INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 154
ACETONITRILE
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
First draft prepared by Dr K. Hashimoto (Kanazawa University, Japan),
Dr. K. Morimoto (National Institute of Hygienic Sciences, Japan) and
Dr. S. Dobson (Institute of Terrestrial Ecology, Monks Wood
Experimental Station, United Kingdom)
World Health Orgnization
Geneva, 1993
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chemicals.
WHO Library Cataloguing in Publication Data
Acetonitrile.
(Environmental health criteria ; 154)
1.Acetonitriles - adverse effects 2.Acetonitriles - toxicity
3.Environmental exposure I.Series
ISBN 92 4 157154 3 (NLM Classification: QV 633)
ISSN 0250-863X
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CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR ACETONITRILE
1. SUMMARY
1.1. Properties, uses and analytical methods
1.2. Environmental levels and sources of human exposure
1.3. Environmental distribution and transformation
1.4. Environmental effects
1.5. Absorption, distribution, biotransformation and
elimination
1.6. Effects on laboratory mammals
1.7. Effects on humans
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND
ANALYTICAL METHODS
2.1. Identity
2.2. Physical and chemical properties
2.2.1. Physical properties
2.2.2. Chemical properties
2.3. Conversion factors
2.4. Analytical methods
2.4.1. Determination of acetonitrile in ambient air
2.4.1.1 Sampling methods
2.4.1.2 Measurement of acetonitrile in
collected air samples
2.4.2. Monitoring methods for the determination of
acetonitrile and its metabolites in
biological materials
2.4.2.1 Acetonitrile in urine
2.4.2.2 Acetonitrile in serum
2.4.2.3 Acetonitrile metabolites in tissues
and biological fluids
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
3.1. Natural occurrence
3.2. Anthropogenic sources
3.2.1. Production levels and processes
3.2.2. Uses
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION
4.1. Transport and distribution between media
4.1.1. Water
4.2. Transformation
4.2.1. Biodegradation
4.2.1.1 Water and sewage sludge
4.2.1.2 Soil
4.2.2. Abiotic degradation
4.2.2.1 Water
4.2.2.2 Air
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
5.1. Environmental levels
5.1.1. Air
5.1.2. Water and bottom sediment
5.1.3. Food
5.1.4. Tobacco smoke
5.1.5. Other sources of exposure
5.2. Occupational exposure
5.3. Acetonitrile in various solvent products
6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS
6.1. Absorption
6.1.1. Human studies
6.1.2. Experimental animal studies
6.1.2.1 Intake through inhalation
6.1.2.2 Dermal absorption
6.1.2.3 Intake via the gastrointestinal tract
6.2. Distribution
6.2.1. Human studies
6.2.2. Experimental animal studies
6.3. Biotransformation and elimination
6.3.1. Human studies
6.3.2. Experimental animal studies and
in vitro studies
6.3.2.1 Cyanide liberation from acetonitrile
6.3.2.2 The oxidative pathway of acetonitrile
metabolism
6.4. Biological monitoring of acetonitrile uptake
7. EFFECTS ON LABORATORY MAMMALS; IN VITRO TEST SYSTEMS
7.1. Acute toxicity
7.1.1. Single exposure
7.1.2. Clinical observations
7.1.2.1 Effect on skin
7.1.2.2 Effect on the eyes
7.1.2.3 Effect on respiration
7.1.2.4 Effect on adrenals
7.1.2.5 Effect on the gastrointestinal tract
7.1.3. Biochemical changes and mechanisms of
acetonitrile toxicity
7.1.3.1 Effect on cytochrome oxidase
7.1.3.2 Effect on glutathione
7.1.4. Antidotes to acetonitrile
7.2. Subchronic toxicity
7.2.1. Inhalation exposure
7.2.2. Subcutaneous administration
7.3. Teratogenicity and embryotoxicity
7.4. Mutagenicity
7.4.1. Bacterial systems
7.4.2. Yeast assays
7.4.3. Drosophila melanogaster
7.4.4. Mammalian in vivo assays
7.4.5. Chromosome aberrations and sister chromatid
exchange
7.5. Carcinogenicity
7.6. Cytotoxicity testing
8. EFFECTS ON HUMANS
8.1. Acute toxicity
8.1.1. Inhalation exposure
8.1.2. Dermal exposure
8.1.3. Oral exposure
8.2. Chronic toxicity
8.3. Mutagenicity and carcinogenicity
8.4. Occupational exposure to cyanide
8.5. Chronic poisoning by cyanides
8.5.1. Ingestion
9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD
9.1. Microorganisms
9.2. Aquatic organisms
10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT
10.1. Evaluation of human health risks
10.2. Evaluation of effects on the environment
11. RECOMMENDATIONS FOR THE PROTECTION OF HUMAN HEALTH
12. FURTHER RESEARCH
13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES
REFERENCES
RESUME
RESUMEN
WHO TASK GROUP ON ENVIRONMENTAL HEALTH
CRITERIA FOR ACETONITRILE
Members
Dr R. Bruce, System Toxicants Assessment Branch, Office of Research
and Development, Environmental Criteria and Assessment Office,
US Environmental Protection Agency, Cincinnati, Ohio, USA
(Joint Rapporteur)
Dr R.J. Bull, College of Pharmacy, Washington State University,
Pullman, Washington, USA
Dr S. Dobson, Institute of Terrestrial Ecology, Monks Wood
Experimental Station, Huntingdon, United Kingdom
(Vice-Chairman)
Dr K. Hashimoto, Department of Hygiene, School of Medicine,
Kanazawa University, Kanazawa, Japan
Dr P. Lauriola, Local Hygiene Unit, Office of Public Hygiene,
Modena, Italy
Dr M. Lotti, Institute of Occupational Medicine, University of
Padua, Padua, Italy (Chairman)
Dr K. Morimoto, Division of Biological Chemistry and Biologicals,
National Institute of Hygienic Sciences, Tokyo, Japan (Joint
Rapporteur)
Dr Y.F. Panga, Department of Standard Setting, Chinese Academy of
Preventive Medicine, Beijing, China
Dr S.A. Soliman, Department of Pesticide Chemistry, College of
Agriculture and Veterinary Medicine, King Saud University,
Al-Qasseem, Bureidah, Saudi Arabia
Secretariat
Dr B.H. Chen, International Programme on Chemical Safety,
World Health Organization, Geneva, Switzerland (Secretary)
Dr E. Smith, International Programme on Chemical Safety,
World Health Organization, Geneva, Switzerland
a Invited but unable to attend.
NOTE TO READERS OF THE CRITERIA MONOGRAPHS
Every effort has been made to present information in the
criteria monographs as accurately as possible without unduly
delaying their publication. In the interest of all users of the
Environmental Health Criteria monographs, readers are kindly
requested to communicate any errors that may have occurred to the
Director of the International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland, in order that they may be
included in corrigenda.
* * *
A detailed data profile and a legal file can be obtained from
the International Register of Potentially Toxic Chemicals, Case
postale 356, 1219 Châtelaine, Geneva, Switzerland (Telephone
No. 9799111).
* * *
This publication was made possible by grant number
5 U01 ES02617-14 from the National Institute of Environmental Health
Sciences, National Institutes of Health, USA.
WHO TASK GROUP ON ENVIRONMENTAL HEALTH
CRITERIA FOR ACETONITRILE
A WHO Task Group on Environmental Health Criteria for
Acetonitrile met in Modena, Italy, from 24 to 28 November 1992.
Mr Giorgio Baldini, the President of the Province of Modena, opened
the meeting and greeted the participants on behalf of the Province
of Modena. Dr B.H. Chen of the International Programme on Chemical
Safety (IPCS) welcomed the participants on behalf of the Director,
IPCS, and the three IPCS cooperating organizations (UNEP/ILO/WHO).
The Task Group reviewed and revised the draft criteria monograph and
made an evaluation of the risks for human health and the environment
from exposure to acetonitrile.
The first draft of this monograph was prepared by Dr K.
Hashimoto, Kanazawa University, Japan, Dr K. Morimoto, National
Institute of Hygienic Sciences, Japan, and Dr S. Dobson, Institute
of Terrestrial Ecology, Monks Wood Experimental Station, United
Kingdom. The second draft was prepared by Dr K. Morimoto
incorporating comments received following the circulation of the
first draft to the IPCS Contact Points for Environmental Health
Criteria monographs. Dr M. Lotti (Institute of Occupational
Medicine, University of Padua, Italy) made a considerable
contribution to the preparation of the final text.
Dr B.H. Chen and Dr P.G. Jenkins, both members of the IPCS
Central Unit, were responsible for the overall scientific content
and technical editing, respectively. The efforts of all who helped
in the preparation and finalization of the document are gratefully
acknowledged.
* * *
Financial support for this Task Group meeting was provided by
the Province of Modena, Communes of Mirandola and Medolla, Local
Hygiene Units N. 16 of Modena and N. 15 of Mirandola, Association of
Business and Industries of the Province of Modena and ENICHEM
(National Organization of Industrialization for Chemistry) in Italy.
ABBREVIATIONS
CLD chemiluminescence nitrogen detector
GC gas chromatography
HPLC high performance liquid chromatography
NPD nitrogen-phosphorus selective detector
TCD thermal conductivity detection
TEA thermal energy analyser
1. SUMMARY
1.1 Properties, uses and analytical methods
Acetonitrile (CH3CN) is a by-product of acrylonitrile
manufacture. It may also be formed by the combustion of wood and
vegetation. It is a liquid with an ether-like odour. Acetonitrile
is a volatile, highly polar solvent used to extract fatty acids and
animal and vegetable oils. It is used in the petrochemical industry
in extractive distillation based on its selective miscibility with
organic compounds. It is used as a solvent for spinning synthetic
fibres and in casting and moulding plastics. In laboratories, it is
widely used in high-performance liquid chromatographic (HPLC)
analysis and as a solvent for DNA synthesis and peptide sequencing.
The most widely used analytical technique for acetonitrile is
gas chromatography.
1.2 Environmental levels and sources of human exposure
Very few data on acetonitrile levels in the environment are
available. Worldwide, acetonitrile concentrations in air of 200 to
42 000 ng/m3 have been reported. Slightly higher values were
obtained for urban than rural air in one study. Single measurements
before and after burning of bush and straw showed a 10-fold increase
in acetonitrile air concentration.
Acetonitrile was not detected in 72 water samples from Japan but
was found in 11 out of 60 aquatic sediment samples at
concentrations between 0.02 and 0.54 mg/kg. Acetonitrile has not
been detected in food.
Tobacco smoke contains acetonitrile and burning polyurethane
foam releases acetonitrile and hydrogen cyanide.
Whilst production of acrylonitrile offers the greatest potential
for exposure, this is carried out in a closed system. Practical
uses of acetonitrile lead to greater exposure.
1.3 Environmental distribution and transformation
Acetonitrile volatilizes from water and would also volatilize
from soil surfaces. It is readily biodegraded by several strains of
bacteria common in sewage sludge, natural waters and soil.
Acclimatization of bacteria to acetonitrile or petroleum wastes
increases the rate of degradation. Anaerobic degradation appears to
be limited or absent.
Hydrolysis of acrylonitrile in water is extremely slow. There
is no significant photodegradation in either water or the
atmosphere. Reaction with ozone is slow as is reaction with singlet
oxygen. The major mechanism for removal of acetonitrile from the
troposphere is reaction with hydroxyl radicals; residence times
have been estimated at between 20 and 200 days.
Acetonitrile does reach the stratosphere where it is
characteristically associated in positive ion clusters in the upper
regions.
1.4 Environmental effects
Acetonitrile has low toxicity to microorganisms (bacteria,
cyanobacteria, green algae and protozoans) with thresholds at
500 mg/litre or more. Freshwater invertebrates and fish acute
LC50s are 700 mg/litre or more. Acute tests have been conducted
under static conditions without analytical confirmation of
concentrations. Similar results obtained from 24- and 96-h tests
suggest volatilization of acetonitrile.
1.5 Absorption, distribution, biotransformation and elimination
Acetonitrile is readily absorbed from the gastrointestinal
tract, through the skin and the lungs. All three routes of exposure
have been reported to lead to systemic effects.
Postmortem examination of tissues from poisoned humans has
revealed that acetonitrile distributes throughout the body. This is
supported by animal studies in which acetonitrile distribution has
been found to be fairly uniform throughout the body. There are no
indications of accumulation in animal tissues following
repeated administrations of acetonitrile.
There are substantial data to suggest that most of the systemic
toxic effects of acetonitrile are mediated through its metabolism to
cyanide, which is catalysed by the cytochrome P-450 monooxygenase
system. Cyanide is subsequently conjugated with thiosulfate to form
thiocyanate which is eliminated in the urine. Peak concentrations
of cyanide in the blood of rats following administration of near
lethal doses of acetonitrile approximate to the concentrations
observed following the administration of an LD50 dose of potassium
cyanide. However, the peak concentration of cyanide after
administration of acetonitrile is delayed by up to several hours as
compared to other nitriles. Moreover, the more rapid rate at which
cyanide is produced in the mouse appears to account for the much
greater sensitivity of this species to the toxic effects of
acetonitrile. Cyanide and thiocyanate have been identified in human
tissues after exposure to acetonitrile. A portion of the
acetonitrile dose is also eliminated unchanged in expired air and in
urine.
1.6 Effects on laboratory mammals
Acetonitrile induces toxic effects similar to those observed in
acute cyanide poisoning, although the onset of symptoms is some-what
delayed compared to inorganic cyanides or other saturated nitriles.
The 8-h inhalation LC50 in male rats is 13 740 mg/m3 (7500 ppm).
The oral LD50 in the rat varies from 1.7 to 8.5 g/kg depending on
the conditions of the experiment. Mice and guinea-pigs appear to be
more sensitive, with an oral LD50 in the range of 0.2-0.4 g/kg.
The main symptoms in animals appear to be prostration followed by
seizures.
Dermal application of acetonitrile causes systemic toxicity in
animals and has been implicated in the death of one child. The
percutaneous LD50 in rabbits is 1.25 ml/kg.
Subchronic exposure of animals to acetonitrile produces effects
similar to those seen after acute exposures.
Acetonitrile is not mutagenic in assays using Salmonella
typhimurium, both with and without metabolic activation. It
induces aneuploidy in a diploid yeast strain at very high
concentrations. No animal studies on chronic or carcinogenic effects
of acetonitrile have been reported.
1.7 Effects on humans
The levels causing toxicity in man are unknown but are
probably in excess of 840 mg/m3 (500 ppm) in air. Symptoms and
signs of acute acetonitrile intoxication include chest pain,
tightness in the chest, nausea, emesis, tachycardia, hypotension,
short and shallow respiration, headache, restlessness,
semiconsciousness, and seizures. Other non-specific symptoms may be
due to the irritant effects of the compound. The systemic effects
appear to be largely attributable to the conversion of acetonitrile
to cyanide. Blood cyanide and thiocyanate levels are elevated
during acute intoxication. Two fatalities after exposure to
acetonitrile vapour in the workplace and one fatal case of a child
ingesting an acetonitrile-containing cosmetic have been reported.
Elevated tissue cyanide concentrations were found in postmortem
examin-ation of these cases.
No epidemiological study of cancer incidence relating to
acetonitrile exposure has been reported.
Acetonitrile can cause severe eye burns. Skin contact with
liquid acetonitrile should be avoided. An employee's exposure to
acetonitrile in any 8-h shift has been recommended in many
countries not to exceed a time-weighted average of 70 mg/m3 air
(40 ppm).
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND
ANALYTICAL METHODS
2.1 Identity
Chemical formula: CH3CN
Chemical structure:
Relative molecular mass: 41.05
CAS chemical name: acetonitrile
CAS registry number: 75-05-8
Synonyms: cyanomethane, ethanenitrile, nitrile of
acetic acid, methyl cyanide, ethyl
nitrile, methanecarbonitrile
Specifications for commercial acetonitrile are given in
Table 1. The principal organic impurity in commercial acetonitrile
is propionitrile, although small amounts of allyl alcohol may also
be present (Grayson, 1985).
2.2 Physical and chemical properties
2.2.1 Physical properties
Acetonitrile is a volatile, colourless liquid with a sweet,
ether-like odour (Grayson, 1985). It is infinitely soluble in water
and readily miscible with ethanol, ether, acetone, chloroform,
carbon tetrachloride and ethylene chloride (Clayton & Clayton,
1982). It is immiscible with many saturated hydrocarbons (petroleum
fractions) (Budavari, 1989).
Important physical constants and properties of acetonitrile are
summarized in Table 2.
Table 1. Commercial acetonitrile specificationsa
Specific gravity (at 20 °C) 0.783-0.787
Distillation range (°C)
initial point, minimum 80.5
end point, maximum 82.5
Purity (minimum), % by weight 99.0
Acidity (as acetic acid, maximum % by weight) 0.05
Copper (maximum), ppm 0.5
Iron (maximum), ppm 0.5
Water (maximum), % by weight 0.3
Colour (maximum), Pt-Co 15
a From: Grayson (1985)
2.2.2 Chemical properties
Although acetonitrile is one of the most stable nitriles, it
undergoes typical nitrile reactions and is used to produce many
types of nitrogen-containing compounds. It can be trimerized to
S-trimethyltriazine and has been telomerized with ethylene and
copolymerized with alpha-epoxides (Grayson, 1985).
Acetonitrile produces hydrogen cyanide when heated to
decomposition or when reacted with acids or oxidizing agents
(Reynolds, 1982).
2.3 Conversion factors
1 ppm = 1.68 mg/m3 (25 °C, 760 mmHg)
1 mg/m3 = 0.595 ppm (25 °C, 760 mmHg) (Clayton & Clayton, 1982)
Table 2. Physical properties of acetonitrile
Properties Value Reference
Appearance colourless liquid Budavari (1989)
Odour ether-like Budavari (1989)
Boiling point 81.6 °C (760 mmHg) Budavari (1989)
Freezing point -45.7 °C Grayson (1985)
-44 to -41 °C Verschueren (1983)
Specific gravity 0.78745 (15/4 °C) Grayson (1985)
0.7138 (30/4 °C) Grayson (1985)
Vapour density 1.42 (air = 1) Clayton & Clayton (1982)
Refractive index (ND) 1.34604 (15 °C) Clayton & Clayton (1982)
1.33934 (30 °C) Clayton & Clayton (1982)
Solubility in water infinitely soluble Clayton & Clayton (1982)
Vapour pressure
at (15.5 °C) 7.32 kPa (54.9 mmHg) US EPA (1984)
at (20.0 °C) (74.0 mmHg) Verschueren (1983)
at (30.0 °C) (115.0 mmHg) Verschueren (1983)
Water azeotrope boiling point 76 °C
water content 16% US EPA (1984)
Log P (octanol/water -0.38 Leo et al. (1971)
partition coefficient -0.34 Verschueren (1983)
Table 2 (contd)
Properties Value Reference
Flash point 5.6 °C (open cup) Reynolds (1982)
12.8 °C (closed cup) Reynolds (1982)
Ignition temperature 524 °C Sax & Lewis (1989)
Explosive limits lower 4.4 Grayson (1985)
in air (% by volume) 3.05 Prager (1985)
upper 16.0 Grayson (1985)
17.0 Prager (1985)
2.4 Analytical methods
2.4.1 Determination of acetonitrile in ambient air
2.4.1.1 Sampling methods
The use of absorption tubes to trap acetonitrile from ambient
air with subsequent thermal or liquid desorption prior to gas
chromatographic (GC) analysis has been reported in many references.
The National Institute of Occupational Safety and Health (NIOSH,
1977, 1984) recommended the use of a glass tube (9 cm long and 6 mm
internal diameter) containing two sections of 20-40 mesh activated
(600 °C) coconut charcoal (front = 400 mg and back = 200 mg)
separated by 3 mm section urethane foam and held in place with plugs
of silanized glass wool. The tube is then flame-sealed at both ends
until it is used for air sampling. Other sampling tubes containing
different sorbents (i.e. porous polymer beads) have also been
recommended (Campbell & Moore, 1979; Berg et al., 1980; Rigby,
1981; Kashihira, 1983; Kashihira et al., 1984; Wood, 1985; Cobb
et al., 1986).
2.4.1.2 Measurement of acetonitrile in collected air samples
Several methods have been used to measure acetonitrile in
environmental samples. Most of the reported methods are based on
the use of GC.
a) Gas chromatography
GC is frequently used for determining acetonitrile using
different kinds of detectors in conjunction with the charcoal or
porous polymer beads sampling technique. A number of detectors have
been recommended. Until recently, almost all of the
published work involved the use of flame ionization detection (FID).
However, it was found that FID did not respond to acetonitrile in a
repeatable way even with the use of internal standards (Joshipura
et al., 1983).
Attention has therefore turned to the use of thermal
conductivity detection (TCD) (Joshipura et al., 1983) and to
nitrogen-phosphorus selective detector, NPD (Cooper et al., 1986).
Rounbehler et al. (1982) described a modification for the thermal
energy analyser (TEA), a highly sensitive nitrosyl-specific GC
chemiluminescence detector, which allows it to be used as a highly
selective one in detecting nitrogen-containing compounds. They
concluded that the modified TEA was as sensitive as the alkali-bead
flame ionization detection (AFID) but had a much higher selectivity
toward nitrogen-containing compounds. Using the TEA, these
investigators were not able to detect any acetonitrile in bacon or
beer. Kashihira et al. (1984) used a chemiluminescence nitrogen
detector GC (CLD-GC) method to measure acetonitrile and
acrylonitrile in air. The method was able to detect as little as
20 ng of acetonitrile per injection.
Cooper et al. (1986) developed a very sensitive method of
measuring nitrogen-containing hazardous pollutants in complex
matrices by GC with NPD and were able to detect 1.5 pg acetonitrile.
Table 3 summarizes the different types of detectors used in GC
analysis of acetonitrile along with the conditions employed and
their corresponding detectability.
b) High-performance liquid chromatography (HPLC)
The use of HPLC to determine trace amount of acetonitrile in
environmental samples has not been reported.
c) Microwave spectrometry
Kadaba et al. (1978) analysed toxic constituents including
acetonitrile in tobacco smoke by microwave spectroscopy and were
able to measure acetonitrile down to 2 ppm.
2.4.2 Monitoring methods for the determination of acetonitrile and
its metabolites in biological materials
2.4.2.1 Acetonitrile in urine
Mckee et al. (1962) determined acetonitrile in urine samples
obtained from 20 male nonsmokers and 40 male smokers by a
modification of the method reported by Rhoades (1958, 1960) for the
analysis of coffee volatiles. The modification permitted the
stripping of urinary volatiles at 37 °C and at reduced pressure.
The stripped volatiles were collected in a liquid nitrogen trap,
vapourized, and analysed by GC with a thermal conductivity detector.
The column, which was packed with 15% Carbowax 1500 and silicone oil
200 (ratio 2:1) on 40-60 mesh Chromosorb P, was operated at 40 °C.
The carrier gas was helium at a pressure of 4 pounds per square
inch. Acetonitrile concentrations as low as 2.9 µg/litre could be
measured in urine using this method.
Table 3. Gas chromatographic conditions for acetonitrile determination
Packing Conditions Detection Reported level of References
detectability
Porapak 250 x 0.25 cm, 160 °C injector FID 10 ppm in Thomson (1969)
150 °C helium, 70 ml/min acrylonitrile
Porapak Q 122 x 0.63 cm, 180 °C injector FID 10 mg/m3 in air NIOSH (1977)
270 °C nitrogen, 50 ml/min (6 ppm)
Porapak Q 305 x 0.32 cm, 200 °C injector FID 0.01 ppm in air Campbell & Moore (1979)
200 °C nitrogen, 20 ml/min
0.1% SP 1000 200 x 0.19 cm, 35-235 °C injector FID 0.07 ppm in air Berg et al. (1980)
on Carbopack C 125 °C nitrogen, 21 ml/min
20% Carbowax 180 x 0.2 cm, 90-145 °C injector TEA 0.041 ppm Rounbehler et al. (1982)
20 M 120 °C
Chromosorb 103 90 x 0.3 cm, 85 °C injector CLD 1 ppb in air Kashihira et al. (1984)
150 °C helium, 60 ml/min
Porapak Q 508 x 0.32 cm, 170 °C injector FID 0.2 ppm in air Wood (1985)
200 °C nitrogen, 30 ml/min
20% SP-1200W/0.1% 305 x 0.32 cm, 180 °C injector NPD 1.5 ppb Cooper et al. (1986)
Carbowax 1500 190 °C nitrogen, 30 ml/min or
helium, 35 ml/min
FID = Flame ionization detection; CLD = Chemiluminescent nitrogen detection; NPD =
Nitrogen-phosphorous detection; TEA = Thermal energy analyser
2.4.2.2 Acetonitrile in serum
Freeman & Hayes (1985a) determined serum acetonitrile
concentrations in rats dosed orally with acetone, acetonitrile, and
a mixture of acetone and acetonitrile by GC equipped with FID. The
analysis was performed isothermally (150 °C) at a helium flow rate
of 30 ml/min using a 2 mm x 1.22 m Chromosob 104 column (100/120
mesh) with a 15-cm precolumn. Propionitrile was added to the serum
samples as an internal standard prior to injection, and the samples
were injected directly into the column. Under the conditions of
this study, the retention times of acetone, acetonitrile and
propionitrile were 2.05, 3.65 and 6.20 min, respectively. The limit
of detection was not reported. However, it was reported that the
serum acetonitrile concentrations of animals in the control group
were all below 1 mg/litre.
2.4.2.3 Acetonitrile metabolites in tissues and biological fluids
a) Cyanide
Since hydrogen cyanide is a reactive and volatile nucleophile,
a variety of problems are encountered in its assay in biological
materials due to tissue binding or diffusibility (Troup &
Ballantyne, 1987). To reduce artefacts due to simple evaporative
losses, cyanide should be extracted under alkaline conditions.
Amdur (1959) determined the cyanide level in the blood of 16
workers, who were accidentally exposed to acetonitrile, by the
method of Feldstein & Klendshoj (1954), which uses a Conway
microdiffusion approach (Conway, 1950). The sensitivity of this
method is as low as 0.1 µg cyanide in a 1 ml sample. Willhite &
Smith (1981) measured cyanide concentrations in the liver and brain
of mice challenged by acetonitrile using the method of Bruce et al.
(1955), which is capable of determining 0.05 µg cyanide in a 1 ml
sample. Haguenoer et al. (1975a,b) determined free cyanide in the
tissues and urine of rats using the pyridine-benzidine method
described by Aldridge (1944); the sensitivity of this method was
0.7 µg hydrogen cyanide in a 1 ml sample. Ahmed & Farooqui (1982)
determined the tissue and blood cyanide levels in rats by the Conway
diffusion method described by Pettigrew & Fell (1973). Willhite
(1983) determined tissue cyanide level in hamsters by the procedure
of Bruce et al. (1955). A combination of the aeration procedure
of Bruce et al. (1955) with the colorimetric method of Epstein
(1947), which can determine 0.2 µg of cyanide in a 1 ml sample, has
been used to determine the cyanide level in brain (Tanii &
Hashimoto, 1984a) and in liver microsomes of mice (Tanii &
Hashimoto, 1984b). The aeration apparatus consists of three serial
tubes containing 25 ml 20% NaOH, 5 ml 20% trichloroacetic acid and
0.5 ml 0.1 N NaOH. An aliquot of samples is added to the tube
containing trichloroacetic acid, which is then aerated at a flow
rate of 600 ml/min, passing from the tube containing 20% NaOH for
10 min toward the tube containing 0.1 N NaOH. An aliquot from the
tube containing 0.1N NaOH is then removed, neutralized with acetic
acid and subjected to analysis for cyanide. Under these conditions,
the recovery of known amounts of cyanide is 97-100%. Freeman &
Hayes (1985a) determined cyanide in the blood of rats by a
microdiffusion method modified from Feldstein & Klendshoj (1954).
Samples were analysed colorimetrically at 586 nm using
pyridine-barbituric acid reagent as described by Blanke (1976).
Cyanide concentrations as low as 0.1 mg/litre could be reproducibly
detected by these methods. Zamecnik & Tam (1987) reported an
improved GC method for cyanide analysis in blood with acetonitrile
as an internal standard. GC with NPD was used with a 180 x 0.2 cm
column packed with 100/120 mesh Porapak Q. Other conditions were:
temperature, column 120 °C, detector 250 °C, and a helium gas flow
rate of 20 ml/min. The blood samples containing cyanide were
pipetted into disposable vials. Samples were then sealed and
glacial acetic acid was injected into the vials. These were then
vortexed and allowed to equilibrate for 30 min at room temperature.
The head space was injected into the gas chromatograph. The typical
retention times for the cyanide and acetonitrile peaks were 0.6 min
and 2.5 min, respectively. The sensitivity for cyanide was
0.05 ppm. Three procedures for the determination of cyanide in
biological fluids have been reported with full detail (Rieders &
Valentour, 1975). The first procedure is qualitative, the second
colorimetric (chloramine-T and barbituric acid and pyridine), and
the third depends on GC using electron capture detection.
Table 4 summarizes the methods which have been used for cyanide
analysis in biological samples.
b) Thiocyanate
Pozzani et al. (1959a) determined urinary thiocyanate levels
in various animals by means of the colorimetric method of Chesley
(1941). Using this method, 25-180 mg thiocyanate/litre urine could
be measured with a ± 4% error. Silver et al. (1982) determined
thiocyanate in the urine of rats dosed with acetonitrile.
Thiocyanate was first isolated from urine by separation on an ion
exchange column (10 x 1 cm) of Amberlite CG-400 as described by
Kanai & Hashimoto (1965) and then measured colorimetrically
according to the method of Epstein (1947). Willhite (1983)
determined the tissue thiocyanate levels in hamsters using the
method described by Bruce et al. (1955).
Table 4. Analysis of cyanide in biological materials
Analytical methods Application
Principle Detectability References Biological materials References
(µg/ml)
Conway diffusion method 0.1 Feldstein & Klendshoj (1954) human blood Amdur (1959)
0.1 Pettigrew & Fell (1973) rat tissues and blood Ahmed & Farooqui (1982)
0.1 Feldstein & Klendshoj (1954); rat blood Freeman & Hayes (1985a)
Blanke (1976)
Benzidine and pyridine 0.1 Aldridge (1944) rat tissues and Haguenoer et al.
methods, colorimetry urine (1975a,b)
Aeration procedure and 0.2 Bruce et al. (1955); mouse brain Tanii & Hashimoto
colorimetry Epstein (1947) (1984a,b)
0.05 Bruce et al. (1955) mouse liver and brain Willhite & Smith (1981)
0.05 Bruce et al. (1955) hamster tissues Willhite (1983)
GC, nitrogen-phosphorus 0.05 - blood Zamecnik & Tam (1987)
detector
Pereira et al. (1984) used the method of Contessa & Santi
(1973) to determine thiocyanate levels in urine samples collected
from rats treated with different nitriles. The method was able to
detect thiocyanate concentrations as low as 100 µg in a 0.2 ml urine
sample.
Table 5 summarizes the methods reported for analysis of
thiocyanate in biological samples.
Table 5. Analysis of thiocyanate in biological materials
Analytical methods Application
Principle Detectability References Biological materials References
(µg/ml)
Colorimetry 25 Chesley (1941) animal urine Pozzani (1959a)
0.6 Bruce et al. (1955) hamster tissues Willhite (1983)
Ion exchange separation 0.5 Kanai & Hashimoto (1965); rat urine Silver et al. (1982)
and colorimetry Epstein (1947)
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
3.1 Natural occurrence
Acetonitrile may be formed by combustion of wood, straw and
other vegetation. However, the rate of formation and the
contribution to atmospheric acetonitrile has not been quantified
(Becker & Ionescu, 1982).
3.2 Anthropogenic sources
3.2.1 Production levels and processes
Acetonitrile is a by-product of acrylonitrile synthesis. This
process is known as the SOHIO (Standard Oil Company of Ohio) process
and involves a high temperature catalytic reaction between propylene
and ammonia. The SOHIO process is the principal route to both
acrylonitrile and acetonitrile, produced in the ratio of 0.035 kg
acetonitrile/kg acrylonitrile (Lowenheim & Moran, 1975).
Acetonitrile can be synthesized by several other routes. Good
yields are obtained by dehydration of an acetic acid and ammonia
mixture, acetamide or ammonium acetate.
CH3COOH + NH3 -> CH3CN + 2H2O
CH3CONH2 -> CH3CN + H2O
CH3CO2NH4 -> CH3CN + 2H20
A 90% yield of acetonitrile is obtained by the reaction of
ethanol and ammonia in the presence of catalyst such as Ag, Cu,
MoO3, and ZnS at moderate temperatures. Acetonitrile is also
produced by the reaction of cyanogen chloride with methane, ketones,
ethanol, alkylene epoxides, and paraffins or olefins.
The principal organic impurity in commercial acetonitrile is
propionitrile, together with a small amount of allyl alcohol (US
EPA, 1992).
Reported production of acetonitrile in the USA during the
period 1980-83 (US EPA, 1985) was:
Year Production (millions of kg)
1980 10.1
1981 9.5
1982 9.4
1983 11.4
3.2.2 Uses
Being a volatile highly polar solvent, acetonitrile finds its
greatest use as an extracting fluid for fatty acids and animal and
vegetable oils.
Acetonitrile has been widely used as an extractive distillation
solvent in the petrochemical industry for separating olefin-diolefin
mixtures and for C4-hydrocarbons. When acetonitrile is used in this
way, recycling is effected by water dilution of the extract and
condensate with subsequent phase separation, after which the
acetonitrile is azeotroped from the aqueous phase.
Acetonitrile has been used as a solvent for polymer spinning
and casting because of the combination of high solubility and
desirable intermediate volatility. It is also used as a solvent for
isolating components from crude products such as crude wool resin.
Acetonitrile is used as a common laboratory solvent for
recrystallizing various chemicals and is widely used as a solvent in
HPLC analysis. Acetonitrile is also used in biotechnology research
as a solvent in the synthesis of DNA and peptide sequencing (Borman,
1990).
Acetonitrile can be used to remove tars, phenols and colouring
matter from petroleum hydrocarbons that are not soluble in
acetonitrile.
Acetonitrile is also used as a starting material for the
synthesis of many chemicals such as acetophenone, alpha-naphthyl
acetic acid, thiamine and acetomidine (Hawley, 1971).
The use patterns of acetonitrile are summarized in Table 6.
Table 6. Main use patterns of acetonitrilea
Extraction of fatty acids and animal and vegetable oils
Extraction of unsaturated petroleum hydrocarbons
Solvent for polymer spinning and casting
Moulding of plastics
Removal of tars, phenols and colouring matter from petroleum
hydrocarbons
Purification of wool resin
Recrystallization of steroids
Starting material for synthesis of chemicals
Solvent in DNA synthesis and peptide sequencing
Medium for promoting reactions
Solvent in non-aqueous titrations
Non-aqueous solvent for inorganic salts
High-pressure liquid chromatographic analysis
Catalyst and component of transition-metal complex catalysts
Extraction and refining of copper
Stabilizer for chlorinated solvents
Perfume manufacture
Pharmaceutical solvents
a From: Veatch et al. (1964); NIOSH (1978); Toxic Substances
Control Act (1979); Smiley (1983); Borman (1990)
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION
4.1 Transport and distribution between media
4.1.1 Water
Hine & Mookerjee (1975) reported that the ratio of
concentration in the water phase to the gas phase of a dilute
aqueous solution of acetonitrile at equilibrium at 25 °C is 891:1.
The inverse of this ratio (1.1 x 10-3) is the unit-less Henry's
Law constant. Conversion to units, using an RT value of 2.4 x
10-2-m3 atm/mol (where R is the gas constant and T is the
temperature in K), yields a Henry's Law constant of
2.6 x 10-5-m3 atm/mol. This value of Henry's Law constant
indicates that the volatilization of acetonitrile is probably
significant for most environmental bodies of water (Lymann et al.,
1982). The concentration of acetonitrile in river water decreased
to 5% of the original level after 72 h in a study carried out under
stable conditions (Chen et al., 1981).
4.2 Transformation
4.2.1 Biodegradation
4.2.1.1 Water and sewage sludge
Ludzack et al. (1958, 1959) measured the biodegradation of
acetonitrile in Ohio River water and in aged sewage by measuring
CO2 production. Degradation in the river water occurred at a
faster rate than in the sewage; the 12-day biological oxygen demand
(BOD) was 40% in river water but only 20% in the sewage.
Acclimatization of microorganisms was examined by reculturing, and
the degradation was found to occur 5 times more rapidly using
acclimatized microorganisms. The effect of temperature on
biodegradation was also studied; degradation at 5 °C took 2.5-5
times longer than at 20 °C. Ludzack et al. (1961) examined the
degradation of acetonitrile by activated sludge in a continuous feed
test at 22-25 °C; 82-94% BOD was removed during 4 weeks of test
operation. Anaerobic digestion does not appear to be an effective
means of removing the compound from waste water (Ludzack et al.,
1961).
Using the Japanese MITI (Ministry of International Trade and
Industry) test, Sasaki (1978) reported that acetonitrile is "readily
biodegradable", meaning that oxygen consumption is > 30% in 2
weeks. Thom & Agg (1975) reported that acetonitrile should be
degradable by biological sewage treatment with appropriate
acclimatization. Mimura et al. (1969) isolated the bacterium
Corynebacterium nitrilophius from activated sludge and found that
this microorganism was capable of assimilating acetonitrile. Kelly
et al. (1967) found virtually no degradation of acetonitrile using
a nitrogenase from Azotobacter chroococcum.
Goud et al. (1985) isolated bacteria of several genera from
various points in an effluent treatment plant at a petrochemical
installation. Azobacter spp and, more particularly, Pseudomonas
spp were able to degrade acetonitrile, added to the culture medium
at 1% as sole carbon source. Aeromonas spp and Bacillus spp,
however, were unable to degrade acetonitrile. The authors pointed
out that many of the bacterial species tested are common in the
environment, and that regular exposure to petrochemicals selects
strains that are able to degrade such compounds.
Chapatwala et al. (1992) investigated mixed cultures of
bacteria isolated from an area contaminated with organic cyanide and
polychlorinated biphenyls and found that they readily utilized
acetonitrile as sole carbon and nitrogen source. Nearly 70% of
14C-labelled acetonitrile was recovered as CO2, the remainder
being incorporated into bacterial growth. The mixed culture lost
its capacity to degrade biphenyl when repeatedly recultured with
acetonitrile, indicating more ready degradation of the nitrile.
Ludzack et al. (1961) observed high levels of nitrates in
effluents from activated sludge degrading acetonitrile. Firmin &
Gray (1976) used a species of Pseudomonas capable of utilizing
acetonitrile as sole carbon source to show that acetonitrile
undergoes direct enzymatic hydrolysis. These authors postulated the
following metabolic pathway based on their results with [2-14C]
acetonitrile: acetonitrile -> acetamide -> acetate -> tricarboxylic
acid cycle intermediates (citrate, succinate, fumarate, malate,
glutamate, etc.).
4.2.1.2 Soil
DiGeronimo & Antoine (1976) isolated Nocardia rhodochrus
Ll100-21 from barnyard soil and demonstrated that the
microorganism was capable of using acetonitrile as a source of
carbon and nitrogen. A decrease in acetonitrile content within the
culture medium was correlated with an increase in acetamide and
acetic acid levels; ammonia was also detected. Under the test
conditions, the initial concentration of acetonitrile was reduced by
14% in 3 h and by 52% in 8 h. Crude cell-free extracts were also
found to degrade acetonitrile by an enzymatic hydrolysis
mechanism that was reported to be inducible. Kuwahara et al.
(1980) found that Aeromonas species BN 7013 could be grown
using acetonitrile as a nitrogen source; the microorganism was
isolated from soil. Harper (1977) isolated a strain of the fungus
Fusarium solani from soil and found that cell-free extracts,
containing the nitrilase enzyme, were capable of hydrolysing
acetonitrile enzymatically.
4.2.2 Abiotic degradation
4.2.2.1 Water
Brown et al. (1975) reported that the hydrolysis rate
constant for acetonitrile in an aqueous solution of pH 10 is 1.195 x
10-8 M-1 sec-1. Assuming a constant pH of 10, the half-life
for this process would be > 18 000 years.
Anbar & Neta (1967) reported that the rate constant for the
reaction of acetonitrile with hydroxyl radicals in aqueous solution
at pH 9 and room temperature is 2.1 x 106 M-1 sec-1; assuming
an environmental hydroxyl radical concentration at 10-17 M, a
half-life of 1042 years can be calculated. Dorfman & Adams (1973)
reported a similar hydroxyl radical rate constant of 3.5 x 106
M-1 sec-1.
The absorption maximum for acetonitrile in the UV range is
< 160 nm (Silverstein & Bassler, 1967); therefore, the direct
photolysis of acetonitrile in the aquatic environment is not
expected to occur.
4.2.2.2 Air
Harris et al. (1981) found in laboratory studies that the
rate of reaction of acetonitrile with ozone was relatively slow, the
rate constant being < 1.5 x 10-19 cm3 molecule-1 sec-1.
Assuming a typical atmosphere concentration of 1012 ozone
molecules/cm3, a half-life of > 54 days can be calculated from
this rate constant.
The reaction rate constant between singlet oxygen and
acetonitrile is reported to be 2.4 x 10-16 cm3 molecule-1
sec-1 (Graedel, 1978); this predicts an atmospheric half-life of >
5000 years for acetonitrile.
Dimitriades & Joshi (1977) reported on the reactivity of
acetonitrile as measured in an US EPA smog chamber with 22
blacklights, 7 sunlamps, 4 ppm acetonitrile and 0.2 ppm NOx.
Acetonitrile was found to be unreactive with respect to ozone yield.
The average rate of disappearance of acetonitrile was found to be
0.02% per hour, i.e. 100 times slower than that measured for
propane. Kagiya et al. (1975) measured the photochemical
decomposition rate of acetonitrile (300-2000 ppm) in air saturated
with water in a reaction cell irradiated with a mercury lamp. No
degradation was observed, however, when chlorine gas (2000 ppm) was
added to the cell, the decomposition rate being 1.32% per second.
Reaction between chlorine radicals and acetonitrile in the
atmosphere is not thought to be significant in relation to hydroxyl
radical reaction (Arijs et al., 1983).
The absorption maximum for acetonitrile in the UV range is
< 160 nm (Silverstein & Bassler, 1967). Therefore, the direct
photolysis of acetonitrile in the ambient atmosphere is not expected
to occur.
The major mechanism for removal of acetonitrile from the
troposphere is reaction with hydroxyl radicals. The rate constant
for the gas-phase reaction of acetonitrile with hydroxyl radicals
has been experimentally determined by Harris et al. (1981) to be
0.494 x 10-13 cm3 molecule-1 sec-1 at 24.2 °C; in the
temperature range 298-424 °K (25-151 °C), the rate constant was
described by the equation k = 5.86 x 10-13 exp (-1500 cal
mole-1/RT). From this rate constant data, Harris et al. (1981)
calculated the tropospheric destruction rate of acetonitrile at
25 °C to be approximately 5 x 10-7 sec-1 for a mean
concentration of 107 hydroxyl radicals/cm3 in a moderately
polluted troposphere; this rate yielded a tropospheric lifetime of
approximately 20 days. In a more average atmosphere of 106
hydroxyl radicals/cm3, the lifetime will be 10 times longer.
Guesten et al. (1981) reported the rate constant for the reaction
between hydroxyl radicals and acetonitrile in the gas phase to be
approximately 0.2 x 10-13 cm3 molecule-1 sec-1 at room
temperature, which agrees reasonably well with the findings of
Harris et al. (1981). The Arrhenius activation energy of
approximately 1500 cal mole-1, as determined by Harris et al.
(1981), indicates that the reaction proceeds largely or entirely by
abstraction of a hydrogen atom.
Acetonitrile does reach the upper atmosphere. It is
characteristically associated in positive ion clusters of the form
H+(CH3CN)m (H2O)n. These ions do not occur in the
ionosphere but become important at 35 km altitude. At lower
altitudes still (about 12 km), acetone ions become evident (Arijs
et al., 1983; Huertas & Marenco, 1986).
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
5.1 Environmental levels
5.1.1 Air
Becker & Ionescu (1982) monitored air near to the ground in
both urban and rural areas and detected acetonitrile at
concentrations of 3360 to 11 960 µg/m3 (2-7 ppb by volume) using
GC/MS. There was some indication that results from city centre
samples were higher than general rural samples; acetonitrile at
concentrations of 7.4 ± 2.4 ppb was reported for the city centre in
Wuppertal, Germany. Given the small number of samples, however, a
comparison of the sites is difficult. A rural site was sampled in
the air before and after burning of bush and grass by farm workers
and results showed an increase in acetonitrile concentration from
4.0 to 34.9 ppb. This seems to be the only reported demonstration
of non-anthropogenic sources of atmospheric acetonitrile.
Acetonitrile has also been reported to be present in the upper
stratosphere (Arijs et al., 1983). It was detected at
concentrations of 210 to 42 000 ng/m3 in the Environmental Survey
of Chemicals in Japan (Office of Health Studies, Environment Agency,
1990).
In the USA, two samplings of air over a period of 24 h in a
rural area gave daily mean levels of 0.048 ppb by volume. A single
sampling of urban air was below the detection limit of the
analytical method (US EPA, 1988).
5.1.2 Water and bottom sediment
Acetonitrile was not detected in water but was detected in
bottom sediment in the Environmental Survey of Chemicals in Japan
(Office of Health Studies, Environment Agency, 1990). The sampling
was conducted in all 47 prefectures of Japan, but no information is
available concerning the nature of the sampling sites. It is not
known, therefore, whether the high ends of the ranges in air and
aquatic sediment were associated with industrial production and
release (Table 7).
5.1.3 Food
No report has been published showing contamination of food by
acetonitrile.
Table 7. Environmental levels in Japan of acetonitrile in 1987a
Concentration Frequency of Detection limit
detectionb
Water not detected 0/72 3 µg/litre
Sediment 0.021 to 0.54 mg/kg 11/60 0.021 mg/kg
Air 210 to 42 000 ng/m3 44/70 200 ng/m3
a From: Office of Health Studies (1990)
b Number of detections/number of samples
5.1.4 Tobacco smoke
The absorption of acetonitrile from smoke has been confirmed by
GC/MS analysis of a composite sample of the urine of 40 smokers
(Mckee et al., 1962). The average acetonitrile level was
117.6 µg/litre urine, while the average level for 20 nonsmokers was
2.9 µg/litre urine.
5.1.5 Other sources of exposure
Nitrogen-containing products such as hydrogen cyanide,
acetonitrile and acrylonitrile, and some other toxic gases have been
detected from the thermal decomposition of flexible polyurethane
foams (Woolley, 1972). The yield of hydrogen cyanide and
acetonitrile, respectively, from 10 mg foam was 26.4 and 21.4 µg at
800 °C, where a volatile yellow smoke was produced, and 522 and
30.5 µg at 1000 °C, where the yellow smoke was decomposed.
5.2 Occupational exposure
Synthesis of acetonitrile is usually carried out in a closed
system. Therefore, occupational exposure would only be accidental.
NIOSH estimated that 23 000 workers may be exposed to acetonitrile
in the USA. Since much of the acetonitrile produced has noncaptive
uses, the general population may also be exposed (NIOSH, 1979).
The occupational exposure limit for acetonitrile in various
countries is shown in Table 8.
Table 8. Occupational exposure limits for various countriesa
Country TWA STEL
(ppm) (mg/m3) (ppm) (mg/m3)
Australia 40 70 60 105
Belgium 40 67 60 101
Denmark 40 70 - -
Finland 40 70 60 105
France 40 70 - -
Germany 40 70 - -
Hungary - 50 - 100
Switzerland 40 70 80 140
United Kingdom 40 70 60 105
USA
(ACGIH) 40 67 60 101
(NIOSH/OSHA) 40 70 60 101
USSR - - - 10
a From: ILO (1991)
5.3 Acetonitrile in various solvent products
After a nationwide survey in Japan of organic solvent
components in various solvent products, acetonitrile was not
detected in either thinners (321 samples) or miscellaneous solvents
(56 samples), but was detected in 1% of the degreasers (145 samples)
(Inoue et al., 1983).
6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS
6.1 Absorption
6.1.1 Human studies
Acetonitrile is well absorbed by inhalation. There is little
information on absorption of inhaled acetonitrile in humans.
Studies on smokers showed that 91 ± 4% of the acetonitrile
inhaled in cigarette smoke was retained (Dalhamn et al., 1968a).
A significant portion of this could have been retained in the mouth,
as 74% of the acetonitrile was retained as a result of holding smoke
in the mouth for 2 sec (Dalhamn et al., 1968b).
There are no absorption studies concerning dermal or oral
exposure. However, human poisoning cases indicate that acetonitrile
is well absorbed by both routes.
6.1.2 Experimental animal studies
6.1.2.1 Intake through inhalation
Although there is information that acetonitrile is easily
absorbed from the lungs of animals exposed to acetonitrile vapour,
no quantitative analytical data is available on the pulmonary
absorption of acetonitrile.
6.1.2.2 Dermal absorption
Pozzani et al. (1959a) studied the skin penetration of
undiluted or diluted acetonitrile under polyethylene sheeting in
rabbits (the site of application was not reported). The dermal
LD50 value was decreased when application was made as a 75% (by
volume) aqueous solution, i.e. from 1.25 (0.84 to 1.85) ml/kg in the
case of the undiluted compound to 0.5 (0.37 to 0.67) ml/kg in the
case of the diluted aqueous solution. These LD50 values are
similar or even lower than those obtained after oral administration
in other animal species, indicating effective skin absorption of
acetonitrile.
6.1.2.3 Intake via the gastrointestinal tract
Although there is information that acetonitrile is easily
absorbed from the gastrointestinal tract, no quantitative analytical
data are available.
6.2 Distribution
6.2.1 Human studies
A postmortem investigation on a man accidentally exposed to
acetonitrile suggested that acetonitrile absorbed through inhalation
or skin contact is distributed in the body as shown in Table 21
(section 8.1.1).
6.2.2 Experimental animal studies
Ahmed et al. (1992) studied by means of whole body
auto-radiography the distribution of radioactivity derived from
2-14C-acetonitrile in the body of ICR mice at time points between
5 min and 48 h after administration of a single intravenous dose.
Irreversible association of label was determined in co-precipitated
protein and nucleic acids and extracted lipid. No attempt was made
to distinguish between metabolically incorporated or adducted label.
The highest concentrations of non-volatile radioactive compounds
were generally found in the liver, kidney and the contents of the
upper gastrointestinal tract. A significant fraction (40-50%) of
the radioactivity found in liver at 24 and 48 h was bound to the
macromolecular fractions of the tissues. The radioactivity contents
of other organs were, in large part (40-50% of total), present in
the lipid fraction of the tissue.
6.3 Biotransformation and elimination
6.3.1 Human studies
There is no specific human study describing acetonitrile
biotransformation and elimination. However, accidental poisoning
cases indicate that acetonitrile is biotransformed to cyanide and
thiocyanate, which are then excreted from urine (see section 9).
6.3.2 Experimental animal studies and in vitro studies
6.3.2.1 Cyanide liberation from acetonitrile
The release of cyanide from acetonitrile and its subsequent
metabolism to thiocyanate have been studied under a number of
experimental conditions and in several animal species.
Biotransformation of acetonitrile to cyanide and thiocyanate
has been demonstrated in a variety of in vitro preparations.
Liver slices obtained from male golden hamsters show an increasing
generation of cyanide and thiocyanate as the concentration of
acetonitrile increases (Willhite, 1983). Release of cyanide from
acetonitrile is also catalysed by liver microsomes of hamster in a
concentration-dependent manner (Willhite, 1983). Production of
cyanide from acetonitrile has been demonstrated in isolated
hepatocytes from female SD rats; the Km and Vmax values (mean ±
SD) were 3.4 ± 0.8 mM and 1.1 ± 0.1 nmol cyanide/106 cells per
10 min, respectively (Freeman & Hayes, 1987). The release of
hydrogen cyanide from acetonitrile has also been demonstrated in
mouse liver microsomes, both with and without NADPH (Ohkawa et al.,
1972). The Km and Vmax values obtained from male ddY mouse
microsomes were 4.19 mM and 14.3 ng cyanide formed in 15 min per mg
protein, respectively (Tanii & Hashimoto, 1984a).
Dahl & Waruszewski (1989) studied the metabolism of
aceto-nitrile to cyanide in rat nasal and liver tissues and found
that the maximum rates of cyanide production from acetonitrile by
nasal maxilloturbinate and ethmoturbinate microsomes and liver
microsomes were 0, 0.9 ± 0.2 and 0.098 ± 0.008 nmol cyanide/mg
protein per min, respectively.
In vivo metabolism of acetonitrile to cyanide and thiocyanate
was first demonstrated by Pozzani et al. (1959a). Studies were
conducted in rats, monkeys and dogs under a number of experimental
conditions. Fifteen male and fifteen female rats were exposed to
acetonitrile vapour (166, 330, and 655 ppm) 7 h/day, 5 days/week for
90 days. During the 5-day sampling period (inhalation days 59 to
63), thiocyanate concentrations in urine ranged from 27 to 79 and 29
to 60 mg/100 ml for the 166 and 330 ppm exposure groups,
respectively. Thiocyanate was not completely eliminated between
daily exposures, but was almost completely excreted during the
2.5-day rest period over weekends. The excretion of thiocyanate in
the higher exposure group was not reported.
The concentrations of thiocyanate in the urine of three dogs
exposed to 350 ppm acetonitrile in air increased from 69 to
252 mg/litre over the same 5-day inhalation period as described
above for rats. Unlike the rats, dogs continued to eliminate
thiocyanate beyond the 2.5-day rest period over the weekend. When
three monkeys were exposed to 350 ppm acetonitrile in the same
manner as the dogs, the urinary thiocyanate concentration ranged
from 60 to 114 mg/litre. Thiocyanate was also excreted after the
2.5-day rest period.
Rhesus monkeys were injected intravenously either with
acetonitrile (0.1 ml/kg) or with thiocyanate (1.55 ml/kg of a 10%
solution in saline). The percentages of the dose excreted as
thiocyanate were 12% and 55%, respectively. It seems therefore that
more than 12% of the injected acetonitrile was converted into
thiocyanate (Pozzani et al., 1959a).
After a single intraperitoneal administration of acetonitrile
(780 mg/kg) in rats, all animals died in 3 to 12 h, and acetonitrile
was found to be distributed in various organs (Dequidt & Haguenoer,
1972). The free cyanide varied from 170 µg/kg in the liver to
3.5 mg/kg in the spleen. Concentrations of combined cyanide in the
liver, spleen, stomach and skin were 3.6, 13.5, 17.6 and 10.5 mg/kg
tissue, respectively.
Haguenoer et al. (1975a,b) studied the pharmacokinetics of
acetonitrile in male Wistar rats after a single intraperitoneal
acetonitrile injection or inhalation exposure. Rats given 2340 or
1500 mg/kg died within 3 to 28 h after the intraperitoneal
injection, but rats given 600 mg/kg survived with no apparent
symptoms. After administration of 2340 mg/kg, concentrations of
acetonitrile and free and combined cyanide in various organs ranged
from 900 to 1700 mg/kg, 200 to 3500 µg/kg, and 3.5 to 17 mg/kg
tissue, respectively. Mean total urinary acetonitrile and free and
combined cyanide (essentially all thiocyanate) excreted during the
11 days following an intraperitoneal injection of 600 mg/kg were 28,
0.2 and 12 mg, respectively. These values were equivalent to 3,
0.035 and 2.3% of the acetonitrile dose, respectively. Urinary
acetonitrile was detectable for 4 days after dosing, whereas free
and combined cyanide were detectable until 11 days, at which time
the animals were sacrificed. Rats inhaling 25 000 ppm died within
30 min from the beginning of exposure. The concentration of
acetonitrile in muscle and kidney ranged from about 1.4 to 24 mg/kg,
and that of free cyanide in liver and spleen from 0.3 to 4 mg/kg
tissue. When three rats were exposed to 2800 ppm (2 h/day for
3-5 days) the concentrations of acetonitrile and free cyanide in
various tissues at the time of death were 1000-2900 mg/kg and
0.5-10 mg/kg tissue, respectively.
The liver and brain cyanide levels of male CD-1 mice (n = 9-10)
that died 2.5 h after intraperitoneal administration of 175 mg
acetonitrile/kg were found to be 47.8 ± 36.1 and 13.4 ± 4.8 µmol/kg,
respectively (Willhite & Smith, 1981). Sprague-Dawley rats
administered an oral LD50 of acetonitrile (2460 mg/kg) were found
to have cyanide levels of 16 ± 6 mg/kg in liver, 102 ± 39 mg/kg in
kidney and 28 ± 5 mg/kg in brain (Ahmed & Farrooqui, 1982).
Freeman & Hayes (1985a) found that the peak blood cyanide
concentration (5.2 ± 0.5 mg/litre) was achieved 35 h after oral
administration of 1470 mg/kg to female SD rats. Silver et al.
(1982) reported that urinary thiocyanate excretion for a 24-h period
following oral or intraperitoneal adminstration of acetonitrile
(30.8 mg/kg) in SD rats was 11.8 ± 2.5 and 4.4 ± 0.5% of the dose,
respectively. Inhalation studies on male and female Wistar rats
exposed to 166 and 330 ppm (660 ppm was fatal) indicated that the
amount of thiocyanate in urine was not proportional to the
concentration of acetonitrile inhaled (Pozzani et al., 1959a).
Table 9 shows that acetonitrile is converted to cyanide at a
slower rate than other nitriles. In fact, one hour after
acetonitrile administration the blood level of cyanide was much
lower than those after acute toxic doses of other nitriles. Peak
concentrations of blood cyanide were found 7.5 h after acetonitrile
dosing and were comparable to those of other nitriles measured one
hour after dosing.
Brain cyanide concentration one hour after acetonitrile dosing
was also lower than those after exposure to potassium cyanide (KCN)
or other nitriles. Urinary excretion of thiocyanate after exposure
to various nitriles indicated that for acetonitrile the percentage
of the dose excreted was lower than for other nitriles, even though
the absolute given amount of acetonitrile, based on its oral LD50
value, was much higher. These data, taken together, indicate that
the toxicity of acetonitrile is lower than those of cyanide and
other nitriles, as shown by oral LD50 values in Table 9. The
reason for this is most probably the slower transformation of
acetonitrile to cyanide and consequently the more efficient
detoxification via thiocyanate excretion.
The relevance of acetonitrile pharmacokinetics is further
illustrated by examining the relationship between symptoms produced
by acetonitrile one hour after exposure and the amounts of cyanide,
as well as the effect on cytochrome c oxidase in the brain
(Table 10). Animals treated with acetonitrile were asymptomatic at
this time, but animals treated with other nitriles or KCN at LD50
doses were symptomatic. In fact, the inhibition of brain cytochrome
c oxidase paralleled brain cyanide concentrations. In the case of
acetonitrile, the brain cyanide concentration was too low to affect
cytochrome c oxidase activity and therefore to cause symptoms.
In conclusion, the data reported in Tables 9 and 10 indicate
that the apparent lack of relationship, assessed shortly after
dosing, between acetonitrile toxicity and cyanide production is due
to the slow transformation of acetonitrile to cyanide.
There is sufficient evidence from all animal species studied
that the toxicity of acetonitrile is due to cyanide. Interspecies
variations, as shown in Tables 11 and 12, are probably related to
the relative speed of cyanide formation from acetonitrile (data of
Willhite & Smith, 1981 in mice versus the data of Ahmed & Farooqui,
1982 in rats).
Table 9. Metabolism of nitriles to cyanide in relation to their lethal effects
Compound Cyanide concentration Urinary thiocyanate Oral LD50
(1 h after an oral LD50) excretion (mg/kg body weight)c
Blood (mg/litre)a Brain (mg/kg)c (% dose/24 h)d
Potassium cyanide 6.3 748 ± 200 not measured 10
Acetonitrile 0.3b 28 ± 5 11.8 ± 2.5 2460
Propionitrile 4.0 508 ± 84 65.1 ± 2.9 40
Butyronitrile 3.8 437 ± 106 64.9 ± 3.5 50
Malononitrile 6.5 649 ± 209 not measured 60
Isobutyronitrile not measured not measured 74.0 ± 2.6 160
Acrylonitrile 4.1 395-106 37.3 ± 1.9 90
a Estimated from: Ahmed & Farooqui (1982)
b 7.5 h after oral administration (1470 mg/kg body weight), the blood cyanide level was found to
be 7.3 mg/litre (Estimated from: Freeman & Hayes, 1985a)
c Ahmed & Farooqui (1982) 1 h after oral LD50
d Silver et al. (1982)
6.3.2.2 The oxidative pathway of acetonitrile metabolism
Following the observation of acetonitrile metabolism to cyanide
and thiocyanate by Pozzani et al. (1959a), many authors reported
the same results in humans as well as in experimental animals both
in vitro and in vivo (Amdur, 1959; Ohkawa et al., 1972;
Willhite & Smith, 1981; Ahmed & Farooqui, 1982; Silver et al.,
1982; Willhite, 1983; Pereira et al., 1984; Tanii & Hashimoto,
1984a,b, 1986; Freeman & Hayes, 1985a,b; Ahmed et al., 1992).
They all suggested a metabolic pathway in which acetonitrile is
bio-transformed by cytochrome P-450 monooxygenase system initially
to cyanohydrin, which then spontaneously decomposes to hydrogen
cyanide and formaldehyde as shown in Fig. 1. Formaldehyde has not
been identified in all of these studies, but this could be due to
its high reactivity and rapid conversion into a simple metabolite
(CO2).
Acetone, an inducer of cytochrome P-450 isozyme LM3a (Koop &
Casazza, 1985; Johannsen et al., 1986), has been demonstrated to
stimulate the metabolism of acetonitrile to cyanide in vivo in
rabbits (Freeman & Hayes, 1985a). In an in vitro study, liver
microsomes were isolated and pooled 24 h after pretreatment of
female Sprague-Dawley rats with acetone. Microsomal metabolism of
acetonitrile to cyanide was found to be NADPH-dependent and
heat-inactivated tissue was unable to catalyse this reaction
(Freeman & Hayes, 1985b). The metabolism of some nitriles,
including acetonitrile to cyanide by mouse hepatic microsome system,
has been shown to be NADPH-dependent and enhanced by pretreatment
with ethanol (Tanni & Hashimoto, 1986). Ohkawa et al. (1972)
found that the amount of hydrogen cyanide released in mouse liver
microsomal preparations was increased greatly by the addition of
NADPH. It is known that treatment of rats with cobalt-heme
effectively depletes liver cytochrome P-450 concentrations (Drummond
& Kappas, 1982). Freeman & Hayes (1987) demonstrated a marked
decrease in acetonitrile metabolism in isolated hepatocytes prepared
from rats pretreated subcutaneously with cobalt-heme (90 µmol/kg)
48 h before killing. However, the rate of acetonitrile
biotransformation into cyanide by liver microsomal preparation
obtained from cobalt-heme-treated rats was 13% of controls, while
the total cytochrome P-450 content was reduced by only 41% compared
to the controls.
Table 10. Biochemical and clinical effects in Sprague-Dawley male rats dosed with cyanide and nitrilesa
Compound Brain cyanide Brain cytochrome c CNS Convulsionb Respiratory
concentration oxidase activity depressionb failureb
(mg/kg) (% of control)
Control 0 100 no no no
Potassium cyanide 748 ± 200 29 4 4 4
Acetonitrile 28 ± 5 92 no no no
Propionitrile 508 ± 54 47 3 1 1
Butyronitrile 437 ± 106 41 2 1 1
Malononitrile 649 ± 209 73 3 3 2
a Measured 1 h after an LD50; data from: Ahmed & Farooqui (1982)
b Physiological changes were graded on a scale of 1 (lowest) to 4 (highest)
NADPH, O2 spontaneous
CH3CN -------> HOCH2CN -------> [HCHO] + CN- (1)
mixed-function
oxidases
rhodanese
CN- -------> SCN- (2)
S2O3
[HCHO] has not been identified
CN- and SCN- have been identified both
in vitro and in vivo
Fig. 1. Oxidation (1) and conjugation (2) reactions in acetonitrile
metabolism
Treatment of rats with inducers of P-450 IIE1, such as
pyrazole, 4-methylpyrazole and ethanol, resulted in a 4- to 5-fold
increase in cyanide production from acetonitrile by isolated
microsomes (Feierman & Cederbaum, 1989). Phenobarbital treatment
had a small stimulatory effect, whereas 3-methylcholan-threne
treatment decreased microsomal oxidation of acetonitrile. Cyanide
production was inhibited by carbon monoxide, ethanol, 2-butanol,
dimethyl sulfoxide (DMSO) and 4-methylpyrazole in vitro.
Oxidation of acetonitrile to cyanide by microsomes from rats treated
with pyrazole or 4-methylpyrazole was nearly completely inhibited by
an antibody (IgG) against P-450 3a.
These results imply a role for P-450 in the oxidation of
acetonitrile to cyanide and suggest that P-450 IIE1 may be the
specific catalyst for this oxidation. Acetonitrile oxidation was
not affected by hydroxyl radical scavengers or by desferrioxamine.
The results of human and animal studies indicate that cyanide
formed in vivo is subsequently conjugated with thiosulfate to form
thiocyanate, which is then eliminated in urine. This conjugation is
catalysed by the enzyme rhodanese (thiosulfate cyanide sulfur
transferase: EC 2.8.1.1) (Pozzani et al., 1959a; Takizaw &
Nakayama, 1979; Silver et al., 1982; Willhite, 1983; Pereira
et al., 1984).
Acetone inhibits acetonitrile metabolism when the two compounds
are administered simultaneously. Blood cyanide concentrations were
maximally elevated 9 to 15 h after female SD rats were dosed with
acetonitrile alone at 1470 mg/kg. In rats dosed concomitantly with
acetonitrile (1470 mg/kg) and acetone (1960 mg/kg), blood cyanide
concentrations measured 0 to 24 h after dosing were much lower than
those in rats given the same dose of acetonitrile alone. Blood
cyanide levels, however, reached peak concentration 39 to 48 h after
dosing with the two compounds and were 50% higher than those
measured in rats treated with acetonitrile only (Freeman & Hayes,
1985a).
From these time courses of blood cyanide it was postulated that
acetone has a biphasic effect on acetonitrile metabolism, causing an
initial inhibition and a subsequent stimulation of cyanide
generation from acetonitrile. Freeman & Hayes (1985b) also found
that the in vitro metabolism of acetonitrile to cyanide by either
hepatic microsomal preparations or by isolated liver cells
(hepatocytes) from rats pretreated with acetone (2.5 ml/kg) was
significantly increased (2 fold). However, when acetone was
incubated with hepatocytes, it inhibited acetonitrile metabolism
without affecting cell viability.
Ethanol has also been shown to affect the in vitro metabolism
of some nitriles, including acetonitrile (Tanii & Hashimoto, 1986).
A 1.8-fold increase in cyanide liberation from acetonitrile was
observed in hepatic microsomes from male ddY mice pretreated with
ethanol (4.0 g/kg) 13 h prior to the study.
Freeman & Hayes (1988) further investigated the metabolism of
acetonitrile in vitro and the effects of acetone and other
compounds. They suggested that the conversion of acetonitrile to
cyanide is mediated by specific acetone-inducible isoforms of
cytochrome P-450 and cytochrome P-450j (LM3, LMeb). Acetone,
dimethylsulfoxide and ethanol competitively inhibited this
conversion. Aniline HCl has been shown to reduce acetonitrile
metabolism.
6.4 Biological monitoring of acetonitrile uptake
Workers accidentally exposed to acetonitrile vapour showed
increased serum cyanide and thiocyanate levels but the exposure
concentrations were unknown (Amdur, 1959). In three human
volunteers exposed at different times to concentrations of up to
160 ppm for 4 h (Pozzani, 1959a), no significant changes in urinary
blood cyanide and thiocyanate levels were observed compared to those
measured prior to exposure. In experimental animal studies using
various routes of exposure, blood cyanide and thiocyanate levels
showed increases but they were not proportional to the exposures
(Pozzani, 1959a). It should be noted that there is a delay of
several hours in the formation of cyanide following exposure to
acetonitrile, and the timing of blood sampling is therefore
critical.
From these data it is not possible to derive biological indices
for exposure monitoring.
7. EFFECTS ON LABORATORY MAMMALS; IN VITRO TEST SYSTEMS
7.1 Acute toxicity
7.1.1 Single exposure
The LD50 values for acetonitrile in mammals are summarized in
Table 11; they range between 175 and 5620 mg/kg body weight. The
mouse and the guinea-pig seem to be the most sensitive species. No
consistent effects of sex, administration route or vehicle were
observed. An experiment using four different age groups of rats
showed that new-born rats (24 to 48 h old, 5-8 g) are the most
sensitive. Significant differences in LD50 values were found
between 14-day-old and adult rats, but not between young adults
(80-160 g body weight) and older adults (300-470 g body weight)
(Kimura et al., 1971).
The acute inhalation toxicity of acetonitrile in various animal
species is shown in Table 12. The LC50 values range between about
2700 ppm for a 1-h inhalation or 2300 ppm for a 2-h inhalation in
mice and 16 000 ppm for a 4-h inhalation or 12 000 ppm for an 8-h
inhalation in rats. Mice appear to be the most sensitive species to
acetonitrile inhalation. In Nelson rats, the LC50 value for an
8-h inhalation was significantly lower in males (7551 ppm with 5975
to 9542 confidence interval) than in females (12 435 ppm with 11 036
to 14 011 confidence interval), while that for a 4-h inhalation was
the same in both sexes (16 000 ppm with 13 070 to 19 636 confidence
interval) (Pozzani et al., 1959a).
Pozzani et al. (1959b) studied the relationship between the
observed and predicted LD50 of acetonitrile given in combination
with other chemicals to rats exposed orally or by inhalation.
Predictions were made using the method of Finney (1952). The
mixture of acetonitrile and acetone seemed to show effects that were
greater than additive. Results are summarized in Table 13.
7.1.2 Clinical observations
Signs and symptoms of acute acetonitrile intoxication are
similar in different animal species. Verbrugge (1899) described
signs of acute acetonitrile toxicity in rabbits. One to three hours
after a subcutaneous acetonitrile injection of 90 to 150 mg/kg,
rabbits showed rapid and irregular respiration, immobilization and
convulsions, and two out of seven animals died. Monkeys exposed to
2510 ppm acetonitrile vapour appeared normal after the first day of
inhalation but showed poor coordination followed by prostration and
laboured breathing during the second day. Death occurred a few
hours later (Pozzani et al., 1959a). Mice exposed to
concentrations of acetonitrile ranging from 500 to 5000 ppm (the
LC50 for a 60-min exposure was 2693 ppm) displayed dyspnoea,
tachypnoea, gasping, tremors, convulsions and corneal opacity
30-300 min after the beginning of the exposure. Exposure of mice to
5000 ppm acetonitrile for 60 min killed all the animals within 2 h.
The syndrome of acute acetonitrile toxicity was indistinguishable
from that observed after exposure to cyanide or other nitriles
(Willhite, 1981; Willhite & Smith, 1981).
In a study by Willhite (1983), pregnant hamsters were exposed
to acetonitrile concentrations from 3800 to 8000 ppm for one hour.
The number of hamsters showing tremors, hypersalivation, ataxia,
hypothermia, lethargy and coma increased with increasing dose.
Hamsters died about 3 h after exposure to 5000 ppm acetonitrile and
within 90 min after exposure to 8000 ppm acetonitrile.
In a study by Johansen et al. (1986), all of five pregnant
rats treated with acetonitrile at doses of 750 mg/kg or more per day
by gavage on gestation days 6-15 died, whereas only three out of
five animals treated with 375 mg/kg per day died. Four out of six
rats treated with 275 mg/kg had reduced body weight at parturition,
while two others died.
Ahmed & Farooqui (1982) measured cyanide levels one hour after
administration of LD50 doses of several saturated and unsaturated
nitriles to male SD rats. Few symptoms were noted with acetonitrile
in this study because little cyanide was released within the first
hour following treatment. The tissue concentrations of cyanide
after lethal doses of propionitrile, butyronitrile and malononitrile
were very similar and approximately those observed with a lethal
dose of KCN.
In female SD rats given an oral dose of acetonitrile
(1770 mg/kg), acute toxic effects appeared after 30 h (Freeman &
Hayes, 1985a).
7.1.2.1 Effect on skin
The skin irritation of acetonitrile in Sherman rats was
reported by Smyth & Carpenter (1948) to be comparable to that of
acetone, although no precise description of the technique used for
testing skin irritation was provided.
7.1.2.2 Effect on the eyes
Eye injury caused by acetonitrile, reported by Smyth &
Carpenter (1948), is of intermediate intensity and similar to that
produced by acetone (Carpenter & Smyth, 1946). Corneal opacity has
been observed after either inhalation or intraperitoneal
administration of acetonitrile in male mice (Willhite, 1981;
Willhite & Smith 1981). Pregnant hamsters exposed to 8000 ppm
acetonitrile via inhalation for 60 min showed eye irritation
(Willhite, 1983).
Table 11. LD50 values of acetonitrile for various species and different routes of administration
Species (strain) Sex Observation Route LD50 (mg/kg or Vehicle References
period (days) ml/kg body weightb
Mouse (Kunming) male -a gavage 453 mg/kg water Chen et al. (1981)
Mouse 1 intraperitoneal 520.79 mg/kg Yoshikawa (1968)
Mouse - intraperitoneal 0.25 ml/kg saline Pozzani et al. (1959a)
Mouse (NMRI) 7 intraperitoneal 400 mg/kg water Zeller et al. (1969)
Mouse (CD-1) male 7 intraperitoneal 175 mg/kg water Willhite & Smith (1981)
Mouse (ddY) male 7 oral 269 mg/kg water Tanii & Hashimoto (1984a)
Rat (Sherman) - oral 3800 mg/kg -a Smyth & Carpenter (1948)
Rat (Wistar) or albino male - gavage 1.68 ml/kg undiluted Pozzani et al. (1959a)
Rat (Wistar) or albino male - gavage 2460 mg/kg water Pozzani et al. (1959a)
Rat (Wistar) or albino male - intravenous 1.68 ml/kg undiluted Pozzani et al. (1959a)
Rat (Wistar) or albino female - gavage 2230 mg/kg 1% Tgc Pozzani et al. (1959a)
Rat (Wistar) or albino female - gavage 1730 mg/kg corn oil Pozzani et al. (1959a)
Rat (Wistar) or albino female - gavage 8.56 ml/kg undiluted Pozzani et al. (1959a)
Rat (Wistar) or albino female - intraperitoneal 7.96 ml/kg undiluted Pozzani et al. (1959a)
Rat (Wistar) or albino female - intraperitoneal 5620 mg/kg saline Pozzani et al. (1959a)
Rat (Wistar) or albino female - intraperitoneal 0.85 ml/kg undiluted Pozzani et al. (1959a)
Rat (Wistar) or albino female - intravenous 1.68 ml/kg undiluted Pozzani et al. (1959a)
Rat (SD) - oral 3200 mg/kg water Zeller et al. (1969)
Rat (SD) 14-day old male - oral 0.2 ml/kg undiluted Kimura et al. (1971)
Rat (SD) young adult male - oral 3.9 ml/kg undiluted Kimura et al. (1971)
Rat (SD) older adult male - oral 4.4 ml/kg undiluted Kimura et al. (1971)
Rat (SD) female 3 oral 4050 mg/kg undiluted Freeman & Hayes (1985a)
Table 11 (contd).
Species (strain) Sex Observation Route LD50 (mg/kg or Vehicle References
period (days) ml/kg body weightb
Guinea-pig male - gavage 0.177 ml/kg undiluted Pozzani et al. (1959a)
Rabbit - skin 5.0 ml/kg undiluted Smyth & Carpenter (1948)
Rabbit male - skin 1.25 ml/kg undiluted Pozzani et al. (1959a)
Rabbit male - skin 0.50 ml/kg water Pozzani et al. (1959a)
a - = not reported
b 1 ml acetonitrile = 783-787 mg at 20 °C
c Tg = Tergitol 7 in water
Table 12. Acute inhalation toxicity of acetonitrilea
Species (strain) Sex Concentration Exposure Mortality
(ppm) time (h) measuresb
Mouse (Kunming) 2300 2 LC50
Mouse (Kunming) 5700 2 LC50
Mouse (CD-1) male 2700 1 LC50
male 5000 1 10/10
Rat (Nelson) male 7551 8 LC50
female 12 435 8 LC50
male 16 000 4 LC50
female 16 000 4 LC50
Rat (Wistar) 12 000 2 MLC
Guinea-pig male + 5655 4 LC50
female
Guinea-pig 7400 2 MLC
Rabbit male 2800 4 LC50
Rabbit 4500 2 MLC
Dog male 32 000 4 1/1
male 16 000 4 3/3
male 8000 4 0/1
male 2000 4 0/2
a From: Willhite (1981), Pozzani et al. (1959a,b), Wang et al. (1964)
b MLC = minimum lethal concentration
Table 13. Predicted and observed LC50 and LD50 values of acetonitrile in
combination with other solvents in rata
4-h inhalation (g/m3) Oral (ml/kg)
PLC50 OLC50 PLD50 OLD50
Acetonitrile - 26.9 - 8.27
Acetonitrile + n-hexane 45.6 74.1 - -
Acetonitrile + acetone 39.7 14.6 9.99 2.75
Acetonitrile + ethyl 32.4 51.4 9.40 14.1
acetate
Acetonitrile + carbon 31.5 45.5 4.35 6.77
tetrachloride
Acetonitrile + toluene 22.3 44.4 8.68 3.73
a From: Pozzani et al. (1959b)
PLC50 or PLD50 = predicted LC50 or LD50
OLC50 or OLD50 = observed LC50 or LD50
7.1.2.3 Effect on respiration
Animals exposed to acetonitrile via different routes of dosing
always showed respiratory symptoms: rapid and irregular respiration
after subcutaneous administration in rabbits (Verbrugge, 1899),
laboured or difficult breathing after inhalation exposure in monkeys
(Pozzani et al., 1959a) or rats (Haguenoer et al., 1975b), and
intense dyspnoea after either inhalation or intraperitoneal
administration in mice (Willhite, 1981; Willhite & Smith, 1981).
Histopathological investigations of rat lungs after acetonitrile
inhalation showed haemorrhage and congestion (Haguenoer et al.,
1975b). After inhaling 660 ppm acetonitrile for 2 h, two monkeys
showed focal areas of emphysema and atelectasis, with occasional
proliferation of alveolar septa (Pozzani et al., 1959a).
7.1.2.4 Effect on adrenals
Szabo et al. (1982) studied structure-activity relationships
of 56 chemicals, including acetonitrile, with respect to their
potential for causing adrenocortical necrosis in rats. The dose was
selected on the basis of preliminary experiments and was aimed to
lead to 70 to 100% mortality in 4 to 5 days. The compounds were
given 3 times per day for 4 days, and surviving animals were
sacrificed on the 5th day. Acetonitrile, along with 13 other
compounds out of 56 test chemicals, did not show any
adrenocorticolytic effect in rats.
7.1.2.5 Effect on the gastrointestinal tract
Rats that inhaled acetonitrile at a concentration of 2800 ppm
(2 h/day for 2 days) showed temporary diarrhoea (Haguenoer et al.,
1975b).
Acetonitrile did not produce duodenal ulcers in female SD rats
after oral or subcutaneous administration 3 times per day for 4
days, the total dose being 432 mmol/kg (Szabo et al., 1982).
7.1.3 Biochemical changes and mechanisms of acetonitrile toxicity
7.1.3.1 Effect on cytochrome oxidase
An in vitro study carried out by Willhite & Smith (1981)
showed that high concentrations of acetonitrile (up to 0.47 M) did
not inhibit cytochrome c oxidase activity. Ahmed & Farooqui
(1982) investigated the ability of acetonitrile and other nitriles
to inhibit cytochrome c oxidase one hour after they were
administered at the LD50 to male SD rats. There was no direct
evidence for the inhibition of cytochrome oxidase after the
administration of acetonitrile. However, very little increase in
tissue or blood cyanide concentrations was observed one hour after
dosing with acetonitrile. Symptoms had not occurred within this
time period, and the evidence from other studies indicates that peak
cyanide levels are achieved much later than one hour (in 9-15 h)
(Freeman & Hayes, 1985a). The need to consider the different
pharmacokinetic and metabolic factors involved in making such
comparisons was emphasized by Willhite & Smith (1981).
7.1.3.2 Effect on glutathione
Levels of glutathione (GSH) in liver, kidney and brain were
unaffected one hour after oral administration of acetonitrile (at
the LD50 level) in male SD rats (Ahmed & Farooqui, 1982). Aitio &
Bend (1979) studied the in vitro effect of 12 organic solvents,
including acetonitrile, on the activity of rat liver soluble
glutathione S-transferase. They demonstrated that in the presence
of 630 mM acetonitrile, the conjugation of styrene oxide,
benzo[ a]pyrene-4,5-oxide and 1,2-dichloro-4-nitrobenzene by GSH
was reduced to 79.0 ± 5.2, 92.6 ± 3.0 and 59.2 ± 1.4%,
respectively, of the control values.
7.1.4 Antidotes to acetonitrile
Multiple intraperitoneal administrations of 1 g sodium
thiosulfate per kg at the rate of one injection every 100 min over a
10-h period or two intraperitoneal injections of 75 mg sodium
nitrite per kg significantly reduced mortality in CD-1 mice exposed
to 3800 or 5000 ppm acetonitrile by inhalation for 60 min (Willhite,
1981). Treatment of animals with thiosulfate at a dose rate of
1 g/kg every 100 min for an 8-h period was effective in providing
significant protection against the lethal effect of an
intraperitoneal injection of acetonitrile (575 mg/kg) in male CD-1
mice (Willhite & Smith, 1981). An intraperitoneal injection of
sodium thiosulfate (300 mg/kg) 20 min prior to inhalation of
8000 ppm acetonitrile in pregnant hamsters abolished the overt signs
of acetonitrile poisoning and reduced mortality from 3 out of 12
hamsters to zero (Willhite, 1983). Repeated intraperitoneal
administrations (6 injections in 10 h) of sodium thiosulfate
(1 g/kg), which started at the onset of acute toxicity about 30 h
after oral administration of acetone (1960 mg/kg) and acetonitrile
(1770 mg/kg) given simultaneously, provided significant protection
against mortality in female SD rats (Freeman & Hayes, 1985a).
Two intraperitoneal injections of 75 mg sodium nitrite did not
provide CD-1 mice with any significant protection against the lethal
effect of acetonitrile (575 mg/kg) (Willhite & Smith, 1981).
7.2 Subchronic toxicity
7.2.1 Inhalation exposure
In a rat study, the body weight gain and organ weights of male
and female rats which inhaled 166, 330 or 655 ppm acetonitrile
(7 h/day, 5 days/week, for a total of 90 days) did not differ
significantly from those of the controls (Pozzani et al., 1959a).
Histopathological examination showed that of the 28 rats that
inhaled 166 ppm, one had histiocyte clumps in the alveoli and
another had atelectasis. Of 26 rats that inhaled 330 ppm, three
showed bronchitis, pneumonia, atelectasis and histiocyte clumps in
the alveoli. After the inhalation of 655 ppm acetonitrile vapour,
10 out of 27 animals showed alveolar capillary congestion and/or
focal oedema in the lung, often accompanied by bronchial
inflammation, desquamation and hypersecretion. Tubular cloudy
swelling of the kidneys in eight rats and swelling of the livers of
seven rats were observed. These effects were statistically
significant (lungs, P < 0.001; kidney, P < 0.005; liver,
P < 0.04) compared with control animals. No lesions were found in
the adrenals, pancreas, spleen, testes or trachea. Focal cerebral
haemorrhage was observed in one of the five brains examined.
Wang et al. (1964) reported that there was no change of
iodine levels in the thyroid of Wistar rats exposed to 80 or 400 mg
acetonitrile/m3 (4 h/day, 6 days/week) for 10 weeks. Degenerative
changes in the epithelial cells of thyroid follicles were observed
in rabbits exposed to 400 mg/m3 (4 h/day, 6 days/week) for 16
weeks.
In an inhalation study (7 h/day) on four Rhesus monkeys, one
female monkey was exposed to 2510 ppm, two females to 660 ppm and
one male to 330 ppm (Pozzani et al., 1959a). The monkey exposed
to 2510 ppm appeared normal during the first inhalation day but on
the second day showed incoordination and laboured breathing and died
a few hours later. In the two monkeys exposed to 600 ppm there was
also incoordination from the second week. One monkey died on day 23
and the other on day 51. The monkey exposed to 330 ppm showed
overextension reflexes and hyperexcitability towards the end of the
99-day inhalation period and was sacrificed then. At autopsy, the
monkey exposed to 2510 ppm had engorgement of the dural capillaries,
and the animals exposed to 660 and 330 ppm showed focal dural or
subdural haemorrhage in the parietal and/or occipital tissues
adjacent to the superior sagittal sinus. The monkey exposed to
2510 ppm had pleural effusion, and those exposed to 660 ppm had
focal areas of emphysema and atelectasis with occasional
proliferation of alveolar septa, and cloudy swelling of the proximal
and convoluted tubules of the kidneys. The monkey exposed to
330 ppm had pneumonitis as shown by diffuse proliferation of
alveolar septa, monocytic infiltration and pleural adhesions.
In another inhalation study (Pozzani et al., 1959a), three
male Rhesus monkeys were exposed to 350 ppm acetonitrile (7 h/day,
5 days/week) for 91 days, and at the end of the study the animals
were sacrificed. At autopsy, haemorrhages of the superior and
inferior sagittal sinuses were found in the brains of all three
monkeys. Small discrete caseous nodules were seen in the lungs of
two monkeys and one monkey had a pale liver. Histological
investigations of the lung showed focal emphysema, diffuse
proliferation of alveolar septa, and focal accumulations of
pigment-bearing macrophages. In two of the monkeys there was cloudy
swelling of the proximal tubules of the kidney.
One female and two male dogs inhaled 350 ppm acetonitrile
(7 h/day, 5 days/week) for 91 days. The haematocrit and haemoglobin
values of the three dogs were depressed by the fifth week of
inhalation, but with the exception of one dog, there was a return to
pre-inhalation values toward the end of the 91-day inhalation
period. No significant deviation of the erythrocyte counts was seen
in any dogs. Histopathological examination of these dogs showed
some focal emphysema and proliferation of alveolar septa.
Roloff et al. (1985) exposed groups of male and female rats
(strain unspecified) to acetonitrile vapour (0, 1038, 3104 and
10 485 mg/m3) for one month (6 h/day, 5 days/week). Death and
reduced body weight gains were observed at the highest exposure
level. Respiratory and/or ocular irritation were noted in animals
exposed to 3104 and 10 485 mg/m3.
In a 13-week inhalation study of acetonitrile (100, 200 and
400 ppm) in 25 male mice and male rats, there were no effects on
body weight or on testicular weight and sperm motility (Morrissey
et al., 1988).
In a 13-week inhalation study on acetonitrile in mice and rats,
ten mice (B6C3F1) and ten rats (F-344/N) of each sex were exposed
to acetonitrile vapour at 0, 100, 200, 400, 800 and 1600 ppm
(6 h/day, excluding weekends and holidays) for 13 weeks (Battelle,
Pacific Northwest Laboratories, 1986a,b). At 400 ppm one female
mouse, at 800 ppm one male and four female mice, and at 1600 ppm ten
female and ten male mice were found dead during the study. The
majority of the mortality occurred after two weeks of exposure.
Clinical signs observed were hypoactivity and a hunched rigid
posture. Body weight gains were comparable to control values for
all surviving mice. An increase in absolute and relative liver
weight was attributed to acetonitrile exposure. The maximum
tolerated concentration determined by this 13-week subchronic study
was 200 ppm. Significant changes were observed in the liver and
stomach of male mice exposed to 400 ppm of acetonitrile and female
mice exposed to 200 ppm or more. At 800 ppm one male rat and at
1600 ppm six male and three female rats were either moribund (and so
sacrificed) or found dead during the study. The clinical signs
observed were hypoactive, abnormal posture, ataxia, bloody crusts on
nose and/or mouth and a rough haircoat. The moribund, sacrificed
rats exhibited tonic/clonic convulsions. Reductions in body weight
gain were observed in rats exposed to 1600 ppm. Minimum to mild
lesions were found in the lungs and brain of some rats exposed to
800 ppm (Table 14).
In a 92-day study, reported as an abstract, acetonitrile was
administered by inhalation to B6C3F1 mice and Fischer-344 rats at
concentrations of (25, 50, 100, 200 and 400 ppm) for a total of 65
days (Hazleton, 1990b). In mice, one male in each of the 50, 200
and 400 ppm groups died. There was an increase in body weight gain
in all males exposed to 50, 100, 200 and 400 ppm acetonitrile and in
the females of the 200 and 400 ppm groups. Body weight gain was
decreased by comparison with controls in the 25, 50 and 100 ppm
female groups. Liver/body weight ratio was increased in males at
400 ppm group and in females at 100, 200 and 400 ppm groups.
Liver/brain weight ratio was increased in males at the 400 ppm and
in female at 100 and 400 ppm groups. There was slight cytoplasmic
vacuolization of hepatocytes in both males and females in the 200
and 400 ppm groups. Mean haemato-crit and erythrocyte counts were
marginally reduced in females at 200 and 400 ppm group. In females
of the 200 and 400 ppm groups haematocrit, haemoglobin, red and
white blood cell counts, and serum IgG were all depressed. In rats,
one male in the 400 ppm group died during the study. There was
slight cytoplasmic vacuolization of hepatocytes in females at
400 ppm. Marginal decreases in mean leucocyte counts were reported
in males at 100 and 200 ppm and in both males and females at
400 ppm.
7.2.2 Subcutaneous administration
Marine et al. (1932a) gave daily subcutaneous injections of
0.1 ml acetonitrile to 4-month-old rabbits for 21 days. Two groups
of four male rabbits developed pronounced (more than twice normal
size) thyroid hyperplasia whereas one group of four females showed
no effect. Allyl-benzyl and phenyl nitriles produced less
pronounced hyperplasia or no effect on thyroids at up to 4 times the
dose of acetonitrile. A further study (Marine et al., 1932b)
suggested that young rabbits were more susceptible than adults and
that the effect varied with the strain.
7.3 Teratogenicity and embryotoxicity
In a study by Berteau et al. (1982), mated rats were
administered daily aqueous solutions of acetonitrile by gavage
(125, 190 and 275 mg/kg) on gestation days 6-19. Although maternal
body weights were reduced and death occurred in the high-dose group,
no other maternal effects were noted in any treated group.
Embryotoxic effects, as shown by increases in early resorptions and
postimplantation losses, were also noted in the high-dose group.
However, no teratogenic responses were observed at any dose level.
Table 14. Subchronic inhalation toxicity of acetonitrile in mice and rats
Spe