United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA-60O/8-86-014A
September 1986
External Review Draft
Research and Development
Health Assessment
Document for
Acrolein
Review
Draft
(Do Not
Cite or Quote)
NOTICE
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
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(Do Not Cite or Quote) EPA-600/8-86-014A
September 1986
External Review Draft
Health Assessment Document
for Acrolein
NOTICE
i
This document is a preliminary drgft. It has not been formally released by the U.S. Environmental
Protection Agency and should not at this stage be construed to represent Agency policy. It is being
circulated for comment on its technical accuracy and policy implications.
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, North Carolina 27711
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DISCLAIMER
This document is an external draft for review purposes only and does not
constitute Agency policy. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
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CONTENTS
Page
LIST OF TABLES vi
LIST OF FIGURES vii
PREFACE '. viii
ABSTRACT ix
AUTHORS, CONTRIBUTORS, AND REVIEWERS x
1. HEALTH EFFECTS SUMMARY AND CONCLUSIONS 1-1
1.1 BACKGROUND INFORMATION 1-1
1.1.1 Properties 1-1
1.1.2 Production 1-1
1.1.3 Use 1-1
1.1.4 Envi ronmental Release 1-1
1.1.5 Envi ronmental Transport and Fate 1-2
1.1.6 Ecosystems and Aquatic Biota 1-2
1.2 MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION 1-2
1.3 MAMMALIAN TOXICITY 1-4
1.4 MUTAGENICITY 1-5
1.5 CARCINOGENICITY 1-6
1.6 REPRODUCTIVE AMD TERATOGENIC EFFECTS 1-6
1.7 REGULATIONS AND STANDARDS 1-6
1.8 CONCLUSIONS 1-7
1.9 RESEARCH NEEDS 1-7
2. INTRODUCTION 2-1
3. BACKGROUND INFORMATION 3-1
3.1 PHYSICAL AND CHEMICAL PROPERTIES 3-1
3.1.1 Synonyms 3-1
3:1.2 Identi fi cati on Numbers 3-1
3.1.3 Significance of Physical/Chemical Properties with
Respect to Environmental Behavior 3-1
3.1.4 Chemical Reactions in the Environment 3-3
3.2 ANALYTICAL METHODOLOGY 3-3
3.2.1 Chemical Analysis in Air 3-3
3.2.2 Chemical Analysis in Water 3-5
3.3 PRODUCTION, USE, AND RELEASES TO THE ENVIRONMENT 3-6
3.3.1 Production 3-6
3.3.2 Use 3-7
3.3.3 Environmental Release 3-8
3.3.3.1 Combustion 3-8
3.3.3.2 Cigarette Smoke 3-8
3.3.3.3 Food Processing 3-9
3.3.3.4 Production Processes 3-9
3.3.4 Environmental Occurrence 3-9
3.4 ENVIRONMENTAL TRANSPORT AND FATE 3-10
3.4.1 Transport 3-10
11 i
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CONTENTS (Continued)
3.4.2 Fate 3-11
3.4.2.1 Atmospheric Fate 3-11
3.4.2.2 Aquatic Fate 3-12
3.4.2.3 Terrestrial Fate 3-14
3.5 ECOSYSTEM CONSIDERATIONS 3-14
3.5.1 Introduction 3-14
3.6 AQUATIC ECOSYSTEMS 3-15
3.6.1 Aquatic Plants, Bacteria, and Algae 3-15
3.6.2 Aquatic Invertebrates 3-16 *
3.6.3 Fish 3-18
3.7 EFFECTS ON TERRESTRIAL LIFE 3-22
3.7.1 Terrestrial Plants '< 3-22 M
3.7.2 Terrestrial Animals 3-23
3.8 BIOCONCENTRATION, BIOACCUMULATION, AND BIOMAGNIFICATION .... 3-23
3.9 REFERENCES 3-24
4. ACROLEIN: MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION 4-1
4.1 INTRODUCTION 4-1
4.2 ABSORPTION 4-1
4.2.1 Oral 4-1
4.2.2 Dermal 4-3
4.2.3 Pulmonary 4-3
4.3 DISTRIBUTION AND EXCRETION 4-4
4.4 METABOLISM 4-5
4.4.1 Quantisation of Metabolism 4-5
4.4.2 Noncatalytic Interaction with Sulfhydryl Groups 4-7
4.4.3 Enzymatic Pathways 4-11
4.4.4 Covalent Binding 4-15
4.5 SUMMARY 4-21
4.6 REFERENCES 4-23
5. MAMMALIAN TOXICITY 5-1 f
5.1 ACUTE TOXICITY 5-1
5.1.1 Inhalation 5-1
5.1.2 Subcutaneous 5-3
5.1.3 Oral 5-4
5.1.4 Intraperitoneal 5-4
5.1.5 Dermal 5-4
5.1.6 Ocular 5-4
5.2 SUBCHRONIC TOXICITY 5-4
5.3 CHRONIC TOXICITY 5-7
5.4 EFFECTS ON THE LIVER, KIDNEYS, AND LUNGS 5-8
5.4.1 Liver 5-8
5.4.2 Kidneys ...: 5-9
5.4.3 Lungs 5-9
5.5 OTHER STUDIES , 5-11
5.5.1 Cardiovascular • 5-11
5.5.2 In Vitro Cytotoxicity 5-12
IV
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CONTENTS (continued)
5.6 EFFECTS ON HUMANS 5-12
5.7 SUMMARY 5-16
5.8 REFERENCES 5-19
6. MUTAGENICITY 6-1
6.1 GENE MUTATIONS 6-1
6.1.1 Salmonella 6-1
6.1.2 E. coli 6-3
6.1.3 YeastT 6-3
6.1.4 Drosophila 6-4
6.2 CHROMOSOMAL EFFECTS 6-4
6.3 SUMMARY OF MUTAGENIC EFFECTS 6-5
6.4 REFERENCES 6-7
7. CARCINOGENICITY 7-1
7.1 ANIMAL STUDIES 7-1
7.1.1 Subcutaneous Injection-Acrolein 7-1
7.1.2 Skin Application-Acrolein 7-2
7.1.3 Inhalation-Acrolein 7-2
7.1.4 Skin Painting Glycidaldehyde (Metabolite of
Acrol ei n) 7-2
7.1.5 Skin Painting and Subcutaneous Injection-
Glycidaldehyde 7-3
7.2 SUMMARY 7-4
7.3 CONCLUSION 7-5
7.4 REFERENCES 7-7
8. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS 8-1
8.1 IN VIVO STUDIES 8-1
8.2 TN VITRO CULTURE STUDIES 8-2
8.3 CONCLUSION ABOUT REPRODUCTIVE EFFECTS 8-4
8.4 REFERENCES ... 8-5
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LIST OF TABLES
Number Page
3-1 Physical and chemical properties of acrolein 3-2
3-2 Methods for acrolein measurement 3-6
3-3 Effects of acrolein on aquatic invertebrates 3-17
3-4 Effects of acrolein on fish 3-19
4-1 Xenobiotics biotransformed jn vivo to acrolein 4-2
4-2 Demonstrated bio-metabolites of acrolein 4-6
4-3 DNA-Protein cross-linking 4-17
4-4 Covalent binding of 3H-Acrolein to RNA, DNA and protein of
regenerating rat 1iver 4-17
4-5 Binding of 14C-Acrolein to hepatic microsomes from pheno-
barbital-treated rats in the absence of NADPH 4-19
4-6 Metabolism-mediated binding of 14C-Acrolein to hepatic micro-
somes phenobarbital-treated rats; NADPH added 4-20
5-1 Acute toxicity of acrolein 5-2
5-2 Acute inhalation toxicity of acrolein 5-3
5-3 Effect of acrolein inhalation on liver alkaline phosphatase
and relative liver weight 5-8
5-4 In vitro cytotoxicity of acrolein in mammalian cell
cultures ; 5-13
5-5 Ocular response to airborne acrolein \ 5-14
5-6 Thresholds of response after exposure to acrolein ., 5-15
VI
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LIST OF FIGURES
Number Page
4-1 Dose-related depletion of liver reduced glutathione (GSH)
in mice after intraperitoneal injections of acrolein and
cyclophosphamide 4-9
4-2 Log-dose relationship of percentage depletion of nonpro-
tein sulfhydryl groups (glutathione) in the nasal respira-
tory mucosa of rats exposed to acrolein for 3 hr 4-10
4-3 Postulated pathways of acrolein metabolism 4-12
4-4 Rate of formation of epoxide from acrolein and from ally!
alcohol by microsomes isolated from rat liver or lung 4-14
VII
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PREFACE
The Office of Health and Environmental Assessment has prepared this health
assessment to serve as a source document for EPA use. The health assessment was
developed for use by the Office of Air Quality Planning and Standards to support
decision making regarding possible regulation of acrolein as a hazardous air
pollutant.
In the development of the assessment document, the scientific literature
has been inventoried, key studies have been evaluated, and summary/conclusions
have been prepared so that the chemical's toxicity and related characteristics
are qualitatively identified. Observed effect levels and other measures of
dose-response relationships are discussed where appropriate, so that the nature
of the adverse health responses is placed in perspective with observed environ-
mental levels. The relevant literature for this document has been reviewed
through July 1, 1986.
Any information regarding sources, emissions, ambient air concentrations,
and public exposure has been included only to give the reader a preliminary
indication of the potential presence of this substance in the ambient air.
While the available information is presented as accurately as possible, it is
acknowledged to be limited and dependent in many instances on assumption rather
than specific data. This information is not intended, nor should it be used,
to support any conclusions regarding risks to public health.
If a review of the health information indicates that the Agency should
consider regulatory action for this substance, a considerable effort will be
undertaken to.obtain appropriate information regarding sources, emissions, and
ambient air concentrations. Such data will provide additional information for
drawing regulatory conclusions regarding the extent and significance of public
exposure to this substance.
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ABSTRACT
This health assessment document on acrolein was undertaken by the Environ-
mental Protection Agency's (EPA) Environmental Criteria and Assessment Office
to provide information necessary to determine if regulation of the release of
acrolein into the environment may be justified. Acrolein, a chemical inter-
mediate in the synthesis of several organic compounds, has an estimated produc-
tion volume of 55-70 million pounds per year and a potential for human exposure
during manufacture and use.
Acrolein is a volatile, reactive chemical. Its estimated half-life in the
atmosphere is less than one day. Studies in animals and the consequences of
accidental human exposure indicate adverse effects on the respiratory tract,
liver, cardiovascular system, and possibly kidneys. Insufficient data were
available to determine dose-response relationships for these effects.
The reactivity and toxicity of acrolein are seen at the principal sites of
exposure, the gastrointestinal and pulmonary tracts. There is no evidence that
below aversive levels, acrolein enters the general circulation. Acrolein is
metabolized by liver and lungs; metabolism is the principal route of elimina-
tion. There is inadequate evidence for acrolein1s mutagenicity or chemical
interaction with mammalian germ cells. Animal evidence for carcinogenicity is
limited. No studies of the results of long-term, occupational exposure of
humans, or other epidemiological studies were available.
Acrolein can produce both embryotoxic and fetotoxic effects, and is tera-
togenic under certain conditions. Studies of ecosystem effects show acrolein
to be highly toxic to aquatic plants, invertebrates, and fish; calculations
suggest that it is not likely to accumulate in the food chain. Recommendations
are offered for research in epidemiology, pharmacokinetics, chronic toxicity,
and human exposure.
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AUTHORS AND CONTRIBUTORS
Dr. Arthur Chiu, Carcinogen Assessment Group, U.S. EPA, Washington, D.C. 20460
Dr. Eric Clegg, Reproductive Effects Assessment Group, Washington, D.C. 20460
Dr. Ivan Davidson, Bowman-Gray School of Medicine, Winston-Sal em, NC 27103
Mr. Chris Dippel, Dynamac Corporation, Rockville, MD 20852
Mr. Lawrence Kaufman, Dynamac Corporation, Rockville, MD 20852
Mr. James Konz, Dynamac Corporation, Rockville, MD 20852
Dr. William McLellan, Dynamac Corporation, Rockville, MD 20852
Dr. Lawrence Valcovic, Reproductive Effects Assessment Group, Washington, D.C. 20460
REVIEWERS
Dr. Joseph Bufalini
Atmospheric Sciences Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Dr. H. C. Cornish
830 West Clark Road
Ypsilanti, MI 48197
Dr. Ivan Davidson
Department of Physiology and Pharmacology
Bowman Gray School of Medicine
Winston-Sal em, NC 27103
Dr. John L. Egle, Jr.
Department of Pharmacology
Medical College of Virginia
Health Sciences Division
Richmond, VA 23298
Dr. Ronald C. Grafstrom
Department of Toxicology
Karolinska Institute
S-104 01 Stockholm, Sweden
Dr. Henry Heck
Chemical Industry Institute of Toxicology
P.O. Box 12137
Research Triangle Park, NC 27709
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Dr. Charles Hobbs
Lovelace Research Institute;
P.O. 5890
Albuquerque, NM 87185
Dr. Neil Kravanic
Haskell Laboratory
E.I. Du Pont de Nemours and Co.
Newark, DE 19714
XI
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1. HEALTH EFFECTS SUMMARY AND CONCLUSIONS
1.1 BACKGROUND INFORMATION
1.1.1 Properties
Acrolein is a colorless, volatile liquid at room temperature and has an
acrid, pungent odor. It is very soluble in water and soluble in many organic
solvents. Acrolein is highly reactive, is unstable in light and air, and can
polymerize spontaneously.
1.1.2 Production
Acrolein is currently produced in the United States by the direct oxidation
of propylene using one of several catalytic systems. Facilities for the commer-
cial production of acrolein are found in the United States, Federal Republic
of Germany, France, and Japan. The sole U.S. manufacturer is Union Carbide;
its manufacturing plant has a capacity of 60-100 million pounds per year. Pro-
duction volume in the United States for 1984 is estimated to be 55-70 million
pounds. No estimates were available on the number of persons exposed to acro-
lein during its manufacture and use.
1.1.3 Use
Acrolein is predominantly used as an intermediate in the synthesis of
acrylic acid, ajlyl alcohol, methionine, 1,2,6-hexanetriol, glutaraldehyde,
and, up until 1980, glycerine. Acrolein is used to modify food starch and to
control the growth of algae, aquatic weeds, and mollusks. It is also used as a
slimicide in the manufacture of paper and as a microbiocide in wastewater
injection systems and liquid fuels.
1.1.4 Environmental Release
The potential for the release of acrolein to the environment exists during
its manufacture, transport, storage, and use in chemical synthesis as well as
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its use as a herbicide, however, no data were available on which estimates of
the potential levels of release could be made. Acrolein has been identified
but not quantified in the process streams of plants manufacturing acrylic acid
and oxygenated organic chemicals and in the emissions of processes that remove
solvents from coatings using heat. Acrolein is also a component of cigarette
smoke, automobile exhaust, wood smoke, emissions from fossil fuel powerplants,
and urban smog, and may be released during the cooking or processing of certain
foods.
•?*
1.1.5 Environmental Transport and Fate
Due to its high vapor pressure and water solubility, acrolein is expected "
to be highly mobile when released into the environment, although degradative
processes are likely to limit its transport. If released into the atmosphere,
it is not expected to persist very long, usually one solar day, and would react
quickly with hydroxyl radicals and with ozone, to a lesser extent; and also
photodissociate. In aquatic systems, acrolein would be removed by chemical and
microbial degradation and volatilization. Estimates of acrolein's half-life in
water range from 4 to 50 hours. In the terrestrial environment, it is estimated
that acrolein would have a low tendency to adsorb to soil and would probably
volatilize into air or be leached from the soil by water.
1.1.6 Ecosystems and Aquatic Biota
Several studies have confirmed the effectiveness of acrolein in control-
ling growth of aquatic plants and bacteria, although effective threshold levels.
were not identified. The acute toxicity of acrolein to certain aquatic in-
vertebrates and LC5Q values for several species of freshwater fish (46-240
ug/1) have also been determined. A bioconcentration factor of 344 with a
half-life in tissues of more than 7 days was derived from a study in bluegill
sunfish. This factor corresponded to a relatively low potential for
bioconcentration.
1.2 MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION ;
Acrolein, is a highly reactive aldehyde which reacts nonenzymatically and
enzymatically to 1) form stable adducts with extracellular and intracellular
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glutathione and other free thiol groups, 2) form adducts with nucleic acids and
proteins, 3) cross-links nucleic acids and proteins, and 4) reacts with enzymes
and membranes to cause a variety of biochemical consequences such as impairment
of DNA replication, inhibition of protein synthesis and mitochondria! respira-
tion, loss of liver and lung microsomal enzyme activities, and other parameters
of cellular integrity. This propensity for covalent binding by acrolein pro-
vides the basis for its cellular toxicity.
The reactivity and toxicity of acrolein is manifested in the
gastrointestinal and pulmonary tracts, the principal sites of exposure. There
is no documented evidence (for instance, blood determinations) that for expo-
sure concentrations below aversive levels, acrolein breaches the protective
mechanisms at these portals to gain entry to the general circulation. Bron-
chial and mucosal secretions, and mucosal and endothelial tissues at these
locations contain high concentrations of free thiols. Lung, gastrointestinal
mucosa and liver also contain effective metabolizing systems with high capacity
to biotransform acrolein. Hence the extent and kinetics of absorption of acro-
lein into the body during oral and inhalation exposure remain to be determined.
Acrolein can be formed in vivo via the metabolism of a number of xenobio-
, tics. For example, allyl alcohol, CH2CHCH2OH, causes intensive hepatic peri-
portal necrosis in rats after oxidation by hepatic alcohol dehydrogenase to
acrolein. Since protection from allyl alcohol toxicity is afforded by
pretreatment with sulfhydryl group donors (cysteine or N-acetylcysteine), it is
presumed that when free thiol groups are depleted by acrolein interaction,
acrolein combines covalently with other nucleophilic groups of cellular
macromolecules and thus leads to cellular damage.
Acrolein has been demonstrated to be metabolized in vivo, particularly by.
liver and lung parenchymal tissues. Two major pathways have been demonstrated:
(1) nonenzymatic and/or glutathione transferase reactions to form stable
adducts with glutathione and other thiols leading to mercapturic acid
metabolites, and (2) oxidative metabolism to (a) acrylic acid via aldehyde
dehydrogenase activity, and (b) to the epoxide glycidaldehyde via microsomal
P450 oxidation system. Glycidaldehyde, a reactive metabolite capable of
covalent binding, is further metabolized to innocuous metabolites by cellular
glutathione epoxide transferase or by epoxide hydrase. The relative importance
of these various pathways has not been assessed, nor has the effect of acrolein
dosage on metabolic disposition. However, acrolein appears to be extensively,
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if not completely, metabolized in mammalian systems; acrolein itself has not
been found in urine or exhaled air of rodents after parenteral administration
of high doses. Hence metabolism appears to be the principal route of elimina-
tion from the body. Mercapturic acid derivatives are found in the urine of
rodents after administration but account for less than 20 percent of dose.
Further information on the distribution of acrolein j_n vivo, dose-metabolism
relationships, profiles of metabolism pathways across species, and relation of
covalent binding to toxicity are needed.
1.3 MAMMALIAN TOXICITY
Studies in animals have indicated that acrolein is highly toxic by the
inhalation and oral routes and is a strong skin and eye irritant. In sub-
chronic inhalation studies, exposures to levels between 3 and 5 ppm (8 and 12
mg/m ) caused severe toxic signs and some degree of respiratory damage in all
species tested. Studies in mice found that levels below those causing overt
toxicity (0.1 ppm) reduced pulmonary compliance and tidal volume. In other
studies, inhibition of ciliary transport and decreased resistance to pulmonary
infection were reported after short-term exposure to acrolein at 6 ppm.
Acrolein has also been found to have effects on the liver (increased enzyme
activity), kidneys and cardiovascular system (transient increase in blood pres-
sure) after intravenous injection or inhalation exposure, Subchronic
inhalation studies in hamsters, rats, and rabbits (6 hours/deiy, 5 days/week, 13
weeks) determined a no observed effect level of 0.4 ppm (1 mg/m3), A chronic
inhalation study in hamsters exposed to 4 ppm (7 hours/day, 5 days/week, 52
weeks) indicated increased lung weights and moderate nasal inflammation and
epithelial metaplasia; no other levels were tested.
Data on the effects of human exposure to acrolein were limited to short-
term irritation studies and to 'reports of accidental exposures. Acrolein was
found to be severely irritating to the mucosa and eyes after a 10-minute expo-
sure at 0.8 ppm (1.8 mg/m ). The threshold for ocular irritation was 0.2 ppm
(0.45 mg/m ). The consequences of accidental exposure were immediate lung
injury and chronic bronchitis and emphysema; two fatalities occurred after
inhalation of smoke from burning vegetable oil that contained acrolein vapor.
Studies on the effects of long-term exposure were not available for review.
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1.4 MUTAGENICITY
The majority of the mutagenicity tests on acrolein have employed bacterial
systems and both positive and negative results have been reported. Differences
in bacterial strains tested, protocols used and the differences in concentra-
tions tested preclude a reconciliation of the apparently conflicting results.
All reports do indicate that acrolein is extremely toxic with significant
toxicity noted between 0.1 and 1 umoles/plate and complete toxicity at less
than 5 (jmoles/plate in Salmonella.
In eukaryotes, acrolein did not induce gene mutations in methionine
requiring strains of yeast, but did induce mitochondria! "petite" mutations
in another yeast strain. Acrolein induced sex-linked recessive lethals in
Drosophila when larvae were treated but not when adult males were treated in
accordance with the currently conventional procedure.
Acrolein did induce sister chromatid exchange in mammalian cells in vitro
but only in the absence of exogenous S9 activation. Extreme toxicity precluded
detection of chromosome aberrations in these cells. Finally, acrolein was
reported negative in a mouse dominant lethal test in which males received a
single IP injection of 1.5 or 2.2 mg/kg.
Applying the weight-of-evidence scheme of the proposed Guidelines for
Mutagenicity Risk Assessment to the acrolein data, results in the classifica-
tion of "Inadequate evidence bearing on either mutagenicity or chemical
interactions with mammalian germ cells." The basis for this conclusion is the
absence of data for mammalian gene mutations and in vivo mammalian cytogenetics
data (other than the very limited dominant lethal test), and the lack of data
on mammalian germ cell interaction.
1.5 CARCINOGENICITY
Animal evidence for carcinogenicity in skin painting studies is considered
to be limited in the evaluation by CAG. There is no epidemiological data. It
was not possible, based on the available data, to perform a quantitative risk
estimation or to calculate potency factors.
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1.6 REPRODUCTIVE AND TERATOGENIC EFFECTS
With respect to reproductive and developmental toxicity acrolein is
embryo/fetotoxic. There is no evidence of direct effects on either the male
or female reproductive systems. However, the evidence is not convincing that
such effects cannot occur since no detailed examination has been done of the
effects of acrolein on either the male or female reproductive system. The
embryo/fetotoxic effects have been observed in iji vitro culture and embryo
injection studies. The effects include reduced viability and growth
retardation. Teratogenic effects can also be produced under specialized
conditions. The important consideration appears to be that a sufficient
amount of acrolein actually reaches the sensitive sites within the embryo or
fetus. Possibly due to binding of acrolein to sulfhydryl and other reactive
sites, as well as metabolism of the compound, fetal effects :have not been
demonstrated in vivo in the absence of maternal toxicity.
1.7 REGULATIONS AND STANDARDS
The current standard for acrolein set by the Occupational Safety and
Health Administration is 0.1 ppm (0.25 mg/m3) over an 8-hour workshift. The
use of acrolein in food packaging materials and as a starch modifier has been
approved by the Food and Drug Administration; the quantity used to modify
starch cannot exceed 0.6 percent. Regulations promulgated by the Department of
Transportation specify labeling and packaging requirements for acrolein. Under
the Clean Water Act, acrolein is a hazardous substance and discharge of more
than 1 pound is prohibited. Disposal of acrolein is regulated under the
Resource Conservation and Recovery Act, and its use as a pesticide is limited
to certified applicators by the Federal Insecticide, Fungicide, and Rodenticide
Act.
1.8 CONCLUSIONS
The Environmental Protection Agency is considering the regulation of
acrolein as a hazardous air pollutant based on its toxic properties and poten-
tial human exposure. The information provided in this document indicates that
acrolein1s persistence in the atmosphere may be limited to a short period of
time and that insufficient data are available to determine current levels of
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exposure. Studies of acrolein's toxicity in animals suggest that respiratory
tract damage occurs after prolonged exposure to low levels of acrolein (4 ppm,
10 mg/m ). Insufficient data were available to determine the effects of long-
term inhalation of acrolein by humans. Further studies are needed to identify
effects in humans and to quantitate levels of acrolein in the environment.
1.9 RESEARCH NEEDS
The research needed to support or strengthen the current data base on
acrolein is outlined below. Of primary importance are studies that investigate
the effects of long-term, low-level exposure in humans.
1. Human Studies - Prospective and/or retrospective cohort epidemic!ogic
studies are needed that consider a range of potential effects on ex-
posed workers and attempt to quantitate exposure.
2. Pharmacokinetics - Studies are needed to verify the reported degree
of absorption after inhalation and ingestion by mammals, to identify
the distribution of acrolein and its metabolites after absorption.
3. Chronic Toxicity - Chronic inhalation studies, using at least two
dose levels, in two species of mammals would be useful to adequately
determine acrolein's toxic and carcinogenic effects.
4. Monitoring - Current data on emissions from acrolein's manufacture,
transport, and use are needed to permit estimates of general popu-
lation exposure.
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2. INTRODUCTION
This health assessment document has been prepared by the Environmental
Criteria and Assessment Office (ECAO) as a basis for its evaluation of acrolein
as a hazardous pollutant. It is intended by the Office to be one of several
information sources to guide regulatory strategies of the EPA program offices.
The preparation of this document involved the participation of the following
groups: ECAO, the Carcinogen Assessment Group (CAG), and the Reproductive
Effects Assessment Group (REAG), of the U.S. Environmental Protection Agency;
and Dynamac Corporation, Rockville, MD. CAG prepared the carcinogenicity sec-
tion of the document, and REAG prepared the reproductive, teratogenic, and
genetic toxicology sections. Dynamac was responsible for the literature search
and retrieval and prepared the other sections of the document.
The basis of this document was a literature search performed by the Envi-
ronmental Control Division of Dynamac Corporation using the health and environ-
mental effects files in the following data base systems: National Library of
Medicine (MEDLARS), Lockheed Information System (DIALOG), and System Development
Corporation (ORBIT). The literature that was identified in the search was in-
ventoried, and relevant studies were retrieved, evaluated, and summarized. Each
chapter was written to include a summary to the significant aspects of acro-
lein1 s production, presence in the environment, and/or toxicity.
The major topics included in the document are physical and chemical proper-
ties, sampling and analytical methods, production and use, levels and sources
in the environment, transport and fate, and biological effects. The discussion
of biological effects includes the areas of metabolism and pharmacokinetics as
well as mammalian toxicity to organ and tissue systems, carcinogenicity, muta-
genicity, teratogenicity, and reproductive effects. Data on the effects of
acrolein in humans are also presented.
In the sections on animal toxicity, key studies are presented in a descrip-
tive manner that includes information on the test species, dose or exposure
regimen, route of exposure, types of effects seen with each dosage, number of
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animals in each test and control group, sex and age of the animals, and statis-
tical significance. Information on the purity of the test material is specified
when the data were available. Emphasis is placed on observed effect levels and
other measures of dose-response relationships.
This document is intended to serve as a basis for decision-making in the
various regulatory offices within EPA as well as to inform the general public
of the nature and extent of information available for assessment of health
hazards resulting from exposure to acrolein.
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3. BACKGROUND INFORMATION
3.1 PHYSICAL AND CHEMICAL PROPERTIES
Acrolein is an unsaturated aldehyde with the chemical formula CH2=CHCHO.
It is a colorless, volatile liquid at room temperature and has an acrid, pun-
gent odor. Acrolein is very soluble in water and soluble in many organic sol-
vents. It is unstable in light and air and can be readily polymerized; hydro-
quinone is usually added to inhibit polymerization. The conversion factor for
acrolein concentration in air is 1 ppm = 2.3 mg/m3 at 760 mmHg and 25°. Table
3-1 lists the physical and chemical properties of acrolein.
3.1.1 Synonyms
Acrolein has the following synonyms:
• 2-Propenal . Prop-2-en-l-al
• Propenal . 2-Propen-l-one
* Acraldehyde . Ethylene aldehyde
• Acrylic aldehyde . Aqua!in
• Ally! aldehyde . Aqualine
• Aery1aldehyde . Acquinite
3.1.2 Identification Numbers
Acrolein has the following identification numbers:
• Chemical Abstracts Service (CAS) No. 107-02-8
.* Registry of Toxic Effects of Chemical Substances (RTECS) No. AS 1050000
• Toxicology Data Bank (TDB) No. 0177.
3.1.3
Significance of Physical/Chemical Properties with Respect to
Environmental Behavior
Parameters that affect environmental behavior include water solubility,
vapor pressure, octanol/water partition coefficient, and degradation rates.
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TABLE 3-1. PHYSICAL AND CHEMICAL PROPERTIES OF ACROLEW
Parameter
Value
Molecular weight
Specific gravity, 20/20 °C
Coefficient of expansion at 20 °C, vol/°C
Boiling point, °C
101.3 kPa (760 mmHg)
1.33 kPa (120 mmHg)
Melting point, °C
Vapor pressure at 20 °C, kPa (mmHg)
Heat of vaporization at 101.3 kPa (1 atm), kJ/kg (Btu/lb)
Critical temperature, °C
Critical pressure, MPa (atm)
Solubility at 20 6C, wt% (mg/1)
in water
water in
Refractive index n20
Viscosity at 20 °C, mPa-s
Weight oer liter at 20 °C, kg (Ib/gal)
Flashpoint, open cup, °C
closed cup, °C
Flammability limits in air, vol%
upper
lower
Autoignition temperature in air, °C
Heat of combustion at 25 °C, kJ/kg (Btu/lb)
Heat of polymerization (vinyl), kJ/mol (kcal/mol)
Heat of condensation (aldol), kJ/mol (kcal/mol)
56.06
0.8427
0.00140
53
-36
-87.0
29.3 (220)
93 (216)
233
5.07 (50)
20.6 (206,000)
6.8
1.4013
0.35
0.842 (7.02)
-18
-26
31
2.8
234
5,383 (12,507)
71.1-79.5 (17-19)
41.8 (10)
Source: Hess et al. (1978).
Because acrolein is very soluble in water and has a high vapor pressure, its
environmental behavior associated with the water and air compartments will
probably be of greater importance than its behavior in soil, due to vapori-
zation from the soil into air and leaching from soil into water. The high
water solubility and low partition coefficient also suggest that soil sorption
may not be an important process (USEPA, 1979). However, since acrolein is very
reactive, it is possible that it will bind to organic constituents in the soil
and not be available for transport to other environmental compartments.
Microbial degradation and reversible hydrolysis appear to be the mechanisms
responsible for removal of acrolein from water. The environmental behavior of
acrolein is more completely discussed in Section 3.4.
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3.1.4 Chemical Reactions In the Environment
Acrolein is a highly reactive chemical because of the carbonyl-double
bond conjugation (Hess et al., 1978) and the presence of both a vinyl group
and an aldehyde group on a low molecular weight compound (USEPA 1980a). When
exposed to light and air, liquid acrolein polymerizes to form disacryl, which
is an inactive plastic substance (Merck, 1983).
Acrolein vapor reacts with hydroxyl radical (Edney et al. 1982; Atkinson,
1986) and with ozone (Atkinson et al., 1981). In general, the promary removal
process for acrolein in the atmosphere is through reaction with hydroxyl,
giving it a lifetime of approximately 14 hours. The compound would be stable
to hydroxyl attach at night but would be subject to reaction with ozone.
3.2. ANALYTICAL METHODOLOGY
3.2.1 Chemical Analysis in Air
Prior to the late 1970's, the methods used for sampling acrolein in air
involved liquid absorbing solutions. The analytical method of the National
Institute for Occupational Safety and Health (NIOSH) is representative of the
liquid sorbent techniques (NIOSH, 1978). In this method, air is drawn through
two midget impingers with fritted glass inlets containing a mixed absorbing
reagent. The reaction of acrolein with 4-hexylresorcinol in the presence of
ethanol, trichloroacetic acid, and mercuric chloride results in a blue-colored
product with a strong absorption maximum at 605 nm. A spectrophotometer is
then used to quantitate the acrolein levels. A concentration of 0.01 ppm
acrolein can be detected in a 50-liter air sample using this method. Slight
interference may occur with dienes.
In reviewing the NIOSH method, Hemenway et al. (1980) pointed out a
potential problem in the sample preparation procedure. The procedure calls for
the use of a mixed reagent, and since the trichloroacetic acid, one of the
components of this reagent, can react with the other reagents in the solution,
it is possible for the mixed reagent to deteriorate over time. According to
the authors, actual levels of acrolein may be underestimated by as much as 35
percent because of this problem.
Liquid sorbent procedures are not convenient for personal sampling in an
industrial setting or for field sampling. As a result, several solid sorbent
sampling methods have been developed that use gas or liquid chromatographic
analysis.
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A personal air sampling method for acrolein has been developed based on
the use of the solid sorbent Amberlite XAD-2 coated with 2,4-dinitrophenyl-
hydrazine (Andersson et al., 1981). Using this method, acrolein in the range
of 0.02-0.52 ppm can be analyzed in 5-liter samples with a recovery of 80-100
percent. The analysis of the chemosorbent for acrolein is performed by high-
performance liquid chromatography (HPLC) using an ultraviolet (UV) adsorbance
detector. Another personal air sampling method using a Porapak N adsorption
tube to trap acrolein with subsequent thermal desorption has been described by
Campbell and Moore (1979). With this method, acrolein concentrations below 1
ppi can be determined with recovery efficiencies approaching 100 percent.
Analysis is performed using a gas chromatograph equipped with a flame-
ionization detector (FID).
Hurley and Ketcham (1978) have described a personal air sampling method
for acrolein based on the use of hydroquinone-treated carbon as the solid
sorbent. Ethylene dichloride is used to desorb acrolein followed by analysis
with a gas chromatograph equipped with a FID. Recovery efficiencies of
approximately 80 percent are routinely obtained. The method has a sensitivity
of 0.02 ppm acrolein for a 5-liter air sample and is useful for measuring
acrolein in the 0.05-5 ppm range.
Another analytical method used to measure acrolein concentrations in air
involves the use of microwave spectroscopy (Tanimoto and Uehara,, 1975). This
method reportedly is more selective and has higher resolution than the col ori-
metric method. Using this method, exhaust samples are collected through a
glass tube packed with phosphorus pentoxide and trapped on a color adsorbent in
an acetone-dry ice bath. The adsorbent is heated, and the desorbed gas is
introduced into the spectrometer. Preconcentration of acrolein is apparently
necessary for levels below 10 ppm. Recovery efficiencies were reported to be
low.
Activated 13X molecular sieves have been used as the sorbent for acrolein
vapor over a wide range, 3-200 ug/g of sieves. Recovery in the 3-8 ug/g range
was 97 percent, while recovery in the 60-200 |jg/g range was 90 percent follow-
ing storage at 0 °C for up to 4 weeks. Analysis involved the desorption of the
acrolein with distilled water followed by gas chromatographic (GC) analysis
using a FID (Gold et al., 1978). A fluorimetric method has also been developed
using molecular sieves to collect acrolein vapor and o-aminobiphenyl as the
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fluorescent reagent. Measurement of the fluorescence intensity with a spec-
trophotometer showed that the excitation wavelength at 345 nm was highly selec-
tive for acrolein. Use of a molecular sieve 3A in combination with molecular
sieve 13X allowed the sampling of large volumes of gas. A recovery efficiency
of almost 100 percent was reported when a two-stage bubble system was used to
collect the desorbed acrolein. The detection limit for this method is 1 ppb
(Suzuki and Imai, 1982).
A C02 laser photoacoustic technique has been suggested for the deter-
mination of ppb levels of acrolein in air (Loper et al., 1982). In this
method, the COg laser absorption spectra for acrolein is measured in a very
selective manner, and absorptive interferences due to water vapor or other
atmospheric compounds are minimized. Based on results of preliminary testing,
the technique appears to be highly suited for detecting acrolein in ambient
air. Detection limits of approximately 40 ppb were suggested.
An analyzer specific for acrolein has recently been developed to con-
tinuously monitor concentrations between 0.01 and 17 ppm in air for periods up
to 8 hours (Reddish, 1982). Although this method does not give "real time"
information, the time between sampling and analysis is less than 20 minutes.
The analyzer consists of six functional units: (1) a gas scrubber/reaction
vessel containing an absorbing reagent similar to that used in the NIOSH
method; (2) a multichannel peristaltic pump; (3) a reaction coil maintained at
60 °C; (4) a flowthrough cell (black polytetrafluoroethylene) with a 4-cm path
length using fiber optics to facilitate measurement of the transmission/
absorbance; (5) a colorimeter capable of measuring the colored complex at 605
nm and capable of accepting fiber-optic light transmission; and (6) a chart
recorder to record the concentration/absorbance profile of acrolein. Using
this technique, 90 percent accuracy has been achieved when compared to results
using the HPLC technique.
3.2.2 Chemical Analysis in Water
Various analytical methods are used to determine acrolein concentrations
in water (Table 3-2). These include the direct measurement of acrolein by UV
spectroscopy, gas-liquid chromatography (GLC), nuclear magnetic resonance (NMR)
spectroscopy, colorimetry, differential pulse polarography, titrimetry, and
direct fluorescence spectroscopy (Brady et al., 1977; Kissel et al., 1978).
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TABLE 3-2. METHODS FOR ACROLEIN MEASUREMENT
Analytical method
Detection limit
Interferences
NMR (aldehydic proton) 100 mg/1
Colorimetry
2,4-DNPH 80 pg/1
4-Hexylresorcinol 700 |jg/l
Fluorimetry
Direct 20 mg/1
J-Acid 20 jjg/1
m-Aminophenol derivative 10 ug/1
Differential pulse polarography 30 ug/1
Gas chromatography
Flame ionization 500 ug/1
Mass spectral 50 ug/1
Few
Many
Many
Very few
Very few
Very few
Few
Very few
Very few
Source: U.S. Environmental Protection Agency (1980b).
A more recent gas chromatographic/mass spectrometric (GC/MS) method allows
the simultaneous analysis of acrolein in a much shorter period of time (Trussel
et al., 1981). This technique uses a single fused silica, open tubular capil-
lary column that improves chromatographic resolution. The column, which
measures 0.25 mm x 30 m, is coated with SE-54 liquid phase and is directly
interfaced to the ion source of the mass spectrometer. The sensitivity of this
technique.is below the 1 ug/1 level. Field applications of this method have
not been documented.
3.3 PRODUCTION, USE, AND RELEASES TO THE ENVIRONMENT '
3.3.1 Production
Acrolein was initially produced by the vapor-phase condensation of
acetaldehyde and formaldehyde. However, since 1959 acrolein has been produced
by the direct oxidation of propylene. Many different catalyst systems have
been used that allow greater than 90 percent conversion of propylene. The
conversion is highly selective'to acrolein with 70-84 percent yields. Other
products include acrylic acid, acetic acid, acetaldehyde, and carbon oxides
(Hess et al., 1978).
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Shell Chemical Company phased out production of acrolein on July 1, 1980,
due to economic reasons, and Union Carbide Corporation is currently the only
;producer of acrolein in the United States (SRI, 1980, 1983). Although the
production volume is not currently available, the Union Carbide plant in Taft,
LA, has a capacity of 60 million pounds per year, which can be expanded to 100
million pounds per year. Production levels in 1984 were estimated to be 55-70
million pounds (SRI, 1980).
3.3.2 Use
Acrolein is predominantly used as an intermediate in the synthesis of
several derivatives including acrylic acid, ally! alcohol, methionine,
1,2,6-hexanetriol, and glutaraldehyde (IARC, 1979). Before 1980, Shell Chemical
Company used its acrolein production to produce synthetic glycerine; however,
since Shell stopped production, acrolein is apparently not used for this
synthesis (SRI, 1977, 1980). In 1979, the demand for methionine in"the United
States was 69 million pounds. Most of this demand (67 percent) was met by
acrolein derivatives. Methionine is predominantly used as a protein supplement
in animal feed as well as for Pharmaceuticals and cosmetics (SRI, 1980).
Other major derivatives include 1,2,6-hexanetriol, which is used as a
humectant and in the manufacture of flexible polyurethane foam, and gluteral-
dehyde, which is used in leather tanning, in photographic chemicals and X-ray
supplies, as a sterilizing agent, and as a tissue fixative for transmission
electron microscopy (SRI, 1980). Additional chemicals and chemical products
produced from acrolein include 2-hydroxyadipaldehyde, quinoline, pentaerythritol,
cycloaliphatic epoxy resins, oil-well additives, and water treatment formulae
(IARC, 1979).
Acrolein is also used to modify food starch; as an aquatic herbicide,
biocide, and slimicide (IARC, 1979); as a microbiocide in wastewater injection
systems; to protect liquid fuels from attack by micro-organisms; to control the
growth of algae, aquatic weeds, and moll usks in recirculating process water
systems; and as a slimicide in paper manufacturing (Brady et al., 1977; Hess et
al., 1978).
Acrolein vapor has been used for tissue fixation for electron microscopy.
Results from preliminary studies suggest that acrolein vapor fixation preserves
more materials in tissue than other methods, such as those that use glutaralde-
hyde, sodium tetroxide, or osmium tetroxide (Kawai et al., 1983).
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3.3.3 Environmental Release
3.3.3.1 Combustion. Acrolein is produced by the incomplete combustion of
gasoline, diesel fuel, and other fuels. Levels ranging from 0.05 to 22.5
O
mg/m have been measured in automobile engine exhaust (Tanimoto and Uehara,
1975; USEPA, 1980), accounting for approximately 3-10 percent of the total
aldehydes emitted (Stahl, 1969).
Acrolein has also been identified in wood smoke. Levels detected in pine
smoke ranged from 0.62 to 0.67 mg/g of wood burned (USEPA, 1980). Einhorn
(1975) reported levels as high as 50 ppm in smoke from the combustion of wood.
Acrolein was also identified (but not quantified) in the air of commercial
smokehouses (Love and Bratzler, 1966).
Acrolein is suspected to be a gaseous emission from fossil fuel power-
plants, as one of the group of chemicals classified as formaldehyde and related
compounds (Natusch, 1978). For this aldehyde group, average emission
levels of 0.002, 0.1, and 0.2 lb/1,000 Ib of fuel have been reported for coal,
oil, and natural gas plants, respectively. Emissions of aldehydes from both
oil- and gas-powered plants have been reported to be 0.5 Ib/ton of fuel. The
actual levels of acrolein contained in these emissions has not been determined.
Acrolein has been identified as both a combustion product and a pyrolysis
product of polyethylene-based materials (Potts et al., 1978). Acrolein con-
centrations resulting from combustion of polyethylene foams ranged from 2 to 23
ppm, while levels measured (by GC/MS analysis) during pyrolysis were between 76
and 180 ppm. Acrolein has also been detected during the incineration of
plastic beverage containers (Wharton, 1978).
3.3.3.2 Cigarette Smoke. Several studies have confirmed the presence of
acrolein in cigarette smoke. Horton and Guerin (1974) reported that a commer-
cial, 85-mm filtered cigarette delivered 102 ug of acrolein (153 jjg/g tobacco)
directly to the smoker. A similar, nonfiltered cigarette delivered 111 ug of
acrolein (135 ug/g tobacco); an experimental 85-mm marijuana cigarette con-
tained 145 ug of acrolein (199 ug/g marijuana); and a commercial 85-mm cigar
contained 70 ug of acrolein (107 ug/g tobacco). An experimental 85-mm cigarette
with a charcoal filter delivered the least amount of acrolein, 62 ug/cigarette
(97 ug/g tobacco). '.
Acrolein is also present in the sidestream smoke of cigarettes, leading to
possible exposure for nonsmokers. Ayer and Yeager (1982) reported acrolein
levels of 0.9-1.3 ppm in the sidestream smoke of three commercial brands of
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cigarettes that were three orders of magnitude above occupational limits. In
experiments to determine the gas-phase components of sidestream smoke, Jermini
et al. (1976) reported that the acrolein concentration in an unventilated 30 m3
room after the simultaneous smoking of 30 cigarettes was 0.37 ppm.
3-3.3.3 Food Processing. Acrolein is produced as a result of the heating of
organic substrates. It has been detected (but not quantified) during the
cooking or processing of foods (Boyd et al., 1965; Grey and Shrimpton, 1967;
Hrdlicka and Kuca, 1965; Izard and Libermann, 1978; Kishi et al:, 1975) and in
the fermentation of alcoholic beverages (Rosenthaler and Vegezzi, 1955).
3.3.3.4 Production Processes. No information was found on the release of
acrolein during its production or use as a chemical intermediate. Acrolein has
been identified in the process streams in acrylic acid plants (Serth et al.,
1978), in industries manufacturing oxygenated organic compounds (e.g.,
aldehydes, alcohols), and in processes that remove solvents from coatings by
the use of drying or heating ovens (Stahl, 1969). Acrolein is also evolved
during the processing of plastics (temperature range 140-340 °C); however,
release rates have not been reported (Reddish, 1982).
3.3.4 Environmental Occurrence
A very limited amount of data is available regarding the environmental
levels of acrolein. Levels between less than 1 and 20 mg/m3 (.44 and 8.7 ppm)
are considered representative of concentrations present in urban air (Carson
et al., 1981; Natusch, 1978).
Acrolein is a component of urban smog and has been measured in the air of
Los Angeles, CA. The average concentration for 10 days during the period
September-November 1960 was 0.005 ppm (.011 mg/m3), and for 7 days during the
same period in 1961 it was 0.008 ppm (.018 mg/m3) (Altschuller and McPherson,
1963). An average concentration of 0.004 ppm (.009 mg/m3) was reported for 10
days between July and November 1960 (Renzetti and Bryan, 1961).
Propylene is oxidized to acrolein in air. A mathematical model developed
by Graedel et al. (1976) has computed the peak concentration for acrolein in
urban,air based on several variables including estimated rate constants for the
reactions leading to the formation of acrolein from the oxidation of propylene
(with ally!ic radicals being the apparent precursors). Based on this model,
the peak concentration for acrolein in urban air (Los Angeles) is estimated to
be 1.3 x 10"2 ppm.
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Acrolein can also be formed in the atmosphere by the photo-oxidation of
diolefins or other hydrocarbon nitrogen oxide mixtures (Stahl, 1969).
Acrolein has not been shown to be a contaminant of drinking vrater or water
supplies (USEPA, 1980b).
Although acrolein has been detected in many foods (see sectiion 3.3.4.3),
the concentrations have not been quantified.
3.4 ENVIRONMENTAL TRANSPORT AND FATE
Acrolein is released into the atmospheric, aquatic, and terrestrial
environments. Acrolein is released into the atmosphere from the manufacture,
transport, use, or combustion of organic substrates; into natural! waters from
manufacturing effluents and direct herbicidal use; and onto land as a result of
accidental spills, indirect herbicidal use, and land disposal. Once released,
the environmental fate of acrolein is determined by a combination of dispersive,
degradative, and accumulative processes. The relationships of these processes
to acrolein1s environmental transport and fate are discussed in the following
sections.
3.4.1 Transport
Acrolein is expected to be highly labile when released into the environ-
ment. Because of its high reactivity, its transport in the environment is
limited. Due to its high vapor pressure, if spilled, it is expected to
rapidly evaporate at ambient temperatures. Once in the atmosphere, it will
react with OH radicals, ozone, and photodissociate.
Acrolein is very soluble in water (206,000 mg/1). Due to rapid degrada-
tion, however, only between 2 and 29 percent of the amount of acrolein released
into a river is estimated to be transported downstream to a distance of 50-250
miles over a 5-day travel period (Falco et a!., 1980). In laboratory experi-
ments, Bowmer ,et al. (1974) found that differences in the chemical properties
of water bodies could affect the loss of acrolein by reaction or degradation,
whereas greater turbulence is expected to increase loss by volatilization.
The extent to which acrolein is transported through the terrestrial
environment is inversely related to the degree to which it is adsorbed to soil.
Soil adsorption can be described by the soil/water partition coefficient as a
function of organic carbon content (K ). This value may be estimated for
i OC
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acrolein by the regression equation of Kenaga and Goring (1980). Using this
equation [log KQC - 3.64 ~ 0.55 (log S), where S = water solubility in mg/1], a
KQC value of 5.2 is estimated, indicating a low soil adsorption potential for
acrolein. Thus, acrolein is expected to volatilize from soil and/or leach from
soil where it may be transported to groundwater.
3.4.2 Fate
3.4.2.1 Atmospheric Fate. Few data were found on the atmospheric fate of
acrplein. The available information suggests that acrolein released into the
atmosphere would persist for a few days only. Aldehydes in the atmosphere, in
general, can be expected to photodissociate into the "R" group and a free
aldehyde group. This "Norrish Type 1" fragmentation typically occurs at 313 nm
irradiation (Calvert and Pitts, 1966).
The primary photochemical process, however, would compete with possible
photophysical deactivation processes to affect the excited-state molecules.
Moreover, the radicals produced may undergo secondary reactions to yield
thermally stable compounds (Calvert and Pitts, 1966).
Cupitt (1980) described two chemical removal processes in air that affect
organic compounds containing double bonds such as acrolein. The first is
reaction with hydroxyl radicals that may be added across the double bond of
acrolein to form oxygenated compounds such as aldehydes, ketones, and dicar-
bonyls. The second chemical removal process is reaction with ozone. This
process (ozonolysis) results in the formation of a carbonyl compound (aldehyde
or ketone) and a percarbonyl biradical that may undergo further rearrangement
to form a variety of products including organic acids or radicals and carbon
dioxide.
In a laboratory study to determine atmospheric hydroxyl reactions, Edney
et al. (1982) reported that both additions and abstractions by OH radicals were
mechanisms involved in the removal of acrolein from air. Reaction products
identified included peroxy nitrates, formaldehyde, and glycoaldehyde. An
atmospheric half-life for acrolein, based on the hydroxyl rate constant was
estimated to be 5.6 hours.
The atmospheric residence time (T) can be estimated for acrolein according
to the method of Lyman et al. (1982). The method estimates residence time as a
function of reaction rates with hydroxyl radicals and ozone but does not
consider reactions with other substances or photodissociation; therefore, T
must be considered a maximum rate.
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Acrolein is removed from the atmosphere thru OH and 0, reactions. The
c
ambient levels of OH are of the order of 10 molecules/cc. Since the rate
—12 ' -1 -1
constant is 20 X 10 cc mol sec , the lifetime is approximately 14 hrs.
Acrolein will also react with ozone to the same extent but since the rate
constant is of the order of 10 cc mol" sec" with ambient levels of 03 at
40-50 ppb, this reaction will not be very important for the removal of signifi-
cant levels of acrolein. Acrolein will also photodissociate with an atmospheric
lifetime of 5.3 days. Thus, the most important removal process of acrolein is
thru reactions with OH radicals (U.S. EPA, 1986).
3.4.2.2 Aquatic Fate. Acrolein may be removed from natural waters through a
combination of physical, chemical, and biological degradative processes and by
volatilization, sorption, and dilution. Information on the relative importance
of the individual processes is limited; therefore, this discussion is supple-
mented with extrapolations from known properties of acrolein.
Aqueous photolysis is a possible degradative mechanism for acrolein in
water. As discussed in the previous section on atmospheric fate, the primary
photochemical reaction would be a "Norrish Type 1" fragmentation, which typical-
ly occurs at 313 nm irradiation. For acrolein in the environmental radiation
range of 290-700 nm, the maximum absorption will occur at 315 nm with a molar
absorptivity (E) equal to 26 liters/mol-cm (Lyman et al., 1982). These data,
however, are not sufficient to predict aqueous photolysis rates, in part
because they may be affected by suspended sediments, surfactants, and sensiti-
zers.
The United States Bureau of Reclamation reported that acrolein dissipation
from flowing water is a first-order process (Bowmer and Saintyj, 1977). The
equation is:
K = (U/AX) In (Ca/Cb) ;
where K is the first-order rate constant of decay; Ca and C. are the con-
a D
centrations observed in the plateau (i.e., stable concentration) region as the
treated water passes stations, at distances X_ and X. downstream from the
a D
injection point; and U is the mean velocity. The first-order rate contants
were determined in eight irrigation canals in Australia and the United States
(Bowmer and Sainty, 1977). K values ranged from 0.104 to 0.211 per hour and
had a mean (± standard deviation) value of 0.16 (± 0.04) per hour. This
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corresponds to a half-life of 4.3 hours. It was hypothesized that acrolein
dissipated by volatilization, degradation, and adsorption.
The primary acrolein reaction products have been identified in laboratory
studies under acidic and alkaline conditions (Bowmer and Higgins, 1976). The
primary reaction of acrolein under acidic conditions' is a reversible hydrolysis
to beta-hydroxypropionaldehyde, whereas under alkaline conditions a polyconden-
sation reaction occurs to form a pentamer. In their study using environmental
pH conditions (pH 5-9), reaction products were not identified. Bowmer and
Higgins predicted that acrolein's half-life in the Australian irrigation canals
would be pH dependent (38 hours at pH 8.6 and 50 hours at pH 6.6).
The third degradative process that may be important to the aquatic fate of
acrolein is biodegradation. Acrolein has been reported to be relatively
nonbiodegradable. In a determination of the oxygen demand (i.e., degrada-
bility) of chemicals of interest to Shell Research Company (Amsterdam), Bridie
et al. (1979) reported that the theoretical, 5-day biochemical (BOD), and
chemical oxygen (COD) demands of acrolein to be 2.0, 0.0, and 1.76 g/g, respec-
tively. According to Lyman et al. (1982), biodegradability may be estimated
from the ratio of 5-day BOD to COD. Acrolein with a value of 0.01 is thus
rated as "relatively undegradable" using the data of Bridie et al. and the
degradability index of Lyman et al. solubility and low partition coefficient
also suggest that soil sorption may not be an important process (USEPA, 1979).
However, since acrolein is very reactive, it is possible that it will bind to
organic constituents in the soil and not be available for transport to other
environmental compartments. Microbial degradation and reversible hydrolysis
appear to be the mechanisms responsible for removal of acrolein from water.
The environmental behavior of acrolein is more completely discussed in Section
3.4.
The "microbial degradation rate" of acrolein in a hypothetical river was
calculated using the EXAMS model by Falco et al. (1980). The rate of 0.08/day
suggests that acrolein is probably somewhat more biodegradable than indicated
above.
Bowmer and Higgins (1976),studied the persistence and fate of acrolein in
several Australian irrigation canals. An unidentified, nonvolatile reaction
product of acrolein was observed when acrolein concentrations fell below 2-3
ppm. Jt was transient and dissipated rapidly following a lag period reportedly
through "microbiological processes," The authors suggested that these processes
might involve microbial oxidation of the aldehyde to carboxylic acid.
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Actual measurements of acrolein volatilization from water were not found
in the literature. Estimates of the liquid and gas exchange coefficients
indicate the importance of volatilization to overall acrolein dissipation.
According to Lyman et al. (1982), when the Henry's Law Constant (H) ranges from
•»£ «>^ ^
10 to 10 atm-m/mol, as with acrolein, both the liquid-phase and gas-phase
resistances are important and volatilization is moderate. If it is assumed
that there are no resistances other than those of the gas and liquid phases,
the half-life of acrolein in water at depth Z (e.g., 100 cm), is 5.5 hours.
However, volatilization would be limited by chemical reactions or degradation
of acrolein in water, whereas turbulence is expected to increase loss by
volatilization (Bowmer et al., 1974).
Acrolein present in natural waters is not expected to adsorb to bottom
sediments or to bioconcentrate in significant quantities. The estimated low
sediment (or soil) adsorption potential has been discussed in section 3.4.1,
Transport. The bioconcentration of acrolein in the bluegill sunfish was
studied and, as predicted from acrolein1s chemical properties, was not found to
be a very significant process (Veith et al., 1980). Bioconcentration and other
accumulation processes are discussed further in chapter 9, Ecosystem Considera-
tions.
3.4.2.3 Terrestrial Fate. As discussed in the transport section, acrolein has
a low soil adsorption potential. Acrolein is expected to volatilize or leach
from the soil where it will be transported either to the atmosphere or to
groundwater.
3.5 ECOSYSTEM CONSIDERATIONS
3.5.1 Introduction
Because of its use as an aquatic herbicide and as an organic intermediate,
acrolein may be potentially harmful to the aquatic and terrestrial ecosystems.
Since the 1950's the effects of acrolein on various target and nontarget
organisms have been considered and reported in the scientific literature. The
effects on residents of the aquatic ecosystem, fish and some aquatic plants in
particular, are best known (Section 3.6). Very little is known about acrolein's
potential effects on terrestrial ecosystems (Section 3.7). Acrolein has a
relatively limited potential for bioconcentration and bioaccumulation (Section
3.8).
September 1986
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DRAFT—DO NOT QUOTE OR CITE
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3.6 AQUATIC ECOSYSTEMS
The effects of acrolein on aquatic life are summarized in the sections
below. The available literature was limited for all groups except fish. The
data indicate that acrolein has acute toxic effects on most fish and aquatic
invertebrates tested at a concentration of 1 mg/1.
3.6.1 Aquatic Plants, Bacteria, and Algae
The few available studies were designed to show the effectiveness of
acrolein for controlling nuisance growths of aquatic plants, bacteria, and
algae. Consequently, there was little concern with identifying either threshold
response levels for nontarget species or overall ecosystem effects. In general,
however, the tested herbicidal or slimicidal levels exceed the safe levels for
fish (see Section 3.6.3).
The growth of two aquatic plants, floating pondweed (Potamogeton
tricarinatus) and ribbonweed (Vallisneria spiral is), in several Australian
irrigation canals was controlled by acrolein treatments (Bowmer and Sainty,
1977). Laboratory studies confirmed field observations that pondweed is 7-10
times more tolerant of acrolein than ribbonweed. Mature plants of each species
growing in buckets of mud were exposed to measured levels of acrolein in
500-liter containers for at least 1 hour followed by "washing and 7 days' growth
in clean water." One-hour treatments with 26 mg/1 acrolein for pondweed and 3.7
mg/1 acrolein for ribbonweed resulted in 80 percent reduction in plant growth,
compared to controls, 1 week from treatment.
Van Overbeek et al. (1959) reported that acrolein, as the active ingredient
in an unspecified product, controlled submersed aquatic weeds in a 20-mile
irrigation canal in Kern County, CA. Application of 1.15 gallons of acrolein
per cubic foot of waterflow per second for 30-45 minutes controlled the growth
of pondweed (P. crispus) and other unspecified weeds for several weeks, and was
sufficient to raise water flow by about 75 percent. Van Overbeek and coworkers
also reported that in laboratory experiments, leaf cells of the water plant
El odea densa were destroyed by acrolein application at 0.5 ppm after 24 hours or
at 5 ppm after 2 hours. 'The authors hypothesized that the mode of action was
mediated by effects on enzyme systems with functional sulfhydryl group, rather
than cell membrane, destruction. This conclusion was based on the observation
that El odea cell contents were destroyed after being dipped in 1,000 ppm
acrolein, while the cells maintained turgor pressure for several hours.
September 1986 3-15 DRAFT—DO NOT QUOTE OR CITE
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The effects of acrolein on aquatic bacteria were reported by Starzecka
(1975), who tested the effectiveness of a powerplant cooling water biocide.
Several bacterial groups (including Pseudomonas and Achromobacter-Alcaligenes)
were isolated from the "unpolluted" Trzebunka River in Poland and tested using
high levels (greater than 62 mg/1) of an unspecified formulation of acrolein.
Bacterial cultures grown for several days in peptone (10 g/1) and glucose (1
g/1) were slightly inhibited by acrolein at 62 mg/1 and severely inhibited at
125-250 mg/1. Levels such as these far exceed the concentrations necessary to
control submersed plants (Bowmer and Sainty, 1975; Van Overbeek et al., 1959).
Only one study was found indicating that acrolein could reduce the growth
of a green filamentous alga, Cladophora sp. (Jordan et al., 1962). Visual
estimates of Cladophora growth were made during 4 months in three replicate
ponds (675 ft ; 7,500 gallons) treated with technical-grade acrolein (at 3 mg/1,
nominal). The estimates of percent pond surface coverage by algae made by two
to seven independent observers were evaluated for statistical significance by
analysis of variance and multiple range tests. In only one of the 4 months was
the acrolein treatment found to be significantly different from controls;
however, the study did not provide sufficient data to determine a threshold of
algal toxicity.
3.6.2 Aquatic Invertebrates
Although acute toxicity data were found for six aquatic invertebrates, only
the waterflea (Daphnia magna) was tested using a well-defined protocol with
measured levels of acrolein. In addition, data from a chronic study with D.
magna showed acrolein to be highly toxic (Table 3-3).
Acute bioassay procedures, recommended by the American Public Health
Association, and chronic testing procedures, developed by Macek et al. and
personnel of EPA's National Water Quality Laboratory, were used to test acrolein
on E). magna (Macek et al., 1976). Proportional dilutions delivered acrolein (99
percent active ingredient) to four replicate jars each containing 2 liters of
one of five test concentrations. For both the acute and chronic studies, 20
test vessels each were used, having 5 or 10 waterfleas in each vessel,
respectively.
In both the acute and chronic studies, young animals (less than 24 hours
old) were used. In the acute studies, survival was measured after 48 hours. In
the chronic studies, survival and production of young were recorded at 1, 2, and
September 1986
3-16
DRAFT—DO NOT QUOTE. OR CITE
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3 weeks. The young at weeks 1 and 2 were discarded. At week 3, 10 young were
selected at random to begin a second-generation test. The same {procedures were
used for second- and third-generation tests. The three generations were studied
for 21-22 days each. Treatment effects were measured by Duncan's multiple range
test.
In selected vessels, acrolein was analyzed by adding 2,4-dinitrophenyl-
hydrazine forming a hydrazone complex, which was extracted with benzene and
determined spectrophotometrically (365 nm). The minimum detectable level was 3
ng/1. Actual acrolein measurements of 3.2-42.7 ug/1 (n=8) agreed fairly well
with the five nominal concentrations (4-60 ug/1). Water quality parameters (the
mean values ± SD) were also measured: alkalinity (33 ± 2.1, n=5); total hardness
(35 ± 2.1, n=5); pH (7.1 ± 0.1, n=5); acidity (4.4 ± 0.8, n=5); temperature (20
± 1 °C); and dissolved oxygen (7.5 ± 0.6, n=29).
The acute 48-hour bioassay yielded an LC5Q (95 percent confidence interval)
of 57 (20-99) ug/1. Based on the three-generation 64-day test,,, the maximum
acceptable toxicant concentration (MATC) ranged from 16.9 to 33.6 ug/1. The
sensitivity of young Daphnia to acrolein was greatest in the second generation,
when survival was significantly reduced at exposures of 16.9 ug/J. In the first
and third generations, however, Daphnia survival was reduced at concentrations
greater than or equal to 33.6 ug/1.
3.6.3 Fish
Acute and chronic studies with several species of fish indicate that
exposures to acrolein for several hours at concentrations below 250 ug/1 are
highly toxic (Table 3-4). At these low levels, acrolein was harmful to all
tested fish species: estuarine and freshwater, coldwater and warawater, juvenile
and adult, and traditionally tolerant and intolerant species. Moreover, all
species showed similarly low acrolein tolerance levels regardless of experi-
mental conditions (e.g., static vs. flowthrough, measured vs. unmeasured
acrolein levels). The 24- to 96-hour LC5Q values for 11 freshwater species
ranged from 46 to 183 ug/1; the single 24-hour LC5Q value for estuarine
killifish was slightly higher, 240 ug/1.
Measured concentrations of acrolein were reported in only two studies. One
of these studies (Macek et al., 1976) is discussed below along with a
comparatively detailed study (Lorz et al., 1979) in which only nominal concen-
trations of acrolein were found.
September 1986
3-18
DRAFT—DO NOT QUOTE OR CITE
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The response of the fathead minnow (Pimephales promelas) to acrolein was
studied using acute and chronic bioassay procedures (Macek et al., 1976).
Mount-Brungs proportional diTutors were used to dose duplicate glass aquaria
(0.08 m ) with flowthrough rates equaling seven aquarium volumes daily. Diluent
well water was analyzed for 28 ions or compounds.
Acrolein (99 percent active ingredient) in 100 percent ethanol was added to
each aquarium to achieve the appropriate test concentration. Acrolein was
analyzed by adding 2,4-dinitrophenylhydrazine to form a hydrazone complex, which
was extracted with benzene and determined spectrophotometrically (365 nm). The
minimum detectable level was 3 ng/1. Mean acrolein concentrations in the
chronic studies ranged from 4.6 ± 2.7 (n=15) ug/1 to 41.7 ± 35.8 (n=28) ug/1.
Total hardness, alkalinity, pH, acidity, temperature, and dissolved oxygen
were measured by APHA (1971) methods.
Fathead minnows exposed to measured acrolein concentrations (0-41.7 ug/1)
for 3-245 days showed no acrolein-related changes in survival, length, or
weight. Eighty fish were tested at each concentration. There were also no
effects on spawning, number of eggs per female, and percent hatchability among
treated and control fish. However, two larval groups of second-generation
fatheads exposed to acrolein at 41.7 pg/1 for 60 days showed only 2 percent
survival, which was significantly lower (p <0.05) than the survival rate for
controls and test concentrations up to 11.4 ug/1 (Macek et al., 1976).
Static 96-hour LC5Q values were reported for yearling coho salmon exposed
to 1 of 12 water-soluble herbicides including acrolein (Lorz et al., 1979). The
estimated 96-hour LC5Q value for acrolein was 68 ug/1 (Table 3-4). Acrolein was
the most toxic, being more than two orders of magnitude more toxic than 10 of
the herbicides including picloram, atrazine, diquat, and paraquat. Lorz and
coworkers concluded that acrolein could kill all salmonid life stages if
normally treated irrigation waters were released into streams before herbicide
inactivation.
Static tests were performed in fiberglass tanks (120 liters, aerated, 85
percent replacement daily) using at least two replicates of seven acrolein
concentrations with 10 fisto per tank. The acrolein used was described only as a
1-liter sample provided by Shell Chemical Company. Acrolein concentrations were
not measured. The fish used were 12- to 17-month-old coho salmon obtained from
Oregon's Fall Creek Salmon Hatchery.
September 1986 3-21 DRAFT—DO NOT QUOTE OR CITE
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Virtually all (119 of 120) coho yearlings exposed to acrolein at 0-50 pg/1
for 144 hours survived, whereas all 40 cohos exposed at 75-100 ng/1 for 144
hours died. There was no increase in mortality in the surviving cohos when
transferred to seawater for 280 hours.
Histologic examination of the gills, kidneys, and liver from the three fish
selected from each group (0, 50, or 100 ug/1) indicated increased Incidences of
tissue lesions that were dose-dependent (Macek et al., 1976).
Lorz et al. (1979) also discussed a large fishkill that occurred in the
Rogue River in Oregon, which was apparently caused by the premature release of
acrolein-treated irrigation water. The water in an irrigation canal, which had
been treated for aquatic plant control with a gaseous form of acrolein
(Magnecide H), was released into the Rogue River after 1 day instead of the
recommended 6 days. For a 10-mile portion of the Rogue River, an estimated
238,000 fish were killed. No other details of this spill were given.
3.7 EFFECTS ON TERRESTRIAL LIFE ;
Although it is released primarily to the aquatic environment, acrolein
contamination of agricultural lands and other terrestrial environments is
possible as a result of irrigation. Although such contamination was identified
as a concern over 20 years ago, actual studies of this phenomenon w
-------
bubbling air through acrolein solutions; acrolein concentrations were measured
by the m-aminophenol method.
Pollen tube elongation was completely inhibited (compared to controls) in
pollen grains exposed to acrolein at 0.4 ppm for 5 hours, 1.3 ppm for 2 hours,
and 1.7 ppm for 1 hour. When exposed at 0.4 ppm for i hour, elongation was only
reduced by 10 percent.
3.7.2 Terrestrial Animals
The toxic effects of acrolein on laboratory rats, cats, rabbits, and dogs
are discussed in chapter 6. No information was found on the compound's effects
on the animals of terrestrial ecosystems.
3.8 BIOCONCENTRATION, BIOACCUMULATION, AND BIOMAGNIFICATION
The environmental hazards posed by accumulation of many chemicals in the
tissues of both aquatic and terrestrial biota are a serious concern.
Environmental levels of certain compounds that appear to be safe even in chronic
toxicity tests may accumulate to harmful levels in many organisms like fish and
birds. Three potentially important accumulation processes include
bioconcentration (direct absorption from the water), bioaccumulation (absorption
from food and/or water), and biomagnification (absorption through the food
chain). .
Available data were limited to a study of the bioconcentration of acrolein
in bluegill sunfish (Lepomis macrochirus). Veith et al. (1980) reported the
bioconcentration factor (BCF) to be 344 with a half-life in tissues of more than
7 days. The fish were exposed to acrolein at 13.1 ± 26 ug/1 for 28 days in a
modified Mount-Brungs intermittent flow proportional dilutor. 14C-labeled
residues in whole fish and water were measured by liquid scintillation after 1,
2, 4, 7, 10, 14, 21, and 28 days of exposure and after 1, 2, 4, and 7 days of
depuration. None of the metabolites was identified.
The measured BCF (344) is higher than would be predicted from acrolein1s
octanol/water partition coefficient (log P), reported to be 1.2 (Veith et al
1980).
No other information was found on acrolein1s bioconcentration in animals or
on its bioaccumulation and biomagnification.
September 1986 3-23 DRAFT-DO NOT QUOTE OR CITE
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3.9 REFERENCES
Alabaster, J. S. (1969) Survival of fish in 164 herbicides, insecticides,
fungicides, wetting agents, and miscellaneous substances. Int. Pest
Control 11: 29-35.
Altshuller, A. P.; McPherson, S. P. (1963) Spectrophotometric analysis of
aldehydes in the Los Angeles atmosphere. J. Air Pollut. Control Assoc. 13:
109-111.
American Conference of Governmental and Industrial Hygienists. (1983) Threshold
limit values for chemical substances and physical agents in the workroom
environment with intended changes for 1983-84. Cincinnati, OH: American
Conference of Governmental and Industrial Hygienists.
Andersson, K.; Hallgren, C.; Levin, J.-O.; Nilsson, C.-A. (1981) Solid
chemosorbent for sampling sub-ppm levels of acrolein and glutaraldehyde in
air. Chemosphere 10: 275-280.
Ayer, H. E.; Yeager, D. W. (1982) Irritants in cigarette smoke plumes. Am. J.
Public Health 72: 1283-1285.
Bond, C. E.; Lewis, R. H.; Fryer, J. L. (1960) Toxicity of various lierbicidal
materials to fishes. Presented at: biological problems in water pollution
seminar; 1959; Washington, DC: U. S. Department of Health, Education, and
Welfare; Public Health Service technical report W60-3; pp. 96-101.
Bowmer, K. H.; Higgins, M. L. (1976) Some aspects of the persistence and fate
of acrolein herbicide in water. Arch. Environ. Contam. Toxicol. 5: 87-96.
Bowmer, K. H.; Sainty, G. R. (1977) Management of aquatic plants with acrolein.
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4. ACROLEIN: MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION
4.1 INTRODUCTION
Acrolein (2-propenal, acrylic aldehyde) is a highly reactive aldehyde
which is used as a synthetic intermediate in a wide variety of industrial
processes. It is also produced in automobile exhaust, tobacco smoke, and as a
degradation product during overheating of oils and fats (Izard and Libermann,
1978; reviewed in Chapter 3). Acrolein is also encountered in mammalian systems
as a metabolite from the metabolic conversion of a variety of other xenobiotics
(Table 4-1).
Acrolein contains a highly reactive, aldehyde-conjugated double bond, with
a strong reactivity in particular toward free sulfhydryl groups or thiols; its
high reactivity has an important relationship to its toxicity (Izard and
Libermann, 1978). Under ambient environmental conditions, acrolein, a volatile
liquid with an acrid and pungent odor, has a high vapor pressure of 400 torr
(at 34.5°C) and hence inhalation is an important route of entry to the body.
It is an irritant and vesicant to the eyes, and to the mucosae of the nose,
bronchial tree and lungs. Acrolein is both soluble in water (about 20 percent,
w/v) and lipid-soluble, and hence may be expected, on the basis of these
physicochemical properties, to diffuse freely across mucosal barriers of the
lungs and gastrointestinal tract into the body. Contrariwise, acrolein's
propensity to react with free thiol groups may effectively restrict free diffu-
sion across membranes at portals of entry into the body as well as across mem-
branes of body compartments within the organism.
4.2 ABSORPTION
4.2.1 Oral
Acrolein is known to be absorbed into the body from the gastrointestinal
tract but the completeness and kinetics of the process have not been quantified.
Rats administered acrolein in corn oil solution by gastric intubation (10 mg/kg
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TABLE 4-1. XENOBIOTICS BIOTRANSFORMED IN VIVO TO ACROLEIN
Compound
Mechanisms:
Experiment Conditions
Investigator
Allyl alcohol
Ally!amine
3-Substituted
Propylamines
2-Substituted Propyl
alcohol
Spermine, Spermidine
2,2,2-THfluoroallyl
ether
Cyclophosphamide,
Isophosphamide
Alcohol dehydrogenase;
isolated hepatocytes
and renal epithelial cells,
rat liver cytosol,
rat liver microsomes
Amine oxidase; rat and
human tissue homogenates
Amine oxidase; cell
culture + serum
Horse liver alco.
dehydrogenase
Amine oxidase; incubation
with calf serum
P450-catalyzed; rat liver
microsomes
P450-catalyzed; rat liver
microsomes
Ohno et al.9 1985
Patel et al.,( 19809 1983
Serafini-Cessi, 1971
Boor and Nel«on, 1981,
1982
Kawase et al,, 1982
Alston et al., 1981
Alarcon, 1964, 1970
Kawase et al., 1982
Murphy et al.., 1983
Alarcon and Meienhofer,
1971
Alarcon et al., 1972
Marinello et al., 1984
body weight) have been found to excrete mercapturic acid metabolites in their
urine, but at such a low yield that the question arises whether incomplete
absorption other unsampled pathways of metabolism was the explanation
(Draminski et al., 1983). Fassett (1962) has reported that, the acute oral
LD5Q for acrolein in rats is 46 mg/kg. Similar oral LD5Q values have been
reported for the rat (42 mg/kg) .and mouse (28 mg/kg) by Albin (1962). These
data presume that death was occasioned by systemic toxicity after absorption.
Albin (1962) records that rats administered acrolein in drinking water up to
200 ppm for 3 mo (about 4,mg/d inges'tion) showed little evidence of toxicity,
either in weight gain or pathological changes.
Acrolein has a great propensity to interact with thiol groups to form con-
jugated compounds, in aqueous solutions at physiological hydrogen ion concen-
trations (Section 4.4.2). The effect on absorption of acrolein by these or
other noncatalytic chemical reactions as induced or modified by the presence
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of food or other conditions of the gastrointestinal tract has not been investi-
gated but these reactions are likely to have a limiting influence on absorption
of the acrolein molecule per se.
4.2.2 Dermal
The acute lethal toxicity of acrolein by the percutaneous route for the
rabbit ranges from LD5Q of 160 to 1000 rag/kg body weight, depending on the
vehicle and concentration (Albin, 1962). It can be inferred from these data
that acrolein is capable of crossing the dermal barrier by diffusion but
further information has not been reported.
4.2.3 Pulmonary
Controlled studies to determine pulmonary uptake rate and absorption of
acrolein into the systemic circulation of man or other mammalian species have
not been conducted. The respiratory mucosa and lung alveolar endothelia may
act as an efficient barrier for low inhaled doses of acrolein because of the
reactivity of acrolein with reduced glutathione content of these cells (McNulty
et a!., 1984; Lam et a!., 1985), and the ability of lung tissue to metabolize
acrolein (Patel et al., 1980).
The acute lethal toxicity (LC5Q) of acrolein in air by inhalation route is:
rat, 8 ppm, 4 hr; dog, 150 ppm, 30 min; and mouse, 175 ppm, 10 min (Albin, 1962).
However, pathological findings were limited principally to the lungs, providing
little indication of absorption into the systemic circulation. Similar observa-
tions have been reported with subchronic and chronic daily inhalation exposures
to rodents (Kutzman et al., 1985; Boulez et al., 1974; Feron et al., 1978; Lyon
et al., 1970; Watanabe and Aviado, 1974).
Egle (1972) has investigated the respiratory uptake of acrolein by
pentobarbital-anesthetized dogs at air exposure concentrations of 0.4 to 0.6
|jg/ml (^ 175 to 265 ppm) during a complete ventilatory cycle. The inhaled
amount of compounds (air concentration times tidal volume) was designed to
miniic the content of acrolein in a 40-ml puff of cigarette smoke (-v 8.2 ug).
The percentage of aldehyde taken up by the respiratory tract of the dog was
calculated using the measured amount inhaled (or exposed to tissues) and the
amount recovered in exhaled air. The retained uptake of acrolein by the total
respiratory tract of dogs (nasal and lung mucosa) at ventilatory rates of 6 to
20 respirations per min averaged 80 to 85 percent and was independent of
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ventilation rate. By trachea! cannulation, uptake and retention by the upper
respiratory tract (between nose and bronchioles) was estimated; it was found
that only about 20 percent of acrolein inhaled reached the lower tract.
Exposure of the lower tract alone (lung mucosa) resulted in about 65 to 70
percent retention of acrolein dose (decreasing slightly with increase of
ventilation rate). Generally, the percent retention of acrolein under these
experimental conditions v/as not affected over a 2 to 3-fold concentration range
but did decrease slightly (85 percent to 77 percent) with increase of tidal
volume (100 ml to 160 ml). These studies do not specifically provide
information on the disposition of the retained acrolein, and because of the
brevity of exposure they do not provide pertinent information on pulmonary
uptake and body disposition for exposure conditions common to the workplace or
ambient environment. However, the results indicate that acrolein, in rela-
tively high air concentrations, is rapidly and extensively removed from inhaled
air by both the- upper and lower respiratory tract, presumably by interacting
with thiol groups of mucus secretions or cell surfaces or by diffusion into
rnucosal and alveolar endothelial cells where the compound may react with
cellular sulfhydryl groups, be metabolized, or be absorbed into the body
(Section 4.4). !
McNulty et al. (1984) and Lam et al. (1985) have exposed rats to acrolein
in air (0.1 ppm to 5 ppm) for 3-hr periods and demonstrated a marked decrease
of non-protein sulfhydryls (glutathione, GSH) in the respiratory nasal mucosa
in a concentration-dependent manner (Figure 4-2). Liver GSH content was also
determined by McNulty and coworkers, but as it did not decrease,! it did not
provide evidence of absorption of acrolein into the systemic circulation for
these experimental conditions.
4.3 DISTRIBUTION AND EXCRETION
The distribution of acrolein into body tissues after entry into the body
from oral or inhalation exposures has not been experimentally documented. Dis-
tribution may be expected to be substantially influenced by the chemical reacti-
vity of acrolein in aqueous solution at physiological hydrogen-ion concentrations
of blood and extracellular fluids. After oral .administration to rats (10 mg/kg
b.w.), mercapturic acid metabolites of acrolein are found in the urine, indica-
ting distribution to the liver and occurrence of hepatic metabolism (Draminski
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et al., 1983). As a dose-dependent depletion of liver reduced glutathione has
been shown to occur with parenteral administration of acrolein (Gurtoo et al.,
1981; Patel and Leibman, 1978), a dose-dependent first-pass effect on hepatic
clearance after oral exposure can be predicted. Similar considerations per-
tain to inhalation exposure. After a 3-hr inhalation exposure of 0.1 to 5 ppm
acrolein in air, a dose-dependent depletion of glutathione of respiratory mucosa
occurs while hepatic glutathione remains unaffected (McNulty et al., 1984; Lam
et al., 1985), indicating that pulmonary clearance and distribution to the liver
are limited. Blood (plasma and red blood cells) normally contains substantial
amounts of reduced glutathione which, acting as a sink for acrolein-glutathione
noncatalytic interactions (Section 4.4.2), may limit acrolein distribution into
body tissues, particularly under conditions of low exposure concentration.
Virtually nothing is known of the elimination or excretion of assimilated
acrolein from the body following oral or inhalation exposures. Of the three
major routes of elimination of xenobiotics from the body—renal, pulmonary, and
metabolic—acrolein has not been found in urine or exhaled breath after oral or
parenteral administration (Draminski et al., 1983; Alarcon, 1976; Kaye, 1973).
Hence the principal route of elimination appears to be by metabolism (Section
4.4). However, quantitative studies designed to determine the disposition of
acrolein have not been carried out after either acute or chronic dosage.
Moreover, blood concentration-time disappearance curves after acrolein dosage
have not been reported, and the kinetics of elimination of acrolein from the
systemic circulation or from the body remain to be determined.
4.4 METABOLISM
4.4.1 Quantitation of Metabolism
The extent to which a given dose of acrolein is metabolized by mammalian
species has not been defined experimentally. Balance studies with labeled or
nonlabeled acrolein administered by any route have not been reported. Hence
dose-metabolism relationships for acrolein are not available.
Table 4-2 gives the known metabolites of acrolein. Of those listed, only
the mercapturic acids S-(hydroxypropyl), S-(carboxyethyl), and S-(propionic
acid methyl ester) have been identified in urine after oral, subcutaneous or
intraperitoneal administration to rats with a yield of 10 to 18 percent of the
given dose (Alarcon, 1976; Kaye, 1973; Draminski, 1983). Kaye and Young (1974)
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TABLE 4-2. DEMONSTRATED BIO-METABOLITES OF ACROLEIN
Metabolite
System
Investigator
S-(hydroxypropyl)-
mercapturic acid
S-(carboxyethyl)-
mercapturic acid
S-(proprionic acid
methyl ester) -
mercapturic acid
Acrylic acid
Glycidaldehyde
Glyceraldehyde
Rat urine
Rat urine
Rat urine
Rat liver
cytosol
Rat liver and
lung microsomes
Rat liver and
lung cytosol
Kaye, 1973
Alarcon, 1976
Draminski et al.,
1983
Draminski et al.,
1983
Pate! et al., 1980
Patel et al., 1980
Pate! et al., 1980
and Alarcon (1976) found significant amounts of S-(hydroxypropyl)-mercapturic
acid in human urine after administration of cyclophosphamide, of which acrolein
is a metabolite. This observation indicates that the mercapturic acid pathway
for acrolein metabolism is not unique to the rodent. Thus at least 80 percent
of an administered dose of acrolein remains unaccounted for. Draminski et
al. (1983) trapped expired air of rats through a charcoal tube after oral ad-
ministration of acrolein (10 mg/kg body weight). GC analysis of the expired
air on Carbowax 20 M-Gas Chromosorb Q revealed the presence of a volatile
compound with a retention time (2.37 min), shorter than acrolein (4.1 min), or
the corresponding alcohol or acid: allyl alcohol (14.9 min), or methyl aerylate
(6.7 min). This fragmentary evidence suggests a portion of the acrolein dose
may be excreted via the pulmonary route in the form of a metabolite, but
apparently not as acrolein itself. There is, however, sufficient reason to
believe, until more complete and satisfactory experimental evidence is avail-
able, that acrolein is extensively, if not completely, biotransformed by all
mammalian species at exposure doses likely to be encountered in the workplace
or ambient environment. As described below (Section 4.4.2), the facile reac-
tivity of acrolein, both noncatalytic and catalytic, in biological systems
provides a potential for extensive biotransformation. It may be noted that
September 1986
4-6
DRAFT—DO NOT QUOTE OR CITE
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acrolein has been demonstrated to be biotransformed by hepatic metabolizing
systems to acrylic acid and to glyceraldehyde (Table 4-2). Acrylic acid may
be incorporated into many endogenous components, including amino acids, fatty
acids and sterols, via the formation of acrylyl CoA by thiokinase, and hence to
propionyl CoA and beta-hydroxypropionyl CoA. In turn, glyceraldehyde is known
to readily enter cellular glycolytic pathways. It is likely, therefore, that a
large portion of a given dose of acrolein may eventually be accounted for
experimentally by expired C02 and/or by incorporation into normal body
constituents.
4.4.2 Noncatalytic Interaction with Sulfhydryl Groups
Acrolein, with a conjugated double bond system and a tendency for its beta
carbon group to be positively charged, has a high chemical reactivity to
reduced sulfhydryl groups (-SH), or thiols at its beta carbon double bond
reactive site (Izard and Libermann, 1978). Acrolein has the potential to react
with these groups in vivo nonenzymatically as well as enzymatically.
Alarcon (1976) investigated the conjugation of acrolein in phosphate
buffer (at pH 7, 7.4, and 8.5, at 23°C) with glutathione (GSH), acetylcysteine
and cysteine by measuring the decrease of acrolein's characteristic UV absor-
bance at 209 nm. With equimolar amounts of acrolein and the sulfhydryl com-
pounds, adduct formation occurred rapidly (dependent on pH), with 50 percent
formation ,at pH 7.4 within 0.5 rain (cysteine), 1.0 min (glutathione), and 3.0
min (acetylcysteine). lodometric titration of the sulfhydryl compounds, before
and after interaction with acrolein, confirmed that the reaction involved the
sulfhydryl anion in these compounds. Furthermore, the adduct could be decom-
posed by incubation at 100°C with regeneration of acrolein in high yield. With
borohydride the adducts were reduced and then chromatographed. Acrolein-
acetylcysteine and acrolein-cysteine adducts yielded on reduction
3-hydroxy-propyl mercapturic acid and 3-hydroxypropyl cysteine, respectively.
Alarcon (1976) notes that 3-hydroxypropyl mercapturic acid was isolated by him
from rat urine after the administration of acrolein subcutaneously, confirming
a prior observation of Kaye (1973).
Esterbauer et al. (1975, 1976) also investigated the kinetics of non-
enzymatic interaction of conjugated carbonyl compounds in aqueous solution with
glutathione and cysteine and found that acrolein reacted more rapidly than
other carbonyls to give very stable adducts. Similar observations have been
September 1986 4-7 DRAFT—DO NOT QUOTE OR CITE
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made by Patel et al. (1984) and Dore and Mental do (1984), who observed also
that acrolein very rapidly conjugates with equimolar amounts of glutathione in
water or buffered solutions. The conjugate(s), however, were not isolated and
identified by these investigators. Gray and Barnsley (1971) have reported
rapid first-order reaction of crotonaldehyde (methylacrolein) with glutathione
in aqueous solution (pH 7.5) that results in one major product upon chromato-
graphing. Ohno et al. (1985) has suggested the possibility that glutathione-
acrolein may also produce a thiohemiacetal adduct. Such an aclduct may be
reversible and form a reservoir for release of acrolein for conversion by
cellular aldehyde dehydrogenase to form acrylic acid, a known metabolite of
acrolein (Patel et al., 1980). The nonenzymatic reactions of aldehydes with
glutathione have been reviewed by Ketterer (1982).
Gurtoo et al. (1981) have demonstrated that acrolein (and cyclophospha-
raide, of which acrolein is a metabolite) injected intraperitoneally into mice
produces a dose-dependent depletion of liver glutathione (Figure 4-1).
However, fairly high doses (500-1000 mg/kg) were required to deplete cellular
glutathione to the 30 percent level. Peak depletion occurred between 2 and 8
hr after injection with a return towards normal levels after 24 hr. Patel and
Liebmann (1978) have observed a similar glutathione depletion in rat liver and
lung after i.p. injection of acrolein. Respiratory mucosal glutathione deple-
tion in rats has also been observed after inhalation of acrolein (McNulty,
1984). These workers exposed rats to 0.1 to 5 ppm acrolein in air for 3 hr and
found a dose-dependent depletion of cellular glutathione (63 percent, at 5 ppm)
of nasal mucosa, even though liver glutathione did not decrease with this expo-
sure. Similar results have been demonstrated by Lam et al. (1985), who exposed
rats to 0.1 to 2.5 ppm for 3 hr and observed a concentration-dependent decrease
in glutathione content of the nasal respiratory mucosa. Their results are
shown in Figure 4-2.
Acrolein added to isolated rat hepatocytes (0.025 to 0.25 mM) induced a
rapid (10 to 30 min), dose-dependent decrease of cellular glutathione (Zitting
and Heinonen, 1980; Dawson et al., 1984). At the highest concentration (0.25
mM), the depletion was virtually complete; at low concentrations recovery of
cellular glutathione occurred gradually over a 2-hr period (Zitting and
Heinonen, 1980). Similar observations with isolated rat hepatocytes have also
been observed with ally! alcohol, the alcohol corresponding to acrolein and
biotransformed by alcohol dehydrogenase to acrolein (Table 4-1). Ohno et al.
September 1986 4-8 DRAFT—DO NOT QUOTE OR CITE
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0)
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oc
Z
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1.5 —
DOSAGE, mg/kg
40 60
100
1.0 -
PHOSPHORAMIDE MUSTARD
• ACROLEIN
• CYCLOPHOSPHAMIDE
*DIETHYLMALEATE
0.5 —
100 200 30O 400 500 600 700 800 900 1000 1100 1200
DOSAGE, mg/kg
Figure 4-1. Dose-related depletion of liver reduced glutathione (GSH) in mice
after intraperitoneal injections of acrolein and cyclophosphamide
(acrolein, a metabolite), and of the classic depletor, diethyl
maleate. The mice were sacrificed 2 hr after injection.
Source: Gurtoo et al. (1981).
September 1986
4-9
DRAFT—DO NOT QUOTE OR CITE
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i—r
80
70
60
c
V
o
o 50
a.
Z
O
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30
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1 |—i—i—i i i i i
% DEPLETION = 30.76 (log [ACROLEIN]) + 47.86
r2 = 0.980
p = 0.0099
0.1
0.2 0.3 0.5 0.8 1
ACROLEIN CONCENTRATION, ppm
1.5 2 2.5 3
Fiaure 4-2 Log-dose relationship of percentage depletion of nonprotein sulfhy-
dryl-groups (glutathione) in the nasal respiratory mucosa of rats
exposed to acrolein for 3 hr. Each data point is the mean of 4
rats ± SE; ^denotes p <0.05 compared to controls.
Source: Lam et al. (1985).
September 1986
4-10
DRAFT—DO NOT QUOTE OR CITE
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(1985) found a rapid (10 to 30 min), dose-dependent decrease of cellular
content of glutathione with addition of 0.1 to 0.5 mM allyl alcohol. Depletion
was blocked by inhibitors of alcohol dehydrogenase (pyrazole or methylpyrazole).
These workers also observed the appearance of acrolein-GSH adducts in the
incubation medium, which, however, were not identified chemically. Kaye
(1973), who found considerable urinary excretion of mercapturates in rats
given ally! alcohol or acrolein subcutaneously, suggested these adducts arise
enzymatically from acrolein-glutathione transferase reactions.- Boyland and
Chasseaud (1967) have demonstrated enzyme-catalyzed conjugations of glutathione
with alpha, beta-unsaturated aldehydes including acrolein-diethylacetal and
crotonaldehyde, a homolog of acrolein.
The observations reviewed above impart considerable uncertainty as to the
extent acrolein reactions with glutathione and other free sulfhydryls in vivo
occur catalytically or noncatalytically (or both). The glutathione trans-
ferases are ubiquitous in vivo, although the evident high reactivity of
acrolein with glutathione and other free sulfhydryls in aqueous solutions at
physiological pH and temperature increases the likelihood that some of these
interactions are not glutathione-transferase-dependent. The further iji vivo
conversion of S-substituted glutathione, whether from enzymatic or nonenzymatic
origins, to the corresponding mercapturic acids is well understood and docu-
mented (Arias and Jakoby, 1976).
*
4.4.3 Enzymatic Pathways
Acrolein appears to undergo biotransformation via two major pathways: 1)
oxidation to acrylic acid and/or conjugations with glutathione; 2) oxidation to
glycidaldehyde and conjugations with glutathione. Figure 4-3 illustrates these
pathways and their interrelationships. The evidence derives primarily from i_n
vitro experiments with liver and lung tissue preparations and from metabolite
identification in urine; no well-designed jn vivo balance studies with labeled
acrolein have been conducted. Consequently the relative importance of the
pathways has not been clearly assessed.
Acrylate pathway: Patel et al. (1980) have demonstrated the conversion of
acrolein to acrylic acid by rat liver (9000 g supernatant fraction, cytosolic
fractions or microsomes fortified with either NAD+ or NADP+). About 20 percent
and 7 percent of added acro'lein was metabolized to acrylic acid by cytosol frac-
tions by these NAD+ and NADP+ dependent aldehyde dehydrogenase reactions in
cytosolic fractions respectively. Disulfiram (at 0.5 and 1.0 mM) completely
September 1986 4-11 DRAFT-DO NOT QUOTE OR CITE
-------
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inhibited acrylic acid formation by 9000 g supernatant fractions, cytosolic
fractions, and by microsomes indicating reaction dependence on aldehyde
dehydrogenase. Similar fractions from rat lung did not form acrylic acid,
presumably because of the absence of both alcohol and aldehyde dehydrogenase
activities in this tissue (Patel and Leibman, 1978). [Acrolein is also a
substrate for human, horse and yeast alcohol dehydrogenase, although for the
pair 2-propenol: 2-propenal, the equilibrium constant favors the conversion of
alcohol to aldehyde (Pietruszko et al., 1973)].
Draminski et al. (1983) isolated S-(proprionic methyl ester) mercapturic
acid in rat urine after oral dosing with 120 mg acrolein/kg. Since, however,
the urine sample was methylated for GC-MS analysis, it was not possible to
exclude the non-methyl ester S-carboxylethyl mercapturic acid as the metabolite
(or both). The acrylic acid ester, methyl acrylate, when administered to rats,
has been shown to yield in rat urine S-carboxylethyl mercapturic acid and its
nonmethyl ester, S-(proprionic acid methyl ester) mercapturic acid in a ratio
20:1 (Delbressine et al., 1981). Thus Draminski et al. suggest that acrylic
acid formed from acrolein is esterified to methyl acrylate which then conju-
gates with glutathione catalyzed by glutathione S-alkenetransferase as de-
scribed by Boyland and Chasseaud (1968). Since there is uncertainty as to the
complete substrate specificity of glutathione S-alkene transferase(s) (Boyland
and Chasseaud, 1968; Delbressine et al., 1981; Kaye, 1973), the possibility
remains that acrylic acid and acrolein themselves may also serve as substrates
for these transferases. Kaye (1973) and also Alarcon (1976) have shown that
rats injected subcutaneously or intraperitoneally with acrolein (16 to 2,500
mg/kg) excreted in the urine 10 percent to 18 percent of the dose (increasing
with dose) as S-(hydroxypropyl)-mercapturic acid. Kaye suggested that this
metabolite was likely the result of acrolein conjugation with glutathione
catalyzed by one of the many glutathione S-alkene transferases described by
Boyland and Chasseaud (1967, 1968). In fact, Boyland and Chasseaud (1967) have
demonstrated that acrylic acid esters, acrylic acid homologs and acrolein
diethylacetal and acrolein homologs are substrates for these transferases. At
some stage during the conversion of the glutathione conjugate into the
mercapturic acid, reduction of the carbonyl moiety to an alcohol group must
occur as well as peptidase conversion of the conjugate to the mercapturic acid.
Glycidaldehyde pathway: Patel et al. (1980) have demonstrated with both
rat liver and lung microsome preparations fortified with NADPH the formation of
the epoxide of acrolein, glycidaldehyde and the hydration of the epoxide to
September 1986 4-13 DRAFT—DO NOT QUOTE OR CITE
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0.5
GLYCIDERALDEHYDE FORMATION
• LUNG MICROSOMES
• LIVER MICROSOMES ;
GLYCIDOL FORMATION
LUNG MICROSOMES
LIVER MICROSOMES
8 12
TIME, minutes
Figure 4-4. Rate of formation of epoxide from acrolein and from ally! alcohol
by microsomes isolated from rat liver or lung.
Source: Patel et al. (1980).
September 1986
4-14
DRAFT—DO NOT QUOTE OR CITE
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glyceraldehyde. Figure 4-4 shows the time course of epoxidation of acrolein
(and of the corresponding alcohol, ally! alcohol) by rat liver and lung micro-
somes. The apparent decline in rate of production of the epoxide after about 6
min incubation can be accounted for by hydration to glyceraldehyde (by epoxide
hydratase) and by inactivation of microsomal cytochrome C reductase and con-
version of P450 to P420 by acrolein or its epoxide at a similar time course
(Patel et al., 1980, 1984; Marinello et al., 1981). Glycidaldehyde was also
found by Patel et al. (1980) to be a substrate for both rat liver and lung
cytosolic activity presumed to be glutathione epoxide transferase(s).
Fjellstedt et al. (1973) previously have shown that glycidaldehyde was a
substrate for purified glutathione epoxide transferase(s) isolated from rat
liver cytosol. While the structure of the product of the enzymatic reaction of
glutathione and the epoxide was not rigorously established, evidence was
obtained to suggest that the product was an unsymmetrical thioether between
glutathione and one of the oxirane carbons. These observations indicate that
glycidaldehyde conjugates with glutathione with subsequent conversion to a
mercapturic acid, possibly S-(hydroxypropyl)-mercapturic acid or S-(carboxy-
ethyl)-mercapturic acid, which are found in rat urine (Table 4-2; Figure 4-3).
Glyceraldehyde, the hydration product of glycidaldehyde, may be expected to
enter the normal cellular energy pathways by conversion to glyceraldehyde-3
phosphate by the enzyme ATP: D-glyceraldehyde 3-phosphate transferase
[E.C.2.T.1.28] and thereby enter the glycolytic pathway to pyruvate or lactate.
This may also represent a principal pathway for acrolein biotransformation.
4.4.4 Covalent Binding
Reactivity to nucleic acid in solution: Acrolein and other alpha,
beta-unsaturated carbonyl compounds such as crotonaldehyde can interact direct-
ly, not only with free thiols (Section 4.4.2), but also with nucleic acids
(Izard and Libermann, 1978). Descroix et al. (1971) have found that even in
aqueous solution jn vitro, acrolein reacts with nucleotides and DNA, using as
a measure of reaction the disappearance of the UV spectra of acrolein. Munsch
et al. (1974) interacted 3H-acrolein (0.6 mM) in tris buffer (pH 7.0; 38°C)
with calf thymus DNA and synthetic nucleotide polymers and found covalent
binding of radioactivity in the ratio of 1 to 2 molecules of acrolein per 1000
nucleotide units. Recently, Chung et al. (1984) have demonstrated direct adduct
formation from acrolein-deoxyguanosine and acrolein-calf thymus DNA interactions
September 1986 4-15 DRAFT—DO NOT QUOTE OR CITE
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in phosphate buffer at pH 7, 37°C. These investigators identified cyclic
l,N2-propanodeoxyguanosine adducts from these interactions, with a possibility
of other adducts with other DNA bases or cross-linking adducts. G'lycidaldehyde,
the bifunctional epoxide metabolite of acrolein (Figure 4-3), has also been
shown to react with guanosine jn vitro to form a cyclic derivative between N
and N2 (Goldschraidt et al., 1978).
Binding in biological systems in vitro and ijn vivo: Lam et al. (1985)
have reported evidence that acrolein can interact with nucleoprotein (like
formaldehyde; Ross and Shipley, 1980; Casanova-Schmitz et al., 1984) to produce
"DNA-protein cross-links" as a result of covalent binding. Cross-linking of
DNA to proteins causes a decrease in the extractability of DNA from tissue pro-
teins. The "absent" DNA can be quantitatively recovered from the proteins after
proteolytic digestion. Acrolein was added to rat respiratory mucosa tissue-
homogenates in vitro (0°C, 10 min incubation) and subsequently DNA was isolated
by extraction with chloroform/iso-amyl alcohol/phenol (14/1/25) solvent mixture
followed by centrifuging. DNA in the aqueous and interface layers was deter-
mined before and after digestion with proteinase K. Table 4-3 shows a concen-
tration-dependent increase in the percentage of interfacial layer DNA, indi-
cating the formation of DNA-protein cross-links. However, similar experiments
with respiratory mucosa taken from rats exposed to acrolein (2 ppm for 6 hr)
in vivo revealed no significant increase in the percent interfacial DNA in com-
parison with unexposed rats. Thus, acrolein appeared to form few, if any, DNA-
protein cross-links with jn vivo exposure conditions. Acrolein did, however,
enhance DNA-protein cross-links in vivo from formaldehyde exposure, presumably
by causing glutathione depletion and thereby decreasing potential for formalde-
hyde glutathione detoxification pathway.
Munsch et al. (1974) have investigated the in vivo binding of H-acrolein
to nucleic acids and protein in the 3-day partial hepatectomized rat model of
liver regeneration. After intraperitoneal injections, partition of radioactiv-
ity of 3H-acrolein in the liver cellular fractions—acid-soluble, lipids,
proteins, RNA and DNA~was approximately 93, 3.5, 1.25, 0.65 and 0.35 percent
respectively of total liver radioactivity, with little change in distribution
percentage at timed intervals 10 min to 24 hr. These results suggest rapid
(within 10 min) and stable binding to cellular macromolecules. Table 4-4 shows
the time course of the binding of H-acrolein to nucleic acid and protein
isolated from liver. The binding of radioactivity to these macromolecules is
September 1986 4-16 DRAFT-DO NOT QUOTE-: OR CITE
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TABLE 4-3. DNA-PROTEIN CROSS-LINKING: QUANTITIES OF DNA RECOVERED IN THE
AQUEOUS PHASE AND IN THE AQUEOUS-ORGANIC INTERFACE OF RAT NASAL MUCOSAL
HOMOGENATES INCUBATED WITH SELECTED CONCENTRATIONS OF ACROLEIN*
Acrolein
Concentration
(mM)
0
0.3
3.0
30.0
DNA (mg/g tissue)
Aqueous Interface
4.69 0.48
4.14 0.43
4.45 0.69
3.64 1.26
Interfacial
DNA
(%)
9.3
9.4
13.4
25.7
*Each homogenate (in 0.1 M phosphate containing 5 mM EDTA pH 8) was pre-
pared from the respiratory mucosa of 3 rats and was incubated with acrolein
at the indicated concentration for 10 min (0°C). Aqueous and interfacial DNA
were isolated by extraction with a phenolic solvent system and protein
digestion.
Source: Lam et al. (1985).
TABLE 4-4. COVALENT BINDING OF 3H-ACROLEIN* TO RNA, DNA AND PROTEIN
OF REGENERATING RAT LIVER
Time After
Injection
10 min
30 min
1 hr
3 hr
5 hr
24 hr
Radioactivity— Acrolein Equivalents
Protein
p mol/mg
4.57**
5.85
6.0
7.6
7.02
7.75
RNA
p mol/ug P
0.6
0.48
0.48
0.48
0.48
0.48
DNA
p mol/ug P
0.7
0.85
0.77
0.67
0.95
0.60
*3H-acrolein injected intraperitoneally into male Wistar rats, 70 hr after
partial hepatectomy (75 mCi/m mole).
**Mean of 4 animals.
Source: Munsch et al. (1974).
September 1986 4-17 DRAFT—DO NOT QUOTE OR CITE
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3
rapid, 80 to 90 percent occurring within 10 min after injection of H-acrolein.
The binding to protein is augmented significantly during the first hour follow-
ing injection, but the binding to DNA and RNA is not significantly increased
with time. This may suggest that the acrolein binding to nucleic acids is in
large part direct, i. e. nonenzymatic, and does not require prior metabolism.
Munsch and Frayssinet (1971) have shown that acrolein (50 to 270 ug/100
gm; i.p.), in a dose-dependent manner, strongly inhibits the de novo synthesis
of DNA and RNA in the liver and lungs of partially hepatectomized rats, as
measured by labeled thymidine, orotic acid and 32P incorporation., Previously,
Leuchtenberger et al. (1968) had observed that acrolein inhibited RNA synthesis
by mouse-kidney epithelial tissue in cell culture. Investigations of this
phenomenon by Munsch and associates (Moule and Frayssinet, 1971; Munsch et al.,
1973, 1974) indicate that acrolein inhibits RNA polymerase transcription by
acting on the enzyme itself rather than on the DNA template. Moule and
Frayssinet (1971) demonstrated that the addition of acrolein to isolated rat
liver nuclei in tris buffer in vitro inhibited in a dose-dependent manner RNA
polymerase-dependent polymerization of ATP, UTP and GTP. Inhibition was
independent of added calf thymus DNA, whereas addition of progressively higher
levels of RNA polymerase enzyme led to a partial recovery. Munsch and
Frayssinet (1973) studied also the effects of acrolein on DNA synthesis In
vitro. These investigators used two different DNA polymerases: regenerating
rat liver.DNA polymerase and E. coli DNA polymerase I, with templates of calf
thymus DNA or linear polymers of adenine-thymidine nucleotidesi. Acrolein
produced an inhibitory effect on rat DNA polymerase activity at concentrations
above 8 x 10"5M (although an activation effect occurred at low concentrations).
In the presence of 2-mercaptoethanol, acrolein inhibitory effect was suppressed
and enzyme activity restored. Hence these workers postulated that acrolein, by
interacting with the active thiol groups of rat DNA polymerase, inhibits enzyme
activity. This hypothesis was consistent with the observation that E. coli
enzyme devoid of SH groups in its active center was not inhibited by acrolein.
Munsch et al. (1974) further explored the effect of acrolein on rat liver DNA
polymerase through binding studies with 3H-acrolein. When DNA jjolymerase was
incubated in vitro with 3H-acrolein (6 x 10"4M), binding to the enzyme in-
creased linearly with 3H-acrolein concentration. Binding to enzyme pre-
incubated with mercaptoethanol was blocked, and addition of mercaptoethanol
after binding occurred could not reverse the covalent binding. Binding of H
September 1986 4-18 DRAFT-DO NOT QUOTE OR CITE
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acrolein to E. coll DNA polymerase also occurred but was 10 to 20 times less
than that with rat liver DNA polymerase. Binding also occurred to calf thymus
DNA or synthetic nucleotide polymer templates but at higher concentrations (6 x
10"4M). These investigators suggest that acrolein has a greater reactive
affinity for rat liver DNA polymerase sulfhydryl groups than to DNA nucleotide
binding.
Marinello et al. (1984) studied the binding of lC-acrolein to rat hepatic
microsomes, and the effect of glutathione and other free sulfhydryl and amino
groups on the binding. The reaction was carried out in the absence of coenzyme
(NADPH) so that binding was not mediated by metabolism. Table 4-5 shows that
in the absence of metabolism significant binding occurred. Glutathione, cys-
teine and acetylcysteine effectively blocked the binding which can therefore
be assumed to be primarily with microsomal sulfhydryl groups. The amino group
in lysine was only marginally effective in blocking binding. Table 4-6 shows
14
the results, with added coenzyme, of metabolism-mediated binding. While C-
acrolein binding per se in the absence of metabolism showed significant binding
to the microsomes (Table 4-5), the binding was substantially enhanced in the
presence of NADPH, suggesting strongly that a metabolite of acrolein (probably
TABLE 4-5. BINDING OF 14C-ACROLEIN TO HEPATIC MICROSOMES FROM
PHENOBARBITAL-TREATED RATS IN THE ABSENCE OF NADPH
Treatment 14C-acrolein bound,
nmol/mg protein
None
Acrolein (4mM) 37
As % of 14C-acrolein
bound
Acrolein (4mM) 100
Plus Glutathione 10
Plus Cysteine 18
Plus N-acetylcysteine 28
Plus Lysine 88
Source: Marinello et al. (1984).
September 1986 4-19 DRAFT—DO NOT QUOTE OR CITE
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TABLE 4-6. METABOLISM-MEDIATED BINDING OF 14C-ACROLEIN TO HEPATIC
MICROSOMES PHENOBARBITAL-TREATED RATS; NADPH ADDED
Radioactivity as
Treatment p mol acrolein/mg protein
Exp. 1 Exp. 2
With added NADPH
minus without NADPH 2707 5700
With added NADPH
minus without NADPH
plus SKF 525A 6 <6
Control microsomes (saline
treated); with NADPH
minus NADPH 23 <10
Source: Marine!lo et al. (1984).
glycidaldehyde) was also binding to the microsomes. The enhancement of binding
is clearly metabolism-dependent since SKF-525A completely abolished the differ-
ence in binding observed between plus and minus NADPH. In addition, compared
to phenobarbital-treated microsomes, noninduced control microsomes generated
considerably less metabolite binding (Table 4-5).
Patel et al. (1984), Gurtoo et al. (1981, 1983), Ivanetich et al. (1978)
and Marine-Ho et al. (1981) have demonstrated total destruction of liver
microsomal NADPH-cytochrome C reductase activity and reduction of P450
cytochrome activity by acrolein covalent interaction and binding to free
cysteine sulfhydryl groups including those in or near the active sites of these
cytochromes. Experiments ir\ vitro with purified cytochromes and liver micro-
somes indicate that acrolein inactivates these enzymes by forming adducts with
sulfhydryl groups in their protein structures by direct, non-catalytic inter-
action. Microsomal protein-bound sulfhydryl groups were decreased upon incu-
bation with acrolein, while addition of chemicals containing a free sulfhydryl
group (glutathione, dithiothreitol, N-acetylcysteine, cysteine) prior to the
addition of acrolein provided significant protection of cytochrome activity.
September 1986 4-20 DRAFT-DO NOT QUOTE OR CITE
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4.5 SUMMARY ..,....__. , ; ; ;
Acrolein, CH2=CHCHO, is a highly reactive aldehyde which reacts nonenzyma-
tically and enzymatically to 1) form stable adducts with extracellular and
intracellular glutathione and other .free thiol groups, 2) form .adducts with
nucleic acids and proteins, 3) cross-links nucleic acids and proteins, and 4)
reacts with enzymes and membranes to cause,a variety of biochemical consequenc-
es such as, impairment of DNA replication, inhibition of protein synthesis and
mitochondria! respiration, loss of liver and lupg microsomal enzyme activities,
and .other parameters of cellular integrity.; This propensity for covalent
binding by acrolein provides the basis for its cellular toxicity.
The reactivity and toxicity of acrolein is manifested in the
gastrointestinal and pulmonary tracts, the principal sites of exposure. There
is no documented evidence (for instance, blood determinations) that for expo-
sure concentrations below aversive levels, acrolein breaches the protective
mechanisms at these portals to gain entry to the general circulation. Bron-
chial and mucosal secretions, and mucosal and endothelial tissues at these
locations contain high concentrations of free thiols. Lung, gastrointestinal
mucosa and liver also contain effective metabolizing systems with high capacity
to biotransform acrolein. Hence the extent and kinetics of absorption of acro-
lein into the body during oral and inhalation exposure remain to be determined.
Acrolein can be formed jji vivo via the metabolism of a number of xenobio-
tics. For example, ally! alcohol, CH2CHCH2OH, causes intensive hepatic peri-
portal necrosis in rats after oxidation by hepatic alcohol dehydrogenase to
acrolein. Since protection from ally! alcohol toxicity is afforded by
pretreatment with sulfhydryl group donors (cysteine or N-acetylcysteine), it is
presumed that when free thiol groups are depleted by acrolein interaction,
acrolein combines covalently with other nucleophilic groups of cellular
macromolecules and thus leads to cellular damage.
Acrolein has been demonstrated to be metabolized jn vivo, particularly by
liver and lung parenchyma! tissues. Two major pathways have been demonstrated:
(1) nonenzymatic and/or glutathione transferase reactions to form stable
adducts with glutathione and other thiols leading to mercapturic acid
metabolites, and (2) oxidative metabolism to (a) acrylic acid via aldehyde
dehydrogenase activity, and (b) to the epoxide glycidaldehyde via microsomal
P450 oxidation system. Glycidaldehyde, a reactive metabolite capable of
covalent binding, is further metabolized to innocuous metabolites by cellular
September 1986 • 4-21 DRAFT—DO NOT QUOTE OR CITE
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glutathione epoxide transferase or by epoxide hydrase. The relative importance
of these various pathways has not been assessed, nor has the effect of acrolein
dosage on metabolic disposition. However, acrolein appears to be extensively,
if not completely, metabolized in mammalian systems; acrolein itself has not
been found in urine or exhaled air of rodents after parenteral administration
of high doses. Hence metabolism appears to be the principal route of elimina-
tion from the body. Mercapturic acid derivatives are found in the urine of
rodents after administration but account for less than 20 percent of dose.
Further information on the distribution of acrolein jji vivo, dose-metabolism
relationships, profiles of metabolism pathways across species, and relation of
covalent binding to toxicity are needed.
September 1986 4-22 DRAFT—DO NOT QUOTE OR CITE
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4.6 REFERENCES
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Alarcon, R. A. (1970) Acrolein IV. Evidence for the formation of the cytotoxic
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Bioche.m. Biophys. 137: 365-372.
Alarcon, R. A. (1976) Formation of acrolein from various amino-acids and
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Alarcon, R. A.; Meienhofer, J. (1971) Formation of the cytotoxic aldehyde
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3-nitro-l-propanol (miserotoxin aglycone) to cytotoxic acrolein by alcohol
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Arias, I. M.; Jakoby, W. B., eds. (1976) Glutathione: metabolism and function.
New York: Raven Press.
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Boyland, E.; Chasseaud, L. F. (1967) Enzyme-catalysed conjugations of gluta-
thione with unsaturated compounds. Biochem. J. 104: 95-102.
Boyland, E.; Chasseaud, L. F. (1968) Enzymes catalysing conjugations of gluta-
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651-661.
Casanova-Schmitz, M.; Starr, T. B.; Heck, H. d'A. (1984) Differentiation
between metabolic incorporation and covalent binding in the labeling of
September 1986 4-23 DRAFT—DO NOT QUOTE OR CITE
-------
macro-molecules in the rat nasal mucosa and bone marrow by inhaled 14C-
and 3H-formaldehyde. Toxicol. Appl. Pharmacol. 76: 26-44.
Chung, F. L.; Young, R.; Hecht, S. S. (1984) Formation of cyclic 1, N2-propano-
deoxyguanosine adducts in DNA upon reaction with acrolein or crotonalde-
hyde. Cancer Res. 44: 990-995. ;
Dawson, J.; Norbeck, K.; Anundi, I.; Moldeus, P. (1984) The effectiveness of
N-acetylcysteine in isolated hepatocytes, against the toxicity of
paracetamol, acrolein, and paraquat. Arch. Toxicol. 55: 11-1!;>.
Delbrissine, L. P. C.; Seuter-Berlag, F.; Seutter, E. (1981) Identification of
urinary mercapturic acid formed from acrylate, methacrylate and crotonate
in the rat. Xenobiotica 11: 241-247.
Descroix, H. S.; Puisseux-Dao, S.; Suard, M. (1981) Mise en evidence par
spectrophotometrie ultraviolette d'in interaction entre racroleine et le
cytidine monophosphate en solution aqueuse. C. R. Acad. Sci. Ser. D. 272:
2472-2475.
Dore, M.; Montaldo, C. (1984) Studies on the in vitro binding of ally! alcohol
and its metabolites to reduced glutathione. Boll. Soc. Ital, Biol. Sper.
60: 1497-1501.
Draminski, W.; Eder, E.; Henschler, D. (1983) A new pathway of acrolein meta-
bolism in rats. Arch. Toxicol. 52: 243-247.
Eder, E.; Neudecker, T.; Lutz, D.; Henschler, D. (1982) Correlation of alkylat-
ing and mutagenic activities of allyl and allylic compounds: standard
alkylation test vs. kinetic investigation. Chem. Biol. Interact. 38:
303-315.
Eder, E.; Henschler, D.; Neudecker, T. (1983) Mutagenic properties of allylic
and alpha, beta-unsaturated compounds: consideration of alkylating
mechanisms. Xenobiotica 12: 831-848.
Egle, J. L., Jr. (1972) Retention of inhaled formaldehyde, proprionaldehyde,
and acrolein in the dog. Arch. Environ. Health 25: 119-124.
Esterbauer, H.; Zollner, H.; Schplz, N. (1975) Reaction of glutathione with
conjugated carbonyls. Z. Naturforsch. C. Biosc. 30: 466-473.
Esterbauer, H.; Ertl, A.; Scholz, N. (1976) Reaction of cysteine with alpha,
beta-unsaturated aldehydes. Tetrahedron 32: 285-289.
Fassett, D. W. (1962) Aldehydes and acetates. In: Patty, F. A., ed. Industrial
hygiene and toxicology, v. 2, New York, NY: John Wiley & Sons; p. 1977.
Feron, V. J.; Kruysse, A.; Til, H. P.; Immel, H. R. (1978) Repeated exposure to
acrolein vapour: subacute studies in hamsters, rats and rabbits.
Toxicology 9: 47-57.
Fjellstedt, T. A.; Allen, R. H.; Duncan, B. K.; Jakoby, W. B. (1973) Enzymatic
conjugation of epoxides with glutathione. J. Biol. Chem. 248: 3702-3707.
September 1986 4-24 DRAFT-DO NOT QUOTE OR CITE
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Goldschmidt, B. M.; Biazej, T. P.; van Duuren, B. L. (1978) The reaction of
guanosine and deoxyguanosine with glycidaldehyde. Tetrahedron Lett. 13:
1583-1587.
Gray; J. M; Barnsley, E. A. (1971) The metabolism of crotyl phosphate, crotyl
alcohol and crotonaldehyde. Xenobiotica 1: 55-67.
Gurtoo, H. L.; Marinello, A. J.; Berrigan, M. J.; Bansal, S. K.; Paul, B.;
Pavelic, Z. P.; Struck, R. F. (1983) Effects of thiols on toxicity and
carcinostatic activity of cyclophosphamide. Sem. Oncol. 10: 35-45.
Gurtoo, H. L.; Marinello, A. J.; Struck, R. F. (1983) Studies on the mechanism
of denaturation of cytochrome P450 by cyclophosphamide and its
metabolites. J. Biol. Chem. 256: 11691-11701.
Ivanetich, K. M.; Lucas, S.; Marsh, J. A.; Zinman, M. R.; Katz, I. D.;
Bradshaw, J. J. (1978) Organic compounds. Their interaction with and
degradation of hepatic microsomal drug-metabolizing enzymes in vitro. Drug
,Metab. Dispos. 6: 218-225.
Izard, C.; Libermann, C. (1978) Acrolein. Mutat. Res. 47: 115-138.
Kaye, C. M. (1973) Biosynthesis of mercapturic acids from ally! alcohol, ally!
esters, and acrolein. Biochem. J. 134: 1093-1101.
Kaye^ C. M.; Young, L. (1974) Acrolein as a possible metabolite of
cyclophosphamide in man. Biochem. Soc. Trans. 2: 308-310.
Ketterer, B. (1982) The role of nonenzymatic reactions of glutathione in xeno-
biotic metabolism. Drug Metab. Rev. 13: 161-187.
Kutzman, R. S.; Popenoe, E. A.; Schmaeler, M.; Drew, R. T. (1985) Changes in
rat lung structure and composition as a result of subchronic exposure to
acrolein. Toxicology 34: 139-151.
Lam, C. W.; Casanova, M.; Heck, H. d'A. (1985) Depletion of nasal glutathione
by acrolein and enhancement of formaldehyde-induced DNA-protein cross-
linking by simulanteous exposure to acrolein. Arch. Toxicol.: in press.
Leuchtenberger, C.; Schumacker, M.; Haldeman, T. (1968) Further cytological and
cytochemical studies on the biological significance of the gas phase of
fresh cigarette smoke. Z. Praeventivmed. 13: 130-141.
Lutz, D.; Eder, E.; Neudecker, T.; Henschler, D. (1982) Structure-mutagenicity
relationship in alpha, Beta-unsaturated carbonylic compounds and their
corresponding allylic alcohols. Mutat. Res. 93: 305-315.
Lyon, J. P.; Jenkins, L. J., Jr.; Jones, R. A.; Coon, R. A.; Siege!, J. (1970)
Repeated and continuous exposure of laboratory animals to acrolein.
Toxicol. Appl. Pharmacol. 17: 726-732.
Marinello, A. J.; Berrigan, M. J.; Struck, R. F.; Guengerich, F. P.; Gurtoo, H.
L. (1981) Inhibition of NADPH-cytochrome P-450 reductase by cyclophospha-
mide and its metabolites. Biochem. Biophys. Res. Comm. 99: 399-406.
September 1986 4-25 DRAFT—DO NOT QUOTE OR CITE
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Marinello, A. J.; Bansal, S. K.; Paul, B.; Koser, P. L.; Love, J.; Struck, R.
F.; Gurtoo, H. L. (1984) Metabolism and binding of cyclophosphamide and
its metabolite acrolein to rat hepatic microsomal cytochrorae P-450. Cancer
Res. 4615-4621.
McNulty, M. J.; Heck, M. d'A.; Casanova-Schmitz, M. (1984) Depletion of gluta-
thione in rat respiratory mucosa by inhaled acrolein. Fed. Proc. 43:
Abstract 1695.
Munsch, N.; Frayssinet, C. (1971) Action of acrolein on nucleic acid synthesis
in vivo. Biochimie 53: 243-248.
Munsch, N.; de Recondo, A.-M.; Frayssinet, C. (1973) Effects of acrolein on DNA
synthesis in vitro. FEBS Lett. 30: 286-290.
Munsch, N.; deRecondo, A. M.; Frayssinet, C. (1974) In vitro binding of 3H-
acrolein to regenerating rat liver DNA polymerase. Experientia 30:
1234-1236.
Munsch, N.; Marano, F.; Frayssinet, C. (1974) Incorporation d'acroleine 3H dans
le foie du rat et chez Dunaliella bioculata [Incorporation of tritium-
labeled acrolein in rat liver and in Dunaliella bioculata]. Biochimie 56:
1433-1436.
Murphy, M. J.; Dunbar, D. A.; Kaminsky, L. S. (1983) Acute toxicity of fluori-
nated ether anesthetics: role of 2,2,2-trifluoroethanol and other metabo-
lites. Toxicol. Appl. Pharmacol. 71: 84-92.
Neudecker, T.; Lutz, D.; Eder, E.; Henscheler, D. (1981) Crptonaldehyde is
mutagenic in a modified S. typhimurium mutagenicity testing system. Mutat.
Res. 91: 27-31.
Ohno, Y.; 'Jones, T. W.; Ormstead, K. (1985) Ally! alcohol toxicity in isolated
epithelial cells: protective effects of low molecular weight thiols. Chem.
Biol. Interact. 52: 289-299.
Ohno, Y.; Ormstad, K.; Ross, D.; Orrenius, S. (1985) Mechanism of ally! alcohol
toxicity and protective effects of low-molecular-weight thiols studied
with isolated rat hepatocytes. Toxicol. Appl. Pharmacol. 78: 169-179.
Patel, J. M.; Leibman, K. C. (1978) Metabolism of ally! alcohol and acrolein by
rat liver and lung preparations. Pharmacologist 20: 181.
Patel, J. M.; Wood, J. C.; Leibman, K. C. (1980) The biotransformation of ally!
alcohol and acrolein in rat liver and lung preparations. Drug Metab.
Dispos. 8: 305-308.
Patel, J. M.; Gordon, W. P.; Nelson, S. D.; Leibman, K. C. (1983) Comparison of
hepatic biotransformation and toxicity of ally! alcohol and [1,1,-2H2]
ally! alcohol in rats. Drug Metab. Dispos. 11: 164-165.
Patel, J. M.; Ortiz, E.; Kolmstetter, C.; Leibman, K. C. (1984) Selective
inactivation of rat lung and liver microsomal NADPH-cytochrome c reductase
by acrolein. Drug Metab. Dispos. 12: 460-463.
September 1986 4-26 DRAFT-DO NOT QUOTE OR CITE
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Pietruszko, R.; Crawford, K.; Lester, D. (1973) Comparison of substrate speci-
ficity of alcohol dehydrogenases from human liver, horse liver and yeast
towards saturated and 2-enoic alcohols and aldehydes. Arch. Biochem.
Biophys. 159: 50-60.
Ross, N. E.; Shipley, N. (1980) Relationship between DNA damage and survival in
formaldehyde-treated mouse cells. Mutat. Res. 79: 277-283.
Serafini-Cessi, F. (1972) Conversions of ally! alcohol into acrolein by rat
liver. Biochem. J. 128: 1103-1107.
Watanabe, T.; Aviado, D. M. (1974) Functional and biochemical effects on the
lung following inhalation of cigarette smoke and constituents. II.
Skatole, acrolein, and acetaldehyde. Toxicol. Appl. Pharmacol. 30:
201-209.
Zitting, A.; Heinonen, T. (1980) Decrease of reduced glutathione in isolated
rat hepatocytes caused by acrolein, acrylonitrile, and the thermal degra-
dation products of styrene copolymers. Toxicology 17: 333-341.
September 1986 4-27 DRAFT—DO NOT QUOTE OR CITE
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5. MAMMALIAN TOXICITY
5.1 ACUTE TOXICITY
The acute toxicity values for acrolein that were reported in the available
literature are compiled in Table 5-1. Several of the values were based on
range-finding studies that did not give complete experimental details as to
age, strain, and number of animals. The early acute toxicity studies have been
reviewed by A!bin (1975).
5.1.1 Inhalation
Skog (1950) studied groups of eight rats exposed for 30 minutes to
2
acrolein vapor at dose ranges between 100 and 200 mg/m and observed the
animals for up to 3 weeks. The LCcn. calculated by probit analysis, was 300
mg/m (130 ppm), with all deaths occurring within 68 hours. Toxic signs
observed in the animals were marked respiratory difficulties, eye irritation,
lacrimation, and nasal discharge. Histopathologic changes in the lungs of the
animals that died were hemorrhages and intraalveolar and perivascular edema.
Salem and Cullumbine (1960) studied the inhalation toxicity of acrolein as
one substance of a series of saturated and unsaturated aldehydes. Groups of 50
mice, 20 guinea pigs, and 5 rabbits (strains unspecified) were exposed to
either an aerosol or vapor of acrolein for up to 10 hours or until the animals
died. Table 5-2 summarizes the results. The aerosol was produced by injecting
air through an all-metal "Collision Spray" into 50 ml of acrolein in an
all-glass container at room temperature; the mean particle size was 0.7 urn
diameter. Vapor was generated by air injection into acrolein warmed to 50 C.
Aerosols and vapors were sampled and analyzed with hydroxylamine. The mice
were found to be more susceptible to the acrolein exposure than the guinea pigs
and rabbits; no differences were noted between administration by aerosol or
vapor. Under the conditions of the study, acrolein was slightly more toxic
than crotonaldehyde and considerably more toxic than acetaldehyde,
propionaldehyde, and butyra'Idehyde; formaldehyde was not toxic. The animals
exhibited eye irritation, labored breathing, signs of bronchial constriction,
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TABLE 5-2. ACUTE INHALATION TOXICITY OF ACROLEIN
Median lethal doses, x 10° mg x
Species VaporAerosol
Mouse
Guinea pig
Rabbit
0.7
1.3
1.4
0.6
1.1
1.2
Mean Concentration, mg/m3
5225
(2271 ppm) (2010 ppm)
SOURCE: Salem and Cullumbine (1960).
and convulsions probably due to anoxia; death followed. On necropsy, the
animals had expanded edematous and hemorrhagic lungs; those dying in the first
5 hours also had distended alveoli and ruptured alveolar septa.
The 4-hour LC5Q in hamsters for inhaled acrolein was reported to be 25.4
ppm (58 mg/m ) by Feron and Kruysse (1977).
An investigation of the interaction of acrolein and formaldehyde as
sensory irritants was made by (Kane and Alarie, 1978). In this study, groups
of four mice (Swiss-Webster strain, 20-30 g each) were exposed in inhalation
chambers to varying concentrations of acrolein, formaldehyde alone, or the two
in combination. Respiratory rates were measured as an index of sensory
irritation; i.e., the greater the irritation, the more the animal suppresses
its inhalations. Eleven 10-minute exposures were conducted in which the
acrolein concentration ranged from 0.12 to 8.97 ppm and the formaldehyde
concentration ranged from 0,33 to 9.73 ppm. The data from these exposures
were used in a mathematical model developed to describe the interaction of
agents acting at a single sensory receptor site. From the results, the
investigators concluded that acrolein and formaldehyde act in competitive
antagonism for the same receptor sites when causing sensory irritation.
•*
5.1.2 Subcutaneous
The subcutaneous LDcns of acrolein in mice and rats were 30 and 50 mg/kg,
OU
respectively. No toxic signs were noted except narcosis. All deaths occurred
within 1 day. Histopathologic changes in the lungs were alveolar and peri-
vascular edema without hemorrhage; these changes were less severe than those
September 1986 5-3 DRAFT—DO NOT QUOTE OR CITE
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following inhalation exposure. Other histologic changes noted were hyperemia and
fatty degeneration in the liver and inflammatory changes in the kidneys (Skog,
1950).
5.1.3 Oral
The oral LDj-gS for mice and rats were 28 and 42-45 nig/kg, respectively
(Table 5-1). Single doses of 10 mg/kg by gavage killed two of two rats, and
doses of 5 mg/kg/day for 9 days were tolerated by six rats (Carl et al.,1939).
5.1.4 Intraperitoneal
Carl et al. (1939) suggested that acrolein was more toxic in rats by the
intraperitoneal route than by oral gavage. Intraperitoneal injection of 2.5
mg/kg/day was lethal in all rats (number not specified) tested two days after
administration of the test substance.
5.1.5 Dermal
When applied to the skin of rabbits, undiluted acrolein caused necrosis
and a 1 percent aqueous solution caused burns.
5.1.6 Ocular
Instillation of a 1 percent solution into the eyes of rabbits caused
severe (grade 10+) injury (Smyth et al., 1951).
5.2 SUBCHRONIC TOXICITY
Lyon et al. (1970) reported the results of four species of animals exposed
to two different regimens: (1) acrolein vapor at levels of 0.7 or 3.7 ppm (1.6
3
and 8.5 mg/m ) for 8 hours/day, 5 days/week for 6 consecutive weeks, and (2)
acrolein vapor continuously for 90 days at levels of 0.21, 0.23, 1.0, and 1.8
3
ppm (0.48, 0.52, 2.3, and 4.1 mg/m ). Test groups consisted of seven male and
seven female NMRIrO Sprague-Dawley rats and Princeton or Hartley derived guinea
pigs, nine male squirrel monkeys (Saimiri sciurea), and two male beagle dogs.
The data for the animals exposed continuously at 0.21 and 0.23 ppm were
combined in reporting the study. Concentrations of acrolein in the exposure
chamber were monitored several times daily. The initial weights of rats and
guinea pigs in the various exposure groups varied widely; however, it appears
that control groups of similar weights were used for comparison with each dosed
September 1986 5-4 DRAFT—DO NOT QUOTE OR CITE
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group. Pre and postexposure body weights and hematology determinations were
recorded, and immediately prior to study termination, blood urea nitrogen
levels and serum alanine and aspartate aminotransferase activities were
determined. Histopathologic examination of the heart, lungs, liver, spleen,
and kidneys were made on all dogs and monkeys and on half of the rats and
guinea pigs.
For the 0.7 ppm exposures, there were no toxic signs after repeated
exposure. However, histologic examination of the lungs revealed mild chronic
inflammatory changes and occasional mild emphysema. Inflammatory changes in
the bronchi (mild infiltration of round cells) were more pronounced in monkeys
and dogs than in other species.
During the exposure at 3.7 ppm, the dogs and monkeys showed eye
irritation, excessive saliveition, and labored breathing; these signs were less
severe after the first week of exposure, although eye irritation persisted
throughout the study (Lyon et al., 1970). Two monkeys died (days 6 and 9);
both had pulmonary lesions. Rats and guinea pigs exposed repeatedly to 3.7 ppm
acrolein vapor exhibited no pharmacotoxic reactions. Weight gains were
signficantly lower in rats exposed at 3.7 ppm, but there were no effects on
hematology or blood chemistry parameters in any species. Nonspecific
inflammatory histologic changes were found in lung, liver, and kidney sections
of all species. Squamous cell metaplasia and basal cell hyperplasia were noted
in the tracheas of dogs and monkeys, and necrotizing bronchitis and squamous
cell metaplasia of the lungs were found in seven of the nine monkeys exposed at
3.7 ppm acrolein.
In the studies using continuous exposure, no toxic effects were noted in
any species at the 0.22 ppm exposure level. At exposure levels of 1.0 and 1.8
ppm, dogs and monkeys exhibited severe irritation as described for the repeated
exposure study. Rats had decreased weight gains at both the 1.0 and 1.8 ppm
exposure levels when, compared to controls. Histologic changes after continuous
exposure at 0.22 ppm acrolein paralleled those at the 0.7 ppm repeated exposure
described above. At the 1.0 ppm level of continuous exposure, guinea pigs
showed varying degrees of pulmonary inflammation and occasional foci of liver
necrosis; three of nine rats examined histologically had foci of liver necrosis
and occasional pulmonary hemorrhages; and the dogs had inflammation of the
lungs, liver, and kidneys. At the highest level of exposure (1.8 ppm), all of
the animals examined histologically had inflammatory changes in the lungs,
liver, kidneys, brain, and heart; the histologic changes in the lungs and
September 1986 5-5 DRAFT—DO NOT QUOTE OR CITE
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bronchi paralleled those found after repeated exposure at 3.7 ppm acrolein
(Lyon et al., 1970).
Bouley et al. (1974, 1975) exposed 110 OFA male rats continuously to
acrolein in air at 1.27 mg/m3 (0.55 ppm) for up to 77 days; a control group of
110 rats was included in the study. Sneezing and nasal irritation were
observed between days 7 and 21, and these signs disappeared thereafter. Mean
body weights and food consumption were lower in the exposed group than in the
control group throughout the study. After 60 days, the mean body weight of
acrolein-exposed rats was approximately 80 percent of controls. The
lung-to-body weight ratios were similar between exposed and control groups
sacrificed at 15 and 32 days but were higher in the acroleinexposed group
sacrificed after 77 days than in corresponding controls. Serum alkaline
phosphatase was slightly but significantly decreased in exposed rats at day 15,
but similar to control levels at 32 and 77 days. The absolute number of
pulmonary macrophages was decreased at day 26 in exposed rats when compared to
controls. There was an accompanying increased susceptibility of acrolein-
exposed animals to experimental aerosol infection with Salmonella enteritis at
day 18, but not day 24; the death rate in rats exposed to acrolein and infected
by airborne Salmonella at day 18 was 15/16 compared to 8/15 for controls.
After 63 days, similar death rates due to experimental Salmonella infection
were found in both exposed and control groups (10/10).
Feron et al. (1978) studied the effects of repeated exposure to acrolein
vapor in Syrian golden hamsters, SPF Wistar rats, and Dutch rabbits. Groups of
20 hamsters, 12 rats, and 4 rabbits of each sex were exposed to acrolein vapor
at 0, 0.4, 1.4, and 4.9 ppm (0, 0.9, 3.2, and 11.3 mg/m3) for 6 hours/day, 5
days/week for 13 weeks. Body weights and food consumption were measured
weekly. Hematology, clinical chemistry, and urinalysis determinations were
performed at 12 weeks. At study termination, necropsies were performed, organ
weights measured, and histopathdlogy performed on all control and high dose
animals. No toxic signs were noted in any species at the 0.4 ppm exposure
level. At the 1.4 ppm level, the rabbits exhibited some sneezing and the rats
and hamsters appeared narcotized. Increased salivation and nasal secretion and
eye irritation were noted in all animals exposed to acrolein at the 4.9 ppm
level. At the highest dose level, deaths were reported in 3/12 male and 3/12
female rats. One hamster in the high dose (4.9 ppm) group was sacrificed
moribund at week 12 with renal failure. There was a decreased weight gain in
September 1986 5-6 DRAFT-DO NOT QUOTE OR CITE
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rats and rabbits exposed at both 1.4 and 4.9 ppm and in hamsters exposed at 1.4
ppm. There were no effects of exposure on hematologic or clinical chemistry
parameters. There were increases in organ-to-body weight ratios in animals
exposed at the highest level. At 4.9 ppm, there were increases in relative
weights of lungs in hamsters, rats, and rabbits; of the heart and kidneys in
hamsters and rats; and of adrenals in rats as compared with controls. At the
highest exposure level, marked histo!ogic changes were seen in the epithelium
of the nasal cavity of all species; there was necrosis and neutrophil
infiltration of the mucosa. At an exposure level of 1.4 ppm acrolein, squamous
metaplasia and neutrophil infiltration of the nasal mucosa of rats was observed
although a similar effect in hamsters was not found. Hyperplasia in the
trachea was seen in all species at the highest exposure level; hyperplastic
effects were more severe in rats than in hamsters or rabbits. Histologic
changes in the lungs and bronchi, hemorrhage, and perivascular and aleveolar
edema were found in rats and rabbits exposed at 4.9 ppm, whereas no effects
were noted in hamsters. On the basis of this study, the descending order of
species sensitivity was rats, rabbits, hamsters.
5.3 CHRONIC TOXICITY
Feron and Kruysse (1977) performed a study to determine if acrolein acted
as a cocatcinogen in respiratory carcinogenesis induced by benzo(a)pyrene or
diethylnitrosamine. They exposed a control group of 18 male and 18 female
7
Syrian golden hamsters at 4 ppm (9.2 mg/m ) acrolein for 7 hours/day, 5
days/week for 52 weeks and observed some of the animals until week 81. Eye
irritation, excessive salivation, and nasal discharge were noted during the
first week of the study; however, the animals adapted and the signs were no
longer observed. The mean body weights of both exposed males and females were
lower than those of the controls during the exposure period. The differences
decreased in the postexposure period. Lung weights were increased in exposed
animals compared to controls, and there was a moderate degree of inflammation
and epithelial metaplasia in the nasal cavity; these changes persisted through
the postexposure period (weeks (56-81). There were no other histologic changes
in the respiratory system related to acrolein exposure. Acrolein did not
augment the effects of benzo(a)pyrene or diethylnitrosamine.
September 1986 5-7 DRAFT—DO NOT QUOTE OR CITE
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5.4 EFFECTS ON THE LIVER, KIDNEYS, AND LUNGS
5.4.1 Liver
Murphy et al. (1964) studied the effects of acrolein on liver enzymes
after it was administered to rats by intraperitoneal injection or when the rats
were exposed to acrolein vapor. Groups of four adult male Sprague-Dawley rats
were injected intraperitoneally with acrolein; the average liver alkaline
phosphatase (AP) levels 24 hours later were 84, 113, 170, and 368 percent of
controls at dose levels of 0.38, 0.75, 1.5, and 3.0 mg/kg, respectively. Table
5-3 shows the effect of acrolein on liver-to-body weight ratios and liver AP
activity. In a separate experiment, continuous exposure at 4 pprn acrolein for
4, 8, and 20 hours resulted in liver AP activities that were 135, 222, and 233
percent of their respective controls. Subsequent studies indicated that the
effect of acrolein on liver AP was secondary to stimulation of hypersecretion
of glucocorticoids by the adrenals. The effect on AP activity elicited by acro-
lein was prevented by adrenalectomy and hypophysectomy. Tyrosine amino Tyrosine
amino transferase activity was also increased in the livers of acrolein-treated
rats (Murphy, 1965; Murphy and Porter, 1966). Szot and Murphy (1970, 1971) stud-
ied the effect of single sublethal doses (intraperitoneal) of acrolein on plasma
and adrenal corticosterone in male Holtzman rats. Doses between 0.1 and 6.0
mg/ral acrolein were given to rats, and corticosteriod levels were measured after
1 hour. Levels of adrenal and plasma corticosterone increased 300500 percent of
control levels at increasing dose levels of acrolein. Dexamethazone partially
reversed the effect of a low dose of acrolein (0.10 or 0.25 mg/kg) but not of
high doses (1.50 or 6.0 mg/kg).
TABLE 5-3. EFFECT OF ACROLEIN ON LIVER ALKALINE PHOSPHATES
AND RELATIVE LIVER WEIGHT
Concentration,
ppm
4.1
2.1
1.0
4.0
3.9
No. of
animals
6
12
12
6
6
Time,
hr
20, continuous
41, continuous
81, continuous
4/day x 5 days
4/day x 9 days
Mean liver AP,
% of control
233
148
104
100
71
Increased
liver-to-body
weight ratio
+
+
i
"•
Source: Murphy et al. (1964).
September 1986
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DRAFT—DO NOT QUOTE OR CITE
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Butterworth et al. (1978) found periportal necrosis in weanling male
Wistar-derived rats 24 hours after intravenous infusion of acrolein (0.85 or
1.70 mg/kg) into the mesenteric vein.
5.4.2 Kidneys
Mild effects on the kidneys were noted in section 6.1.2 (Skog, 1950).
5.4.3 Lungs
Murphy et al. (1963) measured pulmonary function in guinea pigs exposed to
nonlethal doses of acrolein. Random-bred male guinea pigs were exposed at
levels of acrolein vapor between 0.4 and 1.0 ppm via facemask, under dynamic
flow conditions, for 2 hours,, Respiratory rate, tidal volume, and flow resis-
tance were measured with a plethysmograph. There was a dose-related increase
in total respiratory flow resistance accompanied by a decreased respiratory
rate and increased tidal volume. The changes were reversible. Acrolein
appeared to mediate its effects by bronchoconstriction, since anticholinergic
drugs known to relieve bronchoconstriction partially or completely reversed the
effects. The levels of acrolein used were near or below the levels of human
sensory detection.
Watanabe and Aviado (1974) exposed male Swiss mice to acrolein for 5 weeks
at a level of 0.1 mg/1 (0.1 ppm) or for 5 minutes at levels of 0.3 or 0.6 mg/1
(0.3 or 0.6, ppm). The effects were a decreased pulmonary compliance, decreased
tidal volume, and decreased respiratory rate.
Kane and Alarie (1977) studied the effect of exposure to acrolein on the
respiratory rate of Swiss Webster mice. Groups of four mice were exposed at
varying levels of acrolein vapor in a glass chamber in which the heads only
were exposed to the atmosphere and the bodies enclosed in a plethysmograph that
was connected to external recorders. There was a dose-response decrease in
respiratory rate during a 10-minute exposure. The RD™ (50 percent decrease in
3 "
respiratory rate) was 1.7 ppm (3.9 mg/m ). This was calculated by linear
regression of a graph of the logarithm of concentration vs percent decrease in
respiratory rate at levels of acrolein between 0.2 and 10 ppm. Acrolein was
about twice as potent as formaldehyde, which had an RD™ of 3.1 ppm. By
exposing the mice while they were breathing through a tracheal cannula, it was
shown that the primary effect is on the upper respiratory tract. When the mice
were exposed at 0.4 or 1.7 ppm acrolein vapor for 3 hours/day for 4 days, and
then tested on the 5th day for an acute RD^g, Kane and Alarie found that the
September 1986 5-9 DRAFT—DO NOT QUOTE OR CITE
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response was greater than expected (hypersensitivity). However, when the mice
were exposed on 3 consecutive days at one-tenth the RD5Q (0.17 ppm) and tested
on the 4th day, there was a development of tolerance and a decrease in the
expected response.
The mucoid layer of the upper respiratory tract is a complex system of
mucus and cilia that protects the organism by clearing the upper respiratory
tract of foreign material (Carson et al., 1966). Kensler and Battista (1966)
found that acrolein was one of the components of tobacco smoke that inhibited
ciliary transport activity in isolated tracheal preparations from chickens. In
this study, there were no quantitative data on the levels of acrolein causing
an effect. In another study, Battista and Kensler (1970) reported the effects
of cigarette smoke and acrolein on ciliary transport activity in the tracheas
of chickens jn vivo. Smoke was passed through absolute filters to remove
acrolein; measured amounts of acrolein were then added to the smoke, and the
smoke was administered to the chickens. The ED5Q for acrolein based on eight
puffs (e.g., one cigarette equivalent) was 3040 ug acrolein/40 ml puff. A 40
ml puff of unfiltered cigarette smoke contains between 8 and 20 ug of acrolein.
Dalhamn and Rosengren (1970) confirmed the effect of acrolein on cilia of
isolated trachea of cats. Ciliostasis was reported at aerosol levels of 50100
mg/ra3 (2243 ppm) acrolein.
Astry and Jakab (1983) exposed female Swiss mice to acrolein vapor for 8
hours at levels of 3 and 6 ppm (6.9 and 13.8 mg/m3); a control group was also
included in the study. Following the exposure to acrolein, the mice received
an aerosol of 32Plabeled Staphylococcus aureus. Groups of animals were
sacrificed immediately after the bacterial challenge and after 8 hours. The
32
lungs were removed and homogenized. Viable bacterial counts and P levels
were determined at both time intervals, and the percent of initial viable
staphylococci remaining in the lungs was determined. In control animals there
was 95 percent intrapulmonary killing of S. aureus after 8 hours. Approxi-
mately 50 percent of the initial viable bacteria remained in the lungs after
exposure at 3 ppm; after exposure at 6 ppm, the pulmonary antibacterial
defenses were totally repressed, allowing growth of S. aureus to 150 percent of
the initial challenge.
The lowered defense against infection by bacteria noted in the above study
and in the study of Bouley (1974, reviewed in section 6.2) correlate with the
results of two other studies. In the study by Rylander (1973), the author
deduced that acrolein was one of the components of cigarette smoke that caused
September 1986 5-10 DRAFT-DO NOT QUOTE OR CITE
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a decrease in the number of macrophages in the free lung cells of guinea pigs
inhaling cigarette smoke. In the study by Kilburne and McKenzie (1970), the
authors found that acrolein in combination with carbon caused recruitment of
"scavenging" cells to the respiratory system and was cytotoxic to these cells
whose normal homeostatic function is host protection against microbial
infection. Groups of 614 Syrian golden hamsters, weighing approximately 100 g
each, were exposed to acrolein aerosol (6 ppm) and carbon (1.4 urn particle size
diameter, 6 ppm) for 6-96 hours; less than 10 percent of that to which the
hamsters were exposed was inhaled. After various periods of exposure, groups
of animals were sacrificed and the number of leukocytes "recruited" to airway
cells of the trachea and lungs was measured by electron microscopic examination
of tissue sections. Polymorphonuclear leukocytes (PMNs) were recruited to both
tracheal and bronchiolar cells with a peak of accumulation occurring at 24-48
hours and then subsiding. Neither carbon nor acrolein alone caused the effect.
Absorption of carbon was necessary for the chemotactic effect of acrolein on
the respiratory system, but it was shown that acrolein by itself can cause a
cytotoxic effect on airway cells. Cytotoxicity of acrolein to cultured
mammalian cells is discussed in section 6.5.2.6.5
5.5 OTHER STUDIES
5.5.1 Cardiovascular
Egle and Hudgins (1974) studied the sympathomimetic and cardiovascular
effects of injected and inhaled acrolein on anesthetized male Wistar rats. A
low concentration of acrolein (9.25 mg/kg) was administered by intravenous
injection, and a rise in blood pressure (pressor effect) was noted in most
rats, which began within 5 seconds, reached a peak within 30 seconds, and
subsided in approximately 1 minute. Higher loses of acrolein depressed blood
pressure in most animals. Groups of 6-11 animals were exposed 16-28 times each
to acrolein levels ranging from 0.01 to 5.00 ppm. Inhalation of levels of 0.01
ppm acrolein caused a pressor effect; higher doses frequently caused a cardio-
inhibitory effect. It was suggested that the pressor effect was mediated by
release of neurotransmitters from sympathetic nerve endings as well as by an
action on the adrenal medulla.
Green and Egle (1983) studied the effect of acrolein on blood pressure in
an inbred hypertensive strain of Wistar rats and compared the effects with the
September 1986 5-11 DRAFT—DO NOT QUOTE OR CITE
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same strain of rats receiving the antihypertensive agent guanethidine. Doses
of 0.05 and 0.1 mg/kg acrolein administered intravenously to anesthetized rats
caused a pressor effect, whereas decreased blood pressure was noted at doses of
0.3, 0.4, and 0.5 mg/kg acrolein. The responses were not altered by guanethi-
dine. Uniform responses were not observed in all animals in each group.
5.5.2 In Vitro Cytotoxicity
Several studies have investigated the jm vitro cytotoxicity of acrolein in
cultured mammalian cells. Results of the studies are summarized in Table 5-4.
Holmberg and Malmfors (1974) found that acrolein was substantially more
cytotoxic in Ehrlich-Landschultz ascitis cells than was formaldehyde, and that
both aldehydes were among the most toxic organic solvents in the series
studied. The cytotoxic effects of acrolein may be due to effects on nucleic
acid and protein synthesis. Leuchtenberger et al. (1968) studied the uptake
and incorporation of 3H-uridine in primary mouse kidney cultures. The cells
were exposed to 5-second pulses of acrolein vapor (approximately 5 ug/pulse)
every 15 seconds for a total exposure of approximately 400 ug. There was a
progressive dose-related decrease of uptake of H-uridine, an inhibition of RNA
synthesis, and pycnosis of the cell nuclei. Moule and Frayssinet (1971) and
Hunsch et al. (1973) compared the effects of acrolein on DNA and RNA poly-
merases of rat liver and Escherichia coli. It was concluded (Munsch et al.,
1974) that-the effect of acrolein on DNA synthesis was mediated by the reaction
of acrolein with the sulfhydryl group of the polymerase. Rat liver DNA poly-
merase has a functional sulfhydryl group, whereas the E. coli polymerase does
not, and rat liver DNA polymerase bound about 25 times as much H-acrolein as
did the E. coli enzyme (Munsch et al., 1973).
5.6 EFFECTS ON HUMANS
Acrolein vapor has been found to be an extremely potent eye and respira-
tory tract irritant in humans. Table 5-5 summarizes ocular responses to
acrolein in humans, and Table 5-6 summarizes threshold levels for responses.
Sim and Rattle (1957) studied experimental human exposure to several aldehydes.
Extreme irritation was noted in humans exposed to acrolein at levels of 0.80
ppm (1.8 mg/m3) for 10 minutes or 1.22 ppm (2.8 mg/m3) for 5 minutes. Lacrima-
tion occurred after 20 seconds at 0.8 ppm and after 5 seconds at 1.22 ppm. For
September 1986
5-12
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TABLE 5-6. THRESHOLDS OF RESPONSE AFTER EXPOSURE TO ACROLEIN
Concentration,
ppm Response
0.2 Eye irritation threshold
0.33-0.40 Odor threshold
0.40-1.0 Prolonged deep respiration
0.62 Respiratory response threshold
1.0 Immediately detectable
5.5 Intense irritation
SOURCE: National Research Council (1981).
comparison, crotonaldehyde was highly irritating (4.1 ppm for 15 minutes);
acetaldehyde was slightly irritating; and propionaldehyde, butyraldehyde, and
isobutyraldehyde were nonirritating. The threshold level for eye irritation
due to acrolein exposure is 0.2 ppm (0.46 mg/m3) (Table 5-6);
formaldehyde, by comparison, is about 2.5 times less potent (Altschuller,
1978).
Acrolein has been identified as one of the main components of smog that
acts as an eye irritant (Renzetti and Bryan, 1961; Altschuller, 1978).
Acrolein is a component of diesel exhaust and can be produced experimentally by
photooxidation of hydrocarbons in the presence of nitrous oxides (Schuck and
Renzetti, 1960). It is also a component of cigarette smoke and is a major
contributor to the irritant property of cigarette smoke (Weber-Tschopp et al.,
1976).
Two cases of occupational exposure to acrolein (one fatal) have been
reported by NIOSH (National Research Council, 1981). It has been speculated
that the greatest occupational danger of acrolein exposure is associated with
the welding of fat and oil cauldrons. Because of the acutely irritating
properties of acrolein, human exposure is likely to be limited and thus
observed toxic effects rare (National Research Council, 1981).
A case of accidental exposure to acrolein was reported by Champeix et al.
(1966) in which a chemical worker was accidentally sprayed in the face with
acrolein. He had immediate burning of the eyes and face, and within 20 hours
was hospitalized with thoracic constriction, cough, frothy sputum, and
cyanosis. Other signs included swollen eyelids and a rapid respiratory rate.
After 9 days of hospitalization and treatment, the patient was released with a
September 1986 5-15 DRAFT—DO NOT QUOTE OR CITE
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moderate cough. Eighteen months later he developed a chronic bronchitis and
emphysema possibly related to the acrolein exposure and injury to the respira-
tory system.
Gosselin et al. (1979) reported a case of two boys (ages 2 and 4.5 years)
who accidentally inhaled smoke from an overheated fryer for 2 hours. The smoke
contained acrolein. The younger boy was found dead, and the other was hospi-
talized with respiratory difficulties and given oxygen therapy. Death due to
asphyxiation occurred within 24 hours. On autopsy, massive cellular
destruction in the trachea and bronchi and pulmonary infarcts were found.
Bauer et al. (1977) reported a case of acrolein poisoning caused by
inhaling vapors from vegetable cooking oil that had been overheating for 6
hours in a small kitchen. The subject experienced pulmonary changes that were
life threatening. It was concluded by the authors that acrolein was the major
toxic component of the inhaled vapor.
In its liquid form, acrolein has caused severe skin irritation in humans.
Dermal application of a 1 percent solution produced a positive patch test
(National Research Council, 1981).
Some information on the metabolism of acrolein in humans was; produced by
studies of patients receiving cyclophosphamide. This drug, which is used in
cancer chemotherapy, is generally believed to be metabolized to phosphoramide
mustard and acrolein (Alacron, 1976; Low et al., 1983). Two studies have
identified-S-hydroxypropylmercapturic acid (MCA) in the urine of patients given
cyclophosphamide, suggesting that acrolein may in part be converted to the
mercapturate as had been found in rats. In one study (Kaye and Young, 1974),
the urine of two patients receiving cyclophosphamide orally (50 mg twice daily
and 50 mg three times daily, respectively) was collected over 24 hours, and MCA
was identified in both samples by paper chromatography. In the second study
(Alacron, 1976), cyclophosphamide (1 g intravenously, equivalent to 10-18
mg/kg) was given to five patients who were receiving multiple drugs for
unspecified illnesses, and their urine was collected over 6 hours. The amounts
of urinary MCA found varied widely from 6.4 to 50.0 umol; no correlation
between dose and urinary MCA was evident.
5.7 SUMMARY
Toxicity studies with acrolein indicate that it is highly toxic by the
inhalation and oral routes of exposure, moderately toxic through skin
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absorption, and is a strong skin and eye irritant. The descending order of
sensitivity in acute inhalation exposure to acrolein is mouse, rat, guinea pig,
cat. The approximate LC5Q for a lOminute exposure in mice was 175 ppm (493
rng/m ) and in rats 375 (750 mg/m ); however, concentrations on the order of 12
ppm acrolein vapor were irritating to the eyes and nose of experimental animals
after an exposure period of 12 minutes. Mice were more sensitive than rats
when given acrolein orally, approximate LD50s being 28 and 45 rag/kg,
respectively; subcutaneous LD^s were in a similar range. One percent aqueous
solutions of acrolein were extremely irritating when applied to the skin or
instilled in the eyes of rabbits. Acute inhalation exposure to acrolein in
animals caused bronchiolar constriction resulting in anoxia and death.
Histologic changes in animals that died after exposure were found in the nasal
passages, bronchi, trachea, and lungs.
Subchronic inhalation studies of acrolein have been conducted using both
repeated exposure for 6 hours/day, 5 days/week for 6 weeks or using continuous
exposure for 6 weeks with monkeys, dogs, rabbits, guinea pigs, hamsters, and
rats. A no observed effect level (NOEL) in all species was 0.4 ppm (0.92
O
mg/m ). Repeated exposure at 0.7 ppm acrolein for 6 weeks caused no toxic
effects in monkeys, dogs, guinea pigs, or rats, but histologic examination
revealed mild inflammatory changes in the bronchi and lungs that were more
severe in monkeys and dogs than in rats and guinea pigs. Levels between 3.7
o
and 4.9 ppm (8.5 and 11.3 mg/m ) caused severe toxic signs and some degree of
histologic change in tissues of the respiratory tract in all species tested.
Repeated exposure at 3.7 ppm (8.5 mg/m ) acrolein caused toxic effects
(irritation, lacrimation, and nasal discharge) in dogs and monkeys but not in
rats and guinea pigs.
In a study comparing species sensitivity to inhalation exposure to
acrolein, it was found that the descending order of sensitivity was rats,
rabbits, hamsters. After repeated exposure to acrolein at 4.9 ppm (11.3
o
mg/m ), the nasal cavities, trachea, and bronchi showed histologic aberrations
consisting of destruction, hyperplasia, and metaplasia of the epithelia of the
airways and neutrophil infiltration of the mucosa; similar changes were seen in
o
hamsters exposed to acrolein at 4 ppm (9.2 mg/m ) for 7 hours/day, 5 days a
week for 52 weeks.
Continuous exposure at 0.22 ppm (0.5 mg/m ) and 1.0 ppm (2.3 mg/m3)
acrolein produced similar results to repeated exposures at 0.4 ppm and 4.9 ppm,
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respectively. Histologic changes in the respiratory tract were generally more
severe in monkeys and dogs than in rats or guinea pigs.
Reduced pulmonary compliance and tidal volume and a decrease in respira-
tory rate were reported after both acute and long-term exposure of mice to
levels of acrolein below those that cause toxic effects. Inhalation of levels
of acrolein equivalent to those found in cigarette smoke reportedly caused an
inhibition of ciliary transport. This effect correlates with decreased
pulmonary antibacterial defenses in experimental animals exposed to acrolein.
Although the primary target site of acrolein exposure appears to be the
respiratory system, effects have also been noted on the liver, kidneys, and
cardiovascular system. An increase in liver enzymes in rats receiving acrolein
by the intraperitoneal route or by inhalation may be mediated by hypersecretion
of glucocorticoids by the adrenals. Effects on the kidneys are not well
defined. Cardiovascular effects, primarily a transient increase in blood
pressure, may be sympathomimetic in nature.
In vitro studies indicate that the mechanism of the toxic effects of
acrolein may be a binding of essential thiol groups in proteins that affects
macromolecular biosynthesis and causes cell death.
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5.8 REFERENCES
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propylmercapturic acid as an index of cyclophosphamide (NSC-26271) activa-
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Altshuller, A. P. (1978) Assessment of the contribution of chemical species to
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Astry, C. L.; Jakab, G. J. (1983) The effects of acrolein exposure on pulmonary
antibacterial defenses. Toxicol. Appl. Pharmacol. 67: 49-54.
Au, W.; Sokova, 0. I.; Kopnin, B; Arrighi, F. E. (1980) Cytogenetic toxicity of
cyclophosphamide and its metabolites in vitro. Cytogenet. Cell Genet. 26:
108-116. :
Battista, S. P.; Kensler, C. J. (1970) Mucus production and ciliary transport
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326-338.
Bauer, K.; Czech, K.; Porter, A. (1977) Schwere akzidentelle Acroleinvergiftung
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Bouley, G.; Dubreuil, A.; Godin, J.; Boudene, Cl. (1975) Effets, chez le rat,
d'une faible dose d'acroleine inhalee en continu [Effects of a small dose
of acrolein constantly inhaled by rats]. Eur. J. Toxicol. Environ. Hyg. 8:
291-297.
Bouley, G.; Dubreuil, A.; Godin, J.; Boisset, M.; Boudene, C. (1976) Phenomena
of adaptation in rats continuously exposed to low concentrations of
acrolein. Ann. Occup. Hyg. 19: 27-32.
Butterworth, K. R.; Carpanini, F. M. B.; Dunnington, D.; Grasso, P.; Pelling,
D. (1978) The production of periportal necrosis by allyl alcohol in the
rat. In: Proceedings of the British Pharmacological Society; March;
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Carl, M.; Mcknight, R. S.; Scott, B.; Lindegren, C. C. (1939) Physiological
effects of garlic and derived substances. Am. J. Hyg. 29: 32.
Carson, S.; Goldhamer, R.; Weinberg, M. S. (1966) Characterization of physical,
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Catilina, P.; Thieblot, L.; Champeix, J. (1966) Lesions respiratoires experi-
mentales par inhalation d'acroleine chez le rat [Experimental respiratory
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Champelx, J.; Courtail, L.; Perche, E.; Catilina, P. (1966) Acute broncho-pnue-
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mucosa. Arch. Otolaryngol. 93: 496-500.
Egle, J. L., Jr.; Hudgins, P. M. (1974) Dose-dependent sympathomimetic and
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rat. Toxicol. Appl. Pharmacol. 28: 358-366.
Feron, V. J.; Kruysse, A. (1977) Effects of exposure to acrolein vapor in
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J. Toxicol. Environ. Health 3: 379-394.
Feron, V. J.; Kruysse, A.; Til, H. P.; Immel, H. R. (1978) Repeated exposure to
acrolein vapour: subacute studies in hamsters, rats and rabbits.
Toxicology 9: 47-57.
Green, H. A.; Egle, J. L., Jr. (1983) The effects of acetaldehyde and acrolein
on blood pressure in guanethidine-pretreated hypertensive rats. Toxicol.
Appl. Pharmacol. 69: 29-36.
Holmberg, B.; Malmfors, T. (1974) The cytotoxicty of some organic solvents.
Environ. Res. 7: 183-192.
Hussain, J. I.; Smith, C. J.; Allen, J. C. (1983) Polyamine-mediated inhibition
of in-vitro cell proliferation is not due to acrolein. Cell Tissue Kinet.
16: 583-591.
International Technical Information Institute. (1975) Acrolein. In: Toxic and
hazardous industrial chemical safety manual for handling and disposal with
toxicity and hazard data. Tokyo, Japan: International Technical
Information Institute; pp. 13-14.
Kane, L. E.; Alarie, Y. (1977) Sensory irritation to formaldehyde and acrolein
during single and repeated exposures in mice. Am. Ind. Hyg, Assoc. J. 38:
509-522. i
Kaye, C. M.; Young, L. (1974) Acrolein as a possible metabolite of cyclophospha-
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Kensler, C. J.; Battista, S. P. ,(1966) Chemical and physical factors affecting
mammalian ciliary activity. Am. Rev. Respir. Dis. 93: 93-102.
Kilburn, K. H.; McKenzie, W. N. (1978) Leukocyte recruitment to airways by
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Leuchtenberger, C.; Schumacker, M.; Haldeman, T. (1968) Further cytological and
cytochemical studies on the biological significance of the gas phase of
fresh cigarette smoke. Z. Praeventivmed. 13: 130-141.
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Munsch, N.; Frayssinet, C. (1971) Action of acrolein on nucleic acid synthesis
In vivo. Biochimie 53: 243-248.
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synthesis jn vitro. FEBS Lett. 30: 286-290.
Munsch, N.; Marano, F.; Frayssinet, C. (1974) Incorporation d'acroleine 3H dans
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1433-1436.
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Toxicol. Appl. Pharmacol. 7: 833-843.
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Formaldehyde and other aldehydes. Washington, DC: National Academy Press;
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Phillips, B. J. (1974) A simple, small scale cytotoxicity test, and its uses in
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Pilotti, A.; Ancker, K.; Arrhenius, E.; Enzell, C. (1975) Effects of tobacco
and tobacco smoke constituents on cell multiplication jm vrlro. Toxicology
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Rylander, R. (1973) Toxicity of cigarette smoke components: free lung cell
response in acute exposures. Am. Rev. Respir. Dis. 108: 1279-1282.
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Toxicol. Appl. Pharmacol. 2: 183-187.
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20: 263-269.
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6. MUTAGENICITY
6.1 GENE MUTATIONS
6.1.1 Salmonella
There are more than 10 articles in the published literature which report
on the testing of acrolein in one or more strains of Salmonella. Those that
were abstracts or publications reporting the results of a large number of
chemicals in which only statements of positive/negative were reported are not
included in this evaluation.
The most extensive testing of acrolein was reported by Lijinsky and
Andrews (1980) who employed the plate incorporation test using strains TA1535,
TA1537, TA1538, TA98 and TA100 without activation and with added rat or
hamster liver S-9 from animals induced with Aroclor 1254. Acrolein was ini-
tially tested over a broad concentration range both in dimethyl sulfoxide
(DMSO) and distilled water. Acrolein was highly toxic and positive mutagenic
results were seen only i'n strain TA98 in the absence of S-9 when acrolein was
dissolved in distilled water. Representative data from one of the replicate
experiments were reported in which acrolein was tested from 0.001 to 0.1
ul/plate in increments of 0.01 above .01 ul/plate. The response increased with
dose up to 0.03 ul/plate at which the response was approximately 2.5 times the
control level. At higher concentration the number of revertants decreased and
was just twofold above controls at 0.1 pi/plate.
Lutz et al. (1982) used TA100 to test acrolein and several other unsatu-
rated aldehydes in a 90 minute preincubation test. Acrolein was tested at
0.0125, 0.0375, 0.075S 0.1125 and 0.15 micromoles/plate both without and with rat
liver S-9 activation. A positive dose related response was reported only
without S-9 and in least two independent experiments. The data were presented
only in graphical form and interpolation from the graph indicates approximately
430 revertants/plate at the highest concentration compared to approximately 130
revertants/plate for controls. An aliquot of the preincubation suspension was
also diluted and plated on complete medium for determination of survival. While
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it is not possible to interpolate with any accuracy from the toxicity graph it
appears that survival was very low at the high concentration.
Hales (1982) tested acrolein at one log concentration intervals from 0.001
to 10 ug/plate (10 ug/plate = 0.178 micromoles/plate) only in TA1535. Acrolein
was extremely toxic both with and without S-9 activation and was reported not
mutagenic. Because only log intervals were tested and because only TA1535
was used, these results cannot be compared with other reports.
The National Toxicology Program (Haworth et al_. 1983) used..TA1535, TA1537,
TA98 and TA100 in a preincubation assay to test acrolein. Metabolic activation
was provided by both rat and hamster liver S-9. Concentrations of acrolein
tested included half-log increments from 0.03 to 100 ug/plate and several
other concentrations between 10 and 100 ug/plate. Severe toxicity was observed
in all strains at less than 10 ug/plate without activation, with no evidence of
mutagenicity. Higher concentrations were tolerated with both rat and hamster
S-9 but no clear mutagenic response was observed. They did observe a dose-
related trend in TA100 with rat S-9 with a maximum response about 1.8 times
concurrent controls at 50 ug/plate. A repeat of the TA100 assay was later
conducted (Haworth, personal communication) at concentrations of 0, 1, 3.3, 10,
33 and 100 ug/plate. Again negative responses were seen without S-9 and with
hamster S-9. With rat S-9 the number of revertants was not different from
controls up to 10 ug/plate, was 2 times controls at 33 ug/plate, and total
toxicity prevailed at 100 ug/plate.
Marnett et al. (1985) tested acrolein using a new Salmonella strain TA104.
This is a base pair substitution strain which carries a non-sense mutation at
the reversion site and, along with TA102, was designed to detect peroxides and
other oxidants. Concentrations of acrolein were not listed but interpolation
from the graphical presentation of the data indicates that they tested at least
0.1, 0.2 and 0.375 micromoles/plate. While they stated that 0.9 micromoles/
plate was the maximum non-toxic concentration, no mutagenicity data were
reported for this concentration. A dose-related response was observed with a
doubling of control valves occurring at about 0.375 micromoles/plate.
It is clear that the results of these studies, as well as those not
presented due to their sparse reporting, do not present a consistent interpre-
tation of the mutagenic activity of acrolein in Salmonella. To a large extent
this appears to be due to the extreme toxicity of acrolein to bacteria and
therefore the variations in concentrations used and minor differences in
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protocols are especially critical. It is further noted that the maximum
mutagenic response obtainable before cell toxicity predominates was approxi-
mately three-fold over the background rate. This point is raised here because
there is a tendency to compare mutagenic potencies on the basis of induced
mutants per unit of concentration (often revertants per nanomole for Samlonella),
as was done in some of the articles cited above. The value of this comparison
becomes questionable when chemicals with widely varying toxicity are compared
on this basis.
6.1.2 E. coli
Ellenberger and Mohn (1977) used strain K12/343/113, which is capable of
detecting both forward and reverse mutations at several loci, to evaluate the
mutagenicity of acrolein. Cells were incubated with nine concentrations of
acrolein ranging from 0.05 to 0.70 mM for 3 hr and aliquots were plated on
complete and selective media for survival and mutation detection, respectively.
Interpolation from a toxicity curve reported shows a dramatic increase in
toxicity beginning at 0.45 mM (50 percent survival). At 0.7mM survival was
less than 0.5 percent of controls. The actual mutagenicity data were not
reported, but the authors stated that in several experiments acrolein did not
exhibit mutagenic activity.
Hemminki et al. (1980) reported acrolein as a weak mutagen in strain WP2
uvrA (trp-> of E coli. .Acrolein was one of many chemicals reported and the
concentrations were reported only as being in the range of 20 to 10,000
micromolar for all chemicals. Also, the results were reported only as induced
mutant frequency per micromole without reporting the control frequency, or the
level of toxicity. Therefore, it is not possible to verify the conclusion
reported.
6.1.3 Yeast
Izard (1973) reported both positive and negative responses in yeast when
different endpoints were measured. The positive was observed in the induction
of "petite" mutants; a doubling of the control incidence at 50 percent survival
after treatment with acrolein at 320 mg/1 (5.7 mM). Since only the percent
response was reported, it is not clear that a two-fold increase is actually
significantly different from controls. Acrolein, tested at concentrations
ranging from 6.25 mg/1 to 100 g/1, did not induce an increase in either of two
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yeast strains capable of detecting reverse mutations at the methionine locus.
The author did not remark on toxicity in this experiment, but in the light of
the toxicity encountered in the strain used to measure "petite" mutants, a
concentration of 100 g/1 seems implausible (a typographical error in the
report is more likely). Neither of the strains used by Izard have been
extensively used, hence it is not possible to evaluate this study in the
context of current work in yeast.
6.1.4 Drosophila
The first report on the mutagenicity of acrolein was that of Rapaport
(1948). Drosophila larvae were allowed to feed on nutrient medium containing
acrolein or other unsaturated aldeheydes. The incidence of sex-linked reces-
sive lethals in the survivors of acrolein treatment was 2.23 percent as com-
pared to 0.19 percent in controls. The concentration of acrolein used was not
reported but the level used was reported to cause death in greater than 75
percent of the larvae treated. There is also no mention of whether mutational
clusters were considered; this is especially important since larval treatment
involves treatment of only primordial germ cells and clusters are almost
certain to appear. More recently, Zimmering et al. (1985), under contract with
the National Toxicology Program, tested acrolein in the more conventional
protocol for sex-linked recessive lethals by treating adult males. Acrolein
was fed at-3,000 ppm in 5 percent sucrose or injected at 200 ppm in saline with
appropriate vehicle controls and the incidence of recessive lethals was essen-
tially identical to controls (0.05-0.07 percent).
6.2 CHROMOSOMAL EFFECTS
In an investigation of cyclophosphamide and its metabolites Au et al.
(1980) tested acrolein for chromosome breaks and sister chromatid exchange
(SCE) in Chinese hamster ovary cells (CHO). For the analysis of chromosome
breaks, cells were exposed for 5 hr to acrolein at 0, 10, 40 and 100 uM both
without and with rat liver S-9 activation. Extreme toxicity at all levels
without S-9 precluded a measure of breaks and with S-9 they observed a doubling
in the incidence of breaks at 40 uM acrolein, but the high variability and the
fact that the incidence at 10 uM was actually lower than controls led the
authors to conclude that the response was not a true indication of a mutagenic
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effects of acrolein. For SCE analysis cells were exposed for 1 hr at 0, 5, 10,
20 and 40 uM acrolein, washed and incubated with Brd U for 24 hr: for the
visualization of SCE. A dose-related increase in SCE was recorded without S-9;
10 uM was an approximate doubling of controls and 40 uM was toxic to the cells.
Recently, the National Toxicology Program also employed these same assays with
different treatment times and concentrations of acrolein (Zeiger, personal
communication). Acrolein was tested at 0, 0.1, 0.3 and 1.0 ug/ml (approxi-
mately 0, 1.8, 5.4 and 18 uM, respectively) and CHO cells were exposed for 48
hrs without activation and 2 hr with rat liver S-9. For total chromosome
aberrations without S-9 the response at the highest dose was marginally
significantly higher than controls but there was not a dose-related increase.
For SCE without S-9 a dose-related positive response was observed. Both
endpoints were negative in the presence of S-9. Given the differences in
protocols and concentrations, these results of two studies are quite similar.
The only w vivo mammalian study on acrolein was a dominant lethal assay
in ICR male mice reported by Epstein et al. (1972). Males 8-lO^eeks old
received single IP injections of 1.5 or 2.2 mg/kg acrolein and were mated with
3 untreated females per week for 8 weeks. No data were reported but the
authors stated that the incidence of early fetal deaths and preimplantation
losses were within control limits.
6.3 SUMMARY OF MUTAGENIC EFFECTS
The majority of the mutagenicity tests on acrolein have employed bacterial
systems and both positive and negative results have been reported. Differences
in bacterial strains tested, protocols used and the differences in concentra-
tions tested preclude a reconciliation of the apparently conflicting results.
All reports do indicate that acrolein is extremely toxic with significant
toxicity noted between 0.1 and 1 jjmoles/plate and complete toxicity at less
than 5 umoles/plate in Salmonella.
In eukaryotes, acrolein did not induce gene mutations in methionine
requiring strains of yeast, but did induce mitochondria! "petite" mutations
in another yeast strain. Acrolein induced sex-linked recessive lethals in
Drosophila when larvae were treated but not when adult males were treated in
the currently conventional procedure.
Acrolein did induce sister chromatid exchange in mammalian cells ui vitro
but only in the absence of exogenous S-9 activation. Extreme toxicity precluded
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detection of chromosome aberrations in these cells. Finally, acrolein was
reported negative in a mouse dominant lethal test in which males received a
single IP injection of 1.5 or 2.2 mg/kg.
Applying the weight-of-evidence scheme of the proposed Guidelines for
Mutagenicity Risk Assessment (Environmental Protection Agency, 1984) to the
acrolein data, results in the classification of "Inadequate evidence bearing on
either mutagenicity or chemical interactions with mammalian gen cells." The
basis for this conclusion is the absence of data for mammalian |jene mutations
and jn vivo mammalian cytogenetics data (other than the very limited dominant
lethal test), and lack of data on mammalian germ cell interaction!.
September 1986
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6,4 REFERENCES
Au, W.; Sokova, 0. I.; Kopnin, B; Arrighi, F. E. (1980) Cytogenetic toxicity of
cyclophosphamide and its metabolites in vitro. Cytogenet. Cell Genet. 26;
108-116.
Ellenberger, J.; Mohn, G. R. (1977) Mutagenic activity of major mammalian
metabolites of cyclophosphamide toward several genes of Escherichia coli.
J. Toxicol. Environ. Health 3: 637-650.
Epstein, S. S.; Arnold, E.; Andrea, J.; Bass, W.; Bishop, Y. (1972) Detection
of chemical mutagens by the dominant lethal assay in the mouse, Toxicol.
Appl. Pharmacol. 23: 288-325.
Hales, B, F. (1982) Comparison of the mutagenicity and teratogenicity of cyclo-
phosphamide and its active metabolites, 4-hydroxycyclophosphamide, phos-
phoramide mustard, and acrolein. Cancer Res. 42: 3016-3021.
Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.; Zeiger, E. (1983) Salmonella
mutagenicity test results for 250 chemicals. Environ. Mutagen. (suppl. 1):
3-142.
Hemminki, K.; Falck, K.; Vainio, H. (1980) Comparison of alkylation rates and
mutagenicity of directly acting industrial and laboratory chemicals: epox-
ides, glycidyl ethers, methytating and ethylating agents, halogenated
hydrocarbons, hydrazine derivatives, aldehydes, thiuram and dithiocarbamate
derivatives. Arch. Toxicol. 46: 277-285.
Izard, C. (1973) Recherches sur les effects mutagenes de Tacroleine et de ses
deux epoxyeds: le glycidol et le glycidal, sur Saccharamyces cerevisiae.
C. R. Acad. Sci. Ser. D 276: 3037-3040.
Lijunsky, W,; Andrews, A. W. (1980) Mutagenicity of vinyl compounds in Salmo-
nella typhimurium. Carcinog. Mutagen. 1: 259-267.
Lutz, 0.; Eder, E.; Neudecker, T.; Henschler, D. (1982) Structure-mutagenicity
relationship in alpha, Beta-unsaturated carbonylic compounds and their
corresponding allylic alcohols. Mutat, Res. 93: 305-315.
Marnett, L. J.; Hurd, H. K. ; Hollstein, M. C.; Levin, D. E.; Esterbauer, H.;
Ames, B. N. (1985) Naturally occurring carbonyl compounds are mutagens in
Salmonella tester strain TA104. Mutat. Res. 148: 25-34.
Rapaport, I. A. (1948) Mutations induced by unsaturated aldehydes. Dokl. Akad.
Nauk USSR 61: 713-715.
Zimmering, S.; Mason, J. M.; Valencia, R.; Woodruff, R. C. (1985) Chemical muta~
genesis testing in Drosophila. II. Results of 20 coded compounds tested for
the National Toxicology Program. Environ. Mutagen. 7: 87-100.
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Izard, C. (1973) Recherches sur les effects mutagenes de Tacroleine et de ses
deux epoxyeds: le glycidol et le glycidal, sur Saccharamyces cereyisiae.
C. R. Acad. Sci. Ser. D 276: 3037-3040.
Kane, L. E.; Alarie, Y. (1977) Sensory irritation to formaldehyde and acrolein
during single and repeated exposures in mice. Am. Ind. Hyg. Assoc. J. w.
509-522.
Kaye, C. M.; Young, L. (1974) Acrolein as a possible metabolite of cyclophospha-
mide in man. Biochem. Soc. Trans. 2: 308-310.
Lijunsky, W.; Andrews, A. W. (1980) Mutagenicity of vinyl compounds in Salmo-
nella typhimurium. Carcinog. Mutagen. 1: 259-267.
Low, J. E.; Borch, R. F.; Sladek, N. E. (1983) Further studies on the conversion
of 4-hydroxyoxazaphosphorines to reactive mustards and acrolein inorganic
buffers. Cancer Res. 43: 5815-5820.
Lutz, D.; Eder, E.; Neudecker, T.; Henschler, D. (1982) Structure-mutagenicity
relationship in alpha, Beta-unsaturated carbonylic compounds and their
corresponding allylic alcohols. Mutat. Res. 93: 305-315.
Marnett, L. J.; Hurd, H. K.; Hoi 1 stein, M. C.; Levin, D. E.; Esterbauer, H.;
Ames B N. (1985) Naturally occurring carbonyl compounds are mutagens in
Salmonella tester strain TA104. Mutat. Res. 148: 25-34.
National Research Council. (1981) Health effects of some other aldehydes. In:
Formaldehyde and other aldehydes. Washington, DC: National Academy Press,
pp. 8-1 to 8-35.
Rapaport, I. A. (1948) Mutations induced by unsaturated aldehydes. Dokl. Akad.
Nauk USSR 61: 713-715.
Renzetti, N. A.; Bryan, R. J. (1961) Atmospheric sampling for aldehydes and eye
irritation in Los Angeles smog - 1960. J. Air Pollut. Control Assoc. 11:
421-427.
Schuck, E. A.; Renzetti, N. A. (1960) Eye irritants formed during photo-oxida-
tion of hydrocarbons in the presence of oxides of nitrogen. J. Air Pollut.
Control Assoc. 10: 389-392.
Sim V M • Pattle, R. E. (1957) Effect of possible smog irritants on human
' subjects. JAMA J. Am. Med. Assoc. 165: 1908-1913.
Weber-Tschopp, A.; Fischer, T.; Grandjean, E. (1976) Objektlve und subjektive
physio!ogische Wirkungen des Passivrauchens [Physiological and psychologi-
cal effects of passive smoking]. Int. Arch. Occup. Environ. Health 37: 277-
288.
Zimmering, S.; Mason, J. M.; Valencia, R.; Woodruff, R. C. (1985) Chemical muta-
genesis testing in Drosophila. II. Results of 20 coded compounds tested for
the National Toxicology Program. Environ. Mutagen. 7: 87-100.
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7. CARCINOGENICITY
The bioassay literature on acrolein carcinogenicity is limited to skin
painting, and subcutaneous injection studies with mice and an inhalation study
using hamsters, all with essentially negative results, these studies, however,
do not provide sufficient data for a definitive evaluation of the cancer
endpoint. Bioassay evaluations of other substances with related chemical
structures including an acrolein metabolite provides an insight into the
potential for acrolein to elicit a carcinogenic response. Glycidaldehyde,
which is one of the isolated metabolites of acrolein in rodents (see the
metabolism and pharmacokinetics section) has also been tested by skin applica-
tion or subcutaneous injection. Results of these studies indicate a statisti-
cally significant increase of local site tumors.
7.1 ANIMAL STUDIES
7.1.1 Subcutaneous Injection-Acrolein
The earliest study on acrolein carcinogenicity by Steiner et al. (1943)
included several experimental groups. Acrolein (0.2 mg in 0.1 cc sesame oil)
was injected subcutaneously once weekly for 24 weeks in 15 female "partly
inbred albino mice" of the author's own stock. In addition, 15 female and 16
male mice from the same stock received 0.5 cc heated sesame oil three times at
4 week intervals. The "negative, control" include 50 mg cholesterol in 0.5 cc
(non-heated) sesame oil injected subcutaneously five times at 4-week intervals.
The experiment was terminated at the end of two years. Three spindle-cell
sarcoma cases were noted in the "heated sesame oil" groups while none were
noted in the acrolein or "negative control" groups. The number of animals in
this experiment was too small to draw any conclusion regarding the carcinogenic
activity of acrolein in mice.
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7.1.2 Skin Application-Acrolein
Salaman and Roe (1956) have tested 21 chemicals including acrolein for
skin stumor initiating activity in S-strain mice. Fifteen mice (sex and age
not specified) received weekly treatment via skin application of 0.5 percent
acrolein in acetone over a 10 week period. Twenty-five days after the first
acrolein application, the mice received a treatment of 0.17 percent croton oil.
This was followed by 16 more weekly sin application of croton oil. The total
acrolein dose was 12.6 mg per animal. Two of the 15 mice developed a total of
3 papillomas. Four of 19 mice receiving the same regimen of croton oil only
developed 4 papillomas. The tumor incidence in the acrolein treated group is
not a statistically significant response when compared with the croton-oil-only
control. The small number of animals and the short duration of exposure are
major shortcomings for these experiments thus making it impossible to draw a
definite conclusion regarding the presence of absence of a carcinogenic re-
sponse to acrolein treatment.
7.1.3 Inhalation-Acrolein
Eighteen 6-week-old Syrien golden hamster/sex/group were exposed via
inhalation to 0 and 4 ppm (0 and 9.2 mg/m3) acrolein 7 hours/day, 5 days/week
for 52 weeks (Feron and Kruysse 1977). Six animals were killed at 52 weeks and
12 at 81 weeks in both treated and control groups. No increase in mortality
and no statistically significant increase in tumor incidences were found in the
treated animals compared to the control. The duration of the experiment was
too short and the number of aimals too small to draw any conclusion from this
experiment.
In separate experiments these authors tested Benzo(a)pyrene (BaP) and
diethylnitrosamine via intratracheal instillation in addition to administration
of acrolein to test the co-carcinogenic potential of this chemical. The
results, however, fail to show an enhanced tumor response in those animals
exposed to BaP (or DENA) and acrolein in comparison with the control which were
exposed only to BaP (or DENA) and room air.
7.1.4 Skin Painting Glycidaldehyde (Metabolite of Acrolein)
Thirty 55-day old female Swiss albino mice received ither 2.5 mg
glycidaldehyde, or 0.125 mg 7, 12 dimethyl-benz(a)anthracene (DMBA), as a
positive control, in 0.25 ml acetone administered by skin painting. Three
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weeks later, 0.25 ml 0.1 percent croton oil in acetone was applied 5 days/week
for the next 30 weeks. In addition to the one positive control of DMBA, there
were three negative control groups (30 animals per group): 1) no treatment, 2)
croton oil alone, 3) or acetone alone. Forty percent of mice treated with
glycidaldehyde (12 mice) and 93 percent of those treated with DMBA (28 mice)
developed keratoacanthoma after 30 weeks, whereas none were observed in any of
the negative control groups. The time-to-tumor (first tumor) from the begin-
ning of treatment was 5 weeks for DMBA, compared with 16 weeks for glycidalde-
hyde. Although keratocanthoma are benign, there is a potential for their pro-
gression to malignancy in some animals (Shamberger et al., 1974). This tumori-
genic response is suggestive of a carcinogenic potential for glycidaldehyde in
rodents.
7.1.5 Skin Painting and Subcutaneous Injection-Glycidaldehyde
Thirty 8-week old female ICR/Ha Swiss mice were painted with a 3 percent
benzene solution of glycidaldehyde (100 mg solution/application) 3 times weekly
for life. The median survival of these mice was 496 days. This compares
reasonably well with the range of 324-583 days among this strain of mice under
various other treatments. Sixty animals in a "negative-control" group re-
ceiving benzene as a vehicle had a median survival of 498 days. There were no
tumors reported in the negative control group. Sixteen of 30 glycidaldehyde
treated anfmals developed papilloma or carcinoma (8 with papilloma and 8 with
carcinoma). The first papilloma case occurred 212 days after the initial
glycidaldehyde treatment, and the first carcinoma after 338 days. There was
both a statistically significant increase of skin tumor incidence and a short-
ening of latencies in this experiment.
Forty-one 8-week old female ICR/Ha Swiss mice were painted with a 10
percent acetone solution of glycidaldehyde (lOOmg) 3 times weekly for life (598
days). Six developed papilloma; squamous cell carcinoma subsequently developed
in 3 of the 6. The median survival time of the treated rodents was 445 days.
No skin tumors were seen in 300 control animals treated with acetone alone.
The control animals had a median survival of 526 days (Van Duuren et al.s
1967). There was a statistically significant increase in the tumor incidences
with the glycidaldehyde treatment.
Three groups of 8-week old female ICR/Ha Swiss mice (110, 50 and 30
animals per group) were given subcutaneous injection of tricaprylin (vehicle
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control), 0.1 or 3.3 mg glycidaldehyde in 0.05 ml tricaprylin once weekly for
life. Local sarcoma or squamous cell carcinoma occurred in 0/110, 3/50 and
7/30 of the control, low-dose and high-dose groups, respectively, (p for trend
is less than 0.01 showing a "dose effect" response (Van Duuren et al.t 1966).
The median survival times for the control, low-dose and high-dose groups were
560, 593 and 472 days respectively. Among injection site tumors in the treated
animals there were 2 fibrosarcomas and 1 squamous cell carcinoma in the low-
dose group; and there were 3 fibrosarcoma, 1 squamous cell carcinoma, 1 undif-
ferentiated sarcoma and 2 "papillary adenocarcinoma" in the high-dose group.
There were significant increases in tumor incidence among those animals treated
with glycidaldehyde.
Two groups of 6-week old female Sprague-Dawley rats received 1 or 33 mg
glycidaldehyde in 0.1 ml tricaprylin solution subcutaneously weekly for life
(50 and 20 animals each, respectively). Local sarcoma occurred in 1 c«f 50 low-
dose and 2 of 20 high-dose animals. The median survival times were 558 and 539
days, respectively. In two control groups treated with tricaprylin 1 local
sarcoma was seen (with the survival times ranging from 555 to 565 days). In 2
untreated controls (20 and 50 animals), no tumor was seen (Van Duuren et a!.,
1966, 1967).
7.2 SUMMARY
1. No epidemiological studies evaluating potential chronic health effects of
acrolein are available
2. Animal studies on acrolein carcinogen!city are inadequate.
a. Subcutaneous injection and skin painting studies were of inadequate
design to draw any definite conclusions even though the authors'
reported that there were no statistically significant response.
b. An inhalation study in hamsters was negative with a 4 pptn exposure
level although the design and conduct of the study were inadequate as
a chronic bioassay for carcinogenicity. Thus, the results do not
provide a basis for a definite statement about the human carcinogenic
potential.
c. There are no ingestion studies available.
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3. Animal studies with glycidaldehyde, an acrolein metabolite, provide some
evidence of carcinogenicity.
a. Three skin painting studies in mice showed a statistically signifi-
cant response of a benign tumor type which has the potential to
progress to malignant tumors in animals.
b. Subcutaneous injection studies in mice showed a statistically signifi-
cant response for injection site tumors with a dose-effect
relationship.
c. Subcutaneous injection studies in rats showed a statistically signifi-
cant local sarcoma response with a dose-effect relationship.
4. Several classes of chemicals, aldehydes and dienes, which are structurally
or functionally related to acrolein and a metabolite of acrolein,
glycidaldehyde are alkylating agents and show evidence of being animal
carcinogens.
5. There are some positive results from mutagenicity studies in the bacterial
systems, Drosophila larva and sister-chromatid-exchange studies of mam-
malian cells in culture (see mutagenicity section).
7.3 CONCLUSION
There are no epidemiological studies relating acrolein expsoure to
carcinogenicity in the present data base. The skin painting and subcutaneous
studies of acrolein are inadequate to access carcinogenic potential, similar
studies of its metabolite, glycidaldehyde are, however, supportive of a carci-
nogenic potential. There are two different families of chemical compounds
which may be functionally related to acrolein: aldehydes and dienes. The
first group include chemicals such as formaldehyde and acetaldehyde, which are
considered to be probable human carcinogens by EPA. The second group includes
diene-vinyl compounds, such as ethylene oxide, acrylonitrile, vinyl chloride,
and 1,3 butadiene. These have been classified as probable or known (vinyl
chloride) human carcinogens.
Based upon the (1) structural relationship between acrolein and related
compounds which are potentially carcinogenic to humans, (2) animal studies
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which suggest a carcinogenic potential for a known metabolite of acrolein and
(3) the lack of epidemiological data, acrolein is considered to have "limited"
animal evidence for carcinogenicity. The limited evidence designation derives
from EPA's weight-of-evidence criteria in the EPA Guidelines for Carcinogen
Risk Assessment dated August, 1986. With limited animal evidence, acrolein is
classified as a Group C substance, meaning that acrolein should be considered a
possible human carcinogen.
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7.4 REFERENCES
Feron, V. J.; Kruysse, A. (1977) Effects of exposure to acrolein vapor in
hamsters simulanteously treated with benzo[a]pyrene or diethylnitrosamine
J, Toxicol. Environ. Health 3: 379-394.
Salaman, M. H., Roe, F. J. C. (1956) Further tests for tumor initiating acti-
vity: N, N-Di-(2 chloroethyl)-p-aminophenyl-butyric acid (CB1348) as an
initiator of skin tumor formation in the mouse. Br. J. Cancer 10: 363-378.
Shamberger R J.; Andreone T. L.; Willis, C. E. (1974) Antioxidants and can-
cer IV. Initiating activity of malonaldehyde as a carcinogen. JNCI J.
Natl. Cancer Inst. 53: 1771-1773.
Steiner, P. E.; Steele, R.; Koch, F. C. (1943) The possible carcinogenicity of
overcooked meats heated cholesterol, acrolein, and heated sesame oil.
Cancer Res. 3: 100-107.
U. S. Environmental Protection Agency (1980) Ambient water quality criteria
for acrolein. Washington, DC: Office of Water Regulations and Standards;
PB81-117°277n°' EPA'440/5"80-016- Available from:NTIS, Springfield, VA;
Van Duuren, B. L.; Orris, L.; Nelson, N. (1965) Carcinogenicity of epoxides,
707-7175' Per°Xy compounds- Part IL JNCI J- Natl. Cancer. Inst. 35:
Van Duuren, B. L.; Langseth L.; Orris, L.; Teebor, G.; Nelson, N.; Kuschner,
M. (1966) Carcmogemcity of epoxides, lactones, and peroxy compounds.
IM^T , °M J?sp^onse 1n ePithell'al and connective tissue in mice and rats.
JNCI J. Natl. Cancer Inst. 37: 825-838.
Van Duuren, B. L.; Langseth, L.; Orris, L; Baden, M.; Kuschner, M. (1967a)
Carcmogemcity of epoxides, lactones, and peroxy compounds. V. Subcuta-
neous injection in rats. JNCI J. Natl. Cancer Inst. 39: 1213-1216.
Van Duuren, B. L.; Langseth, L.; Goldschmidt, B. M.; Orris, L. (1967b) Carcino-
gemcity of epoxides, lactones, and peroxy compounds. VI. Structure and
carcinogenic activity. JNCI J. Natl. Cancer Inst. 39: 1217-1228
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8. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS
The health effects of acrolein have been reviewed recently (Beauchamp et
al., 1985; Izard and Libermann, 1978; U. S. Environmental Protection Agency,
1980). A U.S. Environmental Protection Agency Health and Environmental Effects
Profile for Acrolein has been prepared recently in draft form. With respect to
reproductive and developmental toxicity, the effects reported indicate that
acrolein is embryo/fetotoxic, with reduced viability and growth retardation as
major effects. Under certain conditions, teratogenic effects can also be
produced. There is no evidence of direct effects on either the male or female
reproductive systems. However, the evidence is not convincing that such
effects cannot occur since no detailed examination has been done of the effects
of acrolein on either the male or female reproductive system.
8.1 IN VIVO STUDIES
Epstein et al. (1972) reported the results of dominant lethal tests for
174 agents. Data for the individual agents were not presented except the
levels of each agent that produced an increase in embryo loss. In that study,
male mice received a single ip injection of acrolein. They were then caged
with three untreated females each week for eight consecutive weeks. Females
were sacrificed 13 days after being removed from the male. Observations were
made on a number of pregnant females (mating not verified), total implants and
early fetal deaths. Acrolein was reported to cause increased early fetal death
and/or preimplantation loss at both levels tested (1.5 and 2.2 mg/kg). No
other data were presented.
Bouley et al. (1976) exposed male and female (SPF OFA) rats continuously,
by inhalation, to 0.55 ppm acrolein for 26 days. Mating was initiated on the
fourth day of dosing and females were examined 22 days after initiation of
cohabitation. No significant difference was detected in number pregnant,
number of fetuses or mean weight of fetuses. No data were presented in that
publication.
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Claussen et al. (1980) tested the effect of acrolein in New Zealand white
rabbits. Pregnant animals received iv injections of 3, 4.5 or 6 mg/kg on day 9
of gestation. On day 29, the fetuses were examined. At levels that cause
maternal toxicity, a dose-dependent, statistically significant increase in
percent resorptions was reported at 6 mg/kg. There were trends that were not
statistically significant for the proportions of retarded and malformed fetuses
to increase.
Claussen et al. (1980) also injected 10, 20, 40 microliters of 0.84
percent acrolein into the yolk sac of day 9 rabbit embryos in utero and exam-
ined the fetuses at day 29. Significant increases were produced in proportions
of resorptions, retarded and malformed fetuses. Observed malformations includ-
ed deformed and asymmetric vertebrae, spina bifida aperta, ribs deformed and
accreted, and lack and fusion of sternum segments.
Using a different approach, Hales (1982, 1983) injected acrolein or
diethylcyclophosphamide (metabolized to acrolein, but not phosphoramide mus-
tard) into the amniotic fluid of day 13 rat embryos. Embryos were then allowed
to develope in utero until day 20. Data presented for the diethylcyclophos-
phamide study indicate that approximately 50 percent of the control-injected
embryos died. When acrolein was injected, 10 or 100 micrograms resulted in
death of 100 percent and 98 percent of the fetuses, respectively. One micro-
gram resulted in death of 65 percent of the fetuses and produced malformations
in 86 percent of the live fetuses, while 0.1 microgram was no different from
the control. Malformations produced by 1 microgram of acrolein included edema,
hydrocephaly, open eyes, cleft palate, omphalocele, tail, and limb defects.
Injection of diethylcyclophosphamide into the amniotic fluid (Hales, 1983)
was not lethal, but did cause malformations in 14 of 40 embryos. The types of
malformations were similar to those produced by acrolein in embryos treated in
the same way. In both of the studies by Hales (1982, 1983), it appears that
the localized dose to the embryo is high compared to that from im vitro culture
or in the in vivo studies in which dosing was done orally or by inhalation.
8-2 IN VITRO CULTURE STUDIES
Spielmann and Jacob-Muller (1981) removed embryos from mice at the 4-8
cell stage and cultured them in vitro with 100 nM to 1 mM acrolein to the
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blastocyst stage. Embryos that reached the blastocyst stage were cultured for
another 120 h in medium that did not contain acrolein. No statistics are
reported in this paper nor are the values for controls. Therefore, it is not
possible to assess the sensitivity of the test system. Compared to controls,
1 pM acrolein appeared to reduce the percent of embryos producing an inner cell
mass with two germ cell layers (79 percent). Ten uM acrolein inhibited attain-
ment of.inner cell mass (74 percent) and inner cell mass with two germ cell
layers (34 percent). At 100 uM acrolein, no embryos reached the blastocyst
stage.
Schmid et al. (1981) cultured more advanced rat embryos in vitro for 48 hr
to assess the effect of acrolein in the medium on both embryo growth and
differentiation. Embryos were expIanted at day 10.5 of gestation. Acrolein
levels of 100 and 150 uM produced a small, but statistically significant
inhibition of growth. At 200 and 250 uM acrolein, growth and differentiation
were severely inhibited, but no gross structural defects were observed.
Culture in the presence of 350 uM cyclophosphamide, liver microsomes and NADPH
(produces both acrolein and phosphamide mustard) produced embryotoxicity that
included teratogenicity.
Mirkes et al. (1981, 1984) have conducted a series of experiments with
rats that jnay explain the discrepancies between the previously-described
studies. First, day 10 embryos were cultured in vitro for 24 hr with acrolein
in the culture medium (Mirkes et al., 1981). Five ug of acrolein/ml reduced
viability to 89 percent (not significant), while 10 ug/ml resulted in 100
percent, embryo death. No malformations were seen. From those results, as
with the results of Schmid et al. (1981), acrolein appeared to be fetolethal,
but not teratogenic.
Subsequently, Mirkes et al. (1984) utilized dechlorocyclophosphamide
(D-CP) and acrolein (separately) in the same culture system (Mirkes et al.,
1981) and with ip injection of rats on day 10 of gestation. D-CP is
metabolized in vivo to acrolein and dechlorophosphamide mustard (D-PM). D-PM
did not produce any embryotoxic effects in the dose range used in these
experiments. In vitro exposure of day 10 embryos to D-CP for 24-26 hr caused a
reduction in growth measurements beginning at 12.5 ug/ml. At 25 ug/ml, the
number of somites was decreased. A dose-dependent increase in incidence of
malformations began at 6.25 ug/ml (33 percent) and reached 100 percent at 25
and 50 ug/ml. With acrolein (0.005-5.0 micrograms/ml), day 10 embryos were
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incubated in serum-free medium to investigate the possibility that acrolein was
bound to macromolecules in the medium (or on membranes outside sensitive
embryonic sites) and thereby prevented from reaching the sensitive sites.
Under those conditions, a 2 hr pulse of 0.05 ug acrolein in the culture medium
produced growth retardation. Decreased viability was also seen at all concen-
trations with complete lethality seen at 0.5 ug/ml and above. Abnormal
embryos (abnormal flexion) were produced at all levels tested.
Mirkes et al. (1984) also injected D-CP (5, 10, 15, 20 and 50 img/kg) ip
into rats at day 11 of gestation. Fetuses were examined at day 20. The only
effect was a reduction in mean fetal weight at 50 mg/kg. No mention was made
about observations for maternal toxicity.
8.3 CONCLUSION ABOUT REPRODUCTIVE EFFECTS
With respect to reproductive and developmental toxicity acrolein is
embryo/fetotoxic. There is no evidence of direct effects on either the male
or female reproductive systems. However, the evidence is not convincing that
such effects cannot occur since no detailed examination has been done of the
effects of acrolein on either the male or female reproductive system. The
embryo/fetotoxic effects have been observed in jjn vitro culture and embryo
injection .studies. The effects include reduced viability and growth
retardation. Teratogenic effects can also be produced under specialized
conditions. The important consideration appears to be that a sufficient
amount of acrolein actually reaches the sensitive sites within the embryo or
fetus. Possibly due to binding of acrolein to sulfhydryl and other reactive
sites, as well as metabolism of the compound, fetal effects have not been
demonstrated i_n vivo in the absence of maternal toxicity.
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8.4 REFERENCES
Beauchamp, R. 0., Jr.; Andjelkoyich, D. A.; Kligerman, A. D.; Morgan, K. T.;
Heck, H. d'A. (1985) A critical review of the literature on acrolein
toxicity. CRC Crit. Rev. Toxicol. 14: 309-380.
Bouley, G.; Dubreuil, A.; Godin, J.; Boisset, M.; Boudene, C. (1976) Phenomena
of adaptation in rats continuously exposed to low concentrations of aero-*
lein. Ann. Occup. Hyg. 19: 27^32.
•,
Claussen, U.; Hellmann, W.; Pache, G. (1980) The embryotoxicity of the cyclor
phosphamide metabolite acrolein in rabbits, tested in vivo by i.v. injec-
tion and by the yolk-sac method. Arzneim. Forsch. Drug Res. 30: 2080-2083,
Epstein, S. S.; Arnold, E.; Andrea, J.; Bass, W.; Bishop, Y. (1972) Detection
of chemical mutagens by the dominant lethal assay in the mouse. Toxicol.
Appl. Pharmacol. 23: 288-325.
Hales, B. F. (1982) Comparison of the mutagenicity and teratogenicity of cyclo-
phosphamide and its active metabolites, 4-hydroxycyclophosphamide, phos-
phoramide mustard, and acrolein. Cancer Res. 42: 3016-3021.
Hales, B. F. (1983) Relative mutagenicity and teratogenicity of cyclophosphamide
and two of its structural analogs. Biochem. Pharmacol. 32: 3791-3795.
Izard, C.; Libermann, C. (1978) Acrolein. Mutat. Res. 47: 115-138.
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U.S. GOVERNMENT PRINTING OFFICE: 1 986 — 748-003'"4003iO
September 1986 8-5 DRAFT—DO NOT QUOTE OR CITE
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