10860

Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary quotation (94) (95) Type: semistatic Species: other: Agonus cataphractus Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring:

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

88 LC50: >= 7900 - 26070

Remark: Seawater Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary quotation (96)

Type: static Species: Alburnus alburnus (Fish, estuary) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no data LC50: = 28000
Method: other: not indicated
Year: 1984
GLP: no data

Test substance: Methanol, no further data. Reliability: (4) not assignable (97) (98)

Type: static Species: Cyprinus carpio (Fish, fresh water) Exposure period: 72 hour(s)
Unit: mg/l Analytical monitoring: no data
EC : >= 790

Method: other: effect on clinical chemical param eters
Year: 1985
GLP: no data Test substance: other TS

Method: 6 fish were used per group. Exposure tim e was 6, 24 or 72 hours. The following parameters w ere examined: serum glucose, serum cortisol, serum pr otein, serum cholesterol, liver glycogen, muscle glycogen.
Result: Methanol treatment did not affect serum glucose but led to increased serum cortisol, decreas ed serum protein, decreasedserum cholesterol, decre ased liver glycogen, increased muscle glycogen. Test substance: Methanol, no further data. Reliability: (2) valid with restrictions 2.1; accpetable study, meets basi c scientific principles (99) Type: static Species: Cyprinus carpio (Fish, fresh water) Exposure period: 72 hour(s)
Unit: mg/l Analytical monitoring: no data
EC : = 790

Method: other: effects on clinical chemical para meters were measured
Year: 1984
GLP: no data Test substance: other TS

Method: Exposure time was 72 hours. Exposure tem perature was 12, 17 or 22 øC. The following parameter s were measured: serum cortisol, serum glucose, liver gl ycogen, muscle glycogen, serum protein, serum cholesterol. Result: The treatment led to increased serum cor tisol and serum

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

89 glucose and to decreased liver gl ycogen, muscle glycogen, serum protein and serum cholester ol. Test substance: Methanol, no further data. Reliability: (2) valid with restrictions 2.1; accpetable study, meets basi c scientific principles (100)

Type: static Species: Lebistes reticulatus (Fish, fresh water) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring:
SG : = 500

Method: other: DIN 38412 T.15
Year: 1973
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: SG = Schaedlichkeitsgrenze (limit of tox icity)
Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary quotation (101) Type: static Species: Leuciscus idus melanotus (Fish, fresh wa ter) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no data LC0: = 7900 LC50: > 10000 LC100: > 10000

Method: other: not indicated
Year: 1978
GLP: no data Test substance: other TS

Test substance: Methanol, no further data. Reliability: (4) not assignable (102)

Type: static Species: Oncorhynchus mykiss (Fish, fresh water) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no data LC50: = 19000

Method: other: not indicated
GLP: no data Test substance: other TS

Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary quotation (103)

Type: static Species: Semolitus atromaculatus (Fish, fresh wat er) Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring: no data
NOEC: < 8000

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

90 EC : >= 8000 - 17000

Method: other: the "critical range of toxicity" was measured
GLP: no data Test substance: other TS

Remark: The test result is described as the crit ical range of toxicity, but the signs of toxici ty examined are not reported in detail. Test substance: Methanol, no further data. Reliability: (3) invalid (104)

Species: Cyprinus auratus Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no data
TLm : = 1659

Method: other: no details indicated
GLP: no data
Test substance: other TS

Remark: The test substance concentration is give n as 0.21 %, the
test result is described as media n tolerance limit without
further details. Test substance: Methanol, no further data. Reliability: (4) not assignable (105)

Species: Cyprinus carpio (Fish, fresh water) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no data
TLm : = 28440

Method: other: no details indicated
GLP: no data
Test substance: other TS

Remark: The test substance concentration is give n as 3.6 % and the
test result is described as media n tolerance limit (no detailed definition). Test substance: Methanol, no further data. Reliability: (4) not assignable (105)

Species: Leuciscus idus (Fish, fresh water) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no data LC0: > 1000

Method: other: DIN 38412 Teil 15
GLP: no data Test substance: other TS

Test substance: Methanol, no further data. Reliability: (4) not assignable (106) Species: Oncorhynchus mykiss (Fish, fresh water) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no data

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

91 LC50: = 10800

Method: other: not indicated
GLP: no data Test substance: other TS

Remark: 13680 ppm (original data)
Test substance: Methanol, no further data. Reliability: (4) not assignable (107)

Species: Oncorhynchus mykiss (Fish, fresh water) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no data
NOEC: = 8530

Method: other: not indicated
GLP: no data Test substance: other TS

Remark: 10800 ppm (original data)
Test substance: Methanol, no further data. Reliability: (4) not assignable (107)

Species: Oryzias latipes (Fish, fresh water) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no data
TLm : = 41080

Method: other: no details indicated
GLP: no data Test substance: other TS

Remark: The test substance concentration is give n as 5.2 %, the
result is described as median tol erance limit without
further details.
Test substance: Methanol, no further data. Reliability: (4) not assignable (105)

Species: Semolitus atromaculatus (Fish, fresh wat er) Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring: no data

: = 6320

Method: other: not indicated
GLP: no data Test substance: other TS

Remark: River (water) is indicated as test envir onment. The concentration indicated is given as toxic threshold.8000 ppm (original data). Test substance: Methanol, no further data. Reliability: (4) not assignable (107)

Species: other: Armed Bullhead Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no data

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

92 LC50: 7900 - 27700

Method: other: not indicated
GLP: no data Test substance: other TS

Remark: 10000 - 35000 ppm (original data)
Test substance: Methanol, no further data. Reliability: (4) not assignable (107)

Type: other Species: other Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no LC50: 10630

Remark: QSAR Estimation of acute toxicity of met hanol to fish
conducted with ECOSAR v.099. ECOSAR Class: Neutral Organics Reliability: (2) valid with restrictions (61) 4.2 Acute Toxicity to Aquatic Invertebrates

Type: static Species: Daphnia magna (Crustacea) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no EC0: > 10000 EC50: > 10000

Method: other: acute Toxicity to daphnia magna
Year: 1989
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: DIN 38412 Teil 11, Bestimmung der Wirkun g von Wasserinhaltsstoffen auf Kleinkre bse Remark: Test procedure: From the solutiom with the pollut ant, dilution series were prepared using dilution water of the specified composition. The concentration steps of the te st solution were selected so as to give 3-4 EC values in a range between EC0 and EC100, of which at least one valu e was below and one above
EC50.
The test vessel were 50 ml beaker s. Two parallel preparations were ma de for each concentration step. Loading amounted to one tes t animal per 2 ml test medium as ten 6-24 h old daphnids were pleaced in each test and control vessel, i.e. 20 anima l per concentration test. After 24 h and again after 48 h t he number of animals in the
control and test solutions that c ould swim was counted. After 48 h the oxygen content wer e determined in order to
ensure that it had not fallen bel ow a minimum oxygen concentration of 2 mg/l. Furtherm ore the ph value was determined at the end of the test

. The test was considered as valid when fewer than 10% of the animals in the control solutions were unable to swim, when the pH value was not below 7.0 an d the oxygen concentration

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

93 was not below 4.0 mg/l.
The EC0 and EC100 values were tak en from the results obtained for the test solution, t he 24 and 48 h EC 50s calculated arithmetically from th e concentration/effect
ratio. Result: EC0 (24 h) > 10000 mg/l EC50 (24 h) > 10000 mg/l
Test condition: 6-24 h old daphnids were used as test animals.

Specific composition of dilution water used: acid capacity: 0.8 mmol/l total hardness: 2.4 mmol/l Calcium to magnesium ratio: 4:1 Sodium to potassium ratio: 10:1 initial pH value: 8.0 +/- 0.2 The test solutions were kept at 2 0 °C in an incubator. The test period lasted 24 and 48 h. N o feed was given during the test period. Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

(108)

Species: Daphnia magna (Crustacea) Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring: EC50: > 10000

Method: other: inhibition of swimming ability
Year: 1977
GLP: no Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable (109)

Species: Daphnia magna (Crustacea) Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring: EC50: > 10000 EC100: > 10000

Method: other: DIN 38412 Teil 11, Bestimmung der Wirkung von
Wasserinhaltsstoffen auf Kleinkre bse
Year: 1982
Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable (110)

Species: Nitocra spinipes (Crustacea) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: LC(I)50 : = 12000

Method: other: acute toxicity
Year: 1979
Test substance: as prescribed by 1.1 - 1.4

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

94 Reliability: (4)not assisnable (98) Type: static Species: Daphnia magna (Crustacea) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no LC50 : > 100

Method: other
Year: 1985
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: nominal concentration Reliability: (4) not assignable

(111)

Species: Artemia salina (Crustacea) Exposure period: 24 hour(s)
Unit: g/l Analytical monitoring: EC50: = 43.6

Method: other
Year: 1994

Result: = 1360 mM Reliability: (4) not assignable (112)

Species: Artemia salina (Crustacea) Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring: LC50 : > 10000

Method: Endpoint evaluated was death.
Remark: Concentrations tested: 100, 1000 and 100 00 mg/l Reliability: (4) not assignable (64) Type: static Species: Asellus intermedius (Crustacea) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no LC50 : > 100

Method: other
Year: 1985
GLP: no

Remark: nominal concentration Reliability: (4) not assignable (111)

Species: Daphnia magna (Crustacea) Exposure period: 24 hour(s)
Unit: g/l Analytical monitoring: EC50: = 21.4

Year: 1994

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

95 Result: = 668 mM
Test substance: methanol, purity > 97 % Reliability: (4) not assignable (112)

Type: other Species: Daphnia sp. (Crustacea) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no EC50: 9374

Remark: QSAR Estimation of acute toxicity of met hanol to daphnid
conducted with ECOSAR v.099. ECOSAR Class: Neutral Organics Reliability: (2) valid with restrictions

(61) Type: static Species: Gammarus fasciatus (Crustacea) Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no LC50 : > 100

Method: other
Year: 1985
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: nominal concentration Reliability: (4) not assignable (111)

Type: static Species: other aquatic mollusc: Helisoma trivolvis Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no LC50 : > 100

Method: other
Year: 1985
GLP: no

Remark: nominal concentration Reliability: (4) not assignable (111)

Type: static Species: other aquatic worm: Dugesia tigrina Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no LC50 : > 100

Method: other
Year: 1985
GLP: no

Remark: nominal concentration Reliability: (4)not assignable

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

96 (111) Type: static Species: other aquatic worm: Lumbriculus variegatu s Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: no LC50 : > 100

Method: other
Year: 1985
GLP: no

Remark: nominal concentration Reliability: (4) not assignable (111)

Species: other: Anodonta imbecilis Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: LC50 : = 37.02

Reliability: (4) not assignable (113)

Species: other: Brachionus clyciflurus Exposure period: 24 hour(s)
Unit: g/l Analytical monitoring: EC50: = 35.9

Method: other
Year: 1994

Result: = 1120 mM Reliability: (4) not assignable (112)

Species: other: Ceriodaphnia dubia Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: LC50 : = 11

Reliability: (4) not assignable (113)

4.3 Toxicity to Aquatic Plants e.g. Algae

Species: Scenedesmus quadricauda (Algae) Endpoint: biomass Exposure period: 8 day(s)
Unit: mg/l Analytical monitoring: no TGK : = 8000

Method: other: growth inhibition
Year: 1978
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: The toxicity threshold of methanol for t he green alga Scenedesmus quadricauda was deter mined in the cell

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

97 multiplication inhibition test.
Culture media and conditions of c ulturing were standardized already for stock and preliminary cultures because a standardized history of the test organisms is essential insofar as history and resistance to pollutants show a close physiological relationship. Compo sition of culture media and conditions of culturing are descr ibed in the paper. Prior of preparations of test cul tures, the pollutant solution of known content was neu tralized.
Dilutions at different volume rat ios with the factor 2 were prepared in flasks from the pollu tant solution and from
bidistillated water under sterile conditions.
In the dilution series to be inoc ulated an addition of 5 ml
of each of the stock nutrient sol ution and of the algal suspension from the preliminary c ulture having a known adjusted extinction value was mad e to obtain the rated value of 50 ml. The quantity of the cel l material to be used for
inoculation was determined turbid imetrically and was
standardized.
The flasks of the non-inoculated dilution series were completed to the rated value of 5 0 ml by addition of 5 ml
each of the stock nutrient soluti on and bidistilled water. While shaking the contents, 10 ml each were taken from each flask of the inoculated dilution series and transferred to 3
culture tubes each and also from each flask of the non-inoculated dilution series, h owever transferred to one culture tube only.
Test cultures and control culture s were kept then under standardized conditions for a per iod of 8 days, exposed to constant lighting at 27 °C and a relative umidity of 50%.
At the end of the test the concen tration of the algal suspension was measured turbidime trically and expressed as the extinction of the primary lig ht of the monocromatic
radiation at 578 nm for a 10 mm l ayer. For graphical evaluation of the t oxicological findings after termination of the test period, t he median value (A) of the
extinction of all test cultures n ot under toxic influence nor stimulated (provided that the ir extinction values were within the range of a <3% standar d deviation) and the median value (B) of the extinction of th e three test cultures having the lowest toxic pollutant concentration were
determined. Reliability: (2) valid with restrictions 2.1; accpetable study, meets basi c scientific principles

(114)

Species: other algae: Plancton algae Endpoint: other: photosynthetic oxygen production Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring: EC10: = 1600 EC50: = 12000

Method: other: assimilation test
Test substance: as prescribed by 1.1 - 1.4

Remark: graphic evaluation (probability distribu tion)
nominal concentration Reliability: (2) valid with restrictions

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

98 (115)

Species: Chlorella pyrenoidosa (Algae) Endpoint: growth rate Unit: g/l Analytical monitoring: no data EC50: = 28.44

Method: other
Year: 1988
GLP: no

Remark: The geen alga Chlorella pyrenoidosa was obtained from the
National Research Council of Cana da, Atlantic Research Laboratory , Halifax, Nova Scotia , Canada. Stock cultures were grown in 250- ml erlenmeyer flasks containing 150 ml of an inorganic , nitrogen-free medium supplemented with 1.5 mg/l of NaN O3. Flasks were incubated at 25 +/-1 °C and a light intens ity of 7000 lux on a 12 hour light-dark cycle. During solvent bioassay experimen t test organisms were cultured in test tubes (150 x 25 mm O.D) in both presence
and absence of solvent. Growth was monitored by following the increase in optical density over time for 10 to 14 da ys by means of a spectrophotometer. The most suita ble wavelength was 660 nm. Cell concentrations were determin ed microscopically using a haemocytometer. A total of 5-10 concentrations be tween 2% and 6% methanol by volume were tested (15.8-47.4 g/l). Each concentration was replicated five to ten times. Appropriate control systems containing no solvent wer e included in each experiment. Bioassay systems contained 9.5 ml of growth medium, an appropriate volume of solvent and 0.5 ml of inoculum standardized to yield an initial cell concentration of 6.5 to 7.8 E+04 cells/ml. In each experiment percent inhibi tion values, relative to growth in control systems, were c alculated daily using spectrophotometric data. The EC50 values (the concentratio ns causing 50% reduction in algal growth) were calculated usi ng linear regression analysis of percentage inhibition values against solvent
concentration data. All correlati ons coefficients were
>0.960.
The EC50 value calculated was 3.6 % v/v methanol (28.44 g/l).
Test condition: Exposure period: 10 - 14 days Reliability: (2) valid with restrictions 2.1; accpetable study, meets basi c scientific principles

(116)

Species: other algae: green algae Endpoint: other Exposure period: 96 hour(s)
Unit: mg/l Analytical monitoring: EC10: calculated EC50: = 4982

Remark: QSAR Estimation of acute toxicity methan ol to green algae
conducted with ECOSAR v.099

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

99 ECOSAR Class: Neutral Organics Reliability: (2) valid with restrictions

(61)

4.4 Toxicity to Microorganisms e.g. Bacteria

Type: aquatic Species: Pseudomonas fluorescens (Bacteria) Exposure period: 16 hour(s)
Unit: mg/l Analytical monitoring: no TGK : = 6600

Method: other: Vereinfachte Modifikation des DEV L8 (1960); Messung der beginnenden Hemmung der Gluco se-Assimilation;
turbidometrische Zellzahlbestimmu ng Year: 1973
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: The toxicity threshold of methanol for P seudomonas fluorescens (bacteria) was determ ined in the glucose-assimilation inhibition t est. Pure cultures of single-cell mode l organism were used in the
test. Culture media and condition s for the prepations of stock cultures, preliminary cultu res and test cultures of the model organism were standardi zed and described in the

paper. Prior of preparations of test cul tures, the pollutant solution of known content was neu tralized (pH: 7.0). From the pollutant solution four parallel dilution series were prepared in 300 ml Erlenmeye r-flasks stoppered with plastic caps. The dilution series were preapare d as follows: the first
flask of each series contained 16 0 ml of pollutant solution. Starting from this flask the subs equent dilution steps were prepared at a constant dilution r atio by consistently mixing 80 ml of preliminary pollutant di lution and 80 ml
bidistilled water. Consequently each flask contained 80 ml of culture liquid at
the start. Each flask of the thre e dilution series to be inoculated was completed to 100 m l by adding 5 ml of each of
the stock solutions and of 10 ml each of the prepared bacterial suspension from the pr eliminary culture having a known adjusted extinction. The quantity of the cell material to be used for inoculation was determined turbidimetrically and was standardized.
The flasks of the non-inoculated dilution series were completed to 100 ml by adding 5 m l each of the stock solutions and 10 ml saline.
Both inoculated and non-inoculate d dilution series were kept at 25 °C for 16 h. After termination of the test per iod the concentration of the bacterial suspension was meas ured turbidimetrically: it is expressed by the extinction of the monochromatic radiation Hg 436 nm.
The concentration at which the in hibitory action of a pollutant starts will be present in that step of a dilution

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

100 series of the pollutant having an extinction value at the
end of the test that is >3% below the mean value of the extinction for non-toxic dilution s of the test cultures. Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment

(117)

Type: aquatic Species: Pseudomonas putida (Bacteria) Exposure period: 16 hour(s)
Unit: mg/l Analytical monitoring: no TGK : = 6600

Method: other: growth inhibition test
Year: 1977
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: The toxicity threshold of methanol for P seudomonas putida (bacteria) was determined in the cell multiplication
inhibition test. Pure cultures of single-cell mode l organism were used in the
test. Culture media and condition s for the prepations of stock cultures, preliminary cultu res and test cultures of the model organism were standardi zed and described in the

paper. Prior of preparations of test cul tures, the pollutant solution of known content was neu tralized. From the pollutant solution four parallel dilution series were prepared in 300 ml Erlenmeye r-flasks stoppered with plastic caps. The dilution series were preapare d as follows: the first
flask of each series contained 16 0 ml of pollutant solution. Starting from this flask the subs equent dilution steps were prepared at a constant dilution r atio by consistently mixing 80 ml of preliminary pollutant di lution and 80 ml
bidistilled water. Consequently each flask contained 80 ml of culture liquid at
the start. Each flask of the thre e dilution series to be inoculated was completed to 100 m l by adding 5 ml of each of
the stock solutions and of 10 ml each of the prepared bacterial suspension from the pr eliminary culture having a known adjusted extinction. The qu antity of the cell material
to be used for inoculation was de termined turbidimetrically
and was standardized.
The flasks of the non-inoculated dilution series were completed to 100 ml by adding 5 m l each of the stock solutions and 10 ml saline.
Both inoculated and non-inoculate d dilution series were kept at 25 °C for 16 h. After termination of the test per iod the concentration of the bacterial suspension was meas ured turbidimetrically: it is expressed by the extinction of the primary light of the
monochromatic radiation at 436 nm for a layer of 10 mm thickness.
The concentration at which the in hibitory action of a pollutant starts will be present in that step of a dilution series of the pollutant having an extinction value at the

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

101 end of the test that is >= 3% bel ow the mean value of the extinction for non-toxic dilution s of the test cultures. Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment

(118)

Type: aquatic Species: activated sludge of a predominantly domes tic sewage Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring:
SG : = 5000

Method: ETAD Fermentation tube method "Determin ation of damage to effluent bacteria by the Fermenta tion Tube Method"
Year: 1973
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: SG = toxicity limit Reliability: (4) not assignable secondary quotation (69)

Type: aquatic Species: Entosiphon sulcatum (Protozoa) Exposure period: 72 hour(s)
Unit: mg/l Analytical monitoring: no TGK : > 10000

Method: other: growth inhibition test
Year: 1978
GLP: no Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable (119) Type: aquatic Species: Microcystis aeruginosa (Bacteria) Exposure period: 192 hour(s)
Unit: mg/l Analytical monitoring: no TGK : = 530

Method: other: growth inhibition test
Year: 1978
GLP: no Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable (114)

Type: aquatic Species: Tetrahymena pyriformis (Protozoa) Exposure period: 48 hour(s)
Unit: mg/l Analytical monitoring: no IC50 : = 18756.34

Method: other: Population Growth Inhibition Test
Year: 1990

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

102 GLP: no data Test substance: other TS

Test substance: purchased from Aldrich Chemical Co., Milwaukee, Wisconsin, USA with a purity of 95% Reliability: (4) not assignable (120)

Type: aquatic Species: Uronema parduzci (Protozoa) Exposure period: 20 hour(s)
Unit: mg/l Analytical monitoring: no TGK : > 10000

Method: other: growth inhibition test
Year: 1980
GLP: no Test substance: as prescribed by 1.1 - 1.4

Reliability: (4) not assignable (121) Type: aquatic Species: Vibrio fisheri (Bacteria) Exposure period: 5 minute(s)
Unit: mg/l Analytical monitoring: no EC50: = 44400 - 88500

Method: other: ToxAlert 10, Microtox and LumiSto x standard assays using the appropriate manufacture rs's luminometer (see respective manual)
GLP: no Test substance: no data

Result: EC50 (5 min): 83600 mg/l (ToxAlert10), 4 4400 mg/l (Microtox), and 88500 mg/l (Lumis tox); data related to nominal concentra tions;
Test condition: temperature: 15 degree C, pH 7 reference standard: 440 mg/l Zinc sulphate in 2% NaCl

The same stock chemical solution was used to prepare the dilution series for each assay (T oxAlert 10, Microtox and Lumistox, respectively) and all t ests were carried out by the same person on the same day.

test criterion: inhibition of lig ht emitted by the
bioluminescent bacteria Vibr io fischeri after 5 min compared with zero dose expr essed as EC (effect concentration); Reliability: (2) valid with restrictions scientifically acceptable, commer cially used assay systems (122)

Type: aquatic Species: other bacteria: Bakterienmischpopulatione n Exposure period: 24 hour(s)
Unit: mg/l Analytical monitoring: EC10: = 13000 EC50: = 30000

Method: other: oxygen consumption

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

1 03 Test substance: as prescribed by 1.1 - 1.4

Remark: graphicevaluation
nominal concentration Reliability: (4) not assignable (115) Type: aquatic Species: other bacteria: Nitrosomonas sp./ Nitroba cter sp. Exposure period: 2 hour(s)
Unit: mg/l Analytical monitoring: EC50: ca. 160

Method: other: inhibition
Year: 1973
GLP: no Test substance: as prescribed by 1.1 - 1.4

Result: Hooper and terry showed methanol to be c ompletely inhibitory to ammonia oxidation by Nitrosomo nas bacteria at a concentration of 5*E-3 M (about 1 60 mg/l). Reliability: (4) not assignable (123)

Type: aquatic Species: other bacteria: activated sludge from a m unicipal sewage treatment plant Unit: g/l Analytical monitoring: EC50: ca. 71.21

Method: other: Activated Sludge Respiration Inhi bition Test

Reliability: (4) not assignable secondary quotation (124)

Type: aquatic Species: other bacteria: cyanobacteria
Unit: g/l Analytical monitoring: EC50: = 20.3 - 43.29

Method: other
Year: 1987
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: A minimum of 10 concentrations were test ed for each species, ranging from 0.6% to 10% methanol by volume (4.74-79.00 g/l). The EC50 values were calcul ated from percentage in- hibition values measured daily re lative to growth in the
controls. Anabaena cylindrica: EC50=2.57% v /v (20.3 g/l); Anabaena inaequalis: EC50=2.68% v/v (21.17 g/l); Anabaena sp.: EC50=3.13% v/v (24.73 g/l); Nosto c sp.: EC50=5.48% v/v (43.29 g/l). Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment. (125) Species: Nitrosomonas sp. (Bacteria) Exposure period: 24 hour(s)

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

104 Unit: mg/l Analytical monitoring: IC50 : = 880

Remark: Inhibition in ammonia consumption Reliability: (4) not assignable (126)

Species: Paramaecium caudatum (Protozoa) Exposure period: 4 hour(s) Unit: mg/l Analytical monitoring: LC50 : = 7690

Remark: Rajini et al. (1989) exposed the ciliate d protozoan
Paramaecium caudatum to methanol. The 4 hour LC50 value was quoted as 1.0% (v/v) methanol or 0.24 M, which corres- ponds to a concentration of 7690 mg/l (calculated from the molar concentration reported). Th e 10 minute LC50 value was quoted as 6.0% (v/v) methanol or 1.4 M, i.e. 44860 mg/l
calculated from the molar concent ration quoted. Reliability: (4) not assignable secondary quotation (127)

Species: Photobacterium phosphoreum (Bacteria) Exposure period: 5 minute(s)
Unit: g/l Analytical monitoring: EC50: = 157

Remark: EC50 (5/15/30 minutes) = 157.00/285.56/3 20.40 g/l. Reliability: (4) not assignable Secondary quotation (128)

Species: Photobacterium phosphoreum (Bacteria) Exposure period: 15 minute(s)
Unit: g/l Analytical monitoring: EC50: = 14.7

Reliability: (4) not assignable (129)

Species: Photobacterium phosphoreum (Bacteria) Exposure period: 24 hour(s)
Unit: g/l Analytical monitoring: EC50: = 29.3

Result: = 916 mM Reliability: (4) not assignable (112)

Species: other bacteria: Steptocephalus proboscide us Exposure period: 24 hour(s)
Unit: g/l Analytical monitoring: EC50: = 1020

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

105 Result: = 1020 mM Reliability: (4) not assignable (112)

Species: other bacteria: aerobic heterotrophs Exposure period: 15 hour(s)
Unit: g/l Analytical monitoring: IC50 : = 20

Remark: Inhibition of oxygen uptake Reliability: (4) not assignable (126)

Species: other bacteria: methanogenes Exposure period: 48 hour(s)
Unit: g/l Analytical monitoring: IC50 : = 22

Remark: Inhibition in gas production Reliability: (4) not assignable (126)

4.5 Chronic Toxicity to Aquatic Organisms

4.5.1 Chronic Toxicity to Fish

4.5.2 Chronic Toxicity to Aquatic Invertebrates

TERRESTRIAL ORGANISMS

4.6.1 Toxicity to Sediment Dwelling Organisms

4.6.2 Toxicity to Terrestrial Plants

Species: other terrestrial plant Expos. period: 14 day(s)

Remark: Plants such as Phaseolus vulgaris, Rapha nus sativus radicula, Lepidum sativum, Trifol ium pratense, Saintpaulia and Petunia hybrids were exposed to air containing methanol
at concentrations between 0.4 and 2.5 mg/m^3 for 14 days. Retarded growth, measured as the effect on both wet and dry weight of the plants, was reporte d for each species. Reliability: (4) not assignable secondary quotation (130)

Species: other terrestrial plant

Remark: US EPA (1976) reported that methanol has not been implicated in vegetation damage as other pol lutants, eg. ethylene, nitrogene oxides, sulphur dioxide and ozone, have. However, a Russian study has indicated tha t plants may be sensitive to methanol vapour at concentrati ons >0.15 ppm (>0.2 mg/m^3). Branches from eight diff erent species of tree were studied and the permissible standard (0.15 ppm or 0.2 mg/m^3) was taken as the conc entration which did not

### OECD SIDS METHANOL

4. ECOTOXICITY ID: 67-56-1 DATE: 26-OCT-2006

106 produce a decrease in photosynthe sis after 5 minutes ex-

posure. Reliability: (4) not assignable secondary quotation (131) (132)

4.6.3 Toxicity to Soil Dwelling Organisms

-

4.6.4 Toxicity to other Non-Mamm. Terrestrial Speci es

-

4.7 Biological Effects Monitoring

-

4.8 Biotransformation and Kinetics

-

4.9 Additional Remarks

Memo: more information see IPCS report No. 1 96

(88)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

107 5.0 Toxicokinetics, Metabolism and Distribution

- [See Section 5.11]

5.1 Acute Toxicity

5.1.1 Acute Oral Toxicity

Type: LD50 Species: rat Value: = 5628 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature

25-OCT-2006 (107)

Type: LD50 Species: rat Strain: Sprague-Dawley
Sex: male/female
No. of Animals: 10
Vehicle: other: as 15 - 35-% aqueous solution
Doses: 7914; 5390 and 3672 mg/kg
Value: > 7914 mg/kg bw

Method: other: BASF-Test
Year: 1975
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: No mortality was observed in any dose gr oup. No pathological findings after po st-observation period of 7 days. key study Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Comparable to guideline study, li mited documentation, acceptable for assessment, key st udy
25-OCT-2006 (133)

Type: LD50 Species: rat Strain: Sprague-Dawley Value: = 7000 mg/kg bw

Year: 1971
GLP: no Test substance: no data

Remark: The LD50 was determined in adult male, o ld rats (300-470 g). non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

108 25-OCT-2006 (134)

Type: LD50 Species: rat Strain: Sprague-Dawley Value: = 10300 mg/kg bw

Year: 1971
GLP: no Test substance: no data

Remark: The LD50 was determined in young adult m ale rats (80-160 g). non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source

25-OCT-2006 (134)

Type: LD50 Species: rat Strain: Sprague-Dawley Value: = 5800 mg/kg bw

Year: 1971
GLP: no Test substance: no data

Remark: The LD50 was determined in rats in 14-d old rats, weight 16-50 g, not yet sexually mature. non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature

25-OCT-2006 (134)

Type: LD50 Species: rat Strain: Wistar
Sex: male/female
No. of Animals: 10
Vehicle: water
Doses: applied in log scale
Value: = 13000 mg/kg bw

Method: other
Year: 1941
GLP: no Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Comparable to guideline study, ho wever, documentation
limited.

25-OCT-2006 (135) (136)

Type: LD50

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

109 Species: mouse Strain: C57BL Value: = 7300 - 10000 mg/kg bw

Method: other: no details reported
Year: 1982
GLP: no data Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature

25-OCT-2006 (88) (137)

Type: LD50 Species: rabbit
Value: = 14400 mg/kg bw

Method: other: no details inidicated
Year: 1972
GLP: no data Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature

25-OCT-2006 (138)

Type: other: LD values Species: rat Strain: no data Vehicle: water
Doses: no data

Year: 1955
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Methanol was administered as 50-% aqueou s solution by

gavage. non-BASF study Result: Severe narcosis was produced without lat ency, however, no acidosis and no changes of the ey e fundus.

Mean LD10 was at 9000 mg/kg mean LD70 was at 9500 mg/kg mean LD100 was at 10000 mg/kg.
Test substance: Methanol, 99.5 % pure from Merck, no further dat a. Reliability: (4) not assignable Secondary source

25-OCT-2006 (139)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

110 Type: other: lethal dose Species: rabbit
Value: = 7000 mg/kg bw

Method: other: no details indicated
Year: 1955
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: 7 g Methanol/kg bw per gavage led to mor tality within 1 - 3 days. All animals showed ataxia, nystagmus and coma but no acidosis and no ophthalmological changes. non-BASF study Test substance: Methanol, 99.5 % pure from Merck, no further dat a. Reliability: (4) not assignable Secondary source

25-OCT-2006 (139)

Type: other: lethal dose Species: dog Value: >= 4000 - 7000 mg/kg bw

Method: other: only single animals were used
Year: 1955
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Methanol was applied orally together wit h each 20 g Saccharose to prevent irritant ef fects. The animals receiving 9000 mg/kg body weight died within 46 hours, one after 4000 mg/kg, whil e 7 other animals receiving between 2500 and 8000 mg/kg bodyw eight survived. All animals showed signs of an in fluence on the central nervous system like ataxia, nysta gmus, coma, narcosis, euphoria and vomiting. No ophthal mological changes occurred and mostly no acidosis. non-BASF study Test substance: Methanol, 99.5 % pure from Merck, Germany, no fu rther data. Reliability: (4) not assignable Secondary source

25-OCT-2006 (139)

Type: LD50 Species: monkey Strain: other: Rhesus macaca
Sex: no data No. of Animals: 12
Vehicle: water
Doses: 500-4000, 5000, 6000, 7000, 8000, and 9 000 mg/kg
Value: ca. 7000 - 9000 mg/kg bw

Year: 1961
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Result: There was no evidence of marked acidosis after high doses (no hyperventilation, no increase in urinary excretion of

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

111 organic acids, or shift in serum bicarbonate). Signs of acidosis in only one monkey recei ving 6 g/kg were not accompanied by the characteristic "Kussmaul respiration" (Cooper and Felig, 1961).

Blindness was not generally noted , but only 4 days after exposure in one very high-dosed m onkey (9 g/kg) who survived. This effect apparently was transient, because the animal showed again normal respon ses to visual stimuli and normal behaviour by the following day (Cooper and Felig, 1961).

Characterisation of typical sympt omatology: - Ataxia, weakness, and lethargy: 1 to 2 h after exposure and disappeard with 24 h. - Transient coma: Usually 0.5 h a fter exposure intoxication evident: progressive ataxia, weak ness, and lethargy resulting in coma 1 to 4 h later for 5 to 12 h, but appeared to fully recover after 24 h. - Death: previous narcosis then d eath within 20 to 30 h. (Cooper and Felig, 1961).

------------------- Test condition: Methanol was administered orally by catheder, di luted to 20
- 30 % in water.
12 animals received doses from 0. 5 to 9 g/kg, with 7 and 8 monkeys at sublethal doses and wi th 6 to 2 monkey at lethal doses. Some animals were re-used after a time of recovery, on the whole 28 applications with 12 animals.

---------------- Test substance: Methanol, 99.9 % pure, Fisher Scientific Comp.
Conclusion: The lethal dose in this study (Cooper and Fe lig, 1961) was significantly higher than publish ed elsewhere for monkeys (minimal lethal dose at approx. 3 000 mg/kg from Potts, 1955; Gilger et al., 1956) and also abo ve reported fatal doses in humans (approx. 3000 mg/kg). In t his study 6000 mg/kg were sublethal, while Gilger et al.(19 59) found a mortality of
78%. The clinical picture was always o ne of intoxication progressing to a narcotic state w hich in fatal cases was
irreversible. The characteristic features of human poisoning, such as a latent perio d, rapid and deep breathing, vomiting, and blindnes s, were almost never be found, but may temporarily occur. Therefore, acute methanol intoxication in monkeys appears t o resemble that in rodents and other lower animals (Cooper a nd Felig, 1961).

------------------ Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, limited, but sufficientl y documented

25-OCT-2006 (140) (141) (142) (143)

Type: other: intoxication and lethality Species: monkey Strain: other: Rhesus No. of Animals: 8
Doses: 6000 mg/kg

Year: 1955
GLP: no

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

112 Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Result: Eight young adult rhesus monkeys receive d 6000 mg/kg methanol by gavage.

After 1 h: narcosis at maximum af ter 4 h. after 15 h: normal appearance wit hout apparent anomalities;
after 20 - 24 h: marked decrease in alkaline blood capacity. Following bicarbonate supplementa tion, 4/8 animals survived.

Clinical observations: Eye fundus: In case of mild intox ication symptoms, no or only transitory alterations of th e eye fundus were observed. In severe cases, an extended edem a of the retina and the optic papilla as well as wide, no n-responsive pupils.

Histological changes: 6/8 animals exhibited cystic degeneration of the outer retinal granular layer, in 1 animal there was evidence of sign ificant demyelinisation of the optic nerve.

In the elektroretinogramme, a-wav e signal was negative, while the b-wave was extinguishe d.

Neurological symptoms were clinic ally expressed as rigor and tremor, histological lesions were seen in the putamen and nucleus caudatus (3/8 animals).

----------------------- Test substance: Methanol, 99.5 % pure from Merck, no further dat a.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

113 Reliability: (2) valid with restrictions

25-OCT-2006 (144) (143)

Type: other: lethal dose Species: monkey Value: >= 3000 - 8000 mg/kg bw

Method: other: only single animals were used
Year: 1955
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Methanol was applied orally to male rhes us monkeys. 1 to 2 g/kg bodyweight did not lead to m ortality while animals receiving 3 to 8 g/kg bodyweight died within two days. Treated animals showed acidosis a nd partly semicoma and ophthalmological changes. 20 g Sa ccharose was applied concurrently to prevent irritant effects (Gilger and Potts, 1955).

[Compare also Cooper and Felig, 1 961 (other entry this Chapter) who found no mortality i n this dose range.] non-BASF study Test substance: Methanol, 99.5 % pure from Merck, no further dat a. Reliability: (4) not assignable Secondary source

25-OCT-2006 (140) (139)

Type: LD50 Species: rat Strain: Sprague-Dawley Value: < 790 mg/kg bw

Year: 1971
GLP: no Test substance: no data

Remark: Neonatal rats, 24 - 48 h old, weight 5 - 8 g: The LD50 could not be exactly det ermined, but was below 790 mg/kg. non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (134)

Type: LD50 Species: rat Value: = 6200 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (107)

Type: LD50 Species: rat Value: = 9100 mg/kg bw

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

114 Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (145)

Type: LD50 Species: rat Value: = 12900 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (146)

Type: LD0 Species: rat No. of Animals: 10
Vehicle: water
Doses: limit test: 6.4 ml/kg
Value: >= 5065 mg/kg bw

Method: other: BASF-Test
Year: 1961
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Result: Oral application of 5065 mg/kg (6.4 ml/k g) did not lead to mortality in any of 10 animals us ed. Only clinical symptoms including staggering, narcotic ef fects. Postobservation period: 7 days Test condition: Methanol was administered as 50-% aqueous soluti on. Test substance: Methanol, containing 1.5 % water, no other conta mination Reliability: (2) valid with restrictions Comparable to guideline study, li mited documentation, acceptable for assessment
25-OCT-2006 (147)

Type: LDLo Species: mouse Value: = 420 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (107)

Type: LD50 Species: rabbit
Value: = 14200 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (148)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

115 Type: LDLo Species: rabbit
Value: = 7500 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (107)

Type: other: ND50 Species: rabbit
Value: = 6000 mg/kg bw

Test substance: no data

Remark: ND50: narcotic dose in 50 % of the anima ls used. non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature
25-OCT-2006 (138)

Type: LDLo Species: dog Value: = 7500 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (107)

Type: other: acute intoxication Species: miniature swine
Strain: other: minipig YU, CR
Sex: female No. of Animals: 3
Vehicle: water
Doses: 1000, 2500, or 5000 mg/kg (20 % solutio n)

Year: 1993
GLP: no data Test substance: no data

Method: Three animals were used per dose group a nd treated by

gavage. Control tests were conducted with formate administration.

------------ Remark: non-BASF study Result: Dose-dependent signs of acute intoxicati on including mild CNS depression, tremor, ataxia, a nd recumbency, developed within 1.5 to 2 h, and resolved b y 52 h. Methanol- and formate-dosed animals did not dev elop optic nerve lesions, toxicologically relevant formate accumulation, and metabolic
acidosis.

Average maximum methanol levels i n blood: 3100 +-700, 6200 +-2300, and 1520 0 +-900 ug/ml, respectively, within 0.5 to 4 h. T/2 ranged from about 9

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

116 (low dose) to about 19 and 22 h f or the higher doses.

Formate plasma levels: transientl y 1.74 - 3.4 mEq/L vs. 0.5 +-0.3 mEq/L (control) within 4 to 30 h post-treatment.

------------ Test substance: Methanol, no further data. Conclusion: The minipig, maintained on a normal folate-d iet, does not appear to be a satisfactory model for human methanol poisoning. Reliability: (2) valid with restrictions
25-OCT-2006 (149)

5.1.2 Acute Inhalation Toxicity

Type: LC50 Species: rat
Sex: male/female
No. of Animals: 100 Exposure time: 4 hour(s)
Value: 128.2 mg/l

Method: other: BASF-Test
Year: 1980
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: 10 male and 10 female Sprague-Dawley rat s (185 +-15 g bw) were tested per dose group. Follo wing analytical / nominal methanol vapor concentrations wer e tested in a head-nose inhalation system: 87.6 / 127 mg/l 115.9 / 159 mg/l 139.0 / 168 mg/l 150.9 / 193 mg/l 151.1 / 242 mg/l.

Mainly in the high concentrations , where also mortality occurred following clinical sympt oms were described: aqueous secretion of eyes and nose, close d eyes, laboring breathing, stagger, apathy, narcosis, prone position, opacity of the cornea. Animals died within 5 day s after substance administration. Necropsy showed c ardiac dilatation and hyperaemic congestion as well as pulmonary edema with extended or scattered hyperaemia.

Some animals, surviving, still sh owed some effect after the 14-day post obse rvation period. key study Test substance: Methanol, purity 99.8 % (Merck) Reliability: (2) valid with restrictions Comparable to guideline study, me eting current standards, acceptable for assessment, key st udy
25-OCT-2006 (150)

Type: LC50 Species: rat
Sex: male/female
No. of Animals: 160 Exposure time: 6 hour(s)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

117 Value: 87.5 mg/l

Method: other: BASF-Test
Year: 1980
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: 10 male and 10 female Sprague-Dawley rat s were tested per dose group. Following analytical / nominal methanol vapor
concentrations were tested in a h ead-nose inhalation system: 31.1 / 36.1 mg/l 64.2 / 93.3 mg/l 65.22 / 81.3 mg/l 66.5 / 103.0 mg/l 83.2 / 104.0 mg/l 88.36 / 121.9 mg/l 94.4 / 113.3 mg/l 160.0 / 197.6 mg/l.

Mainly in the high concentrations following clinical symptoms were described: aqueous secretion of eyes and nose, closed eyes, laboring breathing, stagger, apathy, narcosis, prone position, opacity of the co rnea. Animals were dying within 7 days after substance adm inistration. Necropsy showed cardiac dilatation and hyp eraemic congestion as well as pulmonary edema and scattered hyperaemia. Some animals of high concentratio n groups, surving, still
showed some effect after the 14 d ay post observation period.
No effect was observed in the low est concentration group. key study Test substance: methanol, purity 99.8 % Reliability: (2) valid with restrictions Comparable to guideline study, me eting current standards, acceptable for assessment, key st udy
25-OCT-2006 (151)

Type: LC50 Species: mouse Exposure time: 134 minute(s)
Value: = 79.43 mg/l

Method: other: no details reported
Year: 1994
GLP: no data
Test substance: no data

Remark: The substance concentration is given as 61100 ppm. non-BASF study Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Acceptable study, meets basic sci entific principles

25-OCT-2006 (152)

Type: other: narcotic dose Species: mouse Exposure time: 3.2 hour(s)
Value: = 40 mg/l

Year: 1936

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

118 GLP: no Test substance: other TS

Remark: 40 mg/l for 3.2 hours led to narcosis. non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature

25-OCT-2006 (153)

Type: LC50 Species: cat Exposure time: 6 hour(s)
Value: = 43.68 mg/l

Method: other: no details reported
Year: 1994
GLP: no data Test substance: no data

Remark: The substance concentration is given as 33600 ppm. non-BASF study Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Accpetable study, meets basic sci entific principles

25-OCT-2006 (152)

Type: other: narcotic dose Species: mouse Exposure time: 7 hour(s) Value: = 55 mg/l

Year: 1943
Test substance: no data

Remark: Exposure to 55 mg/l for 7 hours is repor ted as narcotic
dose. non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature

25-OCT-2006 (154)

Type: LC50 Species: cat Exposure time: 4.5 hour(s) Value: = 85.41 mg/l

Method: other: no details reported
Year: 1994
GLP: no data Test substance: no data

Remark: The substance concentration is given as 65700 ppm. non-BASF study Test substance: Methanol, no further data. Reliability: (2) valid with restrictions

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

119 Accpetable study, meets basic sci entific principles

25-OCT-2006 (152)

Type: LC50 Species: cat Exposure time: 6 hour(s)
Value: = 26 - 48 mg/l

Method: other
Year: 1931
Test substance: no data

Remark: Cats were exposed for 6 hours to concent rations of 26; 48; 97; 210 mg/l or for 3.5 hours to 380 mg/l. Concentrations of 26 and 48 mg/l led to 50 % mortality whichincreased with higher concen trations. The number of
animals used is not indicated. non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary literature

25-OCT-2006 (155)

Type: other: lethal dose Species: monkey Strain: other: Rhesus
Sex: male/female
No. of Animals: 11 Exposure time: 18 hour(s)
Value: >= 13 mg/l

Method: other
Year: 1931
GLP: no Test substance: no data

Remark: Exposure to 1.3 mg/l (1000 ppm) for 41 h ours, to 10000 ppm (13 mg/l) for 18 h or to 52 mg/l (40000 ppm) for 1-4 hours led to mortality.

Blindness based on ophthalmology associated with optic atrophy is reported (conditions n ot specified). Recovery
from this lesion is observed. non-BASF study Test substance: Methanol, "synthetic", >95 % pure, no further da ta. Reliability: (2) valid with restrictions Test procedures based on scientif ic principles, limited documentation, acceptable for ass essment

25-OCT-2006 (156)

Type: LC50 Species: rat Exposure time: 4 hour(s) Value: = 83.2 mg/l

Method: other: no details reported
GLP: no data

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

120 Test substance: no data

Remark: Substance concentration is given as 6400 0 ppm. non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (152)

Type: other: Hormone status Species: rat Strain: Long-Evans
Sex: male No. of Animals: 10
Doses: 200, 5000 or 10000 ppm (0.26, 6.5 or 13 mg/l) Exposure time: 6 hour(s)

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Two experiments were conducted to evalua te the acute effects
of inhaled methanol on serum horm ones associated with reproductive function in the male rat: 1. 10 rats per group (60 d old) exposed to methanol (0, 200, 5000 and 10,000 ppm) for 6 h ours were killed at the end of the exposure period or the fol lowing morning. The effect of the handling associated with p lacing the rat in the exposure chamber was also evaluat ed by comparing hormonal changes in sham- and methanol-exp osed groups acclimated for two weeks with groups that were n ot acclimated. [note: Prior handling resulted in an increase in serum lutenizing hormone greater in the non-acclimated gr oups than in the acclimated group.]

2. Groups of acclimated and non -acclimated rats were exposed to 0 or 5000 ppm methanol for 1, 2 and 6 hours and killed immediately after removal from the chamber.

-------------------- Remark: non-BASF study Result: Serum luteinizing hormone, testosterone and follicle stimulating hormone values were n ot different in sham- vs methanol-exposed rats at any time point. An effect of prior handling was noted: In general, the concentrations of these hormones and serum prolactin in t he non-acclimated rats were greater than those observed for a cclimated rats. However, methanol exposure appeared to res ult in increased prolactin
concentrations under both handlin g conditions.

----------------------- Conclusion: Effects on serum hormone levels (luteinizing hormone, follicle stimulating hormone, tes tosterone, prolactine) were inconsistent with partly no effec ts and partly contradictory results. According to the author methanol treatment led to increased prolactin levels in ser um.

------------------------ Reliability: (2) valid with restrictions Test procedures based on scientif ic principles, sufficiently documented, acceptable for assess ment

25-OCT-2006 (157)

Type: other: IRT

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

121 Species: rat Exposure time: 7 hour(s)

Method: other: based on H.F. Smyth et al. Am. In d. Hyg. Ass. J. 23,95-107, (1962)
Year: 1980
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: No mortality was observed when rats were exposed for 7 hours to an atmosphere that has b een saturated at 20 degrees celsius with the volatile part of the compound. Irritant effects on the respiratory tract were observed. Test substance: Methanol, no further data.
25-OCT-2006 (158)

Type: other: IRT Species: rat No. of Animals: 6
Doses: approx. 150 ml/l (estimated from vapour saturation properties
and measured decrease in substanc e during vapour generation) Exposure time: 4 hour(s)

Method: other: BASF-Test
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Mortality was observed when rats were ex posed for more than 1 hour to an atmosphere that had been saturated at room temperature with the volatile par t of the compound (11 - 13 Vol%): 3/6 dead after 2 h; 6/6 de ad after 4 h. No mortality occurred up to 1 hou r. Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Screening test (159)

Type: other: Inhalation Risk Test Species: rat
Sex: male/female Exposure time: 8 hour(s)

Method: other: BASF-Test
Year: 1975
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: No mortaility was observed when 12 rats were exposed for 3 hours to an atmosphere that had b een saturated with the volatile part of the compound. Th e inhalation of this atmosphere over 8 hours caused mo rtality of all 6 rats
tested. Clinical symptoms were strong irr itation of mucosa, opacity
of the cornea and narcosis.

------- Test substance: methanol (160)

Type: other: lethal dose Species: rat

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

122 Exposure time: 18 hour(s)
Value: >= 41 mg/l

Method: other
GLP: no Test substance: no data

Remark: Six concentrations were tested at differ ent exposure times: Rats were exposed to Methanol con centrations of 2.6; 11.5; 30 mg/l 8 hours, 17 mg/l 0.33 hou rs, 65.5 mg/l 2.5 hours and 41 mg/l 18-20 hours. Only 41 mg/l for 18-20 hours was
lethal. non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (161)

Type: other: lethal dose Species: rat Exposure time: 6 hour(s)
Value: >= 41 - 91 mg/l

Method: other: various concentrations and exposu re times were used
Test substance: no data

Remark: Exposure to 65-100 mg/l for 1.5 hours an d 51-70 mg/l for 3 hours did not lead to mortality. Exposure to 70-111 mg/l for 3 hours or 30-120 mg/l for 6 hour s led to a dose related increase in mortality. Cl inical signs indicating effects on the central nervous sy stem occurred in treated
animals. non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source
25-OCT-2006 (162)

Type: LCLo Species: cat Exposure time: 6 hour(s)
Value: = 44 mg/l

Test substance: no data

Remark: non-BASF study
Test substance: Methanol, no further data.
25-OCT-2006 (107)

Type: LC0 Species: dog Exposure time: 1 hour(s)
Value: = 65.5 mg/l

Test substance: no data

Remark: Dogs were exposed for 24 hours to 2.6 mg /l, for 4 hours to 18 mg/l, for 8 hours to 48 mg/l o r for 1 hour to 65.5 mg/l. At 48 or 65.5 mg/l signs of effec ts on the central nervous system occurred. non-BASF study Test substance: Methanol, no further data.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

123 25-OCT-2006 (163)

5.1.3 Acute Dermal Toxicity

Type: other: lethal dose Species: rat Value: >= 45000 mg/kg bw

Method: other
Year: 1955
GLP: no Test substance: no data

Remark: Occlusive application of up to 35000 mg/ kg body weight to rats did not lead to mortality, while 45000 mg/kg was lethal. key study non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source , key study
25-OCT-2006 (164)

Type: LD50 Species: rabbit
Value: = 17100 mg/kg bw

Year: 1981
GLP: no data Test substance: no data

Remark: non-BASF study Result: No detailed data: LD50 given as 20 ml/kg

. Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source

25-OCT-2006 (165)

Type: other: lethal dose Species: monkey Strain: other: Rhesus
Sex: male/female
No. of Animals: 8
Doses: 4x 0.5 and 1.3 ml/(kg*d) under occlusiv e condition
Value: 1600 - 4000 mg/kg bw

Method: other: occlusive patch test
Year: 1931
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Result: Four doses of 400 mg/kg daily caused sic kness within 24 h, and eventually death. At 1000 mg/ kg (4 doses daily), severe illness takes place, followed by death the second day. Test substance: Methanol, "synthetic", >95 % pure, no further da ta. Reliability: (2) valid with restrictions Test procedures based on scientif ic principles, limited

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

124 documentation, acceptable for ass essment

25-OCT-2006 (156)

Type: LD50 Species: rabbit
Value: = 20000 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (107)

Type: LD50 Species: rabbit Value: = 15800 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (148)

Type: LDLo Species: monkey Value: = 393 mg/kg bw

Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data.
25-OCT-2006 (148)

5.1.4 Acute Toxicity, other Routes

Type: LD50 Species: rat Route of admin.: i.p.
Value: = 9540 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (107)

Type: LD50 Species: rat Route of admin.: i.p.
Value: = 7529 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD50 Species: mouse Route of admin.: i.p.
Value: 10500 - 11500 mg/kg bw

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

125 Test substance: other TS

Test substance: Methanol, no further data. (166)

Type: LD50 Species: mouse Route of admin.: i.p.
Value: = 10765 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD50 Species: mouse Route of admin.: i.p.
Value: 7914 mg/kg bw

Method: other: BASF-Test
GLP: no Test substance: as prescribed by 1.1 - 1.4

Test substance: Methanol, no further data. (160)

Type: LD50 Species: rabbit
Route of admin.: i.p.
Value: = 1826 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD50 Species: guinea pig
Route of admin.: i.p. Value: = 3556 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD50 Species: hamster Route of admin.: i.p.
Value: = 8555 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD0 Species: rat
Sex: male

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

126 No. of Animals: 25
Vehicle: physiol. saline
Route of admin.: i.p.
Value: >= 3000 mg/kg bw

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: Methanol was applied to male F344 rats i . p. at dose levels of 25; 125; 600 und 3000 mg/kg. A fter 1; 3; 10; 24; 30 und 48 hours a range of clinical chem ical parameters were determined (formate level, organi c acids, folic acid, pH, pCO2, glucose, urea, Na, K, Cl). The highest dose level led to a slight increased formate lev el in blood, but no decrease in the blood pH. No othe r parameters were changed. Test substance: Methanol, reagent special grade of Junsei Chemic als Co., no
further data. Reliability: (2) valid with restrictions (167)

Type: LDLo Species: mouse Route of admin.: i.p.
Value: = 120 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (107)

Type: other: effective dose Species: rat Route of admin.: i.p.
Value: >= 6000 mg/kg bw

Year: 1975
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Rats were given 6 g/kg bw Methanol i. p. . The treatment led to depressionen of the central ne rvous system for 4-6 hours. Test substance: Methanol, reagent grade, no further data. (168)

Type: other: lethal dose Species: rat Route of admin.: i.p.
Value: = 5065 mg/kg bw

Method: other: BASF-Test
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: The application of 2530 mg/kg did not le ad to mortality in 5 animals but 5065 mg/kg led to mor tality in 10 of 10 rats. Test substance: Methanol, no further data. (169)

Type: other: lethal dose

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

127 Species: mouse Route of admin.: i.p.
Value: >= 5065 mg/kg bw

Method: other: BASF-Test
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: The application of 1270 mg/kg did not le ad to mortality in 5 animals but 5065 mg/kg led to mor tality in 1 of 5 animals and 7910 mg/kg in 6 of 10 animals

. Test substance: Methanol, no further data. (169)

Type: other: lethal dose Species: monkey Strain: other: Macaca fascicularis
Sex: male No. of Animals: 5 Vehicle: physiol. saline

Doses: 0; 25; 125; 600 and 3000 mg/kg.
Route of admin.: i.p.
Value: = 3000 mg/kg bw

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: After 1; 3; 10; 24; 30 and 48 hours the following clinical chemical parameters were examined : pH; pCO2; formate, organ. acids, folate, glucose, ur ea, K, Na, Cl.

No abnormalities were seen up to 600 mg/kg bw. All monkeys exposed to 3000 mg/kg fell into a coma immediately after
administration and remained recum bent for 3 h to 2 d. Additionally, anorexia was observ ed. Autopsy and pathological examination revealed no lesions in the eye, optic nerve or central nervous sy stem except for slight effects in the liver (p. 65). Dur ing another part of the study (p. 66), 3 animal died afte r injection of 3000 mg/kg
within 24 to 48 h.

The highest dose level led to inc reased formate level, decreased pH in blood as well as delayed excretion of methanol and secondary products.

------------------------ Test condition: Results created within a metabolism study (NEDO, 1987, p. 61): Methanol was diluted in wate r and applied to male monkeys (Macaca fascicularis) i.p . A total of 5 monkeys were repeatedly used. Test substance: Methanol, reagent special grade of Junsei Chemic als Co., no
further data. Reliability: (2) valid with restrictions (167)

Type: other: lethal dose / acidosis Species: monkey Strain: other: adult rhesus Macaca mulatta (fema les) and Macaca nemestrina (pigtail monkey) (fema les + 1 male)
Sex: male/female

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

128 No. of Animals: 10
Vehicle: physiol. saline
Doses: 2000, 3000, and 4000 mg/kg bw (as 20- o r 25 % solution)
Route of admin.: i.p.
Value: = 4000 mg/kg bw

Year: 1975
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: Objective of the study was
1. to find the suitable model an imal to represent methanol intoxication in humans (acidose m odel) and 2. to identify those organic aci ds which are responsible for development of acidosis.

Blood buffer status was measured: pH, pCO2, CO2, bicarbonate. Further blood parameters: ion sta tus, glucose, urea. Blood formate was measured by col orimetric analysis. Formate
and formate-borne metabolites wer e analysed in blood, urine and exhaled air by using 14C-form ate. Blood controls were from the same test animals 12 h p rior to methanol exposure.

Organic acids were measured using GC-MS.

-------------------------- Remark: : The pigtail monkey was teste d for effects of
toxic methanol exposure to est ablish an animal model for
human methanol toxicity. Blood acidosis was measured as major toxic effect
of methanol and the responsible acid was
identified. In pigtail monk eys but not in rhesus monkeys significant
blood acidosis was produced by a single i.p. dose of 4 g/kg bodyweight meth anol. Blood concentration
of formate was correlated with acidosis, no other organic
acid was found
atincreased lev els in acidotic monkeys. Test substance: Methanol, reage nt grade, no further data. Mortality occurred at the highest dose level in one monkey, all treated animals showed depression
of the central nervous system and ac idosis Result: 1. Symptomatology and changes in vario us blood parameters

After 4000 mg/kg bw, i.p., 3/4 rh esus macacques exhibited no apparent signs of toxicity within 24 h and later, although a mild acidosis was produced: pH at 7.22 - 7.24, pCO2 at 30 - 34 mmHg vs pH 7.40 and 37 mmHg fo r the control.

1/4 rhesus macacque became progre ssively weaker after treatment and developed a severe metabolic acidosis which
led to coma and death: His blood values were pH = 7.03 and pCO2 = 14.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

129 After 4000 mg/kg bw, i.p., 4/4 pi gtail monkeys exhibited distinct signs of methanol intoxi cation: depression of the CNS within 30 min, thereafter app arently normal apperance, followed by weakness and apathy a t 15 - 18 h later and eventually coma and death.

All displayed a sharp decrease in blood pH (7.03 - 7.10) and in pCO2 (15 to 20, and 33 mmHg): 1/4 died after 4-h coma and 22-h post treatment, 2/4 recovere d from coma, 1/4 in coma
after 32 h and sacrificed after 3 6 h. The acidosis developed within about 10 - 12 h accompanie d and followed by a marked decrease in blood bicarbonate, to tal CO2 and blood pH over about 20 h post-treatent.

The two pigtail monkeys given 200 0 and 3000 mg/kg showed no apparent signs of toxicity, but d isplayed a mild acidosis (pH 7.21 and 7.29; pCO2 30 and 32 mmHg).

Inhibition of ADH prior to methan ol treatment (4- methylpyrazole, 50 mg/kg i.v.) pr evented the occurrence of acidosis in the pigtail monkey at a dose which otherwise routinely produced acidosis.

----------------------------- Blood analysis identified only bi carbonate as the sole ion to change during development of a cidosis. Additionally, an time-dependent increase in blood glucose was evident (<=106 mg/100 ml vs. 73 mg/100 ml of the control).

------------------------------

2. Blood organic acids Bood formate increased steadily i n pigtail monkeys given 4000 mg/kg methanol and reached > 800 mg/l (note: In SD rats given 6000 mg/kg, no increase occ ured. Concomitantly,
bicarbonate declined. Other metabolic organic acids mea sured (lactate, alpha-OH-butyrate, beta-OH-butyra te, alpha-ketobutyrate, etc.) showed a significant increa se versus control values,
but the excess sum in these anion s accounted for only about 0.5 mEq./L anf therefore, for not more than 2 - 3% of the observed decrease in bicarbonate (p. 54).

3. Formate elimination
The calcualted half-lifes for NaF ormate (50 mg - 470 mg/kg bw, i.v.) ranged from 31 min (50 mg/kg) to 51 min (470 mg/kg) in monkey (nor specfied wh ich, and no comparisao between both strains). [note in rat: half-lifes were sig nificant shorter from 12 min (100 mg/kg) to 23 min (670 mg /kg) (p.57)]

----------------------- ----- Test substance: Methanol, reagent grade, no further data. Conclusion: The pigtail macacque appeared to serve as a suitable experimental animal in which to s tudy human methanol acidosis: Biochemical and clinica l parameters as well as the parallelism of the time course fo r the development of symptoms and acidosis observed in human cases of acute intoxication are comparable to th ose found in this monkey.

Results support the view that for mic acid is the major agent to produce acidosis and methanol intoxication in monkeys and

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

130 humans: I appears that formate, a fixed base, replaces bicarbonate, a volatile base, in the blood and that the resultant decrease in blood buffe r capacity renders the organismen incapable of mantainin g blood pH within normal limits (p. 58).

---------------------- Reliability: (2) valid with restrictions 1c: Test procedures in accordance with accepted standard methods, sufficiently documented, acceptable for assessment (168) Type: LD50 Species: mouse Route of admin.: s.c. Value: ca. 4160 mg/kg bw

Test substance: other TS

Remark: LD50 in newborn animals.
Test substance: Methanol, no further data. (170)

Type: LD50 Species: mouse Route of admin.: s.c. Value: = 4100 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (107)

Type: LD50 Species: mouse Route of admin.: s.c. Value: = 9800 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (107)

Type: LD50 Species: rat Route of admin.: i.v.
Value: = 2131 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD50 Species: mouse Route of admin.: i.v.
Value: = 5673 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (107)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

131 Type: LD50 Species: mouse Route of admin.: i.v.
Value: = 5660 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (171)

Type: LD50 Species: mouse Route of admin.: i.v.
Value: = 4710 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD50 Species: mouse Route of admin.: i.v.
Value: = 4710 mg/kg bw

Method: other: no details reported
GLP: no data Test substance: other TS

Test substance: Methanol, no further data. (172)

Type: LD50 Species: rabbit
Route of admin.: i.v.
Value: = 8907 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: LD0 Species: dog
Route of admin.: i.v.
Value: = 4700 mg/kg bw

Test substance: other TS

Remark: Methanol was applied i. v. to 4 female d ogs at dose levels of 2.5 - 4.7 mg/kg bodyweight. No mortality occurred but
ataxia and severe inflammation of the eyes with blindness for some days. Test substance: Methanol, no further data. (173)

Type: LDLo Species: cat Route of admin.: i.v.
Value: = 118 mg/kg bw

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

132 Test substance: other TS

Test substance: Methanol, no further data. (107)

Type: LDLo Species: cat Route of admin.: i.v.
Value: = 4641 mg/kg bw

Test substance: other TS

Test substance: Methanol, no further data. (148)

Type: other: lethal dose Species: rabbit
Route of admin.: i.v.
Value: = 4200 mg/kg bw

Test substance: other TS

Remark: 4200 mg/kg Methanol applied as 50 % solu tion led to mortality after 25 min.. Test substance: Methanol, no further data. (139)

5.2 Corrosiveness and Irritation

5.2.1 Skin Irritation

Species: rabbit
Concentration: 100 % Exposure: Occlusive Exposure Time: 20 hour(s)
No. of Animals: 5 Result: not irritating
EC classificat.: not irritating

Method: other: BASF-Test
Year: 1975
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: The irritation potential of methanol was examined after several exposure periods of 1, 5, and 15 min and 20 h.
Effects were assessed 24 h and 8 days after treatment according to Draize scoring.

A piece of gauze was soaked with an unspecified amount methanol, placed on the shaved sk in and covered by an impervious bandage.

20-h exposure was conducted on th e skin of the back as well
as the ear.

-------------- Remark: key study Result: After 1, 5, 15 min and 20 h of exposure, no signs of irritation were apparent after 24 h and 8 d.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

133 Test substance: Methanol, not further specified Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented, acceptable for assessment , key study
25-OCT-2006 (133)

Species: rabbit
Concentration: 500 mg
Result: moderately irritating

Method: other: no data
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source

25-OCT-2006 (174)

5.2.2 Eye Irritation

Species: rabbit
Concentration: 100 %
Dose: .05 ml
Comment: not rinsed
No. of Animals: 2
Result: irritating

Method: other: BASF-Test
Year: 1975
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: After 1 h, mild erythema and cornea opac ity as well as
moderate edema associated with se cretion were observed. After 24 h, the effects were asse ssed as mild. After 8 days the animals were wit hout symptoms. key study Test substance: methanol, not further specified
Conclusion: This screening test gives evidence of mild t o moderate
irritation of the mucous membrane on contact with pure methanol, but which is reversible and, therefore, gives no rise to classification as eye irr itating.

----------------- Reliability: (2) valid with restrictions Comparable to guideline study and current standards, limited
documentation , key study
25-OCT-2006 (133)

Species: rabbit
Concentration: undiluted
Dose: .1 ml
Comment: not rinsed
No. of Animals: 6
Result: slightly irritating
Method: OECD Guide-line 405 "Acute Eye Irritati on/Corrosion"

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

134 Year: 1990
GLP: no data Test substance: no data

Remark: non-BASF study Result: New Zealand White rabbits treated accord ing to OECD guidelines and assessed based on Draize scoring criteria exhibited the following mean scor es for irritation effects:

Time after application [h]
1 4 24 48 72

--------------------------------- ------------------- Me an scores conjunctivitis 0.89 2.00 1.67 2.28 2.22 chemosis 2.00 2.00 0.67 1.00 0.50 iritis 0.33 1.00 1.00 1.00 0.33 corneal opacity 0.00 0.00 0.50 0.50 0.67

--------------------------------- --------------------

Eight- and 14-d findings not show n. Test substance: Methanol, no further data. Conclusion: The mean scores of the single symptoms did n ot exceed the respective limits for classificat ion: Therefore, this result can be interpreted in terms of a mild irritation potential not requiring classification as e ye irritating.

--------------------------- Reliability: (2) valid with restrictions Guideline study, limited document ation, only abstract , key study
25-OCT-2006 (175)

Species: rat
Dose: other: saturated air (20 °C) Exposure Time: 3 hour(s)
Comment: other: vapour No. of Animals: 12
Result: irritating

Method: other: inhalation risk test
Year: 1975
GLP: no Test substance: as prescribed by 1.1 - 1.4

Result: Clinical symptoms were strong irritation of mucosa, opacity of
the cornea. No mortaility was observed when 1 2 rats (male/female) were exposed for 3 hours to an atmosph ere that had been saturated with the volatile part of the com pound.

------- Reliability: (2) valid with restrictions Acceptable study, meets basic sci entific principles

28-DEC-2004 (160)

Species: rabbit
Result: irritating
EC classificat.: irritating

Method: other: Smyth Carpenter
Year: 1946

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

135 Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source
25-OCT-2006 (176)

5.3 Sensitization

Type: Guinea pig maximization test Species: guinea pig
Concentration 1st: Induction 50 other: % active substance with a nd without Freund ́s Adjuvans (see Method) intracutaneous

2nd: Induction 50 % active substance open epicutan eous 3rd: Challenge 50 % active substance occlusive epi cutaneous No. of Animals: 34
Vehicle: water Result: not sensitizing

Method: other: modified Magnussen-Kligman test
Year: 1979
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: Two studies were conducted using 10 test animals (1st study) and 24 test animals (2nd study) w ith each 5 control animals. During challenge in the first stu dy, a formaldehyde solution (25 %) was applied in order to id entify possible sensitisation effect that might b e caused by metabolism-related transformation of methanol to formaldehyde.

The induction scheme was modified as follows (1 wk between each treatment): 0.1 ml volume, 50 % test substance - 1st intradermal induction (in cluding 6 parallel injections with Freund ́s Adjuvans solely, with methanol solely and both combined) - 1st epidermal induction - 2nd intradermal induction (in cluding 4 parallel injections with methanol solely a nd combined with Freund ́s Adjuvans) - 2nd epidermal induction.

--------------- Remark: key study Result: In the first study, no contact sensitisa tion was observed, in the parallel test with formald ehyde, also a negative
result was seen.

In the second study using 24 fema le animals (two tests à 12 animals), 1/12 (test 1) and 2/12 (test 2) exhibited a slight skin response (score 1) after 24 and 48 h which can be interpreted as a weak sensitising potential.

The intracutane induction produce d necroses and some open ulcerations.

---------------- Conclusion: In summary, the low number of 3/24 animals w ith score 1 gives no evidence of a notable se nsitisation potential of
methanol.

### OECD SIDS METHANOL

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136 Reliability: (2) valid with restrictions , key study
25-OCT-2006 (177) (178)

5.4 Repeated Dose Toxicity

Type: Chronic Species: monkey Sex: no data Strain: Macaca Fascicularis
Route of administration: inhalation Exposure period: a) 7 months; b) 1 year + 7 months; c) 2 ye ars + 5
months Frequency of treatment: 21 h/d Post exposure period: none Doses: 1000; 100; 10 ppm (1.3; 0.13; 0. 013 mg/l) Control Group: yes, concurrent no treatment
NOAEL: 10 ppm
LOAEL: 100 ppm

Method: other: see Method
Year: 1985
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term studies, reproductive a ssays and carcinogenicity
studies: Whole-body exposure: 4 monkeys we re housed in one inhalation
chamber.

8 animals per exposure group were used. Sacrifices were conducted 7 months (2/group), 19 months (3/group), and at 29 months (3/group) (Tab. 3, p. 29). A further group at 1000 ppm was included for recovery (30 d and 6 months) (p. 31 f).

Clinical, clinical-chemical, hema tological, ophthalmological, and pathologica l/histopathological
examiniation were done. Furthermo re, endocrine status was tested after 1 year and 7 months as well as after 2 years
and 5 months.

Analytical control of chamber con centration: Analytical values close to nominal ones. (p. 20 ff)

------------------------- Remark: The blood levels of methanol and formate were not measured. Note: Exposure per day was 20 h, which implies that prolonged steady-state blood lev els were reached which even may have been higher than in stud ies using the same exposure concentration, but shorter exposu re times.

Relevance of observations limited by the low number of animals available at each time in terval.

-------------------- key study non-BASF study Result: The animals showed normal body-weight ga in, food and and water consumption throughout. The monkeys of the 1000-ppm group showed abnormal scratching of the skin and clinical

### OECD SIDS METHANOL

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137 symptoms such as frequent yawning and nasal discharge (see also recovery test, p. 31 f).

In both upper dose groups, the EC G of 1/8 of the 100-ppm exposed monkey and 3/8 of the 100 0-ppm exposed monkeys showed abnormalities (negative T- or Q-wave changes and flattening of T-wave) which indic ated slight myocardial disorder (p. 21).

Examinations of the eye fundus re vealed no changes in any

group.

Haematological examinations revea led no deviations from normal in Hb, Ht, red and white b lood-cell count, or percentage of white blood cells i n any group.

No significant differences in ser um values were seen, but one monkey in the 100-ppm group f or most liver parameters (total protein, GOT, GPT, total c holesterol, free cholesterol, thymol and glucose). This was not considered
treatment-related.

BRAIN: In both upper exposure groups, th e number of responsive stellate cells containing increas ed endoplasmic reticulum (minimal hypertrophy of astroglia ) appeared in the basal ganglions after 1 year and 7 mont hs of exposure. In the cerebral white substance, slight hyperplasia of the astroglia was noted in the 100 an d 1000-ppm groups, in particular in the 100-ppm group a fter 7 months (NEDO, 1987, p.53). Yet, this was not characte ristic of a degenerative process and was unrelated to dosi ng, because no such phenomena were noted after 2 year s and 5 months.

In the cerebral white substance, diffuse increases of responsive stellate cells centere d in the subcortical white
substance and the semioval center of the frontal and parietal lobes was noted at the h igher dose groups, but also emerged in 3/8 monkeys of the 10- ppm group after 29 months (p. 23). This was apparently a re versible effect, as shown from monkeys after recovery.

There were a few cases of degener ation of the optic nerve and the corpus geniculatum latera le after 7 months and 19 months (ophthalmencephalon). In o ne or two cases in the groups of 8 animals exposed for 2 years and 5 months, including the 10-ppm group, slight degeneration of the optic nerve was suspected, but not considered as significant (p. 23).

Slight degeneration processes inc luding increased responsiveness of the astroglia w ere noted in the thalamus after exposure to 100 and 1000 mg for 7 months and longer (p. 24).

Peripheral Nervous System: One monkey at 0.13 mg/L and 2 at 1.3 mg/L showed slight but clear changes in peroneal nerves; the authors concluded that
these effects show that methanol causes damage to peripheral nerves (p. 25/26). There were mild degenerations of the livers and kidneys

### OECD SIDS METHANOL

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138 which showed no clear correlation with exposure levels and exposure time. There was some evi dence of an increase in fatty granules in liver parenchym a at 1000 ppm along with signs of fibrosis in the hepatic cord more pronounced than at the lower concentration. Sudan -positive granules were observed at 100 and 1000 ppm in t he renal tubular epithelium. (NEDO, 1987, p. 26/27).

There was an increase in the Suda n-positive granules in heart tissue at 1000 ppm, but not manifested in any morphological lesion of the heart muscle (e.g. fibrosis).

LUNG and TRACHEA: There were no cases of pneumonia in any of the exposed monkeys. In the lungs, fibrosis w as seen in the interalveolar space. However, no dose response was observed, and the effect was also seen in the controls. In the trachea, atrophy of the epithelium of the
mucous membranes and reduction of goblet cells were observed in a total of 4 cases of exposed animals, but not correlated
with the amount of methanol inhal ed. This was not seen in the controls (p. 28).

--------------------------- Test condition: Exposure time 21 hours per day, significantly lo nger than according to guideline (6 hrs) (s ee p. 5). This does not represent normal industrial and c onsumer use.

--------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride, <3 ppm for maldehyde

------------ Conclusion: Taking into account slight myocardial efects and slight hyperplasia of the astroglia in t he cerebral white substance already noted at 100 ppm, then al ready 100 ppm (130 mg/m3)
have to be defined as LOAEL with a NOAEL of 10 ppm (13 mg/m3).

But no clear adverse effects are reported at 1000 ppm:
no necrotic effects occurred in t he nervous tissue. Hyperplasia of astroglia, not considered as degenerative, might be a transien t methanol-dependent effect which appears to subside upon ces sation of long-term methanol exposure (p. 52/53). But there was no clear correlation to the exposure conce ntration and time. Therefore, the biological relevan ce is questionable.

There was evidence of an increase in mild fatty changes in the liver and kidney upon exposur e to 1000 ppm, which were associated with signs of fibrosis . This effect was still present after recovery (see other entry). Given the small histological effect (p. 27), the pathological relevance appears to be low, but indicates that long-term exposure to 1000 ppm methanol is on the borde rline to toxicologically relevant, histological manifestat ions (authors statement, p. 27).

There were histological signs of diffuse responsiveness of the astroglia in some parts of th e cerebral white substance at all exposure levels, at high e xposure after shorter time period, after 10 ppm after 29 mon ths in a few animals. The relevance was not clearly comment ed by the authors.

### OECD SIDS METHANOL

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139 However, under test conditions (c ontinuous exposure), there
was no evidence of irreversible e ffects arising from long-term exposure to up to 1000 ppm methanol.

Therefore, the NOAEL for continuo us exposure may be established at 1000 ppm (1300 mg/ m3)m, but the low number of animals and limited description d o not allow to draw firm conclusions on the apparent neura l effect.

-------------------------- Note: This study served as basis for risk assessment by Vyskocil and Viau (2000): A 1-h r eference concentration (RfC) was derived to be at about 100 mg/m3, taking into account sensitive people. A very conservative chronic RfC was obtained and proposed at 0.38 mg/m3, based on the assumption that 10 ppm has to be used as NOAEL and 100 ppm as the LOAEL for "neurotoxic effe cts". This appears to be not in compliance with the author s ́ observations and is not suggested in the report.

------------------------------ --- Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, however, documentation and number of anaimals
limited. , key study
25-OCT-2006 (167) (179)

Type: Chronic Species: rat Sex: male/female
Strain: Fischer 344/DuCrj
Route of administration: inhalation Exposure period: 12 months Frequency of treatment: continuous, average about 20 h/d, total exposure time 7318 - 7341 h (males); 747 4 - 7496 h (females) Post exposure period: none Doses: 10; 100; 1000 ppm ( 0.013; 0.13; 1.3 mg/l) Control Group: yes, concurrent vehicle
LOAEL: 1000 ppm
NOEL : 100 ppm

Method: other: in accordance with OECD Guideline Oct. 1980
Year: 1983
GLP: no data
Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity studies (NEDO, 1987): Whole-body exposure: The animals were housed individually in wire-mash cages attached to the i nhalation chamber.

20 animals per sex and dose group were tested. Clinical, ophthalmological, clinical-chemic al, hematological and pathological/histopathological ex aminiation were done. The latter included among others the optic nerve, the eye, and reproductive organs.

Analytical control of chamber con centration: Analytical

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

140 values close to nominal ones.

------------------------- Remark: The blood levels of methanol and formate were not measured. key study non-BASF study Result: One female rat of the 1000 ppm dose grou p and one male rat of the 10 ppm dose group died or were sacrificed in
extremis. This was considered spontaneous o n the basis of the absence of a consistent dose relationship

.

A very slight transient suppressi on of body-weight gain was
observed for males and females of the 1000 ppm dose group from week 27 through 44 (less tha n 5 %). This has to be attributed to temporary occurrenc e of slight diarrhea in these groups during this period. The otherwise addressed significant decreases in body wei ght at the end of the study are not noticable in Fig. 3 repre senting the time-course of body weight development. The high -dose males showed a minimal, but significant decrease in food consumption from week 30 to the end. However, this was not more than about -5
% from the control.

Data of the organ/body weight rat io revealed a dose-related upward tendency in the liver and spleen for females which remained within a 5-% range.

Urinalysis showed no changes sugg esting effects from exposure to methanol. Hematologic examinations revealed no clear changes which could be attributed to exposure t o methanol. Serum biochemical examination sho wed a small tendency to decrease for alkaline phosphatase , the enzymatic activities of GOT, GPT, LDH and gamma-GTP di d not show any differences. Free fatty acid showed lower valu es in all exposure groups without dose-response relationshi p. Cholesterol and triglyceride remained unchanged. Changes of other clinical-chemica l parameters showed no correlation with the methanol exp osure.

Histopathological examinations sh owed a variety of non-tumoral changes in various or gans, many of them were infrequent findings which were po ssibly accidental. For tumoral changes, similarly, almos t all the findings observed were considered spontaneous ones due to aging.

----------------------------- -- Test condition: Exposure time 20 hours per day, significantly lo nger than according to guideline (6 hrs). T his does not represent
normal industrial and consumer us e.

--------------- Test substance: Methanol of first grade by Junsei Chemicals, <1 ppm vinyl chloride, <3 ppm formaldehyde

----------------- Conclusion: According to the authors, only in the 1000 p pm dose-group changes could be considered treat ment-related. But based on the minor degree and severity, th ese changes have no pathological meaning and may be c onsidered as toxicologically
irrelevant.

Levels of 100 ppm or less (at 20 h/d) did not produce any

### OECD SIDS METHANOL

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141 effect (= NOEL). 1000 ppm is adop ted as LOAEL, although it may
be considered as a NOAEL.

(see also IPCS/WHO, 1997)

----------------------------- Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, documentation limited. , key study
25-OCT-2006 (88) (167)

Type: Chronic Species: mouse Sex: male/female
Strain: B6C3F1
Route of administration: inhalation Exposure period: 12 months Frequency of treatment: continous, average ca. 20 h, total 7202 - 7225 h (males); 7352 - 7373 h (fe males) Post exposure period: none Doses: 10; 100; 1000 ppm (0.013; 0.13; 1.3 mg/l) Control Group: yes, concurrent no treatment
NOAEL: 1000 ppm
NOEL : 100 ppm

Method: other: in accordance with OECD Guideline Oct. 1980
Year: 1983
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity studies (NEDO, 1987): Whole-body exposure: The animals were housed individually in wire-mash cages attached to the i nhalation chamber.

30 animals per sex and dose group were tested. Clinical, ophthalmological, clinical-chemic al, hematological and pathological/histopathological ex aminiation were done. The latter included among others the optic nerve, the eye, and reproductive organs.

For interim examination, 10 anima ls each were sacrificed
after 6 months.

Analytical control of chamber con centration: Analytical
values close to nominal ones.

------------------------- Remark: The blood levels of methanol and formate were not measured. Note: Exposure per day was 20 h, which implies that prolonged steady-state blood lev els were reached which even may have been higher than in stud ies using the same exposure concentration, but shorter exposu re times.

------------- key study non-BASF study Result: One female animal of the 100-ppm group d ied on day 174,
another one had to be killed in m oribund state on day 178.

During the whole study, there wer e no changes in the general

### OECD SIDS METHANOL

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142 appearance that could suggest any relation to treatment.

The body-weights of both sexes of the treated groups tended to be higher than the controls in the second half of the study (NEDO, Fig. 3, p. 168).

There was a decreased food consum ption in high-dose females during the first 2 months and aft er 7 months without effect on body-weight gain.

All clinical and blood test faile d to reveal any methanol-related alteration in pa rameters from normal.

In the group of high-dose females , an increase in organ weight with significant differenc es was noted for the liver after 6 months and for the kidney and spleen after 12 months, however, the latter witho ut statistical significance and a clear dose-response.

After 12 months, the incidence of more severe fatty degeneration of hepatocytes was p ronounced in high-dose males: This was graded as moderat e in 8/20, mild in 8/20 and negative in 4/20 surviving males vs. 1/20 (moderate), 9/20 (mild) and 10 negative in the mal e control group. On the other hand, this effect was commo n and found to similar extent in untreated/external male mice. The female mice showed no difference among the gr oups.

-------------------------- Test condition: Exposure time 20 hours per day, significantly lo nger than according to guideline (6 hrs). T his does not represent
normal industrial and consumer us e.

--------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride, <3 ppm for maldehyde

------------------------------ - Conclusion: According to the authors, only in the 1000 p pm dose-group changes could be considered treat ment-related. But based on the minor degree and severity, th ese changes have no pathological meaning and are cons idered as toxicologically
irrelevant.

Levels of 100 ppm or less did not produce any effect (= NOEL). 1000 ppm can be established as NO AEL, while it is the LOEL for non-pathological, apparently treatment-related effects.

(see also IPCS/WHO, 1997)

----------------------------- Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, however, documentation limited. , key study
25-OCT-2006 (88) (167) (180)

Type: Sub-chronic Species: rat Sex: male/female
Strain: Sprague-Dawley Route of administration: gavage Exposure period: 90 days Frequency of treatment: daily

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

143 Doses: 0, 100, 500 or 2500 mg/kg body w eight Control Group: yes, concurrent vehicle
NOAEL: = 500 ml/kg bw
LOAEL: = 2500 mg/kg bw

Method: other: EPA Test protocol
GLP: yes Test substance: no data

Method: 30 animals per sex and group were used. After 6 weeks, 10 rats per sex and dose group were subjected to interim
sacrifice. The following parameters were exa mined: bodyweight, food consumption, clinical signs, opht halmological evaluations, mortality, blood and urine chemis try, gross and microscopic
evaluations.

------------------ Remark: non-BASF study Result: Elevated levels of SGPT, SAP and increas ed (not statistically significant) liver weights in males and females of the high dose group. B rain weights of both high dose males and females were signi ficantly less than those of controls. There were no histop athological effects.
The NOAEL was determined at 500 m g/kg/day, based on increased liver enzyme levels and reduced brain weights.

--------------------- Test substance: Methanol, no further data. Conclusion: The subchronic NOAEL was determined at 500 m g/kg/day, based on increased liver enzyme levels and reduced brain weights. Due to the low pathological relev ance of effects, this value
is considered to be a NOEL.

---------- Reliability: (4) not assignable Guideline study, only abstract av ailable

25-OCT-2006 (181) (182)

Type: Sub-acute Species: rat Sex: male/female
Strain: Sprague-Dawley Route of administration: inhalation Exposure period: 4 weeks Frequency of treatment: 6 h/d, 5 d/wk

Doses: 0, 520, 1980 and 5010 ppm (0.663 ; 2.65; 6.63 mg/l) Control Group: yes, concurrent no treatment
NOAEL: 6.63 mg/l

GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: The study used 5 rats/sex/group. Parame ters evaluated included, ophthalmoscopic exam, b ody weight, clinical signs, organ weights, histopathology and survival.

-------------- Remark: A good screening study. Daily exposure w as 6 hours per day, the normal daily exposure length for inhalation studies evaluating potential workplace ex posure.

-------------- key study non-BASF study

### OECD SIDS METHANOL

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144 Result: All animals survived. In rats nasal and eye discharge (mucoid, nasal, red nasal, lacrim ation) was noted in the treatment groups. Only mucoid nasal discharge appea red to be dose related.
There were no treatment-related e ffects on body weight.
No ocular abnormalities were note d at the terminal ophthalmoscopic exam. No treatmen t related histopathological effects (35 tissues) were noted, but spleen weights were increased in female rats exposed at 2,000 ppm (not at 5,000 ppm). No other organ weight effec ts were noted.

---------------- Test substance: Methanol, 99.85 % pure from Celanese Corp. NY, n o further
data. Conclusion: Rats were exposed to up to 5,000 ppm (6 hr/d , 5d/wk for 4 weeks) showed no treatment relate d histopathological
effects. Inhalation exposure had resulted in some slight treatment-related signs. Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, well documented, accepta ble for assessment , key study
25-OCT-2006 (183)

Type: Sub-acute Species: monkey Sex: Strain: Macaca Fascicularis
Route of administration: inhalation Exposure period: >= 15 d Frequency of treatment: 21 h/d Post exposure period: none Doses: 10000; 7000; 5000; 3000 ppm (abo ut 13; 9.1; 6.5; 3.9 mg/l) Control Group: yes, concurrent no treatment
LOAEL: 3000 ppm

Year: 1987
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity studies: Four animals were used p er test and control group. Exposure period was variable, but was expected to be at least 15 days (p. 12 ff).

Test procedure with respect to ex posure times not well
documented.

--------------- Remark: key study non-BASF study Result: Blood levels (p. 51): 3000 ml/m3 5 000 ml/m3

================================= ============ Methanol: 80 mg/l 5 250 mg/l Formate: 30 mg/l 1 210 mg/l

================================= ============

### OECD SIDS METHANOL

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145 The animals at the top dose showe d lethargy, and after 3 treatment fell into coma and died

. The animals of the 7000-ppm group had to be killed because of suffering. The animals at 5000 and 3000 ppm exhibited weakness and
uncoordinated movement. Food cons umption was reduced at 5000 ppm. The body weight was normal.

Haematological findings displayed no changes, except for a somewhat higher leukocyte count a t the two highest exposure
levels. Blood pH in one animal of the 500 0-ppm and at the highest dosed groups were significantly d ecreased showing acidoses. No changes were found in other cl inical-chemical blood and liver values, but in the 7000-ppm group alkaline phosphatase
increased.

At 5000 ppm, necrosis of the basa l ganglion of the cerebrum is reported, associated with mark ed cerebral edemas.

These effects were not seen at 30 00 ppm.

Effects (mild atrophy) on the vis ual system (retina, optic nerve, state of myelinated fibres ) were hardly noticeable and considered as insignificant a t 3000 ppm and below, but pronounced at 5000 ppm.

Fatty degeneration in the liver o ccurred at 3000 ppm and more pronounced at the higher exp osures, with necrosis after 5000 ppm. Also at this exposure l evel, kidneys were partly affected, showing vacuolated dege neration.

-------------------- Test condition: Exposure time 21 hours per day, significantly lo nger than according to guideline (6 hrs). T his does not represent
normal industrial and consumer us e.

--------------- Test substance: Methanol of first grade by Junsei Chemicals, <1 ppm vinyl chloride, <3 ppm formaldehyde

----------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, limited documented , key study
25-OCT-2006 (167)

Species: monkey Sex: no data Strain: Macaca Fascicularis
Route of administration: inhalation Exposure period: 1000 ppm: 7 months; 2000 ppm + 3000 ppm 20 d; 5000 ppm 6 d + 4000 ppm 6 d Frequency of treatment: 21 h/d Post exposure period: 30 d; 6 months; 10 or 4 months (see Method)
Doses: 1000; 2000; 3000; 5000+4000 ppm (1.3; 2.6; 3.9; 6.5+5.2 mg/l) Control Group: yes, concurrent no treatment

Method: other: Special acute to chronic inhalati on test programme including recovery phases
Year: 1985
GLP: no data

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

146 Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term studies, reproductive a ssays and carcinogenicity
studies: Whole-body exposure: 4 monkeys we re housed in one inhalation
chamber.

Here recovery study with specific focus on neurological effects apart from organ toxicolo gy:

1000 ppm: 7 months exposure + 30 d or 6 months recovery 2000 and 3000 ppm: 20 d exposure + 30 d, 6 or 10 months

recovery 5000 ppm: 6 d exposure + 6 d 4000 ppm exposure with 30 d or 4 months recovery.

Clinical, clinical-chemical, hema tological, ophthalmological, and pathologica l/histopathological
examiniation were done.

Analytical control of chamber con centration: Analytical
values close to nominal ones.

------------------------- Remark: key study non-BASF study Result: At the concentration of 4000 and 5000 pp m, irreversible lesions/degenerations appeared to be likely in the basal ganglions of the cerebrum, becaus e they did not subside within the recovery period, while after 3000 ppm (20 d exposure) the slight necrotic deg enerations were not progressive as evidenced after re covery.

The increase of responsive astrog lia seen in the cerebral white substance was still present after recovery from 3000-ppm exposure.

Degenerative changes in the visua l system were evident after exposure to 3000 ppm and higher: atrophy of the optic nerve and reduction in myelinated fiber s. Recovery was unequivocal at exposures below 1000 and 2000 ppm, but at 3000 ppm, there appeared to be a trend of t hese lesions to progress, although -due to the low number o f animals- this could also reflect individual variability ac c. to authors.

Lesions of the liver occurred in all dose groups with
round-cell infiltration and fibro sis noted in dose-related manner. They were still present h istologically after the recovery phase. Changes of the kidneys were obser ved in all groups: hyalinisation of glomeruli, cell infiltrations into the
renal tube stroma. These effects were no longer noted after

recovery.

----------------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride; <3 ppm for maldehyde

------------ Conclusion: At 1000 ppm (7 months exposure and higher; s ee chronic exposure other entry), some irrev ersibility of degenerative

### OECD SIDS METHANOL

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147 changes (mild fibrosis presumably following fatty degeneration) unrelated to the re covery period was noted in the liver (p.54)[see also p. 27: chronic study]. These effects were histologically small and, therefore, of little pathological relevance.

All other effects -if there were any at all- were not persistent and not considered pat hologically relevant at that level, but were significantl y more pronounced at 3000 ppm and higher after shorter expo sure (20 d), associated with eventual lack of recovery. Between 2000 and 3000 ppm appears to be a threshold concentration where methanol-indu ced neurotoxic effects may become biological relevant (p. 35 ).

-------------- Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, however, documentation and number of animals
limited. , key study
25-OCT-2006 (167)

Type: Sub-acute Species: monkey Sex: male Strain: other: rhesus Macaca mulatta Route of administration: gavage Exposure period: 1.5 to 6 d Frequency of treatment: variable
Doses: initial 2000 mg/kg, thereafter 5 00 or 1000 and 2000 mg/kg (variable frequency) Control Group: other: internal / same animal prior to t reatment

Method: other: (see Method)
Year: 1978
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: Test model in monkeys for methanol-induc ed ocular toxicity to characterise the toxicity synd rome and histological manifestations. Methanol was appl ied as 20% solution by nasogastric tube (see: McMartin e t al., 1975).

Ophthalmoscopic examinations and ultrastructural histopathology (electronmicroscop y) were performed.

----------------------- Remark: non-BASF study Result: 1. Under methanol treatment acc. to t his test design,
formate levels were between min. 7.2 and max. 14.4 mEq/L in blood and cerebrospinal fluid, bl ood bicarbonate min. 4.0 and max. 10.2, and blood pH min. 7.13 and max. 7.28. Methanol levels ranged from 1540 to 2840 mg/L (Martin-Amat et al., 1977).

2. All six animals developed fu ndus changes at the head of the optic nerve (optic disc) with in 43 to 171 h after methanol ingestion, expressed as intraaxonal swellings (Hayreh et al, 1977). Electronmic roscopic studies revealed swelling of the nerve fibers with an accumulation/clustering of swollen mitochondria in the op tic nerve head being maximally in the lamina cribrosa region. Furthermore, in the

### OECD SIDS METHANOL

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148 retrolaminar and intraorbital opt ic nerve, swelling of astrocytes was prominent as well as swelling of the cytoplasm of the oligodendroglial cytoplasm in contact with the axons (Baumbach et al., 1977)

. Alterations were not observed in the retina itself: The ganglion cells of the retina were intact with only minimal swellings of the mitochondria and loss of cristae. But these findings were also present in the control tissue (Baumbach et al., 1977).

---------------------- Test condition: A total of six monkeys was treated such as to ma intain a
state of metabolic acidosis at at tenuated toxicity (arterial blood pH 7.1 to 7.3 and bicarbona te at 10 mEq/L or above) for a prolonged time: A high init ial dose was followed by lower doses depending on the anim al ́s acidotic response in
blood. [note: Experience had told (McMa rtin et al., 1975) that after a single dose of 3000 mg/kg bw., in general, the
animals died within 20 to 30 h wi thout demonstrating ocular abnormalities.] (Martin-Amat et a l., 1977).

---------------------- Test substance: Methanol, highest purity available, no further d ata (see: McMartin etal., 1975) Conclusion: 1. While acute methanol toxicity in monkey s (after a single dose) does not yield ocula r signs, repeated dosing succeeded in producing ocular les ions (Martin-Amat et al, 1977).

The only detectable ocular change was optic disc edema (of the optic papilla) which was simi lar to that seen in raised intracranial pressure in humans, but without this pressure after methanol (Hayreh et al, 197 7). The primary sites of ocular injur y were the optic nerve heads and the anterior segment of the optic nerve rather than the retinal ganglion cells t hemselves. It appears that interference with oxidative phosp horylation causes mitochondrial damage, thus disrup tion of active axoplasmic flow in the retrolaminar optic ne rve (Baumbach et al., 1977; Hayreh et al., 1977).

[note: In humans it has been hypo thised that optic atrophy,
which often follows acute methano l intoxication, is secondary to injury of the retina l ganglion cells.]

2. Mechanistically, there is a c lose causal relationship between the prolonged increase in formic acid from methanol and the development of optic edem a. Similar effects can be produced by i.p. administration o f formate without acidosis (see other entry 5.11: Martin-Ama t et al., 1978).

--------------------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

25-OCT-2006 (184) (185) (186) (187) (188) (189)

Type: Sub-acute Species: rat Sex: male Strain: Sprague-Dawley Route of administration: inhalation

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

149 Exposure period: 1; 2; 4; or 6 Wk Frequency of treatment: 6 h/d, 5 d/Wk Post exposure period: 16 h after the last exposure. One group exposed for 6 wks: recovery 2 wks
Doses: 200, 2,000 or 10,000 ppm (0.265; 2.65; 13.3 mg/l) Control Group: yes, concurrent no treatment
NOAEL: 13.3 mg/l

Year: 1982
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Limited study: Only biochemical/physiolo gical and cytological parameters of the lun g were examined (tissue-specific: lung weight, ti ssue DNA and protein contents, RNase, Protease; in the filtered lavage fluid: surfactant, number of cells, PMN, protein, surface LDH and N-acetyl-beta-glucosaminidase). Histologic analyses of lung tissu e were not conducted. Four rats per group were used. Chamber concentration was analyse d by GC.

-------- Remark: non-BASF study Result: Even the highest concentration caused no signs of lung
inflammation or irritation. Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Special study design, meets gener ally accepted scientific principles, restrictions in asses sment 25-OCT-2006 (190)

Type: Sub-chronic Species: rat Sex: Strain: no data Route of administration: inhalation Exposure period: 3 months Frequency of treatment: 12 h/d, 5 d/wk

Doses: a) 1.77; 49 mg/m3; b) 0.57; 5.3 mg/m3 Control Group: yes, concurrent no treatment

GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: Special neurophysiological study design: chronaximetry =
ratio of the minimum time necessa ry for a stimulus of twice the absolute threshold intensity to evoke a response measured as muscle contractions i n response to an electric current applied to an animal ́s hi nd leg. Normally, the flexor chronaxia is shorter than the extensor chronaxia, and
their ratio is stated to be relat ively stable (NTP-CERHR, 2001, p. 80).

------------ Remark: non-BASF study Result: Low concentrations of methanol inhaled f or 3 months by rats are reported to exhibit changes i n the average motor chronaxia ratio between antagonis tic muscles. No significant effects were noted at 1.77 mg/m3 (Chao, 1959) and 0.57 mg/m3 (Ubaydullayev, 1966). At the othe r doses the chronaxia returned to normal during the rec overy period. Unspecified histopathological cha nges in relation to the

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

150 mucous membranes of the respirato ry tract were seen at 450 mg/m3, but not at the lowest dose s. Some shifts in liver parameters in blood were also men tioned.

------------------- Test substance: Methanol, no further data. Conclusion: The analysis by Kavet and Nauss (1990) indic ated that these studies do not provide adequate e vidence of an association
between neurobehavioral effects a nd low-level exposure to methanol: limited animal number, insufficient reporting of methodss, and unclear statistical analysis. The biological significance of changes in the ch ronaxia ratio is considered as uncertain. (see also NTP-CERHR , 2001, p. 80/81)

------------------- Reliability: (4) not assignable Test methods and performance unce rtain, limited
documentation available
25-OCT-2006 (191) (192) (193)

Type: Sub-acute Species: rat Sex: male
Route of administration: inhalation Exposure period: 1; 2; 4 or 6 weeks Frequency of treatment: 8 h/d 5 d/wk

Doses: 200, 2000, and 10000 ppm (0.265; 2.65; 13.3 mg/l) Control Group: yes, concurrent no treatment

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Study design was to elucidate sexual hor mone status in mature male rats (Sprague-Dawley) : determination in blood after exposure of circulating fre e testosterone, luteinizing hormone and follicle stimulating hormone.

------ Remark: Conflicting results as compared to findi ngs of others (see also other entry 5.1.2: Cooper et al., 1992; and 5.4: Lee et al., 1991). non-BASF study Result: Significantly decreased levels of circul ating free testosterone were observed among rats exposed at 200 ppm for 2 and 6 weeks (32 % of control) a nd 6 weeks at 2,000 ppm. The high dose group (10,000-ppm) showed no change. The authors interpreted this as evide nce that methanol exposure had lowered testicular production of testosterone. In addition, significant increase s in circulating LH were observed after six weeks of expos ure to 10,000 ppm. No changes in follicle stimulating h ormone levels were
observed.

----------------- Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, restrictions in assessment (see Remark)
25-OCT-2006 (194)

Species: rat Sex: male Strain: Sprague-Dawley Route of administration: inhalation

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

151 Exposure period: 1 d or 1 wk Frequency of treatment: 6 h/d Post exposure period: none or 18 h
Doses: 200 ppm (0.265 mg/l) Control Group: yes, concurrent no treatment

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Study design was to elucidate sexual hor mone status in mature male rats (Sprague-Dawley) : determination in blood after exposure of circulating fre e testosterone, luteinizing hormone and follicle stimulating hormone. Remark: Conflicting results as compared to findi ngs of others (see also other entry 5.1.2: Cooper et al., 1992; and 5.4: Lee et al., 1991). non-BASF study Result: SD rats exposed to 200 ppm methanol for 6 h for 1 day or 1x/d for one week showed a signif icant depression of serum testosterone (59 %) immediately a fter the exposure, but not after one-week daily 6-h exposure or after 18-h recovery. There was no evidence of an effec t on LH.

---------------- Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, restrictions in assessment (see Remark)
25-OCT-2006 (195)

Type: Sub-acute Species: rat Sex: male Strain: Sprague-Dawley Route of administration: inhalation Exposure period: a) 1, 2, 4, and 6 wk; b) 13 wk Frequency of treatment: a) 8 hours/day, 5 days/week; b) 20 hours/day, 7 days/week
Doses: a) 200 ppm (260 mg/m3); b) 50, 200, 800 ppm (1040 mg/m3)
NOAEL: = 800 ppm

GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Special study design: The potential toxi c effects of methanol vapors on testicular pro duction of testosterone (a) and the morphology of testes (b) were investigated using
normal or methanol-sensitive fola te-reduced rats.

(a) Nine rats per group were used

. (b) 11 to 12 animals (10 months o ld) for three exposure levels, and 8 animals (18 months old) for a 800-ppm group
were used.

----------- Remark: non-BASF study Result: Methanol inhalation did not reduce serum testosterone levels
in normal rats. Testes isolated from methanol-exposed (200 ppm) rats had the same capability as those from air-exposed rats in synthesizing testosterone , whether testes were

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

152 incubated in the absence or prese nce of hCG.

The testes-to-body weight ratio a nd testicular morphology of rats exposed up to 800 ppm methan ol for up to 13 weeks (20 hours/day, 7 days/week) were not different from control animals, irrespective of the nutr itional state, normally fed
or folate deficient.

A greater incidence of testicular degeneration was seen only in 18 month old, folate-deficient rats exposed to 800 ppm methanol for 13 weeks (20 hours/d ay, 7 days/week). The results are equivocal.

800 ppm, the top level, can be es tablished as subchronic NOAEL based on testicular histopa thology.

-------------- Test substance: Methanol, no further data Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented, acceptable for assessment
25-OCT-2006 (196)

Type: Sub-acute Species: rat Sex: male Strain: Long-Evans Route of administration: gavage Exposure period: 21 d Frequency of treatment: 2 x/d Post exposure period: none Doses: 800; 1600 mg/kg/d Control Group: yes, concurrent no treatment

Method: other: special test on endocrin function and male reproduction parameters
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Result: Treatment with the highest dose produced a transient, but significant decrease in serum tes tosterone. On day 21 of treatment, the testosterone level was normal again, but a significant increase in the lutei nising hormone was analysed. The testis weights were reduced as well as the number of morphologically normal sperms in the epididymis.

The acute treatment with high dos es of 800, 1600, and 3200 mg/kg (2x 12 h apart) had no effe ct on testis weight, and on sperm size. At day 1 after treatm ent, a reduced testosterone value was found in serum and in t he interstitial fluid. After 7, 14, and 21 d post-applic ation, testosterone in both compartments were normal without significant difference from the control. LH was distinctly hi gher on day 21.

(see also other entry 5.1.2: Coop er et al., 1992)

------------------------- Test substance: Methanol, no further data. Reliability: (4) not assignable Only abstract
25-OCT-2006 (197)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

153 Type: Sub-acute Species: rabbit Sex: male Route of administration: gavage Exposure period: 5-7 d Frequency of treatment: daily
Doses: 4.68 g/kg as 30-% solution

Remark: non-BASF study Result: Lethal dose for all 14 rabbits within th e following 5 to 7 days. Test substance: Methanol, no further data.
25-OCT-2006 (198)

Type: Chronic Species: dog Sex: Route of administration: inhalation Exposure period: 379 d Frequency of treatment: 8 h/d, 7 d/wk

Doses: 0.585 - 0.65 mg/l

GLP: no

Remark: non-BASF study Result: Four dogs were tested for a broad spectr um of haematological parameters and ophthalological al terations. There was no
evidence of a treatment-related e ffect. Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source
25-OCT-2006 (199)

Type: Sub-acute Species: monkey Sex: male/female
Strain: Macaca Fascicularis
Route of administration: inhalation Exposure period: 4 wk Frequency of treatment: 6 h/d, 5 d/wk Post exposure period: none Doses: 500, 2000, and 5000 ppm (0.663; 2.65; 6.63 mg/l) Control Group: yes, concurrent no treatment
NOAEL: 6.63 mg/l

Year: 1987
Test substance: as prescribed by 1.1 - 1.4

Method: Three monkeys per sex and dose were used . Chamber methanol concentration was measured regula rly by IR. Histopathological examinations in cluded ophthalmoscopy at the end of exposure, but apparent ly no microscopical inspection of brain and neural ti ssue.

----------- Remark: non-BASF study Result: There were no clinical signds of intoxic ation. There were no
methanol-related abnormalities. Test substance: Methanol, purity 99,85 % (from Celanese Corp., N .Y.) Reliability: (4) not assignable Test procedures in accordance wit h accepted standard methods, insufficiently documente d, limitations in tissue examination (see Method).

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

154 25-OCT-2006 (200)

Species: monkey Sex: male Route of administration: gavage Exposure period: 3 d Frequency of treatment: daily
Doses: 2340 mg/kg as 30-% solution

Test substance: no data

Remark: non-BASF study Result: Lethal dose for all 7 animals under test after 3 days.
Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Meets generally accepted scientif ic standards, sufficiently
documented 25-OCT-2006 (198)

5.5 Genetic Toxicity 'in Vitro'

Type: Ames test System of testing: Salmonella typhimurium TA1535, TA1537, TA153 8, TA98, TA100 (plate incorporation standard test) Concentration: <= 2.5 mg/plate Cytotoxic Concentration: non-toxic within the range of testing
Metabolic activation: with and without
Result: negative

Method: other: acc. to Ames et al., 1975
Year: 1981
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Test condition: S9 mix was prepared from Arochlor-induced SD rat livers.
Test substance: Methanol, from Carlo Erba, no further data. Reliability: (2) valid with restrictions Comprehensive test programme usin g test procedures in accordance with accepted standard methods, but limited documentation

--------------

25-OCT-2006 (201)

Type: Ames test System of testing: Salmonella typhimurium TA97, TA102 (plate in corporation standard system) Concentration: <= 7.5 mg/plate
Metabolic activation: with
Result: negative

Method: other: acc. to Maron and Ames, 1983
Year: 1984
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: key study non-BASF study Result: In TA102, a slightly positive trend was indicated [(+-)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

155 ambiguous], reproducible in 5 par allel independent experiments, but never exceeding the revertant ratio of 2: Spontaneous mutation rate 200 - 3 00/plate, while in the presence of high methanol doses, an increase in the mutation frequency above background of 140 revertants was found. Test condition: S9 mix was prepared from Arochlor-induced SD rat livers.
Test substance: Methanol, from Carlo Erba, no further data. Conclusion: The methanol-related increase in the mutatio n frequency in TA102 did not fulfil the accepted criteria for mutagenic activity. Along with the high met hanol doses required to
induce such a weak effect and the negative results observed in all other Ames tests, the overall evidence clearly
demonstrates that methanol is not mutagenic in bacterial reverse-mutation systems.

------------- Reliability: (2) valid with restrictions Comprehensive test programme usin g test procedures in accordance with accepted standard methods, but limited
documentation

-------------- , key study
25-OCT-2006 (202) (203)

Type: Ames test System of testing: Salmonella typhimurium TA1538, TA98, TA1537, TA1535
Concentration: <= 3.6 mg/plate
Metabolic activation: with and without
Result: negative

Method: other: acc. to Ames
Year: 1981
GLP: no Test substance: as prescribed by 1.1 - 1.4

Remark: Metabolic activation system was derived from liver S-9 fraction of Aroclor-1254 pretreat ed rats. non-BASF study Test substance: Methanol, from Merck, Germany, no further data. Reliability: (2) valid with restrictions Comprehensive test programme usin g test procedures in accordance with accepted standard methods, limited
documented

25-OCT-2006 (204)

Type: Ames test System of testing: Salmonella typhimurium TA100, TA98, TA1537, TA1538, TA1535; Escherichia coli W P2uvrA Concentration: 5, 10, 50, 100, 500, 1000, and 5000 μg/p late Metabolic activation: with and without
Result: negative

Year: 1985
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: Metabolic activation system was derived from liver S-9 fraction of KC500(polychlorinated biphenyl)-pretreated rats. non-BASF study Test substance: Methanol, 99.6 % pure, no further data.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

156 Reliability: (2) valid with restrictions Comprehensive test programme usin g test procedures in accordance with accepted standard methods, sufficiently
documented

25-OCT-2006 (205)

Type: DNA damage and repair assay System of testing: Escherichia coli WP67, WP2, CM871
Concentration: no details Cytotoxic Concentration: > 40000 μg/well (no details, derived from the MIC)
Metabolic activation: with and without
Result: ambiguous

Method: other: no Guideline indicated
Year: 1984
GLP: no data Test substance: no data

Method: E. coli strains used were all trp-depend ent (trp-), carrying
an ochre nonsense mutation blocki ng an intermediate process in the synthesis of tryptophan:

E. coli WP2 (wild-type, repair pr oficient) E. coli WP67 (repair deficient: u vrA-, polA-) E. coli CM8761 (repair deficient: uvrA-, recA-, lexA-).

For each tester strain, the Minim al Inhibitory Concentration (MIC) was determined. The endpoin t is cytotoxicity. The ratio the MIC of the repair proficient to the MIC of the repair
deficient strains was taken as in dicator for genotoxicity. A ratio of greater than 2 was accep ted as significant only if reproducible in 5 parallel indepe ndent experiments.

------------------

Trials were conducted in miniatur e form (liquid micromethod procedure: 350 μl) using microwel l plates that contained nutrient broth, and as 2-h preinc ubation assay ("treat-and-plate method" on nutr ient broth agar).

--------------------

The initial concentration was gov erned by the solubility or by the toxicity of the TS as inferre d from preliminary testing. Starting from this, eight 2-fold dilution steps followed in general.

-------------------

Bacterial inocula were obtained f rom 16-h cultures in nutrient broth with about 10^3 cells per w ell. The exact bacterial density was determined by seeding the same volume of cell culture (0.1 ml) onto a nutrient agar plate after 16-h incubation at 37 °C.

--------------------

Bacterial growth in the test well s was visually evaluated after 16 h at 37°C by observing t he increase in turbidity of the medium and/or formation of pe llets of settled cells.

---------------- Remark: non-BASF study Result: The microwell method resulted in a negat ive result in the

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

157 presence of S9 and in a positive result in the absence of S9 at 20 mg/well (Flora et al., 1990 , 1984). But the preincubation procedure was negat ive without S9, but ambiguous with S9 (Flora et al. 1984).

Note: The MIC concentrations were very high (40 and 20 mg/well = about 120 g/L and 60 g/l).

------------------- Test substance: Methanol, no further data. Conclusion: Given the high concentrations and the confli cting findings, it
is concluded that observations ma de at the margin of significance (ratio = 2) are of l ow reliability and biological
relevance. The weak relative incr ease of toxicity in repair-deficient strains may be a n increase in unspecific cytotoxicity rather than solely " genotoxicity". (Note: A similar result was obtai ned with ethanol at somewhat lower concentrations).

-------------- Reliability: (2) valid with restrictions 2c: Meets generally accepted scie ntific standards, well documented with methodological re strictions (see Conclusions) , key study
25-OCT-2006 (203) (206)

Type: other: Micronucleus Test System of testing: V79 Chinese hamster cells
Concentration: 50 μl/ml (approx. 40 mg/ml)
Metabolic activation: without
Result: negative

Year: 1984
GLP: no data

Remark: Comparative study including alcohols, ac etone, and various alkylating agents. Exposure was 4 8 h for alcohols. 4000 - 7000 interphase cells were scored per treatment.

----------------- key study non-BASF study Result: No MN increases induced by any alcohol o r acetone, whereas the alkylating agents produced si gnificant MN frequences
above medium controls. Test substance: Methanol [CAS 67-56-1], 95 % pure from Prolabo, no further
data. Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented, acceptable for assessment , key study
25-OCT-2006 (207)

Type: other: Micronucleustest in vitr o System of testing: Syrian hamster embryo cells
Concentration: not indicated: derived from range findin g Metabolic activation: without
Result: negative

Method: other: according to Schmuck et al. (1988 )
Year: 1988
GLP: no data Test substance: as prescribed by 1.1 - 1.4

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

158 Method: Comparative study including a broad spec trum of chemicals. Exposure time was 18 hours, no fu rther details of method are reported.

Report refers to: Schmuck, G. et al. (1988): Charac terisation of an in vitro micronucleus assay with Syrian ha mster embryo fibroblasts. Mut. Res., 203, 397-404:

Cell cultures established from 13 -d old embryos. Tests performed with tertiary cultures in a humidified atmosphere with 12 % CO2 in air at 37°C.

Tertiary cell cultures were incub ated for 24 h (1.5 x10exp5 cells on cover slips in 35-mm pet ri dishes). Then various concentrations of th e TS were added in DMSO (final concentration of the solve nt =<0.1%) (Dose ranges had been determined prior to the main test, but no details are given concerning the concentratio ns.) Incubation time 5 h, followed by washing.
Further incubation for 18 h.

Thereafter, the cells were fixed, stained and scored for
micronuclei.

Monitoring of micronuclei: The nu mber of cells containing single or multiple micronuclei wa s counted among 2000 cells
for each dose.

STATISTICAL ANALYSIS: Student ́s t -test

-------------------

---------------------- Remark: non-BASF study Test substance: Methanol, analytical grade Reliability: (4) not assignable
4e: Documentation insufficient fo r assessment, test procedures in accordance with accepted stand ard methods, but limited
documentation.

25-OCT-2006 (208) (209)

Type: Sister chromatid exchange assay System of testing: CHO-Zellen Concentration: up to 0.1%, 1x/d for 8 days
Metabolic activation: without
Result: negative

Method: other
Year: 1977
GLP: no Test substance: no data

Method: Repeated exposure design: ethanol, propa nol, butanol, and acetalaldhyde were also tested. N o standard positive control
substance was included. Test medi um was change every second day, but test substances appeared to be added every day "up to a final concentration of 1 %".

-----------

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

159 Remark: non-BASF study Result: The alcohols produced no increases in th e SCE rate compared to the control, but acetaldehyd i nduced a dose-related, significant increase. Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Only short communication. Test pr ocedure largely accordance with current standards and based on scientific principles. Positive result of acetaldehyde r aises the level of reliability for the proper functi onality of the test system.

25-OCT-2006 (210)

Type: other: Cell transformation assa y System of testing: C3H/10T1/2 Cl 8 cells (mouse embryo fibrobla sts cells) Concentration: 5, 10, 20, 50, 100 mg/ml Cytotoxic Concentration: 50 mg/ml (> 30 % inhibition of growth)
Metabolic activation: with and without
Result: negative

Year: 1981
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: 12-O-Tetradecanoyl-phorbol-13-acetat TP non-BASF study Result: Methanol and formic acid induced no foci type I or III either in the presence or absence of TPA (12-O-Tetradecanoyl-phorbol-13-ac etate) as promoting agent.

[note: Formaldehyde, on the other hand, caused a high number of transformed cells in the prese nce of TPA, but failed to in its absence.] Test substance: Methanol, HPLC grade from Fisher, no further dat a.

----------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

25-OCT-2006 (211)

Type: other: Cell transformation assa y System of testing: BHK 21 C 13 (Baby Syrian Hamster Kidney Cell s) Concentration: 10, 50, and 100 ul/ml (approx. 8, 40, an d 80 mg/ml) Cytotoxic Concentration: LC50 approx. 60 mg/ml
Metabolic activation: with and without
Result: negative

Year: 1981
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: Metabolic activation system was derived from liver S-9 fraction of Aroclor-1254 pretreat ed male NMRI mice (500 mg/kg, i.p.). non-BASF study Result: No increase in transformants, while mito mycin, MMN, cyclophosphamide and dichromate w ere highly active.

--------------- Test substance: Methanol from Merck, Germany, p.a., no further d ata.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

160 Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

25-OCT-2006 (212)

Type: Mammalian cell gene mutation as say System of testing: Chinese Hamster V79 Zellen 8-Azaguanin-, 6-T hioguanin-,
Ouabain-Resistenz Concentration: 15.8, 31.7, 47.4, and 63.3 mg/ml Cytotoxic Concentration: 63.3 mg/ml (approx. 70% inhibition of colony forma tion)
Metabolic activation: with and without
Result: negative

Year: 1987
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: key study non-BASF study Result: No increases in mutant frequency in gene mutation to drug resistance vs. neg. control, wher eas the pos. control DMN produced increases in dose-relate d manner. Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride, <3 ppm for maldehyde

------------------ Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, sufficient documentati on, acceptable for

assessment , key study
25-OCT-2006 (167)

Type: Chromosomal aberration test System of testing: Chinese hamster lung cell
Concentration: 7.1, 14.3, and 28.5 mg/ml Cytotoxic Concentration: 28.5 mg/ml (approx. 50% inhibition)
Metabolic activation: with and without
Result: negative

Year: 1987
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: MNNG was used as pos. control (0.5 and 1 .0 ug/ml) in the
absence of S9 and 2-aminoanthrace ne (2 and 4 ug/ml) in the presence of S), along with two ne gative control (untreated and solvent = medium). 200 metaph ases were scored after 6, 24, and 48-h exposure (p. 242).

------------ Remark: key study non-BASF study Result: No (dose-dependent) increases in any typ e of chromosome and chromatid breaks and gaps were no ted at any time interval, while MNNG and 2-AA produced clea r dose-related increases in breaks and gaps (Tables, p. 250 - 255).

---------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride, <3 ppm for maldehyde

------------------

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

161 Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, sufficient documentati on, acceptable for

assessment , key study
25-OCT-2006 (167)

Type: Mouse lymphoma assay System of testing: mouse lymphoma cells L5178Y
Concentration: 7.9 mg/ml
Metabolic activation: with
Result: positive

Method: other: acc. to Clive and Spector. Mut. R es., 31, 17-29, 1975
Year: 1985
GLP: no data Test substance: no data

Method: Objective: Optimisation of the amount of the S9-mix to be used to obtain maximum yield of m utations. Comparative study including typical mutagens. Exposure time 4 h.

--------------------- Remark: key study non-BASF study Result: In the presence of 10 - 15 ul/ml S9 and methanol (7.9 mg/ml), there was a significant i ncrease in mutation frequency. No detailed results pr esented.

--------------------- Test substance: Methanol, no further data. Reliability: (4) not assignable Only abstract, no detailed data. , key study
25-OCT-2006 (213)

Type: other: Mitotic recombination in ascomycetes System of testing: Aspergillus nidulans diploid strain P1
Concentration: 5.2, 5.6, 6.0, and 7.0 % (v/v) Cytotoxic Concentration: 7.0 % (lowest concentration to arrest conidial germination) Metabolic activation: without
Result: positive

Method: other: no data
Year: 1989
GLP: no data Test substance: as prescribed by 1.1 - 1.4
Method: Mitotic segregants were detected as homo zygous (yAA2/yA2) or hemizygous (yA2/0) yellow sectors or patches in heterozygous (yA2/yA2+) pale-green colonies gr owing on complete medium. Yellow segregants were isolated, analyzed for their nutritional requirements and clas sified as mitotic cross-over, non-disjunctional diploids and ha ploids.

----------------------

Treatment of conidia (10 exp^5/ml ) with the TS at early germination, i.e during the first mitotic divisions, in liquid complete medium at 37 °C under ag itation until emergence of germ tubes (check by light micros copy). Plating onto complete agar for de termination of survival and

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

162 detection of mitotic segregants.

--------------------- Remark: Approx. 3 % chromosomal malsegregation w as induced.

The result was statistically sign ificant at two concentrations and a dose-response relationship was evident (IPSC/WHO 1997). non-BASF study Result: There was a concentration-related increa se in non-disjunctions
with a maximum of about 3 %. No a neuploidies were noted at the
lowest test concentration. No oth er forms of malsegregation were statistically significant (1 /265 cross-over seen at 6.0 %).

The survival was reduced in dose- related manner.

Conc. Survival Scored Aneup loids(yellow segregants) [%] [%] colonies Count s %

================================= ====================== 5.2 26 268 0 0
5.6 19 407 6 1.47*
6.0 10 265 8 3.02**
7.0 5 176 0 0

--------------------------------- ----------------------- Spontaneous control
0 100 2673 7 0.26

================================= ======================= * = P<0.01; ** = P<0.001

Note: Similar results were obtain ed with ethanol and other primary aliphatic alcohols. The a neuploidy rate of ethanol
reached a maximum of about 10 %.

----------------------- Test substance: Methanol [CAS 67-56-1], purity >99.9% Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

25-OCT-2006 (214)

Type: Yeast gene mutation assay System of testing: Schizosaccharomyces pombe (ade 6 locus)
Concentration: <= 5 % Cytotoxic Concentration: 10 %
Metabolic activation: with and without
Result: negative

Year: 1980
GLP: no Test substance: as prescribed by 1.1 - 1.4

Method: Comparative study including various solv ents: As indicator for mutation served the phenotypic change from
the red to the white colour of th e colonies. The P1 strain produces red colonies, owing to a mutation at the ade6 locus which blocks the adenine biosynth esis pathway, associated with the accumulation of a red pigment , 5-amino-4-imidazole ribotide. Any mutation at each 5 different loci blocking the pathway to the pigment will lead to the development of white
colonies.

---------------------

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

163 Remark: There was no increase in mutant frequenc y as compared to the negative controls (buffer and DMS O) at a the maximum conc. of 5 % with or without post-mitc hondrial fraction from phenobarbital-induced mouse liver

.

-------------- non-BASF study Test substance: Methanol, analytical grade, from Carlo Erba, Mil ano, no further data. Reliability: (4) not assignable
4e: Documentation insufficient fo r assessment (see Method)

25-OCT-2006 (215)

Type: Ames test System of testing: Salmonella typhimurium TA98, TA100, TA1535, TA1537 (spot test) Concentration: 3 μmol/plate
Metabolic activation: with and without
Result: negative

Year: 1979
GLP: no Test substance: no data

Remark: Metabolic activation system was derived from liver S-9
fraction of Aroclor-1254 and 3-me thylcholanthrene-pretreated

rats. non-BASF study Test substance: Methanol, no further data. Reliability: (3) invalid Significant methodological defici ency: Test concentration may have been too low, no dose-re sponse relationship given

25-OCT-2006 (216)

Type: Ames test System of testing: Salmonella typimurium TA1535, TA1537, TA1538 , TA98,
TA100 Concentration: <= 5 mg/plate
Metabolic activation: with and without Result: negative

Method: other: acc. to Ames
Year: 1977
GLP: no Test substance: no data

Remark: Metabolic activation system S9 mix was p repared and applied according to Ames non-BASF study Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Comprehensive test programme usin g test procedures in accordance with accepted standard methods, limited
documented
25-OCT-2006 (217)

Type: other: Cell transformation assa y System of testing: Syrian Hamster Embryo Cells
Result: negative

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

164 Year: 1983
GLP: no data Test substance: no data

Remark: non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Secondary source
25-OCT-2006 (218) (219)

Type: Bacterial reverse mutation assa y System of testing: Salmonella typhimurium TA1535, TA 1537, TA15 38, TA100, TA98 + E. coli WP2 uvvA (t rp dependent) Concentration: 10, 50, 100, 500, 1000, and 5000 μg/plat e Metabolic activation: with and without
Result: negative

Method: other: largely in compliance with OECD G uide-lines
Year: 1987
GLP: no data
Test substance: as prescribed by 1.1 - 1.4

Remark: Metabolic activation system was derived from liver S-9 fraction of PCB-pretreated rats ( 500 mg/kg, single i.p.) (p. 241).

--------------- non-BASF study Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride, <3 ppm for maldehyde

------------------ Reliability: (4) not assignable Comprehensive test programme usin g test procedures in accordance with accepted standard methods, only short communication.
25-OCT-2006 (167)

Type: other: mutagenic inactivation o f a bacteriophage

repressor System of testing: E. coli RK+ (lysogenic)
Concentration: up to 23 % (0.23 ml/ml = 0.18 g/ml)
Metabolic activation: without
Result: positive

Method: other
GLP: no data Test substance: no data

Method: The mutagenic inactivation of a bacterio phage repressor gene leading to subsequent replic ative killing of the bacteria was used as mutagenicity screening model. Bacteria were exposed for 10 min. at 30 øC

. Remark: non-BASF study Result: Methanol was toxic and mutagenic at conc entrations of 23 % (v/v) and more (extreme high conc entrations necessary to produce the "mutagenic" effect). Test substance: Methanol, no further data. Reliability: (3) invalid Questionable, not validated test system, extreme high concentrations necessary to produ ce the "mutagenic" effect.
25-OCT-2006 (220)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

165 5.6 Genetic Toxicity 'in Vivo'

Type: other: Cytogenetic assays including ch romosomal aberration, SCE, and micronuclei Species: mouse Sex: no data Strain: no data Route of admin.: oral unspecified Exposure period: no data
Doses: 1000 mg/kg
Result: positive

Method: other: no data
Year: 1982
GLP: no data Test substance: no data

Remark: non-BASF study Result: The incidences of chromosomal aberration s, particularly aneuploidy and SCE in bone marrow cells as well as micronuclei in polychromatic eryt hrocytes are reported to be increased. No detailed results pr esented.

-------------------------- Test substance: Methanol, no further data Reliability: (4) not assignable Only abstract, no detailed data.

25-OCT-2006 (221)

Type: Sister chromatid exchange assay Species: mouse Sex: male Strain: C57BL Route of admin.: inhalation Exposure period: 6 h/day, 5 days

Doses: 800 or 4000 ppm (1.04 or 5.2 mg/l)
Result: negative

Method: other: see Method
Year: 1991
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive test programme (see also o ther entries this chapter): Primary cultures of lun g cells of treated mice and
controls were established and 50 second-division metaphases
were scored for SCE induction. Re plication indices were determined of 200 metaphases and mitotic indices of 1000
cells. Remark: non-BASF study Result: No treatment related SCE induction was s een: SCEs ranged from about 11 - 12 per metaphase irrespective of exposure
and control.

The replicative and mitotic indic es were not significantly
different from the control.

----------------------- Test substance: Methanol, 99.9 % pure from Fisher Scientific Cor poration,
no further data. Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficient documentation, accepta ble for assessment,

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

166 however, missing positive control

25-OCT-2006 (222)

Type: Micronucleus assay Species: mouse Sex: male Strain: ICR Route of admin.: gavage Exposure period: single, 24 h
Doses: 1050; 2110; 4210; 8410 mg/kg
Result: negative

Method: other: see Method
Year: 1987
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Six animals were used per group. A posit ive control group was enclosed (TEM, 0.5 mg/kg). Bo ne-marrow was sampled 24 h post-treatment; 1000 polychromati c erys were scored per mouse for the frequency of micron uclei.

--------------- Remark: non-BASF study Result: There was no methanol-related increase i n the induction of micronuclei: In treated groups, t he mean MN rate varied from 0.5 to 2.2/1000 vs. 1.5/1000 in t he neg. control and 45/1000 in the pos. control group.

--------------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co.,
no further data. Reliability: (2) valid with restrictions Comparable to guideline study and current standards, limited documentation, restriction: only one sampling time

25-OCT-2006 (167)

Type: Micronucleus assay Species: mouse Sex: male/female
Strain: NMRI Route of admin.: i.p. Exposure period: single
Doses: 1920; 3200; 4480 mg/kg
Result: negative

Method: other: see Method
Year: 1981
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comparative study including a broad spec trum of 30 h after a single dose, bone-marrow cells we re isolated from 2 male and 2 female mice per dose, and 1000 polychromatic erythrocytes per mouse scored.

----------------- Remark: non-BASF study Result: There was no significant effect on the f requency of micronuclei in treated as compare d to control animals: 1.3/1000 (control and low dose), 2.0/1000 (2nd dose), and 3.7/1000 (top dose).

----------------

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

167 Test substance: Methanol, from Merck, Germany, no further data. Reliability: (2) valid with restrictions Comparable to guideline study and current standards, limited documentation, restriction: only one sampling time

25-OCT-2006 (223)

Type: Micronucleus assay Species: mouse Sex: female Strain: CD-1
Route of admin.: gavage Exposure period: gd 6 through 10 during pregnancy

Doses: 2500 mg/kg/d
Result: negative

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Study performed during a developmental i nvestigation under
folate-deficient and -sufficient conditions (see other entry 5.8.2).

Maternal peripheral blood was col lected 48 h after the final exposure to methanol (still durin g pregnancy), and fetal blood was taken on gd 18. The fr equency of MN was scored in 1000 reticulocytes.

---------------------- Remark: key study non-BASF study Result: The MN rate in maternal and fetal reticu locytes was similar among the groups. There was no as sociation between either maternal or fetal hepatic folate concentrations. Furthermore, the comparison of MN frequencies in malformed and normal litters gave no eviden ce of an increase of MN.

------------------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented , key study 25-OCT-2006 (224)

Type: Micronucleus assay Species: mouse Sex: male
Strain: Swiss Webster
Route of admin.: i.p. Exposure period: 4 days
Doses: 300, 600, 1200, 2500 mg/kg bw
Result: negative

Method: other: ASTM 1988
Year: 1990
GLP: yes Test substance: as prescribed by 1.1 - 1.4

Remark: Objective of the study: possible effects of methanol were to
be evaluated as function of folat e status, because folate deficiency is known to increase t he spontaneous incidence of micronucleated erythrocytes and s trongly enhance the incidence of micronuclei induced by certain chemicals, and

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

168 because methanol oxidation via fo rmate to CO2 depends on tetrahydrofolate.

--------------------- key study non-BASF study Result: 4/10 folate-deficient animals receiving 2500 mg/kg died between days 2 and 3.

No difference in micronucleus fre quency (MN in PCEs) and in RNA-positive blood erythrocytes w as seen between treated groups and controls while animals treated with the positive control substances responded as e xpected:

+folate: MN 0.17 - 0.23 % vs. 0. 23 % in saline control
-folate: MN 0.31 - 0.37 % Vs. 0. 38 % in saline control.

Caffeine (+folate): MN 0.34 % Caffeine (-folate): MN 2.42 %

Urethane (+folate): MN 2.52 %.

----------

Test condition: Mice were made folate-deficient by maintaining o n a diet
with no folic acid and 1 % succin yl sulfathiazole, inhibitor of the catalase, and another grou p received folate-supplemented diet containi ng 5 mg/kg folic acid. Blood samples were taken at 24 ho urs after the last dosing.
1 10 mice per dose group were used.

Folate limitation resulted in an about 15-20 times lower
folate blood level than in the hi gh-folate groups.

--------------- Test substance: Methanol 99.9 % pure, no further data.
Conclusion: The results indicate that methanol is noncla stogenic to the developing erythroblast in bone m arrow and does not inhibit red blood cell production in eith er normal or folate-deficient mice.

---------- Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment , key study
25-OCT-2006 (225)

Type: Micronucleus assay Species: mouse Sex: male Strain: C57BL Route of admin.: inhalation Exposure period: 6 h/day, 5 days

Doses: 800 or 4000 ppm (1.04 or 5.2 mg/l)
Result: negative
Method: other: see Method
Year: 1991
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive test programme (see also o ther entries this chapter): Five animals per dose w ere used.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

169 1. Peripheral blood MN: Blood smears from tail vein blood were used to score for 2000 polyc hromatic and 2000 normochromatic erythrocytes per a nimal.

2. Primary cultures of lung ce lls: Lung MN were analyzed in 1000 binucleated cells per ani mal after cell isolation from exsanguinated and perfused l ung.

------------------ Remark: key study non-BASF study Result: No treatment related effect on micronucl ei frequency or cell kinetics and no toxicity was seen at any dose level:

Blood erys: Mean MN rates were 3. 1/1000 and 5.2/1000 in PCE, and 3.4/1000 and 3.6/1000 in NCE at either dose, respectively, vs. 5.0/1000 and 3. 7/1000 of respective
controls.

Lung cells: Mean MN rates were a bout 20/1000 to 24/1000 irrespective of a dose or control group. The ratio of bi- to mononucleated cells was not influenced by the treatment, neither was the re evidence of a
treatment-related increase in the incidence of
multi-nucleated cells.

----------------------- Test substance: Methanol, 99.9 % pure from Fisher Scientific Cor poration,
no further data. Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment, however, missing positive control , key study
25-OCT-2006 (222)

Type: Cytogenetic assay Species: mouse Sex: male
Strain: C57BL Route of admin.: inhalation Exposure period: 6 h/day; 5 days

Doses: 800 or 4000 ppm (1.04 or 5.2 mg/l)
Result: negative

Method: other: lung-cell analysis
Year: 1991
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive test programme (see also o ther entries this chapter): Primary cultures of lun g cells): Chromosome aberrations were scored in primar y culture lung cells of treated mice. Primary cultures of lung cells were established from 5 mice per group and 100 first-division metaphases per animal were examined for chromoso me aberrations.

No positive control group was con comitantly examined.

-------------------- Remark: key study non-BASF study Result: No increase in the frequency of aberrati ons per cell and

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

170 percent aberrant cells and no tox ic effects were induced by
the treatment: Aberration rate per cell (includi ng chromatid and chromosome breakage as well as rearrangement events) was from 0.04 to 0.07 in treated animals vs. 0.09 in the negative control. The percentage of aberrant cells ranged from 3.5 to 6.2 % vs. 6.9 in the neg. control. The replicative and mitotic indic es were not significantly
different from the control.

------------ Test substance: Methanol, 99.9 % pure from Fisher Scientific Cor poration,
no further data. Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment, however, missing positive control , key study
25-OCT-2006 (222)

Type: Cytogenetic assay Species: mouse Sex: male Strain: C57BL Route of admin.: inhalation Exposure period: 6 h/d; 5 d

Doses: 1.06; 5.3 mg/l (800; 4000 ppm)
Result: negative

Method: other: spermatocyte analysis
Year: 1991
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive test programme (see also o ther entries this chapter): Spermatocytes of the pa chytene substage of the meiotic prophase after 5 days of last exposure were isolated, and isolates fixed for electronmicroscopy: Several morphological parameters concerni ng the meiotic chromosome structure were examined (synapton emal complex analysis). The so-called "synaptonemal compl ex" is the attachment site of homologues chromosomes during meiotic prophase. An increase in the frequency of syna ptic lesions may be an indicator for exposure to mutagen s.

No positive control group was con comitantly examined.

-------------------- Remark: key study non-BASF study Result: Methanol exposure gave no evidence of a dose-related
increase in structural abnormalit ies of the synaptonemal complex.

------------- Spermatocytes of the pachytene su bstage of the meiotic prophase after 5 days of last exp osure were isolated, and
isolates fixed for electronmicros copy: Several morphological parameters concerning the meiotic chromosome structure were examined (synaptonemal complex an alysis). The so-called "synaptonemal compl ex" is the attachment site of homologues chromosomes during meiotic prophase. An increase in the frequency of syna ptic lesions may be an indicator for exposure to mutagen s.

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

171 -------------------- Test substance: Methanol, purity 99 % Reliability: (2) valid with restrictions Test procedures based on scientif ic principles and standards, sufficiently document ed, acceptable for assessment, however missing pos. control. , key study
25-OCT-2006 (222)

Type: Cytogenetic assay Species: mouse Sex: Strain: no data
Route of admin.: i.p.
Doses: 75 - 300 mg/kg (3x/d)
Result: positive

Method: other: no data
Year: 1983
GLP: no data Test substance: no data

Remark: There is some evidence that bone marrow cytogenetic analysis indicated a dose-related response for structural aberrations, especially centric fusions in mic e treated with three daily doses of methanol (IPCS/WHO 1997)

. non-BASF study Test substance: Methanol, no further data Reliability: (4) not assignable Only abstract, no detailed data

25-OCT-2006 (226) (88)

Type: Drosophila SLRL test Species: Drosophila melanogaster Sex: male Strain: other: Berlin K (wild type, male) and Ba sc (female) Route of admin.: oral feed
Doses: LD50
Result: negative

Method: other: probably largely according to OEC D Guide-line 477
Year: 1981
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: non-BASF study Result: Methanol was not mutagenic in the "Basc- Test in Drosophila".
Test condition: One dose (1000 mM) close to the LD50 was adminis tered to adult male wild-type flies in 5 % sacch arose and mated to virgin
females. About 1200 X-chromosomes were tested per experiment in each of 3 successive broods (3 , 3, and 4 days). Test substance: Methanol from Merck, Germany, no further data. Reliability: (2) valid with restrictions Acceptable study, meets basic sci entific principles

25-OCT-2006 (227)

Type: Micronucleus assay Species: mouse Sex: no data Strain: Swiss
Route of admin.: unspecified

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

172 Exposure period: 4 days
Doses: 300, 600, 1200, 2500 mg/kg bw
Result: negative

Method: other
Year: 1991
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Groups of folate deficient and folate su pplemented mice were compared to clarify the role of tetrahydrofolate mediated formate metabolism in th e genotoxic activity of
methanol.

------------- Ten mice per dose level, no furth er details indicated. Remark: non-BASF study Result: No genotoxic effects were observed in an y treated group. Test substance: Methanol, no further data. Reliability: (4) not assignable Only short communication, abstrac t: But see Amer. Petrol. Inst.: probably the same study.
25-OCT-2006 (228)

5.7 Carcinogenicity

Species: rat Sex: male/female
Strain: Fischer 344/DuCrj
Route of administration: inhalation Exposure period: 24 months Frequency of treatment: continous, 20 h/d Post exposure period: none Doses: 10; 100; 1000 ppm (0.013; 0.13; 1.3 mg/l): 14255 - 14323 h (males); 14407 - 1 4468 h (females) Result: negative Control Group: yes, concurrent no treatment

Method: other: acc. to OECD Guide-line for Chron ic toxicity, Oct. 1980
Year: 1985
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity
studies: Whole-body exposure: The animals were housed individually in wire-mash cages attached to the i nhalation chamber.

52 animals per sex and dose group were tested (initial age 6 weeks). Clinical, ophthalmologica l, clinical-chemical, hematological and pathological/hi stopathological
examiniation were done. Analytical control of chamber con centration: Analytical
values close to nominal ones.

STATISTICS: All data obtained were analysed b y t-test, Fischer ́s exact test or Armitage ́s chi2-test.

------------------------- Remark: The blood levels of methanol were measur ed but not documented

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

173 in detail (reported to be similar to those found in the two-generation study: see there). Formate was not measured.

"Blood dose levels in the low-dos e (10 ppm) and mid-dose (100 ppm) group were almost comparable to those in the control group. In the high-dose group (10 00 ppm), however, they were 54 ppm for males and 88 ppm for f emales." (Discussion part p. 153).

Note: Exposure per day was 20 h, which implies that prolonged steady-state blood levels were re ached which even may have been higher than in studies using the same exposure concentration, but shorter exposu re times.

-------------------- key study non-BASF study Result: 19.2 to 34.6 % of males and 32.7 to 40.4 % of females died
or had to sacrificed in extremis. There were no significant differences from the control grou ps.

No abnormalities of the general s tate could be observed in treated animals. Body weight was somewhat but not significantly retarded between we eks 51 through 72 in the highest female dose-group (1000 p pm). Urinanalysis revealed a positive trend in glucose and a decrease in pH in males of the 10 00-ppm group, but values were within normal range. In 1000 -ppm females, higher bilirubin values were found, yet within normal range. No changes were evident in haemat ological and biochemical parameters.

Non-neoplastic findings were simi lar to those commonly found in the control animals of 24 mont hs (p. 282).

NEOPLASTIC CHANGES: Incidences (number of tumor-beari ng animals/total animals (Tab. 6, p. 146, and Discussion, p. 154/155):

MALES 0 10 100 100 0 ml/m3

================================= ======== A 1/52 5/50 2/52 6/5 2 B 4/52 1/50 5/52 4/5 2

--------------------------------- -------- A+B 5/52 6/50 7/52 10/ 52

--------------------------------- -------- C 7/52 -- -- 4/ 51

================================= ======== -- = not examined A) Papillary adenomas (males) B) Adematoids = hyperplastic chan ges (pre-neoplastic) (males) C) Adrenal chromaffinoma (males)

FEMALES 0 10 100 100 0 ml/m3

================================= ======== A 2/52 -- -- 2/5 2 B 3/52 -- -- 1/5 2

--------------------------------- -------- A+B 5/52 -- -- 3/5 2

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

174 --------------------------------- -------- C 2/50 3/51 2/49 7/5 1

================================= ======== -- = not examined A) Papillary adenomas (males) B) Adematoids = hyperplastic chan ges (pre-neoplastic) (males) C) Adrenal chromaffinoma (females )

There was no dose-dependence of t umour development for types A, B and the sum of A and B in ma les. The spontaneous tumor rate of the Fischer strain in thi s laboratory and in the published literature was in the r ange of 14 %. Therefore, the tumours are unlikely to be treatm ent-related.

No clear dose-dependence was note d for C (chromaffinomas) in females, but at the highest dose the number of animals with this kind of tumors was significa ntly increased and was higher than the spontaneous rate publish ed for the Fischer strain (3.2 - 3.5 %) (p. 155) [Note: not clear whether the given data relate only to females or to both sexes. It should be noted that the spontaneous rate of male rats was high in this test compared with that of females.]

Overall, tumor frequencies showed no significant differences between all groups. There was no evidence of an increase in liver tumors. Specific tumours ap peared at a somewhat higher incidence in high-dose groups of both sexes:

- papillary lung adenomas in ma les (without dose relationship and not statisticall y significant: 6/52 vs. 1/52 in the control ), but not in fema les (Tab. 8, p. 149, see also Takeda and Katoh 1988); - adrenal phaeochromocytomas in females (based on Fisher ́s Exact Test: not statistically sig nificant: 7/51 vs. 2/50), but not in males; - 3/52 and 5/52 metastatic (tra nsition) tumors of common origin in males and female s, respectively (originated from tumours prone to metatasis ( see p. 155/156).

These events having emerged after week 79 and 104 were considered incidental and not tre atment-related.

------------------------------ ------ Test condition: Exposure time 20 hours per day, significantly lo nger than according to guideline (6 hrs). T his does not represent
normal industrial and consumer us e.

--------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride; <3 ppm for maldehyde Conclusion: The results gave no evidence of a clearly ca ncerogenic potential of methanol in rats. Th e biological significance of
the increase in the incidence of the phaeochromocytomas in the aged female high-dose rats is con sidered to be low.

------------------------ Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, documentation limited, acceptable for assessment. , key study
26-OCT-2006 (88) (167) (180)

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

175 Species: mouse Sex: male/female
Strain: B6C3F1
Route of administration: inhalation Exposure period: 18 months Frequency of treatment: continuous, ca. 19 h/d Post exposure period: none Doses: 10; 100; 1000 ppm (0.013; 0.13; 1.3 mg/l: 10436 - 10550 h (males); 10573 - 10642 h (females) Control Group: yes, concurrent no treatment

Method: other: according to OECD Guide-line for chronic toxicity, Oct.
1980
Year: 1985
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity
studies: Whole-body exposure: The animals were housed individually in wire-mash cages installed in the inhalation chamber.

52 animals per sex and dose group were tested (initial age 6 weeks). Clinical, ophthalmologica l, clinical-chemical, hematological and pathological/hi stopathological
examiniation were done.

Analytical control of chamber con centration: Analytical
values close to nominal ones.

------------------------- Remark: The blood levels of methanol and formate were not measured. Note: Exposure per day was 20 h, which implies that prolonged steady-state blood lev els were reached which even may have been higher than in stud ies using the same exposure concentration, but shorter exposu re times.

-------------------- key study non-BASF study Result: Survival was high without significant di ffferences as compared to the control, mortalit y ranging between no deaths and 7.5% deaths by the end of the study: 0% (1000 ppm males, 10 ppm females), 2% (controls), 6 % (10 and 100 ppm males) and 7.5% (100 and 1000 pm females ).

No abnormalities of the general s tate could be observed in treated animals. Body weight was somewhat increased in treated group, significantly in 1 000 ppm males, between months 6 through 12 in males and months 9 through 12 in
females. There was no evidence of a treatm ent-related effect in clinical/blood parameters.

Non-neoplastic organ findings wer e similar to those commonly
found in the control animals of 1 2 months.

Individual tumor frequencies show ed no significant differences between all groups; t here was no difference in the severity of tumors between th e groups. The tumors exclusively observed i n the 1000-ppm group were

### OECD SIDS METHANOL

5. TOXICITY ID: 67-56-1 DATE: 26-OCT-2006

176 all within the spontaneous range and included fatty cell tumor at the submaxillary lymph n ode (1/52 males), chromophobe adenoma of the pituar y gland (3/52 females), adrenal pheochromocytoma (1/52 f emales), dermal fibrosarcoma (1/52 males), and me ningeal sarcoma (1/52 females) vs. 0/52 in all other gr oups. The spontaneous liver-tumors rate naturally high in this species was not increased by meth anol treatment (p. 171; Tab. 5, p. 172).

------------------------ Test condition: Exposure time 19 hours per day, significantly lo nger than according to guideline (6 hrs). T his does not represent
normal industrial and consumer us e.

--------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride; <3 ppm for maldehyde Conclusion: The result gave no evidence of a cancerogeni c potential of
methanol in mice.

--------------------- Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, documentation limited, acceptable for assessment. , key study
26-OCT-2006 (167) (180)

Species: rat Sex: no data Strain: no data Route of administration: oral unspecified Exposure period: no data Post exposure period: no data
Doses: no data Result: negative

Method: other: no data
GLP: no data Test substance: no data

Remark: Tabular compilation of BT Institute data without details: The author reports that Methanol was tested in a carcinogenicity study with oral a pplication in the rat. The substance was clearly not carcino genic according to the
author. No further details are re ported.

------------- non-BASF study Test substance: Methanol, no further data. Reliability: (4) not assignable Only short communication
26-OCT-2006 (229)

5.8.1 Toxicity to Fertility

Type: Two generation study Species: rat
Sex: male/female
Strain: other: Sprague Dawley (Crj:CD)
Route of administration: inhalation Exposure Period: F0: 103 - 108 d; F1: 61-62 d and 145 - 153 d; F2: 54 - 56 d (see Method) Frequency of treatment: continuous (19 - 20 h/d) Premating Exposure Period

### OECD SIDS METHANOL

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177 male: 60 d (approx. 1200 h)
female: 60 d (approx. 1200 h) No. of generation studies: 2
Doses: 0.013; 0.13; 1.3 mg/l (10; 100 ; 1000 ppm) Control Group: yes, concurrent no treatment NOAEL Parental: = 1000 ppm NOAEL F1 Offspring: = 100 ppm NOAEL F2 Offspring: = 100 ppm other: LOAEL F1/F2 offspring : = 1000 ppm

Method: other: in accordance with OECD Guideline s, Oct. 1980
Year: 1985
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity
studies: Whole-body exposure: One multi-st age inhalation chamber was used for each group.

The duration of exposure was as f ollows (p. 184):

F0-Males from age of 8 wk through end of mating (16 wk or after); F0-females from age of 8 wk throu gh mating period (16 wk or after) to end of lactation.

F1-males from birth through end o f mating (at 14 wk or after); F1-females from birth through end of mating (at 14 wk or after) to weaning of F2 pups (21 d after delivery).

F2-Males and -females from birth to 21 d (but to age of 8 wk for 1/sex/litter).

F0 generation consisted of 30 ani mals per sex and group. Additionally, 15 animals were rea red for a second mating (p. 185).

Mating period was maximally 21 d. The pairs without evidence
of insemination within 21 d were again cohabited with untreated animals (2nd mating) to determine the fertility of each animal, in this case without exposure (p.186).

Pathohistology: After mating all males were necro psied, and testes as well as all accessory reproductive org ans preserved. Females either after second mating or not inseminated ones were necropsied and examined for repro ductive organs. Likewise, all other females having delivere d or not were sacrificed for histopathology at due time.

Litters: Part of the pups of each generation were examined for intra-uterine, another one fo r neo- and postnatal development/morphological differe ntiation including behavioral/function testing and h istopathology until one

### OECD SIDS METHANOL

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178 week after weaning (p. 187/188). Gametogenesis: Histological exami nation of morphology of ovaries and sperms was not includ ed.

In 9-week old F1 pups, blood meth anol was measured, but not formate (p. 191).

Analytical exposure control: Anal ytical concentration values
of methanol were close to nominal ones.

-------------------------

Statistical ANALYSIS: All data obtained were analysed b y t-test, Fischer ́s exact test, U-test of Mann-Whitney or A rmitage ́s chi2-test.

------------------------ Remark: Blood levels of methanol measured in the F1 offsprings (age 9 weeks) (NEDO, 1987, p. 191):

controls (baseline): approx. 2 - 3 ug/ml 10 ppm methanol: approx. 3 - 3.5 ug/ml 100 ppm " : approx. 1 - 4.2 ug/ml 1000 ppm " : approx. 53 ( males)-100 (females) ug/ml.

There are no data on formate. Note: Exposure per day was 20 h, which implies that prolonged steady-state blood leve ls were reached which even may have been higher than in stud ies using the same exposure concentration, but shorter exposu re times.

-------------------- key study non-BASF study Result: F0-Generation: No treatment-related alte rations in all reproductive parameters.

F1-Generation: None of the fertil ity indices including sexual cycle, mating time, fertil ity and pregnancy rate showed a significant difference f rom untreated F1 controls.

In male pups of the 1000-ppm grou p, post-natal morphological differentiation appeared to be in fluenced with respect to the descensus testis occurring 0. 5 to 1 d earlier (see same effect in F2 generation) (p. 199/ 200) [not mentioned by Takeda and Katoh, 1988]: This time-depen dent parameter was evaluated by relating the completion of dow nward migration of the testes (final length of th e gubernaculum reached) to the post-natal body-weight gain ( The more reliable body length was not available):

F1-generation: In the F1 pups derived from the 1 000-ppm group(108 males), this process was completed within 16 through 20 post-natal d ays with the climax at day 17 and 18 (32 and 39 %, respectively ), while in the respective control (113 males), descent was complete from 16 through 21 days with the maximum at day 19 (32 %), but also relatively high percentages on th e days before and after: day 18 (22%), day 17 (19%), day 20 (1 8%) (p. 199).

Absolute and relative brain weigh ts were significantly lowered in the high-dose groups of either sex at an age of 8 and 16

### OECD SIDS METHANOL

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179 weeks. This was still found in fe males necropsied after 24 weeks. Also other organs showed slight shifts in weights: thymus, pituitary (lower ), heart, lung, liver (higher). However, there were no histologic al manifestations.
No effects on testes or ovaries r eported.

Likewise, there were no significa nt differences in functional tests (movement, emotion, learnin g) as compared with the control or the other groups.

F2 generation: As in F1 males, an apparently dos e-related earlier descensus testis was noted after 1000-ppm e xposure: F2 (94 males) on day 16 (42%), day 17 (40%), day 18 (1 5%) vs. control (91 males) on day 16 (10%), day 17 (39%), day 1 8 (31%), day 19 (14%) (p. 200). After 100-ppm, "descensus testis" in male F2-progeny was about
0.5 d earlier than in male contro l F2 pups (p. 195). Detailed data not specified and not addres sed under "Discussion".

Organ weights showed similar tend encies as found in the F1-generation without histologica l changes. No effects on testes or ovaries reported.

---------------------- Test condition: Exposure time 19-20 hours per day, significantly longer than according to guideline (6 hrs).

--------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride; <3 ppm for maldehyde Conclusion: No firm conclusions can be drawn about ferti lity of either sex, as the copulation time of 21 d was comfortably long for successful insemination (but in a ccordance with guidelines at that time) and gametogenesis w as not considered.

In the F1 and F2 progeny (both se xes), a decrease in brain weights was evident at 1000 ppm m ethanol, but without noticeable histological changes a nd functional impairments. This phenomenon is believed to re present a change occurring during the perinatal period (Take da and Katoh, 1988).

But no quantitative data and the statistical level are documented for organ weights. The information about organ weigh ts is lacking, with much weight placed on the "descensus t estis".

The meaning of an apparent shift of testis descent in male offspring in relation to body-wei ght development of the pups in the two following generations is unclear and was not directly addressed by Takeda and Katoh (1988), but detailed by NEDO (1987) and considered a s ignificant difference from untreated controls (p. 285). Furt hermore, it is obvious that this parameter showed considerabl e variation also between the control groups of both genera tions (see p. 199/200).

Although there was no obvious pat hological effect in the progeny of 1000-ppm exposed group s, the effects observed may be considered as biologically rel evant under these test conditions and, therefore, 1000 p pm (1.3 mg/L) is established

as LOAEL

### OECD SIDS METHANOL

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180 and 100 ppm (0.13 mg/L) as NOAEL for post-natal development while for parental effects, the NOAEL is 10 00 ppm.

The NOAEL(develop) = 0.13 mg/L is also compatible with a NOEL.

------------------ Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, documentation limited, acceptable for assessment , key study
26-OCT-2006 (167) (180)

Type: Fertility Species: monkey
Sex: female Strain: Macaca Fascicularis Route of administration: inhalation Exposure Period: prebreeding about 120 days; breeding 70 days; pregnancy about 165 days Frequency of treatment: 2.5 h/d Premating Exposure Period
male: none female: prebreeding about 120 days
Duration of test: birth, first 9 months of life of the offs prings
Doses: 200; 600; 1800 ppm (0.262; 0.7 86; 2.358 mg/l) Control Group: yes NOAEL Parental: = 1800 ppm other: NOAEL(Repro) (mother) : = 1800 ppm

GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Study design limited to exposure of fema le animals (pre-breeding, breeding, and preg nancy) and qualitative/clinical signs of tox icity. It comprised

- methanol pharmacokinetics (se e other entry: 5.11) - maternal toxicity - reproductive toxicity (see ot her entry: 5.8.2) - developmental neurotoxicity ( see other entry: 5.8.2).

Specific aims of the study were t o determine whether - repeated exposure to methanol alters methanol metabolism in adult female monkeys, - pregnancy alters methanol met abolism, - long-term exposure to methano l produces overt adult toxicity, or reproductive toxic ity, or both, - long-term exposure to methano l in utero affects offspring development, especi ally neurobehavioral development.

Groups of 11-12 adult female monk eys were exposed to methanol vapor in the concentrati ons of 0; 200; 600 or 1800 ppm, daily for 2.5 hour before br eeding, during breeding and during pregnancy. The study w as performed in 2 cohorts: 24 females/cohort and 2 males/coh ort.

For postnatal developmental evalu ation, 8 to 9 infants were available per group, in total 34, among them 26 in-utero

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181 treated offsprings (see other ent ry: 5.8.2).

------------------------------ ------ Remark: key study non-BASF study Result: Reproductive performance: Methanol exposure had no effect o n most measures of reproductive performance, includi ng menstrual cycles, conception rate, and live-birth d elivery rate. However, all methanol-exposed animals had a de crease of about six to eight days in duration of pregnan cy compared to control animals (Tab. 17, p. 45).
It is not clear whether this decr ease was related to methanol exposure, as there was n o dose response and no differences among offspring group s in body weight or other physical parameters. Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months.

------------------- Test substance: Methanol, no further data Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, well documented, limitat ion due to low number of
animals. , key study
26-OCT-2006 (230)

Type: Fertility Species: mouse Sex: male Strain: B6C3F1
Route of administration: gavage Exposure Period: 5 d Frequency of treatment: 1x/d, 5 consecutive days Premating Exposure Period
male: sperm morphology, no mating
female: no females
Doses: 5 mal 1000 mg/kg

Method: other: sperm morphology
Year: 1984
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Ten male mice (4 months of age) were tre ated and maintained without exposure for 5 weeks (the time needed for testicular spermatogenesis and migration of spermatides to the epididymidis), spermatozoa were i solated from dissected cauda epididymidis and fixed and mounted on slides, and stained for light microscopy. 500 sperms were evaluated per animal.

----------- Remark: non-BASF study Result: In the methanol-treated animals, a sligh t, but statistically insignificant increase in sperms abnormalities (mean factor about 1.7 above neg. control, wit h very high variance: 1.86
+-0.91 vs. 1.12 +-0.39 in the veh icle control) was observed, while the treatment with cyclopho shamide (100 mg/kg)
resulted in an about 6-fold incre ase (5.84 +-1.94).

----------------- Test substance: Methanol, no further data.

### OECD SIDS METHANOL

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182 Reliability: (2) valid with restrictions Test procedures based on scientif ic principles and standards, limited documention, acceptable for assessment

26-OCT-2006 (231)

5.8.2 Developmental Toxicity/Teratogenicity

Species: rat Sex: female Strain: Sprague-Dawley Route of administration: inhalation Exposure period: gd 1-19, at the top dose: gd 7- 15 Frequency of treatment: 7h/d
Duration of test: 21 d
Doses: 6.63; 13.3; 26.6 mg/l (5000; 1 0000; 20000 ppm) Control Group: yes, concurrent no treatment NOAEL Maternal Toxity: = 10000 ppm NOAEL Teratogenicity: = 5000 ppm

Method: other: acc. to standards
Year: 1985
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Whole-body exposure: Mean analytical cha mber concentrations corresponded well to the nominal ones. 15 dams per group were used. Meth anol blood levels were measured in concurrently exposed non-pregnant groups by GC on day 1, 10, and 19.

Dams were sacrificed on gd 20, on e half of the fetuses was subjected to examination for visc eral, the other half for skeletal abnormalities.

---------------- Remark: The biological relevance of this study f or humans has to be questioned for the following cave ats:

1. The exposure concentrations of methanol relate to doses which significantly exceed lethal doses in man: lowest dose (5000 ppm, 7 h) in rats about 180 0 mg/(kg bw*d) based upon calculations (NTP-CERHR, 2001, Ta b. 2-10) [(human lethal dose: 300 - 1000 mg/(kg*d)].

2. This high dose which would be lethal to humans proved to be ineffective in rats.

3. The study was performed in a r ange where methanol metabolism is totally saturated i n rats, not considered as
relevant for humans.

4. Consequently, the blood methan ol levels in rats exposed to 5000 ppm are estimated to be a t a factor of 20-50 higher than in humans exposed e.g. to 10 00 ppm (7-8 h).

5. The differences in metabolism (predominance of catalase in rodents vs. alcohol dehydrogen ase (ADH) in humans) has to be regarded: rate limiting steps are catalase given rise to methanol, but not formate, in blo od of rodents, while in humans rate-limiting is formate d egradation giving rise to
increased formate levels in human blood upon kinetic

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183 saturation.

------------------------------ --- key study non-BASF study Result: Blood levels (dams): 1.0-2.17 mg/ml (5000 ppm); 1.84-2 .24 mg/ml (10000 ppm), 5.25-8.65 mg/ml (20000 ppm). No background levels available.

Maternal toxicity: Only in the high-dose group, slig htly unsteady gait and significantly reduced food consum ption during the first week without adverse effect on body-we ight gain. No signs of maternal toxicity in the other gr oups.

Effects on maternal reproduction: No influence on the number of cor pera lutea and of implantations. No effect on fetal lethality and resorption.

Effects on litters/fetuses: Statistically significant and dos e-related decrease in fetal body weight in the two high-dose groups (p<0.05) [note: The significance is poor for the male /female offsprings of the 10000-ppm group when compared to the control group of other studies (see: ethanol, Nelson et al., Tab. 2, p. 731].

At the highest concentration, an increased number of litters
with skeletal and visceral malfor mations was noted, these included in particular rudimentar y and extra cervical ribs and exencephaly and encephalocele , and, to minor extent, cardiovascular and urinary-tract defects:

Visceral defects: 7/15 litters wi th 15/96 malformed fetuses vs. 0/15 litters and 0/107 fetuse s in the untreated control.

Skeletal defects: 14/15 litters w ith 72/92 malformed fetuses vs. 0/15 litters and 0/98 fetuses in the untreated control.

Similar abnormalities also appear ed in the progeny from the 10000-ppm group, but not statisti cally significant: visceral and skeletal defects 2/15 litter s each with 2/107 and 2/115 fetuses, respectively).

In the 5000-ppm group, visceral d efects were 1/13 litters with 2/90 fetuses, and skeletal defect s were 1/13 with 1/90
fetuses.

-------------------------

There was no evidence of embryoto xic/teratogenic activity of methanol at 5000 ppm.

------------------------ Test substance: Methanol, 99.1 % purity

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184 Conclusion: (For biological relevance for humans: see Re marks.)

1. The CERHR Expert Panel place d high weight on the reliability of results of this pr enatal study (NTP-CERHR, 2001).

2. Based on a logistic quantita tive model of Risk Assessment for Developmental Neur otoxicants (Slikker and Gaylor, 1997, 1998) using the inh alation data in SD rats from Nelson et al., 1995, the fol lowing conclusions were
drawn and estimations made:

The use of continuous foetal weig ht data was as sensitive as
foetal brain malformation data in estimating excess risk to methanol inhalation exposure in d eveloping rat. Excess risk (assumed to be the additional ris k that foetal weight is below the first percentile of con trol animals) was estimated by using a logistic model: 0.1 a t 16000 ppm, 0.01 at 5400 ppm, and 0.001 at 980 ppm.

------------------------ Reliability: (2) valid with restrictions Comparable to guideline study and current standards, sufficiently documented, acceptab le for assessment , key study
26-OCT-2006 (232) (233) (234) (235)

Species: rat Sex: female Strain: Sprague-Dawley Route of administration: inhalation Exposure period: 7 - 17 gd Frequency of treatment: continuous, ca. 22.7 h/d (NEDO, 1987, p. 215)

Doses: 200; 1000; 5000 ppm (0.26; 1.3 ; 6.64 mg/l) Control Group: yes, concurrent no treatment NOAEL Maternal Toxity: > 1000 ppm NOAEL Teratogenicity: > 1000 ppm LOAEL Maternal Toxicity : = 5000 ppm LOAEL Teratogenicity : = 5000 ppm other: NOEL maternal/fetal : = 1000 ppm

Method: other: acc. to national standards (see M ethod)
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity studies (analog Segment II) Whole-body exposure: Pregnant fem ales were housed individually in a cage placed in the inhalation chamber.

36 dams were used per test and co ntrol group, including 12 dams allowed for natural delivery (p. 211). The study comprised of three segm ents: - intra-uterine development/te ratogenicity, - post-natal development F1 (l actation, 21 d, and >=5 d after weaning) (without exposu re) (p. 212), and - reproductive performance of F1 to produce F2.

Analytical exposure control: Anal ytical concentration values

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185 of methanol were close to nominal ones.

------------------------- Remark: Note: Exposure per day was >20 h, which implies that prolonged steady-state blood lev els were reached which even may have been higher than in stud ies using the same exposure concentration, but shorter exposu re times.

-------------------- key study non-BASF study Result: The exposure to 200 and 1000 ppm methano l failed to produce any maternal and embryonal/fetal toxicity or fetal
malformations.

At 5000 ppm, maternal toxicity wa s noted as a decrease in body-weight gain. Food and drink ing water consumption were reduced during gd 7 through 12. One dam died, another one had to be killed before delivery.

After Cesarian section: Embryolethality was increased (la te resorptions: 10.4 % vs. 0.6 % in control, p<0.05), but th e variance between single litters was high (Tab. 2, p. 219) . No differences were evident in the number of corpora lutea, of implantations, and preimplantation resorptions ( p. 219). Mean body weight
of live fetuses after Cesarian se ction was reduced (about -20 %, p<0.001) (p. 220).

Morphological abnormalities in fe tuses: Only after intra-uterine exposure to 5000 ppm, the incidences of various malformatio ns and variations were
increased:
About 50 % of the fetuses with ve ntricular septal defects (visceral malformation in 16/20 l itters or 64/131 fetuses) vs. 0% or near 0% in all other gr oups, and residual thymus (variation in all 20 litter or 70 /131 fetuses) vs. about 2.4
to 2.9 % in 4 litters each of all other groups (p. 22/223). Other changes included increased incidence of skeletal
variations: "atresia of cervical arch/vertebra foramen costotransversarium" (45 %), " bi furcated vertebral center" (14 %) and "cervical rib" (65 %) as well as "excessive sublingual neuropore" (50 %), all of which malformations having no or little relevance in the other group (p. 224/225) except of "atresia foram en" with about 25 % in the
control and about 4 to 8 % in the other exposure groups. After delivery, neo-/postnatal fi ndings: After 5000 ppm, mean gestation ti me was prolonged by 0.7 days (p. 221). Food and drinking water consumption of dams
was reduced also after birth duri ng lactation.

Among descendants from the high-d ose group, mortality was prominent during the first 4 d af ter birth with live fetuses showing poor vitality in this tim e (ca. 10% = 2/12 pups per litter died as compared with 1 to 2% fatal cases in the other groups). Survivors showed normal appearance after day 4. Slight retardation of growth w as still significant at weaning. Water consumption was sl ightly reduced, in particular for females.

Early indicators for post-natal d evelopment: In pups from the 5000-ppm group, eruption of upper incisor

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186 and opening of eyelid for both se xes and descensus testis for males were significantly earl ier than in the controls in relation to term of delivery, but not in relation to the whole gestation time which was pr olonged for this group.

There were no differences in beha vioral and functional tests as compared to control and other test groups.

At the age of 8 weeks, brain, thy roid (males), thymus and testis (males) weights were lower (p<0.01), and pituary-gland weight of males was higher (p<0.05). But histological examination revealed no treatment-related changes.

16.5 % of the offsprings (15/91 i n 8/12 litters) had hemilateral thyroprivia (missing thyroid lobe, mostly left). There was no histopathological le sion in the tissue. The defect was attributed to an impai rment of organogenesis (p. 233).

Reproductive performances of F1 ( from 5000 ppm): No significant effects on sexual cycle, genital function and reproductive performance of the F 1 progeny were noted.

-------------------- Test condition: Exposure time about 22 hours per day, significan tly longer than according to guideline (6 hr s). This does not represent
normal industrial and consumer us e.

--------------- Test substance: Methanol, reagent special grade from Junsei Chem icals Co., <1 ppm vinyl chloride; <3 ppm for maldehyde Conclusion: The exposure to 5000 ppm of pregnant rats du ring gestation (>20 h/d) produces maternal toxic ity, fetal malformation, increased perinatal mortality and developmental delay in surviving progeny. Teratogenic ef fects occurred only at maternally toxic exposure concent ration. Exposure levels of 1000 ppm or le ss did not induce toxic symptoms in maternal animals, str uctural abnormalities or delay in growth or functional dev elopment in the F1-generation. Therefore, the NOEL for maternal and developmental toxicity is considered to be 1000 ppm.

-------------------- Reliability: (2) valid with restrictions Comparable to guideline study and current standards, also largely meeting current standards , documentation limited, acceptable for assessment , key study
26-OCT-2006 (167) (180)

Species: mouse Sex: female Strain: CD-1
Route of administration: inhalation Exposure period: day 6 - 15 of gestation Frequency of treatment: daily, 7 hours/day
Duration of test: up to day 17 of gestation
Doses: 1000, 2000, 5000, 7500, 10000 or 15000 ppm (1.3, 2.6, 6.5, 9.75, 13 or 19.5 mg /l) Control Group: yes, concurrent vehicle NOAEL Maternal Toxity: = 15000 ppm NOAEL Teratogenicity: = 1000 ppm

### OECD SIDS METHANOL

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187 Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Whole-body eposure: Mean measured chambe r concentrations
were close to the nominal ones. An air-exposed and an unhandled control group was inclu ded. Furthermore, for comparison, an orally treated gro up receiving 2x 2000 mg methanol/kg bw/d by gavage (as 12 .5-% solution in water) was
studied.

Blood concentrations were analyse d (gd 6, 10, 15).

60 to 6 litters were produced dep ending on toxicity of the exposure levels.

The following parameters were exa mined: maternal body weight and clinical signs, fetal weight, number of implantation sites, live and dead fetuses, resorptions, gross external anomalies, skeleta l and visceral malformations, blood methanol con centrations in dams.

The 7500- and 10000-ppm group as well the oral group were only examined for resorption, exe ncephaly and cleft palate because earlier studies have demo nstrated that these were the major effects.

--------------------- Remark: The biological relevance of this study f or humans has to be questioned for the following cave ats: 1. The exposure concentrations of methanol relate to doses which significantly exceed lethal doses in man: lowest dose (1000 ppm, 7 h) in mice about 800 mg/(kg bw*d) based upon calculations (NTP-CERHR, 2001, Ta b. 2-10) [(human lethal dose: 300 - 1000 mg/(kg*d)].

2. The minimum dose in mice which is likely to be lethal to humans proved to be ineffective i n mice. Those concentrations which unequivocall y showed malformations were 5000 ppm and above, clearly letha l to humans.

3. The study was performed in a c oncentration range where
methanol metabolism becomes satur ated in mice, not
considered as relevant for humans : 2000 ppm which may be considered as marginally effectiv e (LOAEL) caused already saturation (see: increase in bloo d level of methanol).

4. Consequently, the blood methan ol levels in mice exposed to 1000 ppm are estimated to be a t a factor of approx. 2 to 4, those of mice exposed to 2000 ppm at a factor of 10 to 20 higher, and those of mice exposed to distinctly teratogenic 5000 ppm at a factor of 30 to 60 higher than in humans exposed e.g. to 1000 ppm (7-8 h).

5. The differences in metabolism (predominance of catalase in rodents vs. alcohol dehydrogen ase (ADH) in humans) has to be regarded: rate limiting steps are catalase given rise to methanol, but not formate, in blo od of rodents, while in humans rate-limiting is formate d egradation, giving rise to
increased formate levels in human blood upon kinetic

### OECD SIDS METHANOL

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188 saturation.

---------------- key study non-BASF study Result: Mean blood levels: approx. 100 ug/ml (1000 ppm), 540 (2000 ppm), 1650 (5000 ppm), 3180 (7500 ppm), 4200 (1000 0 ppm), 7300 (15000 ppm) in ug/ml. In the oral group (4000 mg /d), about 4000 ug/ml were found, corresponding about to inh alation exposure of 10000 ppm. The controls had a backgroun d level of about 1.6 ug/ml.

Maternal and fetal effects. 1. There were no signs of materna l toxicity. 2. Significant increases in the i ncidence of exencephaly and cleft palate were observed at 500 0 ppm (approx. 10 % per litter: 14 fetuses/11 litters vs. 0.3 % in the control) and above, increased embryo/fetal dea th at 7500 ppm and above: At the highest dose, almost compl ete resorption of embryos
in most litters occurred. Reduced fetal weight was not note d, but at 10000 ppm and
above. A dose related increase in cervic al ribs or ossification
sites lateral to the seventh cerv ical vertebra was significant at 2000 ppm (approx. 50 %/litter vs. 26 to 34 % in the controls; at 1000 ppm: app rox. 34 %/litter). The NOAEL for developmental toxic ity was determined at 1000 ppm with relation to the most sen sitive defect, the
cervical-rib event.

The calculation of benchmark dose s for a 5-% added risk from
the lower 95-% confidence limit o n the maximum likelihood estimates were generally consiste nt with the NOAELs for various developmental defects: th e 5-% added risk above background level was at 3667 ppm for combined cleft palate, exencephaly, and resorption, for cervical-rib defect at 305

ppm. (see also: NTP-CERHR, 2001)

------------------------------ -- Test substance: Methanol, high purity from Fisher Scientific Reliability: (2) valid with restrictions Comparable to guideline study, al so largely meeting current standards, with acceptable restri ctions (see Method), sufficient documentation, accepta ble for assessment , key study
26-OCT-2006 (233) (236) (237)

Species: mouse Sex: female Strain: no data Route of administration: gavage Exposure period: single treatment on day 7 of gestation ( split into two equal doses) Frequency of treatment: single
Duration of test: up to day 18 of gestation
Doses: 0, 4 or 5 g/kg bodyweight Control Group: yes, concurrent vehicle

Method: other: Special prenatal exposure study
GLP: no data Test substance: no data

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189 Method: Study was to elucidate time-related mani festations and
mechanisms of methanol-induced sk eletal malformations in mouse. Therefore, one single dose prior to gd 9, the sensitive period of skeletal deve lopment. Six and 7 dams were used, representing about 70 to 80 fetuses.

------------------- Remark: non-BASF study Result: The treatment led to dose-related increa sed incidences in
several skeletal malformations su ch as additional rib on C7 (approx. 30 % of fetuses in both dose groups vs. 0 % in control) or split, fused or incom plete vertebra C2 (also about 41 % in the high dose fetus es vs. 7.7 in the control), 25 instead of 26 presacral verteb rae (10 % vs. 2.4 % in control). These effects suggest posterior t ransformation of cervical vertebrae by altering segment pat terning in the developing mouse embryo (posteriorization of cervical vertebrae).

It is assumed that interference o f methanol with retinol conversion to retinoic acid may p lay a crucial role in these

events.

---------------------- Test substance: Methanol, no further data. Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, acceptable for

assessment

26-OCT-2006 (238) (239)

Species: mouse Sex: female Strain: CD-1
Route of administration: inhalation Exposure period: day 6 - 15, 7 - 9 or 9 - 11 of gestation Frequency of treatment: daily, 6 hours/day
Duration of test: up to day 17 of gestation
Doses: 5000, 10000 or 15000 ppm (6.5, 13 or 19.5 mg/l) Control Group: yes, concurrent vehicle NOAEL Maternal Toxity: = 5000 ppm NOAEL Teratogenicity: ca. 5000 ppm LOAEL Embryotoxicity : = 5000 ppm

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Whole-body exposure: The analytical cham ber concentration
was close to the nominal one.

17 - 22 litters per group were ex amined. The following parameters were exa mined: maternal toxicity as clinical signs and bodyweight cha nges, embryo/fetal toxicity as % resorptions, live fetuses an d fetal bodyweight, gross,
skeletal and visceral malformatio ns and histopathology (neural tube defects).

----------------- Remark: non-BASF study Result: Maternal toxicity was seen at 13000 and 19500 mg/m3 as reduced body weight and clinical signs, f etal toxicity occurred as

### OECD SIDS METHANOL

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190 increased resorptions dose-relate d at all dose levels.

Teratogenic activity was observed in dose-related manner at the two upper dose levels as incr eased incidences of neural tube defects and ocular defects ( significant during gd 7-9), hind limb anomalies (during gd 9- 11), cleft palate, renal pelvic dilatation/hydronephrosis and tail anomalies (significant during either gd per iod).

(gd 7-9) 10000 pp m 15000 ppm % lit ters (% fetuses)

--------------------------------- ---------------------- Neural tube defect 30 (3.6 ) 65 (14.7) Cleft palate 50 (14. 6) 88 (50.4)

--------------------------------- ---------------------- At 5000 ppm no difference from co ntrol: no neural tube defect, cleft palate 4 - 9 % lit ters and 0.3 - 0.7 %
fetuses affected.

At 5000 ppm, the incidence of pel vic dilatation (not hydronephrosis) appeared to be in creased although high in the untreated control, too: The i ncrease was statistically significant with p<0.05 in the Sh irley ́s test. Furthermore, post-implantation re sorptions tended to be increased and were significant wi th p<0.05 for the number of litters with >=1 resorption.

But no increases in structural ma lformations were observed.

---------------- Test substance: Methanol, HPLC-grade from J.T. Baker, no further data. Conclusion: The data suggest that the spectrum of terato genic effects is dependent on the number of methan ol exposures and the stage of embryogenesis.

Major methanol-induced events inc luded malformations of the brain, eyes, and the urinary trac t as well as decreased fetal weight. External abnormalit ies of the calvaria, hard palate, jaw, and tail also sugges t a detrimental
methanol-related effect on the ax ial skeleton.

No clear NOAEL can be derived for developmental effects due to increased resorptions and rena l pelvic cavitation after 5000 ppm methanol. Therefore, the LOAEL(embryo-tox) is 5000

ppm.

---------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented, acceptable for assessment

26-OCT-2006 (240)

Species: rat Sex: female Strain: Long-Evans Route of administration: inhalation Exposure period: GD 6 through postnatal day 21 Frequency of treatment: 6 hours daily
Duration of test: until adult phase
Doses: 4500 ppm Control Group: yes

### OECD SIDS METHANOL

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191 Method: other: Neurotoxicological assay after hi gh-dose exposure
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Whole-body exposure design: Four cohorts of pregnant rats (n = 12), each consisting of an expo sure and a control group, were exposed to 4500 ppm methanol vapor for 6 hr daily beginning on GD 6 through post-na tal day (PND) 21. Average analytical chamber values were close to nominal ones from about 4350 to 4580 ppm.

1. Methanol blood levels were det ermined in both dams: blood samples immediately collected fro m dams on gd 7, 13, and 19 after 6-h exposure, and from both dams and pups on PND 7, 14, and 21 during lactation. In t wo cohorts, neuro-histopathological examinati ons were carried out in
neonates on PND 1 and 21. In sele cted adults from one cohort (following long-term behavorial t ests, see below), brain histomorphology and molecular exa mination on NCAM (neural cell adhesion molecule, by immuno blotting) were performed.

In one cohort, post-weaning blood concentrations in relation to increasing age were measured, extended to 52 d of age, including blood sampling (Stern e t al., 1996).

Several behavioral procedures wer e used to assess exposure effects in the offspring (Stern e t al., 1997). Remark: key study non-BASF study Result: 1. Reproductive Performance, Blood Lev els, Neurological

Tests: Control and exposed cohort with s imilar litter sizes, sex distribution, and dam and pup wei ght gains. Any variations were not related to exposure.

Blood levels in dams fairly const ant with averages of 0.55 +- 0.07 and 0.56 +-0.09 mg/ml dur ing gestation and laction. Blood levels in pups about twice as high as in dams, mean 1.26 +- 0.23 mg/ml. In an extra t est, it was conirmed that the dam-pup difference persisted until 48 days of age, i.e. about 4 wk post-weaning (fig. 4, p. 42).

Morphology/light microscopy of br ain tissue: There was no detectable evidence of neuropatho logy. However, NCAM indices revealed a significant treatment difference in the cerebellum of neonates on PND 4, while after 15 months of age previously exposed offsprings , no effects were seen (Stern et al., 1997).

2. Behavioral Tests:
Male-female littermates were stud ied whenever possible to examine sex differences, with one pair from a litter for each procedure. Exposure to metha nol did not affect suckling latency and nipple attachment on PND 5 or perfomance on a aversive olfactory condition proc edure on PND 10. Exposure to methanol did alter performance s in motor activity procedure. Methanol-exposed neonates wereles s active on PND 18, but more active on PND 25 than the eq uivalent control pups. In a

### OECD SIDS METHANOL

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192 conditioning procedure (rotate a running wheel a specified
number of revolutions to obtain f ood-pellet ) males showed decreases, females increases in t he rate of running. In a stochastic spatial discrimination procedure methanol-exposed rats responded less efficienty at asymptotic levels of performance than controls.

-------------------- Test substance: methanol, hplc grade Conclusion: Blood methanol levels in adult rats was simi lar to those found by other. The distinctly hi gher levels in offsprings may be explained by the still imm ature states of the alcohol dehydrogenase system. This may ha ve implications for post-natal neuronal development/s ynaptogenesis and learning profile and behavior. However, no histopathological evi dence was provided, although on the molecular level, a certain deficiency of cell-adhesion molecule pattern (N CAM 180 and 140 -10% to -20 %) appears to reflect a delay in the cell-cell contact formation associated with synapto genesis. But this could no longer be demonstrated in adult, previously post-natally exposed rats any more. The biolog ical meaning remains to be
elucidated.

According to the authors, methano l-exposure was associated with subtle behavioral changes in both neonates and adults.

---------------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, well documented , key study
26-OCT-2006 (241) (242) (243)

Species: monkey Sex: female Strain: Macaca Fascicularis
Route of administration: inhalation Exposure period: prebreeding about 120 days; breeding 70 days; pregnancy about 165 days Frequency of treatment: 2.5 hours daily
Duration of test: birth, first 9 months of life of the offs prings
Doses: 200; 600; 1800 ppm (0.262; 0.7 86; 2.358 mg/l) Control Group: yes NOAEL Maternal Toxity: = 1800 ppm NOAEL Teratogenicity: = 1800 ppm NOAEL Fetotoxicity : = 1800 ppm

Method: other
Year: 1996
GLP: yes Test substance: as prescribed by 1.1 - 1.4

Method: Study design limited to exposure of fema le animals (pre-breeding, breeding, and preg nancy) and qualitative/clinical signs of tox icity. It comprised

- methanol pharmacokinetics (se e other entry: 5.11) - maternal toxicity (see also e ntry: 5.8.1) - reproductive toxicity - developmental neurotoxicity

Specific aims of the study were t o determine whether

### OECD SIDS METHANOL

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193 - repeated exposure to methanol alters methanol metabolism in adult female monkeys, - pregnancy alters methanol met abolism, - long-term exposure to methano l produces overt adult toxicity, or reproductive toxic ity, or both, - long-term exposure to methano l in utero affects offspring development, especi ally neurobehavioral development.

Groups of 11-12 adult female monk eys were exposed to methanol vapor in the concentrati ons of 0; 200; 600 or 1800 ppm, daily for 2.5 hour before br eeding, during breeding and during pregnancy. The study w as performed in 2 cohorts: 24 females/cohort and 2 males/coh ort.

For postnatal developmental evalu ation, 8 to 9 infants were available per group, in total 34, among them 26 in-utero treated offsprings.

------------------------------ ------ Remark: Limitation of the study: low number of a nimals, which precludes weak effects from detec tion with certainty (see also: NTP-CERHR, 2001). non-BASF study Result: A. Blood levels (see details below): Exposure to methanol vapors did n ot affect the health of the adult monkeys prior to or during pregnancy. Single 2.5-hour exposures to methanol vapors caus ed short term elevations in
blood methanol concentrations of approx. 1- to 2-fold in the 200 ppm exposure group, 3- to 4-f old in the 600 ppm group and 13- to 16-fold in the 1800 pp m group.

After long-term exposure, peak bl ood methanol concentrations declined slight over the first mo nth and remained constant
thereafter.

The concentration of plasma forma te remained at baseline levels during the entire course o f the study in all exposure

groups.

Pregnancy had no effect on methan ol disposition. Serum folate concentrations were not affected by pregnancy and methanol exposure.

B. Reproductive performance: Methanol exposure had no effect o n most measures of reproductive performance, includi ng menstrual cycles, conception rate, and live-birth d elivery rate. However, all methanol-exposed animals had a de crease of about six to eight days in duration of pregnan cy compared to control animals.
It is not clear whether this decr ease was related to methanol exposure, as there was n o dose response and no differences among offspring group s in body weight or other physical parameters. Prenatal exposure to methanol had no effect on infant growth and physical development for the first 9 months.

The results or behavioral assessm ents of offspring did not indicate methanol-exposure effect s on most domains of early

### OECD SIDS METHANOL

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194 behavioral development. No consis tent effects due to methanol exposure were observed o n early reflex responses, gross motor development, spatial and concept learning and memory, and social behavior.

Methanol exposure was associated with ratings of "low
arousal" on the Neonatal Behavior al Scale. The effects were
observed when all of the methanol -exposed infants were compared with controls. Further c omparisons, however did not
indicate that the effect was dose -dependent. In addition, many of the methanol-exposed infa nts that were the last to receive optimal scores for the ar ousal items had been delivered via C-section. Thus, th is effect may not be directly related to methanol expo sure independent of mode of delivery.

Methanol exposure was also associ ated with a delay in early sensorimotor development of male infants only (Visually Directed Reaching Test). The eff ect was observed after controlling for the shortened ges tation length observed for the 3 methanol-exposed groups. Th e delay was dose-dependent and ranged from 9 days for the 20 0 ppm exposure group to over 2 weeks for the 600 ppm- and 1800 ppm-exposure groups Part II, p. 9 6). This observation is based on results from group sizes of 8 to 9 infant animals, with 2 to 5 males and 4 to 7 females per group (II, p. 86). The statistical significances (li near contrast test based on ANOVA) of p = or <0.04: This test indicated a significant difference between control infants compared with all methano l-exposed offsprings combined, as well as with the 600 - and 800-ppm groups for males (p<0.04). The comparison be tween the control group and the 200-ppm was nerly significant (p=0.09) (Part II, p. 87).

The results of the Fagan-Test of Infant Intelligence indicated a possible effect of methanol exp osure on visual recognition memory when complex stimuli (soci al problems) were used in testing. Although there were no m ean group differences in the novelty scores across the 4 expos ure groups, only the control group exhibited a significant nov elty preferences for social stimuli.

---------------------------

C. Other observations: Finally, prenatal methanol exposu re was associated with the occurrence of a wasting syndrome in 2/7 female offsprings in the 1800 ppm-exposure group after approximately 1 year of age, both living in the same coho rt. Those females began to show growth retardation with 12 r esp. 17 months of age. They became very weak and had to be eu thanized at the age of 20 resp. 36 months. Assay results fo r viral infection, blood chemistry, complete blood count, and liver, kidney, thyroid, and pancreatic function were with in the normal range.

Necropsies showed signs of severe malnutrition and gastroenteritis. This symptom was not observed in any of the offsprings of the other cohor t.

------------- D. Blood levels: Methanol: No significant differen ces in the blood

### OECD SIDS METHANOL

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195 concentrations between the 4 phas es (measured 0.5 h post-exposure, n = 11 or 12 (non- pregnant); n = 9 or 10 (pregnancy) (Fig 7, Tab. 11, Fig. 14, Part I):

Baseline range approx. 2 ug/ml [approx. 0.06 mM] 200 ppm: range approx. 5 ug/ml [approx. 0.15 mM] 600 ppm: range approx. 10 ug/ml [approx. 0.30 mM] 1800 ppm: range approx. 35 -40 ug /ml [1mM-range]

At 1800 ppm, after 5 h eliminatio n, the residual methanol level was near baseline (max. 2-f old higher).

The mean estimated elimination ha lf-lives (for 600 and 1800 ppm) ranged between about 60 to 9 0 min.

Formate: Irrespective of concentr ation levels or exposure intervals, there was no evidence of a significant increase above the background range of abo ut 0.15 - 0.30 mM (approx. 7 to 14 ug/ml) (Fig. 8, Fig. 15, Tab. 12, Part I).

Folate in serum: There was no sig nificant shift in the folate levels during pregnancy, i ndependent of exposure (Tab. 13, Part I).

------------------- Test substance: methanol, no further data Conclusion: Peer-reviewed conclusion: "The investigators findings suggest that repeated inhalation exposure to methanol vapors as high as 1800 ppm would not result in a ccumulation of
blood formate above baseline leve ls. With the exception of an unexpected shortening of gestatio n, methanol exposure had no effect on reproductive performanc e. The most significant result to emerge from this study was the wasting syndrome observed in two monkeys exposed i n utero to 1800 ppm.

Overall, the results provide no e vidence of a robust effect of prenatal methanol exposure on the neurobehavioral development of nonhuman primate infants durin g the first nine months of life..." (HEI Statement, in Burba cher 1999).

The conclusions are not unanimous about wasting syndrome, gestation length, and sensomotori c and intelligence testing (see NTP-CERHR, 2003, Appendix II -77 to II-81), mainly because interpretation is limited by the normal variance of and by the low number of animals under test, the lack of robust findings, and no control for multiple compa risons in statistical analyses. The neurobehavioral fin dings are considered important from a qualitative pers pective. But the wasting syndrome did not draw much attent ion of the Expert Panel (p. II-79).

--------------

---------------------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, well documented, limitat ion for develomental effects (see Remark)

26-OCT-2006 (230) (233)

Species: mouse Sex: female Strain: CD-1

### OECD SIDS METHANOL

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196 Route of administration: gavage Exposure period: day 6 - 10 of gestation Frequency of treatment: twice daily
Duration of test: up to day 18 of gestation
Doses: 2500 mg/kg bodyweight in water (2x/d) Control Group: yes, concurrent vehicle
other: LOAEL : 5000 mg/kg bw

Method: other: see Method
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Study designed to clarify the role of fo late in the developmental toxicity of methano l in folate-deficient and
folate-sufficient mice. Additiona lly to the developmental toxicity parameters, the micronuc lei formation in maternal and fetal blood was determined (s ee other entry 5.6).

The female animals were fed folat e-free diet supplemented
either with 400 or 1200 nmol foli c acid as well as 1 % succinylsulfathiazole (as inhibit or of intestinal folate synthesis) 5 weeks prior to matin g and throughtout breeding and gestation.

The short dosing interval of gd 6 through 10 was selected because this was the sensitive pe riod for the induction of the observed malformations (Fu et al. 1996, p. 461).

21 - 24 litters were examined per group, representing 215 to 282 fetuses per test and control group.

Folate was determined in maternal and fetal liver and blood plasma and red cells.

------------------- Remark: key study non-BASF study Result: A. Folate levels (measured on gd 18, 8 d after the final methanol treatment): Low-folate diet produced a declin e in folate in the maternal liver (total folate approx. -30%) , in maternal plasma (approx. -30%), in maternal erys (approx. -30%) vs. normal-folate diet and in the fet al liver (approx. -60 to -70%) (Fu et al., 1996, Tab. 2).

Methanol had no marked influence on maternal and folate levels irrespective of folate sup plementation, except in maternal plasma where there was s ome evidence of a reduction
of about 20 % .

Methanol treatment was slighty fe totoxic (reduced mean fetal weight and reduced mean crown-rum p length), but had no impact other reproductive parameters. It showed some evidence of a teratogenic effect (increased inc idences of cleft palate and exencephaly) under folate-adequat e supply, but this was hardly statistically significant: cleft palate (2/222 vs. 0/282) an d execephaly (5/222 vs. 1/282 in the respective high-fola te control).

Likewise, folate deficiency faile d to produce significant malformations (in accordance with previous reports: e.g.

### OECD SIDS METHANOL

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197 Heid et al., 1992): cleft palate (5/215 vs. 0/282) an d execephaly (2/215 vs. 1/282 in the respective high-fola te control).

However, cleft palate, but not ex encephaly was significantly increased in the presence of meth anol: 39/235 vs. 5/215 and 8/235 vs. 2/215 of folate-poor co ntrol, respectively.

Methanol did not induce micronucl ei in maternal or fetal
blood.

-------------------------- B. In pregnant CD-1 mice given methanol (5 g/kg/d) from gestation day 6 - 15, one group r eceiving folate deficient, the other folate supplemented die t, there were no differences in the formate-blood levels between both groups: 5.13 +-0.68 mmol/L (folate-def.) and 3.90 +-0.94 mmol/L (folate-suppl.) vs. 0.36 +-0.13 m mol/L (untreated control). But developmental toxicity was si gnificantly higher in folate-deficient dams (Hong et al . 1997).

The results indicate that increas ed plasma formate levels in
dams do not underlie the increase d developmental toxicity of methanol in mice fed low dietary folate (Hong et al. 1997).

----------- Test substance: Methanol, highest HPLC grade, from Fisher Scient ific Comp.
Conclusion: Folate deficiency enhanced the teratogenic e ffects of
methanol in mice. Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented , key study
26-OCT-2006 (244) (224) (245) (246)

Species: rat Sex: female Strain: Long-Evans Route of administration: inhalation Exposure period: day 7 - 19 of gestation Frequency of treatment: daily for 7 hours/day
Duration of test: up to postnatal day 160
Doses: 19.5 mg/l (15000 ppm) correspo nding to ca. 6100 mg/kg/day Control Group: yes, concurrent vehicle

Method: other: postnatal offspring development a nd behaviour
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Whole-body exposure: The analytical cham ber concentrations varied by less than 5 % and were fairely close to the
nominal ones. Six and 7 dams were used per group.

The following parameters were rec orded: maternal bodyweight, maternal blood methan ol levels, offspring bodyweight, offspring mortality, motor activity (day31-21, 30, 60), olfactory learning (day1 8), behavioural thermoregulation (day 20-21), T-m aze learning (day 23-24), acoustic startle response (day 24 , 60), reflex modification audiometry (day 60), pubertal lan dmarks (day 31-56), passive avoidance (day 72), visual-evoked potentials (day 160). 5 or 6 dams were used per group w ith 10 - 11 offspring.

### OECD SIDS METHANOL

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198 Behavioural tests were performed on 4 - 5 offspring animals.

-------------- Remark: The number of pups available and tested (mostly, 1/sex/litter) was probably too sm all to draw firm conclusions about postnatal devel opmental effects on behavior. Result: Maternal blood methanol levels were betw een 3.8 and 3.1 mg/ml. The treatment led to reduc ed maternal body weight, the only effect noted in dams. No increase in post-implantation loss. Postnatal bodyweight were modestly but statistically lower after tre atment. No increase in postnatal mortality. No other test revealed significan t effects of methanol

treatment. Test substance: Methanol, 99.9 % purity, Fisher Scientific Reliability: (3) invalid Test procedures based on scientif ic principles and standards, well documented, limi ted value for neurological/behavorial assessmen t (247)

Species: mouse Sex: female Strain: CD-1
Route of administration: inhalation Exposure period: day 7 - 9 of gestation Frequency of treatment: daily, 6 hours/day
Duration of test: up to day 8.5, 9.5 or 10.5
Doses: 15000 ppm (19.5 mg/l) Control Group: yes, concurrent vehicle

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Whole-body exposure: The analytical cham ber concentration
was close to the nominal one.

4 - 9 litters and 26 - 99 live fe tuses were examined per group. The following parameters w ere examined: maternal body weightand clinical signs, gross, skeletal and visceral malformations in fetuses, histopa thology and scanning electon microscopy in fetuses.

-------------- Remark: non-BASF study
Result: Maternal toxicity was seen as clinical s igns and reduced bodyweight, in fetuses the treatm ent induced reduced bodyweight and a range of gross n eural defects (anencephaly,encephalocele, holop rosencephaly, ocular anomalies) in up to 41 % of live embryos.

--------- Test substance: Methanol, HPLC gradde, from Baker Conclusion: The result suggest that exposure to high con centrations of methanol injures multiple stem ce ll populations in the neurolating mouse embryo. Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, well documented, accepta ble for assessment
26-OCT-2006 (248) Species: mouse Sex: female Strain: CD-1

### OECD SIDS METHANOL

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199 Route of administration: inhalation Exposure period: 6 hours Frequency of treatment: single treatment on day 8 of gestation
Duration of test: up to day 10 of gestation
Doses: 10000 or 15000 ppm (13 mg/l) Control Group: yes, concurrent vehicle

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: 12 - 14 litters per group were evaluated

.

The effects of methanol and its m ajor metabolite formate as well as the influence of a pretre atment with the alcohol-dehydrogenase inhibitor 4 -methylpyrazole were compared. The following parameter s were examined: crown-rump length, head length, g ross malformations and neural tube defects, methanol con centrations in fetal and
maternal tissue. The folate statu s in RBC and decidual tissue was monitored at 15000 ppm methanol.

---------------------- Remark: Valuable scientific information about me chanisms. However, the biological relevance for huma ns is poor, because the estimated systemic dose im mice [ >=4000 mg/(kg*d)] is distinctly above doses lethal to humans (300 - 1000 mg/kg bw).

------------------ non-BASF study Result: Methanol treatment (10000 ppm) led to a significant increase in the incidence of open neural t ubes (9.65 +-3.13% in 12
litters vs. 2.02 to 2.69 +-1.09% in controls, p<0.05). After 4-methylpyrazole pretreatment (4- MP), the percentage of this effect was 7.21 +-2.65%, but not significant. No such effect was induced by formate given oral ly (750 mg/kg). The other parameters examined were not affe cted by the treatment (Dorman et al., 1995).

Methanol concentrations were equa l in fetal and maternal tissues with maximum maternal pla sma level at 65 to 75 mM, and 62 to 85 mM in decidual swell ing, irresp. of pretreatment with 4-MP. Likewise, mean formate blood and decidual peaks were at 0.5 to 0.6 mM and 1.8 to 2.1 mM, respectively. Furthermore, after 15000 ppm, too, no significant changes of formate we re noted in either compartment.

[That means, 4-MP failed to influ ence methanol and formate levels significantly as well as e mbryonal dysmorhogenesis.] (Dorman et al., 1995).
Folate status: 15000 ppm methanol resulted in tr ansient, albeit statistically insignificant decre ases in total maternal red
blood cell and decidua folate lev els (Dorman et al., 1995).

----------------------------- Test substance: Methanol HPLC-grade from Sigma Chemical Corporat ion, no
further data. Conclusion: Formate seems to play no apparent role in th e development of methanol-induced exencephaly, and methanol itself is the

### OECD SIDS METHANOL

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200 proximate teratogen in pregnant C D-1 mice exposed to high concentration of methanol vapor, because 1. no formate accumulation in me thanol-treated mice, 2. exencephaly (failure of anter ior neuropre to close)
occurred in the absence of increa sed formate levels, 3. no exencephaly after oral tre atment with high doses of formate, 4. methanol, but not formate, c aused neural tube defects in whole-embryo culture.

The results (although only from s ingle dosing) did not provide evidence that folate defi ciency gave rise to methanol-related dysmorphogenesis and appears to be irrelevant at least at low methan ol burdens (compare also test results in monkeys: Dorman e t al., 1994). But based on observations by others (e.g. Saka nashi et al., 1994), an interaction of folate deficiency and methanol cannot be ruled out under certain condition s. (Dorman et al., 1995; Dorman and Welsch, 1996; see also : Medinsky and Dorman, 1995)

------------------------ --- Reliability: (2) valid with restrictions
26-OCT-2006 (249) (250) (251) (252) (253)

Species: mouse Sex: female Strain: CD-1
Route of administration: inhalation Exposure period: see method Frequency of treatment: see method Duration of test: until GD 17
Doses: 10000 ppm (13 mg/l) Control Group: yes

Method: other
GLP: no data
Test substance: as prescribed by 1.1 - 1.4

Method: Critical periods for the developmental t oxicity of methanol were assessed in pregnant CD-1 mi ce exposed to 10000 ppm for 7 hours/day on 2 consecutive days during GD 6-13 (GD 6 and 7; GD 7 and 8; GD 8 and 9, GD 9 a nd 10; GD 10 and 11; GD 11 and 12; GD 12 and 13) or to singl e day (7 hour) exposures during GD 5 and 9. Controls were exposed in the same way to
filtered air. Mice received water but not food during exposure. Maternal blood methanol was determind at times during, at the end of and subsequ ent to a single 7 hr exposure on GD 7. On GD 17 mice were killed and gra vid uteri removed. Live,
dead and resorbed fetuses were co unted, and live fetuses were examined, weighed and preser ved in 70 % ethanol. All fetuses were examined externally and for cleft palate, eviscerated, and stained with Ali zarin red for skeletal
examination. Test and control groups consisted out of 12 to 17 pregnant mice.

----------- Remark: Valuable scientific information about me chanisms. However, the biological relevance for huma ns is poor, because the estimated systemic dose im mice [ some 4000 mg/(kg*d)] is

### OECD SIDS METHANOL

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201 distinctly above doses lethal to humans (300 - 1000 mg/kg bw).

------------------ non-BASF study Result: Peak maternal blood methanol concentrati on at the end of the exposure was about 4 mg/ml, metha nol was cleared from maternal blood within 24 hr. Some fully resorbed litters were observed with 2-day methanol exposure on gd 6-7 (3/12 ) and gd 7-8 (1/16) or 1-day exposure on gd 7 (3/17). Wi th 1-day methanol exposure on gd 7, the number of live fetus es (4.4 per litter) was lower than with exposure on anyot her day. Cleft palate, exencephaly and ske letal defects were the
fetal anomalies observed: cleft palate: occurred with 2-day exposures on gd 6-7 through gd 11-12 (peak on gd 7-8) and with 1-day e xposures on gd 5 through 9 (peak on gd 7)

Exencephaly: occurred with 2-day exposures on gd 6-7 through gd 8-9 (peak on gd 6-7) and with 1-day exposure on gd 5 through 8 (peak on gd 7).

Skeletal elements malformed inclu ded the - exoccipital (peak on gd 6-7 (2 2.5 %); gd 5 (9.9%)) - atlas (peak on gd 6-7 (72.3 %) ; gd 5, 6 (55.5 %, 55.3 %)) - axis (peak on gd 6-7(22.3 %); gd 7 (28.8 %)) - cervical vertebra 7 with a rib (peak on gd 6-7 (73.7 %); gd 7 (45.4 %)) - lumbar vertebra 1 with a rib ( peak on gd 7-8 (68.3 %); gd 7 (39.4 %).

An increase incidence of fetuses with 25 presacral vertebrae (normal 26) was observed wiht met hanol exposure on gd 5; whereas an increased incidence of fetuses with 27 presacral
vertebrae was observed with metha nol exposure on gd 7. According to the authors the resu lts of this study indicate that gastrulation and early organ ogenesis represent a period of increased embryonal sensitivit y to methanol. Test substance: Methanol, high purity from Fisher Scientific Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, well documented, accepta ble for assessment
26-OCT-2006 (254)

Species: rat Sex: female Strain: Long-Evans Route of administration: drinking water Exposure period: gd 15 -17 or gd 17 - 19 Frequency of treatment: continuous
Doses: 2% Methanol in drinking water (ca. 2500 mg/kg/d) Control Group: yes, concurrent no treatment

Method: The study design was to elucidate postna tal neurological effects in rat pups after intraut erin exposure to methanol:
Two behavioral test were selected , 1) the suckling behavior test whi ch measures the latency time to nipple attachment (postna tal day 1) and 2) the nest-seeking/homing behavi or test (postnatal day 10)

### OECD SIDS METHANOL

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202 30 primigravid dams were used, 10 per group.

------------- Remark: non-BASF study Result: Maternal toxicity was not observed. In b oth test groups no
differences were found in litter sizes, neonatal birth weight as well as lethality of of fsprings as compared with the progeny of the untreated cont rol.

Both behavioral tests on post-nat al disorders showed significant retardations in learn ing: Pubs from treated rats required a longer time to begin s uckling on post-natal day 1 (80 to 90 sec. vs. 60 sec of cont rols) and to locate nesting material on post-natal day 10 tha n control pups (3 time- and
distance-related indicators evalu ated).

------------- Test substance: Methanol, no further data. Conclusion: The data suggest that prenatal methanol expo sure may induce neurotoxic effects in the offspri ngs which are not
associated with evident or overt toxicity.

--------------- Reliability: (2) valid with restrictions Test procedures based on scientif ic principles, sufficiently documented, acceptable for assess ment 26-OCT-2006 (255)

Species: rat Sex: female Strain: Long-Evans Route of administration: drinking water Exposure period: day 6-15 of gestation Frequency of treatment: daily
Duration of test: not indicated, presumably to the end of g estation
Doses: 350, 700 or 1400 mg/kg bodywei ght (0.5, 1 or 2% in water) Control Group: yes, concurrent vehicle

Method: other: teratogenicity study with folate deficient animals
GLP: no data

Method: Pilot study/Screening: Rats were rendere d folate deficient by supplying a folate-deficient d iet. 7 to 9 dams were used

per group. Remark: non-BASF study Result: The average blood levels were given as 0 .21, 0.26, and 0.67 mg/L, respectively.

At 1 and 2 % methanol in water fe tal resorptions/deaths as
well as skeletal malformations we re significantly increased. In the 0.5 % group min imal respective changes
indicate a NOEL for maternal and developmental below 0.5 % which corresponds to a blood leve l of 0.21 mg/ml.

No further details are reported. Test substance: Methanol, no further data. Reliability: (4) not assignable Only short communication, abstrac t 26-OCT-2006 (256)

Species: rat Sex: female Strain: Long-Evans Route of administration: drinking water

### OECD SIDS METHANOL

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203 Exposure period: days 6 - 15 of gestation Frequency of treatment: daily
Duration of test: up to day 20 of gestation
Doses: 357.1, 714.3 or 1428.6 mg/kg b odyweight (0.5, 1 or 2%) Control Group: yes, concurrent vehicle NOAEL Maternal Toxity: = 357.1 mg/kg bw

Method: other: comparison with folate deficient dams
GLP: no data Test substance: other TS

Method: 5 dams or litters were used per group, a ll treated groups and one control group were made f olate deficient before
treatment. The examination for te ratogenic effects was not performed at the date of publicat ion. Remark: non-BASF study Result: The highest dose level was highly embryo toxic, of 5 expected litters only one survive d. In the mid dose group resorptions were also significant ly increased. Maternal toxicity as reduced body weight or bodyweight gain was seen in the mid and high dose group. Teratogenic effects were not yet examined at the date of publication. Test substance: Methanol, no further data. Reliability: (4) not assignable Comparable to guideline study, la cking important results,
documentation limited
26-OCT-2006 (257)

Species: rat Sex: female Strain: Long-Evans Route of administration: gavage Exposure period: single application on day 10 of gestatio n Duration of test: not indicated, presumably to the end of g estation
Doses: 1027, 2054 or 4108 mg/kg bodyw eight (1.3, 2.6 or 5.2 ml/kg) Control Group: yes, concurrent vehicle NOAEL Maternal Toxity: = 2054 mg/kg bw

Method: other: no details indicated
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: 10 to 13 dams per group were treated. Wi lson sectioning, gross necropsy and alizarin red s taining of fetuses was performed. No further details are reported. Remark: non-BASF study Result: Maternal toxicity was seen only as body weight loss in the highest dose group (> 20 % decrea se). Methanol at all doses failed to p roduce any significant change in standard reproductive i ndices (e.g. postimplantation loss). A signifi cant decrease in fetal body weight (11 to 19.5 %), however, w as associated with a dose-dependent increase in anomal ies (0.6 % in control, 3.7%; 7% and 16.5% in the dose gr oups). The dose-related anomalies were undescended testes , exophthalmia and anophthalmia.

------------ Test substance: Methanol, HPLC grade

### OECD SIDS METHANOL

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204 Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, test design limited, suf ficiently documented
26-OCT-2006 (258) (259) (260)

Species: rat Sex: female Strain: Wistar
Route of administration: gavage Exposure period: day 6 - 15 of gestation Frequency of treatment: daily
Duration of test: up to day 21 of gestation
Doses: 2.5 g/kg bodyweight Control Group: yes, concurrent vehicle

Method: other
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Well nourished and malnourished groups o f rats were compared. The following parameter s were examined: number of live fetuses, resorptions, corpor a lutea, implantation sites, fetal weight, gross and sk eletal malformations.

12 - 17 dams and 110 - 177 live f etuses per group were examined.

---------------- Remark: non-BASF study Result: The treatment induced maternal toxicity.
The treatment led to increased in cidences of skeletal malformations in well nourished ( 45.4 % compared to 5.6 % in controls) and malnourished rat s (38.8 % compared to 3.8 % in controls). Malformations were primarily found on ribs (extra ribs) and vertebra (irregu lar shape). Malnutrition did not increase the incidence of malformations, but fetal growth w as retarded.

An increase in ske letal malformation, primarily cerivical extra
ribs was noted in the methanol treated rats when compared to controls. Thi s is only a single dose stu dy and the 2.5 g/kg/day
dose is above the
lethal dose in humans. Test substance: Methanol, no further data. Conclusion: This is only a single dose study and the 2.5 g/kg/day dose
is above the lethal dose in human s. Reliability: (3) invalid Unsuitable test design (see Concl usions)
26-OCT-2006 (261)

Species: rat Sex: female Strain: other: Holtzman Route of administration: gavage Exposure period: GD 1-8 Frequency of treatment: daily
Duration of test: up to day 9, 11 or day 20 of gestation
Doses: 1600; 2400 or 3200 mg/kg bw Control Group: yes, concurrent vehicle

### OECD SIDS METHANOL

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205 Method: other: early pregnancy study
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Early pregnancy study: Eight dams per group with 11 - 13 fetuses per dam were used. Animals were killed on days 9, 11 , or 20 of pregnancy, and maternal, embryonic, or fetal par ameters were assessed:

Effects on early pregnancy were d etermined on day 9 as bodyweight, uterine weight, numbe r of implantation sites, number of atypical sites, mean im plantation site weight, ovarian weight and number of corp ora lutea per ovary. Decidual cell response was determ ined on day 9 as uterine weight, uterine function and norm al implantation processes.

Effects on mid pregnancy was dete rmined on day 11 as number of live fetuses, presence of hear tbeat, yolk sac circulation, axial rotation, clos ure of neural tube, presence of limb buds and sensory organs, yolk sac diameter, crown-rump length, head length and somite number.

Effects on late pregnancy were de termined on day 20 of gestation as number of live fetus es, number of resorptions, gross external abnormalities and fetal weight.

Additionally, decidualisation was assessed in pseudopregnant rats using a DCR(decidual cell re sponse) technique.

-------------------

----------------- Remark: non-BASF study Result: The only effect of methanol treatment wa s seen on early pregnancy (day 9) as reduced uter ine and implantation site weight and increased number of at ypical, small implantation sites (with extravasated blood) i n the high-dose group.

All other parameters examined sho wed no statistical significant differences between t reated and control groups at any gestation day. There was n o treatment-related effect on serum estradiol, progesterone, lutenizing hormone and prolactin on day 9 (1 day after a fter cessation of exposure).

No effects on embryo/fetal surviv al or development including
external anomalities were noted a t any gestation day.

The 3200 mg/kg/day dose of methan ol produced a reduction in body weight gain by day 9, but no t day 20, which may be
considered an indication of non-s pecific maternal toxicity.

Decidualisation After methanol treatment of pseud opregnant rats (all 3 doses), a dose-related inhibition of uterine growth was observed (day 9), but not signifi cant at 1600 mg/kg/d.

------------------ Test substance: methanol, high purity grade, no furthr data
Conclusion: Results indicated that effects on uterine we ight and implatantion sites on gd 9 may ha ve resulted from methanol-induced inhibition of th e DCR. However, this apparent potential impact on deci dua development was not

### OECD SIDS METHANOL

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206 expressed by a depression of day- 20 embryo/fetal survival or development, when treatment ended on gd 8.

The biological relevance of these results is unclear, in particular, failing a comprehensi ve data base on hormone status and DCR (see also: NTP-CER HR, 2001).

------------- Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, but technical limitations (see Conclusion).
26-OCT-2006 (262)

Species: rat Sex: female Strain: Long-Evans Route of administration: oral feed Exposure period: day 8 of gestation up to parturition Frequency of treatment: daily
Duration of test: up to some days after parturition
Doses: 0.6, 0.9 or 1.6 % (v/v) (278.6 , 417.9 or 742.9 mg/kg bodyweight) Control Group: yes, concurrent vehicle

Method: other: no details reported
GLP: no data Test substance: other TS

Method: Dams were fed a liquid diet containing m ethanol. Number of animals not indicated, no detaile d description of method. Remark: non-BASF study Result: Methanol did not affect fecundity but re duced maternal weight gain, decreased litter siz es, increased perinatal and postnatal mortality and decre ased weights at weaning. Teratogenic effects are not repor ted. Test substance: Methanol, no further data. Reliability: (4) not assignable Only short communication, abstrac t 26-OCT-2006 (263)

Species: mouse Sex: female Strain: C57BL Route of administration: i.p. Exposure period: 2 injections 4 hours apart on gestationa l day 7 Frequency of treatment: 2 treatments
Duration of test: up to day 17 of gestation
Doses: 1.7 or 2.45 g/kg bodyweight Control Group: no data specified

Method: other: no details reported
GLP: no data Test substance: other TS

Method: Study design was to elucidate pathogenic mechanisms in
methanol intoxication: Ethanol an d methanol were compared with respect to their teratogenic activity on the brain. Ethanol was given at a dose level of 2.45 g/kg bodyweight (Rogers, 1995).

In a further series, so-called co nfocal laser scanning microscopy was used to elucidate the pathogenesis of cranio

### OECD SIDS METHANOL

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207 facial defects (Rogers et al., 19 96).

----------------------- Remark: non-BASF study Result: 1.7 g/kg methanol induced 30 % resorptio ns and midfacial deficiencies and micro/anophthalm ia in over 50 % of live fetuses. 2.45 g/kg resulted in 55 % resorptions and 100 % of live fetuses having malformati ons of the holoprosencephaly spectrum (micro/anophthalmia, ceb ocephaly, absence of forebrain, premaxillary agenesis)

.

Cell death of cells were observed in embryos on gd 8 at the edge s of the neural plate and on gd 9 in the midbrain and visceral arches. On gd 8, sparse mesenchyme and edema were apparent beneath the cranial neur oepithelium. Prosencephalic
deficiences observed included sma ll or telencephalic and optic vesicles (Rogers et al., 19 96).

----------------- Test substance: Methanol, no further data. Conclusion: The pathogenesis of methanol-induced craniof acial
malformations is similar to that previously observed for ethanol (Rogers et al., 1996)(for the ethanol effect: see also review Rogers and Daston, 19 97).

---------------- Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, acceptable for

assessment 26-OCT-2006 (264) (265) (266)

Species: mouse Sex: female Strain: C57BL Route of administration: i.p. Exposure period: gestation day 6, 7, 8, 9 or 10 Frequency of treatment: 2x/d with 4 h apart

Doses: 4900 mg/kg bw., split in 2 equ al doses on resp. gestation day Control Group: yes, concurrent vehicle

Method: other: Screening test for sensitive stag es Year: 1998
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Remark: The results included external and soft t issue defects, still
excluded skeletal malformations. non-BASF study Result: The total incidence of malformations was highest with gestation day 7, indicating that gastrulation is sensitive
to methanol similar to ethanol.

The defects included the eye (an- and microphthalmia, coloboma), the brain and face (ho loprosencephaly, facial clefts) as wel as cardiovascular system (vetricuar septal and great vessel defects).

Contrary to ethanol, no external digit defects were
observed. Reliability: (4) not assignable Only abstract.

### OECD SIDS METHANOL

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208 26-OCT-2006 (267)

Species: other: rat and mouse Sex: female Strain: other: Sprague-Dawley rat and C D-1 mouse
Route of administration: other: in vitro whole embryo culture Exposure period: 24 or 48 hours Frequency of treatment: single
Duration of test: 48 hours
Doses: 0, 8, 12 or 16 mg/ml (rat), 0, 2, 4 or 8 mg/ml (mouse) Control Group: yes, concurrent vehicle

Method: other: in vitro whole embry culture
GLP: no data

Method: Screening test including developmental s tages of embryogenesis: The following parameters were exa mined: viability, yolk sac circulation, heart beat, crown-ru mp length, head length, somite number, Feulgen staining t o localize cell death and proliferation, scanning electon m icroscopy and histology (only rat embryos). Remark: non-BASF study

Result: In rat embryos methanol affected adverse ly yolk sac diameter, head length, developmen tal score, somite number and mortality. At the highest con centration 100 % of embryos were abnormal and cell de ath was increased in
various parts of the nervous system. In mouse embryos methanol affecte d adversely crown rump length, head length, developmenta l score and somite number. At the highest concentration the percentage of abnormal embryos reached 94 %. Cell death was not induced in treated
cultures.

------------------------ Test substance: Methanol, no further data.
26-OCT-2006 (268)

Species: rat Sex: no data Strain: no data Route of administration: other: whole embryo culture Exposure period: 24 hours Frequency of treatment: single
Duration of test: 48 hours
Doses: 0, 4, 6, 8, 12 or 16 mg/ml Control Group: yes, concurrent vehicle

Method: other: whole embryo culture, no details indicated
GLP: no data

Method: Screening test including developmental s tages of embryogenesis: Embryos were explanted on day 9 o f gestation, cultured for 24 hours in methanol supplemented serum and cultured for further 24 hours in pure serum. T he following parameters were measured: embryolethality, s omite number, crown rump length, developmental score, head length. Number of embryos used not indicated. Remark: non-BASF study

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209 Result: 4 and 6 mg/ml was not toxic and did not affect embryonic development. Higher concentration s led to a dose related decrease in somite number and ove rall development. 12 and 16mg/ml led to increased lethalit y. Head length and crown rumplength were not affected by t he treatment. Test substance: Methanol, no further data.
26-OCT-2006 (269)

Species: rat Sex: female Strain: Sprague-Dawley Route of administration: other: whole embryo culture Exposure period: 24 hours Frequency of treatment: single
Duration of test: 48 hours
Doses: 0, 2, 4, 8, 12 or 16 mg/ml Control Group: yes, concurrent vehicle

Method: other: in vitro culture of whole embryos
GLP: no data

Method: Screening test including developmental s tages of embryogenesis: The following parameters were exa mined: somite number, crownrump length, developmental s core, yolk sac diameter, head length, incidence of anomali es. Remark: non-BASF study Result: Rat embryos were explanted and cultured in 0, 2, 4, 8, 12 or 16 mg Methanol/ml rat serum for 2 4 h and then transferred to rat serum alone for 24 h. Embryon ic development of the 2 and 4 mg groups was not significantly different from the controls whereas the higher conce ntrations resulted in a
concentration related decrease in somite number, head length and developmental score. The 12 m g/l dose resulted in some embryolethality as well as dysmor phogenesis, while the highest dose was embryolethal. Me thanol was dysmorphogenic in vitro in rat embryos at a meth anol concentration comparable to that reported in ma ternal serum following teratogenic in vivo exposures. Test substance: Methanol, no further data.
26-OCT-2006 (270)

Species: rat Sex: no data Strain: Sprague-Dawley Route of administration: other: whole embryo culture Exposure period: 48 hours Frequency of treatment: single
Duration of test: 48 hours
Doses: 2.08 - 16.67 μl/ml (1.64 - 13. 17 mg/ml) Control Group: yes, concurrent vehicle

Method: other: in vitro culture of whole embryos
GLP: no data

Method: Screening test including developmental s tages of embryogenesis: 9 - 17 embryos were used per trea tment scheme, the effect ofmethanol and its major metaboli te, formic acid, was compared. The following parameter s were recorded: yolk sac circulation, dorsally bending, he art beat, crown-rump length, somite number and protein content.

### OECD SIDS METHANOL

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210 Remark: non-BASF study Result: Crown-rump length, somite number and pro tein content was dose related decreased firstly at 11.6 ul/ml (9.16 mg/ml), at 8.75 ug/ml (6.91 mg/ml) no tre atment related effects
wereobserved. Test substance: Methanol, no further data.
26-OCT-2006 (271)

Species: mouse Sex: Strain: CD-1
Route of administration: other: whole embryo culture Exposure period: 24 hours Frequency of treatment: single
Duration of test: 24 hours
Doses: 0, 2, 4, 6, 8 mg/ml Control Group: yes, concurrent vehicle

Method: other
GLP: no data

Method: Screening test including developmental s tages of embryogenesis: Day-8 mouse embryos were explante d and cultured in 0, 2, 4, 6 or 8 mg Methanol/ml culture med ium (75 % rat serum, 25 % Tyrode's salt solution) for 24 h. Remark: non-BASF study Result: CD-1 mice: Embryonic development in the 2-mg/ml group was not significantly different from the controls but all higher concentration groups had a signif icant decrease in developmental score and crown-rum p length. The high concentration group als suffered 80 % embryolethality.

C57/BL/6J mice: Somite number was not affected. 2 mg/ml and higher produced a concentration-d ependent increase in dysmorphology in the absence of e mbry-lethality, including open neural tubes, branchial arch hypoplasia, and prosencephalic hypoplasia. Additi onally, 4 and 6 mg/ml caused mes- and rhombencephalic h ypoplasia.

These results indicate that C57 m ice are more sensitive than CD-1 mice, suggesting that differ ences in strain sensitivity to in-vitro methanol exposure ar e more likely due to
intrinsic differences in the deve loping embryo rather than
maternal factors.

----------------------------- Test substance: Methanol, no further data. Reliability: (2) valid with restrictions
26-OCT-2006 (270) (272)

Species: mouse Sex: no data Strain: CD-1
Route of administration: other: whole embryo culture Exposure period: 12 hours Frequency of treatment: single
Duration of test: 45 hours
Doses: 62, 125, 187, 250 or 375 mM (c a. 2, 4, 6, 8 or 12 mg/ml) Control Group: yes, concurrent vehicle

Method: other: in vitro culture of whole embryos

### OECD SIDS METHANOL

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211 GLP: no data Test substance: as prescribed by 1.1 - 1.4

Method: Screening test including developmental s tages of embryogenesis: The following parameters were exa mined: percentage of live embryos, cephalic dysraphism, pro sencenphalic lesions, branchial arch hypoplasia. Remark: non-BASF study Result: Methanol treatment led to dose related i ncreased incidences
of all anomalies examined for. Test substance: Methanol, HPLC-grade from Sigma Chemical Corpora tion, no
further data.
26-OCT-2006 (250)

Species: other: rat and mouse Sex: Strain: other: Sprague-Dawley rat, CD-1 mouse Route of administration: other: whole embryo culture Exposure period: 48 h Frequency of treatment: single
Duration of test: 48 h
Doses: about 2 to 9 mg/ml (also in co mbination with formate) Control Group: yes, concurrent vehicle

Method: other: in vitro culture of whole embryos
GLP: no data

Method: Screening test including developmental s tages of embryogenesis: Follow-up studies based on previo us ones: Methanol and formate as well as combination of both substances were examined.

The following parameters were exa mined: somite number, crownrump length, developmental s core, yolk sac diameter, head length, incidence of anomali es. The developmental scoring system took into account embryonic groth parameters as well as stage s of development. Remark: In a further preliminary study, there wa s some evidence that methanol may interfere with ADH a nd thus prevent oxidative conversion of retinol into retino ic acid, a presumed mediator in embryonic development , which may explain part of methanol-induced disorders (Andre ws et al., 1998b). non-BASF study Result: 1. In rats, a less than additive-dose effect was found:
the combination of methanol and f ormate proved to be less toxic than would be predicted fro m their individual toxicities, suggesting antagonist ic interaction (Andrews et al., 1998a).

A standard methanol dose-response line has been constructed, based on the developmental scorin g system. Combinations with low formate concentrations (<1 mg /ml), along with various methanol concentrations, generall y fell within the 95-%
confidence interval of the standa rd methanol-curve (Andrews et al., 1998a).

2. Formate alone in whole-embry culture, given to the medium of 9-d rat embryos as well as to 8-d mice embryos at 0.2 - 3.0 mg/ml (for 24 or 48 h) exhibited trends towards

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212 reduction of growth and developme nt and increases in the number of dead and abnormal embry os. Formate is embryotoxic
at concentrations >= 4fold lower than those for methanol (Ebron-McCoy et al., 1994) Test substance: Methanol, no further data
26-OCT-2006 (273) (274) (275)

5.8.3 Toxicity to Reproduction, Other Studies

- 5.9 Specific Investigations

Endpoint: Behavioural Effects Species: rat Strain: Long-Evans Sex: male Route of administration: oral, gavage No. of animals: 11 Vehicle: water Frequency of treatment: single dose
Doses: 1000, 2000, and 3000 mg/kg (5 0-% aqueous solution) Control Group: other: test animals = control group ( water) prior to

treatment

Method: other: fixed wheel running ratio test
Year: 1993
GLP: no data Test substance: as prescribed by 1.1 - 1.4

Result: The rats displayed no signs of overt int oxication such as gait disturbance, but a significant, d ose-related reduction in FR20 response was observed at all dose -levels.

A NOAEL for behavioural changes c annot be derived.

-------------------------- Test condition: DOSE selection: 10, 20, and 30 % of the LD50.

TEST ANIMALs: The same animals we re re-used for each dose on different days.

BEHAVORIAL TESTs: Ten minutes after treatment, the animals were subjected to the fixed wheel running ratio test to assess operant running. The test required the animal to run i nside a wheel and rotate it under a fixed ratio of 20 times ( FR20) in order to receive a
food reward.

STATISTICS: Data were evaluated by conducting repeated measures analysis of variance (ANOVA), determining linear trend, correcting for degrees of freedom, and performin g analysis of residuals to
outliers and skewed distribution.

-------------- Test substance: Methanol, HPLC grade, purity 99.93 %, Aldrich

(233) (276)

5.10 Exposure Experience

Type of experience: other: review, general

Remark: Humans (and non-human primates) are u niquely sensitive to

### OECD SIDS METHANOL

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213 methanol poisoning and the tox ic effects in these species
is characterized by formic acidem ia, metabolic acidosis,
ocular toxicity, nervous system depre ssion, blindness, coma and death. Nearly all of the avail able information on methanol toxicity in humans relates to the consequences of acute rather than chronic exposures. A vast majority of poisonings involving methanol have occurr ed from drinking adulterated beverages and from methanol-co ntaining products. Although ingestion dominates as the mos t frequent route of poisoning, inhalation of high concentrati ons of methanol vapor and percutaneous absorption of met hanolic liquids are as
effective as the oral route in producing acute toxic
effects. The most noted health consequence of longer-term exposure to lower levels of me thanol is a broad range of
ocular effects.

The toxic properties of methan ol are based on factors that govern both the conversion of methanol to formic acid and the subsequent metabolism of f ormate to carbon dioxide in the folate pathway. The toxici ty is manifest if formate generation continues at a rate that exceeds its rate of
metabolism.

The lethal dose of methanol fo r humans is not known for
certain. The minimum lethal do se of methanol in the absence
of medical treatment is betwee n 0.3 and 1 g/kg. The minimum dose causing permanent visual defects is unknown.

The severity of the metabolic acidosis is variable and may
not correlate well with the am ount of methanol ingested.
The wide interindividual variabili ty of the toxic dose is a prominent feature in acute met hanol poisoning.

Two important determinants of human susceptibility to methanol toxicity appear to be (1) concurrent ingestion of ethanol, which slows the entra nce of methanol into the metabolic pathway, and (2) hep atic folate status, which governs the rate of formate de toxicification.

The symptoms and signs of meth anol poisoning, which may not

appear until after an asymptom atic period of about 12 to 24 hours, include visual disturba nces, nausea, abdominal and muscle pain, dizziness, weakne ss and disturbances of consciousness ranging from com a to clonic seizures. Visual disturbances generally develop between 12 and 48 h after methanol ingestion and range f rom mild photophobia and misty or blurred vision to markedly reduced visual acuity and complete blindness. In extreme cases death results. The principal clinical feature is severe metabolic acidosis of anion-gap type. The acidosis i s largely attributed to the formic acid produced when meth anol is metabolized.

The normal blood concentration of methanol from endogenous

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214 sources is less than 0.5 mg/li tre (0.02 mmol/litre), but dietary sources may increase b lood methanol levels. Generally, CNS effects appear above blood methanol levels
of 200 mg/litre (6 mmol/litre), a nd fatalities have occurred
in untreated patients with initia l methanol levels in the

range of 1500-2000 mg/litre (47-62 m mol/litre).

Visual disturbances of several types (blurring,
constriction of the visible field, changes in colour perception, and temporary or permanent blindne ss) have been reported in workers who experienced methan ol air levels of about 1500 mg/m3 (1200 ppm) or more.

A widely used occupational exp osure limit for methanol is 260 mg/m3 (200 ppm), which is designed to protect workers from any of the effects of met hanol-induced formic acid
metabolic acidosis and ocular and nervous system toxicity.

No other adverse effects of me thanol have been reported in humans except minor skin and e ye irritation at exposures well above 260 mg/m3 (200 ppm)

. Reliability: (2) valid with restrictions
see remark on selection of lit erature

24-DEC-2004 (88)

Type of experience: other: kinetics, inhalation (Osterloh)

Remark: During a randomized double-blind stud y of the potential
neurobehavioral effects of inh aled methanol at 200 ppm for
4 hours methanol analysis was pe rformed. Methanol was rapidly absorbed by inhalation. Serum methanol conc. were increased by more than fourfold at the e nd of exposure period, as

were urinary methanol excretion rat es, although formate conc. were not increased over backgr ound conc. The overall

- 205/327 -
elimation half-life was 3.2 + 2.3 h. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions

24-DEC-2004 (277)

Type of experience: other: acute toxicity, CNS (Chuwers)

Remark: This study assessed whether acute exp osure to methanol

vapor at the current industrial thre shold limit value of 200 ppm
for 4 hr has adverse effects o n human neurobehavioral performance. Twenty-six health y subjects (15 men, 11 women; ages 26-51 years) were exposed to methanol or water vapor for 4 hr while seated in a cha mber. The subjects served as
their own controls in a random ized, double-blind study design. The variables assessed were serum and urine

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215 methanol and formate levels; visual qua lities (color discrimination and contrast sensitivity); and neurophysiological (auditory evoked potentials) and neurobe havioral qualities. Exposure to methanol increased serum concentrations and urinary excretions of methanol , but did not affect formate levels. Overall visual, neurop hysiological, and
neurobehavioral test outcomes were not significantly affected, unless certain betwe en-subject variables are considered. Slight effects on P-300 amplitude and Symbol Digit testing were noted. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions

24-DEC-2004 (278)

Type of experience: other: review, general (Becker)

Remark: Methanol poisoning is an uncommon but an extremely
hazardous intoxication. Since methanol i s a versatile fuel and is having increasing usage in an energy-conscious society, a high index of suspicion and sw ift laboratory confirmation
is essential in managing this poi soning. Methanol poisoning

may occur in sporadic or epidemic circumstances. Chronic exposure may occur in the occu pational setting. Man is uniquely susceptible to methan ol toxicity, perhaps dependent upon folate metabolism. Classi c symptoms of methanol toxicity can only occur in lab oratory animals who are
rendered folate deficient. Fol ate may be useful in humans enhancing removal of the toxic products of methanol poisoning. The enzyme responsi ble for metabolism of
methanol is alcohol dehydrogenase. Etha nol has a higher affinity for this enzyme and is preferentia lly metabolized. Simultaneous
ethanol and methanol administr ation may confuse the onset
of the intoxication. Pyrazoles ma y also be used to inhibit alcohol dehydrogenase thus pre venting the intoxication. The most important initial symptom of methanol poisoning is visual disturbance. The sympto ms may be delayed up to 24 hours after ingestion due to s imultaneous alcohol administration and metabolic p rocesses. Laboratory evidence
of severe metabolic acidosis w ith increased anion and osmolar gaps strongly suggest the clinical diagnosis. There may be an important associatio n between mean corpuscular volume which is significantly higher in cases of severe methanol poisoning than in mil d cases. Once the diagnosis
is suspected, a blood level from methanol should be returned rapidly. Treatment of methanol toxicity after good supportive care is to diminish the metabolic degradation of
methanol with simultaneous eth anol and then to perform hemodialysis and alkalinizatio n to counteract metabolic
acidosis. Folate should be adm inistered to enhance
metabolic
breakdown of formate. Alcoholi c patients may especially

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216 susceptible to methanol poison ing due to relative folate defiency. Reliability: (2) valid with restrictions
see remark on selectionof lite rature

28-DEC-2004 (279)

Type of experience: other: review, general (Roe 1955)

Remark: During methanol poisoning in man the concentration of
formic acid in the blood is quite var iable. In 5 lethal cases it ranged from 9 to 68 mg per cen t. In three patients who also died it ranged from 5.7 to 19 mg per cent. Furthermore, the methanol concentration in the blood in 23 lethal cases varied between 51 and 274 mg p er cent. It becomes obvious that the mere concentrations o f these substances are not
the only decisive factors in the c linical course of the poisoning. Reliability: (2) valid with restrictions
see remark on selection of lit erature

28-DEC-2004 (280)

Type of experience: other: review, general (Tephly)

Remark: Methanol toxicty in humans and monkey s is characterized by

a latent period of many hours fo llowed by a metabolic
acidosis and ocular toxicity. This is n ot observed in most lower
animals. The metabolic acidosi s and blindness is apparently
due to formic acid accumulatio n in humans and monkeys, a
feature not seen in lower anim als. The accumulation of formate is due to a deficiency in formate metabolism which is, in turn, related, in part, to low hepatic tetrahydrofolate (H4folate). A n excellent correlation between hepatic H4folate and f ormate oxidation rates has
been shown within and across s pecies. Thus, humans and monkeys possess low hepatic H4 folate levels, low rates of formate oxidation and accumula tion of formate after methanol. Formate, itself, pro duces blindness in monkeys in
the absence of metabolic acido sis. In addition to low hepatic H4folate concentration s, monkeys and humans also have low hepatic 10-formyl H4f olate dehydrogenase levels, the enzyme which is the ultima te catalyst for conversion of
formate to carbon dioxide. Reliability: (2) valid with restrictions
see remark on selection of lit erature 28-DEC-2004 (281)

Type of experience: other: review, general (Kavet; Nauss)

Remark: Methanol could become a major automot ive fuel in the U.S., and its use may result in incr eased exposure of the public to methanol vapor. Nearly all of the available information on methanol toxicity in humans relates to the consequences of acute, rather than chronic, exposures. Acute methanol toxicity evaloves in a well-un derstood pattern and consists

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217 of an uncompensated metabolic acidosis with superimposed toxicity to the visual system. The toxic properties of
methanol are rooted in the fac tors that govern both the conversion of methanol to form ic acid and the subsequent
metabolism of formate to carbo n dioxide in the folate pathway. In short, the toxic s yndrome sets in if formte generation continues at a rate that exceeds its rate of
metabolism. Current evidence i ndicates that formate accumulation will not challeng e the metabolic capacity of the folate pathway at the anti cipated levels of exposure to automotive methanol vapor. Reliability: (2) valid with restrictions
see remark on selection of lit erature

28-DEC-2004 (192)

Type of experience: other: kinetics, oral (Ferry)

Remark: Methanol blood and urine concentratio ns were investigated
in four volunteers who had ingest ed small amounts of methanol (0.2 ml hourly for six hours). The methanol urine
concentration did not exceed 8 .0 μg/ml. It was estimated, that at a MAC value of 200 ppm with a total 8 h ventilatory volume of 10 m3 and assuming c omplete absorption and no exhalation 2.6 g methanol woul d be absorbed. The highest urinary concentration attained by oral ingestion of this
amount of methanol at a rate o f 0.5 ml hourly in one of the subjects was 17.6 μg/ml. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles
28-DEC-2004 (282)

Type of experience: other: kinetics, inhalation (Sedivec)

Remark: Five volunteers were exposed to const ant and suitable graded
concentration between 103 to 2 84 mg/m3 of methanol vapours for a period of 8 h. Pulmonary retention ranged from 53.4

to 61.3 %. Elimination half-life was calculated to range
between 1.5 to 2.0 h. Reliability: (2) valid with restrictions aceptable study, meets basic s cientific principles

28-DEC-2004 (283)

Type of experience: other: review, acute toxicity (Roe 1982)

Remark: The minimal lethal dose of methanol i n humans has not been determined. It has been sugges ted that about 1 g/kg can cause death if the patient is untreated and has not
consumed ethanol. In some clinical case s, the blood methanol content is low in the last phase of th e poisoning. In three such
cases blood methanol concentra tion was 0.275, 0.277, and 0.194 g/l, respectively. On th e assumption that the body water in diffusion equilibrium with the blood respresents about 70 % of the body weight, it has been calculated that 0.19, 0.19, and 0.14 g/kg, res pectively, was present in the

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218 body. Data on the rate of meth anol oxidation in humans probably do not exist. Provide d that the rate of methanol oxidation is the same in man a s in rhesus monkeys, the
amount of methanol oxidized du ring 18 hours (the average time needed for development of severe acidosis in clinical cases) would be 0.666 g/kg. It seems reasonable still to regard 1 g/kg of methanol as t he approximate minimal lethal
dose in man. Reliability: (2) valid with restrictions
see remark on selection of lit erature

28-DEC-2004 (284)

Type of experience: other: kinetics, inhalation (Heinrich; Angerer)

Remark: Twenty persons occupationally exposed to methanol were examined according to their me thanol levels in blood and
urine and their formic acid ex cretion. An 8-h exposure to a
methanol concentration of 93 m l/m3 in the air at the working area caused average methanol l evels in blood and urine of 8.9 +- 14.7 mg/l and 21.8 +- 2 0.0 mg/l, respectively, and a
mean formic-acid level in urin e of 29.9 +- 28.6 mg/l. Based on these results a rough estim ate shows the corresponding
methanol content in urine to b e about 40 mg/l for an 8-h exposure at 200 ml/m3. No correlation between exposu re and urinary concentration of formate could be clearly de monstated, although 15 % of
measured values exceeded the n orm Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles
28-DEC-2004 (285)

Type of experience: other: review, acute toxicity (Litovitz)

Remark: Annual report of the American Associa tion of Poison Control
Centers National Data Collecti on System. 3 cases of fatal methanol ingestion were report ed. Blood levels were 175, 402, and 39 mg/dl, respectivel y. Reliability: (2) valid with restrictions
see remark on selection of lit erature

28-DEC-2004 (286)

Type of experience: other: kinetics, inhalation (Baumann; Angerer)

Remark: A study was carried out among 20 work ers employed in a printing office at three diffe rent work places (methanol concentration: 85, 101, and 13 4 ppm) to determine whether
concentration of formic acid i n blood or urine and the
methanol content of aveolar ai r permit the estimation of methanol exposure. The concent ration of formic acid in
blood increased significantly from 3 .2 +- 2.4 mg/ml before to 7.9 +- 3.2 mg/l after the shift in the exposed workers. In 36 non-exposed persons, the blood formate levels ranged from 0 - 20 mg/l. The corresponding concentratio n in urine was increased significantly from 13.1 +- 3.9 mg/l to 20.2 +- 7 mg/l, respectively. On the contrary, in the control groups there

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219 was a small but significant de crease of formic acid
concentration in blood from 5. 6 +- 4.5 mg/l in the morning to 4.9 +- 4.2 mg/l in the afte rnoon. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

28-DEC-2004 (287)

Type of experience: other: acute toxicity, CNS (Muttray)

Remark: Twelve healthy subjects were exposed for 4 h to 200 ppm and to 20 ppm (control) in an expo sure chamber in a cross-over design. The EEG was recorded b efore (reference) and at the end of each exposure with, the subject's eyes closed and opened and during a choice rea ction test (color word stress test). Spectral power was calc ulated by fast Fourier transformation. Subjective sym ptoms and effects of blinding with 20 ppm methanol were asse ssed by questionnaires. The study was a single-blind one. During subjects' exposure to 200 ppm, their scores for pren arcotic and irritating symptoms were not different fr om controls. In the closed-eye condition of subjects, the spe ctral power of the theta-band
and of some electrodes of the delta-band was significantly less at the end of exposure to 200 ppm, than that of controls. In the open-eye cond ition and during the color word stress test no significan t changes were found. The changes in the theta-band sugg est a slight excitatory
effect of 200 ppm methanol. The effec t was weak, as scores of

acute symptoms did not change. Reliability: (2) valid with restrictions acceptable study meets basic s cientific principles

28-DEC-2004 (288)

Type of experience: other: acute toxicity (Jacobsen)

Remark: Eleven patients concomitantly poisone d with methanol are
described. Their whole blood m ethanol conc. ranged from 137.2 mmol/l (4.39 g/l) to 7.4 mmol/l (0.24 g/l). The clinical course in most patien ts was mild, which was
attributed to the concomitant and subsequent ethanol ingestion and rapid transport to dialysing units. One patient suffered permanent vis ual impairment of one eye,
while the others recovered com pletely. Symptoms of poisoning were most clearly correlated t o the degree of metabolic acidosis. All patients were di alysed. Ethanol in conc. even lower than usually recommended may be useful as the only treatment of patients with blo od methanol conc. up to 15 mmol/l (0.5 g/l), provided the re is no acidosis or visual impairment. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles
28-DEC-2004 (289)

Type of experience: other: acute toxicity (Erlanson)

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220 Remark: Four cases of methanol poisoning were treated with alkali, ethyl alcohol, and the haemodi alysis. In 3 cases respirator treatment was also required. S evere acidosis, failing eye-sight, and cerebral damage were present at the start of treatment within 29 to 44 hour s of the methanol ingestion. Three patients, one of whom ha d consumed 90 g, died despite
correction of the acidosis and elimination of methanol. Autopsy in these cases showed massive necrosis of the brain and haemorrhages into the puta mina. The fourth patient, who had consumed 80 g methanol, su rvived but died 1 1/2 year later from pneumonia; autopsy showed slit-shaped cysts in the lateral parts of the putam ina. All the patients had considerable hypopotassaemia.

Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

28-DEC-2004 (290)

Type of experience: other: acute toxicity (Naraqi)

Remark: An outbreak of acute methyl alcohol i ntoxication occurred. Twenty-eight young men attende d a drinking party and drank methyl alcohol. The amount con sumed by each individual ranged from an equivalent of 6 0-600 ml of pure methanol. Three had prior ethanol ingest ion. All 28 became ill 8-36 hours after drinking and were hospitalized. The most commonly observed clinical syn dromes were: acute metabolic acidosis, severe visual impair ment and acute pancreatitis.
Four died within 72 hours afte r admission to the hospital.
All had severe metabolic acido sis and visual impairment and three pancreatitis. Of 24 who recovered, 16 showed no residual complications, six ha d bi-lateral visual impairment and had difficulty in speech a s well as visual impairment.

A three month follow-up examinat ion showed no change in the findings. Coma, seizures and p rolonged acidosis were poor prognostic signs. The estimate d amount of consumed methanol and the rapidity of the appear ance of signs of toxicity following methanol ingestion d id not seem to influence the outcome of poisoning. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

28-DEC-2004 (291)

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221 Type of experience: other: worker exposure (Kawai / Yasugi)

Remark: In methanol-exposed male and female w orkers there was a linear relationship between me thanol exposure and formate excretion in the urine after w orking shift up to an 8-h average of 2000 and 4000 ml/m3 , respectively. In unexposed persons, the baseline urinary excretion of methanol and
formate were 1.9 ±0.8 and 26.2 ±12.2 mg/l urine.

Based upon a regression equati on presented by the authors,
the mean added formate concent ration in the urine amounted to about 180 mg/l (males) and 100 mg/l (females) after an 8-h average of 2000 ml/m3, and to 17 mg/l (males) and about 10 mg/l (females) after 200 ml /m3 (Yasugi et al. 1992).

------------------

The exposure-excretion relatio nship and possible health effects of exposure to methano l vapor were studied in 33 exposed workers during the sec ond half of 2 working weeks. Urinary methanol concentration s were also determined in 91 nonexposed subjects (Kawai et al., 1991). The geometric

mean value for methanol in urine sa mples from the latter was <2 mg/l. Among the exposed worker s, the methanol level in
urine samples collected prior to wor k shift exceeded the 95 % upper limit of normal. Methano l concentrations in urine at the end of shift showed a sign ificant correlation between exposure to methanol vapour at concentrati ons of up to 5,500 ppm and
the
level of methanol measured: Following exposures to mean 16 90 ml/m3, more than 100 mg/l
methanol was found in the urin e of 8 men, at an average exposure level of 550 ml/m3, 3 0 - 100 mg/l methanol were detected in 6 men. The highest 8-h exposures from 4000 ml/m3 and higher corresponded to 30 0 to 500 mg/l (Kawai et al., 1991).

The calculation indicated that a mean level of 42 mg methanol/l urine was excreted in the shift-end urine samples following 8 h exposure to meth anol at 200 ppm.

Highly exposed workers (3500 - 5500 ml/m3) more often complained of blurred vision a nd headache during or after work. Nobody stated to suffer from photophobia. The examination of the eye fun dus failed to reveal retinal changes. Among three workers e mployed in that company between 0.3 and 7.8 years and having been exposed to about 1000 - 1600 and 120 to 3600 mg /m3 (one case), two showed retarded pupil reflex and one mild mydriasis (Kawai et al.

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222 1991). Dimmed vision and nasal irritation were among the most frequent symptoms complai ned during work. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

28-DEC-2004 (292) (293)

Type of experience: other: human exposure (Franzblau)

Remark: Five human subjects were exposed to a n atmospheric concentration of 200 ppm of me thanol in a test chamber for
7 h per day for 5 consecutive da ys. Ambient air in the
chamber was monitored continously for methanol, while urine was
monitored for methanol and for mic acid. Mean urinary methanol concentration were in creased from baseline at the end of each exposure session, but returned to baseline in samples collected 16 h followi ng cessation of exposure. The
concentration of formic acid i n morning urine specimens did not change significantly over the 7 days of the exposure. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

28-DEC-2004 (294)

Type of experience: other: human exposure (Leaf; Zatman)

Remark: The elimination of methanol after dos es of 2.5 to 7 .0 ml
has been studied in five human subjects. At any time the
rate of elimination was found to be proportional to the
concentration of methanol in t he body. Blood levels of 47 to 76 mg/l were measured 2 to 3 h after oral uptake of 71 - 84 mg meth anol/kg bw (6.6 - 7.4 ml per person); methanol disappearanc e obeyed first-order kinetics
with a half-time of about 3 h. Only a very small fraction of ingested methanol (about 2 %) was eliminated via the respira tory and urinary routes. The rates of absorption of methano l by two human subjects during exposure to vapour concentrati ons of 400 - 1000 ppm have been investigated. Over short periods the amount of methanol absorbed appears to be approximately proportion al to the duration of

exposure and to the concentration of va pour in the atmosphere. It is
concluded that accumulation in the body would occur at 3000 ppm an the maximum safe concen tration for occupational exposure is 300 ppm. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

Type of experience: other: acute toxicity (Frederick)

Remark: A health hazard evaluation was conduc ted by the National Institute for Occupational Saf ety and Health (NIOSH) to determine if vapors from dupli cating fluid (99% methyl alcohol) used in direct-proces s spirit duplicating machines were causing adverse health ef fects among teacher aides, or had been responsible for the d eaths of three former teacher

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223 aides. Death certificates and autopsy data were obtained
and evaluated. A self-administered symptom questionnaire was
distributed to current teacher aides (exposed group) and to a comparison group of teachers . Fifteen-minute breathing zone air samples for methyl al cohol vapor were collected at operator stations using an inf rared gas analyzer. No information supported the clai m that the three deaths were related to methyl alcohol expo sure. Teacher aides reported significantly more blurred vis ion, headache, dizziness, and nausea than the comparison gro up. Concentrations of
airborne methyl alcohol ranged from 365 -3080 ppm; 15 of 21
measurements exceeded the NIOS H-recommended 15-minute exposure limit of 800 ppm. A m ean 96% reduction in vapor concentration was accomplished using inexpensive enclosures and existing room exhaust syst ems. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions

28-DEC-2004 (296)

Type of experience: other: human exposure (Batterman)

Remark: Dermal exposures to methanol were adm inistered in a
clinical study designed to compare seve ral biological indicators. Four subjects were exposed in five exposure sessions of varying length. In each sessio n, a sequence of measurements of methanol conc. in blood, br eath, and headspace samples
of air at exposed and unexposed s kin analysed. At exposed skin samples were highly elevated f or at least 8 h following exposure, indicating the prese nce of a methanol reservior
in skin. After exposure, methanol conc. at exposed skin showed a rapid initial decline, then a slower first order
decrease. When transfer was restricted, surface conc. at unexposed skin were similar to levels in breath and were strongly correlated to methanol conc. i n blood. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions
28-DEC-2004 (297)

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224 Type of experience: other: human exposure (skin) (Dutkiewicz)

Remark: Methanol absorption rate through huma n skin (foremarm,
15-60 min.) has been examined and a value of 0.192 mg(cm2/min)
was determined. The excretion of unchanged methanol with urine and exhaled air, after absorption through sk in and administration "per os" amount ed to 16 % and 22 % of the absorbed dose (1.67 g), respec tively. It was estimated, that exposure of one hand to l iquid methanol for only 2 min. would lead to the absorption of as much metha nol (170 mg), as would be taken up by the lungs from an 8 h exposure to MAC of 50 mg/m3. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

28-DEC-2004 (298)

Type of experience: other: kinetics, inhalation, human (Batterman)

Remark: Relationship between methanol concent ration in the blood, urine, and breath of volunteer s exposed to methanol vapor at 800 ppm for periods of 0.5, 1, 2, and 8 h. For methanol, concentrations are not proport ional to the exposure
duration due to metabolic and other elimination processes that occur concurrently with the exposure. The 0.5 to 2-h periods of exposure were used to estimate the half -life of
methanol. Blood data gave a half-life of 1.44 +- 0.33 h. Comparable but slightly more variable res ults were obtained using
urine data corrected for the voiding time (1.55 +- 0.67 h) and
breath data corrected for muco us membrane desorption (1.40

+- 0.38 h). Methanol concentratio ns in blood lagged some 15-30
min. behind the termination of exposure, and concentrations in urine were further delayed. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions
28-DEC-2004 (299)

Type of experience: other: kinetics, inhalation, human (Batterman)

Remark: Experimental study of dermal exposure to methanol in human volunteers estimating percutan eous absorption. 12 volunteers were exposed to met hanol via one hand for
durations of 0 to 16 min in a total of 65 sessions . The pre-exposure methanol conc. in blood was 1.7 mg/l, and subjects had statistically different mean conc. Th e maximum
methanol conc. in blood was reached 1.9 h after ex posure; this is comparable to that reached following inhal ational exposure at a methanol conc. of 200 ppm. Delivery rate from skin into blood lagged exposure by 0.5 h, and methano l continued to enter the systemic circulation for 4 h f ollowing exposure. The mean derived absorption rate was 8. 1 mg/cm2/h. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles
28-DEC-2004 (300)

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225 Type of experience: other: kinetics, inhalation, human (Lee)

Remark: Accumulation of formate, the putative toxic metabolic of methanol, in the blood methano l and the relationship
between pulmonary intake and blood met hanol conc. were investigated
in six human volunteers follow ing a 6-hr exposure to 200

ppm methanol. At the end of a 6-hr exposure to 200 ppm at rest, the blood methanol conc. was i ncreased from a mean of 1.8 μg/ml to 7.0 μg/ml. Under ligh t excercise, the total amount of methanol inhaled during the 6-hr exposure period was 1.8
times that inhaled at rest. Ho wever, no statistically significant increase in blood methanol conc. was observed under exercise: the conc. aver aged 8.1 μg/ml. Formate did
not accumulate in the blood ab ove its background level following the 6-hr exposure to 200 ppm methanol whether subjects were exposed at rest or during excercise. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions
28-DEC-2004 (196)

Type of experience: other: kinetics, inhalation (Kawai)

Remark: Exposure of 34 male workers to combin ed toluene, styrene
and methanol was monitored by pers onal diffusive sampling of solvent vapours in breathing z one air, analysis of
shift-end
blood for the 3 solvents and a nalysis of shift-end urine
for hippuric, mandelic and phenylg lyoxylic acids and methanol. The exposure of most of the wo rkers was below current occupational exposure limits. Regression analysis showed
that a linear correlation exis ts for each of the 3 solvents between any pairs of the conce ntrations in air, blood and urine. Namely, toluene, styren e and methanol concentrations
in blood obtained at the end o f a shift are linearly
related to the time-weighted average i ntensity of exposure to corresponding solvents, and al so hippuric, mandelic and phenylglyoxylic acids as well as methanol in shift-end
urine. The concentrations of h ippuric, mandelic and phenylglyoxylic acids as well as methanol in urine correlated with the respirator y exposure intensity. Comparison of the present resu lts with the exposure-excretion relationshi p after occupational exposure
to the individual solvent show ed that no modification in metabolism is induced by the c ombined exposure when

exposure is low, as in the present case

. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles
28-DEC-2004 (301)

Type of experience: other: acute toxicity, cns (Dethlefs / Naraqi)

Remark: The ocular manifestation of acute met hyl alocohol
intoxication were studied in 2 4 men during an outbreak. The visual acuity, pupillary react ions to light, fundal

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226 appearence and visual fields w ere recorded in all patients within 72 hours of ingestion o f methanol and again three months later. Nine patients ha d no ocular abnormality, 7 had only transient ocular abno rmalities, and eight had permanent ocular abnormalites. Complete blindness occurred in two patients, while severe visual deficit resulted in
four others. The incidence of permanent ocular abnormalities
was found to correlate with th e incidence of metabolic acidosis, and with the stated volume of methanol consumed. An inverse correlation was fou nd between stated volume of
methanol consumed and onset of blurred vision. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles

28-DEC-2004 (302)

Type of experience: other: review, reproductive and developmental eff ects (Lorente)

Remark: Study reviewed a variety of occupatio ns and associated exposures to complex mixtures in women who gave birth to
infants with and without cleft lip or palate.
No association was found betwe en methanol exposure and oral clefts, but several limitation s of the study were raised: small number of subjects expos ed to methanol, lack of individual exposure data, and confounding by other chemical

exposures.

Because of these limitations t he Panel judged the study
results to be uncertain and co ncluded there are insufficient data upon which to evaluate th e developmental toxicity of
methanol in humans. Reliability: (2) valid with restrictions acceptable study, meets bsci s cientific principles

28-DEC-2004 (303) (233)

Type of experience: other: kinetics, nutrition (Davoli)

Remark: Aspartam was administered to humans a t a single dose of 500 mg per individual in 100 ml ta p water.

Four adult volunteers fasted f or 8 h and avoided alcohol, fruits, fruit drinks or vegeta ble for 24 h.

Blood methanol was measured at 0, 30, 45, 60, 90, 120, and 180 min following ingestion.

The dose of aspartame was repr esentative of the daily average sugar consumption and corresonded to about 50 mg methanol = 0.7 - 0.8 mg/kg.
Result: Baseline blood methanol: 1.4 - 2.6 mg /L

Mean incremental increase (max imum after 45 min): <= 1 mg/L

Aspartame consumption by adult s at a dose equivalent to the daily intake of sugar results in methanol blood levels similar to endogenous levels, in particular when divided in

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227 smaller fractions over the day

.

-------------------

28-DEC-2004 (304) (233)

Type of experience: other: kinetics, nutrition (Leon)

Remark: This longer-term study demonstrated t hat ingesting aspartame equivalent to a meth anol dose of 7.5 mg/(kg bw*d) per day resulted in blood meth anol levels around 10 mg/LL
in adults. Reliability: (4) not assignable Secondary literature

28-DEC-2004 (305) (233)

Type of experience: other: remark on selection of literature

Remark: Concerning effects in humans several quality reviews have already been conducted. With a focus on health and safety issues comprehensive reviews i ncluding those of the World Health Organization (WHO), 199 7, Kavet and Nauss, 1990 and the Center for the Evaluation of Risks to Human Reproduction (CERHR), 1988 and single studies cited in
these reviews were selected for this robust summary of methanol. Relevant recent publications h ave also been taken into

account.

Type of experience: other: review, general

Remark: Review of toxicity of methyl aclohol following skin absorption and inhalation in a nimal studies. Reliability: (2) valid with restrictions
see remark on selection of lit erature (156)

Type of experience: other: review, general

Remark: Methyl alcohol is an aliphatic alcoho l with use in a few
cosmetic formulations as a sol vent and denaturant. Concentrations up to 5% are ty pically used to denature ethyl alcohol in cosmetic products. Methyl alcohol is readily absorbed through the skin and from the gastrointestinal and respiratory tracts, is distrib uted throughout all organs
and tissues (in direct relation to the body's water distribution), and is eliminat ed primarily via the lungs. Undiluted Methyl alcohol is an ocular and skin irritant.
Inhalation studies showed a no -effect level for maternal damage of 10,000 ppm and for t eratogenic effects of 5,000 ppm. Overall, methyl alcohol i s not considered mutagenic. Carcinogenicity data were unav ailable. The toxicity of methyl alcohol in humans resul ts from the metabolism of the
alcohol to formate and formic acid through a formaldehyde
intermediate. Formate accumula tion causes metabolic
acidosis and inhibits cellular respirat ion. Methyl alcohol toxicity is time and concentration depe ndent, and its toxic effect

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228 is competitively inhibited with e thyl alcohol. Because of the moderating effect of ethyl alc ohol, it was concluded that methyl alcohol is safe as used to denature ethyl alcohol used in cosmetic products. No conclusion was reached regarding any other use of met hyl alcohol. Reliability: (2) valid with restrictions
see remark on selection of lit erature (306)

Type of experience: other: odour

Remark: Experimental study in two and seven f urther subjects for
detection of odour thresholds in solvents. For methanol the odour threshold was 5,900 ppm and for detection of a distinct odour was 8,800 ppm. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (307) Type of experience: other: odour

Remark: In a group of 25 subjects odour thres hold was 3,4 ppm. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (308)

Type of experience: other: kinetics, inhalation

Remark: Urinary formic acid was studied in fo urteen workers exposed to methanol with 10 +- 5 years in their current occupation. Exposure to methanol vapor at 200 ppm methanol produced 80 mg formic acid/g creatinine. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principle (309)

Type of experience: other: kinetics, inhalation

Remark: Four human subjects were exposed to a pproximately 200 ppm
of methanol in a test chamber for six h. Ambient air in the
chamber was monitored for meth anol, while urine was
monitored for formic acid. Mea n urinary formic acid was
increased from baseline at the end of the exposure session,
but had returned to baseline i n samples collected 16 h following cessation of exposur e. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (310)

Remark: Methanol is slowly eliminated from th e body; hence, repeated exposures result in an increa sing concentration in blood
and tissue. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (311)

Remark: The aim of this study was to determin e the reference values

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229 for methanol in urine of the g eneral population non-occupationally exposed to the xenobiotic under study. Thus, urinary methanol was mea sured in 84 occupationally non-exposed subjects from the city of Sao Paulo, Brazil, by means of headspace gas chromat ography with a flame
ionization detector. The resul ts revealed a mean of 2.26 +/- 1.26 mg methanol/l urine and a geometric mean of 2.10 mg/l for the studied population. Th e reference values varied within the range of 0.50-4.78 mg/l (mean +/- 2 S.D.).
Methanol levels in urine did n ot differ statistically between male and female subjec ts. Urinary methanol in the total population was less than 4.80 mg/l in 95% of cases. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (312)

Remark: One hundred and forty-three workers e xposed to one or more of toluene, xylene, ethylbenze ne, styrene, n-hexane, and methanol at sub-occupational e xposure limits were examined for the time-weighted average intensity of exposure by diffusive sampling, and for bi ological exposure indicators by means of analysis of shift- end blood for the solvent and analysis of shift-end urine fo r the corresponding metabolite(s). Urinalysis was also performed in 20 nonexposed control men to esta blish the "background level."
Both solvent concentrations in blood and metabolite
concentrations in urine correl ated significantly with
solvent concentrations in air. Comparison of blood analysis and urinalysis as regards sens itivity in identifying low solvent exposure showed that b lood analysis is generally superior to urinalysis. It was also noted that estimation
of exposure intensity on an indiv idual basis is scarcely possible even with blood analy sis. Solvent concentration in
whole blood was the same as th at in serum in the case of
the aromatics, except for styrene. It was higher in blood than
in serum in the case of n-hexa ne, and lower in the cases of styrene and methanol. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (313)

Type of experience: other: kinetics, model

Remark: Two patients with extremely high bloo d methanol concentrations (260 and 282 mg /dl) were successfully
treated using pharmacokinetic dosing o f ethanol, hemodialysis and supportive measures. Both pati ents recovered completely without residual ophthalmologi c deficits. Early hemodialysis
and inhibition of methanol met abolism with effective
ethanol concentrations were attributed to the patients' full recovery. Methanol elimination was enhanced by hemodialysis as evidenced by a decrease in half-life from eight to two
and a half hours. Methanol dia lysance was 98 ml/min. A dosage regimen for ethanol was devised, utilizing

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230 dose-dependent pharmacokinetic parameters and the ethanol dialysance (100 to 120 ml/min) from these two patients. An ethanol loading dose of 0.6 g/ kg should be administered to
an adult with an acute methano l ingestion. This dose will produce a blood ethanol concen tration of approximately 100 mg/dl which can be maintained by an ethanol infusion of 66 mg/kg/hour for nondrinkers to 154 mg/kg/hour for chronic ethanol drinkers. Hemodialysis should be initiated if the
blood methanol concentration i s greater than 50 mg/dl. If hemodialysis is initiated, the ethanol infusion should be increased by 7.2 g/hour. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (314)

Type of experience: other: kinetics, nutrition

Remark: Blood methanol concentrations were me asured in 24 1-year-old infants administered aspartame , a dipeptide methyl ester
sweetener. The doses studied i ncluded a dose projected to
be the 99th percentile of daily i ngestion for adults (34 mg/kg body weight), a very high use dose (50 mg/kg body weight)
and a dose considered to be in the abuse range (100 mg/kg body weight). Blood methanol v alues in infants were compared to values observed previously in adults administered equivalent doses of aspartame. Methanol concentrations were below the level of detection ( 0.35 mg/dl) in the blood of
10 infants administered aspartame at 34 mg/kg body weight, but were significantly elevated (P less than or equal to 0.05) after ingestion of aspartame a t 50 and 100 mg/kg body weight. At the latter doses, m ean peak blood methanol concentrations and the area un der the blood methanol concentration-time curve incre ased in proportion to dose. Mean (+/- SEM) peak blood meth anol concentration was 0.30 +/- 0.10 mg/100 ml at a 50 mg/ kg body weight aspartame dose (n = 6) and 1.02 +/- 0.28 mg/m l at the 100 mg/kg body weight dose (n = 8). Blood methanol v alues in infants were similar
to those observed in normal ad ults. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions

(315)

Type of experience: other: kinetics, nutrition

Remark: Blood methanol concentrations were me asured in 30 normal adult subjects administered as partame, a dipeptide methyl
ester. The doses studied inclu ded the 99th percentile of projected daily ingestion (34 mg/kg body weight) and three
doses considered to be in the abuse range (100, 150, and
200 mg/kg body weight). Methanol c oncentrations were below the level of detection (0.4 mg/dl) in the blood of the 12
normal subjects who ingested aspartam e at 34 mg/kg. They were

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231 significantly elevated (p less than or equal to 0 .001) after ingestion of each abuse dose, with the mean peak
blood methanol concentrations and th e areas under the blood methanol concentration-time cu rve increasing in proportion to dose. Mean (+/- SD) peak bl ood methanol concentrations were 1.27 +/- 0.48 mg/dl at th e 100 mg/kg dose, 2.14 +/- 0.35 mg/dl at the 150 mg/kg do se, and 2.58 +/- 0.78 mg/dl

at the 200 mg/kg dose. Blood meth anol concentrations returned to predosing levels by 8 h aft er administration of the 100 mg/kg dose. Methanol was still detected in the blood 8 h after the subjects had ingeste d aspartame at 150 or 200 mg/kg. Blood formate analyses were carried out in the 6 subjects who ingested aspartam e at 200 mg/kg, since recent
studies indicate that the toxi c effects of methanol are due to formate accumulation. No si gnificant increase in blood formate concentrations over pr edosing concentrations was noted. No changes were noted i n any of the blood chemistry profile parameters measured 24 h after aspartame ingestion, compared to values noted befor e administration. Similarly,
no differences were noted in o phthalmologic examinations
carried out before and after a spartame loading. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions

(316)

Type of experience: other: review, acute toxicity

Remark: The fatal dose in humans is between 2 and 8 ounces (1 ounce = 28,35g). Reliability: (2) valid with restrictions
see remark on selection of lit erature 28-DEC-2004 (317)

Type of experience: other: review, acute toxicity

Remark: Most serious cases of methanol poison ing that have been reported (many fatal; others i nvolved permanent or temporary loss of vision) resulted from the ingestion of methanol in
the belief that it was ethanol . Ocular disturbance and blindness in humans resulting from repeated rubbing of the
skin with methanol was also re ported. Reliability: (2) valid with restrictions
see remark on selection of lit erature (135) Type of experience: other: review, acute toxicity

Remark: The signs and symptoms most character istic of methanol poisoning are visual disturban ce and metabolic acidosis. Permanent damage (residual sco tomata) can result even if complete blindness is avoided. Reliability: (2) valid with restrictions
see remark on selection of lit erature (318)

Type of experience: other: review, acute toxicity

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232 Remark: Methanol poisoning with typical distu rbed vision may appear in the occupational setting, b y ingestion after drinking of
methanol instead of ethanol an d continous inhalation when handling the hot product. Reliability: (2) valid with restrictions
see remark on selection of lit erature (319)

Type of experience: other: review, acute toxicity

Remark: Methanol intoxication causes severe m etabolic acidosis and can lead to permanent visual d amage or death. Methanol, readily available in common pr oducts like antifreeze, is ingested accidentally or delib erately as a substitute for ethanol and in suicide attempt s. Because it may become major fuel source in the 21st centur y and because industrial uses are expanding, deliberate and accidental intoxication is likely to increase. Rapid diag nosis is essential so that appropriate treatment can be i nstituted quickly. The
authors review the pharmacology, clini cal and laboratory findings, and pathology and pathophysiol ogy of methanol intoxication. In addition, they discuss the differential diagnosis and
treatment of acute intoxicatio n, including the use of 4-methylpyrazole in preventing the conversion of methanol

to
formate. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (320)

Type of experience: other: review, acute toxicity

Remark: A 41-year-old man ingested orally a l arge quantity of
methanol and was found dead at home. The presence of methanol in body fluids and ti ssues was determined by head-space gas chromatography. The blood ethanol and

acetone were negative. Tissue distribu tion of methanol showed that the kidney presented the highe st content of methanol (5.13 g/kg) followed by liver (4.18 g/kg), vitreous humor (3.96 g/l), heart (3.45 g/kg), urine (3.43 g/l), pericardial
fluid (3.29 g/l), blood (2.84 g/l) a nd finally stomach content (2.21 g/l). Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (321)

Type of experience: other: review, acute toxicity

Remark: Methanol ingestion is an uncommon for m of poisoning that

can cause severe metabolic disturb ances, blindness, permanent neurologic dysfunction and dea th. While methanol itself may be harmless, it is converted i n vivo to the highly toxic formic acid. The diagnosis is sometimes elusive and requires a high index of suspicion. Bec ause antidotal treatment is

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233 available it is important to r ecognize methanol poisoning promptly. The presence of meta bolic acidosis associated
with an increased anion gap and inc reased osmol gap are important laboratory findings. Specific therapeutic measures include
correction of the metabolic ac idosis with sodium
bicarbonate
and administration of enteral or parenteral ethanol to competitively inhibit the meta bolic breakdown of methanol

to formic acid. Hemodialysis acce lerates the elimination of
both methanol and formic acid and also assists in
correction of the metabolic acidosis. Exp erimental data suggests that
administration of folic acid m ay be of benefit by hastening the metabolism of formic acid to carbon dioxide. Prompt institution of specific therap y can probably decrease the morbidity and mortality associ ated with this form of poisoning. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (322)

Type of experience: other: review, acute toxicity

Remark: Review of methanol toxicology incl. c omposition, use/sources, acute toxicity, c hronic toxicity, target organ toxicty, absorption-metablosim -excretion, genotoxicty, neurotoxicity, reproductive to xicity, carcinogenicity, epidemiology, environmetnal fa te, environmental toxicity, and regulatory status. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (152)

Type of experience: other: review, acute toxicity

Remark: There have been about one hundred cas es of amblyopia and
death from inhalation of wood alcohol recorded in the literature to 1912. Reliability: (2) valid with restrictions basic data given, acceptable r strictions (323)

Type of experience: other: acute toxicity

Remark: Case report: a 26-year-old woman inge sted 250 to 500 ml methanol during the 38 th week of pregnancy. The initial
serum methanol conc. was 230 m g/dl and formate was 33.6 mg/dl. A mild metabolic acidos is was present. Therapy included ethanol infusion, bic arbonate administration and three courses of hemodialysis. Delievery occurred six days after methanol exposure, when methanol was no longer detected in maternal blood. No further complications were
noted in the mother and her ne wborn. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (324)

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234 Type of experience: other: acute toxicity

Remark: Case report of a 38-year-old male dev eloping Parkinson
disease after methanol intoxic ation. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (325)

Type of experience: other: acute toxicity

Remark: 64 cases of inhalation toxicity were reported. 6 of them died, 16 were permanently blin d, 25 had permanent impairment of vision, and 8 we re temporarily blind. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (326)

Type of experience: other: acute toxicity

Remark: Clinical and pathologic observations in a group of 320 patients with acute methanol p oisoning after ingestion of bootleg whiskey containing 35 % methanoal and 15 % ethanol were reported. Amount ingested ranged from 30 ml to 260 ml
methanol. There were 37 deaths . Visual symptoms, occuring eighteen to forty-eight hours after ingestion, included blurred vision, loss of centra l vision and complete blindness. Acidosis was presen t in every severely poisoned patient. Partial or complete r ecovery of the initially reduced visual acuity was obse rved in most patients. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (327)

Type of experience: other: acute toxicity

Remark: In four of six patients with methanol intoxication necrotic changes in the area of putamin a were seen. Haemorhages were
also seen in some cases. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (328)

Type of experience: other: acute toxicity

Remark: 14 patients whose vision was affected by methanol intoxication were evaluated by a standard electroretinographic technique . Some increase in mean latencies and peak times appea red and the a- and b-waves amplitude to all parameters od stimuli were significantly below normal range. The change s in the ERG indicate damage to the outer retinal layers in cluding visual cells. The
usual ERG observed in methanol poiso ning is unlike that found
with degeneration of the potic nerv e fibre or ganglion cell layer, and is also unlike that found when damage extends to the bipolar layer. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (329)

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235 Type of experience: other: acute toxicity

Remark: A 54-year-old woman developed acute n ecrotizing pancreatitis following acute methanol poiso ning. She died from multiple organ failure 54 hours after t he ingestion. In a series of 22 consecutive patients admitt ed with a diganosis of acute methanol poisoning, evidence o f pancreatic damage was found in 11 patients. Reliability: (2) valid with restrictions basic data, acceptable restric tions (330)

Type of experience: other: acute toxicity

Remark: Occupational methanol poisoning has f requently caused death
or blindness. Several cases re sulted from work in confined spaces, e.g., varnishing beer vats. Headache and blurred vision were reportedly frequen t symptoms. It is believed, that absorption of 8 grams wou ld seriously affect the eyes
and that such a dose could res ult from inhalation of 800 to 1000 ppm for 8 hours. Work roo m concentration of 500 to 600 ppm were found. It is recommen ded to keep the levels below
1

ppm. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (331)

Type of experience: other: acute toxicity

Remark: In a suicidal attempt by a 13-year ol d white girl, methanol (90 - 240 ml of an antifreeze containing 60 % methanol, 39.25 % potassium phosphate) p roduced classical immediate symptoms and permanent damage to the central nervous system characterized by severe bilate ral optic atrophy, rigidity, spasticity, and hypokinesis. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (332)

Type of experience: other: acute toxicity

Remark: In two case reports, the characterist ic clinical, computertomograph-scan and neu ropathological findings in
acute methanol intoxication ar e described. The significance
of acidosis for the survival i s stressed. After a latency period of several days, one pa tient developed the rare condition of haemorhagic leuco cephalopathy with striking computertomograph-scan and mor phologic findings. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (333)

Type of experience: other: acute toxicity

Remark: A case is presented of a consultant s upervising tank cleaning prior to methanol loa ding. He wore positive pressure breathing apparatus b ut no protective clothing.

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236 After 2-3 hours working in the confined space of the tank,
he worked on deck and continue d to wear his methanol-soaked clothing which eventually drie d out. Visual symptoms of acute methanol toxicity presen ted some 8 hours after exposure. The approriate treat ment (with ethanol provided by the ship bond) was carried out in the hospital and the individual recovered completel y. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (334)

Type of experience: other: acute toxicity

Remark: Case report: loss of vision after chr onic inhalation and skin resorption of methanol. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (335)

Type of experience: other: acute toxicity

Remark: The main toxic effects of methanol is exerted upon the nervous system, particularly t he optic nerves and possibly
the retinae. The condition can progress to permanent blindness. Once absorbed metha nol is only very slowly eliminated. Coma resulting fro m massive exposures may last as long as 2-4 days. In the bo dy, the products formed by its oxidation are formaldehyde and formic acid, both of which
are toxic. Because of the slow elimination, methanol should be regarded as cumulative pois on. Though single exposures

to fumes may cause no harmful eff ect, daily exposure may
result in the accumulation of suffici ent methanol in the body to cause illness. Death from inge stion of less than 30 ml has been reported. Human systemic effects: changes in circulation, cough, dyspnea, h eadache, lacrimation, nausea or vomiting, optic nerve neuro pathy, respiratory effects, visual field changes. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (336)

Type of experience: other: acute toxicity

Remark: Cardinal features in teh mechanism, p athology, sympthomatology and therapy of methyl alcohol poioning are reviewed in the light of a ser ies of 23 cases. The first five of these patien ts died of respiratory arrest
after several hours of coma. T hey were sporadic cases who had ingested relatively hugh a mounts of both ethyl and methyl alcohol. The sixth case was brought to the hospital in profound shock and died wit hin four hours. The remaining
17 cases were all victims of t he same batch of liquor that had killed the six patient, an d were treated with alkalies. None died or sustained permane nt injury. The two basic mechanisms of po isoning are direct chemical irritation of tissues and syst emic acidosis. The former is

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237 influenced by the unlimited mi scibility of methanol with
water and the distribution of water in the tissues of the body. Acidosis is the most imp ortant therapeutic
consideration. Alkalinization should be prompt and vigorous. It is suggested that ketosis m ay play a role in acidosis
and depression, and that intraveno us glucose may be an important adjunct to treatment. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (337)

Type of experience: other: acute toxicity

Remark: A series of 49 cases of acute methyl alcohol poisoing with recovery is reported (no demon strable eye lesion after five months). Gastic lavage was not used in any case, because it is of questionable value when poisoning has occurred.
Plasma
extenders were found valuable in combating peripheral vascular failure. Frequent dra inage of the cerebrospinal fluid does not appear essentia l. The occurrence of hypopotassaemia during managem ent was definetely
established. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (338)

Type of experience: other: acute toxicity

Remark: The clincial course of 4 men who dran k commercial duplicating fluid containing 4 0 % to 60 % methyl alcohol is presented. One of the patients died (ingestion of about 12 ounces), and the necropsy find ings were severe, acute passive congestion of the lung s, kidneys, spleen, liver and brain, as well as congestion w ith petechial hemorrhages of the stomach and segments of th e jejumum. Histologic examination was compatible wit h the cross findings. The other men made a complete reco very. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (339)

Type of experience: other: acute toxicity

Remark: Report of a common-source epidemic of methanol poisoning which involved 18 people, of w hom eight died. Of the 18 known cases, 11 were treated w ith only one death despite several patients with methanol concentrations of 200 mg or more per 100 ml blood. Among d ifferent types of treatment used peritoneal dialysis was s hown to be effective, with a
clearance rate five to ten tim es that obtained with forced
fluid diuresis. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (340)

Type of experience: other: acute toxicity

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238 Remark: Case report of a intoxication in a 55 -year-old woman after
administration of methanol-soa ked patches. Initial symtome were blurred vision, decrease of vision, and neuritis of
the optic nerve. Decrease of visio n was permanent. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (341)

Type of experience: other: acute toxicity

Remark: Report of a child who died after meth anol intoxication, presumably due to percutaneous resorption. Methanol soaked pads had been applied to the c hild ́s chest for appr. twice
12 hours. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (342)

Type of experience: other: acute toxicity

Remark: Case report of fuzzy and heavy feelin g in the head and tendency to sway in a worker e ngaged in antimold spraying

to mildewproof a living room. The antimold agent was diluted
with methanol or ethanol. Sinc e the work generally continues for several days, signs and sy mptoms had persisted. However, within 10 days of completing t he work, these signs any symptoms completely disappeare d. Examination of the urine
disclosed that methanol in uri ne was in the normal range

ten days after discontinuation of the work. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (343)

Type of experience: other: acute toxicity

Remark: Case report on a 50-year-old man with methanol conc. of 5 mg/dl after ingestion of alcoh olic beverages. The ethanol level was 275 mg/dl. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (344)

Type of experience: other: acute toxicity

Remark: Methanol poisoning accounting for sev eral deaths were investigated in an Ontario reg istry. Between 1986 to 1991
43 fatalities were found. Suic ide attempts account for 21
cases while 22 were classified as accidental. 14 of these consumed products labelled as methanol or wood alcohol as a substitue for ethanol. 3 cases ingested methanol being improperly stored in container s normally associated with ethanol, and 5 patients were p oisoned through consumption
of liquor from illicit sources.

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239 Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (345)

Type of experience: other: acute toxicity

Remark: A patient poisoned with methanol was successfully hemodialyzed with an ethanol-e nriched, bicarbonate-based dialysate. Along with a concom itant intravenous infusion of ethanol, the ethanol-enriched dialytic procedure was able

to maintain an intradialytic plas ma ethanol level of 80 to 102 mg/dl. The patient recovered w ithout any sequelae of
methanol intoxication. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (346)

Type of experience: other: acute toxicity

Remark: Observations in 323 patients who had ingested bootleg whiskey containing 35-40 % met hanol and 4 % ethanol were reported. 41 deaths occurred. 115 patients showed acidosis
and visual disturbances when f irst seen in the hospital.
Lowest letal dose was 6 ml met hanol and highest dose
survival
was 200 ml methanol. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (347)

Type of experience: other: acute toxicity

Remark: 21 cases of percutaneous methanol int oxication in children were reported. 12 of these chi ldren died. The children ́s
cloths soaked in methanol were applied overnight and during the day with a rate of one to three applications to each child. Th elapsed time between application and onset of symptoms varied from 1 to 13 h and the time till diagnosis
varied from 4 to 48 h. The ear ly manifestations were centralnervous system depressi on and in some cases hyperpnea and in some cases severe respi ratory depression. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (348)

Type of experience: other: acute toxicity

Remark: Report of 54 cases of death and blind ness from cilombian spirits and other methylated p reparations. As to the general effect: headache was mentioned in 19 cases as a conspicous symptom; gastric pain in 11; n ausea and vomiting in 26; dilated pupils in 20. the resu lts as to visual disturbance were 16 total blindness, 3 tot al blindness of one eye, 15 partial recoveries, 7 recoveri es; 10 remaing cases teminted fatally; sight became dim in t hree hours in one case, six hours in 1 case; twelve hours in 2 cases; twenty hours in 1 case; twenty-four hours in 19 cases; forty-eight hours in 5

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240 cases; three days in 2 cases; six days in 1 case; seven days in 1 case; sight became lost i n twelve hours in 2 cases, in twenty-four hours in 10 cases; in thrity hours in 2 cases; in forty-eight hours in 3 case s; in three days in 3 cases; in four days in 3 cases; in fi ve days in 2 cases; in six days in 1 case; in seventeen d ays in 1 case. Of the remaining 21 facts in this con nection are not definite. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (349) (350)

Type of experience: other: acute toxicity

Remark: Significant toxicity can result from intentional methanol inhalation. We report seven ca ses, involving four patients, of intentional inhalation of C ARB-MEDIC carburetor cleaner containing toluene (43.8%), me thanol (23.2%), methylene chloride (20.5%), and propane (12.5%). Patients arrived at the emergency department with central nervous system depression, nausea, vomiting, shortness of breath, photophobia, and/or decreased visual acuity. Treatment
included correction of acidosi s, leucovorin and/or folic acid, ethanol infusions, and s upportive care. Hemodialysis was necessary in three cases. Measured blood methanol
levels ranged from 50.4 to 128.6 mg/d L. Blood formic acid levels were 120, 193, and 480 microgr ams/mL, respectively, in
three patients. Ophthalmic examinati ons revealed hyperemic discs and decreased visual acuity in one patient. One individual was found pulseless with sever al CARB-MEDIC cans nearby. Attempts at revival were unsuc cessful. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (351)

Type of experience: other: acute toxicity

Remark: In a large epidemic it was demonstrat ed that patients acutely intoxicated with metha nol can be triaged effectively
on the basis of bloodmethanol level, serum bicarbonate concentration, and assessment of specific clinical complications. Additional find ings such as elevated MCV and hematocrit may prove useful as markers for nonspecific
toxic swelling of cells. Ehtanol sho uld be included in the treatment of all suspected cas es, using doses that will
maintain a blood ethanol level of 100 mg/dl until methanol levels fall to the range of 20 mg/dl. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (352)

Type of experience: other: acute toxicity

Remark: A 46-year-old male presented to the e mergency room seven hours after consuming a large container of sterno. He could not see and complained of abdo minal and back pain. He was

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241 tachypneic, tachycardic, hyper tensive and hypothermic. Laboratory results were signif icant for a severe metabolic acidosis, a serum osmolality o f 465 and serum methanol
level of 493 mg/dl. Aggressive treat ment included ethanol drip, bicarbonate and hemodialysis. He survived and regained his eyesight in spite of this degr ee of elevation of the serum
methanol level. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (353)

Type of experience: other: acute toxicity

Remark: A 31-year-old previously healthy man presented with a 7-day history of nausea, intractable vomiting, diffuse abodminal pain, and progressive blurred and tunnel vision. The patient
had visual disturbances and an osmolal gap that suggested methanol intoxication. Reliability: (2) valid with restrictions basic data given, accpetable r estrictions (354)

Type of experience: other: acute toxicity

Remark: A 27-year-old man admitted to the med ical intensive care
unit. A coworker found him sta nding in the bathroom, unresponsive and rigid. The pa tient received standard emergency department treatment for the unconscious patient with suspected methanol poison ing with negative results. The, patient had seizure activ ities with dilated pupils, Clenched aw, and tonic muscle actitivity. Deep tendon reflexes wereabsent symetrical ly. Neurological assessment showed a severely depressed le vel of consciousness. Initial
blood levels showed a methanol concentration of 234 mg/dl and an ethanol level of 74 mg/ dl. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (355)

Type of experience: other: acute toxicity

Remark: A 44-year-old man was found unconscio us beneath an elevated rapid transit right-of-way. On admission to the emergency room, the patient was comatose in metabolic acidosis with high anion and osmolal gaps. T he serum methanol was 583 mg/dL. The serum ethanol and e thylene glycol were negative. The patient was treated with e thanol, bicarbonate, and hemodialysis. He expired 40 h after admission. The postmortem methanol concentrat ions in body fluids were as follows: bile 175 mg/dL, vitre ous humor 173 mg/dL, and
blood 142 mg/dL. Urine was not avail able for analysis. Postmortem
methanol concentrations in bod y tissues are given in decreasing order: brain 159 mg /100 g, kidney 130 mg/100 g, lung 127 mg/100 g, spleen 125 mg/100 g, skeletal muscle 112 mg/100 g, pancreas 109 mg/100 g, liver 107 mg/100 g, and heart 93 mg/100 g. The total a mount of methanol in the gastric contents was 73 mg.

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242 Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (356)

Type of experience: other: acute toxicity

Remark: Methyl ethyl ketone is a common solve nt but data on
overdose in humans are scarce. We repor t a case of co-ingestion of methyl ethyl ketone together w ith methanol associated with

a hyperosmolar coma without anio n gap metabolic acidosis. Blood levels of methyl ethyl k etone and its metabolite, 2-butanol, indicated that this solvent did contribute approximately 20 mosm/L to an observed osmolar gap of 99 mosm/L. At the levels detected , methyl ethyl ketone may
have inhibited methanol metabolism, contributing to the low

serum formate (1.3 mmol/L) and norma l anion gap despite a blood methanol of 67 mmol/L. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (357)

Remark: In 10 adult patients acutely intoxica ted with methanol, the
base deficit and the total blo od CO2 were highly correlated
with blood formate level. No c orrelation was found between the same parameters and blood methanol level. Formate production is the main cause o f acidosis in the early stages of methanol poisoning. The ass ociation of a latency period before treatment exceeding 10 hours and a blood formate level above 0.5 g/l (11.1 mmol /l) is predictive of severe methanol poisoning possibly le ading to permanent sequelae. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions
28-DEC-2004 (358)

Type of experience: other: acute toxicity, cns

Remark: Subarachnoid hemorrhage developed in a patient intoxicated with methanol. Computed tomogr aphy performed at the time of admission suggested this compl ication. The hemorrhage was definite and extensive by the 5th day after admission and was accompanied by left caudat e and pontine hemorrhage, as
well as severe cerebral edema. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (359)

Type of experience: other: acute toxicity, cns

Remark: Bilateral putaminal necrosis is chara cteristic of methanol poisoning. A 31-year-old male alcoholic had headache, impaired consciousness, neck s tiffness, roving eyes with dilated unreactive pupils, pap illoedema, abdominal pain, vomiting, and severe metabolic acidosis after a binge. Abnormalities of the cerebrosp inal fluid included an
initial

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243 pressure of 240 mmH2 O, RBC 28 6/mm3, WBC 8/mm3, and protein 179 mg/dl. Peritoneal dialysis was performed on the 2nd day after drinking. A blood test f or methanol was not performed until the 5th day, and its res ults was negative. However, computed tomography (CT) on th e 3rd day showed necrosis and hemorrhage of bilateral putami na and the cerebral cortex, and post-contrast enhancement of meninges. On the 22nd day, a CT revealed further changes: necrosis of bilateral subcortical white matter, and post-contrast gyral enhancement at the otherwise n ormal-looking areas of the
cerebral cortex. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (360)

Type of experience: other: acute toxicity, cns

Remark: A 37-year-old man with a history of a clohol abuse was brought to the emergency depar tment 1 day after having consumed approximately one hal f gallon of windshield washer fluid. The patient was found t o be unresponsive, with shallow respirations. Toxicolo gical screening revealed a blood methanol level of 331 mg /dl (103 mmol/) and an
ethanol level of 0 mg/dl. MR imaging s howed necrosis of the putamen. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (361)

Type of experience: other: acute toxicity, cns

Remark: In two survivors of severe methanol p oisoning apart from
blindness Parkinsonian-like ex tra-pyramidal syndrome characterized by poor voice vo lume, masked face, mild tremor, rigidity, bradykinesia , hyperreflexia, bilateral Babinski responses, and mild d ementia were reported. CT

scan in both patients demonstrated symmetrical low density
lesion in the fronto-central white ma tter and putamen bilaterally
EMG showed extensive denervati on, mainly involving the legs, but normal motor conduction ve locities. Autopsy in one patient who died from suicide 11 months following poisoning refealed cystic resorption of the putamen and the peripheral
white matter in addition to wi despread neuronal damage throughout cerebrum, cerebellu m, brainstem, and spinal
cord. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (362)

Type of experience: other: acute toxicity, cns

Remark: Five optic nerves from three cases of methanol intoxication were examined. The patients di ed in cardiorespiratory failure thirty hours, two days , and eighteen days, respectively, after presentati on with acute bilateral

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244 visual loss. The retrolaminary nerve segments showed circumscribed demyelination of loss of myeli n staining with relative preservation of axons. Retinal ganglion cells were spared. Electromicroscopy in the case with shortest survival revealed periaxonal intramyeli nic spaces behind the lamina
cribosa. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (363)

Type of experience: other: acute toxicity, cns

Remark: The histopathologic effects of methan ol on the optic nerve were studied in four patients. Circumscribed myelin damage
occurred behind the lamina cri brosa in each nerve. Axons were preserved. Demyelination also occurred in cerebral hemispheric white matter in on e patient. This selective myelinoclastic effect of metha nol metabolism is probably caused by histotoxic anoxia in watershed areas of the cerebral and distal optic nerv e circulations. Juxtabulbar demyelination may cause optic disk edema in methanol poisoning by compressive obstr uction of orthograde axoplasmic flow. Visual loss m ay be due to disruption of saltatory conduction. Retrolam inar demyelinating optic neuropathy is an early morphol ogic correlate of visual loss
in methanol intoxication. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (364)

Type of experience: other: acute toxicity, cns

Remark: Case report of a 40-year-old woman wi th putaminal necrosis
after methanol intoxication. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (365)

Type of experience: other: acute toxicity, cns

Remark: Case report of a 62-year-old plumber with putamen necrosis
after severe methanol intoxica tion. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (366)

Type of experience: other: acute toxicity, cns

Remark: Case report of a case of acute methan ol intoxication with central nervous system damage producing a Parkinson syndrome with persitent sequel. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (367)

Type of experience: other: acute toxicity, cns

Remark: Report of with areas of decreased att enuation symmetrically

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245 localised, especially in the p utamen in the computerised tomography in a 62-year-old pl umber after intake of methanol-containing home-made liquor. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (368)

Type of experience: other: review, therapy

Remark: Review on the roles of alkaline salts and ethyl alcohol in the treatment of methanol pois oning. Reliability: (2) valid with restrictions
see remark on selection of lit erature (369)

Type of experience: other: review, therapy

Remark: Treatment of methanol poisoning is we ll established and consists specifically of the a dministration of alkali and ethanol, and hemodialysis. Sin ce methanol poisoning often
occur as accidents with a lot of patients involved, it is not always possible to follow sophiticated protocols for treatment. If the patients are awake with no severe hyperventilation or acidosis, ethanol adminstration is most important because it is widely available and can be given orally. Under such cicrumstanc es every person suspected of having consumed some of the su spect liquid should be given ethanol orally or intravenousl y aiming at a blood
concentration of about 22 mmol /l. In severely poisoned patients in coma with pronounc ed acidosis and hyperventilation it is vital t o correct the acidosis as

soon as possible. Before any labora tory analyses are available,
at least 200 to 300 mmol of bi carbonate should be infused intravenously, followed by eth anol infusion. If methods of
determination of methanol or f ormate are not available, the magnitude of the osmolal and a nion gaps along with the clinical judgement should prov ide the basis for treatment. Reliability: (2) valid with restrictions
see remark on selectionof lite rature (370)

Type of experience: other: review, therapy

Remark: Twelve cases of methanol poisoning ar e reviewed. The clinical presentation and bioc hemical features are
described
and the results of treatment w ith alkali, ethanol and dialysis reported. The outcome of methanol poisoning

appears to be related more to the inte rval between the time of ingestion and the start of the rapy and to the degree of acidosis than to the initial s erum methanol level. Therefore, early and aggressiv e treatment with bicarbonate and ethanol and subsequent ins titution of hemodialysis are strongly recommended whenever methanol can be detected in the blood, especially when met abolic acidosis of the anion-gap type is present, whe n mental or visual disturbances are present, or w hen more than 30 ml of
absolute methanol has been con sumed.

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246 Reliability: (2) valid with restrictions
see remark on selection of lit erature (371)

Type of experience: other: review, therapy

Remark: A unique opportunity presented itself for reviewing and comparing the results of two d ifferent methods of dialytic therapy for the treatment of m ethanol poisoning. The group
that was treated with hemodial ysis initially had a faster fall in serum methanol level, regained consciousness faster, had a shorter hospital stay, a nd most importantly, had no residual effects when compared with the group treated with peritoneal dialysis. In this g roup, one patient died and another was permanently blinde d. Reliability: (2) valid with restrictions
see remark on selection of lit erature (372)

Type of experience: other: therapy

Remark: A case report of severe methanol pois oning is reported
which required 21 hours of hemodialy sis to bring their serum
methanol levels down to a nont oxic level. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (373)

Type of experience: other: therapy

Remark: Nine patients with methyl alcohol poi soning who were
treated with hemodialysis were describ ed. The time from ingestion

to dialysis varied from 4 to 100 hours. Predialysis blood methanol levels ranged from 3 to 570 mg/dl. All patients
were acidotic and had an incre ased anion gap. Two patients died, seven recovered, but three had permanent visual impairment.
There was little correlation b etween the blood methanol level or anion gap and visual outcome. The interval from ingestion to treatment appears to be more important than
the
initial biochemical status. Pr ompt hemodialysis is recommended, if the blood meth anol level is above 50 mg/dl,
when an amount of methanol exc eeding the minimal lethal
dose (30 ml) is known to have been ingested, when there is
evidence of acidosis or when a n abnormality has developed
in vision, funduscopic examinatio n or mental state. Concurrent therapy with alkali and ethano l is vital. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (374)

Type of experience: other: therapy

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247 Remark: The diagnostic value of determination of the anion and osmolal gaps was studied in 6 patients poisoned with methanol and in 5 poisoned wit h ethylene glycol. Increased osmolal gap was present on adm ission in all patients, whereas increased anion gap wa s present in all except one. In the methanol-poisoned patie nts the mean blood values were: pH 7.27, anion gap 24 mm ol/l, osmolal gap 81 mosmol/kg H2O, methanol 67 mmol/l, ethan ol 11 mmol/l and in the ethylene glycol-poisoned patie nts: pH 6.93, anion gap 38 mmol/l, osmolal gap 35 mosmol/ kg H2O and ethylene glycol 24 mmol/l. In the absence of alco holic acidosis or diabetic coma the finding of a simultan eous increase in both the anion and osmolal gaps indicat es methanol or ethylene glycol poisoning. Thus determinations of the anion and osmolal

gaps are mandatory whenever facing metabolic acidosis of unknown etiology. Reliability: (2) valid with restrictions acceptable study meets basic s cientific principles (375)

Type of experience: other: therapy

Remark: 4-Methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase, may be useful f or the treatment of methanol and ethylene glycol intoxicati ons. A placebo-controlled, double blind, multiple dose, s equential, ascending-dose study has been performed to de termine the tolerance of 4-MP in healthy volunteers. Oral lo ading doses of 4-MP were followed by supplemental doses every 12 h through 5 days, producing plasma levels in the therapeutic range. A slight,
transient elevation in one or both serum transaminase
values was observed in 6 of the 15 su bjects treated with 4-MP.
This
effect was not dose related no r apparently mediated through a hypersensitivity reaction. S erum triglyceride levels were
increased in 30% of 4-MP treat ed subjects, but also in 25% of the placebo subjects. 4-MP treatment did not produce any other significant changes in o bjective clinical parameters nor in subjective side effects . The results suggest that a mild, transient increase in li ver function tests might be observed in some subjects trea ted with multiple doses of 4-MP. Nevertheless, the slower elimination rate and lesser degree of toxicity of 4-MP wou ld make it preferable to ethanol in therapy of these po isonings. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (376)

Type of experience: other: therapy

Remark: 1. Osmolal and anion gaps are helpful in the diagnosis and
evaluation of intoxications wi th methanol and ethylene glycol. Reported reference val ues for osmolal gap and anion gap are -1 (+/- 6) mosm kg-1 H 2O and 16 (+/- 2) mmol l-1, respectively. However, we have repeatedly found unexplained increased gaps in patients adm itted to our department, and

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248 the relevance of the establish ed reference values has been questioned. 2. Osmolal and ani on gaps were determined in an unselected population of patie nts consecutively admitted to an emergency medical departmen t. In the case of unexplained gaps, the blood samples were a nalysed with respect to the presence of alcohols and orga nic acids. 3. We included all accessible patients admitted d uring 14 days. Appropriate blood samples were obtained in 177 patients (88 male, 89 female), with a mean age of 65 years (range 17-94). 4. The mean and (standard deviation) for osmolal and anion gaps in our material were 5.2 mosm kg- 1 H2O (7.0) and 12.9 mmol/l (4.2). Neither methanol nor et hylene-glycol was detected in serum from any patients. Small amounts of ethanol were
found in 5 patients, and high lactat e levels explained in part
the most extensively increased ani on gaps. However, the calculated analytical standard deviation accounted entirely
for the variation in the mater ial. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (377)

Type of experience: other: therapy

Remark: A patient intentionally ingested an u nknown amount of
methanol and was admitted to t he hospital 6 h later. On admission, the methanol concen tration in blood was
estimated as approximately of 134 mmol/L , based on the calculation of the osmolal gap. Intravenous e thanol administration and hemodialysis were promptly sta rted. During hemodialysis, several blood samples were col lected for determination of
methanol and ethanol concentra tions. Initially, we used gas chromatography with split-mode injection of pretreated

serum samples; however, methanol con centrations turned out to be significantly lower than expec ted, based on calculated osmolal gap values. Because no explanation for the excess serum osmolal gap was apparent , we reanalyzed samples, using head-space gas chromatography. The methanol concentrations measured were significantly hi gher and osmolal gap values were no longer excessive. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (378)

Type of experience: other: therapy

Remark: 4-Methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase, may be useful f or the treatment of methanol and ethylene glycol intoxicati ons. A placebo-controlled, double blind, multiple dose, s equential, ascending-dose study has been performed to determin e the tolerance of 4-MP in healthy volunteers. Oral loadi ng doses of 4-MP were
followed by supplemental doses every 12 h through 5 days, producing plasma levels in the therapeut ic range. A slight, transient

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249 elevation in one or both serum transaminase values was observed in 6 of the 15 subjec ts treated with 4-MP. This
effect was not dose related no r apparently mediated through a hypersensitivity reaction. S erum triglyceride levels were
increased in 30% of 4-MP treat ed subjects, but also in 25% of the placebo subjects. 4-MP treatment did not produce any other significant changes in o bjective clinical parameters nor in subjective side effects . The results suggest that a mild, transient increase in li ver function tests might be observed in some subjects trea ted with multiple doses of 4-MP. Nevertheless, the slower elimination rate and lesser degree of toxicity of 4-MP wou ld make it preferable to ethanol in therapy of these po isonings. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (376)

Type of experience: other: irritation

Remark: In an experimental study, 12 voluntee rs were exposed in an
inhalation chamber to 20 and 2 00 ppm methanol. At 200 ppm (20 ppm) 5 (3) volunteers repo rted slight irritation of the nose and 6 (3) of the throat. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (379)

Type of experience: other: irritation

Remark: From 1989 - 1993 two workers with loc al skin- and eye irritation, as well as malaise , after accidental local and inhalative exposure to methano l were send to the local clinic for further treatment. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (380)

Type of experience: other: irritation

Remark: Local eye contact with liquid methano l resulted in severe
chemosis and lesions of the co rnea, which however receded
in a few days when properly treat ed. Reliability: (2) valid with restrictions acceptable study, meets basic scientific principles (381)

Type of experience: other: sensitisation, skin
Remark: In four patients with eczema positive patch-test reactions to methanol (10 %) were descri bed. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (382)

Type of experience: other: review, chronic toxicity

Remark: Cases of chronic poisoning from repea ted exposure to
methanol vapor were manifested by conju nctivitis, headache, giddiness, insomnia, gastric d isturbance, and bilateral

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250 blindness. In one fatal case o f occupational methanol intoxication by inhalation a f emale worker was exposed
about 12 hours. A postevent study of the process evealed methanol vapor concentrations ranging f rom 4,000 to 13,000 ppm. Reliability: (2) valid with restrictions
see remark on selection of lit erature (383)

Type of experience: other: chronic toxicity

Remark: A case of chronic methanol poisoning, with marked
diminution of vision, resulted apparently from the exposure at 1200 to 8000 ppm for 4 years. Also rep orts of headaches among workers exposed at 300 ppm dur ing operation of duplicating machines were recorded. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (384)

Type of experience: other: chronic toxicity

Remark: A case of chronic poisoning from meth anol vapor with marked
diminution of vision and enlar gement of the liver was reported. The employee was eng aged in centrifuging material
which contained 35 to 40 % met hanol. Although he had been employed in this occupation fo r four years, his symptoms
did not develop until blackout con ditions became necessary with
a resultant decrease in ventil ation. Air analysis at the work place revealed levels fro m 1.6 to 10.9 mg/l (appr. 1,200 to 8,300 ppm). Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (385)

Type of experience: other: chronic toxicity

Remark: A study of the wood heel industry in Massachusetts found average methanol vapour concen trations ranging from 160 to 780 ppm, with no definitive ev idence of injury to the exposed workers. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (386)

Type of experience: other: chronic toxicity

Remark: A report indicated severe recurrent h eadaches in workers exposed to methanol in concent rations between 200 to 375

ppm. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (387)

Type of experience: other: chronic toxicity

Remark: From 1978 - 1982 only one new case of methanol-induced occupational disease was compe nsated in Germany.

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251 Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (388)

Type of experience: other: chronic toxicity

Remark: Nineteen workers exposed to the fumes of acetone and methanol in a collar fusing pl ant were studied. Physical
examinations revealed no abnor malities. Blood studies
revealed no disturbances in he matopoesis. Absorption of acetone was demonstrated by fi nding this substance in the urine of every member of the g roup. The concentration of
solvents in the workroom atmos phere (methanol: 22 and 25 ppm, acetone: 45 and 40 ppm) w as apparently not high enough to cause pathological changes. Reliability: (2) valid with restrictions basic data given, acceptable r estrictions (389)

Type of experience: other: carcinogenicity

Remark: No studies have been reported in the literature on the carcinogenicity of methanol in humans. Reliability: (2) valid with restrictions
see remark on selection of lit erature (88)

Type of experience: other: review, reproductive and developmental eff ects

Remark: The Panel reviewed four epidemiologic al studies that examined folate supplementatio n and cleft palate (Peer et al, 1964, Tolarova and Harris, 1995, Shaw et al, 1995, and Cziezel and Dudas, 1992). The Panel concluded that in the aggregate the studies suggest a reduced risk of oral clefts following supplementation with vitamins containing folic acid. The epidemiological find ings weakly support the hypothetical mechanism of acti on that methanol reduction of folate activity in the rodent causes oral cleft defects. However, that extrapolation is difficult due to the various differences, including differe nt folate levels between
rodents and humans.

No human data on the reproduct ive toxicity of methanol have
been located. Reliability: (2) valid with restrictions
see remark on selection of lit erature (233)

Type of experience: other: review, reproductive and developmental eff ects

Remark: No studies have been reported in the peer-reviewed literature on the reproductive and developmental effects of methanol in humans. Reliability: (2) valid with restrictions
see remark on selection of lit erature (88)

5.11 Additional Remarks

Type: other: review

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252 Remark: A. The toxic effects of methanol in h umans and their implications are reviewed. The highest estimated exposure to methanol when used as fuel (EPA 1988) is up to 650 mg/m3.

Acute toxicity in humans affects, as previously reported, the central nervous system and pr imarily the optic nerve.

The background level of methanol in blood was measured as 0.73 mg/l (0.32 - 2.61 mg/l) [Sed ivec et al. 1981: 31 persons) and the background level of formate, the major metabolite, was measured between 3 and 19 mg/l [0.07 - 0.4 mM].

Based on these values a backgroun d body burden of 0.5 mg/kg body weight was estimated and at worst case exposure scenarios additional body burdens between 0.03 and 1.1 mg/kg body weight were calculated (Tab. 6, p. 34).

The clearance of methanol from th e body was calculated as Vmax 48 mg/kg/h in primates and 3 0 mg/kg/h in rats.

Metabolism in humans contributed to 96.9 % of the clearance, whereas renal and pulmonary excre tion contributed only to 0.6 and 2.5 % respectively. Over 90 % of the dose was exhaled as CO2. The half-time was approx. 2.5 - 3 hours.

The contribution of formate to me thanol toxicity is not clear according to the author.

According to the data reviewed no toxic effects of methanol are expected when the substance i s used as fuel even at worst case exposure scenarios.

----------------- B. The minimal lethal dose to h umans is considered to range from 0.3 to 1 g/(kg*d). A blood level of 500 mg/l in acutely poisoned patients general ly is regarded as requiring hemodialysis. This concentration can be achieved by ingestion of 0.4 ml methanol/kg, which is only 28 ml for an adult person (70 kg), but just 4 to 6 ml for small children up to one year old.

Acute methanol intoxication (afte r single dose) evolves in a well-defined pattern: mild depres sion of the CNS, an asymptomatic latent period for se veral hours to >=2 days (most common 12 - 14 hours). The toxicity syndrome that emerge s consists of uncompensated metabolic acidosis with superimpo sed toxicity to the visual system. Clinical symptoms include headache, dizziness, nausea, and vomiting, abdominal p ain, and difficult, periodic breathing (Kussmaul brea thing) which may progress to coma and death from respirator y failure.

In parallel, visual disturbance t o total blindness may

occur.

Ophthalmologic examination would reveal hyperemia of the optic disc, followed by the appea rance of oedema projecting into the surrounding of the retin a from the optic disc,

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253 while the papilla itself is not o edematous.

The severity of retinal oedema is predictive of restoration of vision. Pallor of the optic di sc is an end-stage sign of
irreversible effects of the visua l system and may appear one
to two months after an acute meth anol dosage (or possibly chronic exposure).

Post-mortem signs of damage were observed in the basal ganglia in the brain, specificall y the putamen (which area participates in the control of mo tor activities). Survivors
of severe methanol intoxication m ay continue suffering from residual lesions of the putamen.

----------------------- Test substance: Methanol, no further data. Reliability: (2) valid with restrictions

(192)

Type: other: reviews

(390) (88) (192) (233)

Type: other: Ocular toxicity / folate / rat

Remark: 1. Changes in the electroretinogram (ERG) and the potassium-induced Müller cell dep olarization (KIMD) of
folate-reduced rats have been obs erved after oral methanol administration of 1.5 - 2.5 g/kg whereby formate in blood increased, concentrations ranging from approx. 6 to 15 mM at 48 h (Lee et al., 1994). Moreover , methanol induces a decrease in retinal ATP, which su ggests interference with oxidative phosphorylation. Format e is known as inhibitor of mitochondrial cytochrome oxidase in Müller cells (supporting glial cells).

No effects were noted at 1 g meth anol/kg per os despite folate deprivation. No adverse ef fects are produced by doses of up to 3.5 g/kg in rats maintai ned on normal diet (Garner et al. 1995b).

2. However, creating a blood formate profile by infusion
of formate similar to that observ ed after administration of
a retinotoxic methanol dose faile d to alter the ERG. Retinal
levels of formate after infusion were only 16 % of those measured after methanol administr ation (Garner et al., 1995. p. 105). 3. Intraretinal metabolism of methanol to formate is necessary for the initiation of m ethanol-induced retinal toxicity. Retinal and vitreous hu mor formate levels are approx. 15.3 umol/g and 19.2 mM, resp., after a single oral methanol dose of 3.0 g/kg bw to f olate-reduced rats. The enzymes responsible for methanol metabolism, catalase and aldhyde dehydrogenase, are both p resent in Müller cells.

4. The acute effect of methan ol on the ERG involves a severe decrease in the b-wave amp litude at approx. 48 h following a single dose and has b een observed in humans, in primates and in folate-reduced ra ts (Garner et al., 1995, p.

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254 101). The b-wave is generated by the Müller cell, the primary glial cell, in response t o an increase in extracellular potassium released by the bipolar cells. Exactly parallel, a dose-related evocation loss of the potassium-induced Müller-cell dep olarisation is noted.

5. The electrophysiological a s well as histopathological
effects seen after methanol treat ment correlate closely to those induced by alpha-amino adip ic acid, a specific
Müller-cell toxin.

-------------------------- Conclusion: The inhibition of cytochrome oxidase in Müll er-cell mitochondria by intracellularly g enerated formate may explain the mechanism of methanol -induced retinal toxicity.

------------------ Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

(391) (392) (393)

Type: other: Absorption / excretion / inhala tion / human

Method: Methanol absorption after inhalative exp osure and excretion in urine was measured in groups o f 4 - 5 human volunteers to be able to extrapolate exposure i n the working environment
from excreted urine levels. Exposure concentrations were appr ox. 100 to 300 mg/m3.

Retention of methanol via lung wa s determined from the
concentration difference between inspired and expired air. Methanol in urine (8-h shift) ser ved as a quantitative
measure for retained methanol.
Blood levels were not measured.

------------ Result: The mean normal urine level was 0.73 mg/ l (range from 0.32 - 2.61 mg/l), data selected from a control group of 31
individuals.

A mean of 57.7 % of the inhaled d ose was retained, range 53.4 - 61.3 %, irrespective of th e exposure level of selected magnitude.

Urine excretion represented nearl y 1 % of the retained dose
at a normal diuresis.

Average urine concentrations reac hed a peak after 8 h (Fig. 3) and were fairly proportional t o the exposure levels (approx. 3.3, 7.0, and 9.5 mg/l). After 18 - 24 h from the start of exposure (about >= 12 h after termination), urine methanol has approached baseline level again.

The excretion half-life was about 1.5 to 2 h.

In cross-over drinking experiment s it could be shown that the urinary methanol excretion co rrelated strictly with diuresis, i.e. irrespective of th e urine volume produced at the same exposure level, the urin e concentrations were identical and were dependent onl y on the exposure level.

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255 This suggests that methanol distr ibutes only passively into
the urine in relation to the bloo d level.

This also implies that the total quantity excreted into the
urine cannot be the criterion for the exposure level, but only its concentration.

The mean equation of regression ( Fig. 5) between retained methanol quantitity (body burden) [X in mg] and the whole-shift urine concentration [ Y in mg/l] could be
formulated as

Y = 0.7470 + 0.00763X

-------------- Test substance: Methanol, no further data. Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

(283)

Type: other: Absorption / distribution / rat , mouse

Remark: In mice exposed to methanol in air at co ncentrations of 2500 to 10000 ppm (3.3 - 13 mg/l) absorbed the substance rapidly. Blood methanol concentra tions were approximately 1000 mg/l at 1 hour and 3000 - 40 00 mg/l at 7 hours

exposure.

The fractional absorption was hig her in mice (approx. 85 %) than in rats (56 - 87 %) (p. 251)

.

Comparison of rat and mouse:
The same environmental methanol v apour concentration leads to very different blood methanol level and is in mouse higher although the maximum elimi nation rate is about twice as high in mice as in rat (Pollac k and Brouwer 1996):

At the teratogenic exposure conce ntration in mice of 5000 ml/m3, the following blood levels would be achieved

mouse: 2313 +-338 mg/l(exposed as group); rat: 1047 +-298 mg/l.

In humans: 224 mg/l.

----------------- Test substance: Methanol, no further data. Conclusion: The same environmental methanol vapour conce ntration leads to very different blood methanol level. Therefore, risk assessment for me thanol should be based on blood concentrations following in halation exposure, not on exposure concentrations itself.

The differences between the terat ogenic potential of methanol in rats versus mice, wit h mice being approximately
twofold more sensitive than rats may be due to the differences in blood concentratio ns.

------------------ Reliability: (2) valid with restrictions

### OECD SIDS METHANOL

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256 (394) (395)

Type: other: Toxicokinetics / formate / HEI study monkeys

Method: Comparative study designed to clarify me thanol and formate kinetics in blood at low and high exposure concentrations to methanol in rats and monkeys with extrapolation to humans (Horton et al. 1992):

Animals: Male F344 rats (190 - 2 30 g) and young adult male Rhesus monkeys (Macaca mulatta), 4 rats and 3 monkeys per test group. Inhalation exposure: single for 6 h, but in 3 monkeys also repeated inhalation (2000 ppm, 6 h/d, 5x/wk, 14 d) (not documented: see Horton et al., Di scussion, p.33)

Test concentrations of methanol: nominal 50, 200, 1200, and 2000 ppm (analytical values were close to nominal).

Analytics chamber: continuously b y IR gas analyzer for
methanol.

Analytics blood: for methanol GC, for formate enzymatically coupled to a fluorescence measure ment of a reactant (NADH2).

Sampling up to 12 h post-exposure

.

Blood gas analysis for pH, bicarb onate, and pCO2 was performed.

---------------------- Result: Kinetic key data: Generally, the elimination rate f rom blood decreased with increasing concentrations, in ra ts only at the highest dose significant. At the lowest concen tration, 200 ppm, the elimination was only slightly low er in monkeys than in rats (approx. -30 %), but was distinc tly slower at the higher burdens. No significant increases in blood values above background were evident at 50 ppm methanol.

T1/2[h]

Rat Monkey

--------------------------------- ------------ 200 ppm 0.8 +-0.2 1 .1 +-0.2 1200 ppm 1.0 +-0.1 3 .2 +-1.2 2000 ppm 2.1 +-0.1 2 .9 +-0.6

--------------------------------- ------------

Blood levels after 6-h exposure [ ug/ml]:

Rat Monkey

--------------------------------- ------------ Methanol: 200 ppm 3.1 +-0.4 3.9 +-1.0 1200 ppm 26.6 +-2.0 3 7.6 +-8.5 2000 ppm 79.7 +-6.1 6 4.4 +-10.7

--------------------------------- ------------

Formate: The peak blood levels in methanol-exposed rats and

### OECD SIDS METHANOL

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257 monkeys ranged between 5.4 and 13 .2 ug/ml (approx. 0.25 - 0.6 mM) without showing statisti cally significant differences to control animals [n ote: Data are not documented in Horton et al., 1992 : However, Medinsky and Dorman (1995) present data in Fig . 1 attributed to the studies by Horton et al. (1992).] Blood acidosis: no signs of acido sis which is in line with
the formate blood levels in norma l range.

--------------------------------- ----

Repeated inhalation in monkeys (d ata not shown: see Horton et al., 1992, Discussion, p. 33), carried out as confirmatory test to examine accu mulation toxicology: In blood samples collected after 1 and 2 weeks of exposure, no accumulation of methanol or fo rmate.

--------------------------------- -----

Prediction of in-vivo time-course data by the PBPK model: Below 1500 mg/m3 (1200 ppm), all three species (rat, monkey, human) would exhibit similar post -exposure blood levels of methanol which would be proportio nal to atmospheric concentrations. Above 1200 ppm, i ncrease in blood would
become non-linear for rat and mon key, but remain linear for
human.

------------------ Test substance: Methanol purity >98%
Conclusion: Comparison of monkey blood-methanol eliminat ion rate
constant with those derived for h umans (HEI, 1987) and rats shows an interspecies similarity in the overall elimination of methanol after low doses (Hort on et al., 1992, Discussion).

The dose of methanol required to saturate the folate-dependent metabolism of fo rmate, calculated from an equation derived by the Health Ef fects Institute (HEI, 1987), is about 250 mg/kg. If one assumes a 6-kg monkey breathes 118.8 l/h and 100% absor ption of methanol occurs, a dose equivalent to 308 mg/kg is a bsorbed during a 6-h exposure to 2000 ppm methanol(260 0 mg/m3).

While this amount of methanol cou ld overwhelm the formate metabolism pathway if given as a bolus, the saturation of
metabolism with this amount divid ed over 6 h is unlikely (Horton et al., 1992, Discussion)

.

------------------------------ ------ Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

(396) (397) (252)

Type: other: Absorption / inhalation / human

Method: Six human volunteers (from 29 - 55 years ) were subjected to a controled diet-regimen (without obvious methanol-delivering nutrition and additives) throughout the study and exposed to 200 ppm (260 mg/m3) methanol for 6
hours. Five individuals were each tested at rest or at light
exercise.

### OECD SIDS METHANOL

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258 ------------ Remark: Blood methanol concentrations increased from 1.8 ug/ml (mean endogenous level) to 7.0 ug/ml at rest and to 8.1 ug/ml under light exercise (increase in mean pulmonary ventilation
at a factor of about 2.7 from ave rage 10.5 to 26.6 L/min and increase in respiratory rate at a factor of about 1.7 from 11.2 to 18.6 breathes/min).

Blood formate levels did not incr ease.

Blood levels at rest (from Tab. I II and V):

Test Methanol[ug/ml] Formate [ug/ml] person pre- post- pre- post-

exposure exposure

--------------------------------- ----------------- 1 2.53 5.44 8.11 8.12
2 3.57 7.55 1 0.57 13.06
3 1.32 7.93 1 0.35 7.24
4 0.57 8.08 9.08 8.98
5 1.12 5.83 7.31 6.12

--------------------------------- ----------------- Mean 1.82 6.97 9.08 8.70
+-SD +-1.21 +-1.24 +-1.26 +-2.38

--------------------------------- -----------------

Blood levels at exercise (from Ta b. IV and VI):

Test Methanol[ug/ml] Formate [ug/ml] person pre- post- pre- post-

exposure exposure

--------------------------------- ----------------- 1 1.99 9.82 5.36 8.90
2 2.00 7.40 9.28 11.14
3 3.15 7.47 9.35 9.54
4 0.54 6.40 9.07 9.91
5 1.95 9.57 1 0.83 8.10

--------------------------------- ----------------- Mean 1.93 8.13 8.78 9.52 +-SD +-0.93 +-1.49 +-1.82 +-1.02

--------------------------------- -----------------

The results show that no formate mediated toxicity is expected when workers are expo sed to 200 ppm methanol, the current Occupational Safety a nd Health Administration 8-hr time-weighted average permis sible exposure limit. Test substance: Methanol, no further data. Conclusion: At 200 ppm methanol or below, methanol blood levels may
increase 3.5 to 4-fold above the endogenous methanol concentration in human blood, whi le the formate level remains unchanged, irrespective o f physical activity whether at rest or light exercise. This was confirmed e.g. by D ́Ales sandro et al. (1994) in similar experiments on human volu nteers (comp. also Medinsky and Dorman, 1995).

--------------------- Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted

### OECD SIDS METHANOL

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259 scientific principles, acceptable for assessment.

(398) (196) (252)

Type: other: Risk Assessment Model / inhalat ion

Method: A "multicompartment biologically based d ynamic" inhalation
model based on kinetic methanol i nhalation data from rats (Horton et al., 1992), monkeys (D orman et al., 1994) and humans (Sedivec et al., 1981; Ost erloh et al., 1996; Batterman et al., 1998) was devel oped to describe the time
evolution of methanol and its met abolites in the whole body and in accessible biological matr ices.

--------------- Result: Predictions from simulations (PBPK model ling) of continuous inhalation of 200 ppm methanol in humans for 5 days (Bouchard et al. 2001, p. 177/178 ):

Based on the following assumption s: - a negligible background burden of methanol, - an absorption fraction of 0.57 7 (Sedivec et al. 1981), - a pulmonary ventilation rate o f 10.8 L/min (Sedivec et al. 1981; Batterman et al. 1 998), - an apparent distribution volum e for methanol of 0.7 l/kg (corresponding to human bod y fluid), - an apparent distribution volum e for formate of 4.6 l/kg (estimations by Bouchard et a l. 2001, Tab. 2, p. 173 and p. 180), - a daily urine volume of 1.5 l ,

near steady state will be reached in 20 h. After 5 d,
methanol in blood and urine is es timated at 5.5 mg/l (171 umol/l) and 8.1 mg/l (252 umol/l) ; formate in blood and urine is 1.16 mg/l (3.5 mol/l) an d 1.5 mg/l (31.7 umol/l = 0.97 mg/g creatinine or 2390 umol /mol creatinine).

This shows that exposure concentr ations of <500 ppm are not
sufficient to raise formate level s significantly, while
methanol increases.

The model, adapted to kinetic dat a in humans exposed acutely to methanol, predicts that 8-h in halation exposures ranging from 500 to 2000 ppm, without phy sical activities, are
needed to increase concentrations of blood formate and urinary formic acid above reporte d background values (4.9-10.3 and 6.3-13 mg/l, resp.)

.

Therefore, acc. to authors, blood and urinary methanol
levels are the most sensitive bio markers of absorbed
methanol.

---------------------

Pulmonary retention: Using the ex perimental human data of Osterloh et al. (1996), Sedivec e t al. (1981) and Batterman et al. (1998), the best fit in th e model for the average absorption fraction was higher th an that given by Sedivec et al. (1981), namely about 80 % and corresponded to the retention of 79 % given by Batter man et al. 1998 (Bouchard et al., 2001, p. 177).

### OECD SIDS METHANOL

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260 ----------------- Reliability: (2) valid with restrictions

(299) (399) (251) (397) (277) (283)

Type: other: Absorption / distribution / inh alation / rat

Method: Whole-body exposure system:

1. Adult female Long-Evans rats (n = 12) were exposed to 4500 ppm methanol for 3 consecuti ve days once for 6 h. Blood
methanol levels were measured 1 a nd 6 h during and 1-h post exposure as wel as background lev els before each exposure.

2. Adult female Long-Evans rats (n = 12) were exposed to 4500 ppm methanol during pregnanc y and lactation along with the progeny (gd 6 through post-na tal day[PND] 21).

(see also other entry under 5.8.2 )

-------------------------- Result: exposure time 1 h 6 h 1 h post exp. [in approx. averag es mg methanol/ml]

--------------------------------- -------------------- day 1 0.25 0.90 0.95 day 2 0.40 0.60 0.60 day 3 0.30 0.50 0.60

--------------------------------- --------------------

After the first day, the levels w ere higher which can be explained with adaptation to the new environment (higher activity). The pre-exposure level s (1 h before first exposure and 17 h after each prev ious exposure interval)
were non-detectable.

During pregnancy and lactation, b lood levels in dams were in the range of 0.50 +-0.07 to 0.55 +-0.09 mg/ml. But in offsprings, the level was about 2 x that of dams, at 1.26 +-0.23 mg/ml, and gradually decre ased to that of the dams
after 52 PND.

---------------------- Test substance: Methanol, HPLC grade Reliability: (1) valid without restriction 1c: Test procedures in accordance with accepted standard methods, well documented

(241)

Type: other: Absorption / distribution / ora l / rat

Result: After oral administration of 100 and 250 0 mg methanol/kg to female SD rats, gastrointestinal absorption was 100 % within minutes (abs. half-life 1.5 min a nd 7.6 min, respectively) (p. 16).

The maximum elimination rate is a bout twice as high in mice
as in rat: 117. +-3 and 60.7 +-1. 4 mg/(kg*h) (Pollack and Brouwer 1996, Tab. 7, p. 21; Ward et al. 1995).

### OECD SIDS METHANOL

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261 After inhalation, the mean fracti onal respiratory absorption
of methanol in rats and mice was found to about 85 %, respectively, at exposure concent rations from 1000 to 5000 ml/m3, and tended to be lower (60 - 70 %) at 10000 - 20000 ml/m3 in rats, but not in mice (P ollack and Brouwer 1996, Tab. 12, p. 27; Tab. 15, p. 33).

Blood levels in rats were about 8 00 (calc.) - 1200 mg/l (measured) at 5000 ml/m3 (8 h) (F ig. 37, p. 34).

Blood levels in mice were about 3 000 mg/l at 5000 ml/m3(8 h) (Fig. 37, p. 34).

------------------- Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

(395) (400)

Type: other: Toxicokinetics / formate / fola te deficiency / HEI study monkeys

Method: Pharmacokinetic study to evaluate the co ntribution of
methanol-derived formate to the e ndogenous formate pool in nonprimates under folate-sufficie nt and folate-deficient condition (Medinsky et al., 1995, 1997):

Four 12 year old female cynomolgu s monkeys (Macaca fascicularis) were used: lung-onl y exposure under isoflurane-anaesthesia. Exposure time = 2 h.
14C-radiolabelled methanol was us ed as atmospheric tracer
material.

Exposure concentrations/situation : nominal 10, 45, 200, and 900 ppm (normal folate) and 900 p pm (folate-deficient) in the every same monkeys two months later after folate-deficient diet. Measured c oncentrations corresponded
well to nominal ones. (Achieved folate deficiency corre sponded to stage-III and greater.) Air analytics of methanol by GC.

Blood analysis of methanol and fo rmate by HPLC and liquid scintillation spectroscopy: The m ethanol-dependent fraction of formate was estimated by using 14C-radiolablelled
methanol.

---------------------------- Remark: It is unclear why the endogenous animal- specific total
formate blood levels were not det ermined in the same test
animals. This restriction is not considered to invalidate
the overall result and conclusion

.

------------------------ Result: Methanol levels in blood: Average 2-h concentrations were 0 .65 +-0.3, 3.0 +-0.8, 21 +-16, 106 +-84, and 211 +-71 uM f ollowing 10, 45, 200, 900 and 900 ppm(deficient). AUCs were linearly related to the inhaled methanol dose, the T1/2 were largely unaffected by t he methanol concentrations and ranged from mean t1/2 of 0.56 , 0.88, 0.62, and 0.95 h, respectively, with 1.31 h under f olate deficiency showing a

### OECD SIDS METHANOL

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262 positive increasing trend, althou gh not significant (Dorman et al., 1994; Medinsky et al., 19 97).

Formate levels in blood: Average 2-h concentrations were 0 .07 +-0.02, 0.25 +-0.09, 2.3 +-2.9, 2.8 +-1.7, and 9.5 +-4 .7 uM following 10, 45, 200, 900 and 900 ppm(deficient).

The endogenous formate blood leve l was not measured, but assumed to be 0.1 to 0.2 mM (take n from other sources). The range of the author ́s group is gi ven as 0.29 to 0.56 mM (see Discussion).

---------------------- Test substance: Radiolabelled methanol from Sigma Chemical Corpo ration, >98% pure, methanol HPLC pure. Conclusion: The results suggest that low-dose short-term exposure to methanol would not result in toxi c elevated methanol or formate levels in blood even unde r folate deficiency, because, even under folate defici ency, the methanol-related formate levels remained at a fact or of 10 to 100 below endogenous blood concentrations a nd are several orders of magnitude lower than levels of fo rmate known to be toxic (>7 mM) [see also: Medinsky and Dorma n, 1995, p. 709]. Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

(251) (401) (252)

Type: other: Metabolism / formate / monkey i p

Method: 8 weeks old male Fischer 344 rats and ma le monkeys (Macaca fascicularis) received 14C-labele d methanol per i.p. injection in doses of 25; 125; 60 0 and 3000 mg/kg. 5 animals per dose group were tested (monke ys were repeatedly used). Blood was collected after 6; 12; 24; 30 and 48 hours.

The following parameters were ana lysed: - formic acid - organic acid
- folic acid - pH - biochemistry (glucose, blood ur ea nitrogen, Na, K, Cl).

----------------- Result: Rats: In 25 to 600 mg/kg dosage groups no abnormality was seen and there were hardly any individual difference in blood radioactivity. In the 3000 mg/kg group, however, animals grew weak immediately after administriation , with one death during 48 hours. Moreover, somewhat serious toxic symptoms, such as difficulty in blood collection an d diarrhea, were seen. The disappearance of radioactivity in blood delayed markedly when compared with the other grou ps. The recovery was noted in only on animals which had show n a decrease of the blood level within 48 hours. In animals of 25 to 600 mg/kg dos age groups, the major
excretion route was exhalation: 7 0-80 % of administered dose
was excreted as CO2 and methanol. The excretion of methanol

### OECD SIDS METHANOL

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263 increased with increased dosage.
The excretion into urine and fece s was very low: 6.1-8.3 % and 2.1-2.7 % respectively. The e xcretion was almost completed within 48 hours. At 3000 mg/kg dosage group 2 anim als died within 24 hours,
and one additional animal within 48 hours after administration. The excretion was very slow: about 22 % was excreted into exhaled breath as C O2 or methanol and about 8 % in urine. There were hardly any feces, and therefore the fecal excretion of radioactivity remained very few.

Monkeys: The major route of excretion was exhalation at 25 to 600 mg/kg, 50 % of the administered d ose being excreted as CO2. Even after 48 hours, tota l excretion remained low: about 60 % (probably due to the t est procedure: loss of CO2 during anesthetization and blood collection). Methanol excretion was 0.82-3.8 %, urinary excretion 5.9-7.5 % and
excretion via feces 0.35-0.53 %. At 3000 mg/kg the excretion were 25 % as CO2, 9 % as methanol, 20 % via urine, the exc retion was delayed in this

group.

------------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

(167)

Type: other: Absorption skin / human

Method: a) Exposure of 0.19 - 0.21 ml methanol t o the skin of 6 human volunteers by means of a cl osed application glass chamber (11.2 cm2), absorption wa s measured from 15 to 60 min p.a., 22 determinations in t otal. Residual available
methanol was washed off with wate r and determined sprectrophotometrically (Dutkiewi cz et al. 1980).

b) Exposure of one hand to methan ol (Dutkiewicz et al. 1980).

--------------- Result: a) Following occluded exposure in 6 volu nteers, average skin absorption rate of methanol is re ported to be at 0.192 mg/cm2/min (Dutkiewicz et al. 198 0).

b) Exposure of one hand to liquid methanol for 2 min would result in a body burden of 170 mg , similar to that resulting from exposure to an approximate a ir concentration of 50 mg/m3 (38 ml/m3) for 8 h (Dutkiew icz et al. 1980) (see also: IPSC/WHO, 1997).

c) In 12 humans one hand of whom was exposed to liquid methanol for 2 to 16 min, the ave rage uptake through the skin was 8.1 +-3.7 mg/(cm2*h) = 0 .135 +-0.062 mg/(cm2*min) (Batterman and Franzblau 1997).

Full exposure of one hand for 16 min resulted in a blood level equivalent to that reached after inhalation of 400 ml/m3 for one 8-h working shift w ith a maximal blood level of some 11 mg/l (corrected for ba ckground value) (Franzblau

### OECD SIDS METHANOL

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264 and Batterman 1995; Batterman and Franzblau 1997).

------------- Test substance: Methanol, no further data. Conclusion: The percutaneous absorption may be considera ble on direct
contact of skin to methanol and c onstitute a substantial part of the body burden in relati on to inhalation. Both independent sources arrived at similar quantitative kinetic results for skin permeati on in humans and appear to
be reliable.

--------------- Reliability: (2) valid with restrictions Special study design, sufficientl y documented, meets generally accepted scientific pri nciples, acceptable for

assessment

24-DEC-2004 (300) (390) (298) (402)

Type: other: metabolism / mouse / FDH defici ency

Method: In normal (CB6-F1) and FDH-deficient mic e (NEUT2), acute methanol toxicity and formate eli mination after formate i.p. application were compared. The ho mozygous NEUT2 mice lack cytosolic 10-formytetrahydrofolat e dehydrogenase (FDH). 14CO2 from exhaled air was collec ted after treatment of mice with a low (5 mg/kg) or a high do se (100 mg/kg) of
14C-formate. For discrimination b etween catalase-dependent and other biotransformation route s, 3-aminotriazole (3-AT: 1 g/kg i.p.) was employed as select ive inhibitor of catalase.

--------------------- Result: Acute LD50 were virtually similar in eit her strain at about 6000 mg/kg methanol.

At 5 mg/kg formate, the differenc e in CO2 formation in the
NEUT2 strain was about one half o f the normal mice as expected. However, the inhibitor 3-AT failed to reduce CO2 generation in either case.

At 100 mg/kg formate, CO2 exhalat ion was similar in both strains (which is in line with th e congruent acute toxicity in either strain), but also inhib ited by 3-AT to the same

extent.

There was no significant differen ce in formate-related CO2
release in either strain after 3- AT inhibition, although the
FDH-deficient strain should -in t heory- be unable to oxidise formate under inhibitory conditio n.

--------------------- Conclusion: At low formate burden (here 5 mg/kg), the fu nction of the catalase is poor or not operating , but is active at high doses (here 100 mg/kg). The folat e-dependent FDH system appears to function under normal metabolic conditions.

The residual metabolic capacity o f the FDH-deficient mouse strain to oxidise formate is appa rently a folate-independent alternate oxidation system not ye t characterised.

Therefore, there appear to be at least three metabolic pathways in mice for the oxidatio n of formate arising from
methanol or other sources:

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265 1. the common cytosolic folate -dependent 10-formyltetrahydrofolate dehydr ogenase (FDH) pathway, 2. the catalase-dependent rout e, and 3. a yet unknown route, likely folate-independent.

-------------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

27-DEC-2004 (403)

Type: other: Toxicokinetics / formate / fola te deficiency / monkeys

Method: Sodium formate metabolism was studied in monkeys (Macaca fascicularis) after i.v. infusion usine 14C-radiolabelled formate /doses 1, 2.5, 5 and 10 m mol/kg). Tests were performed under folate-sufficient and folate-deficient
conditions.

---------------- Result: At the highe rdose levels of formate, th e rate of formate
metabolism to CO2 was less than t hat observed in rat (Fig. 3). At lower doses the rate of ox idation to CO2 was about 25
% of that in rat.

The catalase-peroxidase pathway w as shown to play no role in
the oxidation of formate in monke ys (using catalase inhibitor) (Fig 4).

The half-life of formate eliminat ion was about 38 min at 2.5 mmol/kg formate, in the absence o r presence of a catalase inhibitor.

Folate depletion reduced formate metabolism significantly while folate supplementation acce lerated it (Fig. 6 - 9). There was a striking increase in blood formate following methanol application under dietar y folate deficiency (approx. 5 vs. 2 mEq/l blood afte r 0.5 g methanol/kg).

-------------

Elimination half-lives for format e from blood ranged from some 30 - 50 min in the monkey an d about 12 - 23 min in rats, depending on the doses (Cla y et al. 1975, p. 57). Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

24-DEC-2004 (168) (404) Type: other: Metabolism / in vitro / formate / various species

Method: The folate-dependent formate oxidation a nd the activities of several folate-dependent enzym es were measured in vitro with liver homogenate and liver c ytosolic fraction of rat (male SD), mouse (Swiss-Webster), monkey (female Cynomolgus, Macaca fascicularis) and humans ( from victims of traumatic head injuries, without evident li ver diseases).

---------------- Result: Human and monkey liver tissue showed muc h lower 10-formyltetrahydofolate dehydrog enase activity, the enzyme catalyzing the final step of form ate oxidation to CO2, about

### OECD SIDS METHANOL

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266 25 % and 37 %, respectively, than in rat liver. Furthermore, total folate and H4f olate in human liver were only 40 and 50 %, respectively, o f that observed in rat
liver.

In monkey liver, H4folate was com parable to that in humans, but total folate was comparable t o that in rat.

Mouse liver contained much higher H4- and total folate levels than rat and monkey.

The

The in-vitro rates of formate oxi dations were approx. 30 to 40 mg/(kg bw*h) in monkey, about 75 - 80 mg/(kg*h) in rats, and approx. 300 mg/(kg*h) in mice [humans not shown].

-------------- Test substance: Methanol, no further data. Conclusion: The authors explain the higher toxicity of m ethanol in humans and monkeys compared to ro dents by the low H4folate
levels as well as the reduced enz ymic capacity of formate
oxidation.

--------------- Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

24-DEC-2004 (405)

Type: other: Ocular toxicity / folate / huma n / rat

Method: Comparative biochemical study to quantif y folate and 10-formyl tetrahydrofolate dehydr ogenase in human and rat retina and thus explain the highe r ocular toxicity of methanol towards human being.

------------- Result: The cell-specific localisation of the en zyme, 10-FDH, was
found to be similar in rat and hu man retina, preferentially located in the Müller-cell type, the principal glia of the retina (by immunohistochemistry). The amount of 10-FDH found in cyt osolic as well as in the mitochondrial fraction, was about 3x higher in humans than in the SD rats (Western blot anal ysis).

However, the retinal folate level s were lower in humans (about 14 % of that in rats), com pared with the high folate liver pools, the retina contains very much less folate.

--------------------- Conclusion: Formate oxidation was found to be about 50 % lower in human than in rat retina (Eells et al, 1995). This is in line with this finding that lower folate le vels in human retina may
limit conversion of formate into CO2 and result in higher ocular toxicity in humans.

------------------ Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

(406) (407)

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267 Type: other: Risk Assessment / Reproductive parameters

Remark: A range of toxicological investigations, especially teratogenicity studies, was evalu ated to perform a risk assessment for consumer and occup ational exposure
scenarios. The author came to the conclusion that exposure

to the current TWA (time weighted pe rmissible exposure limit) does not pose any risk.

---------------- Test substance: Methanol, no further data. Reliability: (2) valid with restrictions

(249)

Remark: The authors evaluated published data of formate production
in humans and animals after metha nol exposure. They came to the conclusion that even high met hanol exposure up to 2.6 mg/l did not produce toxic blood formate levels and therefore exposure to methanol li kely to be encountered during normal use of methanol-bas ed fuels (up to 0.06 mg/l) will no pose an unacceptable risk

.

-------------- Test substance: Methanol, no further data.

(408)

Type: other: absorption skin human

Remark: Penetration test in male human voluteers : Methanol was applied
to the skin for 4 to 6 h on a soa ked cotton patch under occlusive conditions for 3 days. It was abs orbed through the human skin

at significant amounts, and excretio n into the urine increased
after 2h with the maximum after 6 h.

----------- Test substance: Methanol, no further data.

29-DEC-2004 (409)

Remark: Review on important studies for essentia l endpoint, intended to perform a risk assessment for the general human population: Reference concentrati ons are derived.

The study by NEDO (1987) on monke ys is employed as central
basis for this estimation.

-------------------- Result: A 1-h reference concentration (RfC) was derived to be at about 100 mg/m3, taking into acco unt sensitive people. A very conservative chronic RfC was obtained and proposed at 0.38 mg/m3, based on the assumpti on that 10 ppm has to be used as NOAEL and 100 ppm as the LOAEL for "neurotoxic effects". This appears to be not in compliance with the authors ́ observations and is not suggested in the report.

------------------------------ --- Reliability: (2) valid with restrictions

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268 (167) (179)

Remark: Conclusions on the role of formate:
"From the studies of formate in r odents in vivo and in vitro... it appears that formate is toxic to the developing embryo at concentrations far in e xcess of those achieved after teratogenic methanol exposu re in the mouse, but within the range of levels achieved in h umans after acute high-dose methanol poisoning. Thus, althoug h methanol appears to be the proximate murine teratogen, f ormate is still of concern in terms of potential development al toxicity in humans." (p. 386)

Conclusions on the role of folic acid: "These results (note: including s tudies on folate-deficient diet) suggest that dietary folate intake may be an important consideration in assessing the ri sk of methanol-induced developmental toxicity." (p. 386)

(264)

Type: other: Absorption / inhalation / rat

Method: A flow-through inhalation chamber system (coupled with the
barometric method of evaluation o f the tidal volume) is
introduced which allows the on-li ne monitoring of the essential ventilation parameters, the tidal repiration volume and respiration frequency of the test animal, under
unrestrained conditions as well a s in- and efflux vapor
concentrations. This enables to o btain more accurate and thus representative data about ex traction of the inhalant
from air flow. Concomitant blood sampling via a fixed cannula further allows to analyse blood levels at any time
interval.

Female SD rats were used, exposed to 1000, 5000, 10000, 15000, and 20000 ppm methanol for 8 h.

------------------- Result: Based on this technique, the mean ventil ation volume for 8 h was 280 - 230 L/kg b.w. (mean 250 L/kg), a slight downward trend discernible with increasing exposure concentration. The respiration frequency was fou nd to be significantly lower than commonly published (ab out -20 to -30%), while the
tidal volumes were similar. At 5000 ppm, an extraction (reten tion) factor (absorption) of about 0.9, at 15000 ppm of abo ut 0.6 resulted from the plots of cumulative mass vs. expo sure time. No data given at the lower exposures.

The highest blood concentrations were at about 3500 mg/L (No exposure-blood correlation is sho wn.).

--------------------- Conclusion: Based on these results, the respiratory upta ke of methanol by rats can be assumed to be app rox. 100 % at exposure concentrations of <5000 ppm. Reliability: (2) valid with restrictions

(410)

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269 Type: other: Ocular toxicity / rat model

Method: Nonprimate model in rats in which the fo late-dependent formate oxidation (on the level o f methionine synthetase) has been selectively inhibited by treatment with N2O in
inhaled air.

Animals: Male Long-Evans rats (25 0 - 300 g)

Methanol administered i.p. at an initial dose of 4 g/kg followed by a supplemental dose o f 1 or 2 g/kg 12 h later.

Thus, blood formate levels were f rom 7 - 15 mM or 4 - 6 mM
for 30 - 40 h. The "non-intoxicated" controls re ceived 4 and 2 g/kg methanol without N20, the non-tre ated controls either the vehicle or N2O in air.

Retinal function was assessed by flicker electroretinography (ERG). Histopathology of retina a nd optic nerve was carried out by light microscopic visualis ation.

--------------------- Result: Methanol intoxicated rats developed form ic acidemia,
metabolic acidosis and visual tox icity within 36 hours.

Histopathological effect on retin al structure:

In the high-dose group (7 - 15 mM blood formate vs. methanol-treated control with 0.5 to 2 mM formiate), prominent vacuolation in the phot oreceptors near the junction of inner and outer segme nts, with accumulation of densely stained material in the i nner segments near the outer limiting membrane. Mitochon drial swelling and disruption was noted in the retin al pigment epithelium, photoreceptor inner segments and optic nerve (Eells et al., 2000; Seme et al., 2001). Ultrast ructural studies by electronmicroscopy revealed that the retinal morphology (as represented by the mitochdrial-ri ch, inner segment of the photoreceptor) was similar to the control after recovery of 72 h, but subtle photoreceptor ch anges were still present as a spacing between the cell nuclei of the outer nuclear layer which suggests residual histologi cal alterations from formate-induced, previous edema ( Seme et al., 2001).

In the low-dose group (4 - 6 mM f ormate in blood), no histopathological changes were ap parent at the light-microscopic level (Wallace et al., 1997).

However, visual dysfunction was a lready visible in functional dignostic, occurring a s reductions in the flash evoked cortical potential (FEP) a nd in electroretinogram (ERG) at formate concentrations l ower than those associated with morphological changes and pr ovide functional evidence of direct retinal toxicity in met hanol poisoning (Wallace et al, 1997). Rod- and cone-mediated ERG responses were attenuated in a formate- and time -dependent manner (Seme et al., 1999, 2001).

Biochemical effects: Retinal ATP, ADP, and GSH were si gnificantly depleted

### OECD SIDS METHANOL

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270 following methonol-treatment unde r inhibition of formate
oxidation after 72 and 144 h with GSH levels about 1/2 of controls, and after recovery stil l decreased, while energy
metabolites showed no difference from the control values (Seme et al., 2001).

--------------- Test substance: Methanol, HPLC grade from Sigma

Conclusion: Functional tests provide functional evidence of direct retinal toxicity in methanol pois oning at stages not yet pronounced in histopathological c hanges.

Hypothetical mechanism: Formic ac id binds to cytochrome aa3 and inhibits cytochrome oxidase a ctivity with inhibition
constant values between 5 - 30 mM , which is in the range of
concentrations found in the retin a and vitreous humor of methanol-intoxicated rats. This m ay explain the effect on mitochondria and resulting visual dysfunction (Eells et al., 2000).

The rat model appears to be a use ful tool to also elucidate the sequelae of methanol intoxica tion in humans who are more
sensitive than rodents.

---------------------- Reliability: (2) valid with restrictions 2e: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

(411) (412) (413) (414)

Type: Biochemical or cellular interactions

Remark: The cytotoxicity of methanol on C6 rat g lioma cells was measured by various methods: tryp an blue exclusion, protein synthesis, neutral red assay and induction of heat shock protein synthesis. Methanol showe d a very low toxicity with
first toxic effects in all test s ystems at concentrations between 1 and 10 Mol (ca. 32 - 32 0 g/l).
Test substance: Methanol from Sigma Chemicals, Germany, no furth er data.

(415)

Type: Biochemical or cellular interactions

Remark: The cytotoxicity of methanol was tested in chicken embryo forebrain neuron cultures using t he MTT and the neutral red assay to predict acute toxicity. LC50 in the MTT assay was 482.2 u Mol and in the neutral red assay 340.6 uMol. The toxicity of methanol was low in comparison with the other 49 test substances. Test substance: Methanol from BDH, analar analytical grade, no f urther data.

(416)

Type: Biochemical or cellular interactions

Remark: In neuronal cell cultures (pheochromocyt oma PC12) alcohols in combination with neuronal grow th factor leads to greatly enhanced neurite extension and ce ll differentiation. The effect increases with increasing chain length, methanol

### OECD SIDS METHANOL

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271 being the less potent substance. The role of this effect in alcohol toxicity on brain is not understood. Test substance: Methanol, no further data.

(417)

Type: Biochemical or cellular interactions

Remark: Rat hepatocytes were exposed to methanol for 20 hours at concentrations of up to 39.55 ug/ ml. The treatment had no effect on LDH-leakage, MTT-reduct ion, cellular protein content and neutral red uptake. Test substance: Methanol, no further data.

(418)

Type: Distribution

Remark: After oral administration of a single do se of methanol to dogs (1.97 g/kg) about 10 % was e xcreted unchanged in the
urine. The methanol concentration in the organs was nearly half as high as that found in the urine. About 20 % of the
administered dose was excreted in urine as formic acid.
Formic acid concentrations in tis sues was about half the serum concentration.
After oral administration of 2.38 g/kg methanol to rabbits 10 % were excreted unchanged in u rine. No further details are reported.

-------------- Test substance: Methanol, no further data.

(88) (419)

Type: Distribution

Remark: The examination of the toxicokinetics of intravenously
administered methanol to female r ats as a single dose of 50 or 100 or 2500 mg/kg resulted in a markedly non linear
elimination of methanol from the systemic circulation suggestive of a significant capac ity limited rate of elimination. The data from the 25 00 mg/kg group was described by a kinetic model inco rporating parallel
first-order and saturable elimina tion processes, a portion of this apparent linear eliminati on pathway was due to renalexcretion of the unchanged a lcohol. Test substance: Methanol, no further data.

(88) (420) Type: Metabolism

Remark: The metabolism of methanol (and ethanol) by catalase and alcohol-dehydrogenase was studied in alcohol-dehydrogenase positive and negative deer mice. Metabolism by catalase was the predominant pathway for both alcohols but both enzymes contributed to the total metaboli sm (Bradford et al., 1993).

Methanol metabolism in alcohol-de hydrogenase deficient deer mice was inhibited by 4-methylpyr azole, which inhibited fatty acyl CoA-synthetase, thus r educing the availabilty of H2O2 for catalase dependent alcoh ol metabolism (Bradford et

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272 al., 1992).

----------------------- Test substance: Methanol, no further data.

(421) (422)

Type: Toxicokinetics

Remark: In monkeys, four daily skin application of 0.5 ml (daily dose 1.56 g/kg) could be lethal. Rabbits proved to be distinctly more insensitive to me thanol either dermally applied or also after inhalation exposure. Individual variation was nonetheless large i n either species.
Test substance: Methanol, no further data.

(156)

Type: Toxicokinetics

Method: Comparative toxicity and metabolic study 1. in normal mice a) under fol ate-sufficient, b) under folate-deprived conditions, and
2. in a C57BL mouse strain suff ering catalase deficiency (catalase considered to be the pr ime enzyme oxidising formate) as well as a strain with normal catalase activity.

Methanol and formate levels in bl ood as well as urinary formate excretion were compared w ith acute toxicity (LD50) under respective conditions after oral administration of methanol (2 - 8 g/kg bw).

---------------------- Remark: Those mice with catalase deficiency show ed a somewhat lower LD50(oral and i.p.) than those wi th normal catalase level, irrespective of folate status. F olate- and catalase deficient mice excreted considera bly higher amounts of formate into the urine (30.8-40.8 %), with significantly higher blood formate levels than in those normal, but formate-deficient mice (15 to 17 umol/ml vs. 0.8 - 1.4 umol/ml (24 h after 6 and 5 g/kg methanol oral). In no case, folate deficiency alo ne could not explain the poor decrease in LD50 which was u nexpected in relation to the pronounced increase in the fo rmate level with 24 h.

A low supplementation with methio nine (0.2%) had no
influence on the LD50 under cata lase- and folate-deficient conditions, while at 1.8 % methio nine, there was an apparent LD50-decrease from 7.1 g/kg to 6. 4 g/kg.

----------------- Test substance: Methanol, no further data. Conclusion: The authors concluded that the formate conce ntrations in plasma do not correlate well with methanol-induced
lethalities in catalase-deficient , but folate-sufficient
mice or folate-deficient mice. I n addition, urinary excretion, not oxidation, is the primary means to get rid of high levels of formate.

The results in mice with catalase or folate deficiency -acc. to authors - appear to correspond to conditions in methanol-poisoned monkeys: high f ormate level at low pH in
blood.

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273 -------------------

(137)

Type: Toxicokinetics

Remark: The pharmacokinetics of methanol was com pared in Fisher rats and Rhesus monkeys. After i. v. application to rats 96.6 % were metabolized, 2.6 % exhaled and only 0.8 % excreted in urine. The kinetic parameters mea sured or cited were: AUC at 2000 ppm 623.6 (rats) and 479.8 ( monkeys) ug x h/ml, t1/2(half-life) 2.1 hours in rats and 2.9 hours in monkeys, Vmax15.41 mg/h x kg, Km 33.92 mg/ l. Test substance: Radiolabelled Methanol from Sigma Chemicals Corp oration, no
further data.

(397)

Type: other: Absorption / distribution / inh alation / monkey

Method: Comprehensive study design in analogy to Segment-I study limited to exposure of female mon keys (Macaca fascicularis) and qualitative/clinical signs of toxicity. It comprised

- methanol pharmacokinetics - maternal toxicity (see other entry: 5.8.2) - reproductive toxicity (see ot her entry: 5.8.2) - developmental neurotoxicity ( see other entry: 5.8.2).

Groups of 11-12 adult female monk eys were exposed to methanol vapor in the concentrati ons of 0; 200; 600 or 1800 ppm, daily for 2.5 hour before br eeding, during breeding and during pregnancy.

Blood kinetics for methanol, bloo d concentration of formate
as well as folate were determined 2x/week 30 min after the 2.5-h exposure in female monkeys during the prebreeding period, after 87 d of exposure an d 2x during pregnancy (Kinetic studies 1 to 4). The num ber or animals was 9 to 10

per group.

--------------------- Result: Methanol: No significant differences in the blood concentrations between the 4 phas es (measured 0.5 h post-exposure, n = 11 or 12 (non- pregnant); n = 9 or 10 (pregnancy) (Fig 7, Tab. 11, Fig. 14, Part I):

Baseline range approx. 2 ug/ml [approx. 0.06 mM] 200 ppm: range approx. 5 ug/ml [approx. 0.15 mM] 600 ppm: range approx. 10 ug/ml [approx. 0.30 mM] 1800 ppm: range approx. 35 -40 ug /ml [1mM-range]

At 1800 ppm, after 5 h eliminatio n, the residual methanol level was near baseline (max. 2-f old higher).

The mean estimated elimination ha lf-lives (for 600 and 1800 ppm) ranged between about 60 to 9 0 min.

Formate: Irrespective of concentr ation levels or exposure intervals, there was no evidence of a significant increase above the background range of abo ut 0.15 - 0.30 mM (approx.

### OECD SIDS METHANOL

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274 7 to 14 ug/ml) (Fig. 8, Fig. 15, Tab. 12, Part I).

Folate in serum: There was no sig nificant shift in the folate levels during pregnancy, i ndependent of exposure (Tab. 13, Part I). Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, well documented

(423)

Type: other: Ocular toxicity / animal model

Remark: 1. In a preliminary study, F-344 rats fed control and folate-deficient diets and expose d to 1050 mg/m3 methanol (20 h/d, 7 d/wk, 13 weeks) showed deg eneration of the retina and optic nerve under both conditions , but not yet after 4 eeks of

exposure. However, Long-Evans rats fed eith er diet failed to develop any
ocular lesion. The authors suggest that spontane ous degeneration was specific
for F-344 rats and are therefore not suitable for ocular toxicity studies. (Lee et al. 199 1).

2. To create an animal model for human methanol toxicity folate-deficient rats (male Long- Evans) were exposed to methanol (single oral administrat ion of 2 - 3.5 g/kg) and
formate accumulation in blood was measured. Methanol concentrations in blood did not greatly differ
between normal and folate deficie nt rats, while formate
accumulated in folate-deficient b ut not in normal rats. Inhalation of 1200 or 2000 ppm fo r 6 hours led also to increased blood formate levels on ly in folate deficient

rats.

If in fact formate accumulation i s responsible for methanol toxicity the animal model may be suitable to test these effects. (Lee et al. 1994)

------------- Test substance: Methanol, no further data.

(424) (425)

Type: other: Ocular toxicity / formate /monk ey

Method: Test design to elucidate the mechanism o f methanol-induced toxicity syndrome and ocular toxi city: here influence of formate (McMartin et al., 1978). Four male rhesus monkeys (Macaca mulatta) received buffered Naformate i.v. to a loading of 1. 25 mmol/kg bw., with a mean infusion rate of 3.1 mEq/kg/h. Th is procedure produced no
acidosis.

(see also other investigations of this group under 5.4)

---------------- Result: After 10 h, all animals accumulated form ate in blood between 10 to 30 mEq/L. Blood pHs were ma intained between 7.4 and 7.6. Pupillary reflexes were rapi dly altered, and in most animals no response to light was observed between 24 and 48 h. Ophthalmology revealed marked optic disc edema (mainly in

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275 the prelaminar region, central po rtion of the proximal part of the optic nerve without signif icantly reaching to the distal part. The retina including the ganglion -cell layer were completely normal. (McMartin et al., 1978)

----------- Conclusion: These results with formate were completely c onsistent with those findings after methanol-int oxication (see other entries of this group under 5.4).

Formic acid and not formaldehyde (McMartin et al., 1979) has to be considered the causative ag ent for ocular damage within the methanol-intoxication syndrome, irrespective of
acidosis.

The mechanism of formate toxicity may be seen in the inhibition of oxidative phosphory lation by formate based on the on findings that this substan ce is an efficient inhibitor of cytochrome oxidase.

------------- Reliability: (2) valid with restrictions

(187) (426)

Type: other: Review

Remark: The reviewer describes the effects of me thanol intoxication in humans. Methanol affects prima rily the central nervous system involving the visual syste m up to optic atrophy. The
minimal lethal dose in humans is reported to be 80 g. Methanol causes a variety of hist opathological changes in the central nervous system and es pecially in the optic

nerve. Test substance: Methanol, no further data.

(427)

Type: other: Review

Remark: The toxicity, kinetics and metabolism of methanol is reviewed. According to the review er methanol is readily resorbed by all exposure routes a nd metabolized in the liver firstly to formaldehyde and subse quently to CO2. It is of moderate acute toxicity with the central nervous system and especially the optic nerve as pri mary target organs. Methanol was not carcinogenic in rats and mice in inhalation carcinogenicity studie s but in reproductive toxicity studies it was teratogen ic and embryotoxic in rats
and mice.

---------------- Test substance: Methanol, no further data.

(428)

Type: other: Review

Remark: Potential Health Effects of Gasoline and Its Constituents: A Review of Current Literature (199 0 - 1997) on Toxicological

Data

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276 (429)

Type: other: Risk Assessment Model / inhalat ion

Remark: Quantitative Models of Risk Assessment f or Developmental Neurotoxicants, based on inhalati on data in SD rats from Nelson et al., 1995: The use of continuous foetal weig ht data was as sensitive as
foetal brain malformation data in estimating excess risk to methanol inhalation exposure in d eveloping rat. Excess risk (assumed to be the additional ris k that foetal weight is below the first percentile of con trol animals) was estimated by using a logistic model: 0.1 a t 16000 ppm, 0.01 at 5400 ppm, and 0.001 at 980 ppm.

------------

(430) (234) (235)

Type: other: Toxicokinetics / ethanol influe nce

Method: Comparative study with ADH inhibitors: M ale CD-1 mouse i.p injected 1 g/kg methanol followed by the inhibitor (i.p.).
Elimination of methanol from bloo d was determined (t/2, AUC, mean elemination rate). Result: Ethanol (4 g/kg) doubled the elimination half life as compared with control (approx. 9 vs. approx. 5 h); chloral hydrate (0.4 g/kg) had no signifi cant impact on methanol elimination. But in combination ( 0.2 g/kg) with ethanol (4 g/kg), methanol elimination was r etarded at a factor of >3 as compared with the control, whi ch corresponded to about a twofold decrease of the eliminati on rate as compared with
ethanol alone.

(431)

Type: other: absorption

Remark: In shortly described experiments, monkey s (normal or folate deficient) exposed to low levels of methanol (10 - 900 ppm = 0.013 - 1.17 mg/l) showed a metha nol-derived increment of formate in blood which were 10 - 1000 fold lower than those levels normally present in the bo dy.

It is concluded that exposure to methanol at the threshold limit value of 200 ppm (0.26 mg/l ) is unlikely to pose an unacceptable risk in healthy or e ven moderately folate-deficient individuals.

------------------ Test substance: Methanol, no further data. Reliability: (2) valid with restrictions

24-DEC-2004 (432)

Remark: Acute methanol poisoning in men and monk eys: latency, acidosis, ocular toxicity, not ob served in most other species tested. Formic acid as li kely causative agent is
discussed: formate accumulation m ainly due to limitation/low level in hepatic tetrahydrofolate along with a low 10formyl-THF dehydrogenase.

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277 --------- Test substance: Methanol, no further data. (281)

Method: In anesthetised dogs, the acute cardiova scular effect of methanol was investigated after i .v. infusion (20-% solution).

------------- Remark: Methanol distributed rapidly in the body fluid.

Methanol concentrations of 1.3-2. 0 mg/ ml blood reduced cardiac stroke volume, cardiac ou tput per minute, systemic blood pressure and blood flow thr ough the Arteria femoralis
and Ateria carotis. On the other hand, peripheral
resistance increased. Death cause d by cardiac arrest came
about at a methanol concentration in blood of 4 mg/ml.

-------------- Test substance: Methanol, no further data.

(433)

Remark: In anesthetised dogs, methanol retention was about 85 % of inhaled methanol at exposure conc entrations from 360 to 960 mg/m3. Absorption was dependent o n ventilation rate. Test substance: Methanol, "pure", no further data.

(434)

Remark: In vitro study on enzyme preparations an d in vivo: The influence of glutathione and other SH-compounds on the metabolic inactivation of the alc ohol and aldehyde dehydrogenase by aldhyde and meth anol was examined by poisoning male adult mice by an i .p. dose of 8.6 g/kg methanol. After 10 min, 1, 2 or 3 hours, the mice were treated either with 4 mg/kg 2,4-d imercaptopropanol, 120 mg cysteine or 27 mg mercaptoethanol

. Mean survival time of methanol-tr eated control animals was no longer than about 10 hours. Bu t following preventive treatment, mice survived consider ably longer: the earlier the protective agent was applied, the longer was the survival up to 12-fold compared w ith the control with a few
treated animals alive for 2 month s (end of experiment). Test substance: Methanol, from Fisher Scientific Comp., no furth er data. Conclusion: These findings suggest that part of methanol intoxication in mice may be caused by formaldehyd e-exerted inactivation of alcohol and aldehyde dehydrogenas e, as SH-reagents are also effective in the mitigation of po isoning.

-------------- Method: In this study, enzyme activities respons ible for the
oxidation of methanol to formate were investigated in liver homogenate and the eye of Long-Ev ans rats.

---------------------- Remark: There was no evidence of an alcohol dehy drogenase-dependent
metabolism of methanol in the liv er and in the eye.

In liver, conversion of methanol was catalised by an
acid-labile catalase and acid-sta ble alcohol-oxidising enzyme. In the eye, there was on ly acid-labile catalase activity.

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278 Km value(catalase, eye) = 366 mM , Km value(catalase, liver) = 1.06 mM, Vmax value Werte(catalase, eye) = 19.8 umol/h/g, Vmax value Werte(catalase, liver) = 18.3 umol/h/g

Km value(alc-oxid. enzyme, liver) = 147 mM, Vmax value(alc-oxid. enzyme, live r) = 30.5 umol/h/g tissue.

Metab. capacity(tissue weight xvm ax)[eye] = 2.7 umol/h Metab. capacity(tissue weight xvm ax)[liver catal.] = 255 umol/h Metab. capacity (tissue weight x vmax)[liver alc.oxid.] = 425 umol/h.

------------------------------ Test substance: Methanol, no further data. Conclusion: The results demonstrate that acid-labile cat alase is the sole active system in rat eye to metabolise methanol, while in liver both, catalase and an ac id-stable alcohol-oxidising enzyme system significantly contr ibute to the biooxidation of methanol to formaldhyde.

------------------

(436)

Remark: The toxicity of methanol in relation to formate metabolismus was investigated in isolated hepa tocytes (Sprague-Dawley rats):

In isolated hepatocytes, formate arising from methanol is further oxidised by folate-depend ent pathways (10-formyl-THF synthetase and 10-formyl-THF dehy drogenase) rather than by the catalase-peroxidase reactions

.

In these experiments, oxidation r ate of methanol to CO2 was significantly lower than found in vivo. After addition of methionine, formation of CO2 from methanol increased, which
was ascribed to a restoration and stimulation of the formate
oxidation and is based on the fac t that in in-vitro liver preparations, the methionine cont ent is at a factor of about 10 lower than in vivo. In hepatocytes from folate-defici ent rats, oxidation to formate was poor or absent even a fter addition of 5-formyltetrahydrofolate or folat e, but could be restored by addition of methionine.

The functional role of methionine remains to be elucidated.

-------------- Test substance: Methanol, radiolabelled from New England Nuclear , no furtherdata.

(437)

Method: Methanol was administered by gavage to n ormal and folate-deficient rhesus monkeys ( Macaca fascicularis) at a dose of 3000 mg/kg and 2000 mg/kg bw, respectively. Formaldehyde and formate were ana lyesd in blood, urine, and in the cerebral fluid, the orbit fluid of the eye, liver, kidney, brain, and optic nerve.

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279 ----------------- Result: There was no evidence of any formaldehyd e increase at a time period when signs of acidosis and methanol intoxication occurred, while significant incre ases in formate were found in blood and other body fluids. For verification, one untreated m onkey was infused 14C-radiolabelled formaldehyde (1 mmol/kg bw, 0.2 M solution)
over 3-4 min. The half-life time was 1.5 min.

------------------- Test substance: Methanol, radiolabelled from New England Nuclear , no further data. Conclusion: Formaldehyde cannot be considered as the mai n factor in the development of methanol-induced t oxicity. Reliability: (2) valid with restrictions

(426)

Remark: Human alcohol dehydrogenase exhibited a 10-fold higher Km
value for methanol associated wit h a 10-fold lower activity
than for ethanol. This striking difference suggests that there are different kinetic mechanisms of dehydrogena tion of either alcohol. Test substance: Methanol, purified, from Merck, Germany, no furt her data.

(438)

Remark: Formic acid arising from methanol is eli minated at different rates by different species: most rapidly in rats with T/2 of approx. 12 min, then in rabbits w ith T/2 of about 32 min., dog with T/2 of 77 min., and huma n with T/2 of about 55 min.

Higher folate levels in blood app ear to correlate to lower
formate levels. Pretreatment of d ogs with amethopterin (folic-acid antagonist) induced a pronounced increase in formate of plasma and urine. On c ontrast, prophylactic
administration of folic acid com pletely prevented
formic-acid accumulation in blood

.

------------------- Test substance: Methanol, no further data.

(439)

Test substance: Methanol, no further data.

(279) (230) (429) (440) (441) (442) (44 3) (444) (384) (88) (445) (446) (447) (233) (326) (284) (448) (44 9) (450) (451)

Remark: In alcohol-free persons, the endogenous methanol blood level ranges from 0.5 - 1 mg/l (1.5 mg/ l not exceeded), the urine level from 0.32 - 2.61 mg/l, and the endogenous breath level between 0.21 - 0.79 ug/l. Long-te rm ingestion of significant amounts of ethanol (drinks, wine etc.), the background methanol levels may shift to high er values due to cumulation of endogenously formed methanol, for example through competitively inhibition of metha nol by ethanol.

---------------- Test substance: Methanol, no further data.

(452)

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280 Remark: Laquer thinner containing methanol, whic h is used for inhalation abuse, affected male f ertility with a significant decrease in testicula r and prostatic weights and plasma testosterone levels. Metha nol exposure alone did not produce such effects. Test substance: Methanol, no further data.

(453)

Remark: The metabolism of formate was compared i n monkeys and rats.
Formate oxidation was 50 % lower in monkeys than in rats. The activity of a range of folate -dependent enzymes in liver tissue as well as the hepat ic H4folate levels were compared. H4folate levels were 40 % lower in monkeys than in rats. Enzyme activities were part ly higher and partly lower in monkeys than in rats, and thei r specific contribution to methanol toxicity could not be cl arified. Test substance: Methanol, no further data.

(454)

Remark: A range of publications with respect to the role of formate toxicity in methanol toxicity in humans are reviewed. The difference of methanol toxicity i n primates and rodents is suspected to derive from formate accumulation due to lower
formate metabolism. In fact human s and monkeys possess low hepatic H4folate levels and low r ates of formate oxidation. Formate itself produces blindness in monkeys, the major
toxic effect of methanol. Test substance: Methanol, no further data.

(281)

Remark: The role of liver catalase activity and H2O2 formation on
methanol oxidation in the liver i s discussed. According to
the author the methanol oxidation is mainly dependent on the rate of H2O2 generation, desp ite the fact that catalase plays a role in methanol metaboli sm in liver tissue.

--------------- Test substance: Methanol, no further data.

(455)

Remark: The pigtail monkey was tested for effect s of toxic methanol exposure to establish an animal m odel for human methanol toxicity. Blood acidosis was meas ured as major toxic effect of methanol and the responsible a cid was identified. In pigtail monkeys but not in rhe sus monkeys significant blood acidosis was produced by a single i.p. dose of 4 g/kg bodyweight methanol. Blood concen tration of formate was correlated with acidosis, no othe r organic acid was found
at increased levels in acidotic m onkeys. Test substance: Methanol, reagent grade, no further data. Reliability: (2) valid with restrictions

(168)

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281 Remark: Male rats were exposed to methanol at 20 0 ppm (0.26 mg/l) for up to 6 weeks (8 hours/day, 5 days/week) did not show
an effect on serum testosterone l evels. Exposure to 800 ppm (1.04 mg/l) for up to 13 weeks ha d no effect on testes to body weight ratio and had no effe ct on testicular morphology
in normal and folate deficient ra ts. A greater incidence of testicular degeneration was seen only in 18 month old folate-deficient rats exposed to 800 ppm methanol for 13 weeks (20 hours/day, 7 days/week, 12 - 13 rats/group). The results are equivocal. Test substance: Methanol, no further data.

(456)

Remark: Male rats were exposed to methanol at 20 0 ppm (0.26 mg/l) for 6 hour/day for up to 1 week. Compared to controls
methanol treatment for 1 week led to decreased plasma levels of testosterone, luteinizi ng hormone and corticosterone. The changes were not statistically signficant. Test substance: Methanol, no further data.

(195)

Remark: 81 - 88 % of inhaled methanol was retain ed in the respiratory tract in dogs. The in haled fraction was related
to the concentration in air with a 73 % retention at 390 mg/m3 and a 90 % retention at 720 mg/m3.
Test substance: Methanol, no further data. Reliability: (2) valid with restrictions

(434)

Remark: Methanol is metabolised in rodents and p rimates in different ways and different spee d. In rodents methanol is primarily metabolised to formalde hyde by the catalase-peroxidase system. The V max value was determined at 30 mg/kg/hour. In humans and monk eys the alcohol dehydrogenase oxidizes methanol t o formaldehyde more rapidly. The Vmax value in primat es is 48 mg/kg/hour. Formaldehyde is finally oxidized to CO2 by folate dependent enzymes. Primates have less folat e than rodents and therefore the detoxication of met hanol may be limited by the last steps of metabolism in prima tes.

Test substance: Methanol, no further data.

(457)

Remark: 1. A high dose of methanol (single 3 g /kg, oral) to male Wistar rats gave some evidence of increased LPO with a transient time-dependent increase in MDA in liver and serum (about +20 % and 40 %, respective ly, reversed after 7 d) and wth transient decrease in compone nts of the antioxidant system: GSH in liver after 12 h a nd 2 d, recovered at day 5 (in between no data), various enz ymes also showed a transient decrease (Cu,Zn-Superox ide dismutase ,GSH-Peroxidase, GSSG-Reductase).

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282 Blood methanol peak was ca. 2300 ug/ml after 6 h and rapidly declined (Skrzydlewska et al. 199 7, 1998).

-------------

2. After oral doses (1.5, 3, 6 g/kg bw) to male Wistar rats, the activity of proteases ( elöastase and cathepsin G)
were incresed from 12 h to 5 d in relation to the methanol dose. Concomitantly, the activity of their inhibitors was
reduced. Similar disorders in the proteolytic-antiproteolytic balan ce in favour of protease
action can also be observed in hu man acute intoxications with methanol. The further implic ations are addressed: blood platrelet action, protein degrada tion, release of anaphylotoxins and other peptides (e.g. kinins) incuding vasoactive mediators, activation of other proteases etc. (Skrzydlewska et al. 1999).

----------------

3. The effect of methanol intox ication (6 g/kg bw) on protein
modification in the liver of rats was investigated.
An increase in free radicals were determined 6 and 12 hours
after intoxication. Malondialdehyde and carbonyl grou ps in protein were increased; the levels of amino gr oups and sulphydryl groups and the amount of tryptophan in p roteins were decreased, whereas the amount of bi-tyrosine was increased. According to the authors changes in the protein structure
resulted both from free radical a ction and formaldehyde generation during methanol intoxi cation (Skrzydlewska et al., 2000).

(458) (459) (460) (461) (462) (463)

Remark: The PBPK approach was used to develop a model of methanol disposition during gestation in r ats and mice. To validate this model, concentrations of met hanol in the dams and the conceptus were determined after m ethanol exposure of rats on GD 14 and 20 and of mice on gd 18

. In pregnant animals, conceptal/ma ternal AUC and Cmax-ratios

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283 decreased with increasing dose at both gd 14 and 20 and at gd 18 in the mouse. Additionally, the conceptal/maternal
diffusion-constant ratio consiste ntly decreased with increasing dose in pregnant rats and mice.

These results are consistent with earlier observations that methanol limits its own delivery to the conceptus.

It was further shown that the max imal rate of methanol elimination (vmax) in vivo decrea sed at term in both species with 65 to 80 % of that in non-pr egnant rodents. This was
also found in vitro in rodent liv er homogenates. This appears to suggest that not only altered disposition but also a change in metabolic capaci ties makes the difference in gestation. Fetal rodent liver was capable of metabolizing methanol in vitro, but only at a rate of <5 % of respective adult livers. (Ward and Pollack, 1996; Pollack and Brouwer, 1996) Reliability: (2) valid with restrictions

(395) (464) (465)

Method: In-vitro study on human hepatic enzyme f raction to examine
various inhibitors of ADH with me thanol as substrate Result: In methanol intoxication, cimetidine may potentially be a better antidote than 4-methylpyra zole because of its slightly higher inhibitory actiti vity in particular at higher methanol concentrations.

(466)

Remark: According to Caprino and Togna, 1998:

"Intoxication [of methanol] has d elayed onset characterized by acidosis, mental confusion, oc ular toxicity with visual disturbance [recently suggested a s due to intraretinal metabolism of methanol, rather th an to elevated blood formate levels (Garner et al., 19 95a,b)], reversible or permanent blindness, and, in seve re cases, death. These effects represent a classic examp le of lethal synthesis in
which toxic metabolites can cause fatality after characteristic latency period."

............

"...no interactive effects betwee n methanol and gasoline
were found in a short-term inhala tion study in rats (Poon et al., 1995), and a study on perina tal exposure to low concentrations of methanol result ed... in no significant
abnormalities in the brain of tre ated rats (Stern et al., 1996)."

"There is little evidence from av ailable information of
human health effects from low-lev el exposure which demonstrates that methanol vapors from motor vehicle fuel
can cause acute adverse effects t o health. Lee et al. (1992) demonstrated that after 6-hr expo sure to current methanol threshold limit value (TLV) of 20 0 ppm, the formate does not accumulate in blood in human subj ects at rest and during

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284 exercise. This was confirmed by o ther authors (D ́Alessandro et al., 1994; Osterloh et al., 19 96)."

(429) (391) (392) (196) (277) (467) (241)

Remark: Compilation of human case reports on acu te methanol
intoxications with a collection o f the blood levels found in
sublethal and lethal events at va rious time intervals after ingestion.

A provisional nomogram has been d erived, beginning at a 3-h concentration of 20 mM and ending at a 130-h concentration of 0.5 mM. Concentrations at any time-point above this straight line result either in l ife-threatening poisoning
or a lethal outcome.

----------------

(468)

Result: After oral application of methanol (3 g/ kg bw), the
thiobarbituric acid-reactive subs tances (TAS) indicating lipid peroxidation were significa ntly increased in brain
tissue from 6 h to 7 d after trea tment, also activities of liver GSH peroxidase and GSH redu ctase were reduced after 6 h already.

N-Acetylcysteine exerted an prote ctive antioxidant effect in
the brain and liver of methanol-t reated rats: TAS remained on the background-level after pri or i.p. treatment with N-acetylcysteine (150 mg/kg). It resulted in a lower degree of parenchymal damage (Kasacka an d Skrzydlewska, 2001).

Furthermore, concomitantly, metha nol-induced GSH-depletion, which was significant after 24 h and steadily increased until 7 d, could be mitigated, an d enzyme activities
involved in the removal of reacti ve oxygen species were also
similar to the untreated control while significantly increased without protective coun teraction.

A Trolox-Derivative, an analog of alpha-tocopherol, is
introduced as a beneficial antiox idant which prevents or
reduces methanol-induced LPO in W istar rats (Skrzydlewska and Farbiszewski, 1997).

------------------- (469) (470) (471) (472)

(250) (251) (397) (196) (252) (264)

Method: Comprehensive study programme on three s pecies (rat, mouse, monkey) including metabolic, phar macokinetic and short-, long-term and reproduction studie s and carcinogenicity
studies: Here two-generation reproduction study in rats (see other entry, 5.8.2).

In 9-week old F1 pups, blood meth anol was measured, but not formate (p. 191). Exposure: conti nuous for 19 to 20 h/d.

------------------------- Remark: Blood levels of methanol measured in the F1 offsprings (age

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285 9 weeks) (NEDO, 1987, p. 191):

controls (baseline): approx. 2 - 3 ug/ml 10 ppm methanol: approx. 3 - 3.5 ug/ml 100 ppm " : approx. 1 - 4.2 ug/ml 1000 ppm " : approx. 53 ( males)-100 (females) ug/ml.

There no data on formate.

------------- Reliability: (2) valid with restrictions Test procedures in accordance wit h accepted standard methods, sufficiently documented

(167)

Remark: Under in-vitro conditions, in the presen ce of liver
microsomes or liver nuclei fracti on, hydroxymethyl radicals
were formed from methanol. This b ioactivation was observed only under anaerobic conditions w hich also resulted in the production of formaldhyde. Formal dehyde was the primary intermediate during aerobic incub ation. But both formaldehyde-generating processes appear to be different,
because the anaerobic one was str ongly inhibited by diphenylene iodonium, while the a erobic one was not.

(473) Result: Male rats were exposed to methanol vapou r at concentrations of 200, 5000 or 10000 ppm (0.26, 6.5 or 13 mg/l) for 6 hours and killed immediately after expo sure or at 24 hours. Rats were divided in acclimated (2 wee ks prior to handling) and non-acclimated groups. At 6 h, sh am-exposed rats already showed an increase in the level o f serum LH. Effects on serum hormone levels (luteinizing hormone, follicle stimulating hormone, testosterone , prolactine) were inconsistent with partly no effec ts and partlycontradictory results. According to the author, methanol treatment led only to increased prolactin level s in serum under both
acclimated and non-acclimated con ditions. Test substance: Methanol, no further data.

(157)

Result: The oral administration of 14C-methanol to rats is reported
to have resulted in covalent bind ing to hemoglobin. A linear relationship from 10 to 100 umol/ kg was found.

------------------- Reliability: (4) not assignable Only abstract, no detailed data.

(474)

Remark: In clinical cases, the methanol level is low in the last phase of poisoning.

In Rhesus monkeys given a single methanol dose of 1 g/kg, 37 mg(kg*h) was oxidized (Makar et a l, 1968).

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286 Provided that the rate is the sam e in man, the amount of methanol oxidized during 18 h (th e average time needed for development of severe acidosis in clinical cases) would be 0.7 g/kg.

Acc. to Roe (1982), it seems reas onable to regard 1 g/kg of methanol as the approximate minim al lethal dose in man. Reliability: (4) not assignable Secondary source

(475) (284)

Remark: In Rhesus monkeys given a single methano l dose of 1 g/kg, 37 mg(kg*h) was oxidized (Makar et a l, 1968). Reliability: (4) not assignable

(475) (284)

### OECD SIDS METHANOL

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