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Tert-Butanol - Isobutanol

Tert-Butanol - Isobutanol

Environmental Health Criteria 65

BUTANOLS: FOUR - 1- - 2-Butanol - tert-Butanol -

Please note that the layout and pagination of this web version are not identical with the printed version. - four isomers (EHC 65, 1987)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 65

BUTANOLS: FOUR ISOMERS

- 1-Butanol - 2-Butanol - tert-Butanol - Isobutanol

This report contains the collective views of an international group of experts and does not necessarily represent the decisions or the stated policy of the United Nations Environment Programme, the International Labour Organisation, or the World Health Organization.

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization

World Health Orgnization Geneva, 1987

The International Programme on Chemical Safety (IPCS) is a joint venture of the United Nations Environment Programme, the International Labour Organisation, and the World Health Organization. The main objective of the IPCS is to carry out and disseminate evaluations of the effects of chemicals on human health and the quality of the environment. Supporting activities include the development of epidemiological, experimental laboratory, and risk-assessment methods that could produce internationally comparable results, and the development of manpower in the field of . Other activities carried out by the IPCS include the development of know-how for coping with chemical accidents, coordination of laboratory testing and epidemiological studies, and promotion of research on the mechanisms of the biological action of chemicals.

ISBN 92 4 154265 9

The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available.

Page 1 of 75 Butanols - four isomers (EHC 65, 1987)

(c) World Health Organization 1987

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The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries.

The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR BUTANOLS - FOUR ISOMERS: 1-BUTANOL, 2-BUTANOL, tert-BUTANOL, ISOBUTANOL

INTRODUCTION

1-BUTANOL

2-BUTANOL

tert-BUTANOL

ISOBUTANOL

REFERENCES

WHO TASK GROUP MEETING ON ENVIRONMENTAL HEALTH CRITERIA FOR BUTANOLS - FOUR ISOMERS: 1-BUTANOL, 2-BUTANOL, tert-BUTANOL, ISOBUTANOL

Members

Dr B.B. Chatterjee, Calcutta, India

Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood Experimental Station, Abbots Ripton, Huntingdon, United Kingdom (Rapporteur)

Dr R. Drew, Department of Clinical Pharmacology, Flinders University of South Australia, Bedford Park, South Australia, Australia (Chairman)

Dr M.-S. Galina Avilova, Institute of Occupational and Professional Diseases, Moscow, USSR

Dr A.A.E. Massoud, Department of Community, Environmental and Occupational Medicine, Faculty of Medicine, Ain-Shams University, Abbasia, Cairo, Egypt (Vice-Chairman)

Dr A.N. Mohammed, University of Calabar, Calabar, Nigeria

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Dr C.P. Sadarangani, Bader Al Mulla and Brothers, Safat, Kuwait

Secretariat

Ms B. Bender, International Register of Potentially Toxic Chemicals, United Nations Environment Programme, Geneva, Switzerland

Dr K.W. Jager, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (Secretary)

Ms F. Ouane, International Register of Potentially Toxic Chemicals, United Nations Environment Programme, Geneva, Switzerland

NOTE TO READERS OF THE CRITERIA DOCUMENTS

Every effort has been made to present information in the criteria documents as accurately as possible without unduly delaying their publication. In the interest of all users of the environmental health criteria documents, readers are kindly requested to communicate any errors that may have occurred to the Manager of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda, which will appear in subsequent volumes.

* * *

Detailed data profiles and legal files can be obtained from the International Register of Potentially Toxic Chemicals, Palais des Nations, 1211 Geneva 10, Switzerland (Telephone no. 988400 - 985850).

ENVIRONMENTAL HEALTH CRITERIA FOR BUTANOLS - FOUR ISOMERS: 1-BUTANOL, 2-BUTANOL, tert-BUTANOL, ISOBUTANOL

A WHO Task Group on Environmental Health Criteria for Butanols met in Geneva from 11 to 15 November 1985. Dr K.W. Jager opened the meeting on behalf of the Director-General. The Task Group reviewed and revised the draft criteria document and made an evaluation of the health risks of exposure to butanols.

The first draft of this document was partially based on information contained in Toxicology Data Sheets made available by the Health, Safety and Environment Division of Shell Internationale Maatschappij B.V., and on information obtained by a search of data bases by IRPTC. Additional information supplied by IPCS Participating Institutions was added to the second draft.

The efforts of all who helped in the preparation and finalization of the document are gratefully acknowledged.

* * *

Partial financial support for the publication of this criteria document was kindly provided by the United States Department of Health and Human Services, through a contract from the National Institute of Environmental Health Sciences, Research Triangle Park,

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North Carolina, USA - a WHO Collaborating Centre for Environmental Health Effects. The United Kingdom Department of Health and Social Security generously covered the costs of printing.

INTRODUCTION

The butanol isomers occur naturally as products of and are also synthesized from petrochemicals. They are used widely as and intermediates in chemical industries. Human exposure to high concentrations of the butanol isomers will be primarily occupational while exposure to low concentrations will be mainly through foods in which they occur naturally or as flavouring agents. Apart from slight differences in the point and water , the physical properties of the isomers are similar.

With only minor differences between isomers, the for aquatic organisms of all four butanols is low and none of the compounds shows any capacity for bioaccumulation. Apart from tert-butanol, all isomers are readily biodegradable and would be expected to be fully oxidised by microorganisms within a few days. The tert-butanol is metabolized more slowly and would be degraded within a few weeks. The likely background concentrations of all butanol isomers would not have any impact on the aquatic environment.

In animals, the butanols are readily absorbed through the lungs and gastrointestinal tract. 1-Butanol, 2-butanol, and isobutanol are primarily metabolized by dehydrogenase and are rapidly eliminated from the blood. tert-Butanol is not a substrate for and its elimination is slower than that of the other isomers. On the basis of oral LD50 values in the rat, the butanols can be classified as being slightly or practically non-toxic. In large amounts, all isomers have the ability to induce signs of alcoholic intoxication in both animals and man. Data regarding other biological effects in man and animals cannot be easily compared and symptoms and effects are covered in the separate sections for each .

On the basis of the available data, the Task Group did not expect any adverse effects from occupational exposure under conditions of good manufacturing practice.

The Task Group considered that the available data were inadequate to give guidelines for the setting of occupational exposure limits for any of the butanol isomers.

The effects of long-term exposure to low concentrations of the butanols could not be judged because of lack of information. The Task Group recommended that relevant studies should be conducted so that this could be achieved.

ENVIRONMENTAL HEALTH CRITERIA

FOR

1-BUTANOL

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CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR 1-BUTANOL

1. SUMMARY

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1 Identity 2.2 Physical and chemical properties 2.3 Analytical methods

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

6. KINETICS AND

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1 Aquatic organisms 7.2 Terrestrial organisms 7.3 Microorganisms

8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1 Single exposure 8.1.1 Acute toxicity 8.1.1.1 Signs of intoxication 8.2 Skin, eye, and respiratory tract irritation 8.2.1 Skin irritation 8.2.2 Eye irritation 8.2.3 Respiratory tract irritation 8.3 Repeated and continuous exposure 8.3.1 Inhalation studies 8.3.2 Other routes of administration 8.4 Mutagenicity 8.5 Carcinogenicity 8.6 Reproduction, embryotoxicity, and teratogenicity 8.7 Special studies

9. EFFECTS ON MAN

9.1 Toxicity 9.1.1 Eye irritation 9.1.2 Case reports of occupational exposure

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1 Evaluation of human health risks 10.1.1 Exposure levels 10.1.2 Toxic effects

10.2 Evaluation of effects on the environment 10.2.1 Exposure levels 10.2.2 Toxic effects 10.3 Conclusions

11. RECOMMENDATIONS

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

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1. SUMMARY

1-Butanol is a flammable colourless liquid with a rancid sweet odour. It has a of 118 °C, a water solubility of 77 g/litre and its 1-/water partition coefficient is 0.88. Its vapour is 2.6 times denser than air. It occurs naturally as a product of fermentation of carbohydrates. 1-Butanol is also synthesized from petrochemicals and is widely used as an organic and as an intermediate in the manufacture of other organic chemicals. Human exposure is mainly occupational. Exposure of the general population will be mainly through its natural occurrence in foods and beverages, and its use as a flavouring agent. Exposure may also result from industrial emissions. 1-Butanol is readily biodegradeable and does not bioaccumulate. It is not directly toxic for aquatic animals and practically non-toxic for algae. However, some protozoa are slightly sensitive to 1-butanol and it should be managed in the environment as a slightly toxic compound. It poses an indirect hazard for the aquatic environment because it is readily biodegraded and this may lead to depletion.

In animals, 1-butanol is readily absorbed through the skin, lungs, and gastrointestinal tract. It is rapidly metabolized by alcohol dehydrogenase to the corresponding , via the , and to dioxide, which is the major metabolite. The rat oral LD50 for 1-butanol ranges from 0.7 to 2.1 g/kg body weight. It is, therefore, slightly toxic according to the classification of Hodge & Sterner. It is markedly irritating to the eyes, and moderately irritating to the skin. The primary effects from exposure to vapour for short periods are various degrees of irritation of the mucous membranes, and central nervous system depression. Its potency for intoxication is approximately 6 times that of . A variety of investigations have indicated the non-specific membrane effects of 1-butanol. Effects of repeated inhalation exposure in animals include pathological changes in the lungs, degenerative lesions in the liver and kidneys, and narcosis. However, it is not possible to determine a no-observed-adverse- effect level on the basis of the animal studies available. 1- Butanol has been found to be non-mutagenic. Adequate data are not available on its carcinogenicity, teratogenicity, or effects on reproduction.

The most likely acute effects of 1-butanol in man are alcoholic intoxication and narcosis. Signs of excessive exposure may include irritation of the eyes, nose, throat, and skin, headache, and drowsiness. Vertigo has been reported under conditions of severe and prolonged exposure to vapour mixtures of 1-butanol and isobutanol. However, in this study, it was not possible to attribute the vertigo to a single cause. It has been reported that exposure to 1-butanol may affect hearing and also light adaptation of the eye.

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1. Identity

Chemical structure: CH3-CH2-CH2-CH2OH

Chemical formula: C4H10O

Primary constituent: 1-butanol

Common synonyms: 1-butyl alcohol, butanol-1, normal-butyl alcohol, 1-hydroxy , normal-propyl

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carbinol, butyric alcohol, NBA, butan-1- ol, butyl alcohol

CAS registry number: 71-36-3

2.2. Physical and Chemical Properties

Some physical and chemical properties of 1-butanol are listed in Table 1.

Table 1. Physical and chemical properties of 1-butanol ------(at 20 °C and 101.3 kPa, unless otherwise stated)

Physical state colourless liquid Odour rancid sweet Odour threshold approximately 3.078 mg/m3 Relative molecular mass 74.12 Density (kg/m3) 809 - 811 Boiling point 118 °C Freezing point -89 °C (mPa x s) 2.96 Vapour density (air = 1) 2.55 Vapour pressure (kPa) 0.56 Flashpoint (°C) 33 Autoignition temperature 345 °C Explosion limits in air (v/v) lower = 1.4% upper = 11.2% Solubility (% weight) in water, 7.7; miscible with ethyl alcohol, , and other organic solvents n-octanol/water partition coefficient 0.88

Conversion factors 1 ppm = 3.078 mg/m3 1 mg/m3 = 0.325 ppm ------

2.3. Analytical Methods

NIOSH Method No. S66(321) has been recommended for the determination of 1-butanol. It involves drawing a known volume of air through charcoal to trap the organic vapours present (recommended sample is 10 litres at a rate of 0.2 litre/min). The analyte is desorbed with carbon containing 1% 2-propanol. The sample is separated by injection into a chromatograph equipped with a flame ionization detector and the area of the resulting peak is determined and compared with standards (NIOSH, 1977).

A gas-chromatographic separation and determination method for 1-, sec-, and tert-butanols, with a sensitivity of 1 mg/m3 was reported by Abbasov et al. (1971).

Testing methods for the butanols (ASTM D304-58) are described in ASTM (1977).

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

1-Butanol is used as an ingredient in perfumes and flavours (Mellan, 1950), and for the extraction of: hop, lipid-free protein from egg yolk (Meslar & White, 1978), natural flavouring materials and vegetable oils, perfumes (Mellan, 1950), , and oligosaccharides from plant tissue (Sodini & Canella, 1977), and

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as a solvent in removing pigments from moist curd leaf protein concentrate (Bray & Humphries, 1978). 1-Butanol is also used as: an extractant in the manufacture of antibiotics, hormones, and vitamins (Mellan, 1950; Doolittle, 1954; Yamazaki & Kato, 1978), and of rhenium (Gukosyan et al., 1979); a solvent for , coatings, natural resins, gums, synthetic resins, dyes, , and camphor (Mellan, 1950; Doolittle, 1954); a cleanser for moulded contact lenses (Mizatani et al., 1978); an intermediate in the manufacture of butyl acetate, dibutyl , and dibutyl sebacate (Mellan, 1950; Doolittle, 1954) as well as of the of herbicides (e.g., 2,4-D, 2,4,5-T) (Monich, 1968). Other miscellaneous applications of 1-butanol are as a swelling agent in textiles, as a component of brake fluids, cleaning formulations, degreasers (Monich, 1968; Sitanov et al., 1979), and repellents (Zaikina et al., 1978); and as a component of ore floation agents (Monich, 1968), of protective coatings for glass objects (Artigas Gimenez et al., 1979) and of wood-treating systems (Amundsen et al., 1979). Mixed with xylene, it is used to produce a glass substitute that can be used for sunglasses, safety glasses, windows for airplanes and others (Ferri, 1979). 1-Butanol is also used as an additive to increase the fineness of ground cement (Tavlinova & Dovyborova, 1979) and as a solvent in the purification of polyolefins (Takeuchi et al., 1978). It may be liberated during photographic processing operations.

A further use of 1-butanol is as a flavouring agent in butter, cream, fruit, , rum, and whiskey. Other foods in which it is used include: beverages (12 mg/litre maximum), ice cream and ices (7 mg/kg maximum), candy (34 mg/kg maximum), baked goods (32 mg/kg maximum), cordials (1 mg/litre maximum), and cream (4 mg/kg maximum) (Hall & Oser, 1965).

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

A high rate of degradation of 1-butanol has been found in a wide range of test methods. The data in Table 2 suggest a high proportion of the total oxygen required for its complete oxidation is taken up within a few hours and degradation would be complete within a few days. Its biodegradation in surface waters may present a hazard in terms of oxygen depletion.

At a concentration of 20 mg/litre, butanol gives a strong unpleasant odour to drinking-water. The odour threshold is 1 mg/litre (Nazarenko, 1969).

No data are available on distribution in soil, sediments, or air. Table 2. Biodegradation data for 1-butanol ------activated 36% of ThOD removed in 24 h by Gerhold & Malaney sludge unadapted municipal sludge (1966)

44% of ThOD removed in 23 h by McKinney & Jeris adapted sludge (1955)

biodegradation rate in adapted sludge Pitter (1976) at 20 °C, 84.0 mg COD/g per h

5d BOD 68% of ThOD in fresh water Price et al. (1974)

45% of ThOD in synthetic sea water Price et al. (1974)

5d BOD 33% of ThOD (AFNOR Test) Dore et al. (1974)

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66% of ThOD (APHA Test) Bridie et al. (1979b) anaerobic degraded by acetate-enriched methane Chou et al. (1978a,b) digestion culture after adaptation, 100% of ThOD removed at 100 mg/1itre per day after 4 days of adaptation; 98% of ThOD removed at 80 mg/1itre in anaerobic upflow filters (hydraulic residence time 2 - 10 days) after 52 days of adaptation ------ThOD = theoretical oxygen demand - the calculated amount of oxygen needed for complete oxidation to water and .

COD = chemical oxygen demand - measures the chemically oxidizable matter present.

BOD = biochemical oxygen demand - a simple bioassay measuring the potential deoxygenating effect of biologically oxidizable matter present in an effluent. 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

1-Butanol and other congeners occur naturally as a result of carbohydrate fermentation in a number of alcoholic beverages including beer (Bonte, 1979), grape brandies (Schreier et al., 1979), apple brandies (Woidich et al., 1978), (Bikvaloi & Pasztor, 1977; Bonte, 1978), and whisky (Pastel & Adam, 1978). It has been detected in the volatiles of the following products: hops (Tressl et al., 1978), jack fruit (Swords et al., 1978), heat- treated milks (Juddou et al., 1978), muskmelon (Yabumoto et al., 1978), cheese (Dumont & Adda, 1978), southern pea seed (Fisher et al., 1979), and cooked rice (Yajima et al., 1978). 1-Butanol is also formed during deep frying of corn oil, cottonseed oil, trilinolein, and triolein (Chang et al., 1978). The production or, in some cases, use of the following substances may result in exposure to 1-butanol: artificial leather, butyl esters, rubber cement, dyes, fruit essences, lacquers, motion picture and photographic films, raincoats, perfumes, pyroxylin plastics, rayon, safety glass, shellac varnish, and waterproofed cloth (Tabershaw et al., 1944; Cogan & Grant, 1945; Sterner et al., 1949; Mellan, 1950; Doolittle, 1954). It has also been detected by gas chromatographic methods in waste obtained during the boiling and drying of oil (Novokonskaya et al., 1978), and it is released from linoleum plasticized with poly(dibutyl maleate) (Moshlakova et al., 1976) and from hardened parquet lacquer (Dmitriev & Michahikin, 1979).

Whilst testing the air of mobile homes for the presence of organic chemicals, 1-butanol was found with a frequency of 47%; the mean concentration was 5 ppb and the range 0.07 - 26 ppb (Connor et al., 1985).

An industrial emission study indicated that 616 tonnes of 1- butanol were released into the air, over 1 year, in The Netherlands (Anon, 1983).

6. KINETICS AND METABOLISM

1-Butanol is readily absorbed through the lungs, skin, and intestinal tract (Sander, 1933; Theorell & Bonnichsen, 1951; Winer, 1958; Merritt & Tomkins, 1959; Wartburg et al., 1964) and is primarily eliminated after metabolism by alcohol and aldehyde

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dehydrogenases.

It has been shown that 1-butanol disappeared rapidly from the blood of rats. After an oral dose of 2000 mg/kg body weight, the maximum blood-alcohol concentration was 500 mg/litre after 2 h. The concentration dropped to 150 mg/litre after 4 h and only 0.03% of the dose was excreted in the urine after 8 h (Gaillard & Derache, 1965).

In a study on rabbits, it was stated that aliphatic appeared to be metabolized and eliminated from the body by:

(a) oxidation and elimination of the products (, , , and carbon dioxide) in the urine and expired air;

(b) conjugation as glucuronide or sulfate and elimination of the products in the urine; and

(c) elimination of the unchanged alcohol in the expired air or urine.

In the case of 1-butanol, though no specific numbers concerning the expired air were given, it was found to oxidize to the corresponding acid via the aldehyde, and to carbon dioxide (CO2); 1.8% of the alcohol was excreted conjugated with within 24 h (Kamil et al., 1953).

According to DiVincenzo & Hamilton (1979), rats dosed, by gavage, with 450 mg 1-butanol/kg body weight excreted 83.3% of the dose as carbon dioxide (CO2), at 24 h. Less than 1% was eliminated in the faeces, 4.4% was excreted in the urine, and 12.3% remained in the carcass. Similar excretion patterns were observed at 45 and 4.5 mg/kg body weight. About 75% of 1-butanol excreted in the urine was in the form of o-sulfate (44%) or o-glucuronide (30%). 1-Butanol was absorbed through the skin of dogs at a rate of 8.8 µg/min per cm2; dogs exposed by inhalation to 1-butanol vapour at 53.9 mg/m3 (50 ppm) over 6 h absorbed about 55% of the inhaled vapour. When administered orally to rats, 14C-labelled butanol was found in the liver, kidneys, small intestine, and lungs, 1 h after administration. A decrease in the radioactivity was observed in the organs 4 h later. During the first 3 days, 95% of 14C was excreted from the body; however, only 2.8% of 14C was eliminated in the urine and faeces combined (Rumyanstev et al., 1975).

When administered intraperitoneally (ip) to rats in a single dose, 1-butanol accumulated in the brain nuclei and liver nuclei and, at a slower rate and reaching a lower maximum concentration, in mitochondria (Mikheev et al., 1977).

Excretion of 1-butanol in the breath and urine of rabbits following an oral dose of 2 ml/kg body weight was less than 0.5% of the dose administered in each case (Patty, 1982). In a 1-month study, 1-butanol, administered 5 times/week to mice at 0.1 - 0.5 of the LD50, showed cumulative properties (Rumyanstev, 1976). The elimination of 1-butanol from the perfusate of isolated rat liver was a zero-order process above the concentration of 0.8 mmol and a first-order process below this concentration (Auty & Branch, 1976).

In an in vitro study using rat liver slices, it was reported that 1-butanol was oxidised by alcohol dehydrogenase. At the concentration of 1-butanol tested (25 µl/500 mg liver per 2 ml incubate), CO2 production was decreased by approximately 60% and

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the lactate/pyruvate ratio in the medium was increased ten fold (Forsander, 1967).

The in vitro metabolism of 1-butanol by rat hepatic microsomes has been studied by Teschke et al. (1974) and Cederbaum et al. (1978, 1979). The first authors showed that hepatic microsomes catalysed the oxidation of 1-butanol to its aldehyde by means of a reaction requiring molecular oxygen and NADPH. This reaction was inhibited by carbon monoxide. A direct demonstration of the role of peroxide (H202) in the cytochrome P-450-mediated pathway stems from the observation that reagent H2O2 added to microsomal preparations stimulated the oxidation of butanol (Cederbaum et al., 1978). Indirect evidence was provided by the observation that azide, which prevents the decomposition of H2O2 by , actually stimulated the oxidation of 1-butanol. Thiourea, a compound that reacts with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of 1-butanol to a similar extent, in both the absence and presence of the catalase inhibitor azide (Cederbaum et al., 1979). Achrem et al. (1978) showed that the hydroxyl ion from butanol interacted with the Fe of haem in cytochrome P-450.

Twelve human volunteers were exposed for 2 h to 1-butanol at 300 or 600 mg/m3 in inspired air during rest and during exercise (50, 100, or 150 w) on a bicycle ergometer. At the highest dose level, the difference between levels in inspired and expired air indicated an uptake of 47% 1-butanol at rest, and 37, 40, and 41% at 50, 100, and 150 w, respectively. After 30 min exposure to 300 or 600 mg/m3, the 1-butanol concentrations in the arterial blood were 0.3 and 0.5 mg/litre, respectively. The combination of an apparently high uptake and low concentrations in arterial blood is probably because 1-butanol is dissolved in the water of the dead space mucous membranes (Astrand et al., 1976).

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1. Aquatic Organisms

Toxicity data for aquatic organisms are given in Table 3. Table 3. Table of acute toxicity data for fresh-water organisms ------Species Concentration Parameter Comments Reference (mg/litre) ------Fish

Fresh-water species

Creek chub 1900 - 2300 24-h LC50 Gillette et al (Semotitus atromaculatus)

Golden orfe 1200 48-h LC50 Juhnke & Lüdem (Leuciscus idus (1978) melanotus)

Goldfish 1900 24-h LC50 Bridié et al. (Carassius auratus)

Fathead minnow 1730 - 1910 96-h LC50 Mattson et al. (Pimapheles promelas) Veith et al. 1983)

Bleak 2250 - 2400 96-h LC50 Linden et al.

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(Alburnus alburnus) Bengtsson et

Amphibia

Tadpole 2820 threshold for Münch (1972) ( Rana sp.) narcosis

Invertebrates

Fresh-water species

Water flea 1880 24-h EC50 immobilization Bringmann & Ku (Daphnia magna) (1982)

Harpacticoid copepod 1900 - 2300 96-h LC50 Mattson et al. (Nitocra spinipes) Bengtsson et

Marine species

Brine shrimp 2950 24-h LC50 Price et al. ( (Artemia salina) 2600 excystment Smith & Siege inhibited ------

Table 3. (contd.) ------Species Concentration Parameter Comments Reference (mg/litre) ------Algae (fresh-water)

Green algae (Scenedesmus 875 8-day no- total biomass Bringmann & K quadricauda) observed- (1978a) adverse- effect level

Chlorella 8500 EC50 Jones (1971) pyrenoidosa I chlorophyll content

Blue-green algae (Microcystis 100 8-day no- total biomass Bringmann & K aeruginosa) observed- (1978a) adverse- effect level ------LC50 data for 5 species of fish range from 1000 to 2400 mg/litre and for 3 species of aquatic invertebrates from 1880 to 2950 mg/litre. These concentrations are unlikely to be achieved in the field except locally after accidental spills or through effluence from industrial sites; even under these conditions, the high levels of contamination would not last long. Fresh-water algae are very resistant to the toxic effects of 1-butanol at realistic exposures.

Hill et al. (1981) looked at effects of 1-butanol on goldfish with a conditioned reflex of avoiding light followed by electric shock. The fish were housed in a tank separated into 2 compartments by a plate with a hole big enough for the fish to swim through. Training involved a 10-s light pulse followed by a 20-s shock to one side of the tank. Experimental concentrations of 1-butanol (2.5 - 15 mmol) were applied to the tanks and fish were exposed to step-wise increases (2.5 mmol) in concentration

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with approximately 1.5 h between each step (a 15 mmol concentration of 1-butanol is 58% of the 24 h LC50 for this species). Two responses were scored; "avoidance", defined as the fish leaving the test side of the tank during the light stimulus and before the shock, and "escape", defined as leaving during the 20-s shock. With each step up to 10 mmol butanol, there was a transitory reduction in avoidance. At 10 mmol or higher concentrations, there was also a fall in escape response. Recovery to control scores after first exposure to 10 mmol butanol was slow and incomplete after 2 h. 1-Butanol at 15 mmol, achieved either step-wise or by single application, led to a dramatic and non-recoverable fall in both avoidance and escape to approximately 20% of control values. Escape and avoidance success was correlated with brain levels of butanol. Final concentrations of butanol in the fish brain were

75% of those expected, assuming complete equilibrium with tank water. This compares with 90% for ethanol in the same species. Measuring of brain butanol over a longer period indicated that butanol was metabolized by the goldfish in a similar way to ethanol (Hill et al., 1980). The concentrations of the alcohol that produced effects in these studies are high compared with likely exposure levels in natural waters.

7.2. Terrestrial Organisms

Seed germination in lettuce (Lactuca sativa) was inhibited by 50% at a concentration of 1-butanol of 390 mg/litre (Reynolds, 1977). Seed germination in cucumber (Cucumis sativus) was inhibited at 2500 mg/litre (Smith & Siegal (1975). 1-Butanol had an antisenescence effects on the leaves of oat seedlings (Avena sativa). It both maintained chlorophyll levels and prevented proteolysis in the dark (Satler & Thimann, 1980). There are no relevant data on terrestrial animals; however, as for terrestrial plants, significant exposure to butanol is unlikely.

7.3. Microorganisms

Some toxicity data for microorganisms are given in Table 4. It would be highly unlikely that bacteria would be affected by 1-butanol in the field. Protozoans are more susceptible than bacteria, but only transitory effects on protozoan populations are likely from spills and effluent since the experimental no-observed- adverse-effect levels are high.

1-Butanol at a concentration of 20 mg/litre in water reduced nitrification; a concentration of 5 mg/litre was the no-observed- adverse-effect level for nitrification (Nazarenko, 1969). 1- Butanol does not bioaccumulate (Chiou et al., 1977). Table 4. Toxicity data for microorganisms ------Species Concentration Parameter Comments Refer (mg/litre) ------Protozoa

Uronema parduczi 8 20-h no-observed-adverse- total Brin (ciliate) effect level biomass Kuehn

Chilomonas paramaecium 28 48-h no-observed-adverse- total Brin (flagellate) effect level biomass Kuehn

Entosiphon sulcatum 55 72-h no-observed-adverse- total Brin (flagellate) effect level biomass Kuehn ------

Page 13 of 75 Butanols - four isomers (EHC 65, 1987)

Table 4. (contd.) ------Species Concentration Parameter Comments Refer (mg/litre) ------Bacteria

Pseudomonas putida 650 16-h no-observed-adverse- total Brin effect level biomass Kuehn

Bacillus subtilis 1258 EC50 spore germination Yasu et al

7400 no inhibition of Chou degradation by methane (1978 culture on acetate substrate ------8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1. Single Exposure

8.1.1. Acute toxicity

Some acute toxicity data for experimental animals are given in Table 5.

Table 5. Acute toxicity data for experimental animals ------Species Route LD50 LD100 Reference (g/kg body (g/kg body weight) weight) ------Rabbit dermal 5.3 - Patty (1982) Rabbit dermal 4.2 - Egorov (1972) Rabbit dermal - 7.5 Patty (1963) Hamster oral 1.2 - Dubina & Maksimov (1976) (0.6-2.3)a Mouse oral 2.68 - Rumyanstev et al. (1979) Rabbit oral 3.4 - Münch & Schwartze (1925) Rabbit oral 3.5 - Münch (1972) Rat oral 2.1 - Jenner et al. (1964) Rat oral 0.8-2.0 - Purchase (1969) Rat oral 0.7 - NIOSH (1977a) Rat oral - 4.4 Smyth et al. (1951) Mouse ip 0.1-0.3 - Maickel & McFadden (1979) Rat ip 1.0 - Lendle (1928) Rat ip 0.2 - Macht (1920) Rat ip - 1.0 Browning (1965) Cat iv - 0.24 Macht (1920) Mouse sc - 5.0 Patty (1982) ------a 95% confidence limits for this study.

Rats survived inhalation exposure to 1-butanol at 24 624 mg/m3 (8000 ppm) for 4 h (Smyth et al., 1951). Mice did not show any evidence of toxicity when exposed to 1-butanol at 5078.7 mg/m3 (650 ppm) for 7 h, but exposure to 20 314.8 mg/m3 (6600 ppm) produced signs of marked central nervous system (CNS) depression (narcosis after approximately 2 h) with lethality after 3 h (Patty, 1982).

Male Swiss mice (10 per group) were exposed by inhalation for

Page 14 of 75 Butanols - four isomers (EHC 65, 1987)

4 h to 1-butanol at 1446.7, 1686.7, 2597.8, or 2970.2 mg/m3 (470, 548, 844, or 965 ppm). Following this exposure, the animals were tested in a "behavioural despair" swimming test. Compared with controls, a dose-related decrease in the duration of immobility measured over a 3-min period was observed (de Ceaurriz et al., 1982).

8.1.1.1 Signs of intoxication

The acute toxicity of 1-butanol (Table 5) is moderate in several animal species, and is mainly associated with effects on the CNS. Injection (ip) of 1-butanol in rats induced behavioural effects of intoxication (pronounced ataxia) that were virtually identical to those of ethanol, but the intoxicating potency of 1-butanol was approximately 6 times higher (McCreery & Hunt, 1978). Similarly, after ip administration in mice, 1-butanol was 6 times more potent than ethanol in inducing narcosis (Browning, 1965). The performance in a simple functional test of rats treated with a non-toxic single oral dose of 1-butanol (0.0163 mol/kg body weight) was studied by Wallgren (1960). Rat performance decreased soon after treatment, but recovery was rapid (Wallgren, 1960). In rabbits, the oral administration of 2.1 - 2.44 g 1-butanol/kg body weight caused deep and rapid narcosis; in mice, the narcotic dose by ip injection was 0.76 ml/kg body weight (compared with 4.5 for ethanol) and 20.31 mg/m3 (6.6 ppm) by inhalation (Browning, 1965). In conscious rabbits, the effects of 1-butanol on circulatory variables was investigated in double blind studies. 1-Butanol did not produce any significant effect at an intravenous (iv) dosage of 0.008 g/kg body weight. Doses of 0.1 g/kg body weight and particularly of 0.33 g/kg resulted in a transitory decrease in the heart rate and the systolic and especially diastolic blood pressures. Butanol anaesthetized rats and mice at 15.7 and 15.3 mg/litre, respectively; the anaesthesia transiently lowered the blood levels of erythrocytes and haemoglobin. The animals that died during the treatment showed lung haemorrhages and hyperaemia of other parenchymatous organs. The minimum concentration of 1-butanol disturbing conditioned reflexes was 65 mg/m3 (Rumyanstev et al., 1979). In acute studies, when 1-butanol was administered by the oral or ip route, post-mortem findings included marked hyperaemia of the liver, congestion of several organs in animals that died early, and degenerative signs becoming visible in the liver and kidneys of the rats dying after 5 days. Haemorrhagic areas in the lungs and blood changes were also noted. In the kidney, hyperaemia and cloudy swelling with cast formation in the cortex were seen, the only signs of necrosis were in the medulla (Smyth & Smyth, 1928; Purchase, 1969; Maickel & McFadden, 1979). In normothermic dogs, the mean lethal dose for 1-butanol, administered iv, was 1.26 g/kg body weight and, at a constant infusion rate, the blood alcohol level increased almost linearly with time (McGregor et al., 1964).

8.2. Skin, Eye, and Respiratory Tract Irritation

8.2.1. Skin irritation

In a 24-h patch test, application of 405 or 500 mg 1-butanol to the skin of rabbits resulted in moderate irritation (US DHEW, 1978).

8.2.2. Eye irritation

Instillation of 1.62 mg and 20 mg 1-butanol into rabbit eyes resulted in severe irritation after 72 h and 24 h, respectively (US DHEW, 1978).

Page 15 of 75 Butanols - four isomers (EHC 65, 1987)

1-Butanol is an irritant of mucous membranes, especially of the eye and, in man, it causes an unusual form of keratitis (section 9.1.2).

Instillation of 0.005 ml undiluted 1-butanol or an excess of 40% solution in in the rabbit eye caused severe corneal irritation. A 15% solution in propylene glycol caused minor corneal injury (Patty, 1982).

8.2.3. Respiratory tract irritation

On the basis of the effects of 1-butanol on the respiratory rate in male Swiss OF1 mice, de Ceaurriz et al. (1981) predicted that exposure to a concentration of 40.01 mg/m3 (13 ppm) in air would have only a minimal or no effect on man, a concentration of 390.9 mg/m3 (127 ppm) would be uncomfortable, but tolerable, and 3909 mg/m3 (1268 ppm) would be intolerable.

8.3. Repeated and Continuous Exposure

8.3.1. Inhalation studies

Inhalation studies on the effects of 1-butanol on experimental animals are summarized in Table 6.

According to Rumyantsev et al. (1976), the no-observed-adverse- effect level of 1-butanol can be set at 0.09 mg/m3 (0.03 ppm) for both the rat and the mouse under conditions of long-term continuous exposure.

When 3 groups of 3 guinea-pigs were exposed to 1-butanol in air at 307.8 mg/litre (100 ppm), 4 h per day, for 64 days, the number of red blood cells and relative and absolute lymphocyte counts were decreased. Haemorrhagic areas were observed in the lungs of the exposed animals. There were also early degenerative lesions in the liver, as well as cortical and tubular degeneration in the kidneys (Smyth & Smyth, 1928).

Five white mice were exposed to a concentration of 24.3 mg 1-butanol/litre of air (24 624 mg/m3) for a total exposure time of 130 h (number of h per day not specified). Although the exposed animals were narcotized repeatedly, they gained in weight and survived the exposures. Reversible fatty changes were observed in the livers of the mice (Weese, 1928).

Table 6. Inhalation effects on experimental animals ------Species Dose Duration Effects ------Mouse 24624 mg/m3 repeated exposure narcosis; no deaths; reversible fatty (8000 ppm) for several days infiltrations of the liver and the kidne

Guinea- 307.8 mg/m3 64 days, 4 h/day degenerative lesions in liver, kidney, a pig (100 ppm)

Rat 218 mg/m3 5 h/day, 6 days/ during the first 2 months, a decrease in (71 ppm) week for 6 months consumption and delay in the restoration normal body temperature after cooling; d the next 4 months of long-term exposure, increase in O2 consumption and a return normal body temperature after cooling wa

Page 16 of 75 Butanols - four isomers (EHC 65, 1987)

Rat and 6.8 and 4 months decreased sleeping time; stimulated bloo mouse 40.9 mg/m3 continuously cholinesterase; disturbances of reflexes (2.2 and neuromuscular sensitivity of the nervous 13.3 ppm) system; increased thyroid activity and secretion of thyroxine; increases in eosinophile leukocytes in blood after in of adreno-corticotrophin (ACTH)

Rat 0.09 and 92 days after 4 weeks at 21.8 mg/m3, the amount 21.8 mg/m3 continuously and DNA in blood decreased; there was in (0.03 and leukocyte luminescence, increased diasta 7.1 ppm) activity, decreased catalase activity, increased penetration of butanol across tissue barriers in testis, spleen, and t no effects were observed at 0.09 mg/m3

Mouse 13.6 and 30 days decreased sleeping time 40.01 mg/m3 continuously (2.1 and 13 ppm)

Rabbit -- prolonged mild bronchial irritation with some enla exposure of bronchial lymphnodes ------8.3.2. Other routes of administration

The neuropharmacological effects of 1-butanol were investigated in 31 male Sprague Dawley rats weighing between 200 - 400 g. 1-Butanol was administered iv to the rats at concentrations of 6.7 - 8.1 mmol/kg body weight. Within 20 - 60 s of the iv administration, the rats lost their righting reflexes. Nonconvulsive epileptoid activity was noted in the electroencephalographic tracing compared with the tracing prior to the 1-butanol administration (Marcus et al., 1976).

DiVincenzo & Hamilton (1979) applied 1-14C-1-butanol to the skin of 2 male beagle dogs and observed an absorption rate of 8.8 µg/min per cm2. Application of 42 - 55 ml/kg per day for 1 - 4 consecutive days to the skin of rabbits resulted in 100% mortality. However, repeated applications of 20 ml/kg per day for 30 days over a period of 6 weeks did not produce any fatalities (Patty, 1982).

According to Cater et al. (1977), the daily oral administration of approximately 500 mg 1-butanol/kg body weight dissolved in corn oil, for 4 days, did not affect the testicular tissues of rats. 1-Butanol caused a significant dose-dependent decrease in rat liver contents of , riboflavin, pyrixodine, , and pantothenic acid, after daily oral administration of 1 or 2 ml/kg body weight for 7 days (Shehata & Saad, 1978). Oral administration of butanol (1 or 2 ml/kg of a 10% aqueous solution) for 7 days to rats significantly increased the cerebral GABA levels in the hemispheres (Saad, 1976).

The influence of 1-butanol on the metabolic status of some rat organs and on the rabbit circulation was studied by Geppert et al. (1976). Rats received 1-butanol im at a dose of 0.1 g/kg body weight per day for 50 days. The metabolic status of some organs was examined and compared with that in control rats that had received NaCl solution (9 g/litre) in an equivalent volume (double blind studies). The tissue levels of metabolites of the adenylic acid-creatine phosphate system, glycogen, , and lactate did not differ significantly between the groups. In the liver, the

Page 17 of 75 Butanols - four isomers (EHC 65, 1987)

tissue levels of glycogen, free creatine, and total creatine were significantly elevated in rats that had received 1-butanol.

A group of 30 male Wistar rats was exposed to 1-butanol in the drinking-water (69 g/litre), which also contained (250 g/litre). A control group of equal size was used for comparison. Electron microscopic studies that were carried out after 5, 9, and 13 weeks demonstrated that, within 5 weeks, 1-butanol at this dose level gave rise to the formation of irregularly shaped megamitochondria in liver cells. It was speculated that this was an adaptive process (Wakabayashi et al., 1984). Ethanol and 1-propanol, both at 320 g/litre, produced similar effects under the same test conditions.

8.4. Mutagenicity

1-Butanol was found not to be mutagenic in the Ames Salmonella/ microsome test (McCann et al., 1975). It inhibited the initiation of a new cycle of DNA replication in E. coli but permitted the completion of DNA replication initiated before the addition of 1-butanol to the medium (Patty, 1982). In spite of the fact that the lymphocytes of alcoholic patients exhibit higher incidences of exchange-type aberrations of the chromosome and the chromatid type compared with controls, several alcohols tested including 1-butanol do not produce any effects on the chromosomes of human lymphocytes in culture (Obe et al., 1977). Obe & Ristow (1977) showed that 1-butanol does not affect sister chromatid exchange in Chinese hamster cells in vitro. In the same cell system, ethanol was also found to be inactive, but acetaldehyde induced sister chromatid exchanges; butyric aldehyde was not tested.

1-Butanol was negative in a sister chromatid exchange test using avian embryos (Bloom, 1981).

8.5. Carcinogenicity

Although two long-term studies on rats have been recorded by the US National Cancer Institute, both of these studies were inadequate, by present standards, for the assessment of the carcinogenicity of the substance. No adequate data on carcinogenicity are available.

8.6. Reproduction, Embryotoxicity, and Teratogenicity

No relevant data on the effects of 1-butanol on reproduction, embryotoxicity, and teratogenicity have yet been published. An inhalation teratology study with 1-butanol is in progress in the USA (US EPA, personal communication, 1985).

8.7. Special Studies

Various investigations have indicated that 1-butanol exerts non-specific effects on biological membranes. Evidence of reversible functional derangement of cell membranes by 1-butanol was provided by Stark et al. (1983) and Shopsis & Sathe (1984). The first group of authors was also able to demonstrate cytotoxicity in cultured corneal endothelial and hepatoma cells.

The interaction of 1-butanol with rat liver microsome membranes was studied by Birkett (1974) using a microsome-bound fluorescent probe. 1-Butanol decreased the fluorescent binding to the microsomal membrane, possibly because of a changed net charge on the membrane. 1-Butanol increased the fluidity of Chinese hamster cell plasma membranes (as measured by fluorescence polarization) at

Page 18 of 75 Butanols - four isomers (EHC 65, 1987)

concentrations that inhibited cell adhesion (Juliano & Gagalang, 1979). Moreover, 1-butanol reduced binding to phosphatidylserine or cardiolipin vesicles to the same extent (Puskin & Martin, 1978). These authors also reported that 1-butanol increased cholestane mobility in phosphatidylserine vesicles, thus indicating a more fluid bilayer.

1-Butanol inhibited several rat microsomal metabolic activities in vitro including ethoxycumarin deethylation (Aitio, 1977) and the activity of aldrin epoxidase (Wolff, 1978). A sex difference in the spectral interaction of 1-butanol with liver microsomes from adult mice has been reported by Van den Berg et al. (1979a). In males, a profound reverse type I spectrum was elicited, whereas only a small spectral change of irregular shape was apparent in females. No sex difference was found in immature animals. 1-Butanol also interfered with both type II (aniline) and type I (ethylmorphine) binding in mouse liver microsomes. The apparent dissociation constant of 1-butanol for type I binding was 30 mmol (Van den Berg et al., 1979b).

Prostaglandin biosynthesis requires the presence of a hydroxyl . 1-Butanol was shown to be a hydroxyl radical scavenger and, therefore, an inhibitor of the biosynthesis of prostaglandins. A test system containing microsomes was prepared from bovine vesicular glands. In the presence of epinephrine, incorporation of 14C-eicosa-8,11,14-trienoic acid into prostaglandins was 16.4% for prostaglandin E and 23.4% for prostaglandin F. When 0.025 ml of 1-butanol was added to this incubation system, the amounts incorporated were 7.8% and 9.1%, respectively (Panganamala et al., 1976). In another study using isolated perfused rat lung, the infusion of low concentrations of 1-butanol (0.002 - 0.2 mmol) resulted in maximum release of prostaglandins into the venous effluent at the lowest concentration tested. However, there was a gradual decrease in the prostaglandin output as the concentration of the alcohol increased (Thomas et al., 1980).

Adult male Swiss Cox mice (20 - 25 per group) were dosed orally by intubation with 1-butanol in distilled water at levels of 0.5, 1.0, or 2.0 g/kg body weight in one single dose. This caused a dose-related hypothermia and impairment of rotarod performance. Repetitive doses, at 24 to 72-h intervals did not lead to the development of tolerance in relation to these effects (Maickel & Nash, 1985).

1-Butanol caused relaxation of the canine basilar artery, whereas linear alcohols with fewer carbon atoms above a threshold of 10-2 mol caused contraction (De Felice et al., 1976). 1-Butanol, applied to the lateral olfactory tract of the guinea-pig as a dilute suspension (0.1 - 0.2 mmol), blocked the nerve impulse (Hesketh et all., 1978). The compound also inhibited the contraction of the CaCl2-depolarized guinea-pig ileum (Yashuda et al., 1976) and prolonged frog miniature end-plate currents (Ashford & Wann, 1979).

1-butanol can potentiate the toxicity of carbon tetrachloride (Cornish & Adefuin, 1967) in Sprague Dawley rats.

9. EFFECTS ON MAN

9.1. Toxicity

The most important effects of 1-butanol inhalation are symptoms of alcohol intoxication and narcosis (Smyth, 1956).

Page 19 of 75 Butanols - four isomers (EHC 65, 1987)

Following exposure to 1-butanol vapours, the signs of in human beings, may include irritation of the nose, throat, and eyes, the formation of translucent vacuoles in the superficial layers of the cornea, headache, vertigo, and drowsiness. Defatting of the skin leading to contact dermatitis involving the fingers and hands may also occur, as with other solvents.

9.1.1. Eye irritation

Tabershaw et al. (1944) reported that exposure to levels of more than 153.9 mg/m3 (50 ppm) resulted in irritation of the eyes. However, results of a 10-year study revealed few or no complaints of irritation among workers exposed to an average 1-butanol concentration of 307.8 mg/m3 (100 ppm) (Sterner et al., 1949).

9.1.2. Case reports of occupational exposure

In a raincoat manufacturing plant, the solvent used for the cementing process was 1-butanol, to which various amounts of and denatured alcohol were added. Of the 35 employees working in the department, 28 were found to have from 10 to 1000 vacuoles in the corneal epithelium. The affected workers complained of epiphora and burning and itching of the eyes. Swelling of the eyelids and occasional redness of the eyes were also observed. The symptoms were more severe on awakening in the morning than during the day. When the patients were away from work, the corneal changes were considerably improved and resolved completely in 10 days (Cogan & Grant, 1945). The authors presumed that these symptoms were caused by 1-butanol, but stated that the other components might also be responsible for the symptomatology.

The physical condition of workers exposed to 1-butanol was followed for 10 years. At the beginning of the study, when the concentration of 1-butanol was 615.2 mg/m3 (200 ppm) or more, corneal inflammation was occasionally observed. The symptoms included a burning sensation that could continue for several days after cessation of exposure, blurring of the vision, lachrymation, and photophobia. These symptoms began in the middle of the working week and became more severe towards the end of the week. In addition, the mean erythrocyte count was slightly decreased. Later in the study, after the average concentration was reduced to 307.6 mg/m3 (100 ppm), no systemic effects were observed. Complaints of irritation of the eyes or disagreeable odour were rare at this concentration (Sterner et al., 1949).

Velazquez et al. (1969) reported that prolonged exposure (3 - 11 years) to 1-butanol in a acetate ribbon factory had caused hearing loss in 9 out of 11 exposed workers. Following this finding, the 1-butanol level in the working atmosphere was found to be 246.2 mg/m3 (80 ppm). However, this level may not be representative of the past exposure.

Seitz (1972) reported 7 case histories, which occurred between 1965 and 1971, concerning workers who had been exposed to 1-butanol and isobutanol in a non-ventilated photographic laboratory. They handled the alcohols under intense and hot light without any precautions. Exposure levels were not quantified but must have been excessive, exposure time ranged from 1 1/2 months to 2 years. Two workers had transient vertigo, 3 severe Meniere-like vertigo with nausea, vomiting and/or headache. In one of these cases, hearing was also perturbed. Two workers did not have any signs or symptoms. For ACGIH (1980), the last two papers were the reason for the reduction of the TLV from 307.8 to 153.9 mg/m3 (100 ppm to 50

Page 20 of 75 Butanols - four isomers (EHC 65, 1987)

ppm).

Several papers concerning clinical observations on workers exposed to mixtures of solvents including 1-butanol have been published (Kalekin & Brichenko, 1972; Petrova & Vishnevskii, 1972; Sanatina, 1973; Shalaby et al., 1973; Kudrewicz Hubicka et al., 1978; Zaikov & Bobey, 1978). Pathological observations included effects on the central nervous system, liver, respiration, blood composition, and complications during pregnancy. However, it is not possible to judge whether these effects were due to exposure to 1-butanol.

Baikov & Khachaturyan (1973) recommended the maximum permissible concentration of 1-butanol in ambient air to be set at a level of about 0.09 mg/m3 (0.03 ppm), as a result of studies with 18 volunteers exposed to 1-butanol vapour at levels of between 0.3 and 15 mg/m3. Five concentrations of 1-butanol vapour were tested. At 1.2 mg/m3, 1-butanol changed the light sensitivity of the dark- adapted eye and the electrical activity in the brain. At 1 mg/m3, these parameters were unaffected.

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1. Evaluation of Human Health Risks

10.1.1. Exposure levels

General population levels of exposure to 1-butanol through food and beverages are not available. Occupational levels of exposure to 1-butanol are limited and inadequate.

10.1.2. Toxic effects

1-Butanol is readily absorbed through the skin, lungs, and gastrointestinal tract. In animals, 1-butanol is rapidly metabolized by alcohol dehydrogenase to the corresponding acid, via the aldehyde, and to carbon dioxide, which is the major metabolite. The rat oral LD50 for 1-butanol ranges from 0.7 to 2.1 g/kg body weight; it is, therefore, slightly toxic according to the classification of Hodge & Sterner. It is markedly irritating to the eyes and moderately irritating to the skin. The primary effects from exposure to vapour for short periods are various levels of irritation of the mucous membranes and central nervous system depression. Its potency for intoxication is approximately 6 times that of ethanol. A variety of investigations have indicated non-specific membrane effects of 1-butanol. Effects of repeated inhalation exposure in animals include pathological changes in the lungs, degenerative lesions in the liver and kidneys, and narcosis. However, from the animal studies available, it is not possible to determine a no-observed-adverse-effect level. 1-Butanol has been found to be non-mutagenic. No adequate data are available on carcinogenicity, teratogenicity, or effects on reproduction.

In man, 1-butanol, in the liquid or vapour phase, can cause moderate skin irritation and severe eye irritation manifested as a burning sensation, lachrymation, blurring of vision, and photophobia. Ingestion of the liquid or inhalation of the vapour may result in headache, drowsiness, and narcosis. The occurrence of vertigo under conditions of severe and prolonged exposure to vapour mixtures of 1-butanol and isobutanol has been reported. From this study, it was not possible to attribute the vertigo to a single cause. The symptoms were reversible when exposure ceased.

The minimal information available suggests that occupational

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human exposure to air concentrations below 307.8 mg/m3 (100 ppm) is not associated with any adverse symptoms. However, studies on human volunteers indicate that the light-sensitivity of dark- adapted eyes and electrical activity of the brain may be influenced by air concentrations as low as 0.092 mg/m3 (0.03 ppm).

10.2. Evaluation of Effects on the Environment

10.2.1. Exposure levels

No quantitative data on levels in the general environment are available but, because 1-butanol is readily biodegradable, substantial concentrations are only likely to occur locally in the case of major spillages.

10.2.2. Toxic effects

At background concentrations likely to occur in the environment, 1-butanol is not directly toxic for fish, amphibia, or crustacea and is practically non-toxic for algae. Some protozoa are slightly sensitive to 1-butanol.

1-Butanol should be managed in the environment as a slightly toxic compound. It poses an indirect hazard for the aquatic environment, because it is readily biodegradable, which may lead to oxygen depletion.

10.3. Conclusions

1. On the available data, the Task Group was unable to make an assessment of the health risks of 1-butanol for the general population; however, it was considered unlikely to pose a serious hazard under normal exposure conditions.

2. The Task Group was of the opinion that sufficient data were not available to establish guidelines for setting occupational exposure limits. There are reports of adverse effects resulting from occupational overexposure to levels above 307.8 mg/m3 (100 ppm); therefore, and in line with good manufacturing practice, exposure to 1-butanol should be minimized.

3. The ecotoxicological data available indicate that the impact of background concentrations of 1-butanol on the aquatic environment can be expected to be minimal.

11. RECOMMENDATIONS

1. The Task Group noted that, from the animal studies available, it was not possible to determine a no-observed-adverse-effect level. Relevant studies should be conducted so that this can be achieved.

2. Information on residue and emission levels is desirable.

3. Epidemiological studies, including precise exposure data, would assist in a better assessment of the occupational hazard of 1-butanol.

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

In 1974, the Council of Europe established an acceptable daily intake (ADI) of 1 mg/kg body weight for butan-1-ol.

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More recently (Council of Europe, 1981), specific limits of 30 mg butan-1-ol/kg in beverages and food have been established. The Food Additives and Contaminants Committee (UK MAFF, 1978) recommended that residues in food do not exceed 30 mg/kg and required the results of a 90-day oral toxicity study in the rat within two years.

Butan-1-ol was evaluated by the EEC Scientific Committee for Food in 1980. The Committee agreed on the following evaluation:

The available toxicological data relate to metabolism and short-term oral studies in rats. No long-term oral studies are available. The Committee was therefore unable to establish an ADI. Residues occur in food from use as extraction and carrier solvent as well as from natural occurrence, but adequate residue data are not available. The Committee considers the use of this compound temporarily acceptable as an extraction solvent provided the residues are limited to 30 mg/kg food. The Committee requires the provision of an adequate 90-day oral study in rats as well as information on residue levels by 1983 (CEC, 1981).

At their 23rd meeting, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) reviewed the data on 1-butanol. They concluded that:

"There was a lack of data on the effects of long-term oral exposure to 1-butanol. There were some results of studies on workers exposed for periods of up to 11 years to known vapour concentrations, but these were inadequate for setting an ADI for man. The evaluation of this compound was not possible on the basis of the data available. New specifications were prepared, but no toxicological monograph" (WHO, 1980).

ENVIRONMENTAL HEALTH CRITERIA

FOR

2-BUTANOL

CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR 2-BUTANOL

1. SUMMARY

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1 Identity 2.2 Physical and chemical properties 2.3 Analytical methods

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

Page 23 of 75 Butanols - four isomers (EHC 65, 1987)

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

6. KINETICS AND METABOLISM

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1 Aquatic organisms 7.2 Terrestrial organisms 7.3 Microorganisms

8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1 Single exposure 8.1.1 Acute toxicity 8.1.2 Signs of intoxication 8.2 Skin and eye irritation 8.3 Short-term exposures 8.4 Long-term exposures 8.5 Reproduction, embryotoxicity, and teratogenicity 8.6 Mutagenicity 8.7 Carcinogenicity 8.8 Special studies

9. EFFECTS ON MAN

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1 Evaluation of human health risks 10.1.1 Exposure levels 10.1.2 Toxic effects 10.2 Evaluation of effects on the environment 10.2.1 Exposure levels 10.2.2 Toxic effects 10.3 Conclusions

11. RECOMMENDATIONS

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

1. SUMMARY

2-Butanol is a flammable colourless liquid with a characteristic sweet odour. It has a boiling point of 98.5 °C, a water solubility of 12.5%, and an n-octanol/water partition coefficient of 0.61. Its vapour is 2.6 times denser than air. 2-Butanol occurs naturally as a product of fermentation of carbohydrates. It is used for the extraction of fish meal to produce fish protein concentrate, for the production of fruit essences, and as a flavouring agent in food. Human exposure to 2-butanol is mainly occupational. The general population is exposed through its natural occurrence in food and beverages and its use as a flavouring agent. Exposure may also result through industrial emissions.

2-Butanol is readily biodegradeable by bacteria and does not bioaccumulate. It is not toxic for aquatic animals, algae, protozoa, or bacteria. 2-Butanol should be managed in the environment as a slightly toxic compound. It poses an indirect hazard for the aquatic environment, because it is readily biodegraded, which may lead to oxygen depletion.

Page 24 of 75 Butanols - four isomers (EHC 65, 1987)

In animals, 2-butanol is absorbed through the lungs and gastrointestinal tract. No information is available regarding dermal absorption. Approximately 97% of the dose of 2-butanol in animals is converted by alcohol dehydrogenase to the corresponding , which is either excreted in the breath and urine or further metabolized. The rat oral LD50 for 2-butanol is 6.5 g/kg body weight; it is, therefore, practically non-toxic, according to the classification of Hodge & Sterner. The acute toxic effects are ataxia and narcosis. Its potency for intoxication is approximately 4 times that of ethanol. 2-Butanol is irritating to the eyes and non-irritating to the skin. From the animal studies available, it is not possible to determine a no-observed-adverse-effect level. No adequate data are available on mutagenicity, carcinogenicity, teratogenicity, or effects on reproduction.

In man, the most likely acute effect of 2-butanol is alcoholic intoxication. No published data are available concerning other effects in man.

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1. Identity

Chemical structure: OH | CH3-CH-CH2-CH3

Chemical formula: C4H10O

Primary constituent: 2-butanol

Common synonyms: sec-butyl alcohol, secondary butyl alcohol, butylene hydrate, 2-hydroxy butane, methyl ethyl carbinol, butan-2-ol, sec-butanol, SBA, 2-hydroxybutane, CCS 301

CAS registry number: 78-92-2

2.2. Physical and Chemical Properties

Physical and chemical properties of 2-butanol are given in Table 1.

Table 1. Physical and chemical properties of 2-butanol ------(at 20 °C and 101.3 kPa, unless otherwise stated)

Physical state colourless liquid Odour characteristic sweet odour Odour threshold approximately 7.69 mg/m3 (2.5 ppm) Relative molecular mass 74.12 Density (kg/m3) 806 - 808 Boiling point (°C) initial 98.5 (min) dry point 100.5 (max) Freezing point (°C) -115 Viscosity (mPa x s) 3.54 Vapour density (air = 1) 2.55 Vapour pressure (kPa) 1.66 Flashpoint (°C) 23 Autoignition temperature (°C) 406 Explosion limits in air (%) (v/v) lower = 1.7 upper = 9.0

Page 25 of 75 Butanols - four isomers (EHC 65, 1987)

Solubility (% weight) in water, 12.5; miscible with ethyl alcohol and ether

n-octanol/water partition 0.61 coefficient

Conversion factors 1 mg/m3 = 0.325 ppm 1 ppm = 3.078 mg/m3 ------

2.3. Analytical Methods

2-Butanol is usually determined quantitatively using gas chromatography (Abbasov et al., 1971; Bartha et al., 1978; Beaud & Ramuz, 1978).

NIOSH (1977b) Method No S53 (353) has been recommended. It involves drawing a known volume of air through charcoal to trap the organic vapours present (recommended sample is 10 litres at a rate of 0.2 litre/min). The analyte is desorbed with carbon disulfide containing 1% 2-propanol. The sample is separated by injection into a gas chromatograph equipped with a flame ionization detector and the area of the resulting peak is determined and compared with standards.

Testing methods for the butanols (ASTM D304-58) are described in ASTM (1977).

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

The principal use of 2-butanol is as a chemical intermediate for conversion into methyl ethyl ketone, a solvent with a fairly high boiling point (Monich, 1968).

2-Butanol is used for the extraction of fish meal to produce fish protein concentrate. It is also used for the preparation of fruit essence and as a flavouring agent in food (Federal Register, 1977). Very recently, 2-butanol has proved to be useful as a debittering agent for protein hydrolysates (Latasidis & Sïpberg, 1978).

2-butanol is used, to some extent, as a solvent for lacquers, enamels, vegetable oils, gums, and natural resins; it is also used in hydraulic brake fluids, industrial cleaning compounds, polishes, and penetrating oils, and in the preparation of ore-flotation agents and perfumes (Patty, 1963).

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

A high rate of degradation of 2-butanol has been seen in a wide range of test methods. The data in Table 2 suggest that a high proportion of the total oxygen required for its complete oxidation is used within a few hours, and degradation would be complete within a few days. Its biodegradation in surface waters may present a hazard in terms of oxygen depletion.

No data are available on distribution in soil, sediments, or air.

Biodegradation data are given in Table 2. Table 2. Some biodegradation data for 2-butanol ------5d BOD 33% of ThOD (AFNOR) Dore et al. (1974)

Page 26 of 75 Butanols - four isomers (EHC 65, 1987)

83% of ThOD (APHA) Bridié et al. (1979)b activated sludge 9.3% of ThOD removed in 24 h by Gerhold & Malaney unadapted municipal sludge (1966)

58% of ThOD removed in 23 h by McKinny & Jeris (1955) adapted sludge

biodegradation rate in adapted Pitter (1976) sludge at 20 °C, 55.0 mg COD g per h degraded by acetate-enriched Chou et al. (1978) methane culture after adaptation; 100% of ThOD removed at 342 mg/ litre after 14 days of adaptation

93% of ThOD removed at 110 mg/ Chou et al. (1977) litre in anaerobic upflow filters (hydraulic residence time 2 - 10 days) after 52 days of adaptation bioaccumulation 2-butanol does not bioaccumulate Chiou et al. (1977) ------ThOD = theoretical oxygen demand - the calculated amount of oxygen needed for complete oxidation to water and carbon dioxide.

COD = chemical oxygen demand - measures the chemically oxidizable matter present.

BOD = biochemical oxygen demand - a simple bioassay measuring the potential deoxygenating effect of biologically oxidizable matter present in an effluent. 5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

A residue of between 10 and 70 mg 2-butanol/kg has been reported to remain in the dry fish protein concentrate following extraction with this solvent.

2-Butanol has been found in a number of alcoholic beverages including beer (Bonte et al., 1978), wine (Bikvalvi & Pasztor, 1977; Bonte, 1978), apple brandies (Woidich et al., 1978), and grape brandies (Bonte et al., 1978; Schreier et al., 1979); 2-butanol has also been detected in volatiles of cheese (Dumont & Adda, 1978), southern pea seeds (Fischer et al., 1979), and virgin oil (Olias Jimbnez et al., 1978).

An industrial emission study indicated that 33 tonnes of 2-butanol were released into the air of the Netherlands over a period of 1 year (Anon, 1983).

6. KINETICS AND METABOLISM

In rabbits, 2-butanol was oxidized to methyl ethyl ketone, which could be detected in the expired air, and also conjugated to form 2-butyl glucuronide, which could be isolated from the urine (Williams, 1969). 2,3-Butanediol and 3-hydroxy-2-butanone were the main metabolites of 2-butanol found in the blood of rats given 2.2 ml 2-butanol/kg body weight (Dietz, 1980).

Male rabbits were given 2 ml 2-butanol/kg body weight orally, and venous blood samples were analysed after 1, 2, 3, 4, 5, and 10 h. The concentration of 2-butanol peaked within an hour at

Page 27 of 75 Butanols - four isomers (EHC 65, 1987)

about 1 g/litre and disappeared to a trace after 10 h. Unchanged 2-butanol was excreted to the extent of 3.3% of the dose in the breath and 2.6% in the urine. Methyl ethyl ketone, a metabolite, was detected in the blood and reached a maximum level after 6 h; it was excreted in amounts equivalent to 22.3% of the dose in the breath and 4% in the urine (Saito, 1975).

Dietz et al. (1981) developed a pharmacokinetic model to describe the biotransformation of 2-butanol and its metabolites 2-butanone, 3-hydroxy-2-butanone, and 2,3-butanediol (Fig. 1A). Male Sprague Dawley rats were given 2-butanol (2.2 ml/kg body weight, orally) after an overnight fast; blood concentrations of 2- butanol and its metabolites were estimated at various times up to 30 h. Concentrations of 2-butanol reached a maximum (0.59 g/litre) within 2 h and declined to less than 0.05 g/litre after 16 h. As the blood concentration of 2-butanol fell, the concentrations of 2- butanone, 3-hydroxy-2-butanone, and 2,3-butanediol to maximum levels of 0.78, 0.04, and 0.21 g/litre at 8, 12, and 18 h, respectively. Approximately 97% of the 2-butanol dose was converted 2-butanone by alcohol dehydrogenase; the calculated clearance constant for 2-butanol was 0.40 ml/min. In separate studies, the individual metabolites were administered to rats in order to calculate their clearance constants.

2-Butanol exhibited an apparent blood elimination half-life of 2.5 h in rats treated orally with 2.2 ml/kg body weight. With decline in blood-alcohol concentration (maximum level 800 mg/litre, 1 h after administration), there was a rise in 2-butanone levels with 430 mg/litre detected at 1 h and a maximum of 1050 mg/litre detected 4 h after administration of the alcohol (Traiger & Bruckner, 1976).

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1. Aquatic Organisms

Some toxicity data for 2-butanol in aquatic organisms are given in Table 3. Only two LC50 values are available for fish, and these indicate low toxicity for these species. The toxicity of 2-butanol for aquatic invertebrates is equally low. Table 3. Toxicity data of 2-butanol for aquatic organisms ------Species Concentration Parameter Comments Reference (mg/litre) ------Fish

Fresh-water species

Golden orfe 3520 48-h LC50 Juhnke & Lüdema (Leuciscus idus melanotus)

Goldfish 4300 24-h LC50 Bridié et al. (

Page 28 of 75 Butanols - four isomers (EHC 65, 1987)

(Carassius auratus)

Invertebrates

Fresh-water species

Water flea 2300 24-h EC50 immobilization Bringmann & Kue (Daphnia magna)

Marine species

Brine shrimp 3800 EC50 excystment Smith & Siegel (Artemia salina)

Algae

Green algae

(Scenedesmus 95 8-day no- total biomass Bringmann & Ku quadricauda) observed- (1978a) adverse- effect level

Chlorella 8900 EC50 Jones (1971) pyrenoidosa chlorophyll content ------

7.2. Terrestrial Organisms

An EC50 of 650 mg/litre was reported by Reynolds (1977) for seed germination in lettuce (Lactuca sativa). Inhibition of seed germination in cucumber (Cucumis sativus) was observed at 50 375 mg 2-butanol/litre (Smith & Siegel, 1975). There are no relevant data for terrestrial animals, but, as in the case of terrestrial plants, significant exposure to 2-butanol is unlikely.

7.3. Microorganisms

Some toxicity data for microorganisms are given in Table 4. The toxicity of 2-butanol for both protozoa and bacteria is very low. Table 4. Toxicity of 2-butanol for microorganisms ------Species Concentration Parameter Comments Referenc (mg/litre) ------Protozoa

Uronema parduczi 1416 20-h no-observed- total Bringma (ciliate) adverse-effect level biomass (1981)

Chilomonas paramaecium 745 48-h no-observed- total Bringma (flagellate) adverse-effect level biomass (1981)

Entosiphon sulcatum 1282 72-h no-observed- total Bringma (flagellate) adverse-effect level biomass (1981)

Bacteria

Pseudomonas putida 500 16-h no-observed- total Bringma adverse-effect level biomass (1976)

Bacillus subtilis 1630 EC50 spore germination Yasuda-

Page 29 of 75 Butanols - four isomers (EHC 65, 1987)

al. (197 ------8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1. Single Exposure

8.1.1. Acute toxicity

Acute toxicity data for 2-butanol are given in Table 5. 2-Butanol shows low acute toxicity in rodents.

Table 5. Acute toxicity of 2-butanol ------Species Route of LD50 (g/kg Reference administration body weight) ------Rat oral 6.5 US DHEW (1978) Rabbit oral 4.9 Münch (1972) Mouse intraperitoneal 0.8 US DHEW (1978) ------

Six male albino rats were exposed to 2-butanol vapour at 48.5 mg/litre (16 000 ppm) for 4 h. Within 14 days, 5 of the 6 rats died (Smyth et al., 1954).

8.1.2. Signs of intoxication

Signs of intoxication due to 2-butanol exposure include restlessness, ataxia, prostration, and narcosis (Patty, 1963).

The effects of a moderate non-toxic oral dose of 2-butanol (0.0163 mol/kg body weight) was studied in rats using a simple functional test by Wallgren (1960). The intoxicating effect of 2-butanol, compared with that of ethanol (taken equal to 1), was 4.4 on an equimolar basis. Recovery after the treatment was slow.

The livers of mice that died 1 - 3 days following a single intraperitoneal (ip) dose of 2-butanol showed an abnormal brownish coloration in the peripheral areas, the livers of those that died after 4 - 6 days were uniformally dark brown in colour (Maickel & McFadden, 1979).

8.2. Skin and Eye Irritation

2-Butanol is practically not irritant to the skin of the rabbit, but it is irritant to the rabbit eye (Smyth et al., 1954).

8.3. Short-Term Exposures

White mice (15 - 20 g) were repeatedly exposed to 2-butanol vapour at 16.2 mg/litre (5330 ppm) for a total of 117 h. Although the mice were narcotized, they survived.

Six groups of 2 mice each were exposed to 2-butanol vapour at 5 mg/litre (1650 ppm). After 420 min, no signs of intoxication were observed. When increasing concentrations were used with decreasing durations of exposure, ataxia, prostration, and deep narcosis occurred. The time necessary to induce these symptoms was inversely proportional to the level of exposure. At a concentration of 10 mg/litre (3300 ppm), ataxia occurred in 51 - 100 min, prostration in 120 - 180 min, and narcosis in 300 min. At a concentration of 60 mg/litre (19 800 ppm), these signs appeared in 7 - 8 min, 12 - 20 min, and 40 min, respectively. No deaths

Page 30 of 75 Butanols - four isomers (EHC 65, 1987)

were observed in this study (Starrek, 1938, cited in Patty, 1982).

The neurophysiological effects induced by 2-butanol were investigated in 31 male Sprague Dawley rats (200 - 400 g). 2-Butanol was administered intravenously (iv) to the rats at a concentration of 8.1 mmoles/kg body weight. Within 20 - 60 seconds of the iv administration, the rats lost righting reflexes. Some changes were also noticed in the electroencephalographic tracings compared with the tracings prior to the alcohol administration (Marcus et al., 1976).

8.4. Long-Term Exposures

No long-term exposure studies are available.

8.5. Reproduction, Embryotoxicity, and Teratogenicity

No relevant data on reproduction, embryotoxicity, or teratogenicity have yet been published. However, an inhalation teratology study with 2-butanol is in progress in the USA (US EPA, personal communication, 1985).

8.6. Mutagenicity

2-Butanol did not show any mutagenic activity in the Schizosaccharomyces pombe in both the presence and absence of mouse liver microsomes (Abbondandolo et al., 1980).

8.7. Carcinogenicity

No carcinogenicity studies are available.

8.8. Special Studies

The effects of 2-butanol on cell survival were studied in the yeast S. pombe and in V-79 Chinese hamster cells by Abbondandolo et al. (1980). At 5% concentration, 2-butanol decreased the survival of suspended yeast cells, but did not have any effect on monolayer cultures of V-79 cells.

2-Butanol inhibited the contraction of the depolarized guinea- pig ileum induced by (CaCl2) (Yashuda et al., 1976).

2-Butanol can potentiate the toxicity of carbon tetrachloride (CCl4) (Cornish & Adefuin, 1967). This potentiation may be due to the metabolite 2,3-butanediol, which also has this effect (Traiger & Bruckner, 1976; Dietz & Traiger, 1979).

Inhalation exposure of rats to 2-butanol (1539 mg/m3 (500 ppm) for 5 days) resulted in a 47% increase in the cytochrome P-450 levels of kidney microsomes. A maximal increase of 33% in liver microsomal cytochrome P-450 content was seen after inhalation of 2-butanol at 6156 mg/m3 (2000 ppm) for 3 days. This treatment led to a 77% increase in the formation of the preneurotoxic metabolite 2- from 1- by liver microsomes (Aarstad et al., in press).

Rats receiving a single oral dose of 2-butanol at 2.2 ml/kg body weight were sacrificed 16, 20, or 40 h after dosing. A 50 - 97% increase in microsomal acetanilide hydroxylase activity was found. At 40 h, liver cells showed a marked proliferation of smooth endoplasmic reticulum. This stimulation of the drug- metabolizing system may explain, to a certain extent, the

Page 31 of 75 Butanols - four isomers (EHC 65, 1987)

potentiation of CCl4 hepatoxicity by 2-butanol (Traiger et al., 1975).

9. EFFECTS ON MAN

Excessive exposure may result in headache, dizziness, drowsiness, and narcosis (Muir, 1977). No adverse systemic effects due to exposure to 2-butanol have been reported in man (Patty, 1982).

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1. Evaluation of Human Health Risks

10.1.1. Exposure levels

Levels of exposure of the general population to 2-butanol through food and beverages, and occupational exposure levels are not available.

10.1.2. Toxic effects

In animals, 2-butanol is absorbed through the lungs and gastrointestinal tract. No information is available regarding dermal absorption. Approximately 97% of the dose of 2-butanol in animals is converted by alcohol dehydrogenase to the corresponding ketone, which is either excreted in the breath and urine or further metabolized. The rat acute oral LD50 for 2-butanol is 6.5 g/kg body weight; it is, therefore, practically non-toxic according to the classification of Hodge & Sterner. The toxic effects from acute exposure are ataxia and narcosis. The potency of 2-butanol for intoxication is approximately 4 times that of ethanol. It is irritating to the eyes and non-irritating to the skin. From the animal studies available, it is not possible to determine a no- observed-adverse-effect level. No adequate data are available on mutagenicity, carcinogenicity, teratogenicity, or effects on reproduction.

In man, the most likely acute effect of 2-butanol is alcoholic intoxication. No published data are available concerning other effects on man.

10.2. Evaluation of Effects on the Environment

10.2.1. Exposure levels

No quantitative data relating to levels of 2-butanol in the general environment are available, but, because it is readily biodegradable, substantial concentrations are only likely to occur locally in the case of major spillage.

10.2.2. Toxic effects

At the background concentrations likely to occur in the environment, 2-butanol is not toxic for aquatic animals, algae, protozoa, or bacteria, and it should be managed in the environment as a slightly toxic compound. It poses an indirect hazard for the aquatic environment, because it is readily biodegradable, which may lead to oxygen depletion.

10.3. Conclusions

1. The Task Group was unable to make an assessment of the health risks of 2-butanol for the general population on the basis of

Page 32 of 75 Butanols - four isomers (EHC 65, 1987)

available data. However, it was considered that 2-butanol was unlikely to pose a serious hazard, under normal exposure conditions.

2. The Task Group was of the opinion that available data are not sufficient to establish guidelines for setting occupational exposure limits. In line with good manufacturing practice, exposure to 2-butanol should be minimized.

3. The ecotoxicological data available indicate that the impact of background concentrations of 2-butanol on the aquatic environment can be expected to be minimal.

11. RECOMMENDATIONS

The Task Group recommended that:

1. As it was not possible to determine a no-observed-adverse- effect level on the basis of available animal studies, relevant studies should be conducted so that this could be achieved.

2. Information on residue and emission levels is desirable.

3. Epidemiological studies including precise exposure data would assist in an assessment of the occupational hazards from 2-butanol.

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

The Food Additives and Contaminants Committee (UK MAFF, 1978) recommended that residues of butan-2-ol in food should not exceed 30 mg/kg and required the results of a 90-day oral toxicity study in the rat within 2 years.

This compound could not be included in lists 1 or 2 by the Council of Europe and it was included in list 3A (Council of Europe, 1981).

At their 23rd meeting, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) reviewed the data on 2-butanol. They concluded that "The evaluation of this compound was not possible on the basis of the data available. New specifications were prepared, but no toxicological monograph" (WHO, 1980).

ENVIRONMENTAL HEALTH CRITERIA

FOR

tert-BUTANOL

CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR tert-BUTANOL

1. SUMMARY

Page 33 of 75 Butanols - four isomers (EHC 65, 1987)

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1 Identity 2.2 Physical and chemical properties 2.3 Analytical methods

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

6. KINETICS AND METABOLISM

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1 Aquatic organisms 7.2 Terrestrial organisms 7.3 Microorganisms

8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1 Single exposure 8.1.1 Acute toxicity 8.1.2 Signs of intoxication 8.2 Skin and eye irritation 8.3 Short-term exposures 8.4 Reproduction, embyrotoxicity, and teratogenicity 8.5 Mutagenicity 8.6 Carcinogenicity 8.7 Special studies

9. EFFECTS ON MAN

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1 Evaluation of human health risks 10.1.1 Exposure levels 10.1.2 Toxic effects 10.2 Evaluation of effects on the environment 10.2.1 Exposure levels 10.2.2 Toxic effects 10.3 Conclusions

11. RECOMMENDATIONS

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

1. SUMMARY

tert-Butanol is a colourless liquid or white crystalline solid with a camphor-like odour. It has a of 25 °C, a boiling point of 81.5 - 83 °C, is freely soluble in water, and its n-octanol/water partition coefficient is 0.37. Its vapour is 2.6 times denser than air. It is used primarily as a solvent, a dehydrating agent, and as an intermediate in the manufacture of other chemicals. It is also used as a denaturant for alcohols. Human exposure will be mainly occupational. Data on exposure of the general population are not available, but it may result from industrial emissions. tert-Butanol is inherently biodegradable and does not bioaccumulate. At ambient levels, it is not toxic for fish, amphibia, crustacea, algae, or bacteria.

Page 34 of 75 Butanols - four isomers (EHC 65, 1987)

In animals, tert-butanol is absorbed through the lungs and gastrointestinal tract; no information is available on dermal absorption. tert-Butanol is not a substrate for alcohol dehydrogenase and is slowly metabolized by mammals. Up to 24% of the dose is eliminated in the urine as the glucuronide, and up to 10% of the dose can be excreted in the breath and urine as or carbon dioxide. The rat oral LD50 is 3.5 g/kg body weight; it is, therefore, slightly toxic according to the classification of Hodge & Sterner. The primary acute effects observed in animals are signs of alcoholic intoxication. Its potency for intoxication is approximately 1.5 times that of ethanol. Animal data regarding skin and eye irritation are not available. tert-Butanol produces physical dependance in animals and post-natal effects in offspring exposed in utero. Data concerning the pathological effects of repeated exposure of animals are not available. From the animal studies available, it is not possible to determine a no-observed- adverse-effect level. tert-Butanol has been found not to be mutagenic. Adequate data are not available on carcinogenicity, teratogenicity, or effects on reproduction.

In man, tert-butanol is a mild irritant to the skin. No other effects on man have been reported, and there have been no reports of .

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1. Identity

Chemical structure: CH3 | CH3 - C - CH3 | OH

Chemical formula: C4H10O

Primary constituent: tert-butanol

Common synonyms: 2-methyl-2-propanol, tert-butyl alcohol, tertiary butanol, t. butanol, trimethyl carbinol, TBA, TMA, t. butyl , NCL-C55 367, trimethyl

Cas registry number: 75-65-0

2.2. Physical and Chemical Properties

Some physical and chemical data for tert-butanol are given in Table 1. Table 1. Physical and chemical data for tert-butanol ------(at 20 °C and 101.3 kPa, unless otherwise stated)

Physical state solid (crystals) Odour camphor-like Odour threshold approximately 144.7 mg/m3(47 ppm) Relative molecular mass 74.12 Density (kg/m3) 779 - 782 at 26 °C Boiling point (°C) initial 81.5 (min.); dry point 83.0 (max.) Melting point 25 °C Viscosity (mPa x s) 3.3 at 30 °C Vapour density (air = 1) 2.55

Page 35 of 75 Butanols - four isomers (EHC 65, 1987)

Vapour pressure (mm Hg) 31 (at 25 °C, 42; at 30 °C, 56) Flashpoint (°C, TOC) 16 (°C, TCC) 4 Autoignition temperature 470 °C Explosion limits in air (v/v %) lower 2.35; upper 8.0 Solubility soluble in water; miscible with ethyl alcohol, ether; also soluble in ketones, esters, aromatic and aliphatic n-octanol/water partition 0.37 coefficient

Conversion factors: 1 mg/m3 = 0.325 ppm 1 ppm = 3.078 mg/m3 ------2.3. Analytical Methods

Testing methods for the butanols (ASTM D304-58) are described in ASTM (1977).

It is known that several alcohols, including tert-butanol, give colour reactions with aldehyde in the presence of (Patty, 1963). NIOSH (1977b) describes several methods.

AOAC has finalized the method for detecting tert-butanol in distilled (AOAC, 1975).

tert-Butanol has been determined in the air of industrial premises by Abbasov et al. (1971) using a gas chromatographic method and by Zamarakhina (1973) using a photometric method, and in blood by Wood & Laverty (1976).

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

The primary use of tert-butanol is as a solvent. It is also used as a dehydrating agent, in the extraction of drugs, in the manufacture of perfumes (particularly in the preparation of artificial musk), in the recrystallization of chemicals, and as a chemical intermediate (e.g., in the manufacture of tert-butyl chloride and in the manufacture of tert-butyl ). It is an approved denaturant for ethyl alcohol and for several other alcohols. Catalytic dehydration of tert-butanol is carried out to obtain isobutylene, and it has been patented for use as a antiknock agent.

Moreover, it is used in the purification of polyolefins, for the separation of solids from liquids and as blowing agent for the manufacture of group-containing foams from copolymers of methacrylonitrile and (Patty, 1963; Monich, 1968; Sherman, 1978).

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

No data are available on the distribution of tert-butanol in soil, sediments, or air.

In short-term tests, there was little degradation but over a longer period of about one month, most of the material was fully degraded. Therefore, tert-butanol is inherently rather than readily biodegradable. Some biodegradation data for tert-butanol are given in Table 2.

tert-Butanol does not bioaccumulate (Chiou et al., 1977). Table 2. Biodegradation data for tert-butanol ------

Page 36 of 75 Butanols - four isomers (EHC 65, 1987)

5d BOD 0% of ThOD (AFNOR) Dore et al. (1974) 1% of ThOD (APHA) Bridié et al. (1979b)

30d BOD 0% of ThOD (closed-bottle Gerike & Fischer (1979) test, conventional) 0% of ThOD (closed-bottle Gerike & Fischer (1979) test, preadaptation)

MITI test 0% of ThOD removed after Gerike & Fischer (1979) 14 days (BOD14: 7% of ThOD)

OECD screening 29% of ThOD removed after Gerike & Fischer (1979) test 19 days; no pre-adaptation

Sturm test 32% of ThOD removed, but no Gerike & Fischer (1979) production of CO2

AFNOR T90-302 80% of ThOD removed after Gerike & Fischer (1979) test 28 days 93% of ThOD removed after Gerike & Fischer (1979) 42 days

Zahn-Wellens 96% of ThOD removed after Gerike & Fischer (1979) test 6 days

Couple-units test 33% of ThOD removed after Gerike & Fischer (1979) (conventional) 42 days adaptation

Square-wave 69% of ThOD removed after Gerike & Fischer (1979) feeding 30 days adaptation

Activated 0.8% of ThOD removed in 24 h Gerhold & Malaney sludge by unadapted municipal sludge (1966)

2% of ThOD removed in 23 h by McKinney & Jeris (1955) adapted sludge ------

Table 2. (contd.) ------98% of ThOD removed in 5 days Pitter (1976) by adapted sludge; biodegradation rate at 20 °C 0.03/h

Anaerobic 73% of ThOD removed at Chou et al. (1978) digestion 400 mg/litre in anaerobic up-flow filters (hydraulic residence time 2 - 10 days) after 52 days of adaptation ------ThOD = theoretical oxygen demand - the calculated amount of oxygen needed for complete oxidation to water and carbon dioxide.

COD = chemical oxygen demand - measures the chemically oxidizable matter present.

BOD = biochemical oxygen demand - a simple bioassay measuring the potential deoxygenating effect of biologically oxidizable matter present in an effluent.

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

Page 37 of 75 Butanols - four isomers (EHC 65, 1987)

An industrial emmission study indicated that 207 tonnes of tert-butanol were released into the air in the Netherlands over a period of 1 year (Anon, 1983).

No other data are available.

6. KINETICS AND METABOLISM

tert-Butanol is not a substrate for alcohol dehydrogenase (Derache, 1970; Cederbaum et al., 1983) and is slowly metabolized by mammals (Williams, 1969; Derache, 1970; Beaugé et al., 1981). Possible routes of metabolism are direct conjugation of the hydroxyl group with glucuronic acid and oxidation of one or more of the . Following treatment with tert-butanol, 24% of the dose was detected as the glucuronide conjugate in the urine of rabbits (Kamil et al., 1953) and increased acetone excretion has been observed in the breath and urine of rats treated with tert-butanol (Baker et al., 1982; Yojay et al., 1982).

After a single oral dose of tert-butanol (25 mmol/kg), blood concentrations in female Wistar rats declined slowly from 13.2 ± 0.5 mmol at 2 h to 11.4 ± 0.3 mmol at 20 h (Beaugé et al., 1981). In rats maintained on a liquid diet containing 20 ml tert- butanol/litre for 20 days, the blood level 30 min after withdrawal of the diet of 20 mmol declined to 5 mmol in 8 h. By comparison, blood levels of ethanol of 45 mmol (achieved by a liquid diet of 87 ml ethanol/litre for 20 days) were reduced to zero within 4.5 h (Wood & Laverty, 1979). In female Sprague Dawley rats given single oral doses of either ethanol (5.0 g/kg body weight) or tert- butanol (1.2 g/kg body weight), the rates of elimination were 10.7 ± 0.5 and 0.7 ± 0.1 mmol/kg per h, respectively (Thurman et al., 1980).

Following an oral dose of 2 g tert-butanol/kg body weight to rats, a maximum blood level of 1240 mg/litre (124 mg%) was reached in 2 h; this decreased very slowly to 1200 mg/litre (120 mg%) after 4 h and to 1100 mg/litre (110 mg%) after 8 h; only about 1% of the dose was excreted in the urine (Gaillard & Derache, 1965). tert-Butanol was found in the blood of rabbits 70 h after oral administration of 2 ml/kg body weight (Saito, 1975).

In Long-Evans rats treated with tert-butanol (1 g/kg body weight, route not specified), the rate of disappearance of tert- butanol from the blood was apparently of first order with a half life of 9.1 h (Baker et al., 1982). Using 14C- and 13C- tert-butanol, the same authors investigated the metabolism of tert-butanol to form acetone. It was found that following administration of tert-butanol (0.75 - 2 g/kg body weight), approximately 0.5 - 9.5% of the dose was excreted as acetone in the urine and breath. The total production of acetone varied considerably between animals given the same dose, and, as a result, no correlation between dose and acetone excretion could be established. Evidence was also obtained indicating that carbon dioxide (CO2) was a metabolic product of tert-butanol. The conversion of tert-butanol (possibly via acetone) was not quantified. Yojay et al. (1982) provided evidence that, in rats treated intraperitoneally with tert-butanol at 1 or 2 mg/kg body weight, blood levels of acetone were approximately proportional to the dose of tert-butanol. In support of the in vivo metabolic conversion of tert-butanol to acetone, Cederbaum & Cohen (1980) and Cederbaum et al. (1983) demonstrated the metabolism of

tert-butanol to and acetone in an in vitro system

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consisting of rat liver microsomes and a hydroxyl radical generating system.

Investigations on the induction of tert-butanol metabolism have been conducted by Baker et al. (1982), Thurman et al. (1980), and McComb & Goldstein (1979). In rats, Baker et al. (1982) were unable to demonstrate increased conversion of tert-butanol to acetone in animals in which the hepatic mixed-function oxidase activity had been induced by prior treatment. Thurman et al. (1980) studied the effects on tert-butanol elimination in rats of pre-treatment (oral) with 5.7% (w/v) tert-butanol every 8 h, for either 1 or 2.5 days. After the pre-treatment, animals were given tert-butanol to raise their blood levels to between 1250 and 1500 mg/litre. The rates of elimination were very similar, but there was a suggestion of slightly faster elimination following pre-treatment. The investigators concluded that, unlike ethanol pre-treatment, which induces its own metabolism, tert-butanol pre-treatment had little or no effect on the subsequent rate of tert-butanol elimination in the rat.

In contrast to the results obtained in rats, it appears that tert-butanol elimination in mice can be substantially increased by pre-treatment with tert-butanol. Male Swiss Webster mice were given an ip loading dose of 6.8 mmol tert-butanol/kg body weight and then exposed to various vapour concentrations of tert-butanol for 24 h. It was found that 15 min after the 24-h inhalation period, blood- tert-butanol levels were linearly related to the vapour concentrations. Blood levels ranged from 3 to 14 mmol with increasing vapour concentrations of 30 - 100 µmol/litre air (McComb & Goldstein, 1979). It was also found that the rate of elimination of blood- tert-butanol was significantly increased by inhalation. In mice, after a single ip injection of 8.1 mmol tert-butanol/kg body weight, initial blood levels of 8 mmol took 8 - 9 h for elimination (blood- tert-butanol half-life was approximately 5 h). However, after 3 days, inhalation at a vapour concentration to give levels of 8 mmol/litre blood, tert-butanol disappeared within 3 h of removal of mice from the inhalation chamber (half-life of tert- butanol in blood was approximately 1.5 h) (McComb & Goldstein, 1979).

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1. Aquatic Organisms

The toxicity of tert-butanol has been tested in very few organisms. However, in the few studies performed, the toxicity of the compound has been very low.

Some toxicity data for aquatic organisms are given in Table 3.

Table 3. Toxicity of tert-butanol for aquatic organisms ------Species Concentration Parameter Comments Reference (mg/litre) ------Fish (acute)

Fresh-water species

Creek chub 3000 - 6000 24-h LC50 Gillette (Semotitus et al. atromaculatus) (1952)

Goldfish > 5000 24-h LC50 Bridié

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(Carassius et al. auratus) (1979a)

Invertebrates (acute)

Marine species

Brine shrimp 7800 EC50 excystment Smith & (Artemia salina) Siegel (1975)

Algae

Fresh-water

Green algae Chlorella 24 200 EC50 chlorophyll Jones pyrenoidosa content (1971) ------

7.2. Terrestrial Organisms

An EC50 of 90 800 mg/litre was reported for germination in cucumber (Cucumis sativus) by Smith & Siegel (1975). There are no relevant data for terrestrial animals. However, significant terrestrial exposure to tert-butanol is unlikely for either plants or animals.

7.3. Microorganisms

One study has indicated that Nitrosomonas (nitrifying bacterium) shows a high tolerance for tert-butanol; tert-Butanol inhibits nitrifying activity at 39 400 mg/litre (Blok, 1981).

There was no inhibition of degradation by methane culture on acetate substrate at 7400 mg tert-butanol/litre (Chou et al., 1978).

8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1. Single Exposure

8.1.1. Acute toxicity

Some acute toxicity data for animals are given in Table 4.

Table 4. Acute toxicity of tert-butanol in animals ------Species Route of LD50 (g/kg Reference administration body weight) ------Rat oral 3.5 US DHEW (1979) Rabbit oral 3.6 Münch (1972) Mouse intravenous 1.5 Patty (1982) Mouse intraperitoneal 0.9 US DHEW (1978) ------

8.1.2. Signs of intoxication

Animals exposed to the vapours of tert-butanol may manifest the following signs of intoxication: restlessness, irritation of mucous membranes, ataxia, prostration, and narcosis (Patty, 1963).

The narcotic potency of tert-butanol in rabbits is similar to

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that of other butanols (Münch, 1972). After ip administration to mice, the LD50 was > 1000 mg/kg body weight at 30 min and 441 mg/kg body weight at 7 days; gross post-mortem examination of the livers showed an abnormal dark coloration (Maickel & McFadden, 1979). In rabbits, an oral dose of 1.3 g tert-butanol/kg body weight was shown to be the minimum narcotic dose (Münch & Schwartz, 1925). The minimum oral lethal dose in the rabbit was 6.0 ml/kg body weight (approximately 4.7 g/kg) (Patty, 1963). Injection (ip) of tert-butanol in rats induced a spectrum of intoxication virtually identical to that of ethanol, but its intoxicating potency was approximately 1.5 times that of ethanol (McCreery & Hunt, 1978).

Oral administration of a single dose of 24 mmol tert- butanol/kg body weight caused a temporary disturbance in the metabolism in the liver cells of female Wistar rats. The authors suggest that this may be related to the stress induced by the administration of tert-butanol, which is metabolized very slowly (Beaugé et al., 1981).

8.2. Skin and Eye Irritation

No data are available on skin and eye irritation in animals.

8.3. Short-Term Exposures

Physical dependence following tert-butanol administration was investigated using 12 random-bred male albino rats. A liquid diet containing 20 ml tert-butanol/litre was given to the rats for

4 - 20 days. Docility and slight ataxia were observed in the rats during this period. About 5 - 6 h after removal of the diet, withdrawal-signs such as muscular rigidity, stiff curled tails, abnormal gait, tremor, and irritability became apparant. Four rats exhibited spontaneous forelimb convulsions. Audiogenic convulsions were observed in 5 rats, and 3 of them died as a consequence. tert- Butanol was still detectable in the blood of the rats, 8 h after its withdrawal (Wood & Laverty, 1979).

In another study, 15 adult male Long Evans rats were exposed to 4 different protocols using various concentrations of tert- butanol in water as their only available fluid; 14 animals were used as controls. At concentrations of 3.5 ml/litre, severe toxic reactions were found, including anorexia, self mutilation, and death. When animals consumed at least 3 g tert-butanol/kg body weight per day for 90 days, withdrawal symptoms were observed, independent of dosing conditions. Such an intake occurred only when the concentration of tert-butanol was 3% or greater (Grant & Samson, 1981). In female Sprague Dawley rats, tert-butanol administered by gastric intubation, every 8 h, for up to 6 days, was shown to produce (Thurman et al., 1980).

An investigation was undertaken with 5 to 8-week-old male Swiss-Webster mice weighing 22 - 30 g. A priming dose of 6.8 - 10.1 µmol tert-butanol/kg body weight (10% w/v in 0.9% saline) was administered ip to groups of 24 mice. They were then exposed to vapour concentrations of between 50 and 80 µmol tert-butanol/litre air for 24 h per day; concentrations below or above these limits either did not produce physical dependance or were initially too toxic. Because it was found that continual exposure induced the elimination of tert-butanol, exposure concentrations were increased daily, in order to maintain a steady blood- tert-butanol level of between 5 - 8.5 mmol. Exposure lasted for 1, 3, 6, or 9 days. Withdrawal signs were noted after removing the mice from the

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inhalation exposure. The intensity of the withdrawal reaction increased with the duration of inhalation and with the blood- tert- butanol levels maintained during the intoxication period. The withdrawal syndrome was qualitatively similar to that produced by ethanol. tert-Butanol has been shown to be 4 - 5 times as potent as ethanol in producing physical dependence. Thus, since it is also 4 - 5 times more lipid soluble than ethanol, McComb & Goldstein (1979) concluded that the dose of alcohol necessary to induce physical dependence was inversely proportional to its solubility.

In a study by Bellin & Edmonds (1976), physical dependence was induced in 12 male Sprague Dawley rats (350 - 500 g) and 10 female albino guinea-pigs (200 - 400 g) when given tert-butanol ip (0.8 g/kg body weight as a 10% w/v solution) at 8-h intervals for 4 days. Alteration of body temperature (decreased during intoxication and increased during withdrawal) was more pronounced in the rats than in the guinea-pigs. However, these differences could be due to species or sex.

An impairment of avoidance behaviour was shown following short- term ingestion of tert-butanol in mice (number not stated). Experimental animals received a liquid diet containing tert- butanol at 12.5 ml/litre for 7 days and during this time ingested an average of 3.4 g tert-butanol/kg body weight, per day. One day after cessation of this diet, the animals were exposed to the avoidance training procedure. Avoidance was much less in the treated group than in the controls (Snell & Harris, 1980).

Pregnant Swiss Webster mice (15 per group) were fed liquid diets containing tert-butanol at concentrations of 0, 5.0, 7.5, or 10 g/litre from day 6 to day 20 of gestation. In order to gain insight into the effects of maternal nutrition and behaviour on post-natal pup development, approximately half of the treated maternal animals were replaced, within 24 h of parturition, with untreated females, also recently delivered. tert-Butanol was approximately 5 times more potent than the same dose of ethanol in producing developmental delay in post-parturition physiological, and psychomotor performance, scores. At the higher concentrations, there were also significant postnatal maternal, nutritional, and behavioural factors affecting lactation and/or nesting behaviour, which influenced the development of pups exposed in utero (Daniel & Evans, 1982).

8.4. Reproduction, Embryotoxicity, and Teratogenicity

No relevant data on reproduction, embryotoxicity, or teratogenicity have yet been published. In contrast to ethanol, tert-butanol at concentrations of 1000 - 4000 mg/litre did not reduce the in vitro fertilizing capacity of mouse spermatozoa (Anderson et al., 1982).

An inhalation teratology study is in progress in the USA (US EPA, personal communication, 1985).

8.5. Mutagenicity

At a concentration of 1%, tert-butanol was classified among the chemicals that had no apparent mutagenic effect on formation of antibiotic-resistant mutants in Micrococcus aureus populations (Clark, 1953). tert-Butanol was not mutagenic in Neurospora crassa (Dickey et al., 1949). In short-term tests conducted by the National Toxicology Program (USA), tert-butanol was negative in the Ames test ( Salmonella, +/-activation), mouse lymphoma, and

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in vitro cytogenetics assays (US EPA, personal communication, 1985).

8.6. Carcinogenicity

tert-Butanol is currently being evaluated for carcinogenicity by the US National Cancer Institute using the standard bioassay protocol. Groups composed of 50 male and 50 female B6C3F1 mice were exposed for 2 days per week, from week 9 to 104, to 0, 5000, 10 000, or 20 000 mg tert-butanol/litre drinking-water. Groups comprising 50 male and 50 female Fischer 344 rats were exposed at the same rate to the same levels from weeks 7 to 104. The treatment period has now been completed and histological examinations are in progress (IARC, 1984).

An inhalation carcinogenicity study has been initiated in the USA (US EPA, personal communication, 1985).

8.7. Special Studies

tert-Butanol was investigated for its ability to deplete the cerebral calcium level. An ip dose of 2 g/kg body weight was administered to male Sprague Dawley rats weighing between 150 and 225 g. The animals were sacrificed 30 min after the administration of the alcohol. There was a significant decrease in cerebral calcium contents compared with that in the control animals (35 mg/kg versus 55 mg/kg) (Ross, 1976).

In Sprague Dawley rats (190 - 250 g), ip administration of aqueous tert-butanol (250 g/litre) was shown to increase carbon tetrachloride-induced hepatotoxicity, as evaluated by serum glutamate-pyruvate transaminase levels. However, there was no depletion in hepatic glutathione or loss in body weight (Harris & Anders, 1980). tert-Butanol can potentiate the toxicity of carbon tetrachloride in Sprague Dawley rats (Cornish & Adefuin, 1967).

The inhibition of the synthesis of ornithine decarboxylase (ODC) and tyrosine aminotransferase (TAT) by tert-butanol was studied in partially-hepatectomized female rats (about 238 g) of mixed strains. Immediately after partial hepatectomy, 15% (w/v) aqueous tert-butanol (2.8 g/kg body weight) was given to 6 rats by gastric intubation. In the liver, 4 h after partial hepatectomy, the ODC activity was decreased to 22% and the TAT activity to about 52% of the activities in the control group. In the kidney, 4 h after partial hepatectomy, the ODC activity was decreased to about 31% of the activity in the control group. In the brain, tert- butanol did not induce any significant changes in the ODC activity compared with that in the control group (Poso & Poso, 1980).

The presence of a hydroxy radical is needed for prostaglandin biosynthesis. tert-Butanol was shown to be a hydroxyl radical scavenger and, therefore, an inhibitor of prostaglandin biosynthesis. In the incubation system of microsomes, prepared from bovine vesicular glands containing epinephrine, the 14C incorporation as a percentage of total labelled prostaglandins E and F, respectively, was 16.4% and 23.4%. When 0.025 ml of tert- butanol was added to this incubation system, the values were 15.5% and 13.8%, respectively. Further increases in concentration resulted in decreased incorporation (Panganamala et al., 1976). In another study using isolated perfused rat lungs, the infusion of low concentrations of tert-butanol (0.002 mmol - 0.2 mmol) resulted in the continuous output of prostaglandins into the venous effluent. However, there was a gradual decrease in the

Page 43 of 75 Butanols - four isomers (EHC 65, 1987)

prostaglandin output as the concentration of the alcohol increased (Thomas et al., 1980).

Cation flux across membranes has been investigated in a number of studies. tert-Butanol inhibited, in a dose-dependent manner, the in vitro contraction of depolarized guinea-pig ileum induced by calcium chloride (Yashuda et al., 1976) and at a 34 mmol concentration impaired the response of rat brain cortex slices to electrical stimulation, inhibiting the influx of , but only weakly affecting potassium afflux (Wallgren et al., 1974). Wiesbrodt et al. (1973) studied the effects of tert-butanol on the gastric mucosa of Heidenheim pouches in 4 unanaesthetized dogs. They observed a decrease in the transmucosal potential and an increase in the net flux of Na+ ion into the lumen of the pouch at all concentrations tested (0.5 - 1.5 mol).

The effect of tert-butanol, at 1 mmol concentration, on isolated rat mitochondria and lysosomes was studied by Sgaragli et al. (1975). The alcohol did not affect lipid peroxidation and membrane stability as evaluated by the release of total protein acid and glutamate dehydrogenase.

tert-Butanol decreased the in vitro Km of phosphorilase b for glucose-1-phosphate with saturating glycogen concentrations and markedly increased Vmax (Dreyfus et al., 1978). Akhtem et al. (1978) studied the interaction between tert-butanol and cytochrome P-450 from rat liver microsomes; they revealed spectral shifts indicating the formation of alcohol-cytochrome P-450 complexes. Sugiyama et al. (1976) found that tert-butanol greatly enhanced the side chain cleavage activity of purified P-450.

A 15-min pre-session oral administration of tert-butanol at 0.25 - 3 g/kg body weight to 4 trained male Long Evans rats produced a dose-related decrease in fixed-ratio responding at doses of 0.5 g/kg or more. tert-Butanol was 1.6 times as potent as ethanol, in this respect. The 1 g/kg dose decreased responding by about 20%, 15, 30, or 60 min after dosing (Witkin & Leander, 1982).

tert-Butanol produced a marked dose-dependent activation of locomotor activity in short-sleep mice, selectively bred for relative insensitivity to the properties of ethanol. In alcohol-sensitive long-sleep mice, the locomotor activity was depressed in a dose-dependent fashion. Test doses were 0, 4, 5, or 6 g/kg body weight, administered intravenously in 0.9% saline (Dudek & Philips, 1983).

Twelve neonatal Long Evans rats were given tert-butanol at doses of up to 2.69 g/kg body weight per day in a milk formula through a cannula, from postnatal days 4 to 7 (corresponding to the brain growth spurt). At this point, they were transferred to a plain milk formula, for the next 11 days. After sacrifice on day 18, exposed animals showed significant decreases in absolute and relative brain weights and lower DNA levels in hind-brain samples in comparison with controls, indicating a certain degree of microcephaly (Grant & Samson, 1982).

No effects were found in the basal synaptosomal membrane fluidity or in the activity of Na+ + K+ ATPase in the brain of Sprague Dawley rats given a single oral dose of 1.85 g tert- butanol/kg body weight (Beaugé et al., 1984).

9. EFFECTS ON MAN

tert-Butanol is slightly irritant to the skin. When the

Page 44 of 75 Butanols - four isomers (EHC 65, 1987)

compound was applied to the skin of 5 human volunteers, no reaction other than slight erythema and hyperaemia was observed (Oettel, 1936). However, Edwards & Edwards (1982), described an allergic skin reaction to tert-butanol in a 58-year-old patient who used skin screen containing tert-butanol. A patch test was positive for tert-butanol. There are no other published reports of adverse effects or poisonings in man.

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1. Evaluation of Human Health Risks

10.1.1. Exposure levels

Levels of exposure of the general population through food and occupational exposure levels are not available.

10.1.2. Toxic effects

In animals, tert-butanol is absorbed through the lungs and gastrointestinal tract; no information is available on dermal absorption. tert-Butanol is not a substrate for alcohol dehydrogenase and is slowly metabolized by mammals. Up to 24% of the dose is eliminated in the urine as the glucuronide, and up to 10% of the dose can be excreted in the breath and urine as acetone or carbon dioxide. The rat oral LD50 is 3.5 g/kg body weight; it is, therefore, slightly toxic according to the classification of Hodge & Sterner. The primary acute effects observed in animals are signs of alcoholic intoxication. Its potency for intoxication is approximately 1.5 times that of ethanol. Animal data regarding skin and eye irritiation are not available. tert-Butanol produces physical dependance in animals and post-natal effects in offspring exposed in utero. Data on pathological effects of repeated exposure of animals are not available. From the animal studies available, it is not possible to determine a no-observed-adverse- effect level. tert-Butanol has been found not to be mutagenic. No adequate data are available on carcinogenicity, teratogenicity, or effects on reproduction.

In man, tert-butanol is a mild irritant to the skin. There have not been any reports of poisonings or any other effects in man.

10.2. Evaluation of Effects on the Environment

10.2.1. Exposure levels

No quantitative data relating to levels in the general environment are available, but, because tert-butanol is inherently biodegradable, substantial concentrations are only likely to occur locally in the case of major spillage.

10.2.2. Toxic effects

tert-Butanol is inherently biodegradable and is not toxic for fish, amphibia, crustacea, algae, or bacteria.

10.3. Conclusions

1. On the available data, the Task Group was unable to make an assessment of the health risks from tert-butanol for the general population. However, it was considered unlikely to pose a serious hazard under normal exposure conditions.

Page 45 of 75 Butanols - four isomers (EHC 65, 1987)

2. The Task Group was of the opinion that available data are not sufficient to establish guidelines for setting occupational exposure limits. In line with good manufacturing practice, exposure to tert-butanol should be minimized.

3. The ecotoxicological data available indicate that the impact of background concentrations of tert-butanol on the aquatic environment can be expected to be minimal.

11. RECOMMENDATIONS

1. The Task Group noted that, from the animal studies available, it was not possible to determine a no-observed-adverse-effect level. Relevant studies should be conducted so that this can be achieved.

2. Information on residue and emission levels is desirable.

3. Epidemiological studies, including precise exposure data, would assist in a better assessment of the occupational hazards from tert-butanol.

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

The Council of Europe has recently established (Council of Europe, 1981) a specific limit of 30 mg tert-butanol/kg in candy in confectionery. tert-Butanol was evaluated by the EEC Scientific Committee for Food in 1980. The Committee agreed on the following evaluation:

"The available data are insufficient to establish an ADI. Biochemically, this solvent will behave like other tertiary carbinols, which are generally not very reactive. The residues in food are minimal and are not a hazard to health. The Committee considers the use of this compound temporarily acceptable as an extraction solvent provided residues from use as an extraction solvent in food as consumed do not exceed 10 mg/kg food. The provision of an adequate 90-day feeding study in a rodent species is required by 1983."

ENVIRONMENTAL HEALTH CRITERIA

FOR

ISOBUTANOL

CONTENTS ENVIRONMENTAL HEALTH CRITERIA FOR ISOBUTANOL

1. SUMMARY

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1 Identity

Page 46 of 75 Butanols - four isomers (EHC 65, 1987)

2.2 Physical and chemical properties 2.3 Analytical methods

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

6. KINETICS AND METABOLISM

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1 Aquatic organisms 7.2 Terrestrial organisms 7.3 Microorganisms

8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1 Single exposures 8.1.1 Acute toxicity 8.1.2 Signs of intoxication 8.2 Skin and eye irritation 8.3 Short-term exposures 8.4 Long-term exposures 8.5 Reproduction, embryotoxicity, and teratogenicity 8.6 Mutagenicity 8.7 Carcinogenicity 8.8 Special studies

9. EFFECTS ON MAN

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1 Evaluation of human health risks 10.1.1 Exposure levels 10.1.2 Toxic effects 10.2 Evaluation of effects on the environment 10.2.1 Exposure levels 10.2.2 Toxic effects 10.3 Conclusions

11. RECOMMENDATIONS

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

1. SUMMARY

Isobutanol (2 methyl propanol) is a inflammable colourless liquid with a sweet odour similar to that of . It has a boiling point of 108 °C, a water solubility of 8.7%, and its n-octanol/water partition coefficient is 0.83. Its vapour is 2.6 times denser than air. It occurs naturally as a product of fermentation and is synthesized from petrochemicals. It is used as an organic solvent, as a , in the manufacture of isobutyl esters, in perfumes, and as a flavouring agent. Human exposure is primarily occupational. Exposure of the general population will mainly be from its natural occurrence in food and its use as a flavouring agent, but may also result from industrial emissions.

Isobutanol is readily biodegradable and does not bioaccumulate. It is not directly toxic for fish, crustacea, amphibia, or algae. Protozoa will tolerate levels of isobutanol likely to be found in

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the environment. Isobutanol should be managed in the environment as a slightly toxic compound. It poses an indirect hazard for the aquatic environment, because it is readily biodegraded, which may lead to oxygen depletion.

In animals, isobutanol is absorbed through the skin, lungs, and gastrointestinal tract. It is metabolized by alcohol dehydrogenase to via the aldehyde and may enter the tricarboxylic acid cycle. Small amounts of isobutanol are excreted unchanged (< 0.5% of the dose), or as the glucuronide (< 5% of the dose) in the urine. In rabbits, metabolites found in the urine include acetaldehyde, , isobutylaldehyde, and isovaleric acid. Oral LD50 values (2.5 - 3.1 g/kg body weight) and the inhalation 3 LC50 (19.2 g/m ) in rats classifies isobutanol as slightly toxic according to Hodge & Sterner. The acute toxic effects are alcoholic intoxication and narcosis. Isobutanol is severely irritating to the eyes and moderately irritating to the skin. A group of rats given a solution of isobutanol (1 mol/litre) as their sole drinking liquid for 4 months did not show any adverse effects on the liver, while another group given a 2 mol/litre solution as their sole drinking liquid for 2 months showed reductions in fat, glycogen, RNA content, and overall size of the cells in the liver. Continuous inhalation exposure of rats to 3 mg/m3 for 4 months resulted in depression of leg withdrawal response to electrical stimulation, and minor changes in formed elements of the blood and serum . The estimated no-observed-adverse-effect level was 0.1 mg/m3. In a lifetime carcinogenicity study, groups of rats received isobutanol subcutaneously (0.05 ml/kg, twice a week) or orally (0.2 ml/kg body weight twice a week). The animals exhibited toxic liver damage ranging from steatosis to cirrhosis. Animals showing malignant tumours totalled 8 in the subcutaneous group, 3 in the oral group, and 0 in the control group. The majority of treated animals also showed hyperplasia of blood-forming tissues.

From the animal studies available, it is not possible to determine a no-observed-adverse-effect level for long-term exposure. No adequate data are available to assess the mutagenicity or teratogenicity of isobutanol or effects on reproduction.

The only reported observations in man relate to the production of vertigo under conditions of severe and prolonged exposure to vapour mixtures of isobutanol and 1-butanol. Thus, it is not possible to attribute the vertigo to a single cause.

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1. Identity

Chemical structure: CH3 \ CH-CH2OH / CH3

Chemical formula: C4H10O

Primary constituent: isobutanol

Common synonyms: isobutyl alcohol, isopropylcarbinol, 2-methyl-1-propanol, 2-methylpropyl alcohol, 1-hydroxymethylpropane, 2-methylpropan-1-ol, 1-propanol,-2-methyl, fermentation butyl alcohol

Page 48 of 75 Butanols - four isomers (EHC 65, 1987)

CAS registry number: 78-83-1

2.2. Physical and Chemical Properties

Physical and chemical properties of isobutanol are given in Table 1.

Table 1. Physical and chemical properties of isobutanol ------(at 20 °C and 101.3 kPa, unless otherwise stated)

Physical state colourless liquid Odour sweet, similar to that of amyl alcohol, but weaker Odour threshold approximately 4.6 mg/m3 (1.5 ppm) Relative molecular mass 74.12 Density (kg/m3) 801-803 Boiling point (°C) 107.9 Freezing point (°C) -108 Viscosity (cP) 3.98 Vapour pressure (kPa) 1.17 12.2 at 25 °C Vapour density (air = 1) 2.55 Flashpoint (°C) 27.8 Autoignition temperature (°C) 434 Explosion limits air (% v/v) lower = 1.7, upper = 10.9 Solubility (% weight) in water, 8.7; soluble in alcohol and ether n-octanol/water partition 0.83 coefficient

Conversion factors (25 °C) 1 mg/m3 = 0.324 ppm 1 ppm = 3.083 mg/m3 ------

2.3. Analytical Methods

Testing methods for the butanols (ASTM D304-58) are described in ASTM (1977).

NIOSH (1977) Method No S64 (341) has been recommended. It involves drawing a known volume of air through charcoal to trap the organic vapours present (recommended sample is 10 litres at a rate of 0.2 litre/min). The analyte is desorbed with carbon disulfide containing 1% 2-propanol. The sample is separated by injection into a gas chromatograph equipped with a flame ionization detector, and the area of the resulting peak is determined and compared with standards.

The Association for Official Analytical Chemists has published an Official Final Action for the assay of isobutanol in spirits, based on gas chromatographic analysis (AOAC methods, 1975).

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

The major use of isobutanol is in the manufacture of , which is employed in the lacquer industry. Furthermore, isobutanol is used as a solvent in and varnish removers and in the manufacture of isobutyl esters, which serve as solvents, , flavourings, and perfumes. It is also used as a flavouring agent in butter, cola, fruit, liquor, rum, and whisky (Hall & Oser, 1965). The average maximum levels at which it is used in the USA are listed in Table 2 (Hall & Oser, 1965).

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Table 2. Average maximum use levels of isobutanol in the USA ------Food in which used Approximate average maximum level (mg/kg) ------Beverages 17

Ice cream, ices 7

Candy 30

Baked goods 24 ------

Isobutanol is one of the three main alcohols in fusel oil, and is present in large amounts in some alcoholic beverages (Hedlund Kiessling, 1969).

Natural isobutanol is produced by the fermentation of carbohydrates. Isobutanol is found in fruits: cherry (Postel et al., 1975), raspberry and blackberry (McGlumphy, 1951; Nursten et al., 1967; Broderick, 1976), grape (Bober, 1963), and apple (Schreier et al., 1978). It also occurs in beverages: brandy (Postel et al., 1975), coffee (Walter & Weidemann, 1969), cider (Matthews et al., 1962; Kieser et al., 1964), and gin (Clutton & Evans, 1978). Isobutanol has been identified in sundry other foods including cheddar cheese (Liebich et al., 1970), and hop oil (Lammers et al., 1968).

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

No data are available on distribution in soil, sediments or air.

Isobutanol is readily biodegradable (Table 3). It is degraded in significant amounts within a few hours, and degradation would be expected to be complete within a few days.

Table 3. Biodegradation of isobutanol ------5d BOD 64% of ThOD in fresh water Price et al. (1974)

46% of ThOD in synthetic Price et al. (1974) seawater

5d BOD 16% of ThOD (APHA) Bridié et al. (1979a)

63% of ThOD after adaptation Bridié et al. (1979a) (APHA)

Activated 32.5% of ThOD removed in 24 h by Gerhold & Malaney sludge unadapted municipal sludge (1966)

44% of ThOD removed in 24 h by McKinney & Jeris adapted sludge (1955) ------ThOD = theoretical oxygen demand - the calculated amount of oxygen needed for complete oxidation to water and carbon dioxide.

COD = chemical oxygen demand - measures the chemically oxidizable matter present.

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BOD = biochemical oxygen demand - a simple bioassay measuring the potential deoxygenating effect of biologically oxidizable matter present in an effluent.

Nazarenko (1969) reports an oxygen requirement of approximately 1.4 mg to oxidize 1 mg of isobutanol. Isobutanol at a concentration of 20 mg/litre inhibits nitrification in water (Nazarenko, 1969).

Isobutanol does not bioaccumulate (Chiou et al., 1977).

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

An industrial emission study indicated that 90 tonnes of isobutanol were released into the air over 1 year in the Netherlands (Anon, 1983). Isobutanol produced naturally during the fermentation of carbohydrates can enter the environment by leaching from industrial waste landfills. A concentration of 0.3 g/litre was found to be leaching from a 1-year-old landfill (US EPA, 1980).

6. KINETICS AND METABOLISM

Isobutanol can be absorbed through the lungs and from the gastrointestinal tract (Browning, 1965).

Isobutanol (2 ml/kg body weight) was given to male rabbits by gavage, and blood levels of isobutanol were determined. After 1 h, a maximum concentration of 0.5 g/litre was observed. After 6 h, isobutanol was no longer detectable in the blood. Less than 0.5% of the administered isobutanol was excreted unchanged in the breath or urine within 40 h (Saito, 1975).

Isobutanol can be metabolized to isobutyric acid and then proceed into the tricarboxylic acid cycle, possibly via succinate (Fig. 1B) (Saito, 1975). The urinary metabolites resulting from repeated ingestion of isobutanol were also studied by Saito (1975). Isobutanol (2 ml/kg body weight) was administered by stomach tube to male rabbits. The rabbits were then given water saturated with isobutanol instead of pure water to drink and the urinary metabolites were determined. The length of time over which the urine was collected was not specified. The urinary metabolites were acetaldehyde (80 mg), acetic acid (16 mg), (10 mg), isovaleric acid (128 mg), and unmetabolized isobutanol (40 mg). The origin of urinary isovaleric acid was not discussed by the author. Saito (1975) has proposed the scheme shown in Fig. 1B for the metabolism of isobutanol in rabbits.

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When Kamil et al. (1953) administered isobutanol (25 mmol total) by stomach tube to Chinchilla rabbits, 4.4% of the applied dose was excreted within 24 h, as the glucuronide. After administration of 6 ml isobutanol to the stomach of rabbits (Kamil et al., 1953), no aldehydes or ketones were observed in the expired air within 6 h.

Gaillard & Derache (1965) reported that, following an oral dose of 2 g/kg body weight to rats, 0.27% was excreted in the urine within 8 h.

The oxidation of isobutanol and other lower alcohols by both rat liver homogenates and by the perfusion in situ of rat liver was studied by Hedlund & Kiessling (1969). The authors observed that the alcohols were oxidized at rates that decreased in the following order: 1-propanol, isobutanol, ethanol, and . Furthermore, from their studies with the alcohol dehydrogenase (ADH) inhibitor pyrazole, these authors concluded that all the alcohols studied must be oxidized by ADH in the same manner as ethanol in an NAD-mediated oxidation.

7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT

7.1. Aquatic Organisms

Some data on the toxicity of isobutanol for aquatic organisms are given in Table 4. The high lethal concentrations (1000 - 4000 mg/litre) for fish, amphibia, and crustacea indicate that, at background levels, isobutanol would not be disruptive for an aquatic ecosystem. Table 4. Toxicity of isobutanol for aquatic organisms ------Species Concentration Parameter Comments Reference (mg/litre) ------Fish

Fresh-water species

Bleak 1000-3000 96-h LC50 Lindén et (Alburnus (1979) alburnus)

Golden orfe 1520 48-h LC50 Juhnke & L (Leuciscus (1978) idus melanotus)

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Goldfish 2600 24-h LC50 Bridié et (Carassius (1979a) auratus)

Amphibia

Tadpole 4000 threshold for Münch (197 (Rana sp) narcosis

Invertebrates

Fresh-water species

Water flea 1250 24-h EC50 immobilization Bringmann (Daphnia magna) (1982)

Marine species

Brine shrimp 1400 24-h LC50 Price et a (Artemia salina) 3800 EC50 excystment Smith & S (1975) ------

Table 4. (contd.) ------Species Concentration Parameter Comments Reference (mg/litre) ------Algae

Green algae

Scenedesmus 350 8-day no-observed- total biomass Bringmann quadricauda adverse-effect level (1978a)

Blue-green algae

(Microcystis 290 8-day no-observed- total biomass Bringmann aeruginosa) adverse-effect level (1978a) ------

7.2. Terrestrial Organisms

Toxicity studies in plants indicate that germination will not be affected by exposure to isobutanol at background levels. An EC50 of 760 mg/litre was reported by Reynolds (1977) for seed germination in lettuce (Lactuca sativa). Smith & Siegel (1975) found an EC50 of 40 800 mg/litre for seed germination in cucumber (Cucumis sativus).

There is no information on terrestrial animals, but exposure is unlikely to be significant, except locally after spills.

7.3. Microorganisms

Some data on the toxicity of isobutanol for microorganisms are given in Table 5. Available toxicity data on protozoa bacteria and algae suggest tolerance to exposure to isobutanol. No-observed- adverse-effect levels for various species ranged from 22 to 1180 mg/litre). Exposure to background levels of isobutanol should not be disruptive for the ecosystem.

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Table 5. Toxicity of isobutanol for microorganisms ------Species Concentration Parameter Comments Refer (mg/litre) ------Protozoa

Chilomonas paramaecium 22 48-h no-observed- total biomass Brin (flagellate) adverse-effect level Kuehn

Uronema parduczi 169 20-h no-observed- total biomass Brin (ciliate) adverse-effect level Kuehn

Entosiphon sulcatum 296 72-h no-observed- total biomass Brin (flagellate) adverse-effect level Kuehn

Bacteria

Pseudomonas putida 280 16-h no-observed- total biomass Brin adverse-effect level Kuehn

Bacillus subtilis 1180 EC50 spore Yasu germination et al ------8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS

8.1. Single Exposures

8.1.1. Acute toxicity

The oral EC50 for narcosis in rabbits was 19 mmol isobutanol/kg body weight (Münch, 1972).

Acute toxicity data are given in Table 6.

Table 6. Acute toxicity of isobutanol ------Species Route of Parameter Results Reference administration ------3 Guinea- inhalation LC50 19 900 mg/m Kushneva et al. pig (1983) 3 Mouse inhalation LC50 15 500 mg/m Kushneva et al. (1983) 3 Rabbit inhalation LC50 26 250 mg/m Kushneva et al. (1983) a 3 Rat inhalation LCLo 8000 mg/m (4 h) US DHEW (1978) 3 Rat inhalation LC50 19 200 mg/m Kushneva et al. (1983) a Cat intravenous LDLo 0.018 g/kg US DHEW (1978) a Rabbit oral LDLo 3.75 g/kg US DHEW (1978) Rabbit oral LD50 41 mmol/kg Münch (1972) Mouse oral LD50 3.5 g/kg Kushneva et al. (1983) Rat oral LD50 3.1 g/kg Kushneva et al. (1983) Rat oral LD50 2.46 g/kg US DHEW (1978) Rabbit skin LD50 4.24 g/kg US DHEW (1978) ------a Lo = lowest.

After ip administration of isobutanol to mice, the LD50

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was > 1000 mg/kg at 30 min and 544 mg/kg at 7 days. Gross post- mortem examination of livers showed an abnormal dark coloration (Maickel & McFadden, 1979).

Smyth et al. (1954) reported 100% survival of rats exposed for 2 h to saturated isobutanol vapour (approximately 49 248 mg/m3 or 16 000 ppm) in air, but observed 2 deaths in a group of 6 rats when they were exposed for 4 h to a concentration of 24 624 mg/m3 (8000 ppm) in air.

8.1.2. Signs of intoxication

The toxic effects of isobutanol are mainly alcoholic intoxication and narcosis. Stupor and loss of voluntary movements in animals can be considered signs of intoxication by the oral route (Münch, 1972). Rats and rabbits were exposed, by inhalation, for a period of 4 h to various concentrations of isobutanol. At a concentration of 15 700 mg/m3, there was irritation of the airways. Three days later, symptoms included central nervous depression, a decreased number of lymphocytes in bone marrow, a decreased blood- lactate level, delay in elimination of bromophthalein from blood, and morphological changes including dystrophia of hepatocytes and olfactory neurons in the brain. After exposure to 8000 mg/m3, symptoms were similar but less severe. An isobutanol concentration of 1300 mg/m3 decreased bone marrow lymphocyte numbers. A concentration of 100 mg/m3 only altered breathing frequency (Kushneva et al., 1983).

8.2. Skin and Eye Irritation

Application of 500 mg isobutanol to the skin of rabbits for 24 h was moderately irritating. However, application of 2 mg to the rabbit eye caused severe irritation (US DHEW, 1978).

8.3. Short-Term Exposures

Hillbom et al. (1974a,b) gave one group of 6 male Wister rats a 1 mol/litre solution of isobutanol as their sole drinking liquid for a period of 4 months. Another group of 5 rats of the same species was given a 2 mol/litre solution of isobutanol as their sole drinking liquid for 2 months. Both groups were provided an ad libitum diet of ordinary laboratory food containing about 26% of calories as protein, 55% as carbohydrate, and 19% as fat. At the end of the study, all animals were decapitated, and their livers examined. No adverse effects were detected in the group given the 1 mol/litre solution, while the rats in the group given the 2 mol/litre solution showed a decrease in fat, glycogen, RNA, and overall size of the liver cells.

Oral administration of between 1/10 and 1/5 of the LD50 of isobutanol for 6 days/week over 1 month, did not result in any deaths in rats (Kushneva et al., 1983). Continuous exposure of rats, by inhalation, to isobutanol at 3 mg/m3 over 4 months, caused an increased threshold for leg withdrawal response to electrical stimulation, and depression of haemoglobin content, erythrocyte count, and activities of cholinesterase and catalase in the blood. The activities of -amino and aspartate amino transferase were elevated. At 0.5 mg/m3, numbers of white cells and erythrocytes, haemogloglobin content, and cholinesterase activity were all decreased. No effects were observed at 0.1 mg/m3 (Tsulaya et al., 1978).

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8.4. Long-Term Exposures

With the exception of a carcinogenicity study, no long-term studies have been reported.

8.5. Reproduction, Embryotoxicity, and Teratogenicity

No relevant data are available on reproduction or embryotoxicity and no conclusions can therefore be drawn.

8.6. Mutagenicity

The only reported study on mutagenicity is that of Hillscher et al. (1969) who demonstrated an increased rate of reverse mutation when Escherichia coli CA 274 was treated with 0.7% isobutanol, without metabolic activation. This study in itself is inadequate to assess the mutagenic potential of the compounds.

8.7. Carcinogenicity

Two groups of 19 and 24 male and female Wistar rats (10 weeks old) were administered purified isobutanol. Group one received 0.2 ml/kg body weight, orally, twice weekly. Group two received 0.05 ml/kg, subcutaneously, twice weekly. Two control groups (25 rats each) received 1 ml of 0.9% sodium chloride twice weekly (controls for group one were dosed orally; controls for group two were dosed subcutaneously). All rats were dosed until spontaneous death. Test rats presented liver carcinomas and sarcomas, spleen sarcomas, stomach proventricular carcinomas, and myeoid leukaemia. Tumours of these types did not occur in the control groups (Table 7). In the exposed animals, toxic liver damage was found, ranging from steatosis and cell necrosis to fibrosis and cirrhosis. In addition, most exposed animals had hyperplasia of blood-forming tissues. The carcinogenic response was attributed to isobutanol (Gibel et al., 1974, 1975). No statistical analysis of the data was performed.

Table 7. Summary of tumours in experimental animalsa ------Agent Application Number Average Malignant Benign (twice weekly) of survival tumour tumour animals (days) ------Control oral: 1 ml/kg 25 643 0 3 (0.9% NaCl) body weight solution) subcutaneous: 25 643 0 2 1 ml/kg body weight

Isobutanol oral: 0.2 ml/kg 19 495 3b 9 body weight

subcutaneous: 24 544 8c 3 0.05 ml/kg body weight ------a Adapted from: Gibel et al. (1975). b Proventriculus carcinoma and liver cell carcinoma; proventriculus carcinoma and myeloid leukaemia; myeloid leukaemia. c Proventriculus carcinoma (2); liver sarcoma (2); spleen sarcoma; mesothelioma; retroperitoneal sarcoma (2).

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8.8. Special Studies

In 8 guinea-pigs, serum (SGOT, SGPT, GLDH) activities and levels were measured 16 h after oral administration of 33.5 mg isobutanol/kg olive oil. There was no statistically-significant difference between treated animals and 8 olive-oil treated controls (Siegers et al., 1974).

Isobutanol can potentiate the hepatic toxicity of carbon tetrachloride (Cornish & Adefuin, 1967).

9. EFFECTS ON MAN

Isobutanol may be absorbed through the lungs and the gastrointestinal tract (Browning, 1965).

No data are available on the effects of isobutanol on the skin. However, as with other defatting solvents, isobutanol may cause erythematous skin lesions (Schwarz & Tulipan, 1939).

Immersion of the hands of 5 healthy male volunteers in a 1:1 (v/v) mixture of m-xylene and isobutanol, for 15 min at room temperature, caused only a mild and sometimes barely distinguishable erythema or a burning feeling. Isobutanol (in a 1:1 mixture with m-xylene) dehydrated the skin and decreased the absorption of xylene. However, when water was added as 8% of the mixture, this effect was reduced. The dermal effects of isobutanol only and its absorption were not studied (Riihimaki, 1979).

Seitz (1972) reported 7 case histories, occurring between 1965 and 1971, of workers who had been exposed to 1-butanol and isobutanol in a non-ventilated photographic laboratory. They handled the alcohols under intense and hot light, without any precautions. Exposure levels were not quantified, but must have been excessive; exposure time ranged from 1 1/2 months to 2 years. Two workers had transient vertigo, 3 severe Meniere-like vertigo with nausea, vomiting and/or headache. In one of these cases, hearing was also perturbed. Two workers did not present any signs or symptoms.

Eye irritation, blurred vision, and transient corneal vacuolization have been decribed in another excessive (no measurements done) mixed exposure of workers to isobutanol and butyl acetate. With adequate work room ventilation, it did not recur. The author felt that the more irritant butyl acetate would be the main contributor to this effect (Büsing, 1952).

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1. Evaluation of Human Health Risks

10.1.1. Exposure levels

Levels of exposure of the general population to isobutanol through food and beverages are not available and occupational exposure levels are limited and inadequate.

10.1.2. Toxic effects

In animals, isobutanol is absorbed through the skin, lungs, and gastrointestinal tract. Isobutanol is metabolized by alcohol dehydrogenase to isobutyric acid via the aldehyde and may enter the tricarboxylic acid cycle. Small amounts of isobutanol are excreted unchanged (< 0.5% of the dose), or as the glucuronide (< 5% of

Page 57 of 75 Butanols - four isomers (EHC 65, 1987)

the dose) in the urine. In rabbits, metabolites found in the urine include acetaldehyde, acetic acid, isobutyraldehyde, and isovaleric acid. Oral LD50 values (2.5 - 3.1 g/kg body weight) and inhalation 3 LC50 (19.2 g/m ) in rats classify isobutanol as slightly toxic according to Hodge & Sterner. The acute toxic effects are alcoholic intoxication and narcosis. Isobutanol is severely irritating to the eyes and moderately irritating to the skin. A group of rats given a 1 mol/litre solution of isobutanol as their sole drinking liquid for 4 months did not show any adverse effects in the liver; another group given a 2 mol/litre solution as their sole drinking liquid for 2 months showed a reduction in fat, glycogen, and RNA content, and in the overall size of the cells in the liver. Continuous inhalation exposure of rats to 3 mg/m3 for 4 months resulted in depression of leg withdrawal response to electrical stimulation, minor changes of formed elements of the blood and serum enzymes. The estimated no-observed-adverse-effect level was 0.1 mg/m3.

In a lifetime carcinogenicity study, groups of rats received isobutanol subcutaneously (0.05 ml/kg body weight twice a week) or orally (0.2 ml/kg body weight twice a week). The animals exhibited toxic liver damage ranging from steatosis to cirrhosis. Numbers of animals showing malignant tumours totalled 8 in the subcutaneous group, 3 in the oral group, and 0 in the control group. The majority of treated animals also showed hyperplasia of blood- forming tissues.

Because of lack of mutagenicity studies, the Task Group could not determine whether isobutanol was a genetically active compound. The findings in the carcinogenicity study are a cause for concern. Because of methodological inadequacies and the manner of reporting the data, it was not possible to determine whether isobutanol should be regarded as an animal carcinogen. Thus it is not possible to extrapolate from this study to possible long-term effects in man.

From the animal studies available, it is not possible to determine a no-observed-adverse-effect level for long-term exposure. No adequate data are available to assess mutagenicity or teratogenicity of isobutanol or effects on reproduction.

Exposure of the general population to isobutanol through food and beverages is unlikely to lead to acute toxic effects. The only reported observations in man relate to the production of vertigo under conditions of severe and prolonged exposure to vapour mixtures of isobutanol and 1-butanol. From this study, it is not possible to attribute the vertigo to a single cause.

10.2. Evaluation of Effects on the Environment

10.2.1. Exposure levels

Little quantitative data relating to levels in the general environment are available, but, because isobutanol is readily biodegradable, substantial concentrations are only likely to occur locally in the case of major spillages.

10.2.2. Toxic effects

At background concentrations likely to occur in the environment, isobutanol is not directly toxic for fish, amphibia, crustacea, or algae. Protozoa will be tolerant to levels of isobutanol likely to be found in the environment.

Page 58 of 75 Butanols - four isomers (EHC 65, 1987)

Isobutanol should be managed in the environment as a slightly toxic compound. It poses an indirect hazard to the aquatic environment, because it is readily biodegradable, which may lead to oxygen depletion.

10.3. Conclusions

1. On the basis of available data, the Task Group considered it unlikely that isobutanol would pose a serious acute health risk to the general population under normal exposure conditions. However, the Task Group was unable to make an assessment of the long-term health risk of isobutanol for the general population. It was concluded that the results of the carcinogenicity study need verification by a bioassay of modern standards.

2. The Task Group considered that the data available were inadequate to set an occupational exposure limit. In line with good manufacturing practice, exposure to isobutanol should be minimized.

3. The ecotoxicological data available indicate that the impact of background concentrations of isobutanol on the aquatic environment can be expected to be minimal.

11. RECOMMENDATIONS

1. The Task Group noted that, from the animal studies available, it is not possible to determine a no-observed-adverse-effect level. Relevant studies should be conducted so that this can be achieved.

2. The Task Group considered that adequate studies should be conducted to assess the mutagenicity and carcinogenicity of isobutanol.

3. Epidemiological studies, including precise exposure data, would assist in a better assessment of the occupational hazard of isobutanol.

4. Additional information on environmental pathways (notably emission and leaching) and residues are desirable.

12. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

The Council of Europe (1981) included isobutanol in the list of flavouring substances that can be added to foodstuffs without hazard to public health at a level of 25 mg/kg for beverages and food.

At their 23rd meeting, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) reviewed the data on isobutanol. They concluded that:

"The evaluation of this compound was not possible owing to the paucity of toxicological data. New specifications were prepared, but no toxicological monograph" (WHO, 1980).

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See Also: Toxicological Abbreviations

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