INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 122
n-HEXANE
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. Chipman,
University of Birmingham, United Kingdom
World Health Orgnization
Geneva, 1991
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WHO Library Cataloguing in Publication Data
n-Hexane.
(Environmental health criteria ; 122)
1.Hexanes - adverse effects 2.Hexanes - toxicity
I.Series
ISBN 92 4 157122 5 (NLM Classification: QV 633)
ISSN 0250-863X
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CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR n-HEXANE
1. SUMMARY
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS
2.1. Identity
2.2. Physical and chemical properties
2.3. Conversion factors
2.4. Analytical methods
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
3.1. Natural occurrence
3.2. Production and uses
3.2.1. Production levels and processes
3.2.1.1 Production figures
3.2.1.2 Manufacturing processes
3.2.2. Uses
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION
4.1. Transport and distribution between media
4.2. Biotransformation and photochemical reactivity
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
5.1. Environmental levels
5.1.1. Air
5.1.2. Water
5.1.3. Food
5.2. Occupational exposure during manufacture, formulation, or
use
6. KINETICS AND METABOLISM
6.1. Experimental animals
6.1.1. Absorption, distribution, metabolism, and
excretion
6.1.2. Kinetics of 2,5-hexanedione
6.1.3. In vitro studies
6.1.4. Effects of other chemicals on n-hexane metabolism
6.2. Human beings
7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT
8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS
8.1. Single exposures
8.1.1. Inhalation exposure
8.1.2. Oral administration
8.1.3. Dermal exposure
8.1.4. Parenteral administration
8.1.5. In vitro exposure
8.2. Short- and long-term exposures
8.2.1. Inhalation studies
8.2.1.1 Combined-exposure effects
8.2.1.2 Effects on the respiratory tract
8.2.1.3 Effects on the testes
8.2.1.4 Other effects
8.2.2. Oral studies
8.2.3. Dermal studies
8.2.4. In vitro studies
8.2.5. Parenteral studies
8.3. Reproduction, embryotoxicity, and teratogenicity
8.3.1. Teratogenicity studies
8.3.1.1 Inhalation studies
8.3.1.2 Oral
8.3.2. Fertility studies
8.4. Mutagenicity and related end-points
8.5. Carcinogenicity
8.5.1. Inhalation studies
8.5.2. Skin-painting studies
8.6. Neurotoxicity
8.6.1. Central nervous system effects
8.6.2. Peripheral nervous system effects
8.6.2.1 Clinical and morphological findings
8.6.2.2 Electrophysiological effects
8.6.2.3 Grip strength
8.7. Toxicity of n-hexane metabolites
9. EFFECTS ON MAN
9.1. Single exposures
9.2. Skin and eye irritation; sensitization
9.2.1. Skin irritation
9.2.2. Eye irritation
9.2.3. Skin sensitization
9.3. Short- and long-term exposures
9.3.1. Effects on the nervous system
9.3.1.1 Peripheral neuropathy
9.3.1.2 Effects on vision and optic nerves
9.3.2. Effects on the kidney
9.3.3. Effects on other organs
10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE
ENVIRONMENT
10.1. Animal studies
10.1.1. Short- and long-term exposure
10.1.2. Genotoxicity
10.1.3. Carcinogenicity
10.1.4. Reproductive effects
10.2. Human studies
10.2.1. Effects on the nervous system
10.2.2. Other effects
10.3. Environmental effects
11. RECOMMENDATIONS
11.1. Human health protection
11.2. Environmental protection
12. FURTHER RESEARCH
REFERENCES
RESUME
EVALUATION DES RISQUES POUR LA SANTE HUMAINE ET DES EFFETS SUR
L'ENVIRONNEMENT
RECOMMANDATIONS
RECHERCHES A EFFECTUER
RESUMEN
EVALUACION DE LOS RIESGOS PARA LA SALUD HUMANA Y DE LOS EFECTOS EN
EL MEDIO AMBIENTE
RECOMENDACIONES
NUEVAS INVESTIGACIONES
WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR n-HEXANE
Members
Professor E.A. Bababunmi, Department of Tropical Paediatrics,
Liverpool School of Tropical Medicine, Liverpool, United
Kingdom (Rapporteur)
Dr M. Cikrt, Centre of Industrial Hygiene and Occupational
Diseases, Institute of Hygiene and Epidemiology, Prague,
Czechoslovakia (Vice-Chairman)
Dr S. Dobson, Pollution and Ecotoxicology Section, Institute of
Terrestrial Ecology, Monks Wood Experimental Station,
Huntingdon, United Kingdom
Professor C.L. Galli, Toxicology Laboratory, Institute of
Pharmacological Sciences, University of Milan, Milan, Italy
(Chairman)
Dr S.D. Gangolli, British Industrial Biological Research
Association, Carshalton, Surrey, United Kingdom
Dr C. Konantakieti, Technical Division, Food and Drug
Administration, Ministry of Public Health, Bangkok, Thailand
Dr O. Ladefoged, Laboratory of Pathology, Institute of
Toxicology, National Food Agency of Denmark, Ministry of Health,
Soborg, Denmark
Professor A. Massoud, Department of Community Environmental and
Occupational Medicine, Ainshams Faculty of Medicine, Cairo,
Egypt
Dr V. Riihimäki, Department of Industrial Hygiene and Toxicology,
Institute of Occupational Health, Helsinki, Finland
Observer
Dr H.P.A. Illing, Health and Safety Executive, Bootle, Merseyside,
United Kingdom
Secretariat
Dr P.G. Jenkins, International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland
Ms B. Labarthe, International Register of Potentially Toxic
Chemicals, United Nations Environment Programme, Geneva,
Switzerland
Dr E. Smith, International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland (Secretary)
NOTE TO READERS OF THE CRITERIA DOCUMENTS
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
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.
* * *
A detailed data profile and a legal file can be obtained from
the International Register of Potentially Toxic Chemicals, Palais
des Nations, 1211 Geneva 10, Switzerland (Telephone No. 7988400 or
7985850).
ENVIRONMENTAL HEALTH CRITERIA FOR n-HEXANE
A WHO Task Group on Environmental Health Criteria for n-Hexane
met in Carshalton, United Kingdom, from 12 to 16 March 1990.
Dr E.M. Smith opened the meeting and welcomed the participants on
behalf of the heads of the three IPCS cooperating organizations
(UNEP/ILO/WHO). The Task Group reviewed and revised the draft
criteria document and made an evaluation of the health risks of
exposure to n-hexane.
The first draft of this document was prepared by Dr K. Chipman,
University of Birmingham, United Kingdom. The second draft was
also prepared by Dr Chipman, incorporating comments received
following circulation of the first draft to IPCS contact points for
Environmental Health Criteria monographs. Particularly valuable
comments on the draft were made by the National Food Agency,
Denmark, the National Institute of Public Health and Environmental
Protection, The Netherlands, the European Chemical Industry Ecology
and Toxicology Centre (ECETOC), and the US Environmental Protection
Agency, National Institute of Environmental Health Sciences, Food
and Drug Administration, and Centers for Disease Control.
Dr E.M. Smith and Dr P.G. Jenkins, both members of the IPCS
Central Unit, were responsible for the overall scientific content
and technical editing, respectively, of this monograph.
The efforts of all who helped in the preparation and
finalization of the document are gratefully acknowledged.
* * *
Financial support for the Task Group was provided by the United
Kingdom Department of Health as part of its contributions to IPCS.
Partial financial support for the publication of this monograph 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, North
Carolina, USA - a WHO Collaborating Centre for Environmental Health
Effects.
ABBREVIATIONS
ACGIH American Conference of Governmental Industrial Hygienists
ADI acceptable daily intake
BAER brainstem auditory-evoked response
EEC European Economic Community
EEG electroencephalogram
EMG electromyography
ip intraperitoneal
MEK methyl ethyl ketone
NOEL no-observed-effect level
TWA time-weighted average
1. SUMMARY
n-Hexane (normal hexane) is a colourless, volatile liquid.
Commercial hexane is mainly a mixture of hexane isomers and related
6-carbon compounds, and has an n-hexane content varying between 20
and 80%. Gas chromatography coupled with flame ionization
detection or mass spectroscopy is a suitable technique for the
measurement of n-hexane. Occupational exposure limits range from
100 - 1800 mg/m3 (time-weighted average, TWA) and 400 - 1500 mg/m3
(ceiling value, CLV) in various countries.
n-Hexane can be isolated from natural gas and crude oil. It is
used in food processing, including the extraction of vegetable oil,
and as a solvent in various products and processes.
Once emitted into the environment, n-hexane exists
predominantly in the vapour phase. In the atmosphere its half-life
is estimated to be approximately 2 days, based on its reactivity
with the OH radical alone. Reported LC50 values for aquatic
organisms are few and variable and have been conducted under
inappropriate conditions; an assessment of the toxic effects of
n-hexane in this environment is, therefore, not possible. Low
water solubility and high volatility make exposure of aquatic
organisms unlikely except from uncontrolled discharge into surface
waters.
In mammals, n-hexane is absorbed rapidly through the lungs and
is distributed widely in the adult body, as well as to fetal
tissue. Dermal absorption is limited. n-Hexane is metabolized
oxidatively to a number of compounds, including 2,5-hexanedione,
which is thought to be the ultimate neurotoxic agent. Particularly
high levels of n-hexane and 2,5-hexanedione can occur in the
sciatic nerve of rats. Most n-hexane is excreted unchanged in
exhaled air; some is excreted as metabolites in exhaled air and
urine.
n-Hexane is of low acute toxicity for adult rats by oral
administration or inhalation. Oral LD50 values of 15 - 30 g/kg
have been recorded, and an inhalation LC50 value of 271 040 mg/m3
(77 000 ppm) has been reported for a 1-h exposure. At high vapour
concentrations, animals show ataxia, seizures, and signs of central
nervous system depression.
Testicular lesions and neurotoxicity appear to be the principal
effects of repeated n-hexane exposure in rats. Severe testicular
lesions have resulted from inhalation exposure to n-hexane and oral
exposure to 2,5-hexanedione. Effects have been attributed to
disruption of the cytoskeleton of Sertoli cells. There are
secondary effects on post-spermatogonial germ cells, which
disappear from affected tubules. Testicular effects were
reversible after a single exposure for 24 h to 17 600 mg/m3 (5000
ppm) but irreversible after a 2-week exposure to the same
concentration for 16 h/day, 6 days/week. 2,5-Hexanedione at 1% in
drinking-water produced similar reversible testicular lesions after
2 to 3 weeks of dosing and irreversible effects (within 17 weeks)
after 5 weeks of dosing.
The neurotoxic effect is characterized clinically by hindlimb
weakness, which can progress to paralysis. Axonal swellings
develop in the central and peripheral nervous systems; more severe
lesions (axonal degeneration and loss) can occur, particularly in
the longest, largest-diameter nerves. In essentially continuous
6-month inhalation studies, peripheral and central nervous system
lesions were present at doses of 1760 mg/m3 (500 ppm) or more, but
no clinical or pathological effects were noted at 440 mg/m3 (125
ppm). Only limited recovery of amplitude of the fifth brainstem
auditory-evoked response (believed to reflect central nervous
system activity) and tail nerve action potential was recorded 15 -
22 weeks after cessation of continuous exposure to a vapour
concentration of 3520 mg/m3 (1000 ppm), 5 days/week, for 11 weeks.
Discontinuous exposure of rats to 3168 mg/m3 (900 ppm) for 72 weeks
did not cause any apparent peripheral or central nervous system
lesions, but there was some evidence of electrophysiological
effects on peripheral nerves.
n-Hexane-induced neurotoxicity can be enhanced by combined
exposure to methyl ethyl ketone, methyl isobutyl ketone, and lead
acetate, and decreased by co-exposure to toluene. Toluene and
n-hexane also have a synergistic effect in the disturbance of
dopamine levels.
Severe microscopic lesions were noted in skin when n-hexane
was applied dermally under occlusive conditions for short periods.
Prolonged exposure to an n-hexane vapour concentration of 10 560
mg/m3 (3000 ppm) can cause conjunctival irritation in rats and
marked ocular irritation in rabbits. No skin sensitization data
are available from animal studies.
Chromosomal damage (polyploidy in one study, structural
aberrations in a second study) has been reported in both in vitro
and in vivo studies. No increase in point mutation frequency or
effects in tests for DNA damage has been noted.
There has been one carcinogenicity study with n-hexane (skin
painting on mice), which provided no evidence of carcinogenicity.
The reproductive toxicity of n-hexane has not been studied
adequately. There was no substantial evidence of embryotoxicity or
teratogenicity in rats following inhalation, though concentrations
were relatively low, or in mice after oral dosing. Postnatal
development of rats was transiently delayed when dams were exposed
to an n-hexane vapour concentration of 3520 mg/m3 (1000 ppm).
Very little information is available on the acute toxicity of
n-hexane to humans. Most studies have involved occupational
exposure to solvent mixtures. The available data suggest that
n-hexane has low acute toxicity. Signs of central nervous system
depression, such as drowsiness, vertigo, and giddiness, have been
reported after exposure to a commercial hexane level of 3520 to
17 600 mg/m3 (1000 - 5000 ppm) for 10 - 60 min.
n-Hexane is a mild irritant causing transient erythema when in
contact with human skin for short periods. More severe effects
(erythema and blistering) were documented after occlusive skin
contact for 5 h with commercial grade hexane. There have been no
case reports of sensitization of skin in exposed workers, and no
skin sensitization was noted in a maximization test with n-hexane.
On repeated exposure, n-hexane is neurotoxic, inducing a type
of sensorimotor peripheral neuropathy. Many studies on the
prevalence of n-hexane-induced neurotoxicity have been published;
however, adequate exposure data are often lacking. Exposure to
n-hexane concentrations in air varying from 106 - 8800 mg/m3
(30 - 2500 ppm) has been associated with neuropathy. Cases of
marked peripheral neuropathy were reported among Japanese sandal
workers and Taiwanese press proofers exposed to n-hexane levels of
approximately 176 and 352 mg/m3 (50 and 100 ppm), respectively, for
periods exceeding 8 h per day. In many cases exposure measurements
were recent and probably did not accurately reflect previous
exposures causing neuropathy.
Several cross-sectional studies have independently reported
mild subclinical effects (for example, electrophysiological
changes in peripheral nerves) in workers exposed to 70 - 352 mg/m3
(20 - 100 ppm). However, no clear cases of clinically overt
peripheral neuropathy were identified in any of these studies at
exposure levels of less than 352 mg/m3 (100 ppm).
The effects of n-hexane on the central nervous system have been
investigated only in a few studies. Changes in somatosensory
evoked potentials recorded from workers exposed to n-hexane were
suggested to result from a central nerve conduction block. Altered
visual evoked potentials and EEG traces have also been noted.
These results suggest that n-hexane may produce central nervous
system dysfunction, but the available data provide no information
on related exposure levels.
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS
2.1. Identity
Common synonyms: Hexyl hydride, hexane, Skellysolve B
Chemical structure:
H H H H H H
| | | | | |
| | | | | |
H---C---C---C---C---C---C---H
| | | | | |
| | | | | |
H H H H H H
Chemical formula: C6H14
CAS registry number: 110-54-3
Relative molecular mass: 86.177
2.2. Physical and chemical properties
Some physical and chemical properties of various grades of
n-hexane are given in Table 1.
Table 1. Physical and chemical properties of n-hexanea
------------------------------------------------------------
Boiling point (°C) 68.74b
Melting point (°C) -95.35b
Relative density (20 °C/4 °C) 0.66
Vapour pressure (25 °C) 20 kPa (150 mmHg)
Vapour density 2.97
Autoignition temperature (°C) 225
Explosive limit in air (% by volume) 1.1-7.5
Flash point (°C) -21.7
Closed-cup flash point (°C)c -30.56
Solubility in water (mg/litre at 25 °C) 9.5
Log n-octanol/water partition coefficient
(log Pow at 25 °C) 3.6
Refractive index (20 °C) 1.37
Colour, Saybolt +39
------------------------------------------------------------
a From: Mellan (1977)and IRPTC (1990).
b From: Clayton & Clayton (1981).
c From: ACGIH (1986).
n-Hexane is colourless, highly volatile (NIOSH, 1977a), and
flammable (Dale & Drehman, 1980; ACGIH, 1986). It is poorly
soluble in water but is soluble in most organic solvents including
ethanol and ether (McAuliffe, 1963; NIOSH, 1977a; ACGIH, 1986).
Purified n-hexane contains 95 - 99.5% n-hexane, together with
small amounts of other hexane isomers as impurities (Mellan, 1977;
Baker & Rickert, 1981; Sandmeyer, 1981). Traces of benzene (0.05%)
have been detected (Baker & Rickert, 1981). Commercial hexane is a
mixture of hexane isomers ( n-hexane, 2-methylpentane,
3-methylpentane, 2,3-dimethylbutane), cyclohexane, methyl
cyclopentane and small amounts of pentane and heptane isomers,
acetone, methyl ethyl ketone, dichloromethane, and
trichloroethylene (Perbellini et al., 1981a,b,c; ACGIH, 1986). The
n-hexane content of commercial hexane (Table 2) can vary between
20% and 80% (ACGIH, 1986).
Table 2. Composition (% by weight) of different
grades of n-hexanea
------------------------------------------------
Research Pure Technical
grade grade grade
------------------------------------------------
n-Hexane 99.98 99.5 95-97.7
2-Methylpentane trace trace trace
3-Methylpentane 0.02 0.1 0.2
Methylcyclopentane trace 0.4 2.1
------------------------------------------------
a From: Mellan (1977).
Pure n-hexane contains approximately 0.0005% of non-volatile
material (Mellan, 1977), whereas commercial hexane may contain up
to 0.04% (Patty & Yant, 1929; Vicedo et al., 1985). In commercial
hexane, a number of phthalate esters (including dimethyl, diethyl,
di- n-butyl, di-isobutyl, dihexyl, and diethylhexyl), adipate
esters (dibutyl and dioctyl), and organophosphorus compounds (e.g.,
triphenyl phosphate) have been identified and total about 0.3% of
the distillation residue (Vicedo et al., 1985).
Commercial hexane (containing about 30% n-hexane) has a
slightly disagreeable odour, perceptible at 282 mg/m3 (80 ppm)
(intermittent exposure) or 528 mg/m3 (150 ppm) (continuous
exposure) (Patty & Yant, 1929). An odour threshold of 211 mg/m3
(60 ppm) for hexane (purity not stated) has also been reported
(Laffort & Dravnieks, 1973).
2.3. Conversion Factors
1 ppm n-hexane in air = 3.52 mg/m3
1 mg/m3 = 0.284 ppm n-hexane in air
2.4. Analytical Methods
n-Hexane may be analysed by gas chromatography with flame
ionization detection or mass spectroscopy. A summary of the
techniques employed is given in Table 3.
Carbon cloth can be used for diffusive sampling. It has been
shown that when it is wrapped in aluminium foil there is no
significant loss of n-hexane during storage for at least four days
(Kasahara & Ikeda, 1987).
Table 3. Techniques for the analysis of n-hexane
---------------------------------------------------------------------------------------
Medium Separation Detection Limit of Reference
method system sensitivity
---------------------------------------------------------------------------------------
Air trap with charcoal; flame validated over the NIOSH (1977b)
desorb with CS2; ionization range 877-3679
steel column; gas mg/m3 at 17 °C,
chromatography 764 mmHg; probable
(FFAP) useful range:
180-5400 mg/m3
Air trap with charcoal; flame measurement range: NIOSH (1984)
desorb with CS2; gas ionization 3.56-14.5 mg for
chromatography 5-µl injection
Air diffusive sampling; stain tube detection range: Gentry & Walsh
thermal desorption (Kitagawa 18-500 µg (1987)
133 5B)
Liquid glass column; gas
chromatography; 5%
carbowax (partition) flame not given Franke et al.
or 0.3% carbowax ionization (1988)
(adsorption)
Liquid capillary glass flame 0.05 µg Nomeir &
column; gas ionization Abou-Donia
chromatography (1985)
(OV101)
Biological glass column; gas mass 80 ng/g (biological Tsuruta (1980)
samples chromatography spectrometer sample)
(Porapak Q) (multiple ion
monitoring
m/z 85)
---------------------------------------------------------------------------------------
A high performance liquid chromatographic method using a silica
cartridge has been developed for the analysis of n-hexane
metabolites (2-hexanone, 2,5-dimethylfuran, gamma-valqerolactone,
5-hydroxy-2-hexanone, and 2,5-hexanedione) in chicken plasma
(Nomeir & Abou-Donia 1985). Metabolites of n-hexane have also been
analysed by gas chromatography coupled with mass spectroscopy
(DiVincenzo et al., 1976).
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
3.1. Natural Occurrence
n-Hexane is present in natural gas and crude oil (Guthrie,
1960).
3.2. Production and Uses
3.2.1. Production levels and processes
3.2.1.1 Production figures
It has been estimated that 306 000 tonnes of n-hexane was
recovered from petroleum and natural gas in 1977 in the USA (Dale &
Montgomery, 1983). Babich et al. (1982) cited approximate
estimates of 240 000 to 465 000 tonnes (80 - 155 million gallons)
for the annual production of naphtha (of which hexane is the
principal component) in the USA in 1978/1979, and the total
production in the USA in 1987 was 386 500 tonnes (USITC, 1988).
3.2.1.2 Manufacturing processes
Two-tower distillation of a suitable hydrocarbon feedstock is
used for the manufacture of commercial hexanes. The feedstock may
be straight-run gasolines distilled from crude oil or natural gas
liquids stripped of natural gas. Hexanes can also be obtained from
the remains of catalytic reformates after the removal of aromatics.
Very pure n-hexane can be produced from hexane mixtures by
adsorption on molecular sieves (Dale & Drehman, 1983).
3.2.2. Uses
In most cases, n-hexane is used as a mixture with other hexane
isomers and various solvents. The following uses of n-hexane have
been reported (Dale & Drehman, 1983; CCOHS, 1985):
* in food processing, including the extraction of vegetable oil
from soybeans, flaxseed, peanuts, safflower seed, corngerm, and
cottonseed;
* as a polyolefins solvent and as a cleaning agent;
* as a rubber polymerization solvent;
* as a laboratory chemical;
* in low-temperature thermometers;
* in the manufacture of pharmaceuticals;
* in other products (e.g., adhesives, lacquers).
The consumption of hexane (not specifically n-hexane) in the
USA and Canada during 1975 for the above purposes was estimated to
be 450 000 to 490 000 m3 (120 - 130 million gallons) (Dale &
Drehman, 1983). The consumption of n-hexane in the EEC in 1979 was
10 000 tonnes.
In general, petroleum distillate solvents of a relatively low
boiling point contain a relatively high proportion of n-hexane
(Kasahara et al., 1987). Rubber surface softener samples and
approximately one half of the analysed samples of thinner-cleaner
used for printing and painting were in this category (Kasahara et
al., 1987). Ikeda & Kasahara (1986) found the n-hexane content to
be 0.4 - 9% in four samples of gasoline with boiling points in the
range 30 - 160 °C.
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION
4.1. Transport and Distribution Between Media
There is little information on the transport and mobility of
n-hexane in the environment. It is very insoluble in water (9.5
mg/litre at 25 °C) (McAuliffe, 1963), and transport occurs
predominantly in the vapour phase once it is emitted into the
environment. The solubility of n-hexane in water is increased by
the presence of methanol (Groves, 1988).
Chiou et al. (1988) determined that the sorptive capacity of
soil for hexane is 11 mg/g (0.0167 ml/g).
4.2. Biotransformation and Photochemical Reactivity
Information is only available on the abiotic degradation of
n-hexane. Atmospheric n-hexane is not expected to have a
pronounced effect on the physical properties of the atmosphere, to
participate in the depletion of the ozone layer, or to alter
precipitation patterns (CIIT, 1977). The rate constant for the
reaction of hydroxyl (OH) radicals with n-hexane was determined
(using a smog chamber) to be 6.2 (± 0.6) x 10-12 cm3 sec-1 at 39 °C
(Nolting et al., 1988). Other reported values are 5.63 (± 0.09) x
10-12 cm3 sec-1 at 26 °C (Atkinson et al., 1982) and 5.68 (± 0.04)
x 10-12 cm3 sec-1 (Behnke et al., 1988). In the latter study,
there was additional degradation in the presence of titanium
dioxide aerosol. These rate coefficients are in good agreement
with the value of Klopffer et al. (1988), who measured a KOH of 6.8
x 10-12 cm3 sec-1 in a smog chamber at 27 °C. The half-life of
n-hexane in the troposphere is estimated to be 2 to 2.4 days, based
on degradation by OH radicals alone.
Similar hydrocarbons ( n-pentane and methyl pentane) undergo
photochemical conversion to a "smog" containing
peroxyacetylnitrate and ozone, but n-hexane is one of the least
photochemically reactive hydrocarbons (Katagiri & Ohashi, 1975).
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
5.1. Environmental Levels
5.1.1. Air
Fugitive losses of n-hexane may occur in industries in which it
is used as a solvent or cleaning agent and in the rubber industry.
In 1970, prior to the mandatory use of catalytic converters,
n-hexane was also estimated to represent 1.2% (by volume) of total
emitted hydrocarbons from vehicular exhausts in the USA (equivalent
to approximately 170 000 tonnes of hexane) (CIIT, 1977). Lonneman
et al. (1974) detected n-hexane in air samples collected in the
Lincoln Tunnel, New York, over a 0.5-h period. n-Hexane has been
detected at a level of 0.11 mg/m3 (0.03 ppm) in the ambient
atmosphere of Los Angeles (Nelligan, 1962). Hodgson et al. (1986)
reported that the concentrations of n-hexane in a single open-
office space were 1.97 µg/m3 (0.56 ppb) and 4 µg/m3 (1.14 ppb)
(with 100% ventilation exhaust on and off, respectively), compared
with an outdoor concentration that was below the limit of
quantification.
In the Federal Republic of Germany, n-hexane belongs to class
III of chemical substances, the total emission of which (as the sum
of all compounds in this class) must not exceed 150 mg/m3 at a mass
flow of 3 kg/h or more (IRPTC, 1990). In the USSR, the ambient
vapour concentration of n-hexane is regulated at a maximum of 300
mg/m3 (IRPTC, 1990).
5.1.2. Water
n-Hexane has been detected in the USA in trace quantities in
chlorinated tap water derived from a lake (CEC, 1979).
5.1.3. Food
The Joint FAO/WHO Expert Committee on Food Additives (JECFA)
has not set an ADI for n-hexane but stresses that the solvent
should be used only in accordance with good manufacturing practice
to ensure minimal residues in food (WHO, 1971). In the USA,
cottonseed products and hop extract, modified for human
consumption, may contain no more than 60 mg/kg and 25 mg/kg,
respectively (CFR, 1987a). The latter limit also applies to
certain food colouring agents (CFR, 1987b). The EEC Directive on
Extraction Solvents (June 1988) set maximum residue levels in food
of between 1 and 30 mg/kg depending on the particular use (IRPTC,
1990).
5.2. Occupational Exposure During Manufacture, Formulation, or Use
In addition to the presence of n-hexane in the air during its
manufacture, a study has indicated detectable levels in air samples
from a variety of industrial uses of products containing n-hexane.
The relative abundance (percentage of samples in which it was
detected) was 15% (printing), 7% (painting), 10% (car repair), and
6% (various other operations) (Veulemans et al., 1987). Levels of
hexane exposure in six olive extraction plants in Granada, Spain,
were found to vary from 3 to 341 mg per m3 (0.9 - 97 ppm)
(Medinilla & Espigares, 1989). An Italian worker employed at home
manufacturing shoes was found to have a urinary 2,5-hexanedione
level of 5.7 mg/litre, which exceeded the ACGIH biological exposure
index for hexane of 5 mg/litre (Discalzi et al., 1988).
Concentrations of n-hexane have also been measured in the air to
which petroleum service attendants, transport drivers, and outside
operators were exposed. For outside operators, 54 out of 56
samples contained measurable concentrations of n-hexane, the mean
concentration being 0.473 ± 0.594 mg/m3. All 49 samples of air for
transport drivers contained n-hexane (mean concentration, 1.019 ±
1.953 mg/m3). Service attendants were exposed to a mean
concentration of 1.175 ± 0.894 mg/m3 (detected in 48/49 samples).
These values represented 2.5, 2.2, and 1.7% of the total
hydrocarbon concentration for the three job categories,
respectively (Rappaport et al., 1987). Maximum time-weighted
average (8 h) concentrations of hexane at a hexane extraction
facility were found to be 92 mg/m3 (26 ppm) (NIOSH, 1981a). NIOSH
(1983) reported hexane concentrations in air samples from six
breathing zones at a soybean extraction facility that ranged from
15.5 to 46.5 mg/m3 (4.4 - 13.2 ppm). In area spot samples, the
concentrations were 46.5 - 94.7 mg/m3 (13.2 - 26.9 ppm).
In one metropolitan sewer in Cincinnati, USA, in 1981, the
airspace was found to contain hexane at a concentration greater
than the lower explosive limit (1.2% by volume in air). Two sewers
contained hexane levels of at least 39 600 mg/m3 (11 250 ppm)
(NIOSH, 1981b).
Some limits for occupational exposure in various countries are
shown in Table 4. Some countries (e.g., USA, Belgium, and the
United Kingdom) also recommend an exposure limit (TWA) of 360 mg/m3
for all hexane isomers other than n-hexane.
Table 4. Some national occupational air exposure limit valuesa
-----------------------------------------------------------------------------------
Country/ Exposure limit descriptionb Value Effective
organization datec
-----------------------------------------------------------------------------------
Australia Threshold limit value (TLV)
- Time-weighted average (TWA) 360 mg/m3 1985(r)
Belgium Threshold limit value (TLV)
- Time-weighted average (TWA) 180 mg/m3 1989(r)
Canada Threshold limit value (TLV)
- Time-weighted average (TWA) 180 mg/m3 1989
Finland Maximum permissible concentration (MPC)
- Time-weighted average (TWA) 180 mg/m3 1989(r)
- Short-term exposure limit (STEL) 530 mg/m3
-----------------------------------------------------------------------------------
Table 4. (contd.)
-----------------------------------------------------------------------------------
Country/ Exposure limit descriptionb Value Effective
organization datec
-----------------------------------------------------------------------------------
Germany, Maximum acceptable concentration (MAK)
Federal - Time-weighted average (TWA) 180 mg/m3
Republic of - Short-term exposure limit (STEL) 360 mg/m3 1989(r)
- Biological tolerance value (BAT) urine: 9 mg/litre
hexane-2,5-dione plus 4,5-dihydroxy-2-
haxanone at end of exposure or end of
shift
Germany, Maximum acceptable concentration (MAC)
Democratic - Time-weighted average (TWA) 100 mg/m3
Republic of - Short-term exposure limit (STEL) 400 mg/m3 1988(r)
Italy Threshold limit value (TLV)
- Time-weighted average (TWA) 360 mg/m3 1985(r)
Japan Maximum acceptable concentration (MAC)
- Time-weighted average (TWA) 141 mg/m3 1985
(skin absorption must also be considered)
Poland Maximum permissible concentration (MPC)
- ceiling value (CLV) 400 mg/m3 1986(r)
Romania Maximum permissible concentration (MPC)
- Time-weighted average (TWA) 1200 mg/m3
- Ceiling value (CLV) 1500 mg/m3 1975(r)
Switzerland Maximum acceptable concentration (MAK)
- Time-weighted averge (TWA) 180 mg/m3 1987
Sweden Hygienic limit value (HLV)
- Time-weighted average (TWA) 90 mg/m3
- Short-term exposure limit (STEL) 180 mg/m3 1990(n)
United Guidance limit (under review)
Kingdom - Time-weighted average (TWA) 360 mg/m3
- Short-term exposure limit (STEL) (10 min) 450 mg/m3 1990(n)
USA (ACGIH) Threshold limit value (TLV)
- Time-weighted average (TWA) 176 mg/m3 1990(r)
- Biological exposure index (BEI) 5 mg/litre
2,5-hexanedione in urine (end of shift)
n-hexane in end-exhaled air (during 144 mg/m3
shift)
-----------------------------------------------------------------------------------
Table 4. (contd.)
-----------------------------------------------------------------------------------
Country/ Exposure limit descriptionb Value Effective
organization datec
-----------------------------------------------------------------------------------
USA (OSHA) Permissible exposure limit (PEL) 180 mg/m3 1990(r)
Yugoslavia Maximum permissible concentration (MAC)
- Time-weighted average (TWA) 1800 mg/m3 1971(r)
-----------------------------------------------------------------------------------
a From: IRPTC (1990).
b TWA = a maximum mean exposure limit based generally over the period of a working
day (usually 8 or 12 h)
STEL = a maximum concentration of exposure for a specified time duration
(generally 15 or 30 min).
c When no effective date appears in the IRPTC legal file, the year of the reference
from which the data are taken is shown, indicated by (r); n = notified direct by
country.
6. KINETICS AND METABOLISM
6.1. Experimental Animals
6.1.1. Absorption, distribution, metabolism, and excretion
Radiolabelled 14C- n-hexane was absorbed by F-344 rats
following inhalation of concentrations of 1760, 3520, 10 560, and
35 200 mg/m3 (500, 1000, 3000, and 10 000 ppm) for 6 h daily for 3
days (Bus et al., 1982). The proportion of 14C- n-hexane recovered
in expired air was dose dependent and increased from 12% at 1760
mg/m3 (500 ppm) to 62% at 35 200 mg/m3 (10 000 ppm). There was a
corresponding decrease in the proportion recovered in urine (from
35% at 1760 mg/m3 (500 ppm) to 18% at 35 200 mg/m3 (10 000 ppm), in
faeces (from 4.9% at 1760 mg/m3 (500 ppm) to 0.7% at 35 200 mg/m3
(10 000 ppm), and in the carcass (from 10% at 1760 mg/m3 (500 ppm)
to 1.5% at 35 200 mg/m3 (10 000 ppm). The levels of 14C- n-hexane
in expired air decreased biphasically, with half-lives of 1 and
4.5 h. A biphasic elimination profile was reported for labelled
carbon dioxide in exhaled air, the initial half-life being 4.5 -
7.5 h and 85 - 96% of the exhaled labelled carbon dioxide being
eliminated within 24 h after exposure. Urinary excretion of
metabolites also occurred in two phases, most being excreted in the
initial phase with a half-life of 7 - 8 h. The greater part of the
radioactivity absorbed during inhalation of n-hexane was excreted
within 24 h. The rate of metabolism was non-linear above 1056 mg/m3
(300 ppm) but not below this concentration: it rose from 47 µmol/h
per kg body weight at 1056 mg/m3 (300 ppm) to 245 µmol/h per kg
body weight at 10 560 mg/m3 (3000 ppm). The amount of n-hexane in
the rat increased in a dose-dependent manner to a limiting value of
9.6 relative to the atmospheric concentration (Filser et al.,
1987).
The rate of uptake following inhalation of hexane in male
F-344/N rats was found to be 5 - 7 nmol/kg per min (Dahl et al.,
1988). Absorption and distribution into tissues and organs
(including blood) was rapid following inhalation exposure of F-344
rats to n-hexane (Baker & Rickert, 1981; Howd et al., 1982).
Exposure to concentrations between 3520 mg/m3 and 35 200 mg/m3
(1000 ppm and 10 000 ppm) resulted in plateau levels of n-hexane
being reached within 30 min in blood and within 2 h in the other
tissues examined (liver, kidney, lungs, testis, brain, and sciatic
nerve) (Baker & Rickert, 1981). Acute inhalation exposure to
302 720 mg/m3 (86 000 ppm) resulted in saturated blood levels
within 10 min (Raje et al., 1984).
Dermal absorption of n-hexane by guinea-pigs is poor (Jakobson
et al., 1982). Tsuruta (1982) measured the penetration of
n-hexane through excised rat skin and came to a similar conclusion.
However, skin absorption of n-hexane may be enhanced by other
solvents in the mixture. The highest tissue concentrations of
n-hexane were found in peripheral nerves (sciatic) (Baker &
Rickert, 1981; Bus et al., 1981). Steady-state concentrations in
blood, sciatic nerve, liver, and lung were directly proportional to
the exposure level, but there was some evidence of saturation in
the kidney, brain, and testis.
In female albino rats exposed to a hexane level of 17 000 mg/m3
(50 000 ppm) for up to 10 h, the hexane concentration in the liver
increased with time and steady-state levels were not achieved
(Böhlen et al., 1973). However, kidney, adrenal, blood, brain, and
spleen levels of hexane reached a steady state after approximately
5 h of exposure. The high level of hexane exposure resulted in
lipid accumulation in the liver, and, as hexane is highly lipid
soluble, this could account for the non-saturability of liver for
hexane.
n-Hexane has been detected in the fat, muscle, and bone
(sternum) of F-344 rats exposed to an n-hexane level of 21 123
mg/m3 (6800 ppm) for 4 h and in rat fetuses following inhalation
exposure of pregnant females (Bus et al., 1979). The concentration
in total fetal tissue was similar to that in maternal blood
(Babanov & Babanov, 1981).
Elimination of n-hexane from rat blood and brain was found to
be rapid and multiphasic, with initial half-lives of 2 - 4 and 7
min and a subsequent half-life of 1 - 2 h (Bus et al., 1979; White
et al., 1979; Baker & Rickert, 1981; Howd et al., 1982). In the
rat, elimination from other adult tissues, including sciatic nerve
and liver, and from fetal tissue was also rapid but was slower from
the kidneys (half-life, 6 h). In the guinea-pig, biphasic
elimination from blood (half-lives of 0.5 h and 4 h) has been
reported (Couri et al., 1978).
n-Hexane is oxidized to 1-, 2-, or 3-hexanol. 2-Hexanol may be
further metabolized to 2-hexanone (methyl- n-butyl ketone).
2,5-Hexanediol, 5-hydroxy-2-hexanone, 2,5-hexanedione, gamma-
valerolactone, 2,5-dimethylfuran, 2,5-dimethyl-2,3-dihydrofuran,
2,5-dimethyltetrahydrofuran, and carbon dioxide have been
identified following exposure to n-hexane or 2-hexanone. However,
furan derivatives and gamma-valerolactone are questionable
metabolites of n-hexane because they may be artefacts (DiVincenzo
et al., 1976; Perbellini et al., 1982a; Fedtke & Bolt, 1986b).
2,5-Dimethyl-2,3-dihydrofuran and small amounts of labelled
2-aminohexanoic acid (norleucine), another unidentified amino acid,
and urea have been detected following oral dosing of rats with
1-14C-2-hexanone. Pentanone (isomeric form not specified) has been
detected in rat fetuses from dams exposed to 2-hexanone vapour
(DiVincenzo et al., 1977; Peters et al., 1981). Radiolabelled
2,5-hexanedione is metabolized to carbon dioxide in the rat (Angelo
& Bischoff, 1982). Proposed metabolic pathways for n-hexane in
mammals are shown in Fig. 1.
2-Hexanol was the major urinary metabolite excreted by rats
during n-hexane inhalation exposure for up to 24 h (Perbellini et
al., 1982a; Iwata et al., 1983b). After exposure, the main urinary
metabolites were 2,5-hexanedione, 2,5-dimethylfuran, and gamma-
valerolactone (Baker & Rickert, 1981; Perbellini et al., 1982a;
Iwata et al., 1983b). All the gamma-valerolactone and a
significant proportion of the other metabolites were conjugated;
glucuronides of 2-hexanol and 2,5-dimethylfuran were identified.
Only small amounts of 2-hexanone, 5-hydroxy-2-hexanone, and
3-hexanol were present. 1-Hexanol, mainly as the glucuronide, has
been detected following intraperitoneal injection of n-hexane
(Dolara et al., 1978). With prolonged exposure to 1760 mg/m3 (500
ppm) (3 h/day, for 33 weeks), 80 - 90% of all urinary metabolites
were conjugated, the predominant metabolites being 2-hexanol and
2,5-hexanedione (Iwata et al., 1984). In urine collected from rats
during and up to 24 h after exposure to 3520 mg per m3 (1000 ppm),
the predominant urinary metabolites were 2-hexanol and
2,5-hexanedione, of which 99 and 90%, respectively, were found
(following acid hydrolysis) to be conjugated. In addition, all the
1-hexanol and 3-hexanol and 30% of the 2-hexanol were conjugated.
The hexanol conjugates were considered to be glucuronides and
sulfate esters, but the identity of the other conjugates was
unclear (Fedtke & Bolt, 1986b). Subsequently, Fedtke & Bolt
(1987a) identified 4,5-dihydroxy-2-hexanone as an additional
glucuronic acid conjugate in the urine of male Wistar rats treated
with n-hexane at 7040 mg/m3 (2000 ppm) for 8 h or given a single
ip dose of 2,5-hexanedione (200 mg/kg). This metabolite was
considered to be produced either by hydroxylation of 5-hydroxy-2-
hexanone (which may be produced by reduction of 2,5-hexanedione) or
alternatively by hydroxylation and subsequent reduction of
2,5-hexanedione. Thus, 4,5-dihydroxy-2-hexanone may be a
detoxified product of 2,5-hexanedione. This metabolite was the
second most abundant metabolite in the urine of male Wistar rats
following inhalation of n-hexane (176 - 10 820 mg/m3, 50 - 3074
ppm) for 8 h (Fedtke & Bolt, 1987b).
In studies on the rabbit and monkey (Macaca mulatta), the
urinary excretion of n-hexane metabolites was found to differ from
that of rats (Perbellini et al., 1982b). 2-Hexanol was the main
urinary metabolite during and after inhalation exposure. The other
metabolites present were 3-hexanol and 2,5-hexanedione; gamma-
valerolactone and 2,5-dimethylfuran were not detected. Following
intraperitoneal injection of n-hexane in guinea-pigs, 2-hexanol,
mainly as the glucuronide, and n-hexane itself were identified in
the urine (Couri et al., 1978).
Elimination half-lives in rat urine for individual metabolites
following inhalation of n-hexane have been determined for
2-hexanone (4 h), 3-hexanol and 2,5-hexanedione (7 h),
2,5-dimethylfuran and gamma-valerolactone (11 - 14 h), and
2-hexanol (18 h) (Perbellini et al., 1982a). The half-life for
2-hexanol in the rabbit and monkey was found to be shorter (8 h)
than in the rat.
5-Hydroxy-2-hexanone and 2,5-hexanedione were detected in
guinea-pig serum after intraperitoneal administration of n-hexane
(DiVincenzo et al., 1976). Following administration of 2-hexanol,
a major urinary metabolite of n-hexane in some species, two
additional compounds, 2-hexanone and 2,5-hexanediol, were found. A
similar metabolic profile was found following the administration of
2-hexanone, with 5-hydroxy-2-hexanone, 2,5-hexanedione, and
2-hexanol being identified as metabolites. 2,5-Hexanedione, which
is considered to be the active neurotoxic agent, can thus be formed
from n-hexane, 2-hexanol, or 2-hexanone.
6.1.2. Kinetics of 2,5-hexanedione
A marked increase in blood levels of 2,5-hexanedione on
inhalation exposure to n-hexane at 3168 or 17 600 mg/m3 (900 or
5000 ppm) for 6 - 24 h has been observed in separate studies
(Perbellini et al., 1982a; Kulig, 1983; Kulig et al., 1984). No
evidence of 2,5-hexanedione accumulation in blood was found in
Wistar rats after repeated exposures to n-hexane at 3168 mg/m3 (900
ppm), 8 h/day, for 9 days (Kulig, 1983). 2,5-Hexanedione was
completely eliminated, within 6.25 h of the end of exposure, from
the blood of Wistar rats exposed to an n-hexane level of 3168 mg/m3
(900 ppm) 8 h/day, for 72 weeks (De Groot & Kepner, 1984). There
was no significant difference in 2,5-hexanedione blood levels in
Fischer-344 rats exposed for 1 or 5 days to 3520 mg/m3 (1000 ppm)
for 6 h/day (Bus et al., 1981). In contrast, there were
significantly increased 2,5-hexanedione concentrations in the blood
of Fischer-344 rats after 3 exposures to 14 080 mg/m3 (4000 ppm)
for 8 h/day compared with a single 8-h exposure (Howd et al., 1982).
The elimination half-life for 2,5-hexanedione from blood has
been reported to vary between different strains of rat. Values for
Sprague-Dawley and Fischer-344 rats were 2.3 h and 3.9 - 6 h,
respectively, and an initial half-life of 1 h has been reported for
Wistar rats (Bus et al., 1979; Angelo & Bischoff, 1982; Howd et
al., 1982; Kulig, 1983). It has been suggested that differences in
the rates of elimination of 2,5-hexanedione from the blood may
explain the reported greater susceptibility to n-hexane-induced
neurotoxicity of Fischer-344 rats compared with Wistar rats (Kulig,
1983; Kulig et al., 1984).
In a study by Ladefoged & Perbellini (1986), 2,5-hexanedione
(50 mg/kg) was administered to five male rabbits by intravenous
injection. The pharmacokinetic data fitted a two-compartment model
in which the half-life was 42 (± 11) min (body clearance was 0.0117
(± 0.0026) ml/min per kg).
Blood concentrations of 2,5-hexanedione have been estimated
following continuous exposure of rats to n-hexane (Kulig, 1983;
Kulig et al., 1984). Marked reductions in blood 2,5-hexanedione
levels were reported following exposure to 3168 mg/m3 (900 ppm) for
3 days compared with one day. There were no further decreases
after 9 or 20 days of exposure. Smaller reductions were observed
in short-term studies after 8 or 12 weeks of exposure to 3168 mg/m3
(900 ppm) for either 8 or 24 h/day, 5 days per week, compared with
the blood levels of 2,5-hexanedione recorded after 4 weeks of
exposure to 3168 mg/m3 (900 ppm). No effects were reported in
similar short-term studies at 1056 mg/m3 (300 ppm) (Kulig, 1983).
This may be due to the enhancement of glucuronidation (a major
elimination pathway) by hexane, a phenomenon noted in both in vitro
and in vivo studies in the guinea-pig (Notten & Henderson,
1975a,b). Similar levels of 2,5-hexanedione in blood were found in
weanling and young adult rats following one week of continuous
exposure to 3520 mg/m3 (1000 ppm) (Pryor et al., 1982).
2,5-Hexanedione was detected in the blood, sciatic nerve,
brain, kidneys, liver, and lungs, but not in the testes, following
exposure of Fischer-344 rats to levels of n-hexane between 1760 and
35 200 mg/m3 (500 to 10 000 ppm) for 6 h (Baker & Rickert, 1981).
The relationship between peak tissue concentrations of
2,5-hexanedione and n-hexane exposure levels was complex. In the
kidneys, sciatic nerve, and brain, the highest concentrations were
obtained after exposure to 3520 mg/m3 (1000 ppm). In a further
study, lower tissue levels and greater elimination of
2,5-hexanedione were found in mice than in rats after a single
exposure to 3520 mg/m3 (1000 ppm) for 6 h (Baker et al., 1980).
The elimination of 2,5-hexanedione from the rat liver, kidney,
brain, and sciatic nerve was determined after exposure to 3520
mg/m3 (1000 ppm) 6 h/day, for 1 or 5 days (Bus et al., 1981).
2,5-Hexanedione was selectively retained in sciatic nerve but not
in the brain. However, comparable rates of elimination of
2,5-hexanedione from the brain, sciatic nerve, and blood were
reported in Sprague-Dawley rats given a single oral dose of
2,5-hexanedione (Iwasaki & Tsuruta, 1984). Terminal half-lives of
32 - 33 days were reported in a review of some earlier studies
(O'Donoghue, 1985).
2,5-Hexanedione has also been detected in fetal tissue, at a
level similar to that in maternal blood, following exposure of
F-344 rats to n-hexane (Bus et al., 1979). A fetal half-life of
3 h was calculated.
Less than 10% of 2,5-hexanedione in blood is bound to plasma
components (Angelo & Bischoff, 1982). A study on hens dosed orally
with 2,5-hexanedione showed that it can form a pyrrole adduct with
serum protein (DeCaprio et al., 1982).
6.1.3. In vitro studies
n-Hexane penetrates excised rat skin slowly (Tsuruta, 1977,
1982).
In rat liver and lung preparations, n-hexane has been shown to
be hydroxylated to 1-, 2-, and 3-hexanol (Frommer et al., 1974;
Walseth et al., 1982; Toftgard et al., 1984). 2-Hexanol was found
to be the predominant metabolite, and 2,5-hexanedione was also
formed in studies with rat liver microsomes (Frommer et al., 1974;
Walseth et al., 1982). More than one form of cytochrome P-450 is
involved in n-hexane oxidation (Frommer et al., 1974). In the rat
lung the formation of 2- and 3-hexanol is catalysed by a different
microsomal cytochrome P-450 isoenzyme (cytochrome P-450-PB-B) from
that responsible for the formation of 1-hexanol (Toftgard et al.,
1984). Toftgard et al. (1984, 1986) found that rat liver
microsomes, but not lung microsomes, contained a cytochrome P-450
isoenzyme that converted 2-hexanol to 2,5-hexanediol, and that the
oxidation of hexanols and 2,5-hexanediol by alcohol dehydrogenase
was restricted to the liver. These results suggest that, during
inhalation of n-hexane, the metabolite 2,5-hexanedione is likely to
be formed in the liver but not in the lung.
Preparations of purified rabbit liver cytochrome P-450
hydroxylate n-hexane, and mouse liver microsomes hydroxylate hexane
(Ichihara et al., 1969; Nilsen et al., 1981). Studies using liver
fractions from guinea-pigs demonstrated that n-hexane can be
metabolized to 2-hexanol, 2-hexanone, and 2,5-hexanedione (Couri et
al., 1978). 2-Hexanone was reduced to 2-hexanol by the cytosolic
fraction and oxidized to 2,5-hexanedione by the microsomal
fraction.
In mouse nerve and muscle tissue cultures, n-hexane was
metabolized to 2-hexanediol and 2,5-hexanedione, and 5-hydroxy-2-
hexanone was detected following incubation with 2-hexanol or
2-hexanone (Veronesi et al., 1978, 1980; Spencer et al., 1980).
2,5-Hexanedione has been found to react with primary amino
groups in proteins, such as the epsilon-amino group of lysine, to
yield 2,5-dimethylpyrrole adducts (DeCaprio et al., 1982; Graham et
al., 1982a,b). Pyrrole formation is an obligatory step in the
pathogenesis of neuropathy caused by n-hexane (Sayre et al., 1986).
2,5-Hexanedione appears to be the active neurotoxic metabolite of
n-hexane.
6.1.4. Effects of other chemicals on n-hexane metabolism
Urinary excretion of n-hexane metabolites by rats has been
shown to be increased following pre-treatment with phenobarbital,
an inducer of the microsomal oxidation of foreign compounds by
cytochrome P-450 isoenzymes (Perbellini et al., 1979, 1982a).
In in vitro studies, pre-treatment of rats with phenobarbital
increased the extent of liver microsomal oxidation of n-hexane to
2- and 3-hexanol, and pre-treatment with another inducer,
3,4-benzo[ a]pyrene, enhanced 3-hexanol formation (Frommer et al.,
1974; Näslund & Halpert, 1984). However, phenobarbital pre-
treatment of rats had no effect on n-hexane metabolism by lung
microsomes (Näslund & Halpert, 1984). Phenobarbital pre-treatment
of guinea-pigs increased the metabolism of n-hexane to 2-hexanol
and 2,5-hexanedione by hepatic post-mitochondrial supernatant, and
increased the metabolism of 2-hexanone to 2,5-hexanedione by the
hepatic microsomal fraction (Couri et al., 1978).
Intraperitoneal injection of phthalate esters to rats affects
the metabolism of n-hexane in vitro, presumably by induction of
cytochrome P-450 (Walseth et al., 1982). Treatment with
dimethylphthalate or dibutylphthalate increased significantly the
rate of formation of 2- and 3-hexanol by liver microsomes but
decreased slightly their formation by lung microsomes. A large
increase in 2,5-hexanedione formation by both types of microsomes
was noted following exposure to di(2-ethylhexyl)phthalate. Similar
results have been reported with inhalation exposures to
dibutylphthalate (Walseth & Nilsen, 1984). Male Sprague-Dawley
rats were exposed continuously for 5 days to atmospheric
dibutylphthalate concentrations of 5.7, 28.5, and 79.7 mg/m3 (0.5,
2.5, and 7 ppm), and liver and lung microsomal fractions were
prepared. The in vitro formation of n-hexane metabolites was
assayed by incubating 50 ml of 8% (v/v) n-hexane in sodium
phosphate buffer with 1 mg lung or liver microsomal protein for 10
min. The formation of 1-, 2-, and 3-hexanol was markedly increased
with liver microsomes but not with lung microsomes. At the
intermediate and high exposure concentrations of 28.5 and 79.7
mg/m3 (2.5 and 7 ppm), there was a decrease in in vitro n-hexane
metabolism particularly in lung microsomes.
The formation of 1-, 2-, and 3-hexanol from n-hexane was
significantly increased with kidney and liver microsomes obtained
from rats previously exposed to isopropanol (Zahlsen et al., 1985).
Enhanced formation of 2- and 3-hexanol (greater in male than in
female rats), but not of 1-hexanol, occurred when n-hexane was
incubated with liver microsomes from rats previously exposed to
xylene (mixed isomers) (Toftgard et al., 1983).
Treatment of rats with chloramphenicol (100 mg/kg
intraperitoneal or intravenous) inhibited 2- and 3-hexanol
formation in vitro by lung and liver microsomes derived from these
animals (Näslund & Halpert, 1984).
Toluene has been shown to be a non-competitive inhibitor of
n-hexane metabolism in in vitro studies with rat liver preparations
(Perbellini et al., 1982b). Exposure of rats to mixtures of
n-hexane and toluene resulted in reduced urinary excretion of
n-hexane metabolites (Perbellini et al., 1982b; Iwata et al.,
1983b, 1984). However, no effect on blood n-hexane levels was
reported when rats were given an intraperitoneal n-hexane injection
of 0.91 g/kg with or without 1.18 g toluene/kg (Suzuki et al.,
1974). In a study on the kinetics of 2,5-hexanedione (section
6.1.2), Ladefoged & Perbellini (1986) dosed six male rabbits
intravenously with 50 mg 2,5-hexanedione/kg with or without acetone
(150 mg/kg). A significant reduction of 2,5-hexanedione clearance
was caused by the acetone co-treatment.
Blood levels of 2,5-hexanedione were reduced after a single 6-h
co-exposure to n-hexane (3520 mg/m3, 1000 ppm) and methyl ethyl
ketone (2950 mg/m3, 1000 ppm), but not in animals pre-treated
orally with methyl ethyl ketone. However, pre-treatment resulted
in increases in liver cytochrome P-450 levels and in reactions
mediated by mixed-function oxidase (Robertson et al., 1982;
Robertson et al., 1989). Lower levels of 2,5-hexanedione have been
observed in the rat sciatic nerve after exposure to mixtures of
n-hexane and methyl ethyl ketone, but full data were not presented
(White & Bus, 1980).
In studies by Ralston et al. (1985), the blood and tissue
clearance of 2,5-hexanedione was measured after single or repeated
oral administration of 2,5-hexanedione or of a mixture of equimolar
doses of 2,5-hexanedione and methyl ethyl ketone. There was
reduced blood elimination of 2,5-hexanedione after administration
of the mixture only. The increased blood bioavailability
correlated with neurophysiological findings in co-exposed animals.
The results suggest that methyl ethyl ketone increases the
persistence of 2,5-hexanedione in the blood. Levels of
radiolabelled 2,5-hexanedione in neurofilament-enriched
preparations from the sciatic nerve and spinal cord were generally
unaffected by concurrent exposure to methyl ethyl ketone during the
first two weeks of treatment, and a trend towards decreased tissue
levels was evident after 3 weeks.
Reduced urinary excretion of n-hexane metabolites has been
reported in rats following exposure to mixtures of n-hexane and
methyl ethyl ketone (Perbellini et al., 1982b; Iwata et al., 1983b,
1984). The effect appeared to be transitory and no significant
effects remained after 33 weeks of co-exposure. In contrast,
increased urinary excretion of 2,5-hexanedione was observed in
guinea-pigs exposed to mixtures of methyl ethyl ketone and
2-hexanone (Couri et al., 1978).
6.2. Human Beings
The disposition and metabolism of n-hexane have been studied in
human beings following exposure by inhalation and skin contact.
Most studies were conducted on workers occupationally exposed to
commercial hexane and thus exposed to varying levels of
cyclohexane, 2-methylpentane, and 3-methylpentane, as well as
n-hexane; significant levels of methyl ethyl ketone and toluene
have also been recorded. Three experimental studies on human
volunteers presumably used pure n-hexane (Nomiyama & Nomiyama,
1974a,b; Ralston et al., 1985; Filser et al., 1987). n-Hexane is
absorbed following inhalation (Nomiyama & Nomiyama, 1974a,b;
Brugnone et al., 1978, 1980; Veulemans et al., 1982; Mutti et al.,
1984; Perbellini et al., 1985a). Steady-state pulmonary retention
(calculated by measuring the percentage of hexane in inhaled and
expired air) was in the region of 15 - 30%, and there was no
evidence of saturation at concentrations of up to 704 mg/m3 (200
ppm). Pulmonary retention was greater in more obese individuals,
and, although the alveolar uptake rate decreased during physical
exercise, the total uptake of n-hexane increased slightly as a
result of the higher lung ventilation rate. A net lung uptake of
112 mg in 8 h was reported in workers exposed to an n-hexane level
of 180 mg/m3 (51 ppm) (Perbellini et al., 1985a). Alveolar air
concentrations of n-hexane correlated with blood concentrations in
industrial workers exposed to commercial hexane (Brugnone et al.,
1984).
Some n-hexane is exhaled following cessation of exposure
(Nomiyama & Nomiyama, 1974b), and it has been suggested that this
amounts to about 10% of the net amount absorbed (Mutti et al.,
1984). Elimination was rapid and biphasic, with half-lives of 0.2
and 1.7 h. Steady-state levels of n-hexane in blood were linearly
dose dependent following inhalation of up to 704 mg/m3 (200 ppm)
(Veulemans et al., 1982). Near-plateau levels were obtained within
15 min, both in resting volunteers and in those undergoing physical
exercise. Following the end of exposure, elimination of n-hexane
from blood was rapid and biphasic; two half-lives were obtained,
one of approximately 0.2 h and the other of 1.5 - 2 h.
The percutaneous absorption of n-hexane in humans has not been
well studied, although this route of exposure has been implicated
in case reports of peripheral neuropathy (Nomiyama et al., 1973;
Takahashi et al., 1977). In a limited study, no hexane was
detected in the blood or exhaled air of a volunteer who immersed
one hand in n-hexane for 1 min (Nomiyama & Nomiyama, 1975).
Filser et al. (1987) measured an n-hexane metabolic clearance
of 2.2 litres/min at a concentration of approximately 3.52 mg/m3
(1 ppm) (steady-state concentration), and n-hexane accumulated to a
factor of 2.3. At low concentrations, the clearance of n-hexane
was not limited by saturation of metabolism but rather by the rate
of transport by the blood to the metabolic system. n-Hexane
tissue/gas partition coefficients were determined in vitro using
tissue samples obtained from autopsy cases (Perbellini et al.,
1985b). Values for heart, muscle, brain, kidney, and liver ranged
from 2.8 to 5.2, whereas the fat/air partition coefficient was 104.
The blood/air partition coefficient was 0.8. A half-life of 64 h
for n-hexane in fat has been calculated from a mathematical model
of hexane distribution (Perbellini et al., 1986).
The metabolism of n-hexane in humans is qualitatively similar
to that in the rat. 2,5-Hexanedione, 2,5-dimethylfuran, gamma-
valerolactone, and small amounts of 2-hexanol have been identified
in urine samples from workers exposed to n-hexane (Perbellini &
Brugnone, 1980; Perbellini et al., 1981a,b,c,d; Iwata et al.,
1983b; Mutti et al., 1984). All these compounds were present as
conjugates, together with some free 2,5-hexanedione and
2,5-dimethylfuran (Perbellini & Brugnone, 1980; Perbellini et al.,
1981c). The total amounts of these compounds in urine accounted
for only 15% of the estimated uptake of n-hexane (Mutti et al.,
1984). Fedtke & Bolt (1987b) have also identified 4,5-dihydroxy-2-
hexanone as a major metabolite in the urine of a male volunteer
exposed to n-hexane at a level of 764 mg/m3 (217 ppm) for 4 h.
These authors pointed out that the acid hydrolysis commonly used in
urine analysis may lead to the actual production of 2,5-hexanedione
and 2,5-dimethylfuran from 4,5-dihydroxy-2-hexanone glucuronide.
This finding needs to be taken into account for the measurement of
urinary 2,5-hexanedione in biological monitoring.
Radiolabelled carbon dioxide has been found in the exhaled air
of volunteers after an oral dose of 1-14C-2-hexanone (DiVincenzo et
al., 1978). As 2-hexanone is a metabolite of n-hexane, the latter
may also be converted to carbon dioxide and exhaled.
The end-of-shift urine concentration of n-hexane correlates
strongly with the time-weighted average (TWA) n-hexane air
concentration. Imbriani et al. (1984a,b) reported that 2 h after
exposure had ended, urinary concentrations of n-hexane were reduced
to trace levels. 2,5-Hexanedione was detected in the urine
following exposure to a time-weighted average concentration of
n-hexane in air of more than 53 mg/m3 (15 ppm) (Iwata et al.,
1983b). The end-of-shift urine concentration of 2,5-hexanedione
showed a positive correlation with both the n-hexane time-weighted
average concentration and end-of-shift blood levels of n-hexane
(Perbellini et al., 1981b, 1985a; Iwata et al., 1983b; Mutti et
al., 1984; Ahonen & Schimberg, 1988). A mean half-life of 13 to
14 h for urinary excretion of 2,5-hexanedione has been reported
(Mutti et al., 1980; Perbellini et al., 1985a). Urinary excretion
of 2,5-hexanedione was greatest 3 - 5 h after a shift. The end-of-
shift level was similar to that of the next morning and was highest
at the end of the working week. This finding, and the high
partition coefficient and calculated long half-life of n-hexane in
fat, led to the conclusion that n-hexane may accumulate in the
human body. The level of urinary 2,5-hexanedione excretion was
also positively correlated with the airborne concentration of
methyl ethyl ketone. However, it is likely that the individuals
with the highest exposure to methyl ethyl ketone also had the
highest n-hexane exposure, so that the increased urinary
2,5-hexanedione may not be the result of an effect of methyl ethyl
ketone on n-hexane metabolism. De Rosa et al. (1988) examined 20
workers exposed to n-hexane and other solvents during glueing
operations in shoe factories. The end-of-shift concentrations of
2,5-hexanedione in the urine correlated (r = 0.87) with the 8-h
time-weighted average n-hexane exposure (measured as four
sequential 2-h samples). A biological exposure index of 4.21
mg/litre was obtained for urine collection on a Thursday, but it
was indicated that the index may vary depending on the day of
analysis. Ahonen & Schimberg (1988) estimated a reference value
for 2,5-hexanedione of 10 µmol/litre (approximately 0.7 mmol/mol
creatinine), corresponding to an 8-h time-weighted average
n-hexane concentration of 176 mg/m3 (50 ppm) on the sampling day.
This value of 2,5-hexanedione represents the difference between the
post-shift and pre-shift samples.
Governa et al. (1987) detected 2,5-hexanedione and gamma-
valerolactone in the urine of all of 40 shoe factory workers
exposed to n-hexane. 2,5-Hexanedione was the major metabolite in
39 of the 40 cases. 2-Hexanol was found in 11 cases and in one
case a low level of 2-methyl-2-pentanol was detected. The level of
2,5-hexanedione and gamma-valerolactone gave a statistically
significant correlation with concomitant electroneuromyographic
changes characteristic of neuropathy and the former metabolite was
considered a suitable predictive measurement.
Ghittori et al. (1987) found that the urinary concentration
(Cu) of n-hexane in exposed workers could be expressed by the
following equation:
Cu = (0.05 x Ca) + 3.97
where Ca is the time-weighted average environmental air
concentration. The 4-h exposure urinary concentration value in
workers exposed to a time-weighted average concentration of 180
mg/m3 was 13 µg/litre, whereas the ACGIH biological equivalent
exposure limit is 9 µg/litre.
2,5-Hexanedione has also be detected in the urine (0.45
(± 0.20) mg/litre) of people apparently not exposed to n-hexane.
It was speculated that n-hexane may be produced in the body via
lipid peroxidation (Fedtke & Bolt, 1986a).
7. EFFECTS ON ORGANISMS IN THE ENVIRONMENT
There is a lack of information on the effects of n-hexane on
organisms in the environment. Sax (1984) reported that > 1000
mg/litre is required to kill 50% of exposed "aquatic organisms",
but gave no details of species or exposure. Bringmann & Kühn
(1982) gave an LC50 of > 1000 mg/litre for the waterflea Daphnia
magna. This was a static test with no measurement of actual
exposure concentration. Bobra et al. (1983) reported an LC50 for
Daphnia magna of 3.88 mg/litre, but the test was conducted over
48 h in a vessel closed with an airtight cap. However, it is
unlikely that organisms in the natural environment would ever be
exposed to n-hexane continuously over this period, because the
highly volatile n-hexane with its low water solubility would
rapidly be lost from water. Juhnke & Ludemann (1978) reported LC50
values for golden orfe (Leuciscus melanotus) of 150 - 4480
mg/litre. It appears that this was a static test without
measurement of actual exposure; the test was reported to have
lasted 48 h with "continuous aeration", and results, therefore,
should be treated with caution. Stratton & Smith (1988)
demonstrated a 50% reduction in the growth of a culture of the
green alga Chlorella pyrenoidosa in the presence of hexane at 2.66%
(v/v) (confidence limits 1.97 - 3.35%). Hexane had a knock-down
effect on the house fly (Musca domestica L.) of 7, 20, 47, and 87%
2 h after a topical application of 0.25, 0.5, 1.9, and 2 µl/insect,
respectively (Singh & Jain, 1987). The respective mortality rates
after 5 h were 0, 20, 60, and 80% and at 24 h were 0, 13, 53, and
87%.
8. EFFECTS ON EXPERIMENTAL ANIMALS AND IN VITRO TEST SYSTEMS
8.1. Single Exposures
8.1.1. Inhalation exposure
A 1-h LC50 value of 271 040 mg/m3 (77 000 ppm) has been
reported for adult Fischer male rats (Pryor et al., 1982). A 4-h
LC50 value for hexane of 260 480 mg/m3 (74 000 ppm) was reported in
Long-Evans male rats (Hine & Zuidema, 1970), but it was later
estimated that the concentration of n-hexane in the test material
was probably only about 45% (Delbrück & Kluge, 1982). In mice
(strain unspecified) exposed to pure n-hexane for 2 h, 100 mg per
litre was reported to be the minimum lethal concentration (Lazarev,
1929). Anaesthesia occurred in Swiss mice within 1 min of exposure
to an n-hexane (99% pure) concentration of 225 280 mg/m3 (64 000
ppm) and respiratory arrest occurred within 4.5 min (Swann et al.,
1974). In mice (strain unspecified) exposed to hexane, deaths
occurred at concentrations above 133 760 mg/m3 (38 000 ppm),
normally preceded by loss of postural reflexes. The toxicity of
inhaled hexane, as measured by its lethality, increased with
elevation of temperature from 20 °C to 35 °C (Babanov et al.,
1988).
Male Fischer rats exposed by inhalation to n-hexane showed
myoclonic seizures and ataxia at concentrations above 168 960 mg/m3
(48 000 ppm) (Pryor et al., 1982). Sprague-Dawley rats showed
ataxia and decreased motor activity after 25 - 30 min exposure to
concentrations between 302 720 and 316 800 mg/m3 (86 000 and 90 000
ppm) (Honma, 1983). Sedation, hypothermia, and ptosis followed
exposure of male Sprague-Dawley rats to 7040, 14 080, and 28 160 mg
n-hexane/m3 (2000, 4000, and 8000 ppm) for 8 h (Raje et al.,
1984). No acute behavioural effects were noted in rats exposed to
84 480 mg/m3 (24 000 ppm) for 10 min (Pryor et al., 1982). In
Swiss mice, light anaesthesia followed exposure to 56 320 mg/m3
(16 000 ppm) for 5 min, and deep anaesthesia, with periods of
apnoea, occurred during exposure to 112 640 mg/m3 (32 000 ppm) for
the same length of time (Swann et al., 1974).
Histopathological effects on the lung (lamellar inclusions in
type II pneumocytes) were observed in Wistar rats exposed to an
n-hexane (96 - 99% pure) level of 35 200 mg/m3 (10 000 ppm) for 4
or 8 h or to 24 640 mg/m3 (7000 ppm) for 8 h (Schnoy et al., 1982;
Schmidt et al., 1984). Light and electron microscopy revealed no
effects on intrapulmonary nerves.
Testicular lesions, characterized by degeneration of primary
spermatocytes and mild exfoliation of spermatids, were reported in
a study on Sprague-Dawley rats exposed to 17 600 mg/m3 (5000 ppm)
(99% pure) for 24 h (DeMartino et al., 1987). There were no deaths
or other manifestations of toxicity. Complete recovery had taken
place within 30 days after exposure.
8.1.2. Oral administration
Using a one-week observation period, the LD50 values for
analytical grade n-hexane were estimated to be 28.7 g/kg for older
adult (300 - 470 g) male Sprague-Dawley rats, 32.4 g/kg for young
adult males (80 - 160 g), 15.8 g/kg for 14-day-old rats, and less
than 0.7 g/kg for newborn rats (Kimura et al., 1971).
8.1.3. Dermal exposure
Signs of discomfort and incoordination, but no deaths, were
noted in New Zealand male rabbits dermally exposed under an
occlusive dressing for 4 h to 3 g hexane (45% n-hexane) per kg
(Hine & Zuidema, 1970).
8.1.4. Parenteral administration
Following intraperitoneal injection of 3.6 g n-hexane
(analytical grade) per kg, 8 out of 10 guinea-pigs died within 24 h
(Wahlberg & Boman, 1979). The approximate lethal intraperitoneal
doses of hexane were reported to be 530 mg/kg at an environmental
temperature of 36 °C, 4000 mg/kg at 8 °C, and 9100 mg/kg at 26 °C
in male and female albino Sprague-Dawley rats (Keplinger et al.,
1959). All of 10 male NMRI mice died following slow intravenous
infusion of n-hexane (purity not stated, 830 mg/kg) in a lipid
emulsion. A dose of 750 mg/kg was estimated to be the ED50 value
for loss of righting reflex (Jeppsson, 1975).
8.1.5. In vitro exposure
Gillies et al. (1980) investigated whether or not the
inhibition of sterologenesis induced by chronic ingestion of
2,5-hexanedione could also be induced in vitro. Sterologenesis
was not inhibited in sciatic nerves of rats incubated with
2,5-hexanedione (1 mmol/litre for 3 h). Although it has been shown
that 2,5-hexanedione and other compounds that cause distal
axonopathy inhibit cholesterol biosynthesis, Gillies et al. (1980)
proposed that the neurotoxicity of 2,5-hexanedione is related to
the inhibition of the biosynthesis of ubiquinone rather than that
of cholesterol. Perfusion of isolated New Zealand rabbit hearts
with an n-hexane (laboratory grade) solution of 9.6 mg/litre for
1 h reduced the force of cardiac contraction, but no effects on
heart rate or coronary blood flow were reported. Histopathological
examination showed shrinkage of myocardial fibres (Raje, 1983). The
dose for 50% inhibition (IC50) of cytochrome-P-450-dependent
benzyloxydealkylase activity by n-hexane in lung microsomes from
beta-naphthoflavone-treated rats was found to be 8.8 ± 3.2
µmol/litre (Rabovsky & Judy, 1989).
Decreased oxygen consumption at n-hexane (purity not specified)
plate concentrations of 40 µg/mg or more was reported following
incubation (15 - 38 °C) of rabbit cardiac mitochondria with 0 - 160
µg n-hexane/mg mitochondrial protein (Borgatti et al., 1981).
Adding 1.7 to 20 µl of n-hexane (pure) to a 500-µl suspension of
isolated human erythrocytes resulted in a dose-dependent increase
of specific binding to insulin. In the extracellular medium a
simultaneous increase in the amount of degraded insulin, dependent
on the concentration of n-hexane, was recorded. n-Hexane may
affect the availability of membrane receptors for hormones (Svabova
et al., 1987).
In studies by Notten & Henderson (1975a), male guinea-pigs were
given daily ip injections of n-hexane (purity not specified) of 3
or 60 mg/kg per day in sesame oil for 3 days and killed on day 4.
The livers were removed for homogenization, and the mitochondrial
supernatant was incubated with 0.1 or 0.5% (v/v) n-hexane (purity
not specified). With preparations from animals treated with 60
mg/kg per day, there was a dose-dependent increase in uridine 5'-
diphosphate (UDP) glucuronidation of p-nitrophenol, and a slight
decrease in p-hydroxylation of aniline, but no effect on
N-demethylation of aminopyrine. There was no effect on UDP
glucuronidation of p-nitrophenol when a guinea-pig microsomal
preparation, pre-activated with Triton X-100, was used (Notten &
Henderson, 1975a). In hepatic microsomal preparations (from
untreated guinea-pigs) incubated with up to 7% (v/v) n-hexane
(purity not specified) for 20 min, glucuronidation of
p-nitrophenol and o-aminophenol was increased. Aniline
p-hydroxylation was increased at n-hexane concentrations below
0.1%. N-demethylation of aminopyrine was reduced in a dose-related
manner at n-hexane concentrations above 0.5% (Notten & Henderson,
1975b).
In male Wistar rat hepatic microsome preparations incubated
with n-hexane (analytical grade) at concentrations of 0.44, 1.11,
or 2.21 mol/litre for 15 min, there was an increase in
UDP-glucuronosyl-transferase activity only at 2.21 mol/litre
(Vainio, 1974). Rabovsky et al. (1986) investigated the effect of
n-hexane (2 mmol per litre) on the activities of benzo[ a]pyrene
hydroxylase and 7-ethoxycoumarin de-ethylase in rat liver and lung
microsomes. The approximate extent of enzyme inhibition was 55%
and 30% in the case of benzopyrene hydroxylase and 50% and 35% in
the case of 7-ethoxycoumarin de-ethylase for liver and lung,
respectively. In rat hepatocytes there was a slight increase (14%)
in oxygen consumption and minimal leakage of aspartate
aminotransferase following incubation with n-hexane (20 mmol/litre)
for 1 h (Berger & Sozeri, 1987). At concentrations approaching the
solubility limit (about 0.08 mmol/litre), n-hexane selectively
stimulated the slow phase of cholesterol metabolism in isolated rat
adrenal mitochondria (McNamara & Jefcoate, 1988). This effect was
dependent on mitochondrial integrity and is apparently due to
facilitation of the transfer of cholesterol from the outer membrane
to the inner membrane where the responsible enzymes reside.
8.2. Short- and Long-Term Exposures
8.2.1. Inhalation studies
Combined groups of 10 weanling and 10 young adult male Fischer-
344 rats were exposed to an n-hexane (95% pure) concentration of
3520 mg/m3 (1000 ppm) for 24 h/day, 7 days/week, for 4 weeks and
then for 24 h/day, 6 days per week, for 7 weeks. There was reduced
weight gain, and 5 out of 10 adults died by week 14 (Howd et al.,
1983). When the same strain of rats was exposed for 24 h/day, 5
days per week, to an n-hexane (95% pure) level of 3520 mg/m3, there
was inhibition of body weight gain (Pryor et al., 1982). In both
studies, some recovery of body weight occurred after the end of
exposure. Body weight gain was reduced in Sprague-Dawley rats,
exposed for up to 6 months, 22 h/day, to an n-hexane (95% pure)
concentration of 1760 mg/m3 (500 ppm) (API, 1983a,b).
In Wistar rats exposed to 35 200 mg/m3 (10 000 ppm) n-hexane
(96 - 99% pure), 8 h/day, 7 days/week, for 15 or 19 weeks, there
was loss of body weight and some deaths occurred (Altenkirch et
al., 1978). The same results were reported for male Wistar rats
exposed to 10 563 mg/m3 (3000 ppm) n-hexane (99% pure) for 12
h/day, 7 days/week, for 16 weeks (Takeuchi et al., 1980). Reduced
body weight gain was noted in Wistar rats exposed 12 h/day for 16
weeks to 3520 mg/m3 (1000 ppm) n-hexane (99% pure) (Takeuchi et
al., 1981) and in Fischer-344 male weanling rats exposed 14 h/day,
7 days/week, for 14 weeks to n-hexane (95% pure) (Pryor et al.,
1983). A dose-related reduced weight gain was also reported in
male Wistar rats exposed to 704 or 1760 mg/m3 (200 or 500 ppm)
n-hexane (99% pure), 12 h/day, for 24 weeks (Ono et al., 1982). No
effect on body weight was noted in Sprague-Dawley rats exposed to
n-hexane (analytical grade) (443 mg/m3, 126 ppm), 21 h/day, 7
days/week, for up to 34 weeks (API, 1978), or in male Wistar rats
exposed to 373 mg/m3 (106 ppm) n-hexane, for 12 h/day, 7 days/week,
for 24 weeks (Takeuchi et al., 1983). No statistically significant
effects on body weight were reported in Wistar rats exposed to
concentrations of 352, 1056, or 3168 mg/m3 (100, 300, or 900 ppm)
n-hexane (99% pure) for 8 h/day, 5 days/week, for 72 weeks (De
Groot & Kepner, 1984).
8.2.1.1 Combined-exposure effects
The neuropathic effects of n-hexane on rats are enhanced by co-
exposure to methyl ethyl ketone. Clinical signs and histological
evidence of neuropathy developed earlier and were more severe in
male Wistar rats exposed to n-hexane (31 328 mg/m3, 8900 ppm) with
methyl ethyl ketone (MEK) (3250 mg/m3, 1100 ppm) for 8 h/day, 7
days per week, for 15 weeks, or 31 680 mg n-hexane/m3 (9000 ppm)
with 2950 mg MEK/m3 (1000 ppm) for 19 weeks than in those exposed
to 35 200 mg n-hexane/m3 (10 000 ppm) alone (Altenkirch et al.,
1978). In another study, male Wistar rats were exposed to 1760 or
2646 mg/m3 (500 or 700 ppm) n-hexane (purity not specified) or
n-hexane-MEK mixtures of 1056 mg/m3 (300 ppm) plus 590 mg/m3 (200
ppm), 1408 mg/m3 (400 ppm) plus 295 mg/m3 (100 ppm), or 1760 mg/m3
(500 ppm) plus 590 mg/m3 (Altenkirch et al., 1982). There was a
reduction in the time to onset of hindlimb paralysis in rats
exposed continuously to 1408 mg n-hexane/m3 plus 295 mg MEK/m3
compared with those exposed only to 1760 mg n-hexane/m3.
Hypersalivation occurred in rats exposed to the mixtures in both
studies. Reduced tail motor and mixed nerve conduction velocities
were reported in male Wistar rats after 20 weeks of exposure
(12 h/day) to 352 mg n-hexane/m3 (100 ppm) plus 590 mg MEK/m3 (200
ppm), but there were no toxicologically significant effects with
the same concentration of either chemical alone (Takeuchi et al.,
1983).
Methyl isobutyl ketone (MIBK) enhances n-hexane-induced
neurotoxicity in hens. Abou-Donia et al. (1985a) treated groups of
5 hens for 90 days with 3520 mg n-hexane/m3 (1000 ppm)
simultaneously with 0, 410, 1025, 2050, or 4100 mg MIBK/m3 (0, 100,
250, 500, or 1000 ppm). An untreated control group and a group
treated with MIBK (4100 mg/m3) alone were also included. The
treatments were followed by a 30-day observation period. n-Hexane
alone produced mild ataxia, while hens exposed to mixtures of
n-hexane and MIBK exhibited clinical signs of neurotoxicity
accompanied by large swollen axons and degeneration of the axon and
myelin of the spinal cord and peripheral nerves. The severity of
neurotoxicity depended on the dose of the non-neurotoxic agent
MIBK. The synergistic effect of MIBK may be related to its ability
to induce the cytochrome P-450 system responsible for the
metabolism of n-hexane to neurotoxic metabolites.
A marked increase in the clinical signs of impaired limb
function in male Wistar rats exposed to 2464 mg/m3 (700 ppm)
n-hexane (purity not specified), 23 h/day for 7 - 9 weeks, followed
pre-treatment with, and subsequent co-exposure to, lead acetate.
These effects did not occur with lead acetate alone (Wagner et al.,
1984).
n-Hexane-induced neurotoxicity can be reduced by co-exposure to
toluene. Effects on nerve conduction were less marked when rats
were exposed 12 h/day for 16 weeks to a mixture of 3696 mg/m3 (1050
ppm) n-hexane (99% pure) and 3940 mg toluene/m3 (1050 ppm) than
when they were exposed to 3696 mg n-hexane/m3 alone (Takeuchi et
al., 1981). On the other hand, Ikeda et al. (1986) showed that
co-administration of n-hexane and toluene may potentiate central
nervous system toxicity. Male Wistar rats were exposed to a
mixture of 704 mg n-hexane/m3 and 750 mg toluene/m3 (200 ppm each)
continuously for 30 days. The treatment produced a decrease in the
dopamine levels in the midbrain and hypothalamus and an increase in
the hippocampus. These changes were not found following exposure
to either solvent alone.
The effects of longer exposure (22 h/day, 7 days/week, for 6
months) to mixtures of n-hexane (1760 mg/m3, 500 ppm) and an equal
concentration of other hexane "isomers" (true isomers and related
6-carbon compounds) have been studied (Spencer, 1982; API,
1983a,b). The other hexane isomers appeared to have no effect on
the neuropathic activity of n-hexane in rats. A similar pattern of
neuropathy was seen as a result of exposure to 1760 mg n-hexane/m3
both with (at an equal concentration) or without other hexane
"isomers". There were no neuropathic changes following exposure to
440 mg n-hexane/m3 (125 ppm) either with (at 3 or 11 times the
n-hexane concentration) or without other hexane "isomers". Pellin
et al. (1988) investigated the possibility of an interactive effect
of n-hexane and organophosphorus compounds on neurotoxicity. Hens
that were pretreated with n-hexane (300 mg/kg per day ip for
7 - 15 days) showed a synergistic effect for the inhibition of
neuropathy target esterase (NTE) and acetyl- and butyryl-
cholinesterase induced by a single oral dose of tri- o-cresyl
phosphate (TOCP). At a lower dose of TOCP (20 mg/kg), which was
not neurotoxic, there was still an increase in NTE inhibition to
levels close to the minimum threshold associated with neuropathy.
8.2.1.2 Effects on the respiratory tract
In studies by Dunnick (1989) and Dunnick et al. (1989), B6C3F1
mice were exposed to 0, 1760, 3520, 14 080, and 35 200 mg
n-hexane/m3 (0, 500, 1000, 4000, and 10 000 ppm) for 6 h/day, 5
days/week, for 13 weeks, and also to 3520 mg/m3 for 22 h/day, 5
days/week, for 13 weeks. Body weight gain was reduced at 35 200
mg/m3 (6 h/day) and at 3520 mg/m3 (22 h/day). The final mean body
weights were 17% and 10% lower, respectively, for males and 6% and
0% lower, respectively, for females. Sneezing was also observed
at 35 200 mg/m3. Histopathological changes included mild
inflammatory, erosive, and regenerative lesions in the olfactory
and respiratory epithelium of the nasal cavity at 14 080 and 35 000
mg per 3 (6 h/day) and at 3520 mg/m3 (22 h/day).
In male Wistar rats exposed to n-hexane (96 - 99% pure) 8
h/day, at concentrations of 1760 mg/m3 (500 ppm) for 48 - 70 days,
2464 mg/m3 (700 ppm) for 7 days, or 35 200 mg/m3 (10 000 ppm) for
2 - 4 days, there were ultrastructural changes in the lungs. Light
microscopy did not reveal any confirmed pathological changes in the
lung tissue of animals that had been exposed over 4 - 24 h to
n-hexane or to mixtures of n-hexane and methyl ethyl ketone (MEK).
Cytoplasmic swelling and focal desquamation of alveolar epithelium
followed exposure over 2 - 7 days to 2464 - 35 200 mg/m3 (Schnoy et
al., 1982). Numerous fat-laden alveolar macrophages, degenerate
type 1 pneumocytes and increased numbers of alveolar brush cells
occurred after 7 days of exposure to 1760 mg/m3 (500 ppm). The
basic structures of alveoli and alveolar receptors were preserved.
The authors hypothesized that n-hexane alone or in combination with
methyl ethyl ketone caused fatty degeneration of the alveolar
epithelium due to interference with cellular metabolism. Under
identical test conditions axonal changes in pulmonary nerves were
reported (Schmidt et al., 1984). More pronounced effects,
including degenerative changes in the ciliary cells of small air
passages, followed exposure to mixtures of 1056 - 1760 mg
n-hexane/m3 (300 - 500 ppm) plus 295 - 590 mg MEK/m3 (100 - 200
ppm) for 24 - 89 days or 31 680 mg n-hexane/m3 (9000 ppm) plus 2950
mg MEK/m3 (1000 ppm) for 2 - 14 days. However, no treatment-related
lesions were observed in the lungs of rats (Wistar, Fischer-344,
CD) exposed to n-hexane by a variety of exposure patterns. These
included exposures to up to 35 200 mg/m3 (10 000 ppm) over 13 weeks
and 3168 mg/m3 (900 ppm) over 72 weeks (Kurita, 1974; Toxigenics,
1982; API, 1983a,b; De Groot & Kepner, 1984).
In New Zealand rabbits, severe respiratory tract lesions
followed exposures to an n-hexane (research grade) concentration of
10 560 mg/m3 (3000 ppm), 8 h/day, for 8 days (Lungarella et al.,
1980, 1984; Barni-Comparini et al., 1982). Centriacinar emphysema
and scattered microhaemorrhages were observed, lung damage being
most marked at the transition zone between the terminal bronchioles
and alveolar ducts. Focal subpleural atelectasis, and alveolar and
interstitial oedema were also observed. In another study
(Lungarella et al., 1984), 24 New Zealand male rabbits were exposed
to 10 560 mg/m3, 8 h/day, 5 days/week, for 24 weeks. Clinical
signs of ocular and upper respiratory tract irritation and
difficulty in respiration were reported, but there was no evidence
of neurotoxicity. Animals sacrificed 1 day after the last exposure
showed inflammation of the nasal turbinates and necrotic erosion of
the nasal mucosa. The lungs showed centriacinar emphysema,
scattered foci of pulmonary fibrosis, and papillary proliferations
of non-ciliated bronchiolar cells (described by the authors as
papillary tumours). In a group of rabbits retained following the
end of exposure for a further 120 days, irregular foci of cellular
proliferation, papillary proliferations of non-ciliated bronchiolar
epithelium, scattered pulmonary fibrosis, and centriacinar
emphysema were still present.
Dose-related biochemical changes, indicative of increased
pulmonary secretion and cell damage in the lungs, were reported in
lung lavage fluid of male Sprague-Dawley rats exposed to n-hexane
(purity not stated) concentrations of 1690, 4048, or 5833 mg/m3
(480, 1150, or 1657 ppm) for 6 h/day, 5 days/week, for 4 weeks
(Sahu et al., 1982). There were dose-related increases in protein,
lipid, and sialic content and in acid and alkaline phosphatase,
lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and
angiotensin-converting enzyme activities. Altered enzyme activity
was reported in the lungs of New Zealand rabbits exposed to 10 560
mg n-hexane/m3 (3000 ppm) 8 h/day for 8 days (Barni-Comparini et
al., 1982). The activities of acid phosphatase, beta-
glucuronidase, lactate dehydrogenase, and glucose 6-phosphate
dehydrogenase were significantly increased.
In a study by Hadjiivanova et al. (1987), male Wistar rats were
treated with n-hexane by inhalation at a concentration of 14 995
mg/m3 (4260 ppm, 5 h/day) and pulmonary surfactant was examined at
1 and 15 days. There was a treatment-dependent moderate increase
in the phospholipids of the bronchoalveolar lavage, and an altered
relative concentration of individual phospholipids in lung tissue
homogenate. In combination with irradiation, n-hexane treatment
depleted lung tissue phospholipids due to their release in the
alveoli.
8.2.1.3 Effects on the testes
When Sprague-Dawley rats were exposed to 17 600 mg n-hexane/m3
(5000 ppm) for 16 h/day, 6 days/week, up to the development of
clear symptoms of polyneuropathy, damage to the germinal epithelium
increased with increasing exposure. Early signs were abnormalities
in primary spermatocytes including vacuolation of the cytoplasm and
nuclear pycnosis. Maturing spermatids were also affected and
showed swollen cytoplasm and multinucleated heads with vacuolation.
Sertoli cells revealed vacuolation of the cytoplasm, primarily in
the basal region, and retraction of the apical cytoplasm. Shedding
of damaged spermatocytes and spermatids into the lumen of the
tubule and their appearance in the epididymis were also reported.
Progressive damage continued after cessation of dosing, leading to
tubules devoid of all germinal cells, with the exception of some
spermatogonia, and containing only damaged Sertoli cells. The
authors could not determine whether the primary effect was on the
Sertoli cell, the germinal cells, or both. Signs of testicular
damage were clear before neuropathic symptoms developed in the rats
(DeMartino et al., 1987).
These results are comparable to testicular effects reported
after dosing orally with 2,5-hexanedione, a metabolite of
n-hexane, which is the probable causative agent for these effects
(see section 8.7). A decrease in relative testis weight was
reported in adult and weanling Fischer-344 rats 5 weeks after the
end of exposure to 3520 mg/m3 (1000 ppm) n-hexane (95% pure),
24 h/day, 7 days per week, for 4 weeks followed by the same
concentration, 24 h/day, 6 days/week, for a further 7 weeks; the
testes were not examined histologically (Howd et al., 1983). No
testicular lesions were reported in Wistar or CD rats exposed for
up to 6 months to 1760 mg/m3 (500 ppm) or 18 months to 3168 mg/m3
(900 ppm) n-hexane (99% pure) (API, 1983a,b; De Groot & Kepner,
1984). There was slight congestion in the testes of Wistar rats
following inhalation of 2992 mg/m3 (850 ppm) n-hexane (purity not
specified), 6 days/week, for 20 weeks (Kurita, 1974). There were
no testicular lesions in Fischer-344 rats exposed to 10 560,
22 880, or 35 200 mg/m3 (3000, 6500, or 10 000 ppm) n-hexane (99.5%
pure), 6 h/day, 5 days/week, for 13 weeks (Toxigenics, 1982;
Cavender et al., 1984). However, adult male Sprague-Dawley rats
continuously exposed to n-hexane for 61 days at 3520 mg/m3 (1000
ppm) showed lesions of the tubule. All germinal cells were
progressively lost leaving Sertoli cells (damaged) as the only
component of the tubule. There was no impairment of androgen
synthesis, and circulating androgen levels were not different from
controls. Simultaneous administration of 3520 mg n-hexane/m3 with
4340 mg xylene/m3 (1000 ppm) caused no adverse effects on the
testis (Nylen et al., 1989).
8.2.1.4 Other effects
Panlobular necrosis occurred in the livers of some male CD rats
exposed to n-hexane (99% pure) concentrations of 440 or 1760 mg/m3
(125 or 500 ppm) 22 h/day, 7 days/week, for up to 6 months, and
relative liver weight was increased at 1760 mg/m3 (API, 1983a).
However, no hepatic effects were reported at 1760 mg/m3 in a
further study (API, 1983b). Increased relative liver weight and
increases in hepatic microsomal protein, cytochrome P-450 and
cytochrome b5 levels have been reported in NMRI mice exposed to
88 000 to 105 600 mg/m3 (25 000 to 30 000 ppm) n-hexane (analytical
grade), 23 h/day, for up to 4 days; no histological examination was
performed (Krämer et al., 1974). Howd et al. (1983) found an
increase in relative liver weight in Fischer-344 rats exposed to
3520 mg/m3 (1000 ppm) n-hexane (95% pure), 24 h/day for 4 weeks
followed by 24 h/day (6 days/week) for 7 weeks.
Increased relative kidney weights were reported in two studies
on male CD rats exposed to 1760 mg/m3 (500 ppm) n-hexane (99% pure)
continuously for up to 6 months (API, 1983a,b). In one study,
there were traces of degenerative and regenerative changes in the
kidneys of 4/34 exposed rats (API, 1983a), but in the other no
renal lesions were reported (API, 1983b). In male Fischer-344 rats
exposed to 10 560, 22 880, or 35 200 mg/m3 (3000, 6500, or 10 000
ppm) n-hexane (99.5% pure), 6 h/day, 5 days/week, for 13 weeks,
relative kidney weights were increased in rats exposed to 22 880
and 35 200 mg/m3 and there was a reduction in urinary pH at 35 200
mg/m3, but no treatment-related renal lesions were observed
(Cavender et al., 1984).
A slight increase in giant cell numbers and haemosiderin
precipitation was reported in the spleens of male Wistar rats
exposed to 2992 mg/m3 (850 ppm) n-hexane (purity not stated)
continuously, 6 days/week, for 20 weeks (Kurita, 1974).
No significant haematological changes followed exposure to
n-hexane in studies on Fischer-344, Wistar, and CD rats and New
Zealand rabbits (Kurita, 1974; API, 1978; Rebert et al., 1982;
Cavender et al., 1984; Lungarella et al., 1984). Slight reductions
in blood haemoglobin and in red cell and total white cell counts,
and an increase in immature cells in the bone marrow were reported
in guinea-pigs exposed by inhalation to n-hexane of unstated purity
(Spagna et al., 1967). Exposures were to 4928 mg/m3 (1400 ppm), 2
h/day for 120 days, 29 920 mg/m3 (8500 ppm), 2 h/day for 60 days,
or to 149 952 mg/m3 (42 600 ppm), 2 h/day for 30 days. The
haematological effects occurred at 149 952 mg/m3 and, to a lesser
extent, at 29 920 mg/m3 but not at 4928 mg/m3.
In other studies on Fischer-344, Wistar, and CD rats, no
treatment-related lesions were reported in the spleen (or other
lymphoid organs) or salivary glands following exposures to 1760
mg/m3 (500 ppm) for 6 months, 3168 mg/m3 (900 ppm) for 72 weeks, or
35 200 mg/m3 (10 000 ppm) for 13 weeks (Toxigenics, 1982; API,
1983a,b; Cavender et al., 1984; De Groot & Kepner, 1984).
In Wistar rats exposed to n-hexane (purity not stated)
concentrations of 1760 - 2464 mg/m3 (500 - 700 ppm), 22 h/day, 7
days/week, for up to 9 weeks, there was fatty degeneration and
glandular duct widening of the parotid and salivary glands
(Altenkirch et al., 1982). The effects were more noticeable in the
rats exposed to mixtures of n-hexane and methyl ethyl ketone, and
were associated with hypersalivation.
8.2.2. Oral studies
Body weight gain was reduced in male CD rats given (by gavage)
n-hexane (99% pure) doses of 570 or 1140 mg/kg per day, 5
days/week, for 13 weeks, 4000 mg/kg per day for 17 weeks, or 4000
mg/kg per day technical grade hexane (40% n-hexane), 5 days/week,
for 13 weeks or until hindlimb paralysis was observed. Severe
hindlimb weakness or paralysis (from about day 100), tibial nerve
lesions, and atrophy of testicular germinal epithethelium were
reported in the rats given 4000 mg n-hexane/kg per day (Krasavage
et al., 1980). The tibial nerve lesions (also found in one rat
exposed to the technical grade hexane) included axonal swellings,
adaxonal myelin infolding, and paranodal myelin retraction. A
reduction in tail nerve conduction velocity was reported in male
Wistar rats given daily doses of n-hexane (purity not stated) for 4
weeks (Ono et al., 1979). Doses of 0.5 or 1 g/kg per day in olive
oil were given daily by gavage for 4 weeks and the rats were
observed for a further 4 weeks. At 8 weeks both groups showed
reduced motor and mixed nerve conduction velocities.
A slight reduction in body weight gain (but no clinical or
pathological signs of neurotoxicity or other gross pathological
effects) was reported in male CD rats given a solution (less than
1%) of commercial hexane containing 40% n-hexane as drinking-water
for 10 months (O'Donaghue et al., 1978a; Krasavage et al., 1979).
In a toxicity study in rats, five groups of 30 male and 30
female Wistar rats each received daily doses of 0, 0.04, 0.2, 1, or
5 g commercial hexane/kg body weight by gavage for 13 weeks.
Increased relative kidney weights were observed in the rats that
received 0.2, 1, or 5 g/kg, and histopathological changes occurred
in the kidneys of both male and female rats that received 5 g/kg.
Rats given 0.2, 1, or 5 g/kg also showed increased liver weights.
Changes in plasma enzymes, indicative of liver damage and elevated
cholesterol and triglyceride levels, were detected in the highest-
dose group. Also in this group, histopathological changes were
detected in the adrenals, liver, kidneys, peripheral nerves,
spleen, testes, and thymus. It was concluded that the no-observed-
effect level of technical hexane is 0.04 g/kg body weight per day
but treatment-related effects at the adjacent dose level (0.2 g/kg
per day) were slight and occurred in male rats only (Til et al.,
1989).
8.2.3. Dermal studies
No deaths and no effects on weight gain occurred in guinea-pigs
for up to one month following dermal application (sealed chamber)
of 3.5 g n-hexane (analytical grade) per kg for one week (Wahlberg
& Boman, 1979).
Abou-Donia et al. (1985b) investigated the neurotoxicity of
n-hexane, 2,5-hexanediol, and 2,5-hexanedione in hens with and
without 0-ethyl- 0-4-nitrophenyl phenylphosphonothioate (EPN).
Following a daily dermal application of n-hexane (1 mmol/kg) for 90
days followed by a 30-day observation period, leg weakness was
observed. The other two aliphatic hexacarbons were more toxic at
this dose level causing gross ataxia. Concurrent dermal
application of EPN with n-hexane or 2,5-hexanediol at the same or
different sites produced an additive neurotoxic action with
histopathological changes characteristic of EPN neurotoxicity. The
additive effect of n-hexane and 2,5-hexanediol and a potentiating
effect of 2,5-hexanedione were considered to result from the
enhancing effect of the hexacarbons on EPN absorption and/or
metabolism.
8.2.4. In vitro studies
In mouse spinal cord/dorsal root ganglion/thigh-muscle explants
incubated with n-hexane (97% pure), axonal swellings, degeneration
of central and peripheral nerve fibre, and necrosis of muscle
fibres were reported (Veronesi et al., 1983, 1984). In explants
exposed to 25, 50, 80, 100, or 250 µg n-hexane/ml for up to 8
weeks, giant axonal swellings developed at 100 µg/ml or more and
appeared first in distal, paranodal regions of large diameter
fibres (Veronesi et al., 1984). Explants exposed to 50 - 650 µg/ml
for 3 - 8 weeks showed marked changes in nerve fibres at 400 - 650
µg/ml after 4 - 5 days and progressive axonal changes at 245 - 325
µg/ml after 2 - 6 weeks (Veronesi et al., 1983). In this study,
explants removed from exposure and maintained in plain nutrient
solution for 12 - 15 weeks showed some remyelination of viable
axons. Exposure of explants to n-hexane with non-cytotoxic levels
of methyl ethyl ketone potentiated the axonal effects. Cytoplasmic
bubbling and lysosome proliferation were reported in murine
neuroblastoma cells incubated with up to 1.5% n-hexane (purity not
stated) for up to 10 days (Selkoe et al., 1978).
8.2.5. Parenteral studies
n-Hexane administered subcutaneously to mice at doses of up to
10 ml/kg was found to decrease metallothionein concentrations in
the pancreas. The maximum effect was observed 24 h after
administration. Levels had returned to normal 48 h after
administration (Onosaka et al., 1988).
In male Donryu rats given subcutaneous n-hexane (97% pure)
injections of 330 mg/kg per day, 5 days/week, for 21 weeks, there
was reduced growth rate, reduced movement, disturbed gait, and
decreased amplitude of the tail nerve action potential, but no
effect on nerve conduction velocity (Misumi & Nagano, 1984). In
male Donryu rats injected subcutaneously with n-hexane (purity not
stated; 330 mg/kg per day, 5 days/week, for 5 months), gait
disturbances and decreased sweating response to beta-methylcholine
were reported in all animals (Abe et al., 1980). Peripheral nerve
lesions were reported in Sprague-Dawley rats following subcutaneous
injections of n-hexane (99% pure) (650 - 2000 mg/kg per day, 5
days/week) for up to 35 weeks (Schaumburg & Spencer, 1976). In
male Sprague-Dawley rats given daily intraperitoneal injections of
n-hexane (purity not stated; 540 mg/kg for 5 weeks), reductions in
peak conduction velocity and duration of action potentials in the
sciatic and sural nerves, in the absence of any behavioural or
histological effects, were reported (Anderson & Dunham, 1984).
The potential for n-hexane to affect the haemopoietic system
was indicated by the significant inhibition of uptake of iron by
the bone marrow in rats that had received 1 ml n-hexane/kg (ip) on
two successive days (Goel et al., 1987). The effects of the
metabolite 2,5-hexanediol on the spleen are discussed in section
8.7.
Hepatic necrosis occurred in male rats (strain not specified)
following intraperitoneal n-hexane (analytical grade) injections of
660 mg/kg per day daily for 2 or 7 days or twice weekly for 45 days
(Goel et al., 1982). The activities of serum acetylcholinesterase
and carboxyesterase and serum levels of protein, albumin, and
cholesterol were reduced in all groups.
When female albino rats were treated with n-hexane (1 ml/kg by
intraperitoneal injection) for 1, 2, 7, and 45 days, hepatotoxicity
was evident from a loss of total hepatic sulfhydryl content and
there was a significant increase in lipid peroxidation at 1 and 2
days. There was also a decrease in microsomal drug-metabolizing
activity and microsomal glucose-6-phosphatase activity (Goel et
al., 1988).
Sclerodermatous skin changes were observed in ddy mice after 17
daily intraperitoneal injections of 3.3 mg n-hexane in 1 ml of 0.9%
saline (Yamakage & Ishikawa, 1982). The purity of the n-hexane was
not stated and sclerotic skin changes also occurred in positive
control mice.
Severe lung lesions, including oedema, cellular infiltration,
abcesses, necrosis, fibrosis, and haemorrhage, were reported in
male rabbits given daily intramuscular n-hexane (purity not stated)
injections of 660 mg/kg undiluted for 5 days or 66 mg/kg in olive
oil for 21 - 77 days (Taira, 1975).
Klimes et al. (1987) investigated the effect of intraperitoneal
injections of n-hexane (1 ml/kg daily for 7 days) on the enzymic
degradation of insulin. Insulin degradation in the liver of
treated male Wistar rats was greater than in control rat liver.
However, there was inhibition of insulin degradation in the
erythrocytes of treated rats. There was, therefore, evidence for
some potential disturbance by n-hexane of glucose tolerance. Five
daily intraperitoneal doses of n-hexane (1 g/kg) per week for 2
weeks to female Sprague-Dawley rats (a dose equivalent to one tenth
of the intraperitoneal LD50) had no effect on kidney tubular
function as shown by measurements of urinary N-acetyl-beta-D-
glucosaminidase (NAG), beta2-microglobulin and albumin (Bernard et
al., 1989).
8.3. Reproduction, Embryotoxicity, and Teratogenicity
8.3.1. Teratogenicity studies
8.3.1.1 Inhalation studies
The exposure of groups of 7 - 9 pregnant Fischer-344 rats to
3520 mg/m3 (1000 ppm) n-hexane (purity unspecified) for 6 h/day on
days 8 - 12, 12 - 16, or 8 - 16 of gestation did not result in a
significant increase in resorption rate or in the incidence of
visceral or skeletal malformations (Bus & Tyl, 1979; Bus et al.,
1979). There were slight, but not statistically significant,
increases in minor anomalies such as dilated renal pelvis and
misaligned fourth sternebra in the offspring of all the exposure
groups. No signs of maternal toxicity were reported. Groups of
17 - 20 pregnant CD rats were exposed to 327 or 1436 mg/m3 (93 or
408 ppm) n-hexane (purity not stated) for 6 h/day on days 6 - 15 of
gestation (Litton Bionetics, 1979). There was no embryotoxicity or
increase in the incidence of malformations, but small, not
statistically significant, increases in the incidence of
subcutaneous haematomas and retarded bone ossification were
observed at both exposure levels.
A group of 14 pregnant Fischer-344 rats was exposed to 3520
mg/m3 (1000 ppm) n-hexane (99% pure) for 6 h/day on days 8 - 16 of
gestation and allowed to deliver naturally on day 23 (Bus & Tyl,
1979; Bus et al., 1979). Litters were culled to 6 pups/litter and
postnatal development was followed for 7 weeks with weaning at 4
weeks. There was no statistically significant difference between
the mean litter weights of the exposed and control groups.
However, a significant transient depression in mean litter weight
occurred over the first 6 weeks and was most marked at week 3.
There were no signs of neuropathy in the pups during the 7 weeks of
observation.
In a study on the offspring of Sprague-Dawley rats exposed to
352, 7040, or 35 200 mg n-hexane/m3 (100, 2000, or 10 000 ppm), 7
h/day, from 15 days prior to conception to day 18 of gestation, no
physical malformations or effects on postnatal growth rate or age
at eye opening were reported (Howell, 1979; Howell & Cooper, 1981).
Electrophysiological measurements of visual evoked response (VER)
and inter-hemisphere evoked response (IHR) were performed on
neonatal Sprague-Dawley rats from dams exposed to 0, 352, 7040, or
35 200 mg/m3 (0, 100, 2000, or 10 000 ppm) n-hexane (purity
unknown), 7 h/day, from 15 days prior to conception to day 18 of
gestation (Howell, 1979; Howell & Cooper, 1981). There were no
abnormalities in the VER and IHR at 11, 20, and 60 days. A
significant increase in the amplitude of the early VER peaks was
observed in unanaesthetized pups aged 45 days that had been exposed
in utero to 35 200 mg/m3. However, neither of the studies was
reported in sufficient detail for an assessment of the significance
of these observations to be made.
Exposure of pregnant rats (number and strain not specified) to
1760, 2815, or 5280 mg/m3 (500, 800, or 1500 ppm) n-hexane (purity
not stated) during gestation and lactation resulted in reduced
maternal weight gain, increased resorption rates, reduced pup
weight gain, and retardation of cerebellar histogenesis and
neuronal maturation (Stoltenburg-Didinger et al., 1984).
Peripheral neuropathy (motor weakness) was reported in the dams but
not in the pups. Similar results were obtained following exposure
to a mixture of 5280 mg n-hexane/m3 and 4425 mg MEK/m3 (1500 ppm)
or to MEK alone at concentrations of 2360 (800 ppm) and 4425 mg/m3.
8.3.1.2 Oral
In a study on CD-1 mice, dams were dosed with n-hexane (99%